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The use of proteomic techniques to study the physiology and virulence of _staphylococcus aureus_

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Title:
The use of proteomic techniques to study the physiology and virulence of _staphylococcus aureus_
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Book
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English
Creator:
Rivera, Frances
Publisher:
University of South Florida
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Tampa, Fla
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Subjects / Keywords:
Staphylococcus aureus
Proteomics
Protein extraction
Community-acquired MRSA
Hospital-acquired MRSA
Dissertations, Academic -- Biology -- Masters -- USF   ( lcsh )
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non-fiction   ( marcgt )

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Abstract:
ABSTRACT: Staphylococcus aureus is a bacterial pathogen that is believed to be the most common agent of human infectious disease, causing conditions ranging from common skin lesions to life-threatening illnesses. S. aureus has also shown a remarkable ability to develop resistance to antimicrobial treatment, making infections difficult to treat. In the post-genomic era, proteomic studies analyzing the protein complement of a genome in a particular organism at any given time, have gained real significance. This result is largely due to dynamic changes in protein expression profiles which can lead wide alterations in physiology and behavior. For proteomics, it is necessary to maximize protein concentration and to devise a method that can be easily employed and provide reproducible results. Most proteomic studies of S. aureus involve 2D gel electrophoresis (2-DE); however, 2-DE has many drawbacks. Proteins that are too large, hydrophobic, acidic, or basic are poorly resolved. Multi-dimensional protein identification (MudPIT) allows complex protein samples to be analyzed in solution. As yet, there has not been a study involving solely 2D liquid chromatography followed by mass spectrometric analysis in S. aureus; therefore we sought to catalogue the intracellular proteome and secretome of a commonly used and well-studied lab strain, SH1000. This was conducted during post-exponential and stationary phases of growth so as to understand its adaptation over time by utilizing differential protein synthesis. We found cytoplasmic proteins involved in glycolysis to be highly expressed in post-exponential phase while proteins involved in tricarboxylic acid cycle to be prevalent in stationary phase. We also found x production of agr-regulated secreted toxins and proteases to be upregulated in stationary phase. In addition to this we employed proteomic approaches to quantitatively profile the secretomes of leading clinical isolates of S. aureus, as such a study is currently lacking. These included the two most common hospital-associated S. aureus strains (USA100 and USA200), and the two most common community-associated S. aureus strains (USA300 and USA400). We found agr-regulated proteins are generally upregulated in CA-MRSA strains USA300 and USA400 and surface-associated proteins to be upregulated in HA-MRSA strains USA100 and USA200. This finding concurs with literature regarding transcriptomic studies showing a hyperactive agr in CA-MRSA strains compared to HA-MRSA strains.
Thesis:
Thesis (MS)--University of South Florida, 2010.
Bibliography:
Includes bibliographical references.
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Mode of access: World Wide Web.
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System requirements: World Wide Web browser and PDF reader.
Statement of Responsibility:
by Frances Rivera.
General Note:
Title from PDF of title page.
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Document formatted into pages; contains X pages.

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ABSTRACT: Staphylococcus aureus is a bacterial pathogen that is believed to be the most common agent of human infectious disease, causing conditions ranging from common skin lesions to life-threatening illnesses. S. aureus has also shown a remarkable ability to develop resistance to antimicrobial treatment, making infections difficult to treat. In the post-genomic era, proteomic studies analyzing the protein complement of a genome in a particular organism at any given time, have gained real significance. This result is largely due to dynamic changes in protein expression profiles which can lead wide alterations in physiology and behavior. For proteomics, it is necessary to maximize protein concentration and to devise a method that can be easily employed and provide reproducible results. Most proteomic studies of S. aureus involve 2D gel electrophoresis (2-DE); however, 2-DE has many drawbacks. Proteins that are too large, hydrophobic, acidic, or basic are poorly resolved. Multi-dimensional protein identification (MudPIT) allows complex protein samples to be analyzed in solution. As yet, there has not been a study involving solely 2D liquid chromatography followed by mass spectrometric analysis in S. aureus; therefore we sought to catalogue the intracellular proteome and secretome of a commonly used and well-studied lab strain, SH1000. This was conducted during post-exponential and stationary phases of growth so as to understand its adaptation over time by utilizing differential protein synthesis. We found cytoplasmic proteins involved in glycolysis to be highly expressed in post-exponential phase while proteins involved in tricarboxylic acid cycle to be prevalent in stationary phase. We also found x production of agr-regulated secreted toxins and proteases to be upregulated in stationary phase. In addition to this we employed proteomic approaches to quantitatively profile the secretomes of leading clinical isolates of S. aureus, as such a study is currently lacking. These included the two most common hospital-associated S. aureus strains (USA100 and USA200), and the two most common community-associated S. aureus strains (USA300 and USA400). We found agr-regulated proteins are generally upregulated in CA-MRSA strains USA300 and USA400 and surface-associated proteins to be upregulated in HA-MRSA strains USA100 and USA200. This finding concurs with literature regarding transcriptomic studies showing a hyperactive agr in CA-MRSA strains compared to HA-MRSA strains.
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The Use of Proteomic Techniques to Study the Physiology and Virulence of Staphylococcus aureus by Frances Rivera A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Cellular Molec ular Microbiology College of Arts and Sciences University of South Florida Co Major Professor: Lindsey N. Shaw Ph.D. Co Major Professor: Stanley M. Stevens Ph.D. James T. Riordan Ph.D. Date of Approval: October 22, 2010 Keywords: Staphylococcus aureus proteomics, protein extraction community acquired MRSA, hospital acquired MRSA Copyright 2010 Frances Rivera

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Acknowledgements First of all, I would like to thank Dr. Lindsey Shaw for the unwavering support and motivation to get me thr ough graduate school. It truly is a privilege to have a professor who is proactive and is always trying to find a way to make a project into a successful thesis. I greatly appreciate all the guidance you have provided for me regarding my thesis project a nd all the ceaseless writing in between. I would also like to thanks Dr. Stanley Stevens for teaching me an area of science, mass spectrometry and proteomics, which was completely obscure to me before I began working on this project. Thank you for all th e time you spent teaching me these new techniques and for allowing me unrestricted access to the Proteomics Core Facility in the BiTT Center. Dr. Riordan, thank you for always being available for some much needed advice and cheerfulness and for taking tim e out of your day to be a part of my committee and support me as a graduate student. Then, I would like to thank all of the students, graduate and undergraduate alike, in the Shaw and Stevens labs for moral support, good nature, and a few good uplifting l aughs.

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i Table of Contents List of T ables ................................ ................................ ................................ ..................... v i List of F igures ................................ ................................ ................................ .................. vii i Abstract ................................ ................................ ................................ .............................. ix Introduction ................................ ................................ ................................ .......................... 1 The Staphylococci ................................ ................................ ................................ .... 1 Staphylococcus aureus ................................ ................................ ................. 1 T he Diseases of Staphylococcus aureus ................................ .......... 2 Toxin P roduction by Staphylococcus aureus ................................ ... 2 The Regulation o f Toxin Production ................................ ............... 5 Drug Resistance in Staphylococcus aureus ................................ ..... 6 Trends in S. aureus Infections ................................ ......................... 8 Proteomics ................................ ................................ ................................ ................ 9 Mass Spectrometry ................................ ................................ ................................ 10 Proteome Analysis Techniques ................................ ................................ .............. 11 Proteomic Research and Staphylococcus aureus ................................ ................... 12 Quantitative Proteomic Analysis of S. aureus ................................ ....................... 19 Project Aim ................................ ................................ ................................ ............ 22 Materials and Me thods ................................ ................................ ................................ ....... 24 Buffers ................................ ................................ ................................ .................... 24 Phosphate Buffered Saline ................................ ................................ ......... 24 UDS Buffer ................................ ................................ ................................ 24 Strains, Media, and Growth Conditions ................................ ................................ 24 Cataloguing of Intracellular Proteome and Secretome of S. aureus ...................... 25 Cytoplasmic P rotein E xtraction ................................ ................................ 25 Secreted Protein Extraction ................................ ................................ ........ 26 Trypsin Digestion ................................ ................................ ....................... 26 Mass Spectrometric Analysis of Peptides ................................ .................. 27 Identification of Proteins ................................ ................................ ............ 27 Relative Toxin Production of S. aureus Using iTRAQ ................................ ......... 28 Concentration of Secreted Proteins ................................ ............................ 28 Trypsin Digestion of Secreted Proteins ................................ ..................... 28 iTRAQ Labeling of Peptides ................................ ................................ ..... 29 Mass Spectrometric Analysis of iTRAQ Labeled Peptides ....................... 29 Identification and Quantitation of iTRAQ Labeled Peptides .................... 30 Results ................................ ................................ ................................ ................................ 31

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ii The Application of Mass Spectrometry for Proteome Analysis in S. aureus ........ 31 An Improved Method for the Extraction of Intracellular Proteomes from S. aureus cells ................................ ................................ ................................ ............ 31 Proteomic Analysis of S. aureus SH1000 C ytoplasmic P roteins V ia 1D S DS PAGE C oupled with LC MS/MS ................................ ................................ .. 33 Analysis of the Effects of Solubilization Buffer on Protein Conc entration Yield ................................ ................................ ................................ ....................... 35 The Use of Complex Mixture Analysis Coupled with HPLC Separation to I ncrease the Number of Proteins Identified from Overnight Cultures of S. aureus Cells ................................ ................................ ................................ ........... 36 Specific Cataloging of the S. aureus Intracellular Proteome Using MudPIT anal ysis ................................ ................................ ................................ ................... 38 Derivation of an Improved Method for Extraction of the S. aureus Secretome ................................ ................................ ................................ ............... 41 Specific Cataloging of the S. aureus Secretome Using MudPIT Analysis ............ 43 The Application of Proteomic Methodologies for Quantitative Analysis of Secreted Toxins from a Variety of S. aureus Clinical Isolates .............................. 45 Relative Standard Deviation and Standard Error for the Three Biological Replicates ................................ ................................ ................................ ............... 47 Analysis of Variations in Secretomes of HA MRSA and CA MRSA Strains ................................ ................................ ................................ .................... 47 Gene Ontology Annotations of HA MRSA and CA MRSA clinic al strains ........ 48 Changes in the Expression of M ajor Secreted Proteins between T wo HA M RSA Strains during Post Exponential Growth ................................ ................... 54 Changes in the Expression of Major Secreted Proteins between Two CA MRSA Strains during Post Exponential Growth ................................ ................... 5 5 Changes in the Expression of Major Secreted Proteins between HA MRSA and CA MRSA Strains during Post Exponential Phase ............................ 56 Changes in E xpression of M ajor Secreted Proteins between T wo HA MRSA S trains during S tationary P hase ................................ ................................ 58 Changes in Expression of Major Secrete d Proteins between Two CA MRSA Strains during Stationary Phase ................................ ................................ 59 Changes in Expression of Major Secreted Proteins between HA MRSA and CA MRSA Strains during Stationary Phase ................................ ................... 60 Changes in Expression of Major Secreted Proteins of HA MRSA Strains from Pos t Exponential Phase to Stationary Phase ................................ ................. 62 Changes in Expression of Major Secreted Proteins of CA MRSA Strains from Post Exponential Phase to Stationary Phase ................................ ................. 64 Discussion ................................ ................................ ................................ .......................... 67 Consideration of the Presence of Cytoplasmic Proteins in Secreted Fractions ................................ ................................ ................................ ................. 8 0 Future Directions ................................ ................................ ................................ ............... 82 References ................................ ................................ ................................ .......................... 84

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iii Appendices ................................ ................................ ................................ ....................... 105 Appendix 1 Cytoplasmic P roteins Identified from O vernight C ultures of S. aureus SH1000 S eparated by 1D SDS PAGE ......................... 1 06 Appendix 2. Cytoplasmic P roteins I dentified from O vernight C ultures of S. aureus S H1000 from MudPit A nalysis ................................ .... 116 Appendix 3. Cytoplasmic P roteins I dentified after MudPit A nalysis of SH1000 during P ost E xponential P hase from 2 Biological R eplicates ................................ ................................ ..................... 130 Appendix 4. Cytoplasmic P roteins I dentified after MudPit Analysis of SH1000 during S t ationary Phase from 2 Biological R eplicates ................................ ................................ ..................... 137 Appendix 5. Secreted P roteins Identified after MudPit Analysis of SH1000 during P ost E xponential P hase from 2 B iological R eplicates ................................ ................................ ..................... 14 5 Appendix 6. Secreted P roteins Identified after MudPit Analysis of SH1000 during S tationary P hase from 2 B iological R eplicates ................................ ................................ ..................... 1 4 6 Appendix 7. Chang es in S ecreted Proteins of HA MRSA USA200 C ompared to HA MRSA USA100 during P ost Exponential P hase from 3 B iological R eplicates ................................ ............. 15 3 Appendix 8. Changes in S ecreted P roteins of CA MR SA USA300 C ompared to HA MRSA USA100 during P ost E xponential P hase from 3 B iological R eplicates ................................ ............. 15 8 Appendix 9. Changes in S ecreted P roteins of CA MRSA USA400 C ompared to HA MRSA USA100 during P ost E xponential P hase from 3 B iological R eplicates ................................ ............. 16 3 Appendi x 10. Changes in S ecreted P roteins of HA MRSA USA200 C ompared to HA MRSA USA100 during S tationary Phase from 3 B iological R eplicates ................................ ....................... 16 8 Appendix 11. Changes in S ecreted P roteins of CA MRSA USA300 C ompared to HA MRSA USA100 during S tationary P hase from 3 B iological R eplicates ................................ ....................... 17 9 Appendix 12. Changes in S ecreted P roteins of CA MRSA USA400 C ompared to HA MRSA USA100 during S tationary P hase from 3 B iological R eplicates ................................ ....................... 190

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iv Appendix 13. Statistical Analyses of the F irst Biological R eplicate C omparing HA MRSA U SA100 and HA MRSA USA200 from P ost E xponential P hase ................................ ....................... 20 1 Appendix 14. Statistical A nalyses of the S econd B iological R eplicate C omparing HA MRSA USA100 and HA MRSA US A200 from P ost E xponential P hase ................................ ....................... 20 2 Appendix 15. Statistical A nalyses of the T hird B iologi cal R eplicate C omparing HA MRSA USA100 and HA MRSA USA200 from P ost E xponential P hase ................................ ....................... 2 0 3 Appendix 16. Statistical A nalyses of the F irst B iological R eplicate C omparing HA MRSA USA100 and CA MRSA USA300 from P ost E xponential P hase ................................ ....................... 20 4 Appendix 17. Statistic al A nalyses of the S econd B iological R eplicate C omparing HA MRSA USA100 and CA MRSA USA300 from P ost E xponential P hase ................................ ....................... 20 5 Appendix 18. Statistical A nalyses of the T hird B iological R eplicate C omparing HA MRSA USA100 and CA MRSA USA300 from P ost E xponential P hase ................................ ....................... 20 6 Appendix 19. Statistical A nalyses of the F irst B iological R eplicate C omparing HA MRSA USA100 and CA MRSA USA400 from P ost E xponential P hase ................................ ....................... 20 7 Appendix 20. Statistical A nalyses of the S econd B iological R eplicate C omparing HA MRSA USA100 and CA MRSA USA400 from P ost E xponential P hase ................................ ....................... 20 8 Appendix 21. Statistical A nalyses of the T hird B iological R eplicate C omparing HA MRSA USA100 and CA MRSA USA400 from P ost E xponential P hase ................................ ....................... 2 09 Appendix 22. Statis tical A nalyses of the F irst B iological R eplicate C omparing HA MRSA USA100 and HA MRSA USA200 from S tationary P hase ................................ ................................ .. 2 10 Appendix 23. Statistical A nalyses of the S econd B iological R eplicate C omparing HA MRSA USA100 and HA MRSA USA200 from S tationary P hase ................................ ................................ .. 211 Appendix 24. Statistical A nalyses of the T hird B iological R eplicate C omparing HA MRSA USA100 and HA MRSA USA200 from S tationary P hase ................................ ................................ .. 21 2

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v Appendix 25. Statistical A nalyses of the F irst B iological R eplicate C omparing HA MRSA USA100 and CA MRSA USA300 from S tationary P hase ................................ ................................ .. 21 3 Appendix 26. Statistical A n alyses of the S econd B iological R eplicate C omparing HA MRSA USA100 and CA MRSA USA300 from S tationary P hase ................................ ................................ .. 21 4 Appendix 27. Statistic al A nalyses of the T hird B iological replicate comparing HA MRSA USA100 and CA MRSA USA300 from S tationary P hase ................................ ................................ .. 21 5 Append ix 28. Statistical A nalyses of the F irst B iological R eplicat e C omparing HA MRSA USA100 and CA MRSA USA400 from S tationary P hase ................................ ................................ .. 21 6 Appendix 29. Statistical A nalyses of the S econd B iological R eplicate C omparing HA MRSA USA100 and CA MRSA USA400 from S tatio nary P hase ................................ ................................ .. 21 7 Appendix 30. Statistical A nalyses of the T hird B iological R eplicate C omparing HA MRSA USA100 and CA MRSA USA400 from S tationary P hase ................................ ................................ .. 21 8

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vi List of Tables Table 1. Comparison of D ifferent C ell L ysis T echniques for Extraction of C ytoplasm ic Proteins ................................ ................................ ................. 3 3 Table 2 The T en M ost A bundant C ytoplasmic P roteins I dentified from O vernight C ultures of S. aureus SH1000 S eparated by 1D SDS PAGE ................................ ................................ ................................ ......... 34 Table 3. The T en M ost A bundant C ytoplasmic Proteins I dentified from MudPIT A nalysis of SH1000 ................................ ................................ ..... 38 Table 4. The Ten M ost A bundant Cytoplasmic Proteins I dentified from MudPIT A nalysis of SH1000 during P ost E xponential P hase .................. 40 Table 5. The Ten M ost A bundant C ytoplasmic P roteins I dentified from MudPIT A nalysis of SH1000 during S tationary P hase ............................. 41 Table 6. The Ten M ost Abundant S ecreted P roteins I dentified from MudPIT A nalysis o f SH1000 during P ost E xponential P hase .................. 44 Table 7. The Ten M ost A bundant Secreted Proteins I dentified from MudPIT A nalysis of S H1000 during Stationary P hase ............................. 45 Table 8. A C omparison o f the S ecretomes of USA100 and USA200 during the P ost E xponential P hase of G rowth ................................ ...................... 55 Table 9. Secreted P roteins of USA400 D emonstrating S ignificant C hanges in Expression Compared to USA300 during Post E xponential P hase of G rowth ................................ ................................ ......................... 5 6 Table 10 S ecreted Pro teins of USA300 D emonstrating S ignificant C hanges in Expression C ompared to USA100 during P ost E xponential P hase of G rowth ................................ ................................ ......................... 5 7 Table 11. Secreted P roteins of USA400 D emonstrating S ignificant C hanges in E xpr ession C ompared to USA100 during P ost E xponential P hase of G rowth ................................ ................................ ......................... 5 7 Table 12 Secreted Proteins of USA300 D emonstrating S ignificant C hanges in Expression Compared to USA200 during P ost Exponential P hase G rowth ................................ ................................ ............................. 5 8

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vii Table 13 Secreted P roteins of USA40 0 Demonstrating Si gnificant C hanges in Expression C omp ared to USA200 during P ost Exponential P hase G rowth ................................ ................................ ............................. 5 8 Table 14 Secreted Proteins of USA200 D emonstrating S ignificant C hanges in E xpression C ompared to USA100 during S tationary P hase of G rowth ................................ ................................ ................................ ....... 5 8 Table 15 Secreted P roteins of USA400 Demonstrating S igni ficant C hanges in E xpression C ompared to USA300 during Stationary P hase of G rowth ................................ ................................ ................................ ....... 59 Tab le 16 Secreted Proteins of USA300 Demonstrating S ignificant C hanges in E xpression C ompared to USA100 during S tationary P hase of G rowth ................................ ................................ ................................ ....... 60 Table 1 7 Secreted P roteins of USA400 D emonstrating S ignific ant C hanges in E xpression C ompared to USA100 during Stationary Ph ase of G rowth ................................ ................................ ................................ ....... 61 Table 18 Secreted Proteins of USA300 D emonstrating S ignificant C hanges in E xpr ession C ompared to USA200 during S tationary P hase of G rowth ................................ ................................ ................................ ....... 6 1 Table 19 Secreted Proteins of USA400 D emonstrating S ignificant C hanges in E xpression C ompared to USA200 during S tationary Phase of G rowth ................................ ................................ ................................ ....... 62 Table 20 Secreted P roteins of USA100 D emonstrating S ignificant C hanges in E xpression from P ost E xponential to Stationary P hase ........................ 63 Table 21 Secreted Proteins of USA200 D emonstrating Significant C hanges in E xpression from P ost E xponential to S tationary P hase ........................ 63 Table 22 Secreted Proteins of USA300 D emonstrating S ignificant C hanges in E xpression from P ost E xpone ntial to S tationary P hase ........................ 65 T able 23 Secreted P roteins of USA400 D emonstrating S ignificant C hanges in E xpression from Post Ex ponential to S tationary P hase ........................ 66

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viii List of Figures Figure 1 Workflow of iT RAQ labeling of P eptide S ample ................................ ..... 20 Figure 2. Example of M ass S pectrum R esulting from MS/MS A nalysis of iTRAQ S ample ................................ ................................ ........................... 21 Figure 3. Relative A bundance of R eporter I on I ntensity from iTRAQ S ample ........ 21 Figure 4 1D SDS PAGE A nalysis of Cytoplasmic P roteins from an O vernight C ulture of S. aureus SH1000 ................................ .................... 34 Figure 5. Gene O ntology of HA MRSA USA100 during P ost E xponential P hase ................................ ................................ ................................ .......... 48 Figure 6. Gene O ntology of HA MRSA USA100 during S tationary P hase ............. 49 Figure 7. Gen e O ntology of HA MRSA USA200 during P ost E xponential P hase ................................ ................................ ................................ .......... 50 Figure 8. Gene O ntology of HA MRSA US A200 during S tationary P hase ............. 51 Figure 9. Gene O ntology of CA MRSA USA300 during P ost E xponential P hase ................................ ................................ ................................ .......... 52 Figure10. Gene O ntology of CA MRSA USA300 during S tationary P hase ............. 52 Figure 11. Gene O ntology of CA MRSA USA400 during P ost E xponential P hase ................................ ................................ ................................ .......... 53 Figu re 12. Gene O ntology of CA MRSA USA400 during S tationary P hase ............. 54

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ix Abstract Staphylococcus aureus is a bacterial pathogen that is believed to be the mos t common agent of human infectious disease, causing conditions ranging from common skin lesions to life threatening illnesses S. aureus has also shown a remarkable ability to develop resistance to antimicrobial treatment, making infections difficult to treat. In the post genomic era, proteomic studies analyzing the protein complement of a genome in a particular organism at any given time, have gained real significance. This result is largely due to dynamic changes in protein expression profiles which can lead wide alterations in physiology and behavior. For proteomics, it is necessary to maximize pr otein concentration and to devise a method that can be easily employed and provide reproducible results. Most proteomic studies of S. aureus involve 2D gel electrophoresis (2 DE); however, 2 DE has many drawbacks. Proteins that are too large, hydrophobic acidic, or basic are poorly resolved. Multi dimensional protein identification ( MudPIT ) allows complex protein samples to be analyzed in solution. As yet, t here has not been a study involving solely 2D liquid chromatography followed by mass spectrometr ic analysis in S. aureus ; therefore w e s ought to catalogue the intracellular proteome and secretome of a commonly used and well studied lab strain SH1000 This was conducted during post exponential and stationary phases of growth so as to understand its a daptation over time by utilizing differential protein synthesis. We found cytoplasmic proteins involved in glycolysis to be highly expressed in post exponential phase while proteins involved in tricarboxylic acid cycle to be prevalent in stationary phase. We also found

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x production of agr regulated secreted toxins and proteases to be upregulated in stationary phase. In addition to this we employed proteomic approaches to quantitatively profile the secretomes of leading clinical isolates of S. aureus as suc h a study is currently lacking. These included the two most common hospital associated S. aureus strains (USA100 and USA200), and the two most common community associated S. aureus strains (USA300 and USA400). We found agr regulated proteins are generally upregulated in CA MRSA strains USA300 and USA400 and surface associated proteins to be upregulated in HA MRSA strains USA100 and USA200. This finding concurs with literature regarding transcriptomic studies showing a hyperactive agr in CA MRSA strains com pared to HA MRSA strains.

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1 Introduction The Staphylococci. Staphylococcus is a genus of Gram positive bacteria that have characteristic spherical cells (cocci) in the arrangement of grape like clusters. Indeed the word staphylos is derived from Gre ek, meaning bunch of grapes. These bacteria are facultative anaerobes and are commonly tolerant to high concentrations of salt that normally inhibit the growth of other bacteria [52, 76 ]. Staphylococci include bacteria that are a part of the normal skin flora, such as Staphylococcus epidermidis and others who are medically significant human pathogens, such as Staphylococcus aureus [ 13 ]. Staphylococcus aureu s Staphylococcus aureus is different from other staphylococci due to the rich golden pigmentation of its colonies, as denoted by its name aureus It can be found anywhere in the environment from soil, air, water, and sewage ; as well as inhabiting the skin and mucous membranes of warm blooded animals It is innately resist ant to desiccation, and can live on inanimate objects for extended periods of time [ 105 ]. S. aureus is an opportunistic pathogen, meaning that it is not obligate d to this lifestyle, but more commonly causes disease only upon entering the body via wounds or indwelling devices [ 121 ] The genome of S. aureus commonly consists of a single, circular chromosome of approximately 2.8 million base pairs [ 85 ]. Additionally, strains of S. aureus may also possess mobile genetic elements such as prophages, plasmids and transposons that can be transferred between themselves and other gram positive bacteria [ 162, 114 ].

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2 The diseases of Staphylococcus aureus As a major human pathogen S. aureus is believed to be the most common cause of human disease [ 51 ]. It has the ability to cause a wide range of infections that are both nosocomially and community acquired. These diseases vary from common skin lesions such as wound infections and abscesses to life threatening illnesses, such a s septicemia and endocarditis [ 12 1 ]. Moreover, l ocalized skin infections can spread from the initial site of colonization to other parts of the body via systemic dissemination This can lead to metastatic infections at locations such as the heart muscle, joints, brain, lungs, and bones ; caus ing endocarditis, septic arthritis, brain abscess es pneumonia and ost eomyelitis [ 121 ]. Another subset of disease s caused by S. aureus is toxinos e s. These are caused by particular toxins and include scalded skin syndrome (exfoliative toxins) toxic shock syndrome (toxic shock syndrome toxin) and gastroenteritis (enterotoxins) caused by food poisoning [ 5 ]. Recently, the production of the Panton Valentine leukocidin (PVL) toxin has been associated with fatal necrotizing pneumonia, leading to death w ithin 36 hours [ 70 ]. Toxin production by Staphylococcus aureus S. aureus is an adaptive and versatile pathogen that possesses a diverse arsenal of virulence factors, which it employs to cause disease in a variety of niches within the host. These virulen ce factors are, however, considered accessory elements as they are not crucial to survival. The virulence determinants of S. aureus include cytolytic toxins, sev eral extracellular proteins [152, 91, 146 ], major secreted proteases [ 1 88, 117 178 ], and surf ace associated factors [ 37 ] that work alone or in concert to facilitate disease.

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3 Alpha hemolysin is perhaps the major secreted toxin, and is cytolytic in action, forming pores in the membrane of red blood cells, epithelial cells, endothelial cells, platel ets, and mononuclear immune cells. Membrane damaged caused by direct action of this toxin often results in apoptosis of the target cell, and necrosis of surrounding tissue [ 12 53 19 132 ]. In addition to alpha hemolysin, S. aureus also produces at least 3 other such factors, inc hemolysin is another cytolytic toxin that induces hot cold lysis of red blood cells [ 61 65 ], and is one of the most abundant proteins secreted by S. aureus into the extracellular medium [ 450 hemolysin is a s mall and heat stable protein that can be lytic towards several types of membranes, including those of red blood cells, organelles and even bacterial protoplasts [61 ]. hemolysin and other toxins produced by S. aureus (such as a number of leukocidins) contain two components, commonly termed S and F [ 75, 98 ]. In the case of PVL it has the ability to lyse only neutrophils and macrophages [158 ], whilst the gamma hemolysin can also lyse red blood cells by forming a pore in their membrane [ 98 182 ]. In a ddition to toxins that act directly on the membrane of host cells, S. aureus also produces pyrogenic toxin superantigens that include staphylococcal enterotoxins A E and toxic shock syndrome toxin 1 (TSST 1) [ 17 ]. These superantigens stimulate T cells and cytokine activity that adversely affects the host by causing fever, shock, and immunosuppression [ 137 ]. Exfoliative toxins on the other hand, have a far more specific mode of action, causing scalded skin syndrome via the targeted destruction of human des moglein 1, causing blister formatio n and sloughing of the skin [130 ]. Additionally, there are a total of seven phenol delta toxin), which display leukocidal activity, an d facilitate evasion of the host immune

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4 system. The psm genes have been identified in all sequenced str ains of S. aureus however, the expression of these genes can differ significantly [ 1 94 ]. In addition to specific toxins, S. aureus also produces extracellular enzymes that serve as virulence factors by degrading host tissue, facilitating dissemination and helping to evade the host immune system. Coagulase, for example, converts fibrinogen to fibrin [ 102 ] and is thought to create a clot around localized infections to protect the bacteria from host defenses [ 172 ]. Conversely, staphylokinase is an acti vator of plasminogen [ 32 ] and may serve to release bacteria from these fibrin clots to allow spreading to other sites of the body [ 93 ]. S. aureus also produces a series of extracellular proteases that are commonly produced as a pre proenzyme which are enz ymatically inactive, and require activation in the extracellular millieu [ 178 ]. Perhaps the most abundant of these is the SspA or V8 serine protease, which cleaves immunoglobulins, thereby inhibiting host defense mechanisms [ 157 ]. It also seems to be imp ortant for transition from adhesive to non adhesive phenotypes via degradation of fibronectin binding proteins, and other proteins on the surface of S. aureus cells [ 100 126 ]. In addition to the V8 protease there are many other serine protease like enzy mes ( SplA through S plF ) that have trypsin like catalytic activity, but are not cleaved by other enzymes for activation. S. aureus also secretes two cysteine proteases, known as staphopain A and staphopain B. Staphopain A has strong activity against elast in, suggesting a role in the pathogenesis of S. aureus [ 154 ], whilst staphopain B ( sspB ) cleaves fibronectin, fibrinogen, and kininogen, suggesting that it may serve to allow the spread of S. aureus during infection [ 35, 178 ]. The final major proteolytic enzyme is a metalloprotease, known as aureolysin, which has activity against plasma protease inhibitors, suggesting a role in the pathogenesis of S.

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5 aureus [ 154, 155 ]. A further protease like enzyme, lipase, exists, which is a glycerol ester hydrolase tha t cleaves long chain triacylglycerols [ 109 ], and may have a role in the in vivo nutrition of S. aureus [ 166 ]. The regulation of toxin production The success of S. aureus as a pathogen is commonly attributed to its vast array of virulence determinants, which are tightly controlled by a central global regulator called agr (for accessory gene regulator) [ 4, 91 144, 145, 152 ]. a gr is a quorum sensing, two component regulator that is maximally expressed during post exponential growth, where it represses su rface and attachment proteins and induces the transcription of toxins and exoproteins [ 202 ]. a gr is of particular importance to S. aureus as cells which contain a mutated or nonfunctional agr gene are attenuated in virulence in animal models of infection [ 205 ]. The agr effector molecule is a small regulatory RNA, known as RNAIII, which also encodes the delta hemolysin. RNAIII acts as a regulator of target genes [ 91 14 6 ] by binding to target mRNA molecules and either stabilizing them, or targeting them for destruction [ 135, 113 186 ]. The most significant of these interactions is thought to be for a repressor of toxin production, known as Rot. RNAIII binds to, and inhibits Rot mRNA translation, thereby facilitating the upregulation of extracellular viru lence factor synthesis [ 66 18]. RNAIII also serves to negatively regulate the synthesis of protein A and the fibronectin binding proteins, which are used for adhesion [ 9 1 143, 171 ] In addition to agr there are a number of other important regulators o f toxin production in S. aureus with perhaps the next most important being SarA. SarA ( for staphylococcal accessory regulator ) is a DNA binding protein that acts as a transcriptional regulator of a

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6 variety of different genes that play a role in pathogene sis and metabolic processes [ 9, 29, 30, 49 ]. Specifically, it controls the expression of surface and secreted proteins by binding to the promoter regions of target genes and either repressing them, or bringing about their upregulation. Its targets includ e other regulatory systems, such as agr [ 30, 160 ], and virulence genes, such as protein A ( spa ), fibronectin binding protein A ( fnbA ) and the major extracellular proteases [ 64 20 8 16 ]. The f inal major regulator of toxin production is the two component s ystem SaeRS (for S. aureus exoprotein expression ). SaeRS modulates the production of secreted proteins such as coagulase and alpha hemolysin at the level of transcription [ 721 ] The regulation of these exoproteins by SaeRS overlaps that of the Agr regulon [ 72 ], though it has no direct effect on the expression of agr or sarA [ 71 ]. Drug resistance in Staphylococcus aureus I was introduced for the treatment of bacterial infections; however, s hortly after S. aureus strains appeared that were resistant to this antibiotic. [ 104 ] Since this time, the same has proven true for almost every other antibiotic, including erythromycin, tetracycline, and streptomycin [ 41 7 8 ]. Methicillin introduced in 1960 was used as an effectiv e treatment for infections caused by antibiotic resistant strains of S. aureus This antibiotic differs from penicillin as its unique structure blocks lactamases. H owever, methicillin resistant S. aureus (MRSA) str ains were discovered shortly thereafter in 1961 [ 83 ]. Methicillin resistance is conferred by the mecA gene, which is harbored on a mobile genomic island known as the staphylococcal cassette chromosome mec (SCC mec ) [89 ]. Given the widespread distribution of MRSA strains, and limited therapeutic options, g l ycopeptide

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7 antibiotics, such as v ancomycin (Van), ha ve been commonly used as a last resort drug [ 84 ] It acts by irreversibly binding to D ala D ala residues of N acetylmuramic acid pentapeptide precurs ors Binding of these precursors by Van prevents peptide cross linking between growing layers of peptidoglycan, and consequently weakens the cell wall of gram positive bacteria [ 8 ]. Expectedly, in 1996, a S. aureus isolate in Japan was found to be resist ant to increased concentrations of Van [ 84 ]. Th e new strain was termed Van intermediate S. aureus (VISA) and c ases of VISA soon began making an appearance throughout Europe, Asia, and the US A; and are now present worldwide [ 153 ]. Since 2002, nine cases of Van resistan t Staphylococcus aureus (VRSA) have appeared in the United States [2, 3, 59 196 ] The difference between VISA and VRSA strains can be found at the molecular level, with VISA isolates possessing mutations in existing genes, while VRSA have acquired exogenous genetic material. The intermediary resistance of VISA strains to Van is speculated to be modulated by synthesis of a thicker cell wall that contains an increased number of D ala D ala residues, which trap Van molecules in the outer laye rs of the cell wall, keeping it from accessing cell wall precursor targets in the cytoplasm [ 79 ]. Van resistance in VRSA isolates on the other hand, is attributed to expression of vanA found on a conjugative plasmid that is likely obtained from Van resis tant Enterococcus faecalis (VRE) by horizontal gene transfer [ 196 ]. The Van resistance of VRSA strains is achieved by changing the D ala D ala pentapeptide residues attached to N acetylmuramic acid to D ala D lac. Van having a lower affinity for D ala D lac, consequently does not inhibit synthesis of the altered cell wall in VRSA isolates [ 122 ].

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8 Trends in S. aureus i nfections S. aureus infections have commonly been confined to health care facilities [ 4 3 ]. Historically, such infections affect the im munocompromised, the young or the very old. These hospital acquired methicillin resistant S. aureus (HA MRSA) strains are highly resistant to antibiotics, making HA MRSA infections very difficult to treat. The two most common HA MRSA strains in the Unite d States are CDC PFGE (pulse field gel electrophoresis) types USA100 and USA200 [ 127 ]. Recently, community acquired methicillin resistant S. aureus (CA MRSA) infections have been reported in individuals with no ties to health care facilities [1, 94 138 ]. These CA MRSA strains appear to be far more virulent than HA MRSA, and are especially significant because they cause infections in young, healthy individuals with no predisposing factors [ 24, 54 ] There are a number of genetic differences between HA MRS A and CA MRSA which might explain the vast differences in their transmission, spread and pathogenesis. Specifically, CA MRSA strains usually possess SCC mec types IV, V, or VII [ 194 ], whilst HA MRSA strains commonly harbor SCC mec types I III. There are cu rrently a total of seven SCC mec variations with the common CA MRSA types being the smallest, probably facilitating the expedient transfer of these elements between S. aureus strains [ 164 ] HA MRSA strains usually possess the larger SCC mec types I, II, an d III, which are more burdensome energetically, and result in the slower growth of encoding strains [ 147, 162 ]. Another distinguishable feature of CA MRSA is the presence of the bacteriophage encoded Panton Valentine leukocidin toxin (PVL) [ 193 ] PVL is an exotoxin that creates pores in the membrane of leukocytes, effectively destroying them, and exposing surrounding cells to their damaging contents, commonly leading to tissue necrosis [36,

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9 18 9 ] Recent work has suggested that PVL is not the only toxin i nvolved in CA MRSA hypervirulence, as PVL negative and PVL positive CA MRSA strains have been found to be equally virulent in sepsis and abscess models of infection using mice [ 193 ]. Supporting this finding, Wardenberg et al. recently demonstrated that hy perexpression of hemolysin, not PVL, is the essential factor in necrotizing pneumonia infections [20]. Further to this, a study by Diep et al., showed that CA MRSA USA300 strains contain another mobile genetic element known as the arginine catabolic mob ile element (ACME). This locus has been suggested to contribute to the rapid growth and survival of USA300, giving it a selective advantage in disease causation [ 116 ]. Transcriptome studies have demonstrated that despite sharing a core genome of approxim ately 82%, S. aureus strains demonstrate huge variations in gene expression profiles and pathogenesis [ 116 120 ]. Thus the virulence of S. aureus is likely not determined by the presence or absence of variable genetic elements, but by alterations in the t emporal expression of innate core factors. A key example of this is that the marked hypervirulence of CA MRSA strains appears to be attributable to differential gene expression resulting from elevated activity of regulators such as agr. This regulator ma inly controls the expression of secreted toxins and surface associated proteins that help the organism adhere to the host and become invasive. Despite their increased virulence however, CA MRSA strains currently have reduced antibiotic resistance compared to HA MRSA. With that said, CA MRSA resistance is on the rise, and these strains have recently been reported to be replacing HA MRSA strain in healthcare facilities [ 195 ]. Proteomics. Experimentally, genomics is the study of entire genomes, whilst t ranscriptomics is the study of mRNA transcription giving insight s into the gene

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10 expression of a particular organism at a given time. Proteomics, the identification of entire protein sets present in biological samples complements these approaches, and is a relatively recent development in the post genomic era. Because mRNA levels do not always correlate with the synthesis of proteins proteomics has become an extremely useful tool for the study of differential protein expression as a result of varying ph ysiological conditions. This discipline has gained significance because proteomes are highly dynamic, and protein expression profiles can change relatively quickly. Understanding changes in a given proteome from a particular organism can provide much in sight into its behavior, physiology and interaction with its environment Mass s pectrometry. A major tool used in proteomic studies is mass spectrometry. This technique uses protein samples cleaved by a site specific enzyme, such as trypsin, for the iden tification of proteins present in a biological sample. Following digestion, samples are washed of salts and detergents, which can interfere with the formation of ions in the mass spectrometer, and therefore affect the determination of molecular mass [ 87 ] After de salting, peptides are fractionated by high performance liquid chromatography (HPLC) using a strong cation exchange (SCX) column. Each resulting fraction is then ionized by MALDI (matrix assisted laser desorption ionization) or ESI (electrospray ionization), coupled to a mass spectrometer. A MALDI mass spectrometer utilizes a laser to produce ions from a sample that has been mixed with a matrix and crystallized on a sample plate. The most common matrices used for MALDI TOF peptide analysis incl ude 2,5 cyano 4 hydroxycinnamic acid [174 136 ]. The sample plate is then placed inside the mass spectrometer in a vacuum at high voltage, and the matrix absorbs laser radiation and rapidly breaks down. This causes the m atrix to

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11 expand into gas phase, bringing analyte molecules with it, and the matrix dissociates, leaving a pure and ionized analyte [ 44 ]. Mass analysis of the peptides by time of flight (TOF) entails accelerating ions, resulting in different ion velocities that are inversely proportional to the square root of the mass to charge ratio. These ions are separated in the flight tube and the time of their arrival to the detector is then converted to the mass of the ions [ 3 4, 55 ]. An ESI mass spectrometer involv es the spraying of samples from a needle at high potential, into a chamber at atmospheric pressure. Droplets of the sample in solution are formed, and the solvent is evaporated, creating a stream of ions from the sample to be analyzed by the mass spectrom eter [ 55, 56 ] Electrospray ionization was introduced in the 1980s as a new method for ionizing and introducing peptides into gas phase, and spraying peptides across a potential difference [ 43 ] between a capillary and the opening of a mass spectrometer [ 5 6 ]. Once in the gas phase, peptide fragments enter a collision chamber and are collided with atoms of inert gas, such as argon, producing col lision induced dissociation [197 ]. As a result, peptides are mainly fragmented at amide linkages, and the resulti ng fragments are sent to a mass analyzer to detect the fragments according to mass to charge ratio The resulting mass spectrum is made of ions that are characteristic of a sequence of amino acids in a particular peptide. The mass spectral data from thes e peptides is then compared to protein sequences contained in databases specific to the originating organism [ 43, 174, 204 ]. Proteome analysis t echniques. In order to generate proteome samples for mass spectrometric analysis two major approaches have bee n used. The first, and perhaps most common of these, is two dimensional gel electrophoresis (2 DE). Using this technique, proteins are first separated by differences in electric charge (isoelectric

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12 focusing), and then perpendicularly by SDS PAGE. Separat ed protein spots are then visualized by staining, and those protein spots of interest are excised from the gel and analyzed by mass spectrometry [74 106 ]. Though the data obtained from 2 DE is of significant value, it has many drawbacks. P roteins that a re very large, hydrophobic, acidic, or basic are poorly resolved [ 80 ]. For this reason a newer method, known as multi dimensional protein identification ( MudPIT ) [ 39 ] has become more commonplace. MudPIT combines multi dimensional liquid chromatography w ith tandem mass spectrometry; allowing complex protein samples to be analyzed. Experimentally, proteomes are digested with trypsin to generate peptide fragments. Using HPLC, the peptide fragment mixture is then applied to a column packed with a strong cat ion exchange (SCX) resin. The eluted peptides collected in fractions are then analyzed using a mass spectrometer. After the mass spectral data are obtained, database searches are performed which identify the proteins from the original samples. This tech nique is very efficient because it is a relatively expedient method of analysis, and the two dimensional chromatographic separation of peptides also increases the number of proteins identified [ 80, 148 ]. Proteomic r esearch and Staphylococcus aur eus The first proteomic study of S. aureus protein expression was conducted by Ziebandt et al. almost a decade ago [ 208 ]. The study compared extracellular proteins of two closely related strains, RN6390 (laboratory derived) and COL (clinical isolate) us ing 2D gel electrophoresis and N terminal sequencing, or MALDI TOF, for the identification of proteins. Eighteen secreted proteins were identified in COL [ 208 ], an early hospital acquired methicillin resistant S. aureus isolate [ 69 ], of which nine had not yet been found in the laboratory strain

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13 RN6390. Comparing the cytoplasmic proteins of RN6390 and COL showed only a few differences between the two strains, as opposed to the more striking difference in expression of secreted proteins [ 208 ] This was a p articularly important finding given the importance of secreted proteins in the virulence of S. aureus This study also compared the secretomes of S. aureus mutants lacking the global regulator of virulence, S arA, in an RN6390 background. Five proteins (g lycero hemolysin, lipase, and immunodominant antigen A) were found to be positively regulated by SarA, with a further twelve shown to be negatively controlled by this regulator, including secreted proteases such as staphopain, V8 pr otease, and aureolysin. In this same study, proteomic techniques were employed to understand the role of the major alternative sigma factor, SigmaB. SigmaB is involved in induction of the general stress response of other gram positive bacteria [ 81 ] and i s thought to have a role in the virulence of S. aureus [25 142 ]. In a s igB hemolysin, lipase, and thermonuclease were produced at significantly higher levels than in the wild type [28 111 ]. Conversely, nine other secreted proteins were found to be negatively regulated by SigmaB (including enterotoxin B, serine protea hemolysin, and glycerol ester hydrolase) [ 208 ]. In 2002, Bernardo et al used different forms of SDS PAGE, followed by MALDI TOF analysis, to identify the secreted proteins of three strains: methicillin sensitive ATC29213, methicillin resistant ATC 43300, and a clinical isolate provided from the University Clinic of Cologne in Germany [ 11 ]. What was found was that important proteins such as the major autolysin, staphylococcal nuclease, and three hypothetical proteins could not be reso lved by 2D gel electrophoresis These proteins in particular

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1 4 have high pI values ; 9.65, 9.21, 9.21, 9. 17, 9.28, respectively As mentioned before 2D gel electrophoresis is not an effective technique for resolving proteins that are too basic. Subsequent ly, Bernardo et al found that these proteins could be resolved by conventional 1D gel electrophoresis (SDS PAGE) and identified by MALDI TOF mass spectrometry. Compared to 2D gel electrophoresis, 1D gel electrophoresis requires less protein, yielded more reproducible results, and allowed for the detection of some alkaline proteins. 2D gel electrophoresis, however, had the ability to identify proteins in low abundance when compared to 1D gel electrophoresis [ 11 ]. In a later study by Kohler et al, cytoplas mic proteins of S. aureus COL were analyzed using two methods: 2D gel electrophoresis and multidimensional liquid chromatography followed by mass spectrometry using a matrix assisted laser ionization time of flight (MALDI TOF) mass spectrometer. The comb ination of these two methods identified a total of 1123 cytoplasmic proteins. Four hundred and seventy three proteins were identified by 2D gel electrophoresis and belonged to the transcriptional and translational machinery, aerobic respiration and fermen tation pathways, and some biosynthetic pathways. A gel free method, followed by MALDI TOF mass spectrometry, yielded an additional 650 proteins that mainly belonged to metabolic pathways, and included alkaline and hydrophobic proteins that were not seen i n the 2D g el electrophoresis analysis [107 ]. In S. aureus the expression of virulence genes is regulated in a highly coordinated manner. Cell surface associated proteins are expressed during exponential growth, while secreted virulence factors are e xpressed during post exponential growth [ 202 ] In another study by Z iebandt et al, again using 2 DE, 70 secreted proteins were found to be affected by a mutation in another major global regulator of toxin production, agr Amongst these

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15 affected proteins, many virulence factors such as proteases, toxins, lipases, and staphylokinase were found to be upregulated by agr Apart from upregulating the expression of secreted proteins in late exponential and stationary phases, agr seemingly represses the expressi on of proteins usually present in cells during exponential grow th such as immunodominant antigen A, autolysins, and protein A [ 206 ] In an attempt to quantify the entire S. aureus proteome, Becher et al. analyzed cytoplasmic, membrane bound, surface ass ociated, and secreted proteins in growing and non growing cells of S. aureus COL by a combination of 2 DE and mass spectrometric analysis [ 10 ]. Cytoplasmic proteins were metabolically labeled, and, using a combination of 2 DE and GeLC MS/MS (a fusion of 1 DE and LC MS/MS analysis), a total of 1796 proteins were identified, representing ~67% proteomic coverage of S. aureus In the transition from exponential to stationary phase of growth, it was apparent that ribosomal proteins, translational factors, and some enzymes involved in amino acid synthesis were significantly down regulated. Proteins that were found to be upregulated in stationary phase include PEP carboxykinase (PckA, a gluconeogenetic enzyme), enzymes involved in the TCA cycle, and members of t he phosphate regulon. Membrane proteins were analyzed in this study via a combination of GeLC MS/MS [ 45 ] and trypsin shaving [ 200 ]. The GeLC MS/MS approach resulted in the contamination of membrane proteomes with cytoplasmic proteins, whilst the shaving method, using trypsin, revealed only membrane proteins in the isolated fractions. With the membrane targeted approach, a total of 125 proteins were identified including integral as well as peripheral proteins, and accounted for ~56% of all predicted membr ane proteome. Specifically, proteins associated with phosphotransferase systems (PTS), the glycerol uptake facilitator (GlpF),

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16 and transporters involved in uptake of C 3 carbon sources were found at increased levels during the stationary phase [ 10 ]. Membr ane proteins found to be up regulated in stationary phase included ABC transporters involved in the import of amino acids and oligopeptides, whilst those found in decreased levels included high affinity iron compound ABC transporters. Interestingly, the h ighly conserved Sec secretion machinery was found to be present at constant levels from the exponential to stationary phases of growth [ 10 ]. This study also used a biotinylation approach for surface exposed cell wall associated proteins analysis. The adv antage of this is that such proteomes can be purified with minimal contamination by other subproteomic fractions. A total of 146 surface associated proteins were identified, including membrane proteins, proteins that are covalently attached to the cell wa ll, lipoproteins, cell wall associated proteins containing signal peptides, and cell wall associated proteins that were also found to be secreted [ 10 ]. Proteins involved in adhesion such as fibronect in binding proteins (Fnbps) [171 ] and fibrinogen binding protein (ClfB) [126 ] were mainly upregulated in the exponential phase of growth. Conversely, levels of ClfA and immunodominant protein IsaB were significantly increased in stationary phase [ 10 ]. The last fraction, secreted proteins, was analyzed using a GeLC MS/MS approach. With this technique, a total of 57 secreted proteins were identified, mainly during stationary growth. Specifically, there was an observable switch from the production of proteins involved in adhesion, biofilm formation, and cell in vasion during exponential growth, to the production of virulence factors such as toxins, enzymes, and superantigens in stationary growth [ 10 ]. This study represents the most comprehensive quantification of the S. aureus proteome to date.

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17 In 2010, a st udy by Hempel et al. quantitatively profiled the SigmaB dependent expression of surface associated proteins by metabolic labeling, biotinylation, and GeLC MS/MS A total of 49 proteins were found to be regulated by SigmaB [ 82 ], 21 of which were known to b e dependent or influenced by SigmaB at the transcriptional level [ 14, 14 9 206 ]. The remaining 28 proteins have not been described as being modulated by SigmaB activity. Consistent with the literature, fibrinogen binding proteins ClfA and ClfB were found to be strongly decreased in the sigB mutant, whilst there was an accumulation of surface associated proteins noted, mainly in stationary phase [ 82 ]. This negative influence of SigmaB must be indirect as sigma factors can only act directly as positive reg ulators [ 67 206 ] In a 2010 study by Ziebandt et al., the secretomes of 25 clinical isolates of S. aureus revealed an extreme heterogeneity of secreted protein expression due to genomic plasticity, and differences in regulation. Of the 63 identified se creted proteins, only 7 proteins ( IsaA, Lip, LytM, Nuc, SA0620, SA2097, and SA2437 ) were commonly produced by clinical isolates of different clonal lineages [ 207 ]. This finding is likely explained by the observation that up to 30% of the genomes of differ ent S. aureus isolates consist of variable mobile genetic elements, such as pathogenicity islands, lysogenic bacteriophages plasmids, and transposons [198 ]. Virulence determinants are commonly encoded on these mobile genetic elements, thus facilitating p roteome variability [ 207 ]. Finally, recent proteomic analyses in S. aureus have been focused on characterization of cell surface proteomes. Cell surface proteins are directly in contact with the extracellular environment and might perhaps serve as the lar gest group of targets for vaccine or antibody development. These surface

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18 study by Dreisbach et al. in 2010 [ 45 ]. The aim of this study was to refine and optimize the isolation technique of surface protein s to increase the number of proteins identified and reduce the number of contaminating cytoplasmic proteins due to cell lysis. Seemingly, the most effective method for identifying surface exposed proteins is by shaving these proteins with trypsin followed by MS analysis [ 176, 180, 18 5 ]. The study compared different buffers and trypsin digestion conditions to determine the best method for the isolation of surface exposed proteins. The trypsin shaving technique was used to analyze S. aureus strains from di fferent clonal lineages, including the laboratory strain RN6390, early clinical isolates Newman and COL, and the CA MRSA strain USA300. After mass spectrometric analysis, 39 surface exposed proteins were found in RN6390, 59 in Newman, 47 in COL, and 24 in USA300. Taken together, a total of 96 surface exposed proteins were detected from all four strains, 5 proteins of which contained an LPXTG motif for covalent attachment to the cell wall. A further 17 were predicted to be completely secreted into the extr acellular environment, and 2 other secreted proteins also had motifs for cell wall binding. Intriguingly, there were two toxins that were detected on the surface of the cell that are not predicted to be secreted. Additionally, 16 ribosomal proteins and 17 proteins involved in metabolism or possessing housekeeping functions were also found in the surfacome [ 45 ]. The fact that only 7 proteins (including GAPDH and FBA) were common to all four strains illustrates the heterogeneity of S. aureus strains and the ir associated sub proteomic fractions. In a similar study published at the same time, Ventura et al. analyzed the surface exposed proteome (terming it the surfome ) of the CA MRSA USA300 strain known as LAC (LA County Clone) [ 1 90 ]. A total of 113 surface exposed proteins were identified in LAC during the post

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19 exponential phase of growth, the most abundant of which was Protein A. Surprisingly, a novel uncharacterized two component leukotoxin was also detected in the surfome, termed LukGH. This newly ident ified bipartite toxin was found to have a significant role in pathogenesis, specifically via pore formation in human PMNs [ 1 90 ]. Quantitative proteomic a nalysis of S. aureus There are several methods for determining relative protein expression within a g iven organism, the most popular method being 2 DE. Using this technique, proteins are first separated by differences in electric charge (isoelectric focusing), and then perpendicularly by SDS PAGE. Separated protein spots are then visualized by staining, and those protein spots of interest are excised from the gel and enzymatically digested with trypsin. The resulting peptides are then analyzed by mass spectrometry. This technique, however, has many drawbacks. P roteins that are very large, hydrophobic, acidic, or basic are poorly resolved. Another popular method for determining relative protein expression is known as iTRAQ [ 1 68 ] iTRAQ (isobaric tag for relative and absolute quantitation) is a peptide labeling technique used for identification and rel ative quantification of proteins from different samples in one single experiment. The iTRAQ 4 plex analyzes four different samples in one single experiment after labeling peptides from each sample with an isobaric tag reagent, which includes a reporter mo iety and a balance moiety. The mass of the balance moiety from each reagent is different in order for the total mass of the balance and reporter moieties to equal 145 because mass of each reporter moiety is different [168] A workflow for the iTRAQ label ing of peptides is outlined in Figure 1.

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20 Figure 1. Workflow for iTRAQ labeling of peptide samples After MS/MS analysis, which includes high collision induced dissociation, the balance moieties are lost, leaving only the reporter tags. Becaus e of the unique isotopic distribution of each reporter tag (114, 115, 116, and 117), it is possible to determine relative protein expression across the different samples in a single experiment. An example of a mass spectrum resulting from MS/MS analysis o f an iTRAQ sample is shown in Figure 2 Combination of four samples into one sample 114 115 116 117 MS /MS analysis Labeling of samples with iTRAQ reagents Four separate samples after reduction, alkylation, and trypsinization

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21 Figure 2. Example of mass spectrum resulting from MS/MS analysis of iTRAQ sample Differential expression of a particular protein across the four samples can be determined by the r elative abundance of each rep orter tag resulting from MS/MS analysis. Figure 3 displays an example of the relative intensities of each reporter tag indicating relative protein e xpression. Figure 3. Relative abundance of reporter ion intensity from iTRAQ sample A 2005 study by Ch oe et al., compared 2 DE and shotgun proteomic analysis with isobaric tagged samples (iTRAQ) of Escherichia coli for relative protein expression after induction of rhsA [ 31 ], believed to be involved in biosynthesis and export of capsular polysaccharides [1 29 ] The purpose of this study was to determine the best method for biological reproducibility that minimizes technical variability. It was found that shotgun proteomic analysis with iTRAQ samples provided the best reproducibility with minimal outlier data [ 31 ]. In 2006, Wolff et al. studied the heat shock response of exponentially

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22 growing Bacillus subtilis cells by gel free iTRAQ labeled samples and by the 2 DE approach for quantitation of proteins involved in this response It was found that the proteomi c analysis of gel free iTRAQ label ed protein samples resulted in the identification of more proteins, and was more reproducible [ 201 ]. With regards to S. aureus the first such quantitative proteomic analysis was focused on Van resistance. Van has commonl y been used as a last resort drug to treat MRSA infections [ 84 ]. It acts by preventing peptide cross linking between growing layers of peptidoglycan, consequently weakening the cell wall [ 8 ]. I n 1996, a S. aureus isolate in Japan was found to be resistan t to intermediate concentrations of Van [ 84 ] and are now present throughout Europe, Asia, and the USA [ 153 ]. Drummelsmith et al. in a 2007 study developed a method for the rapid identification of potential biomarkers of VISA strains with the use of 2 DE and iTRAQ tagging. A VISA strain, Mu50, was compared to a Van sensitive MRSA strain, CMRSA 2, for relative protein expression of cytoplasmic proteins. It was found that expression of SAV2095, known as SceD like protein, is consistently and significantly increased in VISA strain s This protein has a potential association with the cell wall which is known to be thickened in such isolates [ 47 ] Project Aim The studies detailed above reveal a concerted effort to develop the most efficient and productive methods for discovering proteins that are differentially expressed in S. aureus Each provides knowledge and insight into the intricate phenotypic switching mechanisms employed by S. aureus that make it such a successful human pathogen. Previous studies have been fraught with problems, including the use of 2D gel electrophoresis that is not efficient for resolving proteins that are too hydrophobic, large, acidic, or basic. To date, there has not been a proteomic study solely based on 2D liquid

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23 chromatog raphy followed by mass spectrometry (2DLC MS) Studies that do involve some 2DLC MS analysis use a urea concentration that is too low for solubilization of insoluble proteins do not include filter sterilization of supernatants to remove bacterial cells a nd ensure purity of secreted proteins, and centrifuge secreted proteins during precipitation at a speed that is too low to ensure maximal protein recovery [ 1 76 206, 208 ]. Appropriate method refinement is therefore important for obtaining reproducible dat a across several biological replicates of a sample In this work we seek to, through method refinement, catalogue the intracellular proteome and secretome of a commonly used and well studied lab strain of S. aureus SH1000 during post exponential and stati onary phases of growth to provid e an insight into its physiology; and how it adapts to its changing environment over time by utilizing differential protein synthesis. Additionally, we aim to profile the secretomes, of clinically significant strains curren tly afflicting individuals in hospitals (USA100 and USA200) and in the community (USA300 and USA400) settings. A proteomic analysis encompassing the entire secretomes of these clinically relevant strains is lacking. With this proteomic approach we hope t o not only identify but also quantify the production of secreted proteins that enable this organism to swiftly infect and cause disease in a patient. It is possible these analyses could lead to the identification of antigens and the development of protect ive vaccines against S. aureus

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24 Materials and Methods Buffers Phosphate buffered saline 0.8% sodium chloride 0.14% disodium phosphate 0.02% potassium chloride 0.02% potassium dihydrogen phosphate UDS buffer 6M urea 5mM DTT 1% SDS 50mM Tris HCl Strai ns media, and growth conditions For cataloguing of the S. aureus intracellular proteome and secretome, the common lab strain SH1000 was used. For relative toxin expression, the strains USA100 (N315), USA200 (MRSA252), USA300 (LAC), and USA400 (MW2) were used. Overnight cultures of the wild types (SH1000, USA100, USA200, USA300, and USA400) were grown in 3% tryptic soy broth (TSB).

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25 To obtain a sample from each strain at a specific phase of growth, 1 ml of the overnight culture was added to 100 ml of 3% TSB. The new culture was allowed to grow for 3 hours and then the optical density of the new culture was standardized to 0.05 in 400 ml of TSB. The new synchronous culture was allowed to grow until the desired phase of growth: post exponential (5 hours) and stationary (15 hours). When incubating, the cultures we 1:2.5 ensuring adequate aeration of cultures. Cataloguing of intracellular proteome and secretome of S. aureus Cytoplasmic protein extraction The synchronized cultures of SH1000 were allowed to grow until post exponential and stationary phases of growth C ultures were then centrifuged for 10 minutes at a speed of 4,150 rpm. The pellets were washed three times with PBS pH 7. 4 and finally resuspended in 1 m l of UDS buffer pH 8, with 0.1 mm disruption glass beads. C ells were then lysed using a BioSpec Mini BeadBeater for a total of 4 minutes with intermittent cooling phases. The lysed cells were then centrifuged for 10 min utes at 13,300 rpm The supernatants were collected and centrifuged again to prevent contamination from non cytoplasmic proteins and ensure purity of the intracellular protein fraction s, for 10 minutes at the same speed and then transferred to new tubes P rotein concentrations were determined using a Pierce 660 nm protein assay kit and then standardized to 1 mg/m l

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26 Secreted protein extraction Synchronized c ultures of SH1000 were allowed to grow until post exponential and stationary time points. The cultures were then centrifuged for 10 minutes at a speed of 4,150 rpm. Supernatants were collected and any contaminating bacterial cells were removed by filter sterilization. Clean supernatants were then concentrated using Millipore Centricon Plus 70 c ent rifugal f ilter u nit s before being precipitated in a final concentration secreted proteins w ere washed with 100% ice cold ethanol and centrifuged for 70 minutes determination of protein concentrations, samples were standardized to 1 mg/mL. Trypsin digestion P rotein samples were reduced with 50l of 200mM dithiothreitol for 1 hour at room temperature followed by alkylation with 200 l of 200 mM iodoacetamide for another hour in the dark at room temperature. Any remaining alkylating reagent was consumed with 2 00l of 200 mM dithiothreitol. The samples were then diluted up to 5ml with 25 mM ammonium bicarbonate and then digested with a ratio of 1:30 by weight of trypsin to protein (33.33l) salted with C 18 Vydac columns. The C 18 Vydac columns were activated with 1 ml of 100% acetonitrile and repeated once. The columns were then equilibrated with 1 ml of 0.1% formic aci d in water and repeated once. The samples were applied to the columns and the peptides were washed twice with 1 ml of 0.1% formic acid in water. The

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27 peptides were eluted from the columns with 300 l of 0.1% formic acid in acetonitrile and repeated for a total of 3 times. Once de salted, the peptide samples were dried using the SpeedVac centrifuge. The peptides were resuspended in 100 l of 0.1% formic acid in water and sonicated for 10 minutes. Mass spectrometric analysis of peptides The peptide sampl es were then placed in the autosampler of the LTQ XL mass spectrometer Each sample was fractionated in the gas phase as opposed to fractionation utilizing multidimensional HPLC. For gas phase fractionation, three separate methods were created. The firs t method scanned ions with a mass to charge ratio within a range of 350 550. The second method scanned ions with a mass to charge ratio within a range of 550 750. Finally, the third method scanned ions with a mass to charge ratio of 750 1500. There were a total of 6 full scan events with the top five most intense ions in a 120 minute HPLC gradient and CID normalized collision energy of 35.0%. Identification of proteins The resulting files from mass spectrometric analysis of the samples were processed using Mascot Daemon software for database searching with a Uniprot database containing sequences specific to the S. aureus COL strain. A maximum of 1 missed cleavage by trypsin was allowed. Peptide tolerance was set to 2.5 Da and the MS/MS tolerance wa s set to 0.6 Da. After alkylation with iodoacetamide, the peptides were quantitatively modified at cysteine residues by carbamidomethylation. Variable modifications include acetylation of the protein at the N terminus, oxidation of methionine, and phosp horylation of serine, threonine, and tyrosine. Proteins were identified and listed

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28 using Scaffold 3 software. A random concatenated database of the S. aureus COL strain was created in Scaffold 3 for the detection of false positive identification of prote ins using a decoy database search strategy. A false positive rate less than 5% was found acceptable. Relative Toxin Production of S. aureus using iTRAQ Concentration of secreted proteins Synchronous cultures of USA100, USA200, USA300, and USA400 wer e grown to post exponential phase (5 hours) or to stationary phase (15 hours). Once the cultures had grown to the desired phase, they were centrifuged at 4,150 rpm for 10 minutes. After centrifugation, the supernatants were filter sterilized and then con centrated using Millipore Centricon Plus Following concentration of the supernatants, the proteins were precipitated with 10% e supernatants were the protein pellet to be washed. The proteins were washed with 100% ice cold ethanol ethanol washing was repeated a total of 3 times. After the last ethanol wash, the pellets were allowed to air dry. Trypsin digestion of secreted proteins instructions. The s ecreted protein pellets were resuspended in dissolution buffer provided by the iTRAQ kit. The Pierce 660 nm Protein Assay was used to determine the

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29 concentrations of the protein samples. The concentrations were standardized to 100 g in a final volume of 20 l of dissolution buffer. One l of denaturant (SDS) was added to for 1 hour. After the incubation period, 1 l of cysteine blocking reagent was added to the sample s followed by a 10 minute incubation period at room temperature. Trypsin was added to the samples in a ratio of 1:30 (3.33l) and the samples were digested for 12 iTRAQ labeling of peptides The iTRAQ reagents were allowed to reach roo m temperature and were mixed with 70 l of ethanol. The peptide samples were individually labeled with the iTRAQ reagents (USA100 114, USA200 115, USA300 116, USA400 117) for 1 hour at room temperature. After the incubation period, the 4 labeled samples were combined into a new tube. To rid the sample of ethanol, the samples were dried using the SpeedVac centrifuge. The samples were then resuspended in 1 ml of 0.1% formic acid in water. The labeled peptides were then de salted using C 18 Vydac columns as previously described. After de salting, the samples were dried using the SpeedVac centrifuge and then resuspended and sonicated in 25 l of 0.1% formic acid in water. Mass spectrometric analysis of iTRAQ labeled peptides The peptide samples were pla ced in the autosampler of the LTQ Orbitrap XL m ass spectrometer. There were a total of 7 full scan events which included a full survey scan (m/z 350 1500) and subsequent MS/MS of the top 3 most intense ions of a range of 350 1500 m/z in a 180 minute gradi ent. There were 3 scan events with CID of 35.0%

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30 normalized collision energy in the linear ion trap followed by 3 scan events with HCD of 40.0% normalized collision energy at a mass resolving power of 30,000 full scan MS with 7,500 high collision induced d issociation scan in the Orbitrap mass analyzer. Identification and Quantitation of iTRAQ labeled peptides The resulting files from mass spectrometric analysis of the samples were processed using Mascot Daemon software for database searching with the USA10 0, USA200, USA300, and USA400 strains. A maximum of 1 missed cleavage by trypsin was allowed. Peptide tolerance was set to 10 ppm and the MS/MS tolerance was set to 0.6 Da. A fixed modif ication of the peptides included methyl methan e thiosulfonation of the cysteine residues of trypsin digested peptides. Variable modifications, on the other hand, include d acetylation of the p roteins at the N terminus and oxidation of methionine. The resulting files from database searching using Mascot were then submitt ed to the HCD merging tool provided by the ExPASy proteomics server ( htt p://www.ex pasy.ch/tools/HCD_CID_ merger.html ) to merge the qualitative peptide sequence ion m/z range of CID with the quantitative reporter ion m/z range of HCD. Following merging of HCD with CID spectra, the resulting files were re searched using Mascot Daemon software. Proteins were identified and quantified using Scaffold 3 Q+ software. A random concatenated database of the S. aureus USA100, USA200, USA300, and USA400 was created in Scaffold 3 for the detection of false positive identification of proteins.

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31 Results The a pplication of mass spectrometry for proteome analysis in S. aureus As a medically significant pathogen, S. aureus employs an arsenal of virulence factors to cause and maintain infection in humans. These virulence factors and other proteins central to the survival of S. aureus are differentially expressed as the organism progresses through the different phases of growth. When used for the study of different t issues and organisms, mass spectrometry can be a useful tool for the identification and quantitation of protein variations within cells. Therefore in this project we aim ed to catalogue the intracellular proteome and secretome of a common lab strain of S. a ureus SH1000 during post exponential and stationary phases of growth to provid e an insight into its physiology and how it adapts to its changing environment over time by utilizing differential protein synthesis. We also aim ed to profile the secretomes of clinically significant strains currently afflicting individuals in hospitals (USA100 and USA200) and in the community (USA300 and USA400) settings as a complete secretomic analysis of these clinically relevant strains is currently lacking. An i mproved me thod for the extraction of intracellular proteomes from S. aureus cells. Traditionally, cytoplasmic protein extraction in S. aureus was performed by boiling cells; allowing them to burst and release cytoplasmic proteins into the buffer. Initially we foll owed such a protocol, extracting cytoplasmic proteins from the S. aureus laboratory strain SH1000. After many repetitions of this approach, coupled with reading protein concentration using a Nanodrop device, we obtained highly inconsistent results.

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32 As su ch, it was important to determine if this resulted from inefficient extraction methods or inaccuracies of the Nanodrop device for determining protein concentration. To resolve this, we used a Pierce 660nm Protein Concentration Assay to calculate protein c oncentration via use of the included protein standards and a BioTek Synergy II plate reader. The subsequent readings obtained revealed far more consistent results from our extraction, however protein yields were consistently low, for example 147.7 g/ml f or a post exponential phase culture and 152.2 g/ml for a stationary phase culture of SH1000. To maximize protein concentrations, different methods for cell lysis were tested. Four different lysis methods were used in quintuplicate: boiling of cells; mech anical shearing of cells by bead beating with 0.1mm glass beads; treatment of cells with a dedicated lytic agent, lysostaphin; and sonication of cells using a disruptor. For each of these methods, a 100 ml overnight culture of SH1000, grown in a 250 ml Er lenmeyer flask, was centrifuged at 4,150 rpm for 10 minutes. The resulting pellets were washed twice with phosphate buffered saline (PBS) to eliminate any remaining growth medium, before being resuspended in 1 ml of fresh PBS. Each of the resuspended SH1 000 cell samples were then subjected to t he various cell lysis methods. For lysis via boiling, samples were placed in a 100C water bath for 10 minutes. Mechanical shearing was achieved with an approximately 0.5 cm depth of 0.1 mm glass disruption beads in the sample tube and a BioSpec Mini BeadBeater programmed for 4 total minutes of lysis, with regu lar cooling intervals. To enzymatically lyse the cells, 100 hour. Finally, for sonication, a disrupting probe was inserted in tubes containing resuspended cells, which were pulsed at an amplification of 70% for 20 seconds, with 10

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33 seconds rest periods between treatments. Cells were sonicated for a total of 3 minutes. Following each different lysis method, samples were centrifuged, and supernatants removed to new 1.5 ml tubes. These tubes were centrifuged further, and the supernatants were again removed to clean 1.5 ml tubes. This process was performed to remove all cell wall and membrane proteins, and ensure the purity of the cytoplasmic fraction. Using each of these me thods for extraction we obtained consistent results for each method, however they varied by process used ( Table 1 ). Boiling Bead beating Lysostaphin treatment Sonication 194.6 g/ml 766.6 g/ml 853.27 g/ml 256.6 g/ml Table 1. Comparison of diff erent cell lysis techniques for extraction of cytoplasmic proteins. Data presented is the average of 5 samples that showed less than 10% variation. The protein yields for boiling and sonication were dramatically low when compared to that from bead beating and lysostaphin treatment. Lysostaphin treatment and bead beating of the cells yielded comparable protein concentrations, although lysostaphin treatment produced consistently higher yields. For our analyses, bead beating was selected as the preferred met hod because of its ease and speed of use, and the low cost associated with it, compared to lysostaphin treatment. Proteomic analysis of S. aureus SH1000 cytoplasmic proteins via 1D SDS PAGE coupled with LC MS/MS. For our initial proteomic analysis of cyto plasmic proteins we grew an overnight culture of SH1000 and extracted proteins via the bead beating method detailed above. These were then resolved via 1D SDS PAGE. After electrophoresis, the gel wa s cut into 7 fragments (Figure 4 ) which were subject ed t o in gel digestion with trypsin.

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34 Figure 4 1D SDS PAGE analysis of cytoplasmic proteins from an overnight culture of S. aureus SH1000. M refers to molecular weight markers in kDa and F1 F7 refer to the excised fragments. After trypsin ization, the fragments were analyzed by a ThermoFinnigan LTQ mass spectrometer. Database searching of the retur ned results for all 7 fragments identified a total of 380 cytoplasmic proteins (Table 2). The database used for searching the mass spectral dat a was the general, non specific Uniprot database (NCBI). This search found many contaminant proteins belonging to organisms other than S. aureus The reason for this is likely that, after trypsin digestion, the resulting S. aureus peptides have incidenta l homology to peptides from organisms unrelated to S. aureus as a result of using a non specific database Identified Proteins (380) Accession # Spectral Counts bifunctional autolysin (atl) SACOL1062 67 ATP dependent Clp protease, putative SACOL2563 56 sdrD protein (sdrD) SACOL0609 43 N acetylmuramoyl L alanine amidase domain protein SACOL2666 36 DNA directed RNA polymerase, beta' subunit (rpoC) SACOL0589 35 translation elongation factor G (fusA) SACOL0593 34 chaperonin, 60 kDa (groEL) SACOL 2016 32 ATP synthase F1, beta subunit (atpD) SACOL2095 32 dnaK protein (dnaK) SACOL1637 31 transketolase (tkt) SACOL1377 30 Table 2. The ten most abundant cytoplasmic proteins identified from overnight cultures of S. aureus SH1000, separated by 1D SDS PAGE SH100 0 M 20 0 0 116 97.2 66.4 44.3 29.0 14.3 6.5

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35 Spectral counts measure r elative protein quantification by comparing the number of identified MS/MS spectra from the same peptide to the total number of identified MS/MS spectra found in a dataset. T he number of spectra matched to peptides fro m a protein is used as a measure of pr otein abundance in the sample [ 118 ]. Amongst the most abundant proteins identified were bifunctional autolysin (Atl), putative, ATP dependent Clp protease, SdrD protein, N acetylmuramoyl L alanine amidase domain protei (F usA), G D naK protein, and transketolase. Some of the identified proteins in Table 2, however, we re not expected to be located in the cytoplasm of S. aureus The bifunctional autolysin, S drD protein, and N acetylmuramoyl L alanine amidase domain protein we re expected to be surface associated proteins; whereas AT s expected to be present in the cell membrane. The c ytoplasmic proteins found in Table 2 are involved in important biological processes. For example, the ATP dependent Clp protease, chaperonin GroEL, and DnaK are involved in protein turnover and folding; whilst the NA transcription. Additionally, translation elongation factor FusA has a role in protein synthesis and the ATP synthase produces ATP from ADP in a proton gradient present across the cell membrane. Finally, transketolase has a role in the pentose phosphat e pathway. Analysis of the effects of solubilization buffer on protein concentration yield. In addition to lysis methods, we also investigated the buffer used to solubilize proteins during extraction. As hydrophobic proteins can be easily lost du ring sample preparation, two different resuspension buffers were compared in an attempt to maximize protein

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36 yield in general, and specifically capture the maximum number of hydrophobic proteins. In our analysis above, PBS was used to resuspend cells for t he extraction of cytoplasmic proteins described in Table 1; however PBS is not efficient at dissolving insoluble proteins, and may not be able to guarantee maximum proteome coverage. Due to its molecular structure, urea is able to solubilize insoluble pro teins by expanding hydrophobic cores via hydrogen bonding, encouraging the solvation of hydrophobic regions and allowing water to compete with intraprotein interactions. UDS buffer (6M urea, 5mM DTT, 1% SDS, 50mM Tris HCl, pH 8) has been used in other stu dies for protein extraction [124 ] and therefore we set out to compare results obtained with this solution, with PBS. Cytoplasmic proteins from overnight cultures of SH1000 were extracted in PBS and in UDS by mechanical shearing of the cells. The concent rations of the cytoplasmic proteins from each sample were determined using a Pierce 660 nm Protein Assay. We found that UDS buffer commonly yielded protein concentrations from SH1000 cultures in stationary phase of around 1256 g/ml compared to PBS, which was onl y around 783 g/ml. UDS appeared to allow for more coverage of the cytoplasmic proteome, including hydrophobic proteins, and was thus chosen as ou r buffer for future analyses. The use of complex mix ture analysis coupled with HPLC separation to in crease the number of proteins identified from overnight cultures of S. aureus cells. Given that the S. aureus genome contains approximately 2800 genes, our analysis above (Figure 2, Table 2) clearly does not represent the entire intracellular proteome of overnight SH1000 cells. Therefore, whilst 380 was a promising initial result, we employed alternative approaches to maximize the total number of proteins we could identify because proteins

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37 that are very large, hydrophobic, acidic, or basic are poorly resol ved by gel electrophoresis. For this reason a newer method was used, known as multi dimensional protein identification ( MudPIT ), which combines multi dimensional liquid chromatography with tandem mass spectrometry; allowing complex protein samples to be a nalyzed. Experimentally, proteomes are reduced and digested with trypsin to generate peptide fragments. Using HPLC, the peptide fragment mixture is then applied to a column packed with a strong cation exchange (SCX) resin. The eluted peptides collected in fractions are then analyzed using a mass spectrometer. This technique is very efficient because it is a relatively expedient method of analysis and the two dimensional chromatographic separation of peptides also increases the number of proteins identif ied [ 145, 76]. Therefore, our new methodology involved using complex mixture analysis of cytoplasmic proteins coupled with HPLC separation, rather than 1D SDS PAGE gel resolution. Thus proteins were again extracted from an overnight culture of SH1000 as described above. These were then reduced, alkylated, and digested overnight with trypsin. The resulting peptide fragments were fractionated via HPLC by applying the peptide fragments to a column packed with a strong cation exchange (SCX) resin. The resul ting fractions containing peptides were then analyzed using a ThermoFinnigan LTQ mass spectrometer. After obtaining the mass spectral data, we analyzed the returned information using a database specific to S. aureus strain COL (a close relative of SH1000) This had the effect of more accurate protein identification and also returned all proteins as belonging to S. aureus This proteomic analysis of overnight S. aureus SH1000 cells yielded a total of 747 cytoplasmic proteins identified (Table 3).

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38 Ident ified Proteins (747) Accession # Spectral counts alkaline shock protein 23 SACOL2173 1129 enolase (eno) SACOL0842 886 translation elongation factor Tu (tuf) SACOL0594 873 glyceraldehyde 3 phosphate dehydrogenase (gapA1) SACOL0838 873 formate ac etyltransferase (pflB) SACOL0204 577 aldehyde dehydrogenase (aldA1) SACOL0154 372 ATP dependent Clp protease, putative SACOL2563 371 DNA binding protein HU (hup) SACOL1513 356 pyruvate dehydrogenase complex E3 component, lipoamide dehydrogenase ( pdhD) SACOL1105 341 dnaK protein (dnaK) SACOL1637 298 Table 3. The ten most abundant cytoplasmic proteins identified from MudPIT analysis of SH1000 Compared to proteins identified from 1D SDS PAGE found in Table 2, proteins identified by MudPIT analy sis in Table 3 were overall more abundant. For example, in Table 1, Clp protease had a total of 56 spectral counts whereas the spectral counts for this particular protein in Table 3 were 371. Also, there was seemingly less contamination by non cytoplasmi c proteins. Specific cataloging of the S. aureus intracellular proteome using MudPIT analysis. Reproducibility using HPLC fractionation proved to be highly inconsistent; therefore a new fractionation method was needed for the cataloging of SH1000. Usua lly, ions with mass to charge ratios used to determine the masses of peptides, between the values of 350 and 1500 are selected in the mass spectrometer for analysis. With a new gas phase fractionation method, three separate protocols were created in the mass spectrometer. The first method scanned ions with a mass to charge ratio within a range of 350 550. The second method scanned ions with a mass to charge ratio within a range of 550 750. Finally, the third method scanned ions with a mass to charge ra tio of 750 1500. Each sample was analyzed using the three methods with three injections each.

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39 H aving derived methodologies that were effective and consistent for the intracellular proteomic analysis of S. aureus we undertook a cataloging project using the laboratory strain SH1000. For this analysis, we chose to isolate intracellular proteomes from two different phases of growth: post exponential (5 hours), and stationary (15 hours). Toxin production begins in the post exponential phase with toxins acc umulating in the stationary phase. For this reason, these two time points are of particular interest when cataloging proteomes of S. aureus Thus, 1 milliliter of an overnight culture of SH1000 was added to a fresh 250 ml flask containing 100 ml of TSB w hich was incubated for 3 (identical conditions) at an optical density of 0.05. These test cultures were then allowed for each growth phase. Cytoplasmic proteins were extracted by mechanically shearing the cells with glass beads in UDS buffer for a total of 4 minutes. Protein yields were calculated using a Pierce 660 nm Assay and standardized to 1 mg/ml for trypsin dig estion. After trypsin digestion, the samples were subsequently analyzed by mass spectrometry for the final identification of cytoplasmic proteins present in the original cultures. The ten most abundant cytoplasmic proteins identified by MudPIT analysis fr om SH1000 during post exponential phase are included in Table 4. A total of 346 proteins were topoisomerase 4, and DNA ligase. Each of these is involved in the central cellular proc ess of DNA synthesis. There was also a presence of DNA directed RNA polymerase also present in SH1000 during post exponential phase, as were proteins involved in

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40 protein s ynthesis, including elongation factor Tu, elongation factor G, and numerous 30S and 50S ribosomal proteins. The majority of the proteins identified such as enolase, pyruvate kinase, pyruvate dehydrogenase, fructose bisphosphate aldolase, dihydrolipoyl deh ydrogenase, phosphoglycerate mutase, are involved in central carbon metabolism and energy generation These proteins would be expected in the post exponen tial phase because cells in this phase have not yet entered the stationary phase and are metabolicall y active and still growing. The complete list of proteins identified can be found in the Appendix. Identified Proteins (346) Accession Number Sample 1 S ample 2 Elongation factor Tu sp|Q5HIC7|EFTU_STAAC 239 119 Probable transglycosylase isaA sp|Q5HCY1| ISAA_STAAC 81 111 Elongation factor G sp|Q5HIC8|EFG_STAAC 89 65 Enolase sp|Q5HHP1|ENO_STAAC 64 39 Pyruvate kinase sp|Q5HF76|KPYK_STAAC 54 27 Dihydrolipoyl dehydrogenase sp|Q5HGY8|DLDH_STAAC 39 36 Pyruvate dehydrogenase E1 component subunit beta sp|Q5H GZ0|ODPB_STAAC 35 38 Bifunctional autolysin sp|Q5HH31|ATL_STAAC 45 24 50S ribosomal protein L30 sp|Q5HDX6|RL30_STAAC 28 25 50S ribosomal protein L15 sp|Q5HDX7|RL15_STAAC 15 45 Table 4. The ten most abundant cytoplasmic proteins identified from MudPIT analysis of SH1000 during post exponential phase The ten most abundant cytoplasmic proteins identified by MudPIT analysis from SH1000 during stationary phase are included in Table 5. A total of 366 proteins were identified, including most of the proteins found in post exponential phase, although in lesser quantities. As will be mentioned later, the method had to be refined yet again due to poor and inconsistent results using the HPLC. The newer gas phase fractionation method that will be mentioned bypass es the need for the HPLC but sacrifices proteome coverage. Proteins involved in DNA replication, transcription, protein synthesis, and central carbon metabolism were present in stationary phase cultures of SH1000. Though toxins such as

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41 phenol soluble mod ulins and delta hemolysin are expected to be secreted, they are still present in high quantities intracellularly during stationary growth, when toxins begin to accumulate. Identified Proteins (366) Accession Number Sample 1 S ample2 Elongation factor Tu sp|Q5HIC7|EFTU_STAAC 246 521 Antibacterial protein (Phenol soluble modulin) tr|Q5HGQ7|Q5HGQ7_STAAC 115 215 Probable transglycosylase isaA sp|Q5HCY1|ISAA_STAAC 140 124 Pyruvate kinase sp|Q5HF76|KPYK_STAAC 115 58 Uracil phosphoribosyltransferase sp|Q5 HE88|UPP_STAAC 66 66 Elongation factor G sp|Q5HIC8|EFG_STAAC 81 67 Dihydrolipoyl dehydrogenase sp|Q5HGY8|DLDH_STAAC 78 48 Bifunctional autolysin sp|Q5HH31|ATL_STAAC 92 31 Cell division protein ftsZ sp|Q5HGP5|FTSZ_STAAC 68 43 Delta hemolysin sp|Q5HE G6|HLD_STAAC 98 18 Table 5. The ten most abundant cytoplasmic proteins identified from MudPIT analysis of SH1000 during stationary phase Derivation of an improved method for extraction of the S. aureus secretome. Given that S. aureus secretes a variety of exoproteins and toxins throughout growth, we determined it of significant importance to globally analyze these proteins at a proteomic level. As such, 100 ml of an overnight culture of SH1000 grown in a 250 ml flask was centrifuged for 10 minutes at a speed of 4,150 rpm. Secreted proteins were precipitated from the supernatant by adding 10% trichloroacetic acid and incubating for 1 hour. After the incubation period, the precipitated secreted proteins were washed with room temperature acetone and then centrifuged for 10 minutes at a speed of 13,300 rpm. Washing with acetone was repeated a total of 3 times and the secreted protein pellet resuspended in UDS buffer. This was then fractionated via HPLC and analyzed using a ThermoFinnigan LTQ mass spectrom eter. Our analysis revealed a total of 728 proteins identified from the supernatants collected from the overnight cultures of SH1000 which unexpectedly contained many cytoplasmic proteins. Thus it would appear that

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42 centrifuging is not sufficient for the removal of bacterial cells that could contaminate the supernatant fraction with cytoplasmic proteins. In order to improve the purity of secreted proteins in supernatants we inserted a filter sterilization step after centrifugation to remove cell contamina tion. Further to this, TCA based precipitation of secretomes is difficult, time consuming and inconsistent, as a result of the large volumes involved (up to 400 ml). In order to maximize efficiency and expedite analysis we employed Millipore Centricon Plus 70 Centrifugal Filter Unit s with a 5 kDa cutoff to concentrate supernatants. This method allowed for reduction in culture volumes; however protein concentrations directly from this process were still lower than required for analysis. Protein precipi tation using trichloroacetic acid was thus still necessary to be able to resuspend the secreted proteins in UDS in an even smaller volume for subsequent mass spectrometric analysis. Therefore, we investigated a new method for precipitating secreted protei ns so as to significantly increase yields and concentrations. After the addition of 10% TCA to the upernatant were discarded and protein pellets washed with 100% ice cold ethanol, before centrifugation total of 3 times to ensure complete removal of TCA. Use of this method resulting in significantly higher proteins yields compared to the former, more rapid method of TCA precipitation. As an average, a stationary phase culture of SH1000 yielded a concentration of 336.6 g/ml of proteins using the new TCA precipitation method compared to the older precipitation method that yielded an average of only 201.8 g/ml.

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43 Specific cataloging of the S. aureus secretome using MudPIT analysis. As with our intracellular investigations, given that we have determined effective meth ods for analysis, we set about cataloging the secretome of S. aureus SH1000. Because HPLC fractionation proved to give inconsistent results, as with the analysis of the intracellular proteome of S. aureus we resorted to the gas phase fractionation method detailed above. We again conducted this study for post exponential and stationary phase cultures to observe the alteration of secreted proteins during growth. Accordingly, we took the supernatants derived from the intracellular cataloging experiment s and filter sterilized them. These were then concentrated using Millipore Centricon Plus 70 c entrifugal f ilter u nit s with a 5 kDa cutoff, before precipitation using the TCA method derived above. After precipitation, the concentrations of secreted protei ns were calculated using a Pierce 660 nm Assay before standardization to 1 mg/ml. Proteins were then digested with trypsin followed by mass spectrometric analysis for the identification of proteins secreted by S. aureus during the different growth phases. The ten most abundant secreted proteins identified by MudPIT analysis from SH1000 during post exponential phase are included in Table 6. A total of 38 secreted proteins were identified; the complete list can be found in the Appendix. Though some cytop lasmic proteins such as enolase, glyceraldehyde 3 phosphate dehydrogenase, and pyruvate dehydrogenase can still be identified in the secreted protein fraction of S. aureus filter sterilization of the supernatant excludes whole cells of S. aureus minimizin g contamination by cytoplasmic proteins. For the most part, secreted proteins of SH1000 identified in the post exponential phase were surface associated proteins. These proteins included immunodominant antigen A, bifunctional autolysin, st aphylococcal se cretory

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44 antigen S saA2, putative surface prote ins, probable transglycosylase S ceD, glycerol phosphate lipoteichoic acid synthase, and lipase 1. Because agr activity increases during post exponential phase, toxin production has just begun and has not yet ac cumulated in the supernatant, accounting for the low number of proteins identified in Table 6. Identified Proteins (38) Accession Number S ample 1 Sample 2 Probable transglycosylase isaA sp|Q5HCY1|ISAA_STAAC 234 186 Bifunctional autolysin sp|Q5HH31 |ATL_STAAC 42 31 Staphylococcal secretory antigen ssaA2 sp|Q5HDQ9|SSAA2_STAAC 46 39 Staphopain A sp|Q5HEL3|SSPP_STAAC 10 6 Enolase sp|Q5HHP1|ENO_STAAC 8 7 Surface protein, putative tr|Q5HDZ9|Q5HDZ9_STAAC 8 2 Probable transglycosylase sceD sp|Q5HE A4|SCED_STAAC 5 3 Glycerol phosphate lipoteichoic acid synthase sp|Q5HHV4|LTAS_STAAC 5 6 Glyceraldehyde 3 phosphate dehydrogenase 1 sp|Q5HHP5|G3P1_STAAC 7 5 Lipase 1 sp|Q5HCM7|LIP1_STAAC 5 0 Table 6. The ten most abundant secreted proteins identifi ed from MudPIT analysis of SH1000 during post exponential phase The ten most abundant secreted proteins identified by MudPIT analysis from SH1000 during stationary phase are included in Table 7, with a total of 346 secreted proteins identified. Overall, t he number of secreted proteins identified was higher in the stationary phase compared to the post exponential phase. Most importantly, the production of secreted toxins and exoenzymes increases immensely in stationary phase. Those identified in SH1000 du ring stationary phase include alpha, delta, and gamma hemolysins, serine proteases SplB and SplC, phenol soluble modulins, leukotoxin LukD, leukocidin like protein 1, and staphopains A and B.

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45 Identified Proteins (346) Accession Number S ample 1 Sa mple 2 Lipase 1 sp|Q5HCM7|LIP1_STAAC 608 361 Bifunctional autolysin sp|Q5HH31|ATL_STAAC 269 229 Probable transglycosylase isaA sp|Q5HCY1|ISAA_STAAC 527 143 Surface protein, putative tr|Q5HDZ9|Q5HDZ9_STAAC 148 114 Putative uncharacterized protein tr|Q5HI54|Q5HI54_STAAC 174 119 Enolase sp|Q5HHP1|ENO_STAAC 70 99 Alpha hemolysin tr|Q5HGS1|Q5HGS1_STAAC 85 82 Lipase 2 sp|Q5HJ48|LIP2_STAAC 105 85 Glyceraldehyde 3 phosphate dehydrogenase 1 sp|Q5HHP5|G3P1_STAAC 93 72 Glycerol phosphate lipoteichoi c acid synthase sp|Q5HHV4|LTAS_STAAC 74 46 Table 7. The ten most abundant secreted proteins identified from MudPIT analysis of SH1000 during stationary phase The application of proteomic methodologies for quantitative analysis of secreted toxin s from a variety of S. aureus clinical isolates. Having refined effective methods for the extraction and mass spectrometric analysis of S. aureus proteomes, we proceeded to apply these methods to a relevant biological question. Clinically significant strains of S aureus currently affecting individuals in healthcare facilities and in the community have thus far only been studied at the genomic and transcriptomic levels. Therefore, we decided to study these strains at the proteomic level, as this analysis has to d ate not been completed. Understanding the differential expression of secreted proteins could give insight into phenotypic switching mechanisms of S. aureus and strain dependent variations in virulence. S. aureus infections have commonly been confin ed to h ealth care facilities [4 1 ] largely affecting immunocompromised, young, or old individuals. These hospital acquired methicillin resistant S. aureus (HA MRSA) strains are highly resistant to antibiotics, making HA MRSA infections very difficult to treat. T he leading HA MRSA strain in the United States is USA100 with HA MRSA s train USA200 a close second [127 ]. Recently, community acquired methicillin resistant S. aureus (CA MRSA) infections

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46 have been reported in individuals with no ties to health care facil ities [1, 94, 138 ]. In the United States, the most common CA MRSA strains are known as USA300 and USA400 [110 17 7 ]. These CA MRSA strains appear to be far more virulent than HA MRSA and are especially significant because they cause infections in young, healthy individuals with no predisposing factors [24, 54 ] Despite their increased virulence, CA MRSA strains currently have reduced antibiotic resistance compared to HA MRSA; however it has recently been reported that CA MRSA are beginning to replace HA MRSA strain in healthcare facilities [ 195 ]. Therefore, s ynchronous cultures of hospital associated methicillin resistant S. aureus ( HA MRSA ) strains (USA100 and USA200) and community associated methicillin resistant S. aureus (C A MRSA ) strains (USA300 and USA400) were grown to the post exponential (5 hours) and stationary phase s of growth (15 hours). After extraction of secreted proteins from the supernatants of the se cultures, the concentrations of these proteins were standardized to 100 g before overnig ht trypsin digestion. The resulting pepti des were labeled using iTRAQ reagents, before mass spectrometric analysis by an LTQ Orbitrap. The reagents (having masses of 114, 115, 116, and 117) were used to label USA100, USA200, USA300, and USA400 respective ly. Each of these analyses was performed separately a total of 3 times for each strain. After database searching of the spectral data using Mascot Daemon, Scaffold 3 was used to determine the secretomes of each clinical strain (USA100, USA200, USA300, US A400). Each strain was analyzed using a database derived from the relevant strains genome sequence. Using Scaffold 3, a t test analysis was performed on the derived data to determine any statistically significant changes in production of major secreted t oxins between the various strains, and between

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47 the two different phases of growth (post exponential and stationary). A p a 95% level of confidence was considered to be statistically significant. Relative standard deviation and standard e rror for the three biological replicates For statistical analyses, each biological sample was replicated a total of three times. To determine variability from sample preparation, relative standard deviation and standard error were calculated for each bi ological replicate from the comparison of one clinical strain to another. The ten most abundant proteins and one low abundance protein were chosen to determine the variability from one biological replicate to another. The relative standard deviation of a ll the proteins in a given biological replicate was then averaged. determines sample variability due to sample preparation is low, meaning the technical replicates were s tatistically sound (See Appendix). Analysis of v ariations in s ecretome s o f HA MRSA and CA MRSA strains. Separate databases were created for each strain: N315 for USA100, MRSA252 for USA200, FPR3757 for USA300, and MW2 for USA400. When database searchin g of the spectral data from each iTRAQ sample, each clinical strain was analyzed by its own database, which can affect the number of proteins identified in Scaffold 3. In the post exponential phase, 109 proteins were identified using the USA100 database, 89 proteins were identified using the USA200 database, 114 proteins were found using the USA300 database, and 119 proteins were found using the USA400 database. In stationary phase, 246 proteins were discovered using the USA100 database, 230 proteins were identified for USA200, 224 proteins were found for USA300, and 240 proteins were identified using the USA400 database. There was an overall increase in the number of proteins

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48 identified in the transition from post exponential phase to stationary phase, a s one might expect, resulting from toxin accumu lation and/or cellular lysis. Gene Ontology Annotations of HA MRSA and CA MRSA clinical strains. Using the complete proteome s et for each clinical strain found at Uniprot.org in conjunction with JCVI CMR, a w ebsite containing complete genome sets of prokaryotic organisms, the ro le of each protei n identified from each strain was annotated. Gene ontology of HA MRSA strain USA100 during post exponential phase is outlined in Figure 5. Proteins abundant during th is phase include those involved in protein synthesis, energy metabolism such as glycolysis, gluconeogenesis, tricarboxylic acid cycle, pentose phosphate pathway, fermentation etc., and proteins involved in the biosynthesis and degradation of the cell envel ope. Figure 5. Gene ontology of HA MRSA USA100 during post exponential phase Figure 6 displays gene ontology of USA100 during stationary phase growth. The majority of the proteins involved found during stationary phase are involved in energy

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49 metabol ism, protein synthesis and metabolism, purine and pyrimidine metabolism, cell envelope, as well as proteins of unknown function and those that are conserved hypothetical proteins Figure 6 Gene ontology of HA MRSA USA100 during stationary phase The gen e ontology of the other HA MRSA known as USA200 during post exponential phase is demonstrated in Figure 7. Most of the proteins expressed at this growth phase are involved in protein synthesis and energy metabolism.

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50 Figure 7. Gene ontology of HA MRSA USA200 during post exponential phase The gene ontology of USA200 during stationary phase is demonstrated in Figure 8 It seems USA200 upregulates the production of proteins involved in purine and pyrimidine metabolism toxin production, protein metabolis m, and detoxification. These proteins would be expected during stationary phase as nutrients become scarce and S. aureus upregulates the production of proteins involved in creating a nutrient source.

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51 Figure 8. Gene ontology of HA MRSA USA200 during st ationary phase Figure 9 outlines the gene ontology of the leading CA MRSA strain known as USA300. The proteins identified are from post exponential phase of growth. USA300 mainly expresses proteins involved in protein synthesis, energy metabolism, cell e nvelope, protein metabolism, protein folding, and other uncharacterized proteins, as well as those that are hypothetical conserved proteins.

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52 Figure 9. Gene ontology of CA MRSA USA300 during post exponential phase During stationary phase, USA300 gener ally produces more proteins involved in energy metabolism and protein synthesis. Interestingly, USA300 significantly upregulates the production of certain conserved hypothetical proteins. Proteins involved in p urine and pyrimidine metabolism, toxin produ ction and pathogenesis are generally upregulated during statio nary phase (Figure 10). Figure 10. Gene ontology of CA MRSA USA300 during stationary phase

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53 The other CA MRSA strain USA400 seems to produce proteins involved in protein synthesis, energy me tabolism, toxin production and resistance, protein metabolism, and in the cell envelope. There also seems to be a significant presence of proteins with unknown functions. Figure 11. Gene ontology of CA MRSA USA400 during post exponential phase After transitioning from post exponential phase to stationary phase, USA400 upregulates the expression of even more proteins involved in protein synthesis, energy metabolism, and those with unknown functions. Also, proteins involved in toxin production and resi stance, fatty acid and phospholipid metabolism, and purine and pyrimidine metabolism are also upregulated in the later phase of growth.

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54 Figure 12. Gene ontology of CA MRSA USA400 during stationary phase C hanges in the expression of major secreted prot eins between two HA MRSA strains during post exponential growth. Analysis of the secreted protein s of HA MRSA USA200 compared to the leading HA MRSA USA100 strain at this time point revealed there were limited variations in protein levels Seemingly USA 200 produces 6.8 fold more mecA gene, at this time point when compared to USA100. Further to this, the foldase protein PrsA, which is involved in controlling the rate of protein folding [ 192 ] is expressed at levels 3.2 fold higher in USA200 when compared to USA100. The production of putative surface protein SA2285, however, is 20.34 times greater in USA100 than in USA200. This p utative surface protein SA2285 is large (1370 aa) and contains domains involved in adhes ion, and for the cleavage of human IgA suggesting a role in pathogenesis [ 1 87 ] Because toxin production begins during the post exponential growth, not much variation would be expected at this time point.

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55 Identified Proteins Accession Number P Val ue USA100 USA200 Putative surface protein SA2285 P61598|PLS_STAAN 0.0000073 1 0.049164184 Penicillin binding protein 2' Q7A8C6|Q7A8C6_STAAN 0.000000000074 1 6.806270155 Foldase protein prsA P60748|PRSA_STAAN 0.000035 1 3.236942617 Table 8. A comparison of the secretomes of USA100 and USA200 during the post exponential phase of growth Changes in the expression of major secreted proteins between two CA MRSA strains during post exponential growth. Table 9 depicts the significant changes in secre ted protein production between the two CA MRSA strains, USA300 and USA400. There is a downregulation (0.58 fold) of putative lipoprotein Q2FIT2 in USA300 when compared to USA400. Putative lipoprotein Q2FIT2 contains a potential signal peptide sequence an d a conserved DM13 domain that possibly functions as a sugar kinase in bacterial two component systems [ 90 ]. On the other hand, the leading CA MRSA strain USA300 upregulates the production of alpha hemolysin (1.8 fold) phenol soluble (3.4 fold) a putative uncharacterized protein SAUSA300_pUSA010004 (10.9 fold) that has no known homologs, and an uncharacterized protein Q2FFS8 that is homologous to a beta lactamase protein (10.8 fold) The presence of thermonuclease, a heat stable DNase, d id not change significantly in either strain.

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56 Identified Proteins Accession Number P Value USA300 USA400 Alpha hemolysin Q2FHS2|Q2FHS2_STAA3 0.00018 1 0.550173608 Thermonuclease Q2FIK2|Q2FIK2_STAA3 0.0016 1 1.219681925 Putative uncharacterized prote in SAUSA300_pUSA010004 Q2FDE2|Q2FDE2_STAA3 0.025 1 0.091960838 Putative uncharacterized protein Q2FFS8|Q2FFS8_STAA3 0.019 1 0.092562355 Putative lipoprotein Q2FIT2|Q2FIT2_STAA3 0.042 1 1.712581503 Phenol soluble modulin alpha 1 peptide P0C7Y0|PSMA1_S TAA3 0.046 1 0.306315112 Table 9. Secreted proteins of USA400 demonstrating significant changes in expression compared to USA300 during post exponential phase of growth Changes in the expression of major secreted proteins between HA MRSA and CA MRSA stra ins during post exponential phase The secreted proteins of the leading CA MRA strain ( USA300 ) are compared to HA MRSA USA100 during post exponential phase of growth, in Table 10 Overall, USA100 produces almost 52 times more putative surface protein SA2 285 and 1.55 times more foldase. Immunodominant antigen A and SasD are both upregulated about 13 fold more in USA100 compared to USA300. The immunodominant antigen A a p robable transglycosylase (IsaA), contains a potential signal peptide for secretion a nd is likely involved in peptidoglycan turnover [ 170 ]. The Serine aspartate repeat containing protein D, also known as SasD is likely to have a signal peptide sequence as well as conserved domains involved in fibrinogen binding. Interesting ly, the data p rovided in Table 10 correlates well with the literature concerning HA MRSA and CA MRSA. Surface proteins are expected to be downregulated in CA MRSA due to hyperactivity of ag r that negatively affects production of these proteins as it upregulates product ion of secreted toxins and proteases It is unsurprising, therefore, that CA MRSA strains such as USA300 do not produce surface associated proteins as much as HA MRSA strains such as USA100.

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57 Identified Proteins Accession Number P Value USA100 USA300 Putative surface protein SA2285 P61598|PLS_STAAN 0.0000019 1 0.019251877 Probable transglycosylase isaA P99160|ISAA_STAAN 0.030 1 0.075564941 Serine aspartate repeat containing protein D Q7A780|SDRD_STAAN 0.0086 1 0.0732664 Foldase protein prsA P6 0748|PRSA_STAAN 0.049 1 0.643330071 Table 10. Secreted proteins of USA300 demonstrating significant changes in expression compared to USA100 during post exponential phase of growth When compared to USA100 during post exponential growth, USA400 significan tly upregulates the expression of staphopain A (4.1 fold) Interestingly, this correlates with the observation that CA MRSA strains have higher agr activity and therefore produce more secreted toxins and proteases than HA MRSA [ 195 ] USA400 also seems to (1.7 fold) when compared to USA100, but there is a marked downregulation of putative surface protein SA2285 in USA400 (0.04 fold) Identified Proteins Accession Number P Value USA100 USA400 Putative surface prot ein SA2285 P61598|PLS_STAAN 0.00014 1 0.040001113 Staphopain A P65826|SSPP_STAAN 0.0046 1 4.10146935 Penicillin binding protein 2' Q7A8C6|Q7A8C6_STAAN 0.025 1 1.711120053 Table 11. Secreted proteins of USA400 demonstrating significant changes in exp ression compared to USA100 during post exponential phase of growth (0.35 fold) in the leading CA MRSA strain USA300, when compared to the HA MRSA USA200. This observation correlate s with the finding that HA MRSA strains seem to be more resistant to antibiotic treatment than CA MRSA strains.

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58 Identified Proteins Accession Number P Value USA200 USA300 Penicillin binding protein 2' Q6GKQ7|Q6GKQ7_STAAR 0.047 1 0.353094018 Table 12. Secreted proteins of USA300 demonstrating significant changes in expression compared to USA200 during post exponential phase of growth CA MRSA USA400 may produce more penicillin binding protein MRSA USA100 (Table 12 ), but HA MRSA USA 200 produce s 2.8 fold more of this protein than USA3 00. Because HA MRSA strains are known to b e more antibiotic resistant [127 ], it is expected to find a HA MRSA strain such as USA200 producing significantly more MRSA strain s uch as USA300. The same relationship between HA MRSA and CA MRSA can be seen in Table 13 as USA200 produces 2.2 Identified Proteins Accession Number P Value USA200 USA400 Penicillin binding protei n 2' Q6GKQ7|Q6GKQ7_STAAR 0.035 1 0.45315937 Table 13. Secreted proteins of USA400 demonstrating significant changes in expression compared to USA200 during post exponential phase of growth Changes in expression of major secreted proteins between two HA M RSA strains during stationary phase When compared to USA100, USA200 seems to produce significantly more zinc metalloprotease (5.52 fold) less lipase 1 (0.2 fold) and foldase (0.66 fold) Compared to the post exponential phase of growth, there seems to be a decreased presence of foldase protein PrsA in USA200 during stationary phase. Identified Proteins Accession Number P Value USA100 USA200 Zinc metalloproteinase aureolysin Q7A378|Q7A378_STAAN 0.012 1 5.521879552 Lipase 1 P65289|LIP1_STAAN 0.040 1 4.922412009 Foldase protein prsA P60748|PRSA_STAAN 0.045 1 1.512870373 Table 14. Secreted proteins of USA200 demonstrating significant changes in expression compared to USA100 during stationary phase of growth

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59 Changes in expression of major secreted p roteins between two CA MRSA strains during stationary phase There is a marked upregula tion of phenol soluble modulin (16.1 fold) antib acterial protein SAUSA300_1067 (2.0 fold), SAUSA300_1068 (10.6 elastin binding protein EbpS (4.8 fold) a putative cell wall surface anchor family protei n (1.4 fold) and a CHAP domain family protein (5.1 fold) in USA400 when compared to USA300. It is predicted to have a signal peptide sequence and it also has a conserved domain involved in cell wall degradation. On the other hand, there is an upregulati on of alpha hemolysin (4.7 fold) an ABC transporter substrate binding protein (1.8 fold) and putative uncharacterized protein SAUSA300_pUSA010004 (7.8 fold) in USA300 in USA300 in the post exponential phase of growth than in USA400 however the reverse is true during post exponential growth Identified Proteins Accession Number P Value USA300 USA400 Alpha hemolysin Q2FHS2|Q2FHS2_STAA3 0.0000000026 1 0.210747 Phenol soluble modulin alpha 1 peptide P0C7Y0|PSMA1_STAA3 0.0000018 1 16.10036 Antibacterial protein SAUSA300_1067 Q2FHR4|Q2FHR4_STAA3 0.0012 1 2.022183 CHAP domain family Q2FIX4|Q2FIX4_STAA3 0.0016 1 5.090964 Elastin binding p rotein ebpS Q2FGW1|EBPS_STAA3 0.0033 1 4.839727 Putative uncharacterized protein SAUSA300_pUSA010004 |Q2FDE2_STAA3 0.0054 1 0.12851 Putative cell wall surface anchor family protein Q2FE08|Q2FE08_STAA3 0.0074 1 1.362511 Antibacterial protein SAUSA300_1 068 Q2FHR3|Q2FHR3_STAA3 0.017 1 10.55861 Phenol soluble modulin alpha 4 peptide P0C817|PSMA4_STAA3 0.022 1 2.464562 ABC transporter, substrate binding protein Q2FJ07|Q2FJ07_STAA3 0.029 1 0.552823 Table 15. Secreted proteins of USA400 demonstrating sig nificant changes in expression compared to USA300 during stationary phase of growth

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60 Changes in expression of major secreted proteins between HA MRSA and CA MRSA strains during stationary phase Table 16 demonstrates changes in secreted protein production of CA MRSA USA300 compared to HA MRSA USA100. Parallel to transcriptomic studies comparing HA MRSA and CA MRSA, there is a significant upregulation of gamma (2.3 fold) and alpha hemolysins (12.7 fold) in USA300 compared to USA100. These observations are associated with a hyperactivity of agr in CA MRSA strains compared to HA MRSA strains. On the other hand, production of the putative surface protein SA2285 is 33.8 times greater in USA100. Identified Proteins Accession Number P Value USA100 USA300 Putati ve surface protein SA2285 P61598|PLS_STAAN 0.00069 1 0.02960504 Gamma hemolysin component C Q7A3S2|HLGC_STAAN 0.030 1 2.30802049 Alpha Hemolysin Q7A632|Q7A632_STAAN 0.032 1 12.6955356 Table 16. Secreted proteins of USA300 demonstrating significant cha nges in expression compared to USA100 during stationary phase of growth There is an enormous upregulation of secreted toxins such as lipase 1 (10 fold) lipase 2 (4.6 fold) enterotoxin C (21.9 fold), the zinc metalloprotease aureolysin (1.8 fold) and ela stin binding protein EbpS (18.1 fold) in CA MRSA USA400 when compared to HA MRSA USA100. There is also a significant upregulation the SA2006 (8 fold) and SA0841 (5.1 fold) protein s in USA400. These two proteins contain conserved MAP domains involved in a dherence.

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61 Identified Proteins Accession Number P Value USA100 USA400 Lipase 2 Q7A7P2|LIP2_STAAN 0.0022 1 4.611289 Enterotoxin type C P0A0L4|ENTC3_STAAN 0.0033 1 21.88768 Elastin binding protein ebpS Q7A5I6|EBPS_STAAN 0.0033 1 18.1368 Lipase 1 P 65289|LIP1_STAAN 0.0047 1 10.02552 SA2006 protein Q7A483|Q7A483_STAAN 0.020 1 8.034026 Zinc metalloproteinase aureolysin Q7A378|Q7A378_STAAN 0.031 1 1.837579 SA0841 protein Q7A6G0|Q7A6G0_STAAN 0.040 1 5.091233 Table 17. Secreted proteins of USA400 de monstrating significant changes in expression compared to USA100 during stationary phase of growth When comparing the production of secreted proteins between HA MRSA USA200 and CA MRSA USA300, it is evident USA200 produces significantly more zinc metallopr otease (3.6 fold) and a putative exported protein Q6GI28 (2.5 fold) than USA300. Putative exported protein Q6GI28 contains a predicted signal peptide sequence and is homologous to a LytR transcriptional regulator I t also has a conserved domain putative ly involved in transcriptional attenuation [ 7 7 ]. Production of gamma hemolysin component C, on the other hand, does not significantly change between USA200 and USA300. Identified Proteins Accession Number P Value USA200 USA300 Zinc metalloproteinase aure olysin Q6GDG5|Q6GDG5_STAAR 0.00038 1 0.279331 Putative exported protein Q6GI28|Q6GI28_STAAR 0.014 1 0.394861 Gamma hemolysin component C Q6GE13|HLGC_STAAR 0.047 1 1.379588 Table 18. Secreted proteins of USA300 demonstrating significant changes in expre ssion compared to USA200 during stationary phase of growth Side by side, CA MRSA USA400 produces significantly more elastin binding protein EbpS (14.8 fold) and an uncharacterized protein SAR1965 (5.2 fold) than USA200. A signal peptide sequence is not be en predicted for uncharacterized protein SAR1965 and

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62 appears to be homologous to intracellular protease s This finding is odd because cytoplasmic proteins are not expected to be identified in the supernatant with secreted proteins. HA MRSA USA200, howev er, produces 14.45 times more anti sigma factor B antagonist protein, also known as anti anti sigma factor B, than USA400. The high presence of anti sigma factor B antagonist in HA MRSA USA100 would lead to higher SigmaB activity in this strain. Because SigmaB has been shown to have a repressive effect on agr activity [15] it is of no surprise CA MRSA strain USA400, due to its hyperactive agr would produce SigmaB at significantly reduced levels SigmaB, being a cytoplasmic protein, is not expected to be identified in the supernatant. Identified Proteins Accession Number P Value USA200 USA400 Elastin binding protein ebpS Q6GGT1|EBPS_STAAR 0.00040 1 14.76076 Anti sigma B factor antagonist Q6GF07|RSBV_STAAR 0.026 1 0.069202 Uncharacterized protein SA R1965 Q6GFI2|Y1965_STAAR 0.042 1 5.188532 Table 19. Secreted proteins of USA400 demonstrating significant changes in expression compared to USA200 during stationary phase of growth Changes in expression of major secreted proteins of HA MRSA strains from post exponential phase to stationary phase In the transition from post exponential phase to stationary phase, it is apparent HA MRSA USA100 upregulates the production of (2.4 fold) putative surface protein SA2285 (9.2 fold) immunodominant antigen A (5.7 fold) and enterotoxin type C 3 (1.4 fold) On the other hand, proteins downregulated in USA100 from post exponential phase include foldase (0.26 fold) lipase 1 (0.14 fold) and 2 (0.12 fold) alpha hemolysin (0.03 fold) S A0841 protein (0.24 fold) and SA2006 protein (0.07 fold)

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63 Identified Proteins Accession Number P Value USA100 5hr USA100 15hr Penicillin binding protein 2' Q7A8C6|Q7A8C6_STAAN 0.0000014 1 2.427176057 Putative surface protein SA2285 P61598|PLS_STAAN 0.0 00055 1 9.192129976 Foldase protein prsA P60748|PRSA_STAAN 0.0048 1 0.264551095 SA0841 protein Q7A6G0|Q7A6G0_STAAN 0.016 1 0.238025085 Lipase 1 P65289|LIP1_STAAN 0.021 1 0.140118214 Alpha Hemolysin Q7A632|Q7A632_STAAN 0.02 1 0.028642902 Probable tra nsglycosylase isaA P99160|ISAA_STAAN 0.033 1 5.734707283 Lipase 2 Q7A7P2|LIP2_STAAN 0.0011 1 0.12382117 Enterotoxin type C 3 P0A0L4|ENTC3_STAAN 0.0012 1 1.40410766 SA2006 protein Q7A483|Q7A483_STAAN 0.015 1 0.070417081 Table 20. Secreted proteins o f USA100 demonstrating significant changes in expression from post exponential to stationary phase When switching from post exponential phase to stationary phase, HA MRSA USA200 (11 fold) Lipase 1 and a putative exported protein ( Q6GIA6 ) on the other hand, are downregulated in the stationary phase 0.4 fold and 0.6 fold, respectively Putative exported protein Q6GIA6 is predicted to have a signal peptide sequence and contains a MAP cons erved domain putatively involved in cell adherence. Downregulation of this putative exporter protein can perhaps be associated with the fact that S. aureus cells transition from adhesion in the earlier phases of growth to toxin production in the stationar y phase. Identified Proteins Accession Number P Value USA200 5hr USA200 15hr Penicillin binding protein 2' Q6GKQ7|Q6GKQ7_STAAR 0.01135 1 10.90122846 Putative exported protein Q6GIA6|Q6GIA6_STAAR 0.025 1 0.588479043 Lipase 1 Q6GDD3|LIP1_STAAR 0.042 1 0.399557859 Table 21. Secreted proteins of USA200 demonstrating significant changes in expression from post exponential to stationary phase

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64 Changes in expression of major secreted proteins of CA MRSA strains from post exponential phase to stationary pha se Numerous proteins have been found to be significantly changed in CA MRSA USA300 during the transition from post exponential phase to stationary phase. A few proteins including, 50S ribosomal protein L11 (1.8 fold) p (29.6 fold) immunodominant antigen A (9.5 fold) antibacterial protein Q2FHR3 (2.1 fold) and a putative cell wall surface anchor family protein (1.6 fold) were prevalent during stationary growth of USA300. Proteins that were significantly downregulated in s tationary phase include: adenylate kinase (0.09 fold) alpha hemolysin (0.2 fold) antibacterial protein Q2FHR4 (0.5 fold) fructose bisphosphate aldolase (0.04 fold) Panton Valentine leukocidin LukS (0.05 fold) peptidoglycan hydrolase (0.04 fold) pheno l soluble modulin (0.02 fold) putative lipoprotein (0.05 fold) putative uncharacterized protein SAUSA300_pUSA010004 (0.2 fold) putative uncharacterized protein SAUSA300_1759 (0.03 fold) putative uncharacterized protein SAUSA300_2164 (0.2 fold), s erine protease S plB (0.4 fold) and triacylglycerol lipase (0.3 fold) There was no significant change in thermonuclease production between the two phases. The presence of 50S ribosomal protein L11, adenylate kinase, and fructose bisphosphate is unexpect ed because these are cytoplasmic proteins and should not be identified in the supernatant as secreted proteins.

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65 Identified Proteins Accession Number P Value USA300 5hr USA300 15hr 50S ribosomal protein L11 Q2FJA3|RL11_STAA3 0.026 1 1.797912631 Ad enylate kinase Q2FER0|Q2FER0_STAA3 0.0138 1 0.086472995 Alpha hemolysin Q2FHS2|Q2FHS2_STAA3 1.07517E 05 1 0.154783038 Antibacterial protein Q2FHR4|Q2FHR4_STAA3 0.009 1 0.543067428 Antibacterial protein Q2FHR3|Q2FHR3_STAA3 0.033 1 2.0973797 Fructose bisphosphate aldolase Q2FF03|Q2FF03_STAA3 0.01485 1 0.039119603 Panton Valentine leukocidin, LukS Q2FGU9|Q2FGU9_STAA3 0.024 1 0.049059483 Penicillin binding protein 2' Q2FKM6|Q2FKM6_STAA3 1.4E 10 1 29.55802797 Peptidoglycan hydrolase Q2FJZ4|Q2FJZ4_STA A3 0.039 1 0.039707336 Phenol soluble modulin alpha 1 peptide P0C7Y0|PSMA1_STAA3 0.014 1 0.018357338 Probable transglycosylase isaA Q2FDT8|ISAA_STAA3 0.016 1 9.526965248 Putative cell wall surface anchor family protein Q2FE08|Q2FE08_STAA3 1.1E 14 1 1. 56173773 Putative lipoprotein Q2FIT2|Q2FIT2_STAA3 0.015 1 0.048020221 Putative uncharacterized protein SAUSA300_1759 Q2FFS8|Q2FFS8_STAA3 0.019 1 0.026126756 Putative uncharacterized protein SAUSA300_2164 Q2FES8|Q2FES8_STAA3 0.031 1 0.209015255 Putativ e uncharacterized protein SAUSA300_pUSA010004 Q2FDE2|Q2FDE2_STAA3 0.0048 1 0.115393064 Serine protease splB Q2FFT0|SPLB_STAA3 0.00435 1 0.413303495 Thermonuclease Q2FIK2|Q2FIK2_STAA3 0.000636 1 0.848936408 Triacylglycerol lipase Q2FDJ1|Q2FDJ1_STAA3 0.0 046 1 0.334270278 Table 22. Secreted proteins of USA300 demonstrating significant changes in expression from post exponential to stationary phase When switching from post exponential growth to stationary growth, CA MRSA USA400 upregulates the production o f surface protein MW2416 (1.6 fold) which is putatively

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66 involved in cleaving human IgA and in adhesion, enterotoxin type C (5.8 fold) and (49.5 fold) Proteins downregulated by USA400 during the stationary phase include: lipa se 1 (0.5 fold) and 2 (0.2 fold) MW1056 protein (0.1 fold) MW0863 protein (0.5 fold) MW2130 protein (0.4 fold) and alpha hemolysin (0.002 fold) MW0863 and MW2130 proteins contain conserved MAP domains and are homologous to cell adherence proteins. I dentified Proteins Accession Number P Value USA400 5hr USA400 15hr Lipase 2 Q8NYC2|LIP2_STAAW 0.00044 1 0.230035026 Lipase 1 Q8NUI5|LIP1_STAAW 0.0043 1 0.453603588 ENTEROTOXIN TYPE C Q8NXJ6|Q8NXJ6_STAAW 0.020833 1 5.7943177 MW1056 protein Q8NX40|Q8N X40_STAAW 0.017 1 0.132835969 MW0863 protein Q8NXE3|Q8NXE3_STAAW 0.019 1 0.518113878 MW2130 protein Q7A090|Q7A090_STAAW 0.024 1 0.368062689 Putative surface protein MW2416 Q8NUV0|PLS_STAAW 0.032 1 1.554528324 Penicillin binding protein 2' Q7A209|Q7A2 09_STAAW 0.019 1 49.50848166 Alpha Hemolysin Q8NX49|Q8NX49_STAAW 0.00089 1 0.001530173 Table 23. Secreted proteins of USA400 demonstrating significant changes in expression from post exponential to stationary phase

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67 Discussion Proteomics, the identi fication of entire protein sets present in biological samples is a useful complementary experimental approach to genomics and transcriptomics. P roteomics has become an extremely useful tool for the study of differential protein expression gain ing signif icance due to highly dynamic protein expression profiles Understanding changes in a given proteome from a particular organism can provide much insight into its behavior, physiology and interaction with its environment Most of the proteomic studies publ ished thus far involve 2D gel electrophoresis with very little emphasis on proteomic analysis of complex protein mixtures. For a proteomic study, it is necessary to maximize protein concentration in order to facilitate the identification of proteins after mass spectrometric analysis. It is also necessary to devise a method that can be easily employed and provide reproducible results. Several published proteomic studies of S. aureus involved methods that have not been optimized to provide comprehensive co verage of the proteome. Previous studies have been fraught with problems, including the use of a urea concentration that is too low for the solubilization of insoluble proteins; a lack of filter sterilization for supernatants to remove bacterial cells and ensure purity of secreted proteins; and secreted proteins centrifuged during precipitation at a speed too low to ensure maximal protein recovery [ 176, 206, 208 ]. Appropriate method refinement is therefore important for obtaining reproducible data across s everal biological replicates of a sample. For the extraction of cytoplasmic proteins, we chose to compare various cell lysis techniques and solubilization buffers.

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68 We also focused on concentration, precipitation, and washing of secreted proteins for a hi gh yield of protein and subsequent mass spectrometric analysis. In addition, apart from traditional 1D or 2D gel electrophoresis, we performed in solution trypsin digestion of extracted proteins followed by mass spectrometry. After our refinement of the methods for extracting cytoplasmic and secreted proteins, we used these newly developed tools to catalogue the intracellular proteome and secretome of a commonly used and well studied lab strain of S. aureus known as SH1000 [ 86 ]. We focused on proteome c overage at two different phases of growth: post exponential phase and stationary phase. We did this in order to provid e an insight into the physiology of S. aureus and how it adapts to its changing environment over time by utilizing differential protein s ynthesis. From our study we found that there is a clear prevalence of ribosomal proteins, involved in protein synthesis, during post exponential growth when compared to SH1000 during stationary phase; likely due to the scarcity of nutrients in the later g rowth phase, resulting in a reduction of translation occurring within the cell. In a study by Becher et al., several subproteomic fractions of a S. aureus strain known as COL were analyzed by mass spectrometry [ 10 ]. When studying the change of cytoplasmi c proteins of COL in the transition from post exponential to stationary phase, Becher et al. also found ribosomal proteins were no longer being synthesized, and were potentially degraded in stationary phase [ 10 ]. Moreover, in our study, the glycolytic en zymes glyceraldehyde 3 phosphate, fructose bisphosphate aldolase, phosphoglycerate kinase, phosphoglycerate mutase, and enolase are generally found to be downregulated in stationary phase when compared to post exponential phase. This would perhaps be expe cted as the availability of carbon sources

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69 becomes limited during stationary growth, and as such, enzymes involved in the catabolism of glucose would no longer be required in the absence of primary carbon sources. On the other hand, proteins involved in t he tricarboxylic acid (TCA) cycle, such as aconitate dehydrogenase, isocitrate dehydrogenase, components of the 2 oxoglutarate dehydrogenase complex, components of succinate dehydrogenase, fumarate hydratase, and malate dehydrogenase are upregulated during stationary phase growth of S. aureus This could result from the catabolism of any remaining pyruvate molecules generated by glycolysis during post exponential phase. Also, phosphoenolpyruvate carboxykinase (PckA), a protein involved in gluconeogenesis, is upregulated in the stationary phase of SH1000 and was not detected in post exponential growth phases. This observation concurs with the 2009 study by Becher et al., where it was suggested that the presence of PckA could be indicative of carbon source starvation, as expected in stationary phase, when nutrients become scarce [ 10 ]. In addition to proteins involved in carbon utilization, the production of Clp protease subunits is also higher in stationary phase than in the post exponential phase. These p roteases which respond to heat, osmotic, and oxidative stresses enable S. aureus to survive in these situations and they tend to accumulate in stationary phase [ 26, 27,62 ]. Furthermore, according to our data, anti sigma factor B antagonist protein is up regulated in the stationary phase whilst the anti sigma factor of Sigma B known as RsbW is downregulated in the stationary phase. This correlates with reports that Sigma B activity is upregulated in stationary phase [ 62, 112 ]. Specifically, the anti sigma factor B antagonist, also known as anti anti sigma factor RsbV, functions to liberate Sigma B

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70 from its anti sigma factor, RsbW [134 ], to regulate the transcription of certain genes in stationary phase [ 111 ]. When analyzing the secretome of SH1000 in the transition from post exponential to stationary phase, there is a clear upregulation of toxins and secreted proteases in the later phase of growth. These proteins include alpha hemolysin [ 146], gamma hemolysin [49], staphopain A and B [25, 99, 203 ], de lta hemolysin [ 146 ], ser ine proteases SplB and SplC ]. Interestingly, some of these secreted proteins are not detected at all during the post exponential phase of growth. Upregulation of these secreted proteins is expec ted in stationary phase of growth due to increased agr activity as a direct result of higher cell density [ 139, 152 ]. A gr is a quorum sensing, two component regulator that is expressed in the post exponential phase of S. aureus growth. During the post e xponential phase, agr represses surface and attachment proteins and induces the transcription of secreted toxins and proteases [ 202 ]. As the population density of S. aureus increases, it secretes an auto inducing peptide that accumulates in the extracellu lar environment. When S. aureus senses the concentration of the AIP has reached a threshold level it induces the transcription of virulence determinants, by the use of an effector molecule, as a response to stress such as nutrient limitation and high popu lation density during stationary growth [ 139,152 ]. The agr effector molecule is a small regulatory RNA, known as RNAIII which act s as a regulator of target genes [ 91, 145 ] by bind ing to target mRNA molecules and either stabiliz ing them or targe ting them for destruction [ 135, 113, 186 ]. RNAIII facilitat es the upregulation of secreted virulence factors [ 18, 6 6 ] and negatively regulate s the synthesis of surface proteins such as protein A and the fibronectin binding proteins,

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71 which are used for adhesion [9 1 143 171 ] Therefore, the upregulation in production of secreted toxins and proteases such as alpha hemolysin, delta hemolysin, gamma hemolysin, staphopains A and B, delta hemolysin, serine proteases SplB and SplC, the phenol agr activity, which is expected in stationary phase. We also observed accumulation of lipase 1 and 2 with the onset of stationary grow th. Though it is not clear if the transcription of these enzymes is regulated by agr they are found to be downregulated in agr mutants of S. aureus [95 ]. From this observation, it is possible to speculate the potentially positive effect of agr activity o n the production of lipases 1 and 2. Though the production of agr regulated proteins is increased in stationary phase; overall agr activity would ultimately be expected to be reduced in the strain SH1000. SH1000 is a strain that descended from another S aureus strain known as 8325 4, whose Sigma B activity is diminished by a natural deletion in one of its positive effectors ( rsbU ). SH1000 was derived from 8325 4 via a full restoration of this deletion, and thus Sigma B activity [ 86 ]. Because Sigma B i s an antagonist of agr agr regulated secreted toxins are downregulated in SH1000 as opposed to 8325 4 and other strains of S. aureus [ 15, 68, 8 6 ]. In accordance with this observation, production of the agr regulated protein V8 protease appears to be dimi nished in SH1000. In our findings, this protein was not detected in the post exponential phase and was only detected at very low levels in stationary growth, when agr activity is at its highest. Furthermore, staphyloxanthin, an orange carotenoid pigment of S. aureus [ 11 1 ] is highly pronounced in strain SH1000 [ 86 ] due to Sigma B activity [ 111 ]. Our data indicate that staphyloxanthin biosynthesis protein is greatly upregulated from post exponential phase to stationary phase. This

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72 finding concurs with th e observation that cultures of SH1000, and therefore the resulting cell pellets, acquire a darker orange color later in growth when Sigma B activity is upregulated [ 86 ]. Having determined the reproducibility of our protein extraction protocols, we ne xt set out to characterize the secretomes of clinically relevant strains of S. aureus Specifically, two HA MRSA (USA100 and USA200) and two CA MRSA (USA300 and USA400) strains were analyzed via quantitative methods at post exponential and stationary phase s of growth. We focused on these growth phases in particular because toxins are produced beginning in the post exponential phase and accumulate throughout stationary phase. We undertook a complete proteomic analysis of differential protein production in the secretomes of these clinically significant strains using the popular quantification technique iTRAQ [ 47 ]. After extraction of the secretomes, proteins were digested with the protease trypsin to produce peptides. The resulting peptides were isobarical ly labeled using iTRAQ reagents for mass spectrometric analysis of three biological replicates for subsequent identification and relative quantification of the proteins secreted by these four strains. With this approach we sought to identify and also quan tify the production of secreted proteins that enable this pathogen to swiftly infect and cause disease in patients. When comparing HA MRSA USA100 to CA MRSA USA300 during post exponential phase of growth, it is evident USA100 produces more surface associ ated proteins than USA300. Putative surface protein SA2285, s erine aspartate repeat containing protein D and immunodominant antigen A, are both significantly upregulated in the HA MRSA strain USA100. Immunodominant antigen A, involved in peptidoglycan h ydrolysis [48 ], has been identified as a secreted and c ell wall associated protein [170 ]. A study by

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73 Dubrac et al. in 2007 found that transcription of the isaA gene, encoding the immunodominant antigen A protein, is directly activated by the essential two component system known as WalKR (also known as YycFG) [48 ]. Interestingly, the activity of WalKR, is upregulated during colonization of human nares in carriers of S. aureus Seemingly, colonizing S. aureus preferentially expresses proteins involved in a dherence to tissues, while at the same time downregulating the production of secreted toxins [21, 22]. Interestingly, agr does not appear to be active during nasal colonization, therefore S. aureus would be expected to increase production of surface prote ins and decrease production of secreted toxins and proteases that are regulated by agr concurring with the observation of increased WalKR activity during nasal colonization. Therefore, it is unsurprising to find a surface protein such as immunodominant a ntigen A to be significantly upregulated in a HA MRSA strain such as USA100 where agr is not as active as it is in a CA MRSA strain such as USA300. On another note, many surface proteins covalently linked to the cell wall require a peptide sorting signal containing an LPXTG motif located at th e C terminus of the protein [175 ]. Proteins containing the LPXTG motif are often attached to the cell wall by a protease known as sortase [ 126, 140 ]. Instead of the traditional LPXTG motif contained by many surfac e proteins of S. aureus p utative surface protein SA2285 and serine aspartate repeating protein D, known as SdrD, both contain YSIRK signal peptides [ 38 ]. YSIRK is a variation of the peptide sequence that marks a protein destined for the cell wall to be s ecreted in a ring like manner near the site of cell division [ 38 ]. Proteins containing the YSIRK motif have been associated with adhesion to bones during infection [ 187 ]. A study by Trad et al. found a correlation between the prevalence of the

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74 sdrD gene and strains of S. aureus that commonly cause bone infections [ 187 ]. In fact, a study by Sabat et al. found that a mutation in sdrD and sdrE a gene tandemly encoded with sdrD in the sdr locus, resulted in significantly decreased potential for S. aureus to cause bone infec tions such as osteomyelitis [169 ]. Interestingly, HA MRSA strains are known to commonly cause chronic infections such as osteomyelitis [108 119 ] whereas CA MRSA strains are known to cause acute infections such as s kin and soft tissue dis eases [24, 94, 103 ]. It is no surprise then that a HA MRSA strain such as USA100 upregulates the production of surface associated proteins such as immunodominant A, SdrD, and protein SA2285 that could enable the pathogen to infect the host and lead to a c hronic disease. Though it is not known whether these surface associated proteins are agr regulated, this observation concurs with the fact that CA MRSA strains such as USA300, having higher agr activity, would therefore express less surface associated pro teins than HA MRSA strains such as USA100 [ 116, 119 ]. When comparing HA MRSA USA100 to CA MRSA USA400 in the post exponential phase of growth we observed that USA100 produced 25 times more putative surface protein SA2285 than USA400. As stated e arlier, because agr activity is expected to be decreased in HA MRSA than in CA MRSA, the expression of surface associated proteins would be upregulated in HA MRSA as agr is a negative effector of surface proteins during the later stages of bacterial growth [ 116, 119 ]. On the same note, agr is a positive effector on the presence of proteases such as staphopain A [117 ], which is produced in elevated quantities in CA MRSA USA400 compared to HA MRSA USA100 in our study. In a 2007 study by Vincents et al., the extracellular proteases staphopain A and staphopain B were found to have a role in the downregulation of human cystatin

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75 activity [191 ]. Cystatins are a family of cysteine protease inhibitors that potentially protect against proteases secreted by invading pathogenic microorganisms. Though body fluids may have high concentrations of these protease inhibitors, they are still vulnerable t o attack by other proteases [188 ]. These proteases, such as staphopain A, may have a role in evasion of the host immune s ystem allowing S. aureus to survive and cause disease in the individual. Additionally, it was shown by Imamura et al. in a 2005 study that staphopain A can instigate vascular leakage consequently leading to septic s hock in affected individuals [88 ]. Beca use CA MRSA strains are not only known for causing cutaneous infections but also sepsis [ 63, 110 17 7 ], our observation of elevated Staphopain A production in these strains appears to fit with previous studies on this enzyme. All strains of S. aureu s regardless of antibiotic resistance, have penicillin binding proteins known as PBP1, PBP2, and PBP3. These penicillin binding proteins, having lactam antibiotics, are bound by these antimicrobial agents, in turn compromising the int egrity of the cell wall by preventing cross linking across the layers of peptidoglycan [ 122 ]. PBP2A, encoded by the mecA lactams, therefore it is not targeted by these antibiotics; consequently preventing weakening of the ce lactam antibiotics in CA MRSA, however, is conferred by PBP4 [ 131 lactamase [ 141 ]. On the other hand, loss of PBP2A, also MRSA reduces antibiotic resistance whereas loss of PBP4 in HA MRSA strains has little effe ct on antibiotic resistance [101 ]. quantities in HA MRSA strains of S. aureus than in CA MRSA. Our data suggests that

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76 this is true a MRSA USA100 compared to CA MRSA strains USA300 and USA400. Overall, toxin production in S. aureus is dramatically upregulated during stationary phase [ 18, 66 ]. When compared side by side, the production of gamma hemolysin component C and alpha hemolysin are drastically upregulated in CA MRSA USA300 as opposed to HA MRSA USA100. As stated earlier, agr activity in CA MRSA strains is expected to be higher than in HA MRSA strains. This increase in agr activity leads to elevated expression of major secreted virulence factors, such as gamma and alpha hemolysins [ 18, 66 7 5 13 3 ]. In a 2010 study by Pang et al., agr activity of a USA300 strain was measured by RT PCR after it had been phago cytosed by polymorphonuclear neutrophils (PMN). PMNs are white blood cells used by the host immune system to ingest and degrade invading pathogenic microorganisms. A USA300 strain containing an agr mutation lost significant viability within the PMN as op posed to the USA300 wild type strain containing an intact and fully functional agr [ 14 7 ]. It was found that once inside the PMN, agr activity by USA300 increased signific antly, leading to an increased hemolysin by USA300 in part was responsible for the lysis of PMNs, allowing for evasion of the host immune system, as opposed to a USA300 agr mutant strain [ 147 ]. Our data c oncurs with the findings by Pang et al. at the proteomic level, by showing CA MRSA USA300 upregulates the production of alpha hemolysin and other toxins due to a higher agr activity compared to a HA MRSA strain such as USA100, where agr activity is diminis hed, and therefore the production of alpha hemolysin and other secreted virulence factors is reduced.

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77 S. aureus produces superantigens such as enterotoxins, which are known to result in gastroenteritis caused by food poisoning [ 7, 11 5 ]. As reported by Baba et al., staphylococcal enterotoxin C is encoded on a pathogenicity island [ 6 ], and is not typically found to be expressed in HA MRSA strains [58 ]. Unsurprisingly, as shown by our data, CA MRSA USA400 produced 21.88 times more enterotoxin C than HA M RSA USA100. In a case report from 2002, an outbreak of gastroenteritis occurred within a family that was caused by a CA MRSA strain producing enterotoxin C. This report became the first case of CA MRSA as the sole culprit o f a gastroenteritis outbreak [9 6 ]. In addition, CA MRSA USA400 has been found to express the collagen adhesion gene ( cna ), which is associated with increased binding of S. aureus to host membrane proteins for the pathogenesis of necrotizing pneumonia [ 6, 43 83 ]. Interestingly, when c ompared to HA MRSA USA100, the production of surface proteins such as elastin binding protein, SA2006 protein, and SA0841 involved in adhesion is dramatically upregulated in USA400. Elastin binding protein is also drastically upregulated in USA400 when co mpared to HA MRSA USA200, as shown by our data. Not surprisingly, USA400 is known to be a strong former of biofilms [97 ]. It is possible to speculate that these adhesion proteins may have a strong role in biofilm formation, and perhaps explain the elevat ed levels of this aggregation phenotype in USA400 strains [ 95 ]. Additionally, when compared to USA100 during stationary phase, USA400 also significantly upregulates lipase 1 and lipase 2. In a recent study [ 95 ] both lipase 1 and lipase 2, which facilita te tissue invasion [167 ], were found in low quantities in an agr mutant of S. aureus [ 95 ]. Likewise, a study of HA MRSA and CA MRSA strains from Brazil showed that toxins such as enterotoxin C and PVL were rarely detected in HA

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78 MRSA isolates [181 ]. Accor ding to our study, the production of lipase 1 and lipase 2 is remarkably higher in CA MRSA strains than HA MRSA strains. Further to this, in a study by Abdelnour et al., an agr mutation in S. aureus led to a marked reduction of toxin and enzyme production, including the production of lipases [ 4 ]. In a mouse model of septic arthritis infection, an agr mutant of S. aureus was significantly less virulent than the wild type counterpart [ 4 ]. Also, lipase was found to be important in preventing the phagocytic k illing of S. aureus after engulfment by granulocytes, also known as po lymorphonuclear neutrophils [165 ]. This observation indicates that lipases can be considered virulence factors, which may be responsible for the pathogenesis of S. aureus As mentioned earlier, having higher agr activity, CA MRSA strains express agr regulated proteins such as lipases 1 and 2 significantly more than HA MRSA, potentially contributing to its hypervirulence. Conversely, the high presence of anti sigma factor B antago nist, also known as anti anti sigma factor B, in HA MRSA USA100 would lead to higher SigmaB activity in this strain. Because SigmaB has been shown to have a repressive effect on agr activity [15], it is of no surprise CA MRSA strain USA400, due to its hyp eractive agr would produce SigmaB at reduced levels. Though the presence of an intracellular protein such as anti sigma factor B antagonist in the secretome of S.aureus may be puzzling, the resulting quantitation of this protein is exactly what is expecte d when comparing HA MRSA and CA MRSA strains. As indicated by our data, there is an astonishing 14.5 increase of anti sigma factor B antagonist production in the HA MRSA USA200 compared to CA MRSA USA400. Anti sigma factor B antagonist is a positive effe ctor of Sigma B activity. The positive effect of Sigma B on surface proteins contributes to adhesion of the

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79 cell to host tissue, whilst at the same time negatively impacting the production of secreted virulence factors when S. aureus enters stationary pha se [ 206, 208 ] As expected, Sigma B activity is reduced in CA MRSA, as these strains of S. aureus are known for the increased secretion of virulence factors, such as toxins and proteases. The secretion of the zinc metalloprotease aureolysin wa s significantly upregulated in the CA MRSA strain USA400 when compared to the HA MRSA strain USA100. Because this protease is positively regulated by agr [ 30, 49, 183 ], it is expected to be highly expressed in strains with a hyperactive agr regulon, such as a CA MRSA isolates. Indeed, we have conducted assays in our laboratory to determine the presence of secreted proteases such as aureolysin in HA MRSA and CA MRSA strains. A total of 24 USA100 strains and 158 USA300 strains were studied, and it was dete rmined that the CA MRSA USA300 isolates produced far more secreted proteases than HA MRSA USA100 isolates (Rivera and Shaw, unpublished observation). This observation correlates with the fact that CA MRSA strains of S. aureus having higher agr activity, secrete significantly more proteases than HA MRSA strains. Because most of the scientific studies involving HA MRSA and CA MRSA strains are at the genomic and transcriptomic level, our study aimed to compare clinically significant strains of S. aureus currently afflicting individuals in healthcare facilities and in the community at the proteomic level. The reproducibility of protein extraction and iTRAQ labeling followed by mass spectrometric analysis can be used to corroborate present li terature and to speculate and answer relevant biological questions. With our study, we found many differences in protein secretion amongst the four clinical strains that support studies of these proteins at the genomic and transcriptomic levels. Overall, we identified

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80 many secreted toxins and proteases that were upregulated in hypervirulent CA MRSA strains such as USA300 and USA400, typical of increased agr activity. On the same token, we found surface associated proteins to be upregulated in HA MRSA suc h as USA100 and USA200, concurring with the finding that agr activity in these particular strains is diminished in comparison to CA MRSA strains. Consideration of the presence of cytoplasmic proteins in secreted fractions. Even after having refined m ethods for the extraction of secreted proteins from supernatants of S. aureus cultures, contamination by cytoplasmic proteins is a common problem. From our study in particular, we have identified several classes of cytoplasmic proteins in the supernatants of S. aureus such as those involved in central carbon metabolism and protein synthesis. Though the identification of cytoplasmic proteins in the extracellular milieu of S. aureus would be unexpected, a few cytoplasmic proteins have been discovered exist a study by Pasztor et al., a wild type strain of S. aureus known as strain 22 secreted cytoplasmic proteins into the supernatant, whereas secretion of cytoplasmic proteins in an atl (coding for major autol ysin) mutant was attenuated [151 ]. In addition, the presence of cytoplasmic proteins, clearly missing a signal peptide sequence for secretion, has been observed in the culture supernatants of Bacillus subtilis and S. aureus [ 179, 1 84, 206 ]. Because the most abundant cytoplasmic proteins are not necessarily seen in supernatants, it is possible the cell secretes these proteins by as yet unknown mechanisms, instead of their being present in supernatants as a result of cell lysis. The study by Pasztor et al., found the protein major autolysin to be responsible for the secretion of a common cytoplasmic protein, glyceraldehyde 3 phos phate dehydrogenase (GAPDH) [1451 ]. As

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81 another example, enolase was found to have a role in S. aureus adh esion by binding to h uman laminin and plasminogen [73 ]. It is thus possible for cytoplasmic proteins, consistently identified as secreted proteins, to have extracellular functions. Because surface associated proteins mediate host bacterial interactions, these newly discovered antimicrobial treatment that could perhaps alleviate the rampant spread of the extremely adaptive and pathogenic S. aureus

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82 Future Direction s W ith our currently refined method for the extraction of secreted proteins, it is possible we are excluding certain proteins with low molecular weight. The Centricon columns used for the concentration of secreted proteins have a cutoff of 5 kDa. Any protei n with a molecular weight less than 5.0 kDa may not be retained by the concentration column, and could thus be omitted from subsequent mass spectrometric analysis. The molecular weights of phenol soluble modulins produced by S. aureus for example, range from 2.17 kDa to 4.5 kDa. Phenol soluble modulins, being important virulence factors of S. aureus which are highly expressed in clinical strains, could potentially be omitted from a proteomic study if Centricon columns are used for the concentration of s ecreted proteins. The method could be further refined by excluding the concentration step altogether and precipitating secreted proteins from the supernatant with TCA immediately after filter sterilization. Though, with this revision, it is possible to s acrifice overall protein concentration, it could provide full coverage of all the proteins secreted by S. aureus at a particular growth phase, including proteins with low molecular weights. Another important modification of the refined method for extractio n of secreted proteins involves fractionation of the sample prior to MS analysis. Our first MudPIT analysis of SH1000 cytoplasmic proteins produced the highest yield of identified proteins. This first MudPIT analysis involved the fractionation of the com plex sample using the HPLC. Due to the inconsistent results from HPLC fractionation, we resorted to gas fraction ation of the samples. Gas fractionation, however, yielded low protein identifications compared to

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83 HPLC fractionation. Ideally, we would like to fractionate samples using a new HPLC for future proteomic studies. The next step, after having analyzed the secretomes of HA MRSA and CA MRSA strains, would be comparing and analyzing the surface associated proteomes, known as as well a s the membrane associated proteins, of these same clinical strains. To date, proteomic studies of surface exposed proteins have been completed using only CA MRSA strains of S. aureus A proteomic analysis of surface associated proteins produced by CA MRS A compared to HA MRSA could lead to a better understanding of any difference in the pathogenesis amongst these strains. Apart from adhesion to the host tissue, surface associated proteins can also mediate evasion of the host immune system and invasion of host cells [ 60 14 2 ]. Though surface associated proteins have consistently been identified in the secretome, a proteomic method targeted to the surface of the cell could lead to the identification of even more surface associated proteins that could potent ially be specific to a given lineage. Understanding any differences in surface proteins associated with adhesion of the cell to tissue or in dwelling devices amongst clinical strains could lead to a better understanding of the host pathogen physiology, an d potentially provide novel vaccine candidates. Also, many conserved hypothetical proteins were found upregulated in the HA MRSA and CA MRSA strains. These proteins, not known to be controlled by the central global regulator agr are currently uncharacter ized. I would suggest studying and characterizing these proteins in an attempt to determine if they are unique to any particular clinical strain that could re sult in differential pathogeneses of these strains.

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84 References 1. (1999). "From the Centers f or Disease Control and Prevention. Four pediatric deaths from community acquired methicillin resistant Staphylococcus aureus -Minnesota and North Dakota, 1997 1999." JAMA 282(12): 1123 1125. 2. (2002). "Staphylococcus aureus resistant to vancomycin -United S tates, 2002." MMWR Morb Mortal Wkly Rep 51(26): 565 567. 3. (2002). "Vancomycin resistant Staphylococcus aureus -Pennsylvania, 2002." MMWR Morb Mortal Wkly Rep 51(40): 902. 4. Abdelnour, A., S. Arvidson, et al. (1993). "The accessory gene regulator (agr) contr ols Staphylococcus aureus virulence in a murine arthritis model." Infect Immun 61(9): 3879 3885. 5. Archer, G. L. (1998). "Staphylococcus aureus: a well armed pathogen." Clin Infect Dis 26(5): 1179 1181. 6. Baba, T., F. Takeuchi, et al. (2002). "Genome and vir ulence determinants of high virulence community acquired MRSA." Lancet 359(9320): 1819 1827. 7. Balaban, N. and A. Rasooly (2000). "Staphylococcal enterotoxins." Int J Food Microbiol 61(1): 1 10. 8. Barna, J. C. and D. H. Williams (1984). "The structure and mo de of action of glycopeptide antibiotics of the vancomycin group." Annu Rev Microbiol 38: 339 357. 9. Bayer, M. G., J. H. Heinrichs, et al. (1996). "The molecular architecture of the sar locus in Staphylococcus aureus." J Bacteriol 178(15): 4563 4570. 10. Beche r, D., K. Hempel, et al. (2009). "A proteomic view of an important human pathogen -towards the quantification of the entire Staphylococcus aureus proteome." PLoS One 4(12): e8176.

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85 11. Bernardo, K., S. Fleer, et al. (2002). "Identification of Staphylococcus au reus exotoxins by combined sodium dodecyl sulfate gel electrophoresis and matrix assisted laser desorption/ ionization time of flight mass spectrometry." Proteomics 2(6): 740 746. 12. Bhakdi, S. and J. Tranum Jensen (1991). "Alpha toxin of Staphylococcus aure us." Microbiol Rev 55(4): 733 751. 13. Bibel, D. J., J. H. Greenberg, et al. (1977). "Staphylococcus aureus and the microbial ecology of atopic dermatitis." Can J Microbiol 23(8): 1062 1068. 14. Bischoff, M., P. Dunman, et al. (2004). "Microarray based analysis of the Staphylococcus aureus sigmaB regulon." J Bacteriol 186(13): 4085 4099. 15. Bischoff, M., J. M. Entenza, et al. (2001). "Influence of a functional sigB operon on the global regulators sar and agr in Staphylococcus aureus." J Bacteriol 183(17): 5171 5179 16. Blevins, J. S., A. F. Gillaspy, et al. (1999). "The Staphylococcal accessory regulator (sar) represses transcription of the Staphylococcus aureus collagen adhesin gene (cna) in an agr independent manner." Mol Microbiol 33(2): 317 326. 17. Bohach, G. A., D. J. Fast, et al. (1990). "Staphylococcal and streptococcal pyrogenic toxins involved in toxic shock syndrome and related illnesses." Crit Rev Microbiol 17(4): 251 272. 18. Boisset, S., T. Geissmann, et al. (2007). "Staphylococcus aureus RNAIII coordinately re presses the synthesis of virulence factors and the transcription regulator Rot by an antisense mechanism." Genes Dev 21(11): 1353 1366. 19. Boyle, T., V. Lancaster, et al. (1994). "Method for simultaneous isolation and quantitation of platelet activating fact or and multiple arachidonate metabolites from small samples: analysis of effects of Staphylococcus aureus enterotoxin B in mice." Anal Biochem 216(2): 373 382. 20. Bubeck Wardenburg, J., T. Bae, et al. (2007). "Poring over pores: alpha hemolysin and Panton Va lentine leukocidin in Staphylococcus aureus pneumonia." Nat Med 13(12): 1405 1406.

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86 21. Burian, M., M. Rautenberg, et al. (2010). "Temporal expression of adhesion factors and activity of global regulators during establishment of Staphylococcus aureus nasal col onization." J Infect Dis 201(9): 1414 1421. 22. Burian, M., C. Wolz, et al. (2010). "Regulatory adaptation of Staphylococcus aureus during nasal colonization of humans." PLoS One 5(4): e10040. 23. Carleton, H. A., B. A. Diep, et al. (2004). "Community adapted me thicillin resistant Staphylococcus aureus (MRSA): population dynamics of an expanding community reservoir of MRSA." J Infect Dis 190(10): 1730 1738. 24. Chambers, H. F. (2001). "The changing epidemiology of Staphylococcus aureus?" Emerg Infect Dis 7(2): 178 18 2. 25. Chan, P. F., S. J. Foster, et al. (1998). "The Staphylococcus aureus alternative sigma factor sigmaB controls the environmental stress response but not starvation survival or pathogenicity in a mouse abscess model." J Bacteriol 180(23): 6082 6089. 26. Cha tterjee, I., P. Becker, et al. (2005). "Staphylococcus aureus ClpC is required for stress resistance, aconitase activity, growth recovery, and death." J Bacteriol 187(13): 4488 4496. 27. Chatterjee, I., S. Schmitt, et al. (2009). "Staphylococcus aureus ClpC A TPase is a late growth phase effector of metabolism and persistence." Proteomics 9(5): 1152 1176. 28. Cheung, A. L., Y. T. Chien, et al. (1999). "Hyperproduction of alpha hemolysin in a sigB mutant is associated with elevated SarA expression in Staphylococcus aureus." Infect Immun 67(3): 1331 1337. 29. Cheung, A. L., J. M. Koomey, et al. (1992). "Regulation of exoprotein expression in Staphylococcus aureus by a locus (sar) distinct from agr." Proc Natl Acad Sci U S A 89(14): 6462 6466. 30. Chien, Y. and A. L. Cheung (1998). "Molecular interactions between two global regulators, sar and agr, in Staphylococcus aureus." J Biol Chem 273(5): 2645 2652.

PAGE 99

87 31. Choe, L. H., K. Aggarwal, et al. (2005). "A comparison of the consistency of proteome quantitation using two dimensional electrophoresis and shotgun isobaric tagging in Escherichia coli cells." Electrophoresis 26(12): 2437 2449. 32. Collen, D. (1998). "Staphylokinase: a potent, uniquely fibrin selective thrombolytic agent." Nat Med 4(3): 279 284. 33. Cornish, T. J. and R. J. Cott er (1997). "High order kinetic energy focusing in an end cap reflectron time of flight mass spectrometer." Anal Chem 69(22): 4615 4618. 34. Cotter, R. J., W. Griffith, et al. (2007). "Tandem time of flight (TOF/TOF) mass spectrometry and the curved field refl ectron." J Chromatogr B Analyt Technol Biomed Life Sci 855(1): 2 13. 35. Coulter, S. N., W. R. Schwan, et al. (1998). "Staphylococcus aureus genetic loci impacting growth and survival in multiple infection environments." Mol Microbiol 30(2): 393 404. 36. Cribier B., G. Prevost, et al. (1992). "Staphylococcus aureus leukocidin: a new virulence factor in cutaneous infections? An epidemiological and experimental study." Dermatology 185(3): 175 180. 37. DeDent (2006). Staphylococcal Sortases and Surface Proteins. Gram positive pathogens. R. N. VA Fischetti, JJ Ferretti, DA Portnoy, JI Rood. Washington, DC, ASM Press: 486 495. 38. DeDent, A., T. Bae, et al. (2008). "Signal peptides direct surface proteins to two distinct envelope locations of Staphylococcus aureus." EMBO J 27(20): 2656 2668. 39. Delahunty, C. M. and J. R. Yates, 3rd (2007). "MudPIT: multidimensional protein identification technology." Biotechniques 43(5): 563, 565, 567 passim. 40. Demerec, M. (1948). "Origin of bacterial resistance to antibiotics." J Bacteriol 56( 1): 63 74. 41. Deresinski, S. (2005). "Methicillin resistant Staphylococcus aureus: an evolutionary, epidemiologic, and therapeutic odyssey." Clin Infect Dis 40(4): 562 573.

PAGE 100

88 42. Diep, B. A., S. R. Gill, et al. (2006). "Complete genome sequence of USA300, an epid emic clone of community acquired meticillin resistant Staphylococcus aureus." Lancet 367(9512): 731 739. 43. Dongre, A. R., J. K. Eng, et al. (1997). "Emerging tandem mass spectrometry techniques for the rapid identification of proteins." Trends Biotechnol 15 (10): 418 425. 44. Doroshenko, V. M. and R. J. Cotter (1999). "Ideal velocity focusing in a reflectron time of flight mass spectrometer." J Am Soc Mass Spectrom 10(10): 992 999. 45. Dreisbach, A., K. Hempel, et al. (2010). "Profiling the surfacome of Staphylococ cus aureus." Proteomics 10(17): 3082 3096. 46. Dreisbach, A., A. Otto, et al. (2008). "Monitoring of changes in the membrane proteome during stationary phase adaptation of Bacillus subtilis using in vivo labeling techniques." Proteomics 8(10): 2062 2076. 47. Dru mmelsmith, J., E. Winstall, et al. (2007). "Comparative proteomics analyses reveal a potential biomarker for the detection of vancomycin intermediate Staphylococcus aureus strains." J Proteome Res 6(12): 4690 4702. 48. Dubrac, S., I. G. Boneca, et al. (2007). "New insights into the WalK/WalR (YycG/YycF) essential signal transduction pathway reveal a major role in controlling cell wall metabolism and biofilm formation in Staphylococcus aureus." J Bacteriol 189(22): 8257 8269. 49. Dunman, P. M., E. Murphy, et al. ( 2001). "Transcription profiling based identification of Staphylococcus aureus genes regulated by the agr and/or sarA loci." J Bacteriol 183(24): 7341 7353. 50. Dziewanowska, K., V. M. Edwards, et al. (1996). "Comparison of the beta toxins from Staphylococcus aureus and Staphylococcus intermedius." Arch Biochem Biophys 335(1): 102 108. 51. Emori, T. G. and R. P. Gaynes (1993). "An overview of nosocomial infections, including the role of the microbiology laboratory." Clin Microbiol Rev 6(4): 428 442.

PAGE 101

89 52. Erwin, D. G. and R. D. Haight (1973). "Lethal and inhibitory effects of sodium chloride on thermally stressed Staphylococcus aureus." J Bacteriol 116(1): 337 340. 53. Essmann, F., H. Bantel, et al. (2003). "Staphylococcus aureus alpha toxin induced cell death: predominant necrosis despite apoptotic caspase activation." Cell Death Differ 10(11): 1260 1272. 54. Etienne, J. (2005). "Panton Valentine leukocidin: a marker of severity for Staphylococcus aureus infection?" Clin Infect Dis 41(5): 591 593. 55. Fancher, C. A., A. S. Woods et al. (2000). "Improving the sensitivity of the end cap reflectron time of flight mass spectrometer." J Mass Spectrom 35(2): 157 162. 56. Fenn, J. (2002). "Electrospray ionization mass spectrometry: How it all began." J Biomol Tech 13(3): 101 118. 57. Fenn, J B., M. Mann, et al. (1989). "Electrospray ionization for mass spectrometry of large biomolecules." Science 246(4926): 64 71. 58. Fey, P. D., B. Said Salim, et al. (2003). "Comparative molecular analysis of community or hospital acquired methicillin resista nt Staphylococcus aureus." Antimicrob Agents Chemother 47(1): 196 203. 59. Finks, J., E. Wells, et al. (2009). "Vancomycin resistant Staphylococcus aureus, Michigan, USA, 2007." Emerg Infect Dis 15(6): 943 945. 60. Foster, T. J. and M. Hook (1998). "Surface prot ein adhesins of Staphylococcus aureus." Trends Microbiol 6(12): 484 488. 61. Freer, J. H. and J. P. Arbuthnott (1982). "Toxins of Staphylococcus aureus." Pharmacol Ther 19(1): 55 106. 62. Frees, D., A. Chastanet, et al. (2004). "Clp ATPases are required for stre ss tolerance, intracellular replication and biofilm formation in Staphylococcus aureus." Mol Microbiol 54(5): 1445 1462.

PAGE 102

90 63. Gales, A. C., H. S. Sader, et al. (2006). "Emergence of linezolid resistant Staphylococcus aureus during treatment of pulmonary infect ion in a patient with cystic fibrosis." Int J Antimicrob Agents 27(4): 300 302. 64. Gao, J. and G. C. Stewart (2004). "Regulatory elements of the Staphylococcus aureus protein A (Spa) promoter." J Bacteriol 186(12): 3738 3748. 65. Gase, K., J. J. Ferretti, et al (1999). "Identification, cloning, and expression of the CAMP factor gene (cfa) of group A streptococci." Infect Immun 67(9): 4725 4731. 66. Geisinger, E., R. P. Adhikari, et al. (2006). "Inhibition of rot translation by RNAIII, a key feature of agr function ." Mol Microbiol 61(4): 1038 1048. 67. Gertz, S., S. Engelmann, et al. (2000). "Characterization of the sigma(B) regulon in Staphylococcus aureus." J Bacteriol 182(24): 6983 6991. 68. Giachino, P., S. Engelmann, et al. (2001). "Sigma(B) activity depends on RsbU in Staphylococcus aureus." J Bacteriol 183(6): 1843 1852. 69. Gill, S. R., D. E. Fouts, et al. (2005). "Insights on evolution of virulence and resistance from the complete genome analysis of an early methicillin resistant Staphylococcus aureus strain and a bi ofilm producing methicillin resistant Staphylococcus epidermidis strain." J Bacteriol 187(7): 2426 2438. 70. Gillet, Y., B. Issartel, et al. (2002). "Association between Staphylococcus aureus strains carrying gene for Panton Valentine leukocidin and highly le thal necrotising pneumonia in young immunocompetent patients." Lancet 359(9308): 753 759. 71. Giraudo, A. T., A. L. Cheung, et al. (1997). "The sae locus of Staphylococcus aureus controls exoprotein synthesis at the transcriptional level." Arch Microbiol 168( 1): 53 58. 72. Giraudo, A. T., C. G. Raspanti, et al. (1994). "Characterization of a Tn551 mutant of Staphylococcus aureus defective in the production of several exoproteins." Can J Microbiol 40(8): 677 681.

PAGE 103

91 73. Glowalla, E., B. Tosetti, et al. (2009). "Proteomi cs based identification of anchorless cell wall proteins as vaccine candidates against Staphylococcus aureus." Infect Immun 77(7): 2719 2729. 74. Gorg, A., W. Postel, et al. (1988). "Horizontal two dimensional electrophoresis with immobilized pH gradients usi ng PhastSystem." Electrophoresis 9(1): 57 59. 75. Gravet, A., D. A. Colin, et al. (1998). "Characterization of a novel structural member, LukE LukD, of the bi component staphylococcal leucotoxins family." FEBS Lett 436(2): 202 208. 76. Greenwood, D. and F. O'Gra dy (1972). "Scanning electron microscopy of Staphyloccus aureus exposed to some common anti staphylococcal agents." J Gen Microbiol 70(2): 263 270. 77. Griffin, A. M., V. J. Morris, et al. (1996). "The cpsABCDE genes involved in polysaccharide production in S treptococcus salivarius ssp. thermophilus strain NCBF 2393." Gene 183(1 2): 23 27. 78. Haight, T. H. and M. Finland (1952). "Resistance of bacteria to erythromycin." Proc Soc Exp Biol Med 81(1): 183 188. 79. Hanaki, H., K. Kuwahara Arai, et al. (1998). "Activate d cell wall synthesis is associated with vancomycin resistance in methicillin resistant Staphylococcus aureus clinical strains Mu3 and Mu50." J Antimicrob Chemother 42(2): 199 209. 80. Haynes, P. A. and J. R. Yates, 3rd (2000). "Proteome profiling pitfalls an d progress." Yeast 17(2): 81 87. 81. Hecker, M. and U. Volker (1998). "Non specific, general and multiple stress resistance of growth restricted Bacillus subtilis cells by the expression of the sigmaB regulon." Mol Microbiol 29(5): 1129 1136. 82. Hempel, K., J. Pane Farre, et al. (2010). "Quantitative cell surface proteome profiling for SigB dependent protein expression in the human pathogen Staphylococcus aureus via biotinylation approach." J Proteome Res 9(3): 1579 1590.

PAGE 104

92 83. Highlander, S. K., K. G. Hulten, et al. (2007). "Subtle genetic changes enhance virulence of methicillin resistant and sensitive Staphylococcus aureus." BMC Microbiol 7: 99. 84. Hiramatsu, K., N. Aritaka, et al. (1997). "Dissemination in Japanese hospitals of strains of Staphylococcus aureus heter ogeneously resistant to vancomycin." Lancet 350(9092): 1670 1673. 85. Holden, M. T., E. J. Feil, et al. (2004). "Complete genomes of two clinical Staphylococcus aureus strains: evidence for the rapid evolution of virulence and drug resistance." Proc Natl Acad Sci U S A 101(26): 9786 9791. 86. Horsburgh, M. J., J. L. Aish, et al. (2002). "sigmaB modulates virulence determinant expression and stress resistance: characterization of a functional rsbU strain derived from Staphylococcus aureus 8325 4." J Bacteriol 184( 19): 5457 5467. 87. Hunt, D. F., J. R. Yates, 3rd, et al. (1986). "Protein sequencing by tandem mass spectrometry." Proc Natl Acad Sci U S A 83(17): 6233 6237. 88. Imamura, T., S. Tanase, et al. (2005). "Induction of vascular leakage through release of bradykini n and a novel kinin by cysteine proteinases from Staphylococcus aureus." J Exp Med 201(10): 1669 1676. 89. Ito, T., K. Okuma, et al. (2003). "Insights on antibiotic resistance of Staphylococcus aureus from its whole genome: genomic island SCC." Drug Resist Up dat 6(1): 41 52. 90. Iyer, L. M., V. Anantharaman, et al. (2007). "The DOMON domains are involved in heme and sugar recognition." Bioinformatics 23(20): 2660 2664. 91. Janzon, L. and S. Arvidson (1990). "The role of the delta lysin gene (hld) in the regulation o f virulence genes by the accessory gene regulator (agr) in Staphylococcus aureus." EMBO J 9(5): 1391 1399. 92. Janzon, L., S. Lofdahl, et al. (1989). "Identification and nucleotide sequence of the delta lysin gene, hld, adjacent to the accessory gene regulato r (agr) of Staphylococcus aureus." Mol Gen Genet 219(3): 480 485.

PAGE 105

93 93. Jin, T., M. Bokarewa, et al. (2004). "Staphylococcus aureus resists human defensins by production of staphylokinase, a novel bacterial evasion mechanism." J Immunol 172(2): 1169 1176. 94. John son, J. K., T. Khoie, et al. (2007). "Skin and soft tissue infections caused by methicillin resistant Staphylococcus aureus USA300 clone." Emerg Infect Dis 13(8): 1195 1200. 95. Jones, R. C., J. Deck, et al. (2008). "Relative quantitative comparisons of the e xtracellular protein profiles of Staphylococcus aureus UAMS 1 and its sarA, agr, and sarA agr regulatory mutants using one dimensional polyacrylamide gel electrophoresis and nanocapillary liquid chromatography coupled with tandem mass spectrometry." J Bact eriol 190(15): 5265 5278. 96. Jones, T. F., M. E. Kellum, et al. (2002). "An outbreak of community acquired foodborne illness caused by methicillin resistant Staphylococcus aureus." Emerg Infect Dis 8(1): 82 84. 97. Joshi, S. G., M. Paff, et al. (2010). "Control of methicillin resistant Staphylococcus aureus in planktonic form and biofilms: a biocidal efficacy study of nonthermal dielectric barrier discharge plasma." Am J Infect Control 38(4): 293 301. 98. Kaneko, J., T. Ozawa, et al. (1997). "Sequential binding of Staphylococcal gamma hemolysin to human erythrocytes and complex formation of the hemolysin on the cell surface." Biosci Biotechnol Biochem 61(5): 846 851. 99. Karlsson, A. and S. Arvidson (2002). "Variation in extracellular protease production among clinical isolates of Staphylococcus aureus due to different levels of expression of the protease repressor sarA." Infect Immun 70(8): 4239 4246. 100. Karlsson, A., P. Saravia Otten, et al. (2001). "Decreased amounts of cell wall associated protein A and fibronectin b inding proteins in Staphylococcus aureus sarA mutants due to up regulation of extracellular proteases." Infect Immun 69(8): 4742 4748. 101. Katayama, Y., H. Z. Zhang, et al. (2003). "Effect of disruption of Staphylococcus aureus PBP4 gene on resistance to be ta lactam antibiotics." Microb Drug Resist 9(4): 329 336.

PAGE 106

94 102. Kawabata, S., T. Morita, et al. (1985). "Enzymatic properties of staphylothrombin, an active molecular complex formed between staphylocoagulase and human prothrombin." J Biochem 98(6): 1603 1614. 103. King, M. D., B. J. Humphrey, et al. (2006). "Emergence of community acquired methicillin resistant Staphylococcus aureus USA 300 clone as the predominant cause of skin and soft tissue infections." Ann Intern Med 144(5): 309 317. 104. Kirby, W. M. (1944). "Extr action of a Highly Potent Penicillin Inactivator from Penicillin Resistant Staphylococci." Science 99(2579): 452 453. 105. Kloos, W. (1991). Staphylococcus. Washington, DC, American Society for Microbiology. 106. Klose, J. and U. Kobalz (1995). "Two dimensional el ectrophoresis of proteins: an updated protocol and implications for a functional analysis of the genome." Electrophoresis 16(6): 1034 1059. 107. Kohler, C., S. Wolff, et al. (2005). "Proteome analyses of Staphylococcus aureus in growing and non growing cells: a physiological approach." Int J Med Microbiol 295(8): 547 565. 108. Kollef, M. H., G. Sherman, et al. (1999). "Inadequate antimicrobial treatment of infections: a risk factor for hospital mortality among critically ill patients." Chest 115(2): 462 474. 109. Kotti ng, J., H. Eibl, et al. (1988). "Substrate specificity of Staphylococcus aureus (TEN5) lipases with isomeric oleoyl sn glycerol ethers as substrates." Chem Phys Lipids 47(2): 117 122. 110. Kourbatova, E. V., J. S. Halvosa, et al. (2005). "Emergence of communit y associated methicillin resistant Staphylococcus aureus USA 300 clone as a cause of health care associated infections among patients with prosthetic joint infections." Am J Infect Control 33(7): 385 391. 111. Kullik, I., P. Giachino, et al. (1998). "Deletion of the alternative sigma factor sigmaB in Staphylococcus aureus reveals its function as a global regulator of virulence genes." J Bacteriol 180(18): 4814 4820.

PAGE 107

95 112. Kullik, I. I. and P. Giachino (1997). "The alternative sigma factor sigmaB in Staphylococcus au reus: regulation of the sigB operon in response to growth phase and heat shock." Arch Microbiol 167(2/3): 151 159. 113. Kumar, C. C. and R. P. Novick (1985). "Plasmid pT181 replication is regulated by two countertranscripts." Proc Natl Acad Sci U S A 82(3): 63 8 642. 114. Lacey, R. W. and I. Chopra (1975). "Effect of plasmid carriage on the virulence of staphylococcus aureus." J Med Microbiol 8(1): 137 147. 115. Le Loir, Y., F. Baron, et al. (2003). "Staphylococcus aureus and food poisoning." Genet Mol Res 2(1): 63 76. 116. Li, M., B. A. Diep, et al. (2009). "Evolution of virulence in epidemic community associated methicillin resistant Staphylococcus aureus." Proc Natl Acad Sci U S A 106(14): 5883 5888. 117. Lindsay, J. A. and S. J. Foster (1999). "Interactive regulatory pathway s control virulence determinant production and stability in response to environmental conditions in Staphylococcus aureus." Mol Gen Genet 262(2): 323 331. 118. Liu, H., R. G. Sadygov, et al. (2004). "A model for random sampling and estimation of relative prote in abundance in shotgun proteomics." Anal Chem 76(14): 4193 4201. 119. Lodise, T. P., P. S. McKinnon, et al. (2003). "Outcomes analysis of delayed antibiotic treatment for hospital acquired Staphylococcus aureus bacteremia." Clin Infect Dis 36(11): 1418 1423. 120. Loughman, J. A., S. A. Fritz, et al. (2009). "Virulence gene expression in human community acquired Staphylococcus aureus infection." J Infect Dis 199(3): 294 301. 121. Lowy, F. D. (1998). "Staphylococcus aureus infections." N Engl J Med 339(8): 520 532. 122. Low y, F. D. (2003). "Antimicrobial resistance: the example of Staphylococcus aureus." J Clin Invest 111(9): 1265 1273.

PAGE 108

96 123. Mallorqui Fernandez, G., A. Marrero, et al. (2004). "Staphylococcal methicillin resistance: fine focus on folds and functions." FEMS Microb iol Lett 235(1): 1 8. 124. Maresso, A. W., T. J. Chapa, et al. (2006). "Surface protein IsdC and Sortase B are required for heme iron scavenging of Bacillus anthracis." J Bacteriol 188(23): 8145 8152. 125. Mazmanian, S. K., G. Liu, et al. (1999). "Staphylococcus a ureus sortase, an enzyme that anchors surface proteins to the cell wall." Science 285(5428): 760 763. 126. McAleese, F. M., E. J. Walsh, et al. (2001). "Loss of clumping factor B fibrinogen binding activity by Staphylococcus aureus involves cessation of transc ription, shedding and cleavage by metalloprotease." J Biol Chem 276(32): 29969 29978. 127. McDougal, L. K., C. D. Steward, et al. (2003). "Pulsed field gel electrophoresis typing of oxacillin resistant Staphylococcus aureus isolates from the United States: est ablishing a national database." J Clin Microbiol 41(11): 5113 5120. 128. McGavin, M. J., C. Zahradka, et al. (1997). "Modification of the Staphylococcus aureus fibronectin binding phenotype by V8 protease." Infect Immun 65(7): 2621 2628. 129. McNulty, C., J. Thomp son, et al. (2006). "The cell surface expression of group 2 capsular polysaccharides in Escherichia coli: the role of KpsD, RhsA and a multi protein complex at the pole of the cell." Mol Microbiol 59(3): 907 922. 130. Melish, M. E. and L. A. Glasgow (1970). "T he staphylococcal scalded skin syndrome." N Engl J Med 282(20): 1114 1119. 131. Memmi, G., S. R. Filipe, et al. (2008). "Staphylococcus aureus PBP4 is essential for beta lactam resistance in community acquired methicillin resistant strains." Antimicrob Agents Chemother 52(11): 3955 3966. 132. Mempel, M., C. Schnopp, et al. (2002). "Invasion of human keratinocytes by Staphylococcus aureus and intracellular bacterial persistence represent haemolysin independent virulence mechanisms that are followed by features of ne crotic and apoptotic keratinocyte cell death." Br J Dermatol 146(6): 943 951.

PAGE 109

97 133. Menestrina, G., M. D. Serra, et al. (2001). "Mode of action of beta barrel pore forming toxins of the staphylococcal alpha hemolysin family." Toxicon 39(11): 1661 1672. 134. Miyazak i, E., J. M. Chen, et al. (1999). "The Staphylococcus aureus rsbW (orf159) gene encodes an anti sigma factor of SigB." J Bacteriol 181(9): 2846 2851. 135. Mizuno, T., M. Y. Chou, et al. (1984). "A unique mechanism regulating gene expression: translational inhi bition by a complementary RNA transcript (micRNA)." Proc Natl Acad Sci U S A 81(7): 1966 1970. 136. Mohr, M. D., K. O. Bornsen, et al. (1995). "Matrix assisted laser desorption/ionization mass spectrometry: improved matrix for oligosaccharides." Rapid Commun M ass Spectrom 9(9): 809 814. 137. Monday, S. R. and G. A. Bohach (2001). "Genes encoding staphylococcal enterotoxins G and I are linked and separated by DNA related to other staphylococcal enterotoxins." J Nat Toxins 10(1): 1 8. 138. Moran, G. J., R. N. Amii, et al (2005). "Methicillin resistant Staphylococcus aureus in community acquired skin infections." Emerg Infect Dis 11(6): 928 930. 139. Morfeldt, E., I. Panova Sapundjieva, et al. (1996). "Detection of the response regulator AgrA in the cytosolic fraction of Staphylococcus aureus by monoclonal antibodies." FEMS Microbiol Lett 143(2 3): 195 201. 140. Navarre, W. W. and O. Schneewind (1994). "Proteolytic cleavage and cell wall anchoring at the LPXTG motif of surface proteins in gram positive bacteria." Mol Microbio l 14(1): 115 121. 141. Navratna, V., S. Nadig, et al. (2010). "Molecular basis for the role of Staphylococcus aureus penicillin binding protein 4 in antimicrobial resistance." J Bacteriol 192(1): 134 144. 142. Nicholas, R. O., T. Li, et al. (1999). "Isolation and characterization of a sigB deletion mutant of Staphylococcus aureus." Infect Immun 67(7): 3667 3669. 143. Nilsson, M., L. Frykberg, et al. (1998). "A fibrinogen binding protein of Staphylococcus epidermidis." Infect Immun 66(6): 2666 2673.

PAGE 110

98 144. Novick, R. P. (2003 ). "Autoinduction and signal transduction in the regulation of staphylococcal virulence." Mol Microbiol 48(6): 1429 1449. 145. Novick, R. P., S. J. Projan, et al. (1995). "The agr P2 operon: an autocatalytic sensory transduction system in Staphylococcus aureus ." Mol Gen Genet 248(4): 446 458. 146. Novick, R. P., H. F. Ross, et al. (1993). "Synthesis of staphylococcal virulence factors is controlled by a regulatory RNA molecule." EMBO J 12(10): 3967 3975. 147. Oliveira, D. C. and H. de Lencastre (2002). "Multiplex PCR s trategy for rapid identification of structural types and variants of the mec element in methicillin resistant Staphylococcus aureus." Antimicrob Agents Chemother 46(7): 2155 2161. 148. Opiteck, G. J., K. C. Lewis, et al. (1997). "Comprehensive on line LC/LC/MS of proteins." Anal Chem 69(8): 1518 1524. 149. Pane Farre, J., B. Jonas, et al. (2006). "The sigmaB regulon in Staphylococcus aureus and its regulation." Int J Med Microbiol 296(4 5): 237 258. 150. Pang, Y. Y., J. Schwartz, et al. (2010). "agr Dependent Interacti ons of Staphylococcus aureus USA300 with Human Polymorphonuclear Neutrophils." J Innate Immun 2(6): 546 559. 151. Pasztor, L., A. K. Ziebandt, et al. (2010). "The staphylococcal major autolysin (ATL) is involved in excretion of cytoplasmic proteins." J Biol Ch em. 152. Peng, H. L., R. P. Novick, et al. (1988). "Cloning, characterization, and sequencing of an accessory gene regulator (agr) in Staphylococcus aureus." J Bacteriol 170(9): 4365 4372. 153. Ploy, M. C., C. Grelaud, et al. (1998). "First clinical isolate of van comycin intermediate Staphylococcus aureus in a French hospital." Lancet 351(9110): 1212. 154. Potempa, J., A. Dubin, et al. (1988). "Degradation of elastin by a cysteine proteinase from Staphylococcus aureus." J Biol Chem 263(6): 2664 2667.

PAGE 111

99 155. Potempa, J., D. F edak, et al. (1991). "Proteolytic inactivation of alpha 1 anti chymotrypsin. Sites of cleavage and generation of chemotactic activity." J Biol Chem 266(32): 21482 21487. 156. Potempa, J., W. Watorek, et al. (1986). "The inactivation of human plasma alpha 1 pro teinase inhibitor by proteinases from Staphylococcus aureus." J Biol Chem 261(30): 14330 14334. 157. Prasad, L., Y. Leduc, et al. (2004). "The structure of a universally employed enzyme: V8 protease from Staphylococcus aureus." Acta Crystallogr D Biol Crystall ogr 60(Pt 2): 256 259. 158. Prevost, G., B. Cribier, et al. (1995). "Panton Valentine leucocidin and gamma hemolysin from Staphylococcus aureus ATCC 49775 are encoded by distinct genetic loci and have different biological activities." Infect Immun 63(10): 4121 4129. 159. Queck, S. Y., M. Jameson Lee, et al. (2008). "RNAIII independent target gene control by the agr quorum sensing system: insight into the evolution of virulence regulation in Staphylococcus aureus." Mol Cell 32(1): 150 158. 160. Rechtin, T. M., A. F. Gil laspy, et al. (1999). "Characterization of the SarA virulence gene regulator of Staphylococcus aureus." Mol Microbiol 33(2): 307 316. 161. Reed, S. B., C. A. Wesson, et al. (2001). "Molecular characterization of a novel Staphylococcus aureus serine protease op eron." Infect Immun 69(3): 1521 1527. 162. Richmond, M. H. and R. W. Lacey (1973). "Gene transfer between strains of Staphylococcus aureus." Contrib Microbiol Immunol 1: 135 143. 163. Robinson, D. A. and M. C. Enright (2003). "Evolutionary models of the emergence of methicillin resistant Staphylococcus aureus." Antimicrob Agents Chemother 47(12): 3926 3934. 164. Robinson, D. A. and M. C. Enright (2004). "Evolution of Staphylococcus aureus by large chromosomal replacements." J Bacteriol 186(4): 1060 1064.

PAGE 112

100 165. Rollof, J., J H. Braconier, et al. (1988). "Interference of Staphylococcus aureus lipase with human granulocyte function." Eur J Clin Microbiol Infect Dis 7(4): 505 510. 166. Rollof, J. and S. Normark (1992). "In vivo processing of Staphylococcus aureus lipase." J Bacteri ol 174(6): 1844 1847. 167. Rosenstein, R. and F. Gotz (2000). "Staphylococcal lipases: biochemical and molecular characterization." Biochimie 82(11): 1005 1014. 168. Ross, P. L., Y. N. Huang, et al. (2004). "Multiplexed protein quantitation in Saccharomyces cerevi siae using amine reactive isobaric tagging reagents." Mol Cell Proteomics 3(12): 1154 1169. 169. Sabat, A., D. C. Melles, et al. (2006). "Distribution of the serine aspartate repeat protein encoding sdr genes among nasal carriage and invasive Staphylococcus au reus strains." J Clin Microbiol 44(3): 1135 1138. 170. Sakata, N., S. Terakubo, et al. (2005). "Subcellular location of the soluble lytic transglycosylase homologue in Staphylococcus aureus." Curr Microbiol 50(1): 47 51. 171. Saravia Otten, P., H. P. Muller, et al (1997). "Transcription of Staphylococcus aureus fibronectin binding protein genes is negatively regulated by agr and an agr independent mechanism." J Bacteriol 179(17): 5259 5263. 172. Sawai, T., K. Tomono, et al. (1997). "Role of coagulase in a murine model of hematogenous pulmonary infection induced by intravenous injection of Staphylococcus aureus enmeshed in agar beads." Infect Immun 65(2): 466 471. 173. Scherl, A., P. Francois, et al. (2005). "Correlation of proteomic and transcriptomic profiles of Staphyloc occus aureus during the post exponential phase of growth." J Microbiol Methods 60(2): 247 257. 174. Scherl, A., P. Francois, et al. (2004). "Nonredundant mass spectrometry: a strategy to integrate mass spectrometry acquisition and analysis." Proteomics 4(4): 9 17 927. 175. Schneewind, O., D. Mihaylova Petkov, et al. (1993). "Cell wall sorting signals in surface proteins of gram positive bacteria." EMBO J 12(12): 4803 4811.

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101 176. Severin, A., E. Nickbarg, et al. (2007). "Proteomic analysis and identification of Streptococ cus pyogenes surface associated proteins." J Bacteriol 189(5): 1514 1522. 177. Seybold, U., E. V. Kourbatova, et al. (2006). "Emergence of community associated methicillin resistant Staphylococcus aureus USA300 genotype as a major cause of health care associat ed blood stream infections." Clin Infect Dis 42(5): 647 656. 178. Shaw, L., E. Golonka, et al. (2004). "The role and regulation of the extracellular proteases of Staphylococcus aureus." Microbiology 150(Pt 1): 217 228. 179. Sibbald, M. J., T. Winter, et al. (2010) "Synthetic effects of secG and secY2 mutations on exoproteome biogenesis in Staphylococcus aureus." J Bacteriol 192(14): 3788 3800. 180. Solis, N., M. R. Larsen, et al. (2010). "Improved accuracy of cell surface shaving proteomics in Staphylococcus aureus us ing a false positive control." Proteomics 10(10): 2037 2049. 181. Souza, R. R., L. R. Coelho, et al. (2009). "Biofilm formation and prevalence of lukF pv, seb, sec and tst genes among hospital and community acquired isolates of some international methicillin resistant Staphylococcus aureus lineages." Clin Microbiol Infect 15(2): 203 207. 182. Sugawara, N., T. Tomita, et al. (1997). "Assembly of Staphylococcus aureus gamma hemolysin into a pore forming ring shaped complex on the surface of human erythrocytes." FEBS Lett 410(2 3): 333 337. 183. Tegmark, K., E. Morfeldt, et al. (1998). "Regulation of agr dependent virulence genes in Staphylococcus aureus by RNAIII from coagulase negative staphylococci." J Bacteriol 180(12): 3181 3186. 184. Tjalsma, H., H. Antelmann, et al. (2 004). "Proteomics of protein secretion by Bacillus subtilis: separating the "secrets" of the secretome." Microbiol Mol Biol Rev 68(2): 207 233. 185. Tjalsma, H., L. Lambooy, et al. (2008). "Shedding & shaving: disclosure of proteomic expressions on a bacterial face." Proteomics 8(7): 1415 1428.

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102 186. Tomizawa, J., T. Itoh, et al. (1981). "Inhibition of ColE1 RNA primer formation by a plasmid specified small RNA." Proc Natl Acad Sci U S A 78(3): 1421 1425. 187. Trad, S., J. Allignet, et al. (2004). "DNA macroarray for id entification and typing of Staphylococcus aureus isolates." J Clin Microbiol 42(5): 2054 2064. 188. Travis, J. and J. Potempa (2000). "Bacterial proteinases as targets for the development of second generation antibiotics." Biochim Biophys Acta 1477(1 2): 35 50 189. Tristan, A., M. Bes, et al. (2007). "Global distribution of Panton Valentine leukocidin -positive methicillin resistant Staphylococcus aureus, 2006." Emerg Infect Dis 13(4): 594 600. 190. Ventura, C. L., N. Malachowa, et al. (2010). "Identification of a nov el Staphylococcus aureus two component leukotoxin using cell surface proteomics." PLoS One 5(7): e11634. 191. Vincents, B., P. Onnerfjord, et al. (2007). "Down regulation of human extracellular cysteine protease inhibitors by the secreted staphylococcal cystei ne proteases, staphopain A and B." Biol Chem 388(4): 437 446. 192. Vitikainen, M., I. Lappalainen, et al. (2004). "Structure function analysis of PrsA reveals roles for the parvulin like and flanking N and C terminal domains in protein folding and secretion i n Bacillus subtilis." J Biol Chem 279(18): 19302 19314. 193. Voyich, J. M., M. Otto, et al. (2006). "Is Panton Valentine leukocidin the major virulence determinant in community associated methicillin resistant Staphylococcus aureus disease?" J Infect Dis 194(1 2): 1761 1770. 194. Wang, R., K. R. Braughton, et al. (2007). "Identification of novel cytolytic peptides as key virulence determinants for community associated MRSA." Nat Med 13(12): 1510 1514. 195. Webb, G. F., M. A. Horn, et al. (2009). "Competition of hospital acquired and community acquired methicillin resistant Staphylococcus aureus strains in hospitals." J Biol Dyn 48: 271.

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103 196. Weigel, L. M., D. B. Clewell, et al. (2003). "Genetic analysis of a high level vancomycin resistant isolate of Staphylococcus aureus." Science 302(5650): 1569 1571. 197. Whalen, K., J. S. Grossert, et al. (1995). "Ion dissociation reactions induced in a high pressure quadrupole collision cell." Rapid Commun Mass Spectrom 9(14): 1366 1375. 198. Witney, A. A., G. L. Marsden, et al. (2005). "Design, validation, and application of a seven strain Staphylococcus aureus PCR product microarray for comparative genomics." Appl Environ Microbiol 71(11): 7504 7514. 199. Woese, C. R. (1987). "Bacterial evolution." Microbiol Rev 51(2): 221 271. 200. Wolff, S., H. Hahne et al. (2008). "Complementary analysis of the vegetative membrane proteome of the human pathogen Staphylococcus aureus." Mol Cell Proteomics 7(8): 1460 1468. 201. Wolff, S., A. Otto, et al. (2006). "Gel free and gel based proteomics in Bacillus subtilis: a c omparative study." Mol Cell Proteomics 5(7): 1183 1192. 202. Wright, J. S., 3rd, R. Jin, et al. (2005). "Transient interference with staphylococcal quorum sensing blocks abscess formation." Proc Natl Acad Sci U S A 102(5): 1691 1696. 203. Xiong, Y. Q., A. S. Bayer et al. (2004). "Impacts of sarA and agr in Staphylococcus aureus strain Newman on fibronectin binding protein A gene expression and fibronectin adherence capacity in vitro and in experimental infective endocarditis." Infect Immun 72(3): 1832 1836. 204. Yates J. R., 3rd, J. K. Eng, et al. (1995). "Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database." Anal Chem 67(8): 1426 1436. 205. Yoshikawa, M., F. Matsuda, et al. (1974). "Pleiotropic alteration of activi ties of several toxins and enzymes in mutants of Staphylococcus aureus." J Bacteriol 119(1): 117 122.

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104 206. Ziebandt, A. K., D. Becher, et al. (2004). "The influence of agr and sigmaB in growth phase dependent regulation of virulence factors in Staphylococcus a ureus." Proteomics 4(10): 3034 3047. 207. Ziebandt, A. K., H. Kusch, et al. (2010). "Proteomics uncovers extreme heterogeneity in the Staphylococcus aureus exoproteome due to genomic plasticity and variant gene regulation." Proteomics 10(8): 1634 1644. 208. Zieban dt, A. K., H. Weber, et al. (2001). "Extracellular proteins of Staphylococcus aureus and the role of SarA and sigma B." Proteomics 1(4): 480 493.

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105 Appendices

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106 Appendix 1. Cytoplasmic proteins identified from overnight cultures of S. aureus SH1000 separated by 1D SDS PAGE Identified Proteins (380) Accession Number Spectral Counts bifunctional autolysin (atl) SACOL1062 67 ATP dependent Clp protease, putative SACOL2563 56 sdrD protein (sdrD) SACOL0609 43 N acetylmuramoyl L alanine amidase domain protein SACOL2666 36 DNA directed RNA polymerase, beta' subunit (rpoC) SACOL0589 35 translation elongation factor G (fusA) SACOL0593 34 chaperonin, 60 kDa (groEL) SACOL2016 32 ATP synthase F1, beta subunit (atpD) SACOL2095 32 dnaK protein (dnaK) SACOL1637 31 transketolase (tkt) SACOL1377 30 DNA directed RNA polymerase, beta subunit (rpoB) SACOL0588 29 lipase (geh) SACOL2694 28 enolase (eno) SACOL0842 25 ribosomal protein S1 SACOL1516 24 fructose bisphosphate aldolase, class I (fdaB) SACOL2622 23 Aerolysin Leukocidin family protein SACOL2006 23 glutamyl tRNA(Gln) amidotransferase, B subunit (gatB) SACOL1960 23 phosphoenolpyruvate protein phosphotransferase (ptsI) SACOL1092 23 FeS assembly ATPase SufC SACOL0914 22 pyruvate kinase (pyk) [2.7.1.40] SACOL1745 22 cell wall surface anchor family protein (sasG) SACOL2505 22 translation elongation factor Tu (tuf) SACOL0594 21 glyceraldehyde 3 phosphate dehydrogenase (gapA1) SACOL0838 19 lipoprotein, putative SACOL0444 19 FeS assembly protein SufD (sufD) SACOL0915 19 sdrC protein (sdrC) SACOL0608 19 polyribonucleotide nucleotidyltransferase (pnp) SACOL1293 19 succinyl CoA synthase, beta subunit (sucC) SACOL1262 18 alkyl hydroperoxide reductase, subunit F (ahpF) SACOL0451 18 aldehyde dehydrogenase SACOL2114 18

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107 Appendix 1. (Continued) sulfatase family protein SACOL0778 18 pyruvate dehydrogenase complex E2 component, dihydrolipoamide acetyltransferase (pdhC) SACOL1104 17 clumping factor A (clfA) SACOL0856 17 cell division protein FtsZ (ftsZ) SACOL1199 17 pyruvate dehydrogenase complex E1 component, beta subunit (pdhB) SACOL1103 16 fumarylacetoacetate hydrolase family protein SACOL0973 16 acetolactate synthase, catabolic (budB) SACOL2199 16 fibronectin binding protein A (fnbA) SACOL2511 16 cell division protein FtsA (ftsA) SACOL1198 15 protein export protein PrsA, putative SACOL1897 15 peptide chain release factor 1 (prfA) SACOL2110 15 conserved hypothetical protein SACOL1767 15 amino acid ABC transporter, amino acid binding protein SACOL2412 14 DNA polymerase III, beta subunit (dnaN) SACOL0002 14 phosphoglucosamine mutase GlmM (glmM) SACOL2151 14 pyruvate dehydrogenase complex E3 component, lipoamide dehydrogenase (pdhD) SACOL1105 13 cysteine synthase (cysK) SACOL0557 13 trigger factor (tig) SACOL1722 13 seryl tRNA synthetase (serS) SACOL0009 13 map protein, programmed frameshift (map) SACOL2002 13 conserved hypothetical protein TIGR00092 SACOL0435 13 alkaline shock protein 23 SACOL2173 12 triosephosphate isomerase (tpiA) SACOL0840 12 DNA directed RNA polymerase, alpha subunit (rpoA) SACOL2213 12 phosphate acetyltransferase (pta) SACOL0634 12 ATP synthase F1, alpha subunit (atpA) SACOL2097 12 pyruvate carboxylase (pyc) SACOL1123 12 phosphoglycerate mutase, 2,3 bisphosphoglycerate independent (pgm) SACOL0841 12 fibronectin binding protein B (fnbB) SACOL2509 12 clumping factor B (clfB) SACOL2652 12 3 oxoacyl (acyl carrier protein) synthase II (fabF) SACOL0988 11 pyridoxine biosynthesis protein SACOL0564 11 hydroxymethylglutaryl CoA synthase SACOL2561 11 glucosamine 6 phosphate isomerase, putative SACOL1912 11 penicillin binding protein 2 (pbp2) SACOL1490 11 formate acetyltransferase (pflB) SACOL0204 10 3 oxoacyl (acyl carrier protein) reductase (fabG1) SACOL1245 10 rod shape determining protein MreC (mreC) SACOL1704 10 conserved hypothetical protein SACOL2136 10

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108 Appendix 1. (Continued) lipoprotein, putative SACOL1101 10 GMP synthase (guaA) SACOL0461 10 LPXTG cell wall surface anchor family protein (sasF) SACOL2668 10 phosphoribosylformylglycinamidine synthase II (purL) SACOL1078 10 phenylalanyl tRNA synthetase, beta subunit (pheT) SACOL1149 10 alkyl hydroperoxide reductase, C subunit (ahpC) SACOL0452 9 staphyloxanthin biosynthesis protein SACOL2291 9 glucosamine -fructose 6 phosphate aminotransferase (isomerizing) (glmS) SACOL2145 9 pyruvate dehydrogenase complex E1 component, alpha subunit (pdhA) SACOL1102 9 ribosomal protein L4 (rplD) SACOL2238 9 phosphoglycerate kinase (pgk) SACOL0839 9 oxidoreductase, short chain dehydrogenase reductase family SACOL2321 9 2 oxoglutarate dehydrogenase, E2 component, dihydroipoamide succinyltransferase (sucB) SACOL1448 9 manganese dependent inorganic pyrophosphatase (ppaC) SACOL1982 9 branched chain amino acid aminotransferase (ilvE) SACOL0600 9 D alanine aminotransferase (dat) SACOL1800 9 LysM domain protein SACOL0507 9 hypoxanthine phosphoribosyltransferase (hpt) SACOL0554 9 N utilization substance protein A, putative SACOL1285 9 conserved hypothetical protein SACOL1792 9 S adenosylmethionine synthetase (metK) SACOL1837 9 translation elongation factor Ts (tsf) SACOL1276 8 aconitate hydratase (acnA) SACOL1385 8 transaldolase (tal) SACOL1831 8 ferritins family protein SACOL1952 8 conserved hypothetical protein SACOL0597 8 L lactate dehydrogenase (ldh1) SACOL0222 8 acid phosphatase5' nucleotidase, lipoprotein e(P4) family SACOL0303 8 aerobic glycerol 3 phosphate dehydrogenase (glpD) SACOL1321 8 glutamyl tRNA(Gln) amidotransferase, A subunit (gatA) SACOL1961 8 dihydroorotase (pyrC) SACOL1213 8 surface protein, putative SACOL0479 8 leukocidin subunit precursor, putative SACOL2004 8 catalase (kataA) SACOL1368 7 glucose 6 phosphate isomerase (pgi) SACOL0966 7 conserved hypothetical protein SACOL1902 7 universal stress protein family SACOL1759 7 acyl carrier protein (acpP) SACOL1247 7 ribosomal protein L10 (rplJ) SACOL0585 7

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109 Appendix 1. (Continued) ribosomal Protein L25 (rplY) SACOL0545 7 ribosomal protein L1 (rplA) SACOL0584 7 aminotransferase, class II SACOL0596 7 conserved hypothetical protein SACOL1985 7 malonyl CoA acyl carrier protein transacylase (fabD) SACOL1244 7 ATP synthase F1, gamma subunit (atpG) SACOL2096 7 DAK2 domain protein SACOL1240 7 acetyl CoA acetyltransferase SACOL0426 7 conserved hypothetical protein SACOL0455 7 alpha hemolysin precursor (hlY) SACOL1173 7 DNA binding protein HU (hup) SACOL1513 6 immunodominant antigen A (isaA) SACOL2584 6 inosine 5' monophosphate dehydrogenase (guaB) SACOL0460 6 fructose bisphosphate aldolase, class II (fbaA) SACOL2117 6 ribosomal protein L21 (rplU) SACOL1702 6 FeS assembly protein SufB (sufB) SACOL0918 6 threonyl tRNA synthetase (thrS) SACOL1729 6 ribosomal protein L6 (rplF) SACOL2224 6 ribosomal protein S3 (rpsC) SACOL2233 6 ribosomal protein S4 (rpsD) SACOL1769 6 ribosomal protein L3 (rplC) SACOL2239 6 deoxyribose phosphate aldolase (deoC2) SACOL2129 6 3 oxoacyl (acyl carrier protein) synthase III (fabH) SACOL0987 6 6 phosphofructokinase (pfkA) SACOL1746 6 conserved hypothetical protein SACOL0669 6 conserved hypothetical protein SACOL1447 6 sasB protein (sasB) SACOL2150 6 conserved hypothetical protein SACOL0912 5 ornithine aminotransferase (rocD2) SACOL0960 5 ribosomal protein L2 (rplB) SACOL2236 5 glycyl tRNA synthetase (glyS) SACOL1622 5 malate:quinone oxidoreductase (mqo2) SACOL2623 5 formate dehydrogenase, alpha subunit, putative SACOL2301 5 ribosomal protein L20 (rplT) SACOL1725 5 NADP dependent malic enzyme, putative SACOL1749 5 conserved hypothetical protein TIGR01033 SACOL0727 5 L lactate dehydrogenase (ldh2) SACOL2618 5 anti sigma B factor (rsbW) SACOL2055 5 immunodominant antigen B (isaB) SACOL2660 5

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110 Appendix 1. (Continued) acetoin reductase SACOL0111 5 serine hydroxymethyltransferase (glyA) SACOL2105 5 glutamyl aminopeptidase, putative SACOL1402 5 DNA directed RNA polymerase, delta subunit (rpoE) SACOL2120 5 NAD(P)H dehydrogenase (quinone), putative SACOL0190 5 oxidoreductase, short chain dehydrogenase reductase family SACOL2488 5 Staphylococcus aureus sex pheromone (camS) SACOL1964 5 dephospho CoA kinase (coaE) SACOL1735 5 lipoprotein, putative SACOL0449 5 IgG binding protein SBI SACOL2418 5 cysteine protease precursor SspB (sspB2) SACOL1970 5 ribosomal protein L15 (rplO) SACOL2220 4 hydrolase, alpha beta hydrolase fold family SACOL2597 4 succinyl CoA synthase, alpha subunit (sucD) SACOL1263 4 valyl tRNA synthetase (valS) SACOL1710 4 superoxide dismutase (sodA2) SACOL1610 4 peptidase, M20 M25 M40 family SACOL1801 4 conserved hypothetical protein SACOL1020 4 conserved hypothetical protein SACOL1789 4 pyrimidine nucleoside phosphorylase (pdp) SACOL2128 4 conserved hypothetical protein SACOL1788 4 naphthoate synthase (menB) SACOL1054 4 arginyl tRNA synthetase (argS) SACOL0663 4 iron compound ABC transporter, iron compound binding protein SACOL2277 4 translation elongation factor P (efp) SACOL1587 4 staphylococcus tandem lipoprotein SACOL0486 4 2 oxoisovalerate dehydrogenase, E1 component, beta subunit SACOL1561 4 single stranded DNA binding protein (ssb2) SACOL0438 4 hydrolase, haloacid dehalogenase like family SACOL0602 4 transcriptional regulator, putative SACOL1065 4 penicillin binding protein 3 (pbp3) SACOL1609 4 antibacterial protein (phenol soluble modulin) SACOL1186 3 ThiJ PfpI family protein SACOL1933 3 antibacterial protein (phenol soluble modulin) SACOL1187 3 hexulose 6 phosphate synthase, putative SACOL0617 3 2 oxoglutarate dehydrogenase, E1 component (sucA) SACOL1449 3 purine nucleoside phosphorylase (deoD2) SACOL2130 3 NADH dehydrogenase, putative SACOL0944 3 ribosomal protein S2 (rpsB) SACOL1274 3

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111 Appendix 1. (Continued) acetate kinase (ackA) SACOL1760 3 adenylosuccinate synthetase (purA) SACOL0018 3 ribosomal protein S7 (rpsG) SACOL0592 3 glucose 6 phosphate 1 dehydrogenase (zwf) SACOL1549 3 ribosomal protein S5 (rpsE) SACOL2222 3 lysyl tRNA synthetase (lysS) SACOL0562 3 uracil phosphoribosyltransferase (upp) SACOL2104 3 ribosomal protein L5 (rplE) SACOL2227 3 peptidyl prolyl cis trans isomerase, cyclophilin type SACOL0957 3 cytochrome aa3 quinol oxidase, subunit II (qoxA) SACOL1070 3 CTP synthase (pyrG) SACOL2119 3 4 diphosphocytidyl 2C methyl D erythritol synthase, putative SACOL0240 3 ribosomal protein L17 (rplQ) SACOL2212 3 NAD(P)H flavin oxidoreductase (frp) SACOL2534 3 cysteine desulfurase, SufS subfamily SACOL0916 3 ribosomal protein L13 (rplM) SACOL2207 3 ribosomal protein S12 (rpsL) SACOL0591 3 elastin binding protein, putative SACOL1522 3 carbamoyl phosphate synthase, large subunit (carB) SACOL1215 3 ribosomal protein L22 (rplV) SACOL2234 3 D alanine activating enzyme D alanine D alanyl carrier protein ligase (dltA) SACOL0935 3 phosphomethylpyrimidine kinase (thiD1) SACOL0626 3 methionine aminopeptidase, type I SACOL1946 3 ATP dependent Clp protease, ATP binding subunit ClpX (clpX) SACOL1721 3 hydrolase, haloacid dehalogenase like family SACOL0931 3 UTP glucose 1 phosphate uridylyltransferase family protein SACOL2161 3 1 phosphofructokinase (fruK) SACOL0758 3 LysM domain protein SACOL0723 3 ATP dependent Clp protease, proteolytic subunit ClpP (clpP) SACOL0833 3 xanthine phosphoribosyltransferase (xpt) SACOL0458 3 peptide ABC transporter, peptide binding protein SACOL2476 3 conserved hypothetical protein SACOL1464 3 penicillin binding protein 1 (pbp1) SACOL1194 3 phage infection protein, putative SACOL2665 3 protein phosphatase 2C domain protein SACOL1231 3 secretory extracellular matrix and plasma binding protein (empbp) SACOL0858 3 thymidylate kinase (tmk) SACOL0524 3 phospholipase C (hlb) SACOL2003 3 6,7 dimethyl 8 ribityllumazine synthase (ribH) SACOL1817 2

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112 Appendix 1. (Continued) 6 phosphogluconate dehydrogenase, decarboxylating (gnd) SACOL1554 2 delta 1 pyrroline 5 carboxylate dehydrogenase, putative SACOL2569 2 D isomer specific 2 hydroxyacid dehydrogenase family protein SACOL2296 2 alcohol dehydrogenase, zinc containing SACOL0660 2 adenylosuccinate lyase (purB) SACOL1969 2 cold shock protein, CSD family SACOL1437 2 ribosomal subunit interface protein SACOL0815 2 phosphocarrier protein HPr (ptsH) SACOL1091 2 asparaginyl tRNA synthetase (asnS) SACOL1494 2 conserved hypothetical protein SACOL1992 2 fumarate hydratase, class II (fumC) SACOL1908 2 mannitol 1 phosphate 5 dehydrogenase (mtlD) SACOL2149 2 immunoglobulin G binding protein A precursor (spa) SACOL0095 2 conserved hypothetical protein SACOL2379 2 adenylate kinase (adk) SACOL2218 2 aspartyl tRNA synthetase (aspS) SACOL1685 2 DNA gyrase, A subunit (gyrA) SACOL0006 2 oxidoreductase, aldo keto reductase family SACOL0763 2 lipoprotein, putative SACOL2365 2 ribosomal protein L11 (rplK) SACOL0583 2 isocitrate dehydrogenase, NADP dependent (icd) SACOL1741 2 cell division protein FtsH, putative SACOL0555 2 translation initiation factor IF 3 (infC) SACOL1727 2 molybdenum ABC transporter, molybdenum binding protein ModA (modA) SACOL2272 2 tyrosyl tRNA synthetase (tyrS) SACOL1778 2 ribose phosphate pyrophosphokinase (prsA) SACOL0544 2 DNA repair exonuclease family protein SACOL1900 2 epimerase dehydratase, putative SACOL2446 2 UDP N acetylglucosamine 1 carboxyvinyltransferase 2 (murAB) SACOL2116 2 DHH subfamily 1 protein SACOL1751 2 copper ion binding protein SACOL2573 2 pyrroline 5 carboxylate reductase (proC) SACOL1546 2 ferrochelatase (hemH) SACOL1888 2 conserved hypothetical protein SACOL1426 2 thiamine phosphate pyrophosphorylase (thiE) SACOL2083 2 cell division initiation protein, putative SACOL1205 2 phenylalanyl tRNA synthetase, alpha subunit (pheS) SACOL1148 2 decarboxylase family protein SACOL0740 2 hydrolase, haloacid dehalogenase like family SACOL0606 2

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113 Appendix 1. (Continued) conserved hypothetical protein SACOL2020 2 hydrolase, haloacid dehalogenase like family SACOL1365 2 ABC transporter, substrate binding protein SACOL0217 2 transcriptional regulator, putative SACOL1398 2 acetyl CoA carboxylase, biotin carboxyl carrier protein (accB) SACOL1572 2 chaperonin, 33 kDa SACOL0556 2 ferredoxin (fer) SACOL1525 2 lipoprotein, putative SACOL1589 2 serine protease SplB (splB) SACOL1868 2 formate -tetrahydrofolate ligase (fhs) SACOL1782 1 conserved hypothetical protein SACOL2163 1 PTS system, IIA component SACOL1457 1 thiol peroxidase, putative SACOL1762 1 thioredoxin (trxA) SACOL1155 1 translation initiation factor IF 2 (infB) SACOL1288 1 Dps family protein SACOL2131 1 ABC transporter, substrate binding protein SACOL0688 1 ribosomal protein L16 (rplP) SACOL2232 1 catabolite control protein A (ccpA) SACOL1786 1 conserved hypothetical protein SACOL1630 1 N acetylglucosamine 6 phosphate deacetylase (nagA) SACOL0761 1 ATP dependent Clp protease, ATP binding subunit ClpB (clpB) SACOL0979 1 ribosomal protein S11 (rpsK) SACOL2214 1 pyruvate oxidase SACOL2553 1 phosphoribosylamine -glycine ligase (purD) SACOL1083 1 acetyltransferase, GNAT family SACOL1189 1 quinol oxidase, subunit I (qoxA) SACOL1069 1 conserved hypothetical protein SACOL1802 1 oxidoreductase, aldo keto reductase family SACOL1835 1 thioredoxin, putative SACOL0875 1 glutamate 1 semialdehyde 2,1 aminomutase (hemL1) SACOL1714 1 hydroxyethylthiazole kinase (thiM) SACOL2084 1 prolyl tRNA synthetase (proS) SACOL1282 1 alanyl tRNA synthetase (alaS) SACOL1673 1 imidazolonepropionase (hutI) SACOL2323 1 ribonucleoside diphosphate reductase, alpha subunit SACOL0792 1 ribosomal protein L23 (rplW) SACOL2237 1 heat shock protein GrpE (grpE) SACOL1638 1 conserved hypothetical protein SACOL1670 1

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114 Appendix 1. (Continued) 4 oxalocrotonate tautomerase (dmpI) SACOL1399 1 ABC transporter, ATP binding protein SACOL1427 1 heat shock protein HslVU, ATPase subunit HslU (hslU) SACOL1271 1 DNA polymerase I (polA) SACOL1737 1 conserved hypothetical protein SACOL2143 1 serine protease HtrA, putative SACOL1777 1 aminotransferase, putative SACOL2000 1 D alanine -D alanine ligase SACOL2074 1 chaperonin, 10 kDa (groES) SACOL2017 1 glucokinase (glk) SACOL1604 1 polypeptide deformylase (def1) SACOL1100 1 glutamate 1 semialdehyde 2,1 aminomutase (hemL2) SACOL1922 1 ribosome binding factor A (rbfA) SACOL1289 1 pyridine nucleotide disulfide oxidoreductase SACOL1520 1 ribonucleoside diphosphate reductase 2, beta subunit (nrdF) SACOL0793 1 enoyl (acyl carrier protein) reductase (fabI) SACOL1016 1 phosphoribosylformylglycinamidine synthase I (purQ) SACOL1077 1 fatty acid phospholipid synthesis protein PlsX (plsX) SACOL1243 1 ATP synthase F0, B subunit (atpF) SACOL2099 1 signal peptidase IB (spsB) SACOL0969 1 RNA methyltransferase, TrmH family SACOL0578 1 NifU domain protein SACOL0917 1 3,4 dihydroxy 2 butanone 4 phosphate synthase GTP cyclohydrolase II (ribBA) SACOL1818 1 phosphoribosylformylglycinamidine cyclo ligase (purM) SACOL1080 1 glyoxalase family protein SACOL2533 1 DNA binding response regulator SrrA (srrA) SACOL1535 1 phosphoribosylglycinamide formyltransferase (purN) SACOL1081 1 UDP N acetylmuramoyl tripeptide -D alanyl D alanine ligase (murF) SACOL2073 1 tellurite resistance protein, putative SACOL1441 1 degV family protein SACOL1460 1 conserved hypothetical protein SACOL0742 1 deoxyribose phosphate aldolase (deoC1) SACOL0123 1 femB protein SACOL1411 1 glycerol 3 phosphate dehydrogenase, NAD dependent (gpsA) SACOL1514 1 GTP binding protein, GTP1 OBG family SACOL1699 1 urease accessory protein UreG (ureG) SACOL2285 1 DAK2 domain protein SACOL0708 1 HAM1 protein SACOL1162 1 conserved hypothetical protein SACOL1558 1

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115 Appendix 1. (Continued) FtsK SpoIIIE family protein SACOL1791 1 conserved hypothetical protein SACOL2605 1 peptide chain release factor 2, programmed frameshift (prfB) SACOL0818 1 cytidylate kinase (cmk) SACOL1518 1 Gid protein (gid) SACOL1268 1 conserved hypothetical protein SACOL1120 1 general stress protein 13 SACOL0552 1 conserved domain protein SACOL2557 1 hypothetical protein SACOL0272 1 conserved hypothetical protein SACOL1885 1 uroporphyrinogen decarboxylase (hemE) SACOL1889 1 transcriptional regulator, putative SACOL2302 1 YlmF protein (ylmF) SACOL1202 1 dnaJ protein (dnaJ) SACOL1636 1 degV family protein SACOL0812 1 PTS system, mannitol specific IIBC components SACOL2146 1 orotidine 5' phosphate decarboxylase (pyrF) SACOL1216 1 lipoprotein, putative SACOL0851 1 cobyric acid synthase, putative SACOL1950 1 glycerophosphoryl diester phosphodiesterase GlpQ, putative SACOL0962 1 thioredoxin, putative SACOL0881 1 5 methyltetrahydropteroyltriglutamate -homocysteine methyltransferase (metE) SACOL0428 1 response regulator related protein SACOL2360 1 hydrolase, haloacid dehalogenase like family SACOL0619 1 conserved hypothetical protein SACOL0579 1 staphyloxanthin biosynthesis protein, putative SACOL2295 1 LPXTG cell wall surface anchor protein (sasE) SACOL1140 1 16S rRNA processing protein RimM (rimM) SACOL1255 1 conserved hypothetical protein SACOL1373 1 conserved hypothetical protein SACOL1375 1 oxygen independent coproporphyrinogen III oxidase, putative SACOL1640 1 iron compound ABC transporter, iron compound binding protein SACOL2010 1 acetolactate synthase, large subunit, biosynthetic type (ilvB) SACOL2043 1 monooxygenase family protein SACOL2297 1 staphylococcus tandem lipoprotein SACOL2497 1 membrane protein, putative SACOL2554 1

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116 Appendix 2. Cytoplasmic proteins identified from overnight cultures of S. aureus SH1000 from MudPit analysis Identified Proteins (747) Accession Number Spectral Counts alkaline shock protein 23 SACOL2173 1129 enolase (eno) SACOL0842 886 translation elongation factor Tu (tuf) SACOL0594 873 glyceraldehyde 3 phosphate dehydrogenase (gapA1) SACOL0838 873 formate acetyltransferase (pflB) SACOL0204 577 aldehyde dehydrogenase (aldA1) SACOL0154 372 ATP dependent Clp protease, putative SACOL2563 371 DNA binding protein HU (hup) SACOL1513 356 pyruvate dehydrogenase complex E3 component, lipoamide dehydrogenase (pdhD) SACOL1105 341 dnaK protein (dnaK) SACOL1637 298 translation elongation factor Ts (tsf) SACOL1276 294 antibacterial protein (phenol soluble modulin) SACOL1186 283 translation elongation factor G (fusA) SACOL0593 281 fructose bisphosphate aldolase, class I (fdaB) SACOL2622 281 alkyl hydroperoxide reductase, C subunit (ahpC) SACOL0452 265 ThiJ PfpI family protein SACOL1933 251 conserved hypothetical protein SACOL0912 240 phosphoglycerate mutase (gpm) SACOL2415 219 formate -tetrahydrofolate ligase (fhs) SACOL1782 208 conserved hypothetical protein SACOL2163 196 pyruvate dehydrogenase complex E2 component, dihydrolipoamide acetyltransferase (pdhC) SACOL1104 192 cysteine synthase (cysK) SACOL0557 192 c atalase (kataA) SACOL1368 187 ornithine aminotransferase (rocD2) SACOL0960 182 DNA directed RNA polymerase, beta' subunit (rpoC) SACOL0589 175 DNA directed RNA polymerase, beta subunit (rpoB) SACOL0588 172 acyl CoA dehydrogenase family protein SACOL0213 171 ribosomal protein S1 (rpsA) SACOL1516 169 ribosomal protein L2 (rplB) SACOL2236 166 phosphoenolpyruvate carboxykinase (ATP) (pckA) SACOL1838 162 pyruvate dehydrogenase complex E1 component, beta subunit (pdhB) SACOL1103 156 chaperonin, 60 kDa (groEL) SACOL2016 155 glucosamine -fructose 6 phosphate aminotransferase (isomerizing) (glmS) SACOL2145 153 transketolase (tkt) SACOL1377 146 thioredoxin disulfide reductase (trxB) SACOL0829 146 pyruvate kinase (pyk) SACOL1745 143 antibacterial protein (phenol soluble modulin) SACOL1187 143 6,7 dimethyl 8 ribityllumazine synthase (ribH) SACOL1817 143 succinyl CoA synthase, beta subunit (sucC) SACOL1262 139 glucose 6 phosphate isome rase (pgi) SACOL0966 138 glycyl tRNA synthetase (glyS) SACOL1622 130 6 phosphogluconate dehydrogenase, decarboxylating (gnd) SACOL1554 130 hydrolase, alpha beta hydrolase fold family SACOL2597 128 aconitate hydratase (acnA) SACOL1385 124

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117 Appendix 2. (Continued) inosine 5' monophosphate dehydrogenase (guaB) SACOL0460 124 succinyl CoA synthase, alpha subunit (sucD) SACOL1263 124 transaldolase (tal) SACOL1831 123 hexulose 6 phosphate synthase, putative SACOL0617 123 ribosomal protein L15 (rplO) SACOL2220 120 cell division protein FtsZ (ftsZ) SACOL1199 116 ATP synthase F1, beta subunit (atpD) SACOL2095 115 pyruvate dehydrogenase complex E1 component, alpha subunit (pdhA) SACOL1102 114 thioredoxin (trxA) SACOL1155 111 ribosomal protein S8 (rpsH) SACOL2225 111 glutamine synthetase FemC (femC) SACOL1329 111 triosephosphate isomerase (tpiA) SACOL0840 110 delta 1 pyrroline 5 carboxylate dehydrogenase, putative SACOL2569 107 phosphate acetyltransferase (pta) SACOL0634 104 ribosomal protein S6 (rpsF) SACOL0437 103 malate:quinone oxidoreductase (mqo2) SACOL2623 101 seryl t RNA synthetase (serS) SACOL0009 97 formate dehydrogenase, alpha subunit, putative SACOL2301 97 PTS system, IIA component SACOL1457 95 3 oxoacyl (acyl carrier protein) reductase (fabG1) SACOL1245 94 conserved hypothetical protein SACOL0597 92 alkyl hydroperoxide reductase, subunit F (ahpF) SACOL0451 91 phosphoglycerate kinase (pgk) SACOL0839 89 ferritins family protein SACOL1952 87 alcohol dehydrogenase, zinc containing SACOL0660 84 D isomer specific 2 hydroxyacid dehydrogenase family protein SACOL2296 84 3 hydroxyacyl CoA dehydrogenase protein SACOL0212 84 ornithine carbamoyltransferase (arcB2) SACOL2656 84 thiol peroxidase, putative SACOL1762 83 universal stress protein family SACOL1759 81 staphylococcal accessory regulator A (sarA) SACOL0672 81 aldehyde dehydrogenase SACOL2114 80 conserved hypothetical protein SACOL1484 80 trigger factor (tig) SACOL1722 79 3 oxoacyl (acyl carrier protein) synthase II (fabF) SACOL0988 78 2 oxoglutarate dehydrogenase, E1 component (sucA) SACOL1449 77 NADH dehydrogenase, putative SACOL0944 77 peptidase, M20 M25 M40 family SACOL1801 75 fructose bisphosphate aldo lase, class II (fbaA) SACOL2117 74 glutamate dehydrogenase, NAD specific (gluD) SACOL0961 74 bifunctional autolysin (atl) SACOL1062 71 hydroxymethylglutaryl CoA synthase SACOL2561 70 L lactate dehydrogenase (ldh1) SACOL0222 70 purine nucleoside phosphorylase (deoD2) SACOL2130 69 NADP dependent malic enzyme, putative SACOL1749 69 glycine cleavage system H protein (gcvH) SACOL0877 67 isoleucyl tRNA synthetase (ileS) SACOL1206 66 methylenetetrahydrofolate dehydrogenase methenyltetrahydrofolate cyclohydrolase (folD) SACOL1072 65 ATP synthase F1, alpha subunit (atpA) SACOL2097 64 adenylosuccinate lyase (purB) SACOL1969 64 ribosomal protein L1 (rplA) SACOL0584 63 pyrimidine nucleoside phosphorylase (pdp) SACOL2128 63 conserved hypothetical protein SACOL1902 62 pyruvate carboxylase (pyc) SACOL1123 62 fumarylacetoacetate hydrolase family protein SACOL0973 61

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118 Appendix 2. (Continued) pyridoxine biosynthesis protein SACOL0564 61 ribosomal protein L7 L12 (rplL) SACOL0586 61 conserved hypothetical protein SACOL1680 61 superoxide dismutase (sodA2) SACOL1610 60 translation initiation factor IF 2 (infB) SACOL1288 59 arginine deiminase (arcA) SACOL2657 59 DNA directed RNA polymerase, alpha subunit (rpoA) SACOL2213 58 ribosomal protein S2 (rpsB) SACOL1274 58 aminotransferase, class II SACOL0596 58 aerobic glycerol 3 phosphate dehydrogenase (glpD) SACOL1321 58 Dps family protein SACOL2131 58 alkylhydroperoxidase, AhpD family SACOL2484 58 lipoprotein, putative SACOL0444 57 glutamyl tRNA(Gln) amidotransferase, B subunit (gatB) SACOL1960 57 2 oxoglutarate dehydrogenase, E2 component, dihydroipoamide succinyltransferase (sucB) SACOL1448 57 ribosomal subunit interface protein SACOL0815 56 glutamyl tRNA synthetase (gltX) SACOL0574 56 ribosomal protein S13 S18 (rpsM) SACOL2215 56 ribosomal protein L20 (rplT) SACOL1725 55 adenylosuccinate synthetase (purA) SACOL0018 55 oligoendopeptidase F (pepF) SACOL1005 54 flavohemoprotein, putative SACOL0220 54 urocanate hydratase (hutU) SACOL2324 54 FeS assembly protein SufB (sufB) SACOL0918 53 spoVG protein (spoVG) SACOL0541 52 phosphoenolpyruvate protein phosphotransferase (ptsI) SACOL1092 51 ribosomal protein L21 (rplU) SACOL1702 51 acetate kinase (ackA) SACOL1760 51 glucosamine 6 phosphate isomerase, putative SACOL1912 50 conserved hypothetical protein SACOL1789 50 conserved hypothetical protein SACOL1020 50 valyl tRNA synthetase (valS) SACOL1710 50 metallo beta lactamase family protein SACOL1098 50 conserved hypothetical protein SACOL0457 49 ribosomal Protein L25 (rplY) SACOL0545 48 threonyl tRNA synthetase (thrS) SACOL1729 48 succinate dehydrogenase, flavoprotein subunit (sdhA) SACOL1159 48 conserved hypothetical protein SACOL2136 47 conserved hypothetical protein SACOL1992 47 glucose 6 phosphate 1 dehydrogenase (zwf) SACOL1549 46 fumarate hydratase, class II (fumC) SACOL1908 46 glutamyl tRNA(Gln) amidotransferase, A subunit (gatA) SACOL1961 45 uracil phosphoribosyltransferase (upp) SACOL2104 45 aldehyde dehydrogenase (aldA2) SACOL1984 45 D isomer specific 2 hydroxyacid dehydrogenase family protein SACOL2535 44 conserved hypothetical protein SACOL2711 44 alcohol dehydrogenase, zinc containing SACOL2178 44 acyl carrier protein (acpP) SACOL1247 43 ribosomal protein L16 (rplP) SACOL2232 43 mannitol 1 phosphate 5 dehydrogenase (mtlD) SACOL2149 43 ribosomal protein S19 (rpsS) SACOL2235 43 methionyl tRNA synthetase (metS) SACOL0533 43 ribosomal protein S5 (rpsE) SACOL2222 42 ABC transporter, substrate binding protein SACOL0688 42 L lactate dehydrogenase (ldh2) SACOL2618 42

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119 Appendix 2. (Continued) asparaginyl tRNA synthetase (asnS) SACOL1494 42 conserved hypothetical protein SACOL0633 42 FeS assembly protein SufD (sufD) SACOL0915 41 phosphoglycerate mutase, 2,3 bisphosphoglycerate independent (pgm) SACOL0841 41 proline dipeptidase SACOL1588 41 cell division protein FtsA (ftsA) SACOL1198 40 deoxyribose phosphate aldolase (deoC1) SACOL0123 40 catabolite control protein A (ccpA) SACOL1786 40 conserved hypothetical protein SACOL1630 40 PTS system, mannitol specific IIA component SACOL2148 40 ribosomal protein L4 (rplD) SACOL2238 39 polyribonucleotide nucleotidyltransferase (pnp) SACOL1293 39 manganese dependent inorganic pyrophosphatase (ppaC) SACOL1982 39 conserved hypothetical protein SACOL1985 39 lysyl tRNA synthetase (lysS) SACOL0562 39 phosphocarrier protein HPr (ptsH) SACOL1091 39 N acetylglucosamine 6 phosphate deacetylase (nagA) SACOL0761 39 deoxyribose phosphate aldolase (deoC2) SACOL2129 38 branched chain amino acid aminotransferase (ilvE) SACOL0600 38 glycine cleavage syste m P protein, subunit 2 SACOL1593 38 glycine cleavage system P protein, subunit 1 SACOL1594 38 D alanine aminotransferase (dat) SACOL1800 37 ribosomal protein S7 (rpsG) SACOL0592 37 conserved hypothetical protein SACOL1788 37 serine hydroxymethyltransferase (glyA) SACOL2105 37 adenylate kinase (adk) SACOL2218 37 naphthoate synthase (menB) SACOL1054 36 peptidyl prolyl cis trans isomerase, cyclophilin type SACOL0957 36 FeS assembly ATPase SufC SACOL0914 35 ribosomal protein L10 (rplJ) SACOL0585 35 protein export protein PrsA, putative SACOL1897 35 ribosomal protein S4 (rpsD) SACOL1769 35 ribosomal protein L5 (rplE) SACOL2227 35 oxidoreductase, aldo keto reductase family SACOL1835 35 fructose 1,6 bisphosphatase, putative SACOL2527 35 oxidoreductase, short chain dehydrogenase reductase family SACOL2321 34 ribosomal protein L17 (rplQ) SACOL2212 34 acetyl CoA synthetase (acs) SACOL1783 34 conserved hypothetical protein SACOL2175 34 alanine dehydrogenase (ald2) SACOL1758 34 acetolactate synthase, catabolic (budB) SACOL2199 33 ribosomal protein L22 (rplV) SACOL2234 33 CTP synthase (pyrG) SACOL2119 33 acetyltransferase, GNAT family SACOL1189 33 phosphoribosylaminoimidazolecarboxamide formyltransferase IMP cyclohydrolase (purH) SACOL1082 33 glutamyl aminopeptidase, putative SACOL1402 32 cytochrome aa3 quinol oxidase, subunit II (qoxA) SACOL1070 32 pyruvate oxidase SACOL2553 32 thioredoxin, putative SACOL0875 32 coenzyme A disulfide reductase SACOL0975 32 phosphoribosylamine -glycine ligase (purD) SACOL1083 31 acetyl CoA synthetase, putative SACOL2624 31 universal stress protein family SACOL1753 31 phosphoglucosamine mutase GlmM (glmM) SACOL2151 30 conserved hypothetical protein TIGR01033 SACOL0727 30

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120 Appendix 2. (Continued) anti sigma B factor (rsbW) SACOL2055 30 3 oxoacyl (acyl carrier protein) synthase III (fabH) SACOL0987 30 acetoin reductase SACOL0111 30 arginyl tRNA synthetase (argS) SACOL0663 30 transcriptional regulator CodY (codY) SACOL1272 30 argininosuccinate synthase (argG) SACOL0964 30 clumping factor A (clfA) SACOL0856 29 DNA polymerase III, beta subunit (dnaN) SACOL0002 29 NAD(P)H flavi n oxidoreductase (frp) SACOL2534 29 conserved hypothetical protein SACOL1802 29 indole 3 pyruvate decarboxylase (ipdC) SACOL0173 29 ribosomal protein S11 (rpsK) SACOL2214 28 6 phosp hofructokinase (pfkA) SACOL1746 28 ribosomal protein L11 (rplK) SACOL0583 28 ATP dependent Clp protease, ATP binding subunit ClpB (clpB) SACOL0979 28 metallo beta lactamase family protein SACOL1294 28 GMP synthase (guaA) SACOL0461 27 ribosomal protein S3 (rpsC) SACOL2233 27 immunoglobulin G binding protein A precursor (spa) SACOL0095 27 conserved hypothetical protein SACOL2379 27 aminopeptidase PepS (pepS) SACOL1937 27 cysteine desulfurase, SufS subfamily SACOL0916 26 4 diphosphocytidyl 2C methyl D erythritol synthase, putative SACOL0240 26 glutamate 1 semialdehyde 2,1 aminomutase (hemL1) SACOL1714 26 hydroxyethylthiazole kinase (thiM) SACOL2084 26 bacterioferritin comigratory protein (bcp) SACOL1921 26 conserved hypothetical protein SACOL2596 26 long chain fatty acid -CoA ligase, putative SACOL0214 26 conserved hypothetical protein SACOL1975 26 ribosome recycling factor (frr) SACOL1278 26 RNAIII activating protein TRAP SACOL1891 25 phosphopentomutase (deoB) SACOL0124 25 acetyl CoA acetyltransferase SACOL0211 25 ribosomal protein L6 (rplF) SACOL2224 24 N utilization substance protein A, putative SACOL1285 24 leucyl tRNA synthetase (leuS) SACOL1808 24 anti anti sigma factor RsbV (rsbV) SACOL2056 24 glutamyl aminopeptidase (pepA1) SACOL1795 24 ribosomal protein L27 (rpmA) SACOL1700 24 ribosomal protein S16 (rpsP) SACOL1254 24 conserved hypothetical protein SACOL1115 23 thioredoxin, putative SACOL1794 23 HPr kinase phosphatase (hprK) SACOL0825 23 phenylalanyl tRNA synthetase, beta subunit (pheT) SACOL1149 22 oxidoreductase, aldo keto reductase family SACOL0763 22 DNA gyrase, A subunit (gyrA) SACOL0006 22 imidazolonepropionase (hutI) SACOL2323 22 conserved hypothetical protein TIGR00103 SACOL0521 22 molybdopterin biosynthesis MoeA protein, putative SACOL2266 22 glutamyl aminopeptidase (pepA2) SACOL2463 22 ribosomal protein S20 (rpsT) SACOL1642 22 3 methyl 2 oxobutanoate hydroxymethyltransferase (panB) SACOL2615 22 autoinducer 2 production protein LuxS (luxS) SACOL2126 22 preprotein translocase, SecA subunit (secA) SACOL0816 22 arsenate reductase, putative SACOL0876 22 lipase (geh) SACOL2694 21

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121 Appendix 2. (Continued) conserved hypothetical protein SACOL1792 21 phosphoribosylformylglycinamidine synthase II (purL) SACOL1078 21 DNA directed RNA polymerase, delta subunit (rpoE) SACOL2120 21 heat shock protein GrpE (grpE) SACOL1638 21 delta aminolevulinic ac id dehydratase (hemB) SACOL1715 21 phosphoribosylformylglycinamidine synthase, PurS protein (purS) SACOL1076 21 ornithine carbamoyltransferase (arcB1) SACOL1181 21 cold shock protein, CSD family SACOL1437 20 hypoxanthine phosphoribosyltransferase (hpt) SACOL0554 20 translation elongation factor P (efp) SACOL1587 20 carbamate kinase (arcC2) SACOL2654 20 NAD NADP octopine nopaline dehydrogenase family protein SACOL2293 20 formiminoglutamase (hutG) SACOL2327 20 ribosomal protein L29 (rpmC) SACOL2231 20 ribosomal protein L3 (rplC) SACOL2239 19 ribosomal protein L13 (rplM) SACOL2207 19 acetyl CoA acetyltransferase SACOL0426 19 aspartyl tRNA synthetase (aspS) SACOL1685 19 ribosomal protein S18 (rpsR) SACOL0439 19 ribosomal protein S9 (rpsI) SACOL2206 19 peptidase, M20 M25 M40 family SACOL0085 19 conserved hypothetical protein SACOL2609 19 N acetylmuramoyl L alanine amidase domain protein SACOL2666 18 acid phosphatase5' nucleotidase, lipoprotein e(P4) family SACOL0303 18 DAK2 domain protein SACOL1240 18 dihydroorotase (pyrC) SACOL1213 18 oxidoreductase, short chain dehydrogenase reductase family SACOL2488 18 tyrosyl tRNA synthetase (tyrS) SACOL1778 18 p rolyl tRNA synthetase (proS) SACOL1282 18 alanyl tRNA synthetase (alaS) SACOL1673 18 ribosomal protein L30p L7e (rpmD) SACOL2221 18 cysteinyl tRNA synthetase (cysS) SACOL0576 18 proline dipeptidase (pepQ) SACOL1756 18 ribosomal protein L31 (rpmE) SACOL2112 18 HIT family protein SACOL1894 18 cytosol aminopeptidase SACOL0945 18 isocitrate dehydrogenase, NADP dependent (icd) SACOL1741 17 glyoxalase family protein SACOL2533 17 NH(3) dependent NAD+ synthetase (nadE) SACOL1974 17 UDP N acetylglucosamine 1 carboxyvinyltransferase 1 (murAA) SACOL2092 17 phosphoribosylaminoimidazole succinocarboxamide synthase (purC) SACOL1075 17 phosphoribosylaminoimidazole carboxylase, catalytic subunit (purE) SACOL1073 17 conserved hypothetical protein SACOL1987 17 elastin binding protein, putative SACOL1522 16 methionine aminopeptidase, type I SACOL1946 16 D alanine activating enzyme D alanine D alanyl carrier protein ligase (dltA) SACOL0935 16 UDP N acetylglucosamine 1 carboxyvinyltransferase 2 (murAB) SACOL2116 16 glutamate 1 semialdehyde 2,1 aminomutase (hemL2) SACOL1922 16 ribosomal protein L18 (rplR) SACOL2223 16 conserved hypothetical protein SACOL0738 16 sigma factor B regulator protein (rsbU) SACOL2057 16 conserved hypothetical protein SACOL1350 16 translation initiation factor IF 3 (infC) SACOL1727 15 quinol oxidase, subunit I (qoxA) SACOL1069 15 polypeptide deformylase (def1) SACOL1100 15 transcriptional regulator, MarR family (norR) SACOL0746 15

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122 Appendix 2. (Continued) oligoendopeptidase F, putative SACOL1419 15 betaine aldehyde dehydrogenase (betB) SACOL2628 15 nucleoside diphosphate kinase SACOL1509 15 DNA binding response regulator YycF (yycF) SACOL0019 15 conserved hypothetical protein SACOL1306 15 heat shock protein HslVU, ATPase subunit HslV (hslV) SACOL1270 15 glyceraldehyde 3 phosphate dehydrogenase (gapA2) SACOL1734 15 ribosomal protein S12 (rpsL) SACOL0591 14 phosphomethylpyrimidine kinase (thiD1) SACOL0626 14 ribonucleoside diphosphate reductase, alpha subunit SACOL0792 14 conserved hypothetical protein SACOL1670 14 DNA polymerase I (polA) SACOL1737 14 peptidase, M20 M25 M40 family SACOL1555 14 recA protein SACOL1304 14 conserved hypothetical protein SACOL2174 14 GTP binding protein TypA (typA) SACOL1118 14 oxidoreductase, putative SACOL0399 14 succinate dehydrogenase, iron sulfur protein (sdhB) SACOL1160 14 alanine dehydrogenase (ald1) SACOL1478 14 PTS system, IIABC components SACOL0175 14 ScdA protein (scdA) SACOL0244 14 amino acid ABC transporter, amino acid binding protein SACOL2412 13 conserved hypothetical protein TIGR00092 SACOL0435 13 malonyl CoA acyl carrier protein transacylase (fabD) SACOL1244 13 conserved hypothetical protein SACOL1447 13 lipoprotein, putative SACOL2365 13 carbamoyl phosphate synthase, large subunit (carB) SACOL1215 13 RNA polymerase sigma factor RpoD (rpoD) SACOL1618 13 ABC transporter, ATP binding protein SACOL1427 13 conserved hypothetical protein SACOL2143 13 glycosyl transferase, group 2 family protein SACOL0243 13 ribosomal protein L36 (rpmJ) SACOL2216 13 NADH dependent flavin oxidoreductase, Oye family SACOL0959 13 ribosomal protein L35 (rpmI) SACOL1726 13 conserved hypothetical protein SACOL1672 13 pyrroline 5 carboxylate reductase (proC ) SACOL1546 12 ribosomal protein L23 (rplW) SACOL2237 12 cell division protein FtsH, putative SACOL0555 12 epimerase dehydratase, putative SACOL2446 12 fatty acid phospholipid synthesis protein PlsX (plsX) SACOL1243 12 aminotransferase, putative SACOL2000 12 enoyl (acyl carrier protein) reductase (fabI) SACOL1016 12 transcription elongation factor GreA (greA) SACOL1665 12 threonine dehydratase, catabolic (ilvA1) SACOL1477 12 transcriptional regulator, TenA family SACOL2086 12 alcohol dehydrogenase, zinc containing SACOL0241 12 glycerol kinase (glpK) SACOL1320 12 dihydroxyacetone kinase family protein SACOL0707 12 conserved hypothetical protein SACOL0455 11 molybdenum ABC transporter, molybdenum binding protein ModA (modA) SACOL2272 11 serine protease HtrA, putative SACOL1777 11 glucokinase (glk) SACOL1604 11 glutathione peroxidase (gpxA1) SACOL1325 11 excinuclease ABC, A subunit (uvrA) SACOL0824 11 DNA directed RNA polymerase, omega subunit (rpoZ) SACOL1222 11 OsmC Ohr family protein SACOL0872 11

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123 Appendix 2. (Continued) SIS domain protein SACOL0618 11 conserved hypothetical protein SACOL2456 11 conserved hypothetical protein SACOL2300 11 conserved hypothetical protein SACOL1767 10 NAD(P)H dehydrogenase (quinone), putative SACOL0190 10 hydrolase, haloacid dehalogenase like family SACOL0931 10 staphylococcus tandem lipoprotein SACOL0486 10 ribose phosphate pyrophosphokinase (prsA) SACOL0544 10 DNA repair exonuclease family protein SACOL1900 10 heat shock protein HslVU, ATPase subunit HslU (hslU) SACOL1271 10 D alanine -D alanine ligase SACOL2074 10 ribonucleoside diphosphate reductase 2, beta subunit (nrdF) SACOL0793 10 phosphoribosylformylglycinamidine synthase I (purQ) SACOL1077 10 femX protein (femX) SACOL2253 10 malate:quinone oxidoreductase (mqo1) SACOL2362 10 peptidase T (pepT) SACOL0806 10 para nitrobenzyl esterase (pnbA) SACOL2459 10 transcription antitermination protein NusG (nusG) SACOL0582 10 conserved hypothetical protein TIGR00294 SACOL0613 10 superoxide dismutase (sodA1) SACOL0118 10 adenine phosphoribosyltransferase (apt) SACOL1690 10 ribosomal protein L24 (rplX) SACOL2228 10 sdrD protein (sdrD) SACOL0609 9 penicillin binding protein 2 (pbp2) SACOL1490 9 conserved hypothetical protein SACOL0669 9 ATP dependent Clp protease, proteolytic subunit ClpP (clpP) SACOL0833 9 ATP synthase F0, B subunit (atpF) SACOL2099 9 chaperonin, 10 kDa (groES) SACOL2017 9 pyridine nucleotide disulfide oxidoreductase SACOL1520 9 3,4 dihydroxy 2 butanone 4 phosphate synthase GTP cyclohydrolase II (ribBA) SACOL1818 9 phosphoribosylformylglycinamidine cyclo ligase (purM) SACOL1080 9 exoribonuclease, VacB RNase II family SACOL0846 9 chorismate mutase phospho 2 dehydro 3 deoxyheptonate aldolase SACOL1787 9 glycine cleavage system T protein (gcvT) SACOL1595 9 conserved hypothetical protein SACOL1099 9 dihydrofolate reductase (folA) SACOL1461 9 ribosomal protein S10 (rpsJ) SACOL2240 9 accessory gene regulator protein A (agrA) SACOL2026 9 phosphoribosylaminoimidazole carboxylase, ATPase subunit (purK) SACOL1074 9 alanine racemase (alr) SACOL2060 9 D alanyl carrier protein (dltC) SACOL0937 9 conserved hypothetical protein SACOL2288 9 staphylococcal accessory regulator S (sarS) SACOL0096 9 ATP dependent RNA helicase, DEAD DEAH box family SACOL2072 9 acetyltransferase, GNAT family SACOL2532 9 urease accessory protein UreE (ureE) SACOL2283 9 lipoate protein ligase A family protein SACOL1591 9 map protein, programmed frameshift (map) SACOL2002 8 ATP dependent Clp protease, ATP binding subunit ClpX (clpX) SACOL1721 8 1 phosphofructokinase (fruK) SACOL0758 8 single stranded DNA binding protein (ssb2) SACOL0438 8 UTP glucose 1 phosphate uridylyltransferase family protein SACOL2161 8 cell division initiation protein, putative SACOL1205 8 RNA methyltransferase, TrmH family SACOL0578 8 NifU domain protein SACOL0917 8 amidophosphoribosyltransferase (purF) SACOL1079 8

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124 Appendix 2. (Continued) DNA gyr ase, B subunit (gyrB) SACOL0005 8 epidermin immunity protein F (epiF) SACOL1873 8 cell division protein FtsY, putative SACOL1251 8 arginase (rocF) SACOL2154 8 GTP binding protein, Era TrmE family SACOL1515 8 thymidylate synthase (thyA) SACOL1462 8 repressor of toxins (rot) SACOL1812 8 ATP binding protein, Mrp Nbp35 family SACOL2156 8 ATP synthase F1, delta subunit (atpH) SACOL2098 8 ABC transporter, ATP binding protein SACOL1994 8 UDP N acetylmuramoylalanine -D glutamate ligase (murD) SACOL1196 8 beta hydroxyacyl (acyl carrier protein) dehydratase FabZ (fabZ) SACOL2091 8 GMP reductase (guaC) SACOL1371 8 phosphoglucomutase phosphomannomutase family protein SACOL2501 8 drP35 protein (drp35) SACOL2712 8 conserved hypothetical protein SACOL0525 8 conserved hypothetical protein TIGR00253 SACOL1651 8 ribosomal protein L32 (rpmF) SACOL1137 8 azoreductase SACOL0607 8 DNA binding response regulator VraR (vraR) SACOL1942 8 peptide chain release factor 1 (prfA) SACOL2110 7 S adenosylmethio nine synthetase (metK) SACOL1837 7 4 oxalocrotonate tautomerase (dmpI) SACOL1399 7 2 oxoisovalerate dehydrogenase, E1 component, beta subunit SACOL1561 7 DHH subfamily 1 protein SACOL1751 7 DNA binding response regulator SrrA (srrA) SACOL1535 7 degV family protein SACOL1460 7 conserved hypothetical protein SACOL0742 7 HAM1 protein SACOL1162 7 conserved hypothetical protein SACOL1558 7 UDP N acetylglucosamine pyrophosphorylase (glmU) SACOL0543 7 conserved hypothetical protein SACOL1483 7 tRNA pseudouridine 55 synthase (truB) SACOL1290 7 alcohol dehydrogenase, zinc containing SACOL2177 7 staphylococcal accessory regulator R (sarR) SACOL2287 7 conserved hypothetical protein SACOL0467 7 conserved hypothetical protein SACOL0409 7 conserved hypothetical protein SACOL0565 7 glyoxalase family protein SACOL1553 7 riboflavin biosynthesis protein RibD (ribD) SACOL1820 7 conserved hypothetical protein SACOL1836 7 uridylate kinase (pyrH) SACOL1277 7 femA protein (femA) SACOL1410 7 5' methylthioadenosine S adenosylhomocysteine nucleosidase (mtn) SACOL1655 7 molybdenum cofactor biosynthesis protein B (moaB) SACOL2268 7 delta hemolysin (hld) SACOL2022 7 UDP N acetylmuramate -alanine ligase (murC) SACOL1790 7 conserved hypothetical protein SACOL1310 7 conserved hypothetical protein SACOL1895 7 esterase, putative SACOL2549 7 staphyloxanthin biosynthesis protein SACOL2291 6 ATP synthase F1, gamma subunit (atpG) SACOL2096 6 dephospho CoA kinase (coaE) SACOL1735 6 copper ion binding protein SACOL2573 6 thiamine phosphate pyrophosphorylase (thiE) SACOL2083 6 ferrochelatase (hemH) SACOL1888 6

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125 Appendix 2. (Continued) phenylalanyl tRNA synthetase, alpha subunit (pheS) SACOL1148 6 femB protein SACOL1411 6 general stress protein 13 SACOL0552 6 urease accessory protein UreG (ureG) SACOL2285 6 DAK2 domain protein SACOL0708 6 ribosomal protein L19 (rplS) SACOL1257 6 translation initiation factor IF 1 (infA) SACOL2217 6 NifU domain protein SACOL0939 6 glucose inhibited division protein A (gidA) SACOL2737 6 conserved hypothetical protein TIGR01777 SACOL0834 6 phosphomethylpyrimidine kinase (thiD2) SACOL2085 6 sucrose 6 phosphate hydrolase (cscA) SACOL2029 6 cmp binding factor 1 (cbf1) SACOL1898 6 conserved hypothetical protein SACOL0157 6 teichoic acid biosynthesis protein, putative SACOL0242 6 D isomer specific 2 hydroxyacid dehydrogenase family protein SACOL0932 6 isochorismatase family protein SACOL2667 6 iron compound ABC transporter, iron compound binding protein SACOL2167 6 histidine ammonia lyase (hutH) SACOL0008 6 lipoate protein ligase A family protein SACOL1034 6 queuine tRNA ribosyltransferase (tgt) SACOL1694 6 conserved hypothetical protein SACOL1980 6 phytoene dehydrogenase SACOL2579 6 FMN reductase related protein SACOL0410 6 Rrf2 family protein SACOL1681 6 glutamyl tRNA(Gln) amidotransferase, C subunit (gatC) SACOL1962 6 conserved hypothetical protein SACOL1387 6 phosphosugar binding transcriptional regulator, RpiR family SACOL2308 6 alpha acetolactate decarboxylase (budA1) SACOL2198 6 DNA topoisomerase I (topA) SACOL1267 6 proline dehydrogenase (putA) SACOL1816 6 conserved hypothetical protein SACOL1090 6 conserved hypothetical protein TIGR00043 SACOL1627 6 Aerolysin Leukocidin family protein SACOL2006 5 ribosome binding factor A (rbfA) SACOL1289 5 conserved hypothetical protein SACOL1426 5 hydrolase, haloacid dehalogenase like family SACOL0606 5 UDP N acetylmuramoyl tripeptide -D alanyl D alanine ligase (murF) SACOL2073 5 tellurite resistance protein, putative SACOL1441 5 GTP binding protein, GTP1 OBG family SACOL1699 5 conserved hypothetical protein SACOL2605 5 peptide chain release factor 2, programmed frameshift (prfB) SACOL0818 5 conserved hypothetical protein SACOL1120 5 2 oxoisovalerate dehydrogenase, E1 co mponent, alpha subunit SACOL1562 5 conserved hypothetical protein SACOL1940 5 S1 RNA binding domain protein SACOL2053 5 tryptophanyl tRNA synthetase (trpS) SACOL1001 5 HD HDIG KH domain protein SACOL1305 5 histidyl tRNA synthetase (hisS) SACOL1686 5 conserved hypothetical protein SACOL1620 5 2 succinyl 6 hydroxy 2,4 cyclohexadiene 1 carboxylic acid synthase 2 oxoglutarate decarboxylase (menD) SACOL1052 5 conserved hypothetical protein SACOL2381 5 ribosomal protein L14 (rplN) SACOL2229 5 citrate synthase (gltA) SACOL1742 5 excinuclease ABC, B subunit (uvrB) SACOL0823 5

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126 Appendix 2. (Continued) NAD(P)H flavin oxidoreductase, putative SACOL0453 5 peptide methionine sulfoxide reductase (msrA) SACOL1397 5 alpha glucosidase (malA) SACOL1551 5 conserved hypothetical protein SACOL2035 5 signal recognition particle protein (ffh) SACOL1253 5 tRNA (5 methylaminomethyl 2 thiouridylate) methyltransferase (trmU) SACOL1676 5 GTP binding protein Era (era) SACOL1624 5 2 oxoisovalerate dehydrogenase, E2 component, dihydrolipoamide acetyltransferase SACOL1560 5 conserved hypothetical protein SACOL2132 5 conserved hypothetical protein SACOL1358 5 MutT nudix family protein SACOL1542 5 YihY family protein (yihY) SACOL1941 5 aspartate carbamoyltransferase (pyrB) SACOL1212 5 conserved hypothetical protein SACOL0152 5 MutT nudix family protein SACOL1724 5 conserved hypothetical protein SACOL1002 5 arginine repressor (argR) SACOL1565 5 lipoprotein, putative SACOL1101 4 iron compound ABC transporter, iron compound binding protein SACOL2277 4 hydrolase, haloacid dehalogenase like family SACOL0602 4 conserved hypothetical protein SACOL1464 4 phosphoribosylglycinamide formyltransferase (purN) SACOL1081 4 xanthine phosphoribosyltransferase (xpt) SACOL0458 4 hydrolase, haloacid dehalogenase like family SACOL1365 4 acetyl CoA carboxylase, biotin carboxyl carrier protein (accB) SACOL1572 4 dnaJ protein (dnaJ) SACOL1636 4 DltD protein (dltD) SACOL0938 4 GTP pyr ophosphokinase (relA2) SACOL1689 4 primosomal protein N` (priA) SACOL1224 4 dihydroorotate dehydrogenase (pyrD) SACOL2606 4 porphobilinogen deaminase (hemC) SACOL1717 4 exonuclease RexA (rexA) SACOL0971 4 glycerophosphoryl diester phosphodiesterase, putative SACOL1130 4 DNA topoisomerase IV, A subunit (parC) SACOL1390 4 sulfite reductase (NADPH) flavoprotein alpha component (cysJ) SACOL2639 4 argininosuccinate lyase (argH) SACOL0963 4 dehydrosqualene desaturase (crtN) SACOL2576 4 heat shock protein, Hsp20 family SACOL2385 4 oxidoreductase, aldo keto reductase family SACOL1543 4 glycosyl transferase, group 1 family protein SACOL1043 4 DNA dependent DNA polymerase family X SACOL1153 4 serine acetyltransferase (cysE) SACOL0575 4 S adenosyl methyltransferase MraW SACOL1192 4 N acetyltransferase family protein SACOL2722 4 phosphotyrosine protein phosphatase SACOL1939 4 exodeoxyribonuclease VII, large subunit (xseA) SACOL1568 4 deoxyribonuclease, TatD family SACOL0534 4 pur operon repressor (purR) SACOL0539 4 glycerate kinase family protein SACOL0805 4 carbamoyl phosphate synthase, small subunit (carA) SACOL1214 4 GTP binding protein LepA (lepA) SACOL1641 4 oxidoreductase, aldo keto reductase family SACOL2192 4 conserved hypothetical protein SACOL2318 4 conserved hypothetical protein TIGR00282 SACOL1307 4 conserved hypothetical protein SACOL0271 4

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127 Appendix 2. (Continued) conserved hypothetical protein SACOL1284 4 conserved hypothetical protein SACOL1899 4 conserved hypothetical protein SACOL0615 4 inositol monophosphatase family protein SACOL1116 4 conserved hypothetical protein TIGR00147 SACOL1958 4 trans sulfuration enzyme family protein SACOL0503 4 GTP pyrophosphokinase (relA1) SACOL1010 4 UTP glucose 1 phosphate uridylyltransferase (galU) SACOL2508 4 transcriptional regulator, Fur family SACOL1541 4 sulfatase family protein SACOL0778 3 immunodominant antigen B (isaB) SACOL2660 3 FtsK SpoIIIE family protein SACOL1791 3 cytidylate kinase (cmk) SACOL1518 3 Gid protein (gid) SACOL1268 3 conserved hypothetical protein SACOL1885 3 uroporphyrinogen decarboxylase (hemE) SACOL1889 3 transcriptional regulator, putative SACOL2302 3 YlmF protein (ylmF) SACOL1202 3 orotidine 5' phosphate decarboxylase (pyrF) SACOL1216 3 UDP N acetylglucosamine -N acetylmuramyl (pentapeptide) pyrophosphoryl undecaprenol N acetylglucosamine transferase (murG) SACOL1453 3 mevalonate kinase (mvk) SACOL0636 3 pyrimidine operon regulatory protein (pyrR) SACOL1210 3 lipoate synthase (lipA) SACOL0927 3 rhodanese like domain protein SACOL1807 3 DNA ligase, NAD dependent (ligA) SACOL1965 3 transferrin receptor SACOL0799 3 pyridine nucleotide disulfide oxidoreductase SACOL0640 3 conserved hypothetical protein SACOL0614 3 peptide chain release factor 3 (prfC) SACOL1025 3 conserved hypothetical protein SACOL2436 3 DNA topoisomerase IV, B subunit (parE) SACOL1389 3 N utilization substance protein B (nusB) SACOL1569 3 rhodanese like domain protein SACOL1592 3 ribosomal protein S21 (rpsU) SACOL1632 3 DNA 3 met hyladenine glycosylase SACOL1711 3 conserved hypothetical protein SACOL0198 3 conserved hypothetical protein SACOL1297 3 ph osphomevalonate kinase SACOL0638 3 conserved hypothetical protein SACOL1286 3 capsular polysaccharide biosynthesis protein Cap5O (cap5O) SACOL0150 3 RNA polymerase sigma 37 factor (rpoF) SACOL2054 3 glycosyl transferase, group 1 family protein SACOL0612 3 conserved hypothetical protein TIGR01741 SACOL0282 3 dihydrodipicolinate reductase (dapB) SACOL1431 3 conserved hypothetical protein SACOL0599 3 formate dehydrogenase, NAD dependent SACOL0162 3 PTS system, IIBC components SACOL0516 3 conserved hypothetical protein SACOL0401 3 phosphopantothenoylcysteine decarboxylase phosphopantothenate -cysteine ligase (coaBC) SACOL1223 3 mevalonate diphosphate decarboxylase (mvaD) SACOL0637 3 conserved hypothetical protein SACOL0804 3 HD domain protein SACOL0821 3 conserved hypothetical protein SACOL1011 3 hypothetical protein SACOL1042 3

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128 Appendix 2. (Continued) aminotransferase, class V SACOL1677 3 protein export membrane protein SecDF SACOL1692 3 amino acid ABC transporter, ATP binding protein SACOL2453 3 glutamate racemase (murI) SACOL1161 3 HD domain protein SACOL0658 3 staphylococcal accessory protein X (sarX) SACOL0726 3 6 pyruvoyl tetrahydrobiopterin synthase, putative SACOL0771 3 diaminopimelate decarboxylase (lysA) SACOL1435 3 PhoH family protein SACOL1628 3 hypothetical protein SACOL0156 3 conserved hypothetical protein SACOL1239 3 DNA repair protein RecN (recN) SACOL1564 3 acetyl CoA carboxylase, carboxyl transferase, beta subunit (accD) SACOL1748 3 sdrC protein (sdrC) SACOL0608 2 Staphylococcus aureus sex pheromone (camS) SACOL1964 2 signal peptidase IB (spsB) SACOL0969 2 conserved hypothetical protein SACOL2020 2 decarboxylase family protein SACOL0740 2 ferredoxin (fer) SACOL1525 2 ABC transporter, substrate binding protein SACOL2403 2 conserved domain protein SACOL1466 2 type I restriction modification system, M subunit (hsdM1) SACOL0476 (+1) 2 exonuclease RexB (rexB) SACOL0970 2 adenosylmethionine -8 amino 7 oxononanoate aminotransferase (bioA) SACOL2427 2 oxidoreductase, Gfo Idh MocA family SACOL0196 2 conserved hypothetical protein SACOL2489 2 riboflavin biosynthesis protein RibF (ribF) SACOL1291 2 UDP N acetylglucosamine 2 epimerase Cap5G (cap5G) SACOL0142 2 conserved domain protein SACOL0445 2 conserved hypothetical protein SACOL0830 2 transcriptional regulator, Fur family SACOL1611 2 protoporphyrinogen oxidase (hemG) SACOL1887 2 hydrolase, haloacid dehalogenase like family SACOL0976 2 hypothetical protein SACOL0182 2 iron dependent repressor (sirR) SACOL0691 2 alkaline phosphatase synthesis transcriptional regulatory protein PhoP (phoP) SACOL1740 2 peptidase, M16 family SACOL1298 2 UDP N acetylmuramoylalanyl D glutamate -2,6 diaminopimelate ligase (murE) SACOL1023 2 hydrolase, alpha beta hydrolase fold family SACOL0668 2 thymidine kinase (tdk) SACOL2111 2 transcriptional regulator, putative SACOL2650 2 conserved hypothetical protein SACOL0058 2 PTS system, IIBC components SACOL0178 2 phosphoglycerate mutase family protein SACOL0447 2 conserved hypothetical protein SACOL0753 2 conserved hypothetical protein SACOL1112 2 fibronectin fibrinogen binding related protein SACOL1220 2 ribosomal protein L28 (rpmB) SACOL1238 2 conserved hypothetical protein SACOL1376 2 transcriptional antiterminator LicT, putative SACOL1393 2 aspartate semialdehyde dehydrogenase (asd) SACOL1429 2 dihydrodipicol inate synthase (dapA) SACOL1430 2 conserved hypothetical protein TIGR00256 SACOL1688 2 cytosolic long chain acyl CoA thioester hydrolase family protein SACOL1936 2 conserved hypothetical protein SACOL1993 2

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129 Appendix 2. (Continued) isopentenyl diphosphate isomerase (fni) SACOL2341 2 staphylococcal accessory protein Z (sarZ) SACOL2384 2 ABC transporter, ATP binding protein SACOL2462 2 gluconokinase (gntK) SACOL2515 2 galactoside O acetyltransferase SACOL2570 2 hydrolase, CocE NonD family SACOL2612 2 tributyrin esterase EstA, putative SACOL2651 2 rod shape determining protein MreC (mreC) SACOL1704 1 LysM domain protein SACOL0507 1 LPXTG cell wall surface anchor family protein (sasF) SACOL2668 1 lipoprotein, putative SACOL0449 1 penicillin binding protein 1 (pbp1) SACOL1194 1 protein phosphatase 2C domain protein SACOL1231 1 secretory extracellular matrix and plasma binding protein (empbp) SACOL0858 1 chaperonin, 33 kDa SACOL0556 1 immunodominant antigen A (isaA) SACOL2584 0 cell wall surface anchor family protein (sasG) SACOL2505 0 fibronectin binding protein B (fnbB) SACOL2509 0 fibronectin binding protein A (fnbA) SACOL2511 0 clumping factor B (clfB) SACOL2652 0 LysM domain protein SACOL0723 0 leukocidin subunit precursor, putative SACOL2004 0 alpha hemolysin precursor (hlY) SACOL1173 0 surface protein, putative SACOL0479 0 IgG binding protein SBI SACOL2418 0 cysteine protease precursor SspB (sspB2) SACOL1970 0 sasB protein (sasB) SACOL2150 0 transcriptional regulator, putative SACOL1065 0 phage infection protein, putative SACOL2665 0 transcriptional regulator, putative SACOL1398 0 penicillin binding protein 3 (pbp3) SACOL1609 0 phospholipase C (hlb) SACOL2003 0 lipoprotein, putative SACOL1589 0 peptide ABC transporter, peptide binding protein SACOL2476 0 ABC transporter, substrate binding protein SACOL0217 0 thymidylate kinase (tmk) SACOL0524 0 glucose inhibited division protein A (gidA) SACOL2737 R 0 fibronectin binding protein B (fnbB) SACOL2509 R 0 serine protease SplB (splB) SACOL1868 0

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130 Appendix 3. Cytoplasmic proteins identified after MudPit analysis of SH1000 during post exponential phase from 2 biological replicates Identified Proteins (346) Accession Number Sample 1 Sample 2 Elongation factor Tu Q5HIC7|EFTU 239 119 Probable transglycosylase isaA Q5HCY1|ISAA 81 111 Elongation factor G Q5HIC8|EFG 89 65 Enolase Q5HHP1|ENO 64 39 Pyruvate kinase Q5HF76|KPYK 54 27 Dihydrolipoyl dehydrogenase Q5HGY8|DLDH 39 36 Pyruvate dehydrogenase E1 component subunit beta Q5HGZ0|ODPB 35 38 Bifunctional autolysin Q5HH31|ATL 45 24 50S ribosomal protein L30 Q5HDX6|RL30 28 25 50S ribosomal protein L15 Q5HDX7|RL15 15 45 Pyruvate dehydrogenase E1 component subunit alpha Q5HGZ1|ODPA 34 25 Dihydrolipoyllysine residue acetyltransferase component of pyruvate dehydrogenase complex Q5HGY9|ODP2 30 23 2,3 bisphosphoglycerate dependent phosphoglycerate mutase Q5HDD9|GPMA 34 13 Phosphate acetyltransferase Q5HI88|PTA 29 21 Alkyl hydroperoxide reductase subunit C Q5HIR5|AHPC 27 16 Transketolase Q5HG77|TKT 26 21 30S ribosomal protein S8 Q5HDX2|RS8 15 17 50S ribosomal protein L13 Q5HDZ0|RL13 25 14 Glycyl tRNA synthetase Q5HFJ5|SYG 29 11 50S ribosomal protein L1 Q5HID7|RL1 22 15 Alkaline shock protein 23 Q5HE23|ASP23 24 11 ATP synthase subunit beta Q5HE97|ATPB 26 16 Pyridoxal biosynthesis lyase pdxS Q5HIF5|PDXS 21 16 Staphylococcal secretory antigen ssaA2 Q5HDQ9|SSAA2 22 17 L lactate dehydrogenase 1 Q5HJD7|LDH1 26 13 50S ribosomal protein L27 Q5HFB8|RL27 23 13 ATP synthase subunit alpha Q5HE95|ATPA 25 14 Cell division protein ftsZ Q5HGP5|FTSZ 14 18 3 oxoacyl [acyl carrier protein] reductase Q5HGK2|FABG 18 17 Probable malate:quinone oxidoreductase 2 Q5HCU5|MQO2 18 9 Succinyl CoA ligase [ADP forming] subunit beta Q5HGI7|SUCC 20 14 GMP synthase [glutamine hydrolyzing] Q5HIQ6|GUAA 20 10 50S ribosomal protein L6 Q5HDX3|RL6 19 11 Glutamine synthetase Q5HGC3|GLNA 21 7 Uracil phosphoribosyltransferase Q5HE88|UPP 17 13 Glyceraldehyde 3 phosphate dehydrogenase 1 Q5HHP5|G3P1 23 7 DNA binding protein HU Q5HFV0|DBH 20 11 Antibacterial protein (Phenol soluble modulin) Q5HGQ7|Q5HGQ7 18 6 30S ribosomal protein S6 Q5HIS9|RS6 22 10 60 kDa chaperonin Q5HEH2|CH60 16 9 UPF0365 protein Q5HFI7|Y1630 19 11 DNA directed RNA polymerase subunit omega Q5HGM2|RPOZ 13 12 DNA directed RNA polymerase subunit beta' Q5HID2|RPOC 20 8 Fructose bisphosphate aldolase class 1 Q5HCU6|ALF1 16 10

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131 Appendix 3. (Continued) DNA directed RNA polymerase subunit beta Q5HID3|RPOB 17 9 Glucosamine 6 phosphate isomerase, putative Q5HES0|Q5HES0 17 12 6 phosphogluconate dehydrogenase, decarboxylating Q5HFR2|6PGD 17 9 50S ribosomal protein L5 Q5HDX0|RL5 22 8 Putative universal stress protein Q5HF64|Y1759 15 12 ATP dependent Clp protease ATP binding subunit clpL Q5HD02|CLPL 16 7 Antibacterial protein (Phenol soluble modulin) Q5HGQ8|Q5HGQ8 16 5 Acetate kinase Q5HF63|ACKA 16 7 50S ribosomal protein L11 Q5HID8|RL11 11 9 Ribosome recycling factor Q5HGH2|RRF 11 13 30S ribosomal protein S19 Q5HDW2|RS19 14 11 Succinyl CoA ligase [ADP forming] subunit alpha Q5HGI6|SUCD 10 9 Putative uncharacterized protein Q5HIT1|Q5HIT1 10 13 Bifunctional purine biosynthesis protein purH Q5HH11|PUR9 18 6 Carbamoyl phosphate synthase large chain Q5HGM9|CARB 12 12 Probable thiol peroxidase Q5HF61|TPX 14 7 Chaperone protein hchA Q5HIC4|HCHA 15 11 Conserved domain protein Q5HH57|Q5HH57 15 9 Fructose bisphosphate aldolase Q5HE75|ALF2 12 6 Pyruvate carboxylase Q5HGX0 11 5 Ornithine aminotransferase 2 Q5HHC8|OAT2 12 6 Ribose phosphate pyrophosphokinase Q5HIH5|KPRS 15 8 Chaperone protein dnaK Q5HFI0|DNAK 13 8 Elongation factor Ts Q5HGH4|EFTS 16 5 UPF0342 protein Q5HET0|Y1902 13 10 Putative 2 hydroxyacid dehydrogenase Q5HDQ4|Y2296 11 10 Oxidoreductase, putative Q5HIW6|Q5HIW6 10 4 Formate acetyltransferase Q5HJF4|PFLB 14 1 N utilization substance protein A, putative Q5HGG5|Q5HGG5 13 6 30S ribosomal protein S11 Q5HDY3|RS11 14 4 50S ribosomal protein L32 Q5HGV6|RL32 12 6 Naphthoate synthase Q5HH38|MENB 14 6 D alanine -D alanine ligase Q5HEB7|DDL 14 8 Cell cycle protein gpsB Q5HFX8|GPSB 13 7 Uncharacterized protein Q5HGK7|Y1240 10 3 30S ribosomal protein S5 Q5HDX5|RS5 10 9 ATP dependent Clp protease ATP binding subunit clpC P0C281|CLPC 12 6 50S ribosomal protein L10 Q5HID6|RL10 12 4 Alcohol dehydrogenase Q5HI63|ADH 13 4 Bifunctional protein folD Q5HH21|FOLD 12 2 Universal stress protein family Q5HF68|Q5HF68 9 6 Phosphoenolpyruvate protein phosphotransferase Q5HH01|PT1 10 6 30S ribosomal protein S2 Q5HGH6|RS2 10 4 NADH dehydrogenase like protein Q5HHE4|Y944 11 7 Ribonucleoside diphosphate reductase Q5HHU0|Q5HHU0 11 7 Hydroxymethylglutaryl CoA synthase Q5HD04|Q5HD04 9 7 Arginyl tRNA synthetase Q5HI60|SYR 9 7 Dihydrolipoyllysine residue succinyltransferase component of 2 oxoglutarate dehydrogenase complex Q5HG07|ODO2 9 4 30S ribosomal protein S7 Q5HIC9|RS7 11 4 30S ribosomal protein S16 Q5HGJ4|RS16 12 4 Thioredoxin Q5HGT9|THIO 11 5 Phosphoribosylformylglycinamidine synthase, PurS protein Q5HH17|Q5HH17 10 5 30S ribosomal protein S1 Q5HFU7|RS1 11 5 UPF0051 protein Q5HHG8|Y918 10 3 50S ribosomal protein L2 Q5HDW1|RL2 7 5

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132 Appendix 3. (Continued) NADP dependent malic enzyme, putative Q5HF72|Q5HF72 7 7 Translation initiation factor IF 2 Q5HGG2|IF2 7 2 Putative uncharacterized protein Q5HJK1|Q5HJK1 7 5 3 oxoacyl [acyl carrier protein] synthase 2 Q5HHA1|FABF 6 5 Cell division protein FtsH, putative Q5HIG4|Q5HIG4 9 5 Asparaginyl tRNA synthetase Q5HFW9|SYN 9 4 50S ribosomal protein L21 Q5HFB6|RL21 8 6 Phenol soluble modulin alpha 1 peptide P0C7Y4|PSMA1 7 3 30S ribosomal protein S10 Q5HDV7|RS10 7 7 N acetylmuramoyl L alanine amidase domain protein Q5HCQ3|Q5HCQ3 6 5 Uncharacterized protein Q5HHB6|Y973 8 5 Putative uncharacterized protein Q5HHZ0|Q5HHZ0 7 5 Prolyl tRNA synthetase Q5HGG8|SYP 7 0 50S ribosomal protein L17 Q5HDY5|RL17 11 1 Imidazolonepropionase Q5HDM7|HUTI 8 5 50S ribosomal protein L7/L12 Q5HID5|RL7 6 5 Glutamyl tRNA(Gln) amidotransferase subunit A Q5HEM2|GATA 6 3 Queuine tRNA ribosyltransferase Q5HFC4|TGT 7 5 DNA polymerase III subunit beta Q5HJZ4|DPO3B 8 2 ATP dependent protease ATPase subunit HslU Q5HGH8|HSLU 8 2 1 pyrroline 5 carboxylate dehydrogenase Q5HCZ6|ROCA 8 2 UvrABC system protein A UVRA 3 1 Serine hydroxymethyltransferase Q5HE87|GLYA 9 4 Aspartyl/glutamyl tRNA(Asn/Gln) amidotransferase subunit B Q5HEM3|GATB 5 3 Uncharacterized protein Q5HEP9|Y1933 6 4 Threonyl tRNA synthetase Q5HF90|SYT 8 2 Thioredoxin, putative Q5HF30|Q5HF30 6 4 50S ribosomal protein L18 Q5HDX4|RL18 7 3 GTP sensing transcriptional pleiotropic repressor codY Q5HGH7|CODY 6 4 Protein grpE Q5HFH9|GRPE 7 4 Seryl tRNA synthetase Q5HJY7|SYS 6 2 Oxidoreductase, short chain dehydrogenase/reductase family Q5HDM9|Q5HDM 9 7 5 UDP N acetylglucosamine 1 carboxyvinyltransferase 1 Q5HEA0|MURA1 6 3 Ferritin Q5HEN0|FTN 7 4 50S ribosomal protein L29 Q5HDW6|RL29 5 3 Probable catabolite control protein A Q5HF38|CCPA 7 5 50S ribosomal protein L33 1 Q5HFK9|RL331 (+1) 7 4 50S ribosomal protein L36 Q5HDY1|RL36 3 2 Thioredoxin reductase Q5HHQ4|TRXB 10 0 Phosphoglucosamine mutase Q5HE43|GLMM 6 2 Transaldolase Q5HEZ4|Q5HEZ4 8 3 6 phosphofructokinase Q5HF75|K6PF 5 4 50S ribosomal protein L23 Q5HDW0|RL23 4 4 UPF0082 protein Q5HHZ9|Y727 7 3 Formate -tetrahydrofolate ligase Q5HF42|FTHS 8 1 ABC transporter, ATP binding protein Q5HG28|Q5HG28 9 2 NifU domain protein Q5HHG9|Q5HHG9 7 3 Valyl tRNA synthetase Q5HFA8|SYV 5 3 GTP binding protein engA Q5HFU8|ENGA 5 3 Deoxyribose phosphate aldolase 1 Q5HJN0|DEOC1 5 2 Chorismate mutase/phospho 2 dehydro 3 deoxyheptonate aldolase Q5HF37|Q5HF37 5 4 Putative aldehyde dehydrogenase AldA Q5HJK3|ALDA 7 3 HTH type transcriptional regulator sarR Q5HDR3|SARR 5 3 Orotidine 5' phosphate decarboxylase Q5HGM8|PYRF 6 4

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133 Appendix 3. (Continued) UPF0355 protein Q5HIR0|UP355 4 4 Adenylosuccinate synthetase Q5HJX8|PURA 6 2 Proline dipeptidase Q5HFM9|Q5HFM9 7 1 Putative uncharacterized protein Q5HET6 5 2 Probable manganese dependent inorganic pyrophosphatase Q5HEK1|PPAC 7 3 UPF0435 protein Q5HEP4|Y1938 6 4 Putative NAD(P)H nitroreductase Q5HD30|Y2534 3 5 Probable acetyl CoA acyltransferase Q5HIU0|THLA 6 1 Isoleucyl tRNA synthetase Q5HGN8|SYI 5 2 Alcohol dehydrogenase, zinc containing Q5HE18|Q5HE18 3 3 Hydroxymethylglutaryl CoA reductase, degradative Q5HD05|Q5HD05 3 2 Putative dipeptidase Q5HF23|PEPVL 4 3 3 hexulose 6 phosphate synthase Q5HIA5|HPS 5 1 Dephospho CoA kinase Q5HF85|COAE 5 1 Aminoacyltransferase femA Q5HG45|FEMA 4 3 FeS assembly ATPase SufC Q5HHH2|Q5HHH2 7 1 NifU domain protein Q5HHE8|Q5HHE8 4 4 Delta hemolysin Q5HEG6|HLD 5 4 Hypoxanthine guanine phosphoribosyltransferase Q5HIG5|HPRT 3 4 Inositol monophosphatase family protein Q5HGX7|Q5HGX7 5 3 Phosphoglycerate kinase Q5HHP4|PGK 5 3 30S ribosomal protein S4 Q5HF54|RS4 6 0 Protein translocase subunit secA 1 Q5HHR7|SECA1 4 0 CTP synthase Q5HE73|PYRG 3 2 Serine protein kinase rsbW Q5HED6|RSBW 3 2 Anti sigma B factor antagonist P60071|RSBV 5 2 50S ribosomal protein L14 RL14 3 1 S1 RNA binding domain protein Q5HED8|Q5HED8 2 4 3 oxoacyl [acyl carrier protein] synthase 3 Q5HHA2|FABH 5 1 tRNA uridine 5 carboxymethylaminomethyl modification enzyme mnmG Q5HCI4|MNMG 2 2 Acetyl coenzyme A carboxylase carboxyl transferase subunit beta Q5HF73|ACCD 4 2 Polyribonucleotide nucleotidyltransferase Q5HGF7|PNP 2 1 N acetylmuramoyl L alanine amidase sle1 Q5HIL2|SLE1 3 2 D alanine aminotransferase Q5HF24|DAAA 3 2 ATP synthase subunit delta Q5HE94|ATPD 6 1 2 oxoglutarate dehydrogenase E1 component Q5HG06|ODO1 4 3 30S ribosomal protein S20 Q5HFH5|RS20 4 0 Succinate dehydrogenase, iron sulfur protein Q5HGT4|Q5HGT4 4 3 Rrf2 family protein Q5HFD6|Q5HFD6 3 2 Probable DEAD box ATP dependent RNA helicase Q5HEB9|Y2072 3 3 Putative uncharacterized protein Q5HEF7|Q5HEF7 3 3 Phosphoribosylglycinamide formyltransferase Q5HH12|PUR3 2 3 50S ribosomal protein L25 Q5HIH4|RL25 3 2 Tyrosyl tRNA synthetase Q5HF45|SYY 2 2 Deoxyribonuclease, TatD family Q5HII5|Q5HII5 2 1 Aconitate hydratase Q5HG69|ACON 3 1 Low molecular weight protein tyrosine phosphatase ptpA Q5HEP3|PTPA 1 4 Peptide chain release factor 1 Q5HE82|RF1 2 3 Peroxide responsive repressor perR Q5HER3|PERR 1 2 Mannitol specific phosphotransferase enzyme IIA component PTMA 3 1 PhoH family protein Q5HFI9|Q5HFI9 1 2 Mannitol 1 phosphate 5 dehydrogenase MTLD 4 0 Lysyl tRNA synthetase Q5HIF7|SYK 3 1 UPF0297 protein Q5HFE5|Y1672 6 1 6,7 dimethyl 8 ribityllumazine synthase Q5HF08|RISB 4 2

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134 Appendix 3. (Continued) Putative uncharacterized protein Q5HEL5|Q5HEL5 4 2 Probable transglycosylase sceD Q5HEA4|SCED 2 2 Guanylate kinase Q5HGM3|KGUA 4 2 2 C methyl D erythritol 4 phosphate cytidylyltransferase 2 Q5HJC1|ISPD2 3 3 Oxidoreductase, aldo/keto reductase family Q5HHW7|Q5HHW 7 3 0 50S ribosomal protein L9 Q5HJY1|RL9 4 0 Ribonuclease J 1 Q5HGZ5|RNJ1 5 0 33 kDa chaperonin Q5HIG3|HSLO 5 0 Putative uncharacterized protein Q5HGE0|Q5HGE0 3 3 Phosphoribosylamine -glycine ligase Q5HH10|PUR2 3 1 ATP synthase gamma chain Q5HE96|ATPG 2 1 Trigger factor Q5HF97|TIG 3 0 Nitrite reductase [NAD(P)H], large subunit Q5HDF6|Q5HDF6 2 2 Penicillin binding protein 2 Q5HFX3|Q5HFX3 4 0 Riboflavin biosynthesis protein RibF Q5HGF9|Q5HGF9 2 0 UPF0122 protein Q5HGJ6|Y1252 3 0 Uridylate kinase Q5HGH3|PYRH 3 2 Phosphoribosylformylglycinamidine synthase 2 Q5HH15|PURL 1 1 Catalase Q5HG86|CATA 3 1 Phosphate acyltransferase Q5HGK4|PLSX 3 2 Phosphoribosylformylglycinamidine synthase 1 Q5HH16|PURQ 4 2 Probable cysteine desulfurase Q5HHH0|CSD 5 1 30S ribosomal protein S18 Q5HIS7|RS18 3 1 Molybdopterin molybdenumtransferase Q5HDT4|MOEA 3 2 MutT/nudix family protein Q5HF95|Q5HF95 3 0 5 methyltetrahydropteroyltriglutamate -homocysteine methyltransferase Q5HIT8|METE 2 0 Putative uncharacterized protein Q5HIN0|Q5HIN0 3 0 Malonyl CoA acyl carrier protein transacylase Q5HGK3|FABD 6 0 Uncharacterized protein Q5HG10|Y1445 2 1 Alcohol dehydrogenase, zinc containing Q5HJC0|Q5HJC0 1 3 50S ribosomal protein L3 Q5HDV8|RL3 2 1 ComE operon protein 2 Q5HFH2|RANDO M_Q5HFH2 R 1 1 Xanthine phosphoribosyltransferase Q5HIQ9|XPT 2 1 Chaperone protein clpB Q5HHB0|CLPB 4 1 Fumarate hydratase class II Q5HES4|FUMC 3 1 Copper chaperone copZ Q5HCZ2|COPZ 1 2 Probable glycine dehydrogenase [decarboxylating] subunit 1 Q5HFM3|GCSPA 3 2 Glucose specific phosphotransferase enzyme IIA component Q5HFZ9|PTGA 4 1 UDP N acetylglucosamine -N acetylmuramyl (pentapeptide) pyrophosphoryl undecaprenol N acetylglucosamine transferase Q5HG02|MURG 3 1 Methionyl tRNA formyltransferase Q5HGL6|FMT 4 1 Putative trmH family tRNA/rRNA methyltransferase Q5HIE3|TRMHL 3 1 Translation initiation factor IF 3 Q5HF92|IF3 2 2 30S ribosomal protein S3 Q5HDW4|RS3 2 1 Alcohol dehydrogenase, zinc containing Q5HDI5|Q5HDI5 2 2 Histidyl tRNA synthetase Q5HFD2|SYH 2 2 Aspartate carbamoyltransferase Q5HGN2|PYRB 2 2 Glutamate 1 semialdehyde 2,1 aminomutase 1 Q5HFA5|GSA1 2 2 Putative uncharacterized protein Q5HGK8 2 2 Pyrimidine nucleoside phosphorylase Q5HE64|PDP 2 0 PTS system, IIBC components Q5HJD5|Q5HJD5 1 0 50S ribosomal protein L4 RANDOM_RL4 R 1 0 Elastin binding protein ebpS Q5HFU2|EBPS 2 0

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135 Appendix 3. (Continued) Extracellular matrix binding protein ebh Q5HFY8|EBH 1 0 Septation ring formation regulator ezrA Q5HF56|EZRA 4 0 Phosphopentomutase Q5HJM9|DEOB 5 0 Phosphoribosylaminoimidazole carboxylase ATPase subunit Q5HH19|PURK 3 0 Aspartate semialdehyde dehydrogenase Q5HG26|Q5HG26 2 0 Bacterioferritin comigratory protein Q5HER1|Q5HER1 1 0 50S ribosomal protein L24 Q5HDW9|RL24 2 0 Dps family protein Q5HE61|Q5HE61 3 0 Uroporphyrinogen decarboxylase Q5HEU2|DCUP 4 0 DNA polymerase I Q5HF83|Q5HF83 4 0 UPF0133 protein Q5HIJ8|Y521 2 1 Putative hemin transport system permease protein hrtB RANDOM_HRTB R 1 0 Glutamate 1 semialdehyde 2,1 aminomutase 2 Q5HER0|GSA2 2 2 Phosphoribosylformylglycinamidine cyclo ligase Q5HH13|PUR5 3 1 30S ribosomal protein S12 Q5HID0|RS12 2 1 Putative septation protein spoVG Q5HIH8|SP5G 3 1 Inosine 5' monophosphate dehydrogenase Q5HIQ7|IMDH 1 3 Putative uncharacterized protein Q5HFG6|Q5HFG6 2 2 Pyruvate carboxylase Q5HGX0|Q5HGX0 12 5 Putative uncharacterized protein Q5HFX9|Q5HFX9 1 1 S adenosylmethionine synthase Q5HEY9|METK 2 1 Peptidase, M20/M25/M40 family Q5HJR7|Q5HJR7 2 1 Iron compound ABC transporter, iron compound binding protein Q5HDS3|Q5HDS3 2 1 S ribosylhomocysteine lyase Q5HE66|LUXS 1 1 Putative uncharacterized protein Q5HI16|Q5HI16 1 0 DNA topoisomerase 4 subunit B Q5HG65|PARE 2 0 30S ribosomal protein S21 Q5HFI5|RS21 0 1 Peptide deformylase Q5HGZ3|DEF 0 2 Acetyl coenzyme A carboxylase carboxyl transferase subunit alpha Q5HF74|ACCA 3 0 UDP N acetylmuramoylalanine -D glutamate ligase Q5HGP8|MURD 1 0 Trans sulfuration enzyme family protein Q5HIL6|Q5HIL6 2 0 Dehydrosqualene synthase Q5HCY8|CRTM 1 0 Serine aspartate repeat containing protein C Q5HIB4|SDRC 4 0 Transcription antitermination protein nusG Q5HID9|NUSG 3 0 Bifunctional protein glmU Q5HIH6|GLMU 3 0 GTP binding protein TypA Q5HGX5|Q5HGX5 3 0 Sun protein Q5HGL5 2 0 Aspartyl tRNA synthetase Q5HFD3|SYD 2 0 Lipoate protein ligase A family protein Q5HH58|Q5HH58 1 0 Coenzyme A disulfide reductase Q5HHB4|CDR 3 0 Cysteine synthase Q5HIG2|CYSK 0 3 Acetyl CoA carboxylase, biotin carboxylase Q5HFP5|Q5HFP5 2 0 N acetylglucosamine 6 phosphate deacetylase Q5HHW9|Q5HHW 9 4 0 Probable branched chain amino acid aminotransferase Q5HIC1|ILVE 2 0 Probable malate:quinone oxidoreductase 1 Q5HDJ0|MQO1 1 2 LexA repressor Q9L4P1|LEXA 2 1 FtsK/SpoIIIE family protein Q5HF33|Q5HF33 1 1 Menaquinone biosynthesis methyltransferase ubiE Q5HFV2|UBIE 1 1 Adenylate kinase Q5HDX9|KAD 1 0 UPF0403 protein Q5HFZ5|Y1464 1 0 Conserved virulence factor B Q5HG29|CVFB 1 0 UTP -glucose 1 phosphate uridylyltransferase Q5HD54|GTAB 2 0 Urease accessory protein ureE Q5HDR7|UREE 2 0 UDP N acetylenolpyruvoylglucosamine reductase Q5HHT2|MURB 1 0

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136 Appendix 3. (Continued) Ribonuclease J 2 Q5HGF6|RNJ2 1 0 Aerobic glycerol 3 phosphate dehydrogenase Q5HGD1|GLPD 1 0 Glutamate synthase, small subunit Q5HIK4|Q5HIK4 1 0 Alanyl tRNA synthetase Q5HFE4|SYA 3 0 Aminotransferase, putative Q5HEI3|Q5HEI3 3 0 Putative uncharacterized protein Q5HEU6|Q5HEU6 3 0 Hydrolase, haloacid dehalogenase like family Q5HHB3|Q5HHB3 3 0 Acid phosphatase5' nucleotidase, lipoprotein e(P4) family Q5HJ61|Q5HJ61 3 0 DNA ligase Q5HEL8|DNLJ 2 0 Amino acid ABC transporter, ATP binding protein Q5HDA5|Q5HDA5 1 0 Ribonucleoside diphosphate reductase 2, beta subunit Q5HHT9|Q5HHT9 2 0 Probable ctpA like serine protease Q5HG01|CTPAL 1 0 Capsular polysaccharide biosynthesis protein Cap5F Q5HJL6|Q5HJL6 2 0 Aminomethyltransferase Q5HFM2|GCST 1 0 Lipase 1 Q5HCM7|LIP1 2 0 Methionine aminopeptidase Q5HEN6|AMPM 2 0 Thymidylate synthase Q5HFZ6|TYSY 2 0 Serine protease HtrA, putative Q5HF46|Q5HF46 2 0 Putative uncharacterized protein Q5HIE4|Q5HIE4 2 0 Putative uncharacterized protein Q5HI54|Q5HI54 1 0 Peptidase, M20/M25/M40 family Q5HFR1|Q5HFR1 1 0 Leucyl tRNA synthetase Q5HF16|SYL 1 0 UDP N acetylmuramoyl tripeptide -D alanyl D alanine ligase Q5HEB8|Q5HEB8 1 0 Putative uncharacterized protein Q5HE21|Q5HE21 0 1 Glutamyl tRNA synthetase Q5HIE7|SYE 0 1 Cobyric acid synthase, putative Q5HEN2|Q5HEN2 0 1 Putative phosphotransferase Q5HFJ7|Y1620 1 0 ATP dependent protease subunit HslV Q5HGH9|HSLV 1 0 Glycerol kinase Q5HGD2|GLPK 1 0 77 kDa membrane protein Q5HEI2|RANDOM _OMP7 R 1 0 Protein esaA Q5HJ90|RANDOM _ESAA R 1 0 ATP dependent Clp protease proteolytic subunit Q5HHQ0|CLPP 1 0

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137 Appendix 4. Cytoplasmic proteins identified after MudPit analysis of SH1000 during stationary phase from 2 biological replicates Identified Proteins (366) Accession Number Sample 1 Sample 2 Elongation factor Tu Q5HIC7|EFTU 246 521 Antibacterial protein (Phenol soluble modulin) Q5HGQ7|Q5HGQ7 115 215 Probable transglycosylase isaA Q5HCY1|ISAA 140 124 Pyruvate kinase Q5HF76|KPYK 115 58 Uracil phosphoribosyltransferase Q5HE88|UPP 66 66 Elongation factor G Q5HIC8|EFG 81 67 Dihydrolipoyl dehydrogenase Q5HGY8|DLDH 78 48 Bifunctional autolysin Q5HH31|ATL 92 31 Cell division protein ftsZ Q5HGP5|FTSZ 68 43 Delta hemolysin Q5HEG6|HLD 98 18 Pyruvate dehydrogenase E1 component subunit beta Q5HGZ0|ODPB 36 59 Antibacterial protein (Phenol soluble modulin) Q5HGQ8|Q5HGQ8 40 31 50S ribosomal protein L1 Q5HID7|RL1 59 30 50S ribosomal protein L30 Q5HDX6|RL30 45 31 Cysteine synthase Q5HIG2|CYSK 43 39 Inosine 5' monophosphate dehydrogenase Q5HIQ7|IMDH 47 28 Pyruvate dehydrogenase E1 component subunit alpha Q5HGZ1|ODPA 44 26 50S ribosomal protein L6 Q5HDX3|RL6 42 24 Pyridoxal biosynthesis lyase pdxS Q5HIF5|PDXS 36 31 60 kDa chaperonin Q5HEH2|CH60 46 15 DNA binding protein HU Q5HFV0|DBH 37 17 Bifunctional purine biosynthesis protein purH Q5HH11|PUR9 35 21 Staphylococcal secretory antigen ssaA2 Q5HDQ9|SSAA2 32 26 30S ribosomal protein S5 Q5HDX5|RS5 32 23 Bifunctional protein folD Q5HH21|FOLD 40 9 Enolase Q5HHP1|ENO 36 15 Ornithine aminotransferase 2 Q5HHC8|OAT2 25 26 Phenol soluble modulin alpha 1 peptide P0C7Y4|PSMA1 22 11 Putative universal stress protein Q5HF64|Y1759 17 24 50S ribosomal protein L27 Q5HFB8|RL27 27 12 Dihydrolipoyllysine residue acetyltransferase component of pyruvate dehydrogenase complex Q5HGY9|ODP2 19 22 Chaperone protein hchA Q5HIC4|HCHA 25 18 Alkaline shock protein 23 Q5HE23|ASP23 29 12 Alkyl hydroperoxide reductase subunit C Q5HIR5|AHPC 23 18 30S ribosomal protein S1 Q5HFU7|RS1 34 9 50S ribosomal protein L5 Q5HDX0|RL5 25 17 Carbamoyl phosphate synthase large chain Q5HGM9|CARB 19 21 30S ribosomal protein S8 Q5HDX2|RS8 7 17 Phosphoribosylformylglycinamidine synthase 2 Q5HH15|PURL 24 14 Phosphate acetyltransferase Q5HI88|PTA 20 20 Fructose bisphosphate aldolase class 1 Q5HCU6|ALF1 23 9 Ribose phosphate pyrophosphokinase Q5HIH5|KPRS 20 16 Probable malate:quinone oxidoreductase 2 Q5HCU5|MQO2 20 18 UPF0365 protein Q5HFI7|Y1630 17 13

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138 Appendix 4. (Continued) N acetylmuramoyl L alanine amidase domain protein Q5HCQ3|Q5HCQ3 14 18 3 oxoacyl [acyl carrier protein] reductase Q5HGK2|FABG 23 14 50S ribosomal protein L13 Q5HDZ0|RL13 20 16 50S ribosomal protein L21 Q5HFB6|RL21 23 7 Glycyl tRNA synthetase Q5HFJ5|SYG 19 11 D alanine -D alanine ligase Q5HEB7|DDL 20 10 Chaperone protein dnaK Q5HFI0|DNAK 23 6 ATP dependent Clp protease ATP binding subunit clpL Q5HD02|CLPL 15 12 Acetate kinase Q5HF63|ACKA 17 13 sp|Q5HG77|TKT_STAAC Q5HG77|TKT 20 11 Oxidoreductase, short chain dehydrogenase/reductase family Q5HDM9|Q5HDM9 14 15 Translation initiation factor IF 2 Q5HGG2|IF2 17 11 Ribonucleoside diphosphate reductase Q5HHU0|Q5HHU0 17 8 ATP synthase subunit beta Q5HE97|ATPB 13 11 DNA directed RNA polymerase subunit beta Q5HID3|RPOB 12 13 UDP N acetylglucosamine 1 carboxyvinyltransferase 1 Q5HEA0|MURA1 18 7 Succinyl CoA ligase [ADP forming] subunit beta Q5HGI7|SUCC 9 15 ATP synthase subunit alpha Q5HE95|ATPA 12 17 Dihydrolipoyllysine residue succinyltransferase component of 2 oxoglutarate dehydrogenase complex Q5HG07|ODO2 14 14 Oxidoreductase, putative Q5HIW6|Q5HIW6 18 8 DNA directed RNA polymerase subunit beta' Q5HID2|RPOC 13 7 1 pyrroline 5 carboxylate dehydrogenase Q5HCZ6|ROCA 8 14 Imidazolonepropionase Q5HDM7|HUTI 14 8 Cell cycle protein gpsB Q5HFX8|GPSB 19 6 N utilization substance protein A, putative Q5HGG5|Q5HGG5 13 10 UvrABC system protein A UVRA 5 2 Putative 2 hydroxyacid dehydrogenase Q5HDQ4|Y2296 16 8 Uncharacterized protein Q5HHB6|Y973 13 11 GMP synthase [glutamine hydrolyzing] GUAA 14 2 Succinyl CoA ligase [ADP forming] subunit alpha Q5HGI6|SUCD 14 7 NADH dehydrogenase like protein Q5HHE4|Y944 15 9 Uncharacterized protein Q5HGK7|Y1240 13 10 Pyruvate carboxylase Q5HGX0 9 2 Pyrimidine nucleoside phosphorylase Q5HE64|PDP 9 9 Uncharacterized protein Q5HEP9|Y1933 10 7 Probable catabolite control protein A Q5HF38|CCPA 14 8 GTPase obg Q5HFB9|OBG 8 6 Putative uncharacterized protein Q5HIT1|Q5HIT1 10 11 ATP dependent Clp protease ATP binding subunit clpC P0C281|CLPC 8 12 3 oxoacyl [acyl carrier protein] synthase 2 Q5HHA1|FABF 13 8 Alanine dehydrogenase 2 Q5HF65|DHA2 14 4 Bifunctional protein glmU Q5HIH6|GLMU 15 4 30S ribosomal protein S10 Q5HDV7|RS10 8 13 50S ribosomal protein L15 Q5HDX7|RL15 5 12 Elongation factor Ts Q5HGH4|EFTS 12 7 30S ribosomal protein S11 Q5HDY3|RS11 12 4 Glucosamine 6 phosphate isomerase, putative Q5HES0|Q5HES0 12 5 Catalase Q5HG86|CATA 13 6 FeS assembly ATPase SufC Q5HHH2|Q5HHH2 9 10 30S ribosomal protein S2 Q5HGH6|RS2 9 8 NADP dependent malic enzyme, putative Q5HF72|Q5HF72 7 10 DNA directed RNA polymerase subunit omega Q5HGM2|RPOZ 7 10 Naphthoate synthase Q5HH38|MENB 7 8 Phosphoglucosamine mutase Q5HE43|GLMM 10 5 Aspartate semialdehyde dehydrogenase Q5HG26|Q5HG26 9 4

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139 Appendix 4. (Continued) Ribosome recycling factor Q5HGH2|RRF 6 10 6 phosphogluconate dehydrogenase, decarboxylating Q5HFR2|6PGD 11 2 UPF0342 protein Q5HET0|Y1902 13 5 Dephospho CoA kinase Q5HF85|COAE 8 5 L lactate dehydrogenase 1 Q5HJD7|LDH1 5 8 Probable manganese dependent inorganic pyrophosphatase Q5HEK1|PPAC 7 5 30S ribosomal protein S7 Q5HIC9|RS7 4 9 30S ribosomal protein S19 Q5HDW2|RS19 10 6 ATP dependent protease ATPase subunit HslU Q5HGH8|HSLU 6 9 30S ribosomal protein S4 Q5HF54|RS4 10 4 Glutamyl tRNA(Gln) amidotransferase subunit A Q5HEM2|GATA 7 5 Protein recA Q5HGE6|RECA 10 3 Conserved domain protein Q5HH57|Q5HH57 9 5 Fructose bisphosphate aldolase Q5HE75|ALF2 12 2 Transcriptional regulator sarA Q5HI51|SARA 10 5 Putative uncharacterized protein Q5HG68|RANDOM _Q5HG68 R 2 0 Urocanate hydratase Q5HDM6|HUTU 8 3 Hydroxymethylglutaryl CoA reductase, degradative Q5HD05|Q5HD05 6 5 Aconitate hydratase Q5HG69|ACON 8 3 Probable thiol peroxidase Q5HF61|TPX 5 7 S adenosylmethionine synthase Q5HEY9|METK 7 5 Phenol soluble modulin alpha 4 peptide P0C821|PSMA4 8 5 Universal stress protein family Q5HF68|Q5HF68 6 2 50S ribosomal protein L14 Q5HDW8|RL14 4 4 Transaldolase Q5HEZ4|Q5HEZ4 6 3 Valyl tRNA synthetase Q5HFA8|SYV 10 3 GTP binding protein engA Q5HFU8|ENGA 6 4 Prolyl tRNA synthetase Q5HGG8|SYP 6 3 2 C methyl D erythritol 4 phosphate cytidylyltransferase 2 Q5HJC1|ISPD2 7 4 DNA polymerase III subunit beta Q5HJZ4|DPO3B 5 7 50S ribosomal protein L16 Q5HDW5|RL16 9 3 50S ribosomal protein L33 2 Q5HG85|RL332 (+1) 8 3 PTS system, IIBC components Q5HJD5|Q5HJD5 1 0 Alcohol dehydrogenase, zinc containing Q5HE18|Q5HE18 8 3 Asparaginyl tRNA synthetase Q5HFW9|SYN 4 2 6 phosphofructokinase Q5HF75|K6PF 5 5 Aspartyl/glutamyl tRNA(Asn/Gln) amidotransferase subunit B Q5HEM3|GATB 7 4 Putative aldehyde dehydrogenase Q5HE78|ALD1 4 4 Acid phosphatase5' nucleotidase, lipoprotein e(P4) family Q5HJ61|Q5HJ61 7 2 UPF0082 protein Q5HHZ9|Y727 7 2 Aminotransferase, putative Q5HEI3|Q5HEI3 7 3 Glutamine synthetase Q5HGC3|GLNA 6 0 Capsular polysaccharide biosynthesis protein Cap5B Q5HJM0|Q5HJM0 6 6 HTH type transcriptional regulator sarR Q5HDR3|SARR 9 1 Glutamate 1 semialdehyde 2,1 aminomutase 2 Q5HER0|GSA2 3 6 Polyribonucleotide nucleotidyltransferase Q5HGF7|PNP 8 3 Inositol monophosphatase family protein Q5HGX7|Q5HGX7 8 4 sp|Q5HGP8|MURD_STAAC Q5HGP8|MURD 3 2 UPF0051 protein Q5HHG8|Y918 3 5 HTH type transcriptional regulator mgrA Q5HHY2|MGRA 6 5 50S ribosomal protein L10 Q5HID6|RL10 2 6 Putative aldehyde dehydrogenase AldA Q5HJK3|ALDA 5 2 50S ribosomal protein L2 Q5HDW1|RL2 6 3 Queuine tRNA ribosyltransferase Q5HFC4|TGT 4 6

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140 Appendix 4. (Continued) Putative uncharacterized protein Q5HEL5|Q5HEL5 7 3 S1 RNA binding domain protein Q5HED8|Q5HED8 4 5 Putative dipeptidase Q5HF23|PEPVL 6 4 Putative uncharacterized protein Q5HI16|Q5HI16 6 4 Hypoxanthine guanine phosphoribosyltransferase Q5HIG5|HPRT 3 5 Penicillin binding protein 2' Q5HJW3|RANDO M_Q5HJW3 R 2 0 Deoxyribose phosphate aldolase 2 DEOC2 0 1 Putative uncharacterized protein Q5HF32|Q5HF32 8 0 Threonyl tRNA synthetase Q5HF90|SYT 8 2 ABC transporter, ATP binding protein Q5HG28|Q5HG28 4 5 30S ribosomal protein S12 RS12 4 2 50S ribosomal protein L3 Q5HDV8|RL3 4 2 Succinate dehydrogenase, iron sulfur protein Q5HGT4|Q5HGT4 2 6 Extracellular matrix binding protein ebh Q5HFY8|RANDO M_EBH R 1 0 Serine hydroxymethyltransferase Q5HE87|GLYA 4 3 Glyceraldehyde 3 phosphate dehydrogenase 1 Q5HHP5|G3P1 4 3 FtsK/SpoIIIE family protein Q5HF33|Q5HF33 5 3 Rrf2 family protein Q5HFD6|Q5HFD6 4 5 Putative uncharacterized protein Q5HHZ0|Q5HHZ0 4 6 Pyruvate oxidase Q5HD12|Q5HD12 5 2 Uncharacterized protein Q5HG10|Y1445 2 4 50S ribosomal protein L23 Q5HDW0|RL23 4 5 50S ribosomal protein L17 Q5HDY5|RL17 5 4 Formate acetyltransferase Q5HJF4|PFLB 3 7 Phosphoribosylformylglycinamidine synthase, PurS protein Q5HH17|Q5HH17 4 6 Molybdopterin molybdenumtransferase Q5HDT4|MOEA 5 3 Isocitrate dehydrogenase [NADP] Q5HF79|IDH 1 7 ATP synthase subunit delta Q5HE94|ATPD 3 4 Formate -tetrahydrofolate ligase Q5HF42|FTHS 5 1 Xanthine phosphoribosyltransferase Q5HIQ9|XPT 8 1 50S ribosomal protein L18 Q5HDX4|RL18 4 2 Hydroxymethylglutaryl CoA synthase Q5HD04|Q5HD04 4 0 ATP dependent Clp protease ATP binding subunit clpX Q5HF98|CLPX 2 5 50S ribosomal protein L4 Q5HDV9|RL4 2 2 Iron compound ABC transporter, iron compound binding protein Q5HE28|Q5HE28 3 3 30S ribosomal protein S16 Q5HGJ4|RS16 3 3 Cell division protein FtsH, putative Q5HIG4|Q5HIG4 6 3 ABC transporter, ATP binding/permease protein Q5HI25 1 0 Teichoic acids export ATP binding protein TagH TAGH 0 2 Tetrapyrrole methylase family protein Q5HII8|Q5HII8 2 2 Putative uncharacterized protein Q5HGX3|Q5HGX3 7 1 Elastin binding protein ebpS Q5HFU2|EBPS 6 1 Methylenetetrahydrofolate -tRNA (uracil 5 ) methyltransferase trmFO Q5HGI1|TRMFO 3 4 50S ribosomal protein L9 Q5HJY1|RL9 6 2 Alcohol dehydrogenase, zinc containing Q5HJC4|Q5HJC4 2 5 sp|Q5HHP4|PGK_STAAC Q5HHP4|PGK 5 2 Transcription termination factor Rho Q5HE79|Q5HE79 2 4 Uridylate kinase Q5HGH3|PYRH 4 4 CTP synthase Q5HE73|PYRG 3 2 Aerobic glycerol 3 phosphate dehydrogenase Q5HGD1|GLPD 3 2 Dihydroorotate dehydrogenase Q5HCW1|Q5HCW1 4 3 50S ribosomal protein L32 Q5HGV6|RL32 3 4 Putative pyridoxine kinase Q5HI96|PDXK 4 3

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141 Appendix 4. (Continued) Serine protease HtrA, putative Q5HF46|Q5HF46 3 4 2 oxoglutarate dehydrogenase E1 component Q5HG06|ODO1 4 2 Probable DEAD box ATP dependent RNA helicase Q5HEB9|Y2072 1 5 Extracellular matrix binding protein ebh Q5HFY8|EBH 0 1 N acetylglucosamine 6 phosphate deacetylase Q5HHW9|Q5HHW 9 3 1 Seryl tRNA synthetase Q5HJY7|SYS 5 2 Mannitol 1 phosphate 5 dehydrogenase MTLD 3 0 30S ribosomal protein S13 Q5HDY2|RS13 5 1 Putative NAD(P)H nitroreductase Q5HD30|Y2534 4 3 Low molecular weight protein tyrosine phosphatase ptpA Q5HEP3|PTPA 5 2 Acetyl coenzyme A carboxylase carboxyl transferase subunit beta Q5HF73|ACCD 4 4 GTP sensing transcriptional pleiotropic repressor codY Q5HGH7|CODY 5 2 Phosphoribosylaminoimidazole succinocarboxamide synthase Q5HH18|PUR7 3 2 Probable endonuclease 4 Q5HFK3|END4 5 0 GTP binding protein TypA Q5HGX5|Q5HGX5 6 0 Lysyl tRNA synthetase Q5HIF7|SYK 6 0 Chaperone protein clpB Q5HHB0|CLPB 1 4 Orotidine 5' phosphate decarboxylase Q5HGM8|PYRF 4 3 Alcohol dehydrogenase, iron containing Q5HJM2|Q5HJM2 0 1 Putative uncharacterized protein Q5HJK1|Q5HJK1 4 1 UDP N acetylenolpyruvoylglucosamine reductase Q5HHT2|MURB 4 2 Succinate dehydrogenase, flavoprotein subunit Q5HGT5|Q5HGT5 4 1 Putative uncharacterized protein Q5HET6 1 3 Phosphopantothenoylcysteine decarboxylase/phosphopantothenate cysteine ligase Q5HGM1|Q5HGM1 0 2 Primosomal protein DnaI Q5HF89 3 0 UPF0297 protein Q5HFE5|Y1672 6 1 Alcohol dehydrogenase Q5HI63|ADH 4 3 Lipoate protein ligase A family protein Q5HH58|Q5HH58 3 3 Membrane protein, putative Q5HDB3|RANDO M_Q5HDB3 R 1 1 Phosphoribosylaminoimidazole carboxylase ATPase subunit Q5HH19|PURK 5 0 Dihydropteroate synthase Q5HIG1|DHPS 3 0 Probable transglycosylase sceD Q5HEA4|SCED 3 0 3 hydroxyacyl CoA dehydrogenase protein Q5HJE6|Q5HJE6 3 0 sp|Q5HGG0|TRUB_STAAC Q5HGG0|TRUB 5 0 Adenylosuccinate synthetase Q5HJX8|PURA 5 0 Guanylate kinase Q5HGM3|KGUA 7 0 Putative septation protein spoVG Q5HIH8|SP5G 6 0 Pseudouridine synthase Q5HGN5|Q5HGN5 2 2 Translation initiation factor IF 3 Q5HF92|IF3 3 3 Protein nagD homolog Q5HHF6|NAGD 3 3 Ferritin Q5HEN0|FTN 2 3 Signal recognition particle protein Q5HGJ5|Q5HGJ5 2 2 Acetyltransferase, GNAT family Q5HD32|Q5HD32 2 2 Phosphoribosylglycinamide formyltransferase Q5HH12|PUR3 0 4 Aspartyl tRNA synthetase Q5HFD3|SYD 2 1 UPF0355 protein Q5HIR0|UP355 4 2 Putative uncharacterized protein Q5HE56|Q5HE56 2 1 Uroporphyrinogen decarboxylase Q5HEU2|DCUP 3 3 Probable cysteine desulfurase Q5HHH0|CSD 2 4 N acetylmuramoyl L alanine amidase sle1 Q5HIL2|SLE1 3 2 NifU domain protein Q5HHG9|Q5HHG9 3 3 Transferrin receptor Q5HHT4|Q5HHT 2 2

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142 Appendix 4. (Continued) Acetyl CoA carboxylase, biotin carboxylase Q5HFP5|Q5HFP5 4 0 Spermidine/putrescine import ATP binding protein PotA Q5HGY5|RANDO M_POTA R 0 1 NH(3) dependent NAD(+) synthetase Q5HEK9|NADE 5 0 R ibonuclease J 1 Q5HGZ5|RNJ1 5 0 33 kDa chaperonin Q5HIG3|HSLO 2 0 tRNA uridine 5 carboxymethylaminomethyl modification enzyme mnmG Q5HCI4|MNMG 5 0 D alanine aminotransferase Q5HF24|DAAA 0 4 Penicillin binding protein 2 Q5HFX3|Q5HFX3 3 0 Thioredoxin reductase Q5HHQ4|TRXB 4 0 Fumarate hydratase class II Q5HES4|FUMC 3 1 UvrABC system protein A Q5HHQ9|UVRA 4 2 Phosphoenolpyruvate protein phosphotransferase Q5HH01|PT1 2 1 Putative uncharacterized protein Q5HFY2|Q5HFY2 1 1 Probable branched chain amino acid aminotransferase Q5HIC1|ILVE 3 1 Pyruvate carboxylase Q5HGX0|Q5HGX0 9 3 Peptide deformylase Q5HGZ3|DEF 1 1 50S ribosomal protein L29 Q5HDW6|RL29 2 3 Serine protease htrA like Q5HH63|HTRAL 3 1 Arginyl tRNA synthetase Q5HI60|SYR 4 1 NADH dependent flavin oxidoreductase, Oye family Q5HHC9|Q5HHC9 4 1 Glucosamine -fructose 6 phosphate aminotransferase [isomerizing] Q5HE49|GLMS 3 1 Putative uncharacterized protein Q5HET6|Q5HET6 2 3 Phosphoribosylamine -glycine ligase Q5HH10|PUR2 2 1 Acetyl CoA synthetase, putative Q5HCU4|Q5HCU4 2 1 Putative uncharacterized protein Q5HGK8 2 2 Phosphoglycerate mutase family protein Q5HIS0|Q5HIS0 2 0 50S ribosomal protein L36 Q5HDY1|RL36 0 2 10 kDa chaperonin Q5HEH1|CH10 4 0 Glucokinase Q5HFL3|Q5HFL3 2 0 Prophage L54a, major capsid protein, putative Q5HIZ5|RANDOM _Q5HIZ5 R 1 0 UPF0637 protein Q5HGX8|Y1115 2 0 FeS assembly protein SufD Q5HHH1|Q5HHH1 3 0 Uncharacterized oxidoreductase Q5HD73|Y2488 0 4 Triosephosphate isomerase Q5HHP3|TPIS 2 0 NAD specific glutamate dehydrogenase Q5HHC7|DHE2 3 0 Acetoin(diacetyl) reductase Q5HJP2|BUTA 5 0 Enoyl (Acyl carrier protein) reductase Q5HH75|Q5HH75 1 2 Isoleucyl tRNA synthetase Q5HGN8|SYI 2 1 Putative uncharacterized protein Q5HIN0|Q5HIN0 2 1 Putative phosphotransferase Q5HFJ7|Y1620 3 1 Protein translocase subunit secA 1 Q5HHR7|SECA1 2 1 Putative uncharacterized protein Q5HE21|Q5HE21 2 1 Putative uncharacterized protein Q5HGR9|Q5HGR9 1 3 50S ribosomal protein L25 Q5HIH4|RL25 1 1 Probable tautomerase Q5HG56|Y1399 2 1 Protoporphyrinogen oxidase Q5HEU4|Q5HEU4 1 2 Thioredoxin, putative Q5HHL0|Q5HHL0 1 2 UPF0403 protein Q5HFZ5|Y1464 2 0 Phage infection protein, putative Q5HCQ4|Q5HCQ4 2 0 tr|Q5HEB8|Q5HEB8_STAAC Q5HEB8|Q5HEB8 2 0 sp|Q5HFM2|GCST_STAAC Q5HFM2|GCST 0 2 ATP dependent DNA helicase RecQ Q5HFU1|Q5HFU1 2 0 DNA translocase ftsK Q5HGF5|FTSK 2 0

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143 Appendix 4. (Continued) Threonine dehydratase catabolic Q5HFY5|THD2 1 0 Transcription repair coupling factor Q5HIH2|RANDOM _MFD R 1 0 Ribonuclease J 2 Q5HGF6|RNJ2 2 0 DNA ligase Q5HEL8|DNLJ 2 0 Glycerol phosphate lipoteichoic acid synthase Q5HHV4|LTAS 4 0 Proline dipeptidase Q5HFM9|Q5HFM9 3 0 UDP N acetylmuramate -L alanine ligase Q5HF34|MURC 4 0 Amino acid ABC transporter, ATP binding protein Q5HDA5|Q5HDA5 0 4 NAD/NADP octopine/nopaline dehydrogenase family protein Q5HDQ7|Q5HDQ7 4 0 50S ribosomal protein L19 Q5HGJ1|RL19 1 1 30S ribosomal protein S3 Q5HDW4|RS3 1 1 Uncharacterized hydrolase Q5HCW9|Y2597 1 1 S1 RNA binding domain protein Q5HED8|RANDO M_Q5HED8 R 1 1 Signal peptidase IB Q5HHB9|LEP 1 1 Dehydrosqualene desaturase Q5HCY9|CRTN 0 1 Sortase Q5HD25|Q5HD25 1 0 Glucose specific phosphotransferase enzyme IIA component Q5HFZ9|PTGA 2 0 Cobyric acid synthase, putative Q5HEN2|Q5HEN2 0 2 6,7 dimethyl 8 ribityllumazine synthase Q5HF08|RISB 2 0 Putative uncharacterized protein Q5HIQ0|Q5HIQ0 1 0 Exodeoxyribonuclease 7 large subunit Q5HFP8|RANDOM _EX7L R 0 1 Anti sigma B factor antagonist P60071|RSBV 3 0 2,3 bisphosphoglycerate dependent phosphoglycerate mutase Q5HDD9|GPMA 3 0 Delta aminolevulinic acid dehydratase Q5HFA4|HEM2 2 0 Uncharacterized protein Q5HG67|Y1387 2 0 Dihydroorotase Q5HGN1|PYRC 3 0 Thioredoxin Q5HGT9|THIO 3 0 Transcription antitermination protein nusG Q5HID9|NUSG 3 0 2 dehydropantoate 2 reductase Q5HCV2|Q5HCV2 3 0 Acetolactate synthase, catabolic Q5HDZ7|Q5HDZ7 0 2 Chorismate mutase/phospho 2 dehydro 3 deoxyheptonate aldolase Q5HF37|Q5HF37 0 3 OsmC/Ohr family protein Q5HF52|Q5HF52 2 0 Phosphomevalonate kinase Q5HI84|Q5HI84 1 0 Lipoate protein ligase A family protein Q5HFM6|Q5HFM6 2 0 L serine dehydratase, iron sulfur dependent, beta subunit Q5HD20|Q5HD20 0 1 Putative uncharacterized protein Q5HIY4|RANDOM _Q5HIY4 R 0 1 Acyl CoA dehydrogenase family protein Q5HJE5|Q5HJE5 1 0 Dehydrosqualene synthase Q5HCY8|CRTM 2 0 Elongation factor P Q5HFN0|EFP 2 0 50S ribosomal protein L11 Q5HID8|RL11 2 0 Oxidoreductase, aldo/keto reductase family Q5HFS2|Q5HFS2 1 0 PhoH family protein Q5HFI9|Q5HFI9 1 0 2 oxoisovalerate dehydrogenase, E2 component, dihydrolipoamide acetyltransferase Q5HFQ6|Q5HFQ6 1 0 Transcriptional regulator, DeoR family Q5HHX1|Q5HHX1 0 1 Protein export membrane protein SecDF Q5HFC6|Q5HFC6 1 0 IS1272, transposase Q5HJW0|RANDO M_Q5HJW0 R 1 0 ABC transporter, substrate binding protein Q5HJE1|Q5HJE1 0 1 Formate dehydrogenase, NAD dependent Q5HJJ6|Q5HJJ6 1 0 Lysostaphin resistance protein A Q5HDM2|LYRA 1 0

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144 Appendix 4. (Continued) Peptidase, M20/M25/M40 family Q5HFR1|Q5HFR1 1 0 Putative 8 amino 7 oxononanoate synthase/2 amino 3 ketobutyrate coenzyme A ligase Q5HIC5|BIKB 0 1 Aminoacyltransferase femA FEMA 0 1 ABC transporter, ATP binding protein Q5HI35|Q5HI35 1 0 Pur operon repressor Q5HII0|Q5HII0 1 0 Ornithine cyclodeaminase, putative Q5HJQ2|Q5HJQ2 1 0

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145 Appendix 5. Secreted proteins identified after MudPit analysis of SH1000 during post exponential phase from 2 biological replicates Identified Proteins (38) Accession Number Sample 1 Sample 2 Probable transglycosylase isaA Q5HCY1|ISAA 234 186 Bifunctional autolysin Q5HH31|ATL 42 31 Staphylococcal secretory antigen ssaA2 Q5HDQ9|SSAA2 46 39 Staphopain A Q5HEL3|SSPP 10 6 Enolase Q5HHP1|ENO 8 7 Surface protein, putative Q5HDZ9|Q5HDZ9 8 2 Probable transglycosylase sceD Q5HEA4|SCED 5 3 Glycerol phosphate lipoteichoic acid synthase Q5HHV4|LTAS 5 6 Glyceraldehyde 3 phosphate dehydrogenase 1 Q5HHP5|G3P1 7 5 Lipase 1 Q5HCM7|LIP1 5 0 N acetylmuramoyl L alanine amidase domain protein Q5HCQ3|Q5HCQ3 4 2 Lipase 2 Q5HJ48|LIP2 4 4 50S ribosomal protein L17 Q5HDY5|RL17 3 2 Hyaluronate lyase Q5HE02|Q5HE02 0 2 Putative uncharacterized protein Q5HI54|Q5HI54 3 0 30S ribosomal protein S18 Q5HIS7|RS18 3 4 50S ribosomal protein L28 Q5HGK9|RL28 5 5 SdrH protein, putative Q5HEG9|RANDO M_Q5HEG9 R 0 1 6 phosphogluconate dehydrogenase, decarboxylating Q5HFR2|6PGD 3 2 Penicillin binding protein 3 Q5HFK8|Q5HFK8 5 3 Elastin binding protein ebpS Q5HFU2|EBPS 0 1 UvrABC system protein A Q5HHQ9|RANDO M_UVRA R 1 0 Gamma hemolysin component B Q5HDD3|HLGB 1 0 50S ribosomal protein L22 Q5HDW3|RL22 3 3 Helicase, putative Q5HD63|RANDO M_Q5HD63 R 1 0 Penicillin binding protein 1 Q5HGQ0|RANDO M_Q5HGQ0 R 0 1 tRNA modification GTPase mnmE Q5HCI3|RANDOM _MNME R 0 1 Type 1 restriction enzyme R protein Q5HJH8|RANDOM _HSDR R 1 0 Pyruvate dehydrogenase E1 component subunit beta Q5HGZ0|ODPB 2 0 N acetylmuramoyl L alanine amidase sle1 Q5HIL2|SLE1 2 1 Putative uncharacterized protein Q5HI78|Q5HI78 0 1 Leukotoxin LukE Q5HEU9|RANDO M_Q5HEU9 R 1 0 Siderophore biosynthesis protein, IucC family Q5HJQ1|RANDOM _Q5HJQ1 R 0 2 Phosphonate ABC transporter, phosphonate binding protein Q5HJM5|RANDO M_Q5HJM5 R 0 1 Cell division protein sepF Q5HGP2|SEPF 1 1 Bifunctional protein folD Q5HH21|FOLD 2 0 Staphyloxanthin biosynthesis protein, putative Q5HDQ5|Q5HDQ5 0 1

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146 Appendix 6. Secreted proteins identified after MudPit analysis of SH1000 during stationary phase from 2 biological replicates Identified Proteins (346) Accession Number Sample 1 Sample 2 Lipase 1 Q5HCM7|LIP1 608 361 Bifunctional autolysin Q5HH31|ATL 269 229 Probable transglycosylase isaA Q5HCY1|ISAA 527 143 Surface protein, putative Q5HDZ9|Q5HDZ9 148 114 Putative uncharacterized protein Q5HI54|Q5HI54 174 119 Enolase Q5HHP1|ENO 70 99 Alpha hemolysin Q5HGS1|Q5HGS1 85 82 Lipase 2 Q5HJ48|LIP2 105 85 Glyceraldehyde 3 phosphate dehydrogenase 1 Q5HHP5|G3P1 93 72 Glycerol phosphate lipoteichoic acid synthase Q5HHV4|LTAS 74 46 Glutamine synthetase Q5HGC3|GLNA 12 58 LysM domain protein Q5HI03|Q5HI03 56 41 N acetylmuramoyl L alanine amidase domain protein Q5HCQ3|Q5HCQ3 36 40 Inosine 5' monophosphate dehydrogenase Q5HIQ7|IMDH 33 35 Dihydrolipoyl dehydrogenase Q5HGY8|DLDH 30 25 Phospholipase C Q5HEI1|PHLC 7 29 Formate -tetrahydrofolate ligase Q5HF42|FTHS 28 23 Staphopain A Q5HEL3|SSPP 42 15 N acetylmuramoyl L alanine amidase sle1 Q5HIL2|SLE1 23 31 Gamma hemolysin component B Q5HDD3|HLGB 33 10 Fructose bisphosphate aldolase Q5HE75|ALF2 16 18 Alkyl hydroperoxide reductase subunit C Q5HIR5|AHPC 15 26 Staphylococcal secretory antigen ssaA2 Q5HDQ9|SSAA2 47 12 Phosphate acetyltransferase Q5HI88|PTA 16 32 6 phosphogluconate dehydrogenase, decarboxylating Q5HFR2|6PGD 32 16 Probable transglycosylase sceD Q5HEA4|SCED 19 31 Staphopain B Q5HH36|SSPB 27 12 Catalase Q5HG86|CATA 30 15 Clumping factor A Q5HHM8|CLFA 33 8 Glutamyl tRNA synthetase Q5HIE7|SYE 19 16 Delta hemolysin Q5HEG6|HLD 6 24 Succinyl CoA ligase [ADP forming] subunit alpha Q5HGI6|SUCD 4 26 Transcriptional regulator, putative Q5HH28|Q5HH28 5 4 Penicillin binding protein 3 Q5HFK8|Q5HFK8 30 4 Alkaline shock protein 23 Q5HE23|ASP23 13 30 Succinyl CoA ligase [ADP forming] subunit beta Q5HGI7|SUCC 1 12 DNA binding protein HU Q5HFV0|DBH 4 25 ABC transporter, substrate binding protein Q5HI37|Q5HI37 3 24 2,3 bisphosphoglycerate independent phosphoglycerate mutase Q5HHP2|GPMI 28 10 Pyruvate carboxylase Q5HGX0 1 23 3 oxoacyl [acyl carrier protein] synthase 2 Q5HHA1|FABF 24 13 50S ribosomal protein L9 Q5HJY1|RL9 2 5 ATP synthase subunit beta Q5HE97|ATPB 3 29 GTP sensing transcriptional pleiotropic repressor codY Q5HGH7|CODY 9 15 Foldase protein prsA Q5HET4|PRSA 0 20

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147 Appendix 6. (Continued) ATP synthase subunit alpha Q5HE95|ATPA 8 17 Seryl tRNA synthetase Q5HJY7|SYS 11 14 Antibacterial protein (Phenol soluble modulin) Q5HGQ7|Q5HGQ7 7 10 1 pyrroline 5 carboxylate dehydrogenase Q5HCZ6|ROCA 1 29 Elastin binding protein ebpS Q5HFU2|EBPS 12 3 Pyruvate kinase Q5HF76|KPYK 2 19 Staphyloxanthin biosynthesis protein, putative Q5HDQ5|Q5HDQ5 15 15 Phosphoenolpyruvate protein phosphotransferase Q5HH01|PT1 13 10 Glycerophosphoryl diester phosphodiesterase GlpQ, putative Q5HHC6|Q5HHC6 3 16 3 oxoacyl [acyl carrier protein] reductase Q5HGK2|FABG 13 6 DNA directed RNA polymerase subunit beta Q5HID3|RPOB 3 7 Glycyl glycine endopeptidase lytM Q5HJ99|LYTM 12 12 Putative uncharacterized protein Q5HIS3|Q5HIS3 7 15 Serine aspartate repeat containing protein C Q5HIB4|SDRC 14 8 Elongation factor Ts Q5HGH4|EFTS 4 11 GMP synthase [glutamine hydrolyzing] GUAA 9 9 Putative uncharacterized protein Q5HHW2|Q5HHW 2 5 2 Transketolase Q5HG77|TKT 4 14 Purine nucleoside phosphorylase Q5HE62|Q5HE62 10 2 Primosomal protein N` Q5HGM0|RANDO M_Q5HGM0 R 2 0 Chaperone protein dnaK Q5HFI0|DNAK 2 11 Triosephosphate isomerase Q5HHP3|TPIS 8 5 2,3 bisphosphoglycerate dependent phosphoglycerate mutase Q5HDD9|GPMA 1 10 Gamma hemolysin component C Q5HDD4|HLGC 2 8 50S ribosomal protein L7/L12 Q5HID5|RL7 0 1 Cell wall surface anchor family protein Q5HJN4|Q5HJN4 9 13 SdrH protein, putative Q5HEG9|Q5HEG9 19 0 Glucose 6 phosphate isomerase Q5HHC2|G6PI 8 13 L lactate dehydrogenase 1 Q5HJD7|LDH1 6 11 Phosphoribosylaminoimidazole carboxylase, catalytic subunit Q5HH20|Q5HH20 12 6 Surface protein, putative Q5HHA4|Q5HHA4 8 5 Elongation factor Tu Q5HIC7|EFTU 6 11 Chaperone protein hchA Q5HIC4|HCHA 10 10 Urocanate hydratase Q5HDM6|HUTU 16 0 Phosphoglycerate kinase Q5HHP4|PGK 0 20 Gamma hemolysin component A Q5HDD6|HLGA 8 8 Thioredoxin reductase Q5HHQ4|TRXB 4 9 Hydroxymethylglutaryl CoA synthase Q5HD04|Q5HD04 6 3 Transaldolase Q5HEZ4|Q5HEZ4 3 9 DNA directed RNA polymerase subunit beta' Q5HID2|RPOC 5 11 30S ribosomal protein S12 RS12 14 2 Fructose bisphosphate aldolase class 1 Q5HCU6|ALF1 6 10 Uncharacterized lipoprotein Q5HDI7|Y2365 3 9 50S ribosomal protein L27 Q5HFB8|RL27 7 5 Trigger factor Q5HF97|TIG 5 0 Deoxyribose phosphate aldolase 2 DEOC2 0 3 50S ribosomal protein L11 Q5HID8|RL11 5 2 Acetoin(diacetyl) reductase Q5HJP2|BUTA 5 6 6 phosphofructokinase Q5HF75|K6PF 3 8 30S ribosomal protein S16 Q5HGJ4|RS16 1 9 50S ribosomal protein L21 Q5HFB6|RL21 10 8 Leucyl tRNA synthetase Q5HF16|SYL 4 4 3 oxoacyl [acyl carrier protein] synthase 3 Q5HHA2|FABH 1 6 3 hydroxyacyl CoA dehydrogenase protein Q5HJE6|Q5HJE6 3 1

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148 Appendix 6. (Continued) Putative aldehyde dehydrogenase AldA Q5HJK3|ALDA 1 8 Acetate kinase Q5HF63|ACKA 2 14 NA polymerase III subunit beta Q5HJZ4|DPO3B 13 1 Phenylalanyl tRNA synthetase beta chain Q5HGU5|SYFB 4 4 50S ribosomal protein L30 Q5HDX6|RL30 1 8 50S ribosomal protein L3 Q5HDV8|RL3 0 7 LPXTG cell wall surface anchor family protein Q5HCQ1|Q5HCQ1 2 9 Dihydrolipoyllysine residue acetyltransferase component of pyruvate dehydrogenase complex Q5HGY9|ODP2 0 9 Methionyl tRNA synthetase Q5HII6|SYM 0 9 Elongation factor G Q5HIC8|EFG 2 5 Arginase P60086|ARGI 14 0 33 kDa chaperonin Q5HIG3|HSLO 7 1 Amino acid ABC transporter, amino acid binding protein Q5HDE2|Q5HDE2 13 2 Serine hydroxymethyltransferase Q5HE87|GLYA 1 10 Phosphate acyltransferase Q5HGK4|PLSX 9 2 Phosphoenolpyruvate carboxykinase [ATP] Q5HEY8|PCKA 0 14 50S ribosomal protein L17 Q5HDY5|RL17 3 2 Antibacterial protein (Phenol soluble modulin) Q5HGQ8|Q5HGQ8 2 7 50S ribosomal protein L14 RL14 7 0 Uncharacterized hydrolase Q5HCW9|Y2597 8 6 Putative uncharacterized protein Q5HIY1 3 0 50S ribosomal protein L22 Q5HDW3|RL22 5 3 Phenol soluble modulin alpha 1 peptide P0C7Y4|PSMA1 3 7 S adenosylmethionine synthase Q5HEY9|METK 9 0 Pyruvate dehydrogenase E1 component subunit alpha Q5HGZ1|ODPA 0 13 Cell wall surface anchor family protein Q5HD57|Q5HD57 10 5 NAD specific glutamate dehydrogenase Q5HHC7|DHE2 3 8 Superoxide dismutase [Mn/Fe] 1 Q5HFK7|SODM1 4 10 Alanine dehydrogenase 2 Q5HF65|DHA2 1 9 Alcohol dehydrogenase Q5HI63|ADH 1 7 50S ribosomal protein L25 Q5HIH4|RL25 1 3 Glucose specific phosphotransferase enzyme IIA component Q5HFZ9|PTGA 6 8 30S ribosomal protein S2 Q5HGH6|RS2 4 4 Mannitol 1 phosphate 5 dehydrogenase MTLD 0 2 Probable thiol peroxidase Q5HF61|TPX 3 8 Putative septation protein spoVG Q5HIH8|SP5G 1 6 Uncharacterized protein Q5HEP9|Y1933 6 3 Glutamyl endopeptidase Q5HH35|SSPA 0 8 Succinate dehydrogenase, iron sulfur protein Q5HGT4|Q5HGT4 6 6 Diaminopimelate decarboxylase Q5HG20|Q5HG20 1 0 Isoleucyl tRNA synthetase Q5HGN8|SYI 0 4 Immunoglobulin G binding protein A Q5HJQ8|Q5HJQ8 1 10 Fumarate hydratase class II Q5HES4|FUMC 4 1 Adenylate kinase Q5HDX9|KAD 3 6 Naphthoate synthase Q5HH38|MENB 4 2 50S ribosomal protein L15 Q5HDX7|RL15 2 7 30S ribosomal protein S8 Q5HDX2|RS8 3 4 Glucosamine 6 phosphate isomerase, putative Q5HES0|Q5HES0 1 2 N acetylglucosamine 6 phosphate deacetylase Q5HHW9|Q5HHW 9 5 2 60 kDa chaperonin Q5HEH2|CH60 0 7 Polyribonucleotide nucleotidyltransferase Q5HGF7|PNP 0 10 Putative 2 hydroxyacid dehydrogenase Q5HDQ4|Y2296 2 3 2 oxoglutarate dehydrogenase E1 component Q5HG06|ODO1 2 2 Formate acetyltransferase Q5HJF4|PFLB 3 2

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149 Appendix 6. (Continued) Putative uncharacterized protein Q5HEP7 1 0 Putative uncharacterized protein Q5HF32|Q5HF32 2 1 Chitinase related protein Q5HH22|Q5HH22 3 1 AcrB/AcrD/AcrF family protein Q5HDU7|RANDO M_Q5HDU7 R 2 0 Dihydroorotase Q5HGN1|PYRC 7 4 Putative uncharacterized protein Q5HIA8|Q5HIA8 0 3 Uncharacterized leukocidin like protein 1 P21224|LUKL1 3 2 50S ribosomal protein L6 Q5HDX3|RL6 2 5 Bifunctional protein folD Q5HH21|FOLD 2 5 CTP synthase Q5HE73|PYRG 0 4 Glutathione peroxidase homolog BsaA Q5HGC7|BSAA 0 7 Alkyl hydroperoxide reductase subunit F Q5HIR6|AHPF 3 4 Universal stress protein family Q5HF68|Q5HF68 3 3 50S ribosomal protein L13 Q5HDZ0|RL13 7 1 UPF0312 protein Q5HCL0|Y2711 5 5 Cell division protein ftsZ Q5HGP5|FTSZ 7 1 Pyruvate dehydrogenase E1 component subunit beta Q5HGZ0|ODPB 0 7 Threonyl tRNA synthetase Q5HF90|SYT 0 8 Staphylococcus aureus sex pheromone Q5HEL9|Q5HEL9 1 3 Ribonuclease J 1 Q5HGZ5|RNJ1 1 6 Valyl tRNA synthetase Q5HFA8|SYV 3 5 Adenylosuccinate synthetase Q5HJX8|PURA 1 4 Immunodominant staphylococcal antigen B ISAB 0 1 Iron compound ABC transporter, iron compound binding protein Q5HDS3|Q5HDS3 3 3 Chorismate mutase/phospho 2 dehydro 3 deoxyheptonate aldolase Q5HF37|Q5HF37 1 4 Phosphopentomutase Q5HJM9|DEOB 0 6 Fibrinogen binding protein Q5HGS6|FIB 5 0 Serine protein kinase rsbW Q5HED6|RSBW 2 7 FtsK/SpoIIIE family protein Q5HF33|Q5HF33 0 1 Penicillin binding protein 1 Q5HGQ0|Q5HGQ0 0 3 Putative uncharacterized protein Q5HFP6|Q5HFP6 2 3 Rod shape determining protein MreC Q5HFB4|Q5HFB4 5 3 Putative uncharacterized protein Q5HG81|Q5HG81 2 3 Probable acetyl CoA acyltransferase Q5HIU0|THLA 0 1 Cysteine synthase Q5HIG2|CYSK 2 5 S ribosylhomocysteine lyase Q5HE66|LUXS 2 2 Competence protein ComEC/Rec2, putative Q5HFH3|Q5HFH3 1 0 Phosphoribosylformylglycinamidine cyclo ligase Q5HH13|PUR5 0 5 Putative uncharacterized protein Q5HGE4|Q5HGE4 5 0 10 kDa chaperonin Q5HEH1|CH10 3 1 Putative dipeptidase Q5HF23|PEPVL 1 4 Ornithine aminotransferase 2 Q5HHC8|OAT2 0 6 Putative uncharacterized protein Q5HET6 0 3 2 C methyl D erythritol 4 phosphate cytidylyltransferase 2 Q5HJC1|ISPD2 5 2 Thioredoxin, putative Q5HHK5|Q5HHK5 0 4 UPF0133 protein Y521 0 2 Putative uncharacterized protein Q5HDH4|Q5HDH4 0 5 Elongation factor P Q5HFN0|EFP 2 7 Probable branched chain amino acid aminotransferase Q5HIC1|ILVE 5 2 HIT family protein Q5HET7|Q5HET7 1 1 Aminopeptidase PepS Q5HEP5|Q5HEP5 0 5 Respiratory nitrate reductase, alpha subunit Q5HDF9|Q5HDF9 1 0 Adenylosuccinate lyase Q5HEL4|PUR8 0 5 Uracil phosphoribosyltransferase Q5HE88|UPP 0 5 Alcohol dehydrogenase, zinc containing Q5HE18|Q5HE18 2 2

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150 Appendix 6. (Continued) Ribosomal large subunit pseudouridine synthase, RluD subfamily Q5HES5|Q5HES5 0 1 Cell division protein FtsY, putative Q5HGJ7|Q5HGJ7 0 3 30S ribosomal protein S1 Q5HFU7|RS1 0 4 Putative uncharacterized protein Q5HCV8|Q5HCV8 1 5 Ribosome recycling factor Q5HGH2|RRF 3 2 Phosphoglucomutase Q5HD61|PGCA 4 1 ATP dependent Clp protease ATP binding subunit clpL Q5HD02|CLPL 0 1 Putative acetyl CoA C acetyltransferase vraB Q5HIA0|VRAB 1 0 Thioredoxin, putative Q5HHL0|Q5HHL0 0 6 Protein essC Q5HJ86|ESSC 0 1 Uncharacterized protein Q5HHB6|Y973 2 3 30S ribosomal protein S9 Q5HDZ1|RS9 1 2 Putative uncharacterized protein Q5HFN8|RANDO M_Q5HFN8 R 0 1 Xanthine permease Q5HIQ8 0 1 5' nucleotidase family protein RANDOM_Q5HH6 1 R 0 1 Phage infection protein, putative Q5HCQ4|Q5HCQ4 1 3 50S ribosomal protein L24 Q5HDW9|RL24 2 4 Putative uncharacterized protein Q5HGA4|Q5HGA4 1 1 Transcriptional regulator, putative Q5HDP8|Q5HDP8 0 2 1 phosphatidylinositol phosphodiesterase Q5HJS4|Q5HJS4 0 4 Iron regulated surface determinant protein A Q5HGV4|ISDA 0 1 Flavohemoprotein, putative Q5HJD8|Q5HJD8 0 5 Coenzyme A disulfide reductase Q5HHB4|CDR 1 1 Putative uncharacterized protein Q5HGZ2|Q5HGZ2 2 0 Glutamyl aminopeptidase, putative Q5HG53|Q5HG53 0 2 5' nucleotidase family protein Q5HJX2|Q5HJX2 0 2 50S ribosomal protein L23 Q5HDW0|RL23 1 1 50S ribosomal protein L18 Q5HDX4|RL18 1 4 Serine protease splB Q5HEW1|SPLB 2 1 Indole 3 pyruvate decarboxylase Q5HJI5|Q5HJI5 4 1 Glucosamine -fructose 6 phosphate aminotransferase [isomerizing] Q5HE49|GLMS 4 1 50S ribosomal protein L35 Q5HF93|RL35 1 3 Sensor protein srrB Q5HFT1|SRRB 1 0 Delta aminolevulinic acid dehydratase Q5HFA4|HEM2 0 5 Iron compound ABC transporter, iron compound binding protein Q5HE28|Q5HE28 0 1 Arginyl tRNA synthetase Q5HI60|SYR 0 4 ATP dependent Clp protease ATP binding subunit clpC P0C281|CLPC 0 2 50S ribosomal protein L14 Q5HDW8|RL14 9 0 Putative peptidyl prolyl cis trans isomerase Q5HHD1|PPI1 0 4 30S ribosomal protein S7 Q5HIC9|RS7 0 2 Inosine uridine preferring nucleoside hydrolase Q5HJD4|RANDOM _Q5HJD4 R 1 0 Formimidoylglutamase Q5HDM3|HUTG 0 1 3 methyl 2 oxobutanoate hydroxymethyltransferase Q5HCV3|PANB 0 4 50S ribosomal protein L19 Q5HGJ1|RANDOM _RL19 R 2 0 Glucose 6 phosphate 1 dehydrogenase Q5HFR7|Q5HFR7 0 4 3 phosphoshikimate 1 carboxyvinyltransferase Q5HFV9|AROA 1 0 Uncharacterized protein Q5HHR8|Y815 0 4 2 oxoisovalerate dehydrogenase, E1 component, alpha subunit Q5HFQ4|Q5HFQ4 2 0 Uncharacterized N acetyltransferase Q5HGQ5|Y1189 0 4 Probable manganese dependent inorganic pyrophosphatase Q5HEK1|PPAC 1 1 Serine aspartate repeat containing protein D Q5HIB3|SDRD 0 1 Probable ctpA like serine protease Q5HG01|CTPAL 0 1

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151 Appendix 6. (Continued) Pathogenicity island protein, integrase Q5HHK1|Q5HHK1 0 1 Aminoacyltransferase femA FEMA 0 1 Serine protease splC Q5HEW2|SPLC 2 2 D lactate dehydrogenase Q5HD29|LDHD 1 2 ATP dependent Clp protease proteolytic subunit Q5HHQ0|CLPP 1 2 Single stranded DNA specific exonuclease RecJ Q5HFC7|RANDO M_Q5HFC7 R 1 0 Putative uncharacterized protein Q5HE42|Q5HE42 0 2 Ribose phosphate pyrophosphokinase Q5HIH5|KPRS 1 0 ABC transporter, ATP binding protein, putative Q5HEQ3|RANDO M_Q5HEQ3 R 1 0 Histidine ammonia lyase Q5HJY8|HUTH 0 2 Probable DNA directed RNA polymerase subunit delta Q5HE72|RPOE 0 1 Putative uncharacterized protein isdD Q5HGV2|Q5HGV2 0 1 DNA dependent DNA polymerase family X Q5HGU1|Q5HGU1 1 0 Porphobilinogen deaminase Q5HFA2|HEM3 0 2 Signal transduction protein TRAP Q5HEU0|TRAP 0 3 Putative NAD(P)H nitroreductase Q5HD30|Y2534 0 2 Putative uncharacterized protein Q5HF22|Q5HF22 0 3 Pseudouridine synthase Q5HFS9|Q5HFS9 0 3 sp|Q5HEL0|Y1973_STAAC Q5HEL0|Y1973 1 0 sp|Q5HE32|Y2163_STAAC Q5HE32|Y2163 0 2 Oligoendopeptidase F Q5HH84|Q5HH84 0 1 Lipoprotein, putative RANDOM_Q5HHT 0 R 0 2 DNA directed RNA polymerase subunit omega Q5HGM2|RPOZ 0 4 Dihydrolipoyllysine residue succinyltransferase component of 2 oxoglutarate dehydrogenase complex Q5HG07|ODO2 0 3 UPF0477 protein Q5HH71|Y1020 0 1 Putative uncharacterized protein Q5HE56|Q5HE56 0 3 Putative uncharacterized protein Q5HIR8 0 1 sp|Q5HHF6|NAGD_STAAC Q5HHF6|NAGD 1 2 Malonyl CoA acyl carrier protein transacylase Q5HGK3|FABD 1 2 Deoxyribose phosphate aldolase 1 Q5HJN0|DEOC1 0 3 Anthranilate synthase component I Q5HG52|RANDO M_Q5HG52 R 0 1 Oxidoreductase, short chain dehydrogenase/reductase family Q5HDM9|Q5HDM 9 0 2 50S ribosomal protein L33 2 Q5HG85|RL332 0 1 Metallo beta lactamase family protein Q5HJT8|Q5HJT8 0 1 Peptide chain release factor 1 Q5HE82|RF1 0 1 Glutamyl aminopeptidase Q5HF29|Q5HF29 0 1 High affinity nickel transport protein Q5HCK0|RANDO M_Q5HCK0 R 0 2 Hypoxanthine guanine phosphoribosyltransferase Q5HIG5|HPRT 1 0 Methionine import ATP binding protein MetN 2 Q5HHK4|METN2 0 1 D alanine -poly(phosphoribitol) ligase subunit 1 Q5HHF2|DLTA 2 0 Glycerol 3 phosphate acyltransferase Q5HG66|RANDO M_PLSY R 1 0 Extracellular matrix protein binding protein emp Q5HHM6|EMP 0 4 Pyruvate carboxylase Q5HGX0|Q5HGX0 0 24 Urease subunit alpha Q5HDR8|URE1 0 3 Uncharacterized lipoprotein Q5HIN1|Y486 0 3 Probable malate:quinone oxidoreductase 1 Q5HDJ0|MQO1 2 0 Putative uncharacterized protein Q5HF36|RANDOM _Q5HF36 R 1 0

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152 Appendix 6. (Continued) 5' methylthioadenosine/S adenosylhomocysteine nucleosidase Q5HFG2|MTNN 0 4 Dps family protein Q5HE61|Q5HE61 0 3 NADP dependent malic enzyme, putative Q5HF72|Q5HF72 0 4 tr|Q5HH17|Q5HH17_STAAC Q5HH17|Q5HH17 0 3 Ribonucleoside diphosphate reductase 2, beta subunit Q5HHT9|Q5HHT9 0 3 Putative formate dehydrogenase Q5HDP9|FDHL 0 1 Putative uncharacterized protein Q5HFT3|RANDOM _Q5HFT3 R 0 1 Putative uncharacterized protein Q5HJ95|RANDOM _Q5HJ95 R 0 1 Leukotoxin LukD Q5HEV0|Q5HEV0 22 0 tr|Q5HDI9|Q5HDI9_STAAC Q5HDI9|Q5HDI9 1 1 3 hexulose 6 phosphate synthase Q5HIA5|HPS 0 1 Putative uncharacterized protein Q5HF36|Q5HF36 0 1 sp|Q5HIE5|SYC_STAAC Q5HIE5|SYC 0 2 Organic hydroperoxide resistance protein like Q5HHL3|OHRL 1 0 Transcription repair coupling factor Q5HIH2|MFD 0 1 GMP synthase [glutamine hydrolyzing] Q5HIQ6|GUAA 9 9 Glycerol phosphate lipoteichoic acid synthase Q5HHV4|RANDO M_LTAS R 0 1 Dihydrolipoyl dehydrogenase Q5HFQ3|Q5HFQ3 0 2 Peptidase, M20/M25/M40 family Q5HFR1|Q5HFR1 0 3 Putative uncharacterized protein Q5HJT9|RANDOM _Q5HJT9 R 1 0 Clumping factor B Q5HCR7|CLFB 0 2 Probable glycine dehydrogenase [decarboxylating] subunit 1 Q5HFM3|GCSPA 0 1 Arsenate reductase, putative Q5HHK9|Q5HHK9 0 2 6,7 dimethyl 8 ribityllumazine synthase Q5HF08|RISB 0 1 Chaperone protein dnaJ Q5HFI1|DNAJ 0 2 Amidophosphoribosyltransferase Q5HH14|PUR1 0 2 Copper exporting P type ATPase A Q5HCZ3|COPA 0 1 UvrABC system protein A Q5HHQ9|UVRA 1 0 Mannitol specific phosphotransferase enzyme IIA component Q5HE46|PTMA 0 2 Pyrroline 5 carboxylate reductase Q5HFR9|P5CR 0 1 Pyridoxal biosynthesis lyase pdxS Q5HIF5|PDXS 0 2 Thioredoxin Q5HGT9|THIO 0 1 NADH dependent flavin oxidoreductase, Oye family Q5HHC9|Q5HHC9 0 2 sp|Q5HDW6|RL29_STAAC Q5HDW6|RL29 0 1 Transcription repair coupling factor Q5HIH2|RANDOM _MFD R 0 1 Mevalonate kinase Q5HI86|RANDOM _Q5HI86 R 0 1 Putative 8 amino 7 oxononanoate synthase/2 amino 3 ketobutyrate coenzyme A ligase Q5HIC5|BIKB 0 1 Phosphate import ATP binding protein pstB P69881|RANDOM_ PSTB R 1 0 Putative uncharacterized protein Q5HGB8|RANDO M_Q5HGB8 R 0 1

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153 Appendix 7. Changes in secreted proteins of HA MRSA USA200 compared to HA MRSA USA100 during post exponential phase from 3 biological replicates Identified Proteins (109) Accession Number USA100 # 1 USA100 # 2 USA100 # 3 USA200 # 1 USA200 #2 USA200 #3 Lipase 1 P65289|LIP1 Ref 2.8 2.6 1.6 2.6 0.1 Putative surface protein SA2285 P61598|PLS Ref 3.9 0.4 4 0.5 4.3 Bifunctional autolysin Q99V41|ATL Ref 0.5 1.6 0.6 0.3 1 Lipase 2 Q7A7P2|LIP2 Ref 0.6 0.8 1.3 1.8 3 SA0841 protein Q7A6G0|Q7A 6G0 Ref 2.3 1 2.5 0.3 1.5 Penicillin binding protein 2 prime Q7A8C6|Q7A8 C6 Ref 0.9 3.5 3 1.2 0.4 Glycerol phosphate lipoteichoic acid synthase Q7A6U1|LTA S Ref 1.2 0.2 1 1.3 1.4 Staphopain B Q7A6A7|SSPB Ref 5 2.5 2.3 4.8 0.4 Thermonuclease Q7A6P2|NUC Ref 3.8 2.7 1.6 4.7 4.6 Alpha Hemolysin Q7A632|Q7A6 32 Ref 2.5 2.5 1.5 2 3.5 Probable transglycosylase isaA P99160|ISAA Ref 2.7 1 0.6 2.2 1 SA2437 protein Q7A371|Q7A3 71 Ref 1.2 0.7 3 2 3.3 SA0620 protein Q7A6Y9|Q7A 6Y9 Ref 2.8 3.1 1.3 1.1 2.6 Glutamyl endopeptidase Q7A6A6|SSPA Ref 4.5 3 3 1.8 0.8 Probable beta lactamase Q7A4X8|Q7A 4X8 Ref No Values 6.9 Reference Missing Reference Missing 2.8 Immunoglobulin G binding protein A P99134|SPA Ref 5.9 4.9 2.6 3 1.9 Enterotoxin type C 3 P0A0L4|ENTC 3 Ref 0.5 0.8 0.1 0.3 0.9 Putative uncharacterized protein SA0359 Q7A7J8|Q7A7 J8 Ref 2.3 2.1 0.9 2.2 1.6 SA2006 protein Q7A483|Q7A4 83 Ref 2.5 1.3 1.4 2 0.4 Staphopain A P65826|SSPP Ref 1.9 1.9 0.8 2.8 1.9 Alkyl hydroperoxide reductase subunit C P99074|AHPC Ref 2 4.3 0.5 2.8 4.9 Putative uncharacterized protein SA0570 Q7A735|Q7A7 35 Ref 9.8 4.3 0.6 10.9 5.4 Elongation factor Tu P99152|EFTU Ref 1.7 0.6 0.5 2.1 2.3

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154 A ppendix 7. (Continued) Glycyl glycine endopeptidase lytM Q7A7T0|LYT M Ref 5.4 1.7 0.7 6.6 3.7 Immunoglobulin binding protein sbi Q99RL2|SBI Ref 2.4 0.3 0.1 1.4 0.1 Truncated beta hemplysin Q99QR7|Q99Q R7 Ref No Values 3.2 Reference Missing No Values 7.2 Uncharacterized leukocidin like protein 1 Q7A4L0|LUK L1 Ref 4.6 1.4 0.5 4.6 2.6 Enolase P99088|ENO Ref 1.5 0.2 0.6 1.6 0.3 Fructose bisphosphate aldolase P99075|ALF2 Ref 4.2 1.6 2.4 4 1 Probable transglycosylase sceD Q7A4F2|SCED Ref 3.9 1.6 1.7 6.3 1.3 Serine aspartate repeat containing protein D Q7A780|SDR D Ref 0.8 3.1 4.4 5 3.8 SA0587 protein Q7A719|Q7A7 19 Ref 0 0 No Values 0.3 0.1 SA0914 protein Q99V35|Q99V 35 Ref 5 3.6 0.1 4.8 4.5 60 kDa chaperonin P99083|CH60 Ref 4 0.2 0.5 3.2 0.8 Glyceraldehyde 3 phosphate dehydrogenase 1 P99136|G3P1 Ref No Values 2.1 0.6 No Values 2.6 Zinc metalloproteinase aureolysin Q7A378|Q7A3 78 Ref 0 0 No Values 1.6 2.4 Virulence factor esxA Q7A7S4|ESX A Ref 0 0 No Values 0.5 0.6 Serine protease splB Q7A4Y1|SPLB Ref 0.1 1.3 No Values 0.1 0.8 Foldase protein prsA P60748|PRSA Ref 1.3 2.4 1.4 0.9 0.6 50S ribosomal protein L7/L12 P99154|RL7 Ref No Values 4 0.7 No Values 2.8 Iron regulated surface determinant protein A Q7A655|ISDA Ref 0.8 0.3 0.9 1.1 0 DNA binding protein HU Q7A5J1|DBH Ref 8.2 3.5 1.4 9.6 5.3 Staphylokinase Q99SU7|SAK Ref 1.2 0.4 2.3 2.7 0.5 Pyruvate dehydrogenase E1 component subunit alpha Q820A6|ODP A Ref 0 0 No Values 2.6 0.4 Putative uncharacterized protein SA0663 Q7A6V1|Q7A 6V1 Ref 3.1 2.7 0.6 2.8 3.3 Chaperone protein dnaK P99110|DNAK Ref No Values 0.3 2.4 No Values 0.2 Adenylate kinase P99062|KAD Ref 6.5 1.2 1.1 5.6 1 Putative uncharacterized protein SA0908 Q7A6A3|Q7A 6A3 Ref 0.3 1.2 0.8 1.7 1

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155 Appendix 7. (Continued) 50S ribosomal protein L30 P0A0G0|RL30 Ref 0 0 No Values 1.2 1.1 Uncharacterized leukocidin like protein 2 Q99SN7|LUK L2 Ref 3.8 1.8 1.3 4.1 2.7 Dihydrolipoyl dehydrogenase P99084|DLDH Ref No Values 3.8 0.3 No Values 5.2 Phenol soluble modulin alpha 1 peptide P0C7Y7|PSM A1 Ref 1.3 2.2 0.2 3.2 3.3 Serine protease splF Q7A4Y4|SPLF Ref 0 No Values No Values 1 No Values Pyruvate dehydrogenase E1 component subunit beta P99063|ODPB Ref 7.3 8.3 2.5 7.8 10.6 50S ribosomal protein L17 Q7A469|RL17 Ref 2.7 0.2 1.2 3.3 2.2 Glutamine synthetase P99095|GLNA Ref No Values 0.4 0.7 No Values 0.5 N acetylmuramoyl L alanine amidase sle1 Q7A7E0|SLE1 Ref No Values 4.5 0.5 No Values 4.6 Alkaline shock protein 23 P99157|ASP23 Ref 1.7 0.4 0.5 1.3 0.3 Dihydrolipoyllysine residue acetyltransferase component of pyruvate dehydrogenase complex P65636|ODP2 Ref 1.8 2.5 1.6 1.7 Value Missing SA2097 protein Q7A418|Q7A4 18 Ref 0 0 No Values 1.4 0.9 Ribosome recycling factor P99130|RRF Ref No Values 0.7 0.4 No Values 0.3 Elongation factor G P68789|EFG Ref No Values 0.7 0.6 No Values 0.8 Serine protease splC Q7A4Y2|SPLC Ref 0 No Values No Values 0.2 No Values 50S ribosomal protein L11 P0A0F2|RL11 Ref 2.6 0.5 2 2.9 0.4 Thioredoxin P99122|THIO Ref No Values No Values 1.7 No Values No Values Trigger factor P99080|TIG Ref No Values 2.4 0.6 No Values 1 30S ribosomal protein S9 P66646|RS9 Ref No Values 1 2.2 No Values 0.2 50S ribosomal protein L13 Q7A473|RL13 Ref No Values 1.8 0.4 No Values 1 Phosphate acetyltransferase P99092|PTA Ref 3.4 1.4 1.7 2.8 1.2 Acyl carrier protein P0A002|ACP Ref No Values 2.7 1.9 No Values 1.5 Serine aspartate repeat containing protein E Q99W46|SDR E Ref No Values 2 0.5 No Values 2.9

PAGE 168

156 Appendix 7. (Continued) Beta lactamase Q9AC80|Q9A C80 Ref 1.1 1.2 0 1.6 1.2 Cell division protein Q7A620|Q7A6 20 Ref No Values 2.3 1.7 No Values 0.8 Fructose bisphosphate aldolase class 1 P99117|ALF1 Ref No Values 0 No Values No Values 0.3 50S ribosomal protein L14 Q7A463|RL14 Ref No Values 0.1 2.9 No Values 0 Phenol soluble modulin alpha 4 peptide P0C824|PSMA 4 Ref 0 0 No Values 0.9 0.7 Staphylococcal secretory antigen ssaA2 Q7A423|SSAA 2 Ref No Values 2.4 0.8 No Values 2.7 Glycerophosphoryl diester phosphodiesterase Q7A6H7|Q7A 6H7 Ref 0 0 No Values 1 1.7 SA2202 protein Q99RL6|Q99R L6 Ref 0 0 No Values 0.2 1 50S ribosomal protein L22 Q7A460|RL22 Ref 3.6 No Values 1 2.7 No Values Elastin binding protein ebpS Q7A5I6|EBPS Ref 0 0 No Values 0.6 0.4 Triosephosphate isomerase P99133|TPIS Ref No Values 0.3 0.6 No Values 2.5 P99156|GREA_STA AN P99156|GREA Ref No Values 3.4 1.3 No Values 4.9 Cysteine synthase P63871|CYSK Ref No Values 0.1 2.7 No Values Reference Missing Elongation factor Ts P99171|EFTS Ref No Values No Values 1.2 Reference Missing No Values 30S ribosomal protein S6 P99142|RS6 Ref No Values No Values 2 No Values No Values Leukotoxin, LukD Q99T54|Q99T 54 Ref 0 No Values No Values 0.5 No Values Inosine 5' monophosphate dehydrogenase P99106|IMDH Ref 1.8 1.2 1.1 1.5 0.9 Protein grpE P99086|GRPE Ref No Values 0.3 1 No Values 0.3 50S ribosomal protein L24 P60735|RL24 Ref No Values 0 No Values No Values 1.1 L lactate dehydrogenase 1 P65256|LDH1 Ref 1.4 No Values 3 1.1 No Values Superoxide dismutase [Mn/Fe] 1 P99098|SODM 1 Ref No Values No Values 2.2 No Values No Values Transketolase P99161|TKT Ref No Values 0 No Values No Values 0.4 30S ribosomal protein S16 P66440|RS16 Ref 1.8 No Values 0.4 2.5 No Values 10 kDa chaperonin P99104|CH10 Ref No Values 2.9 1.9 No Values 5.5 SA0295 protein Q7A7Q2|Q7A 7Q2 Ref No Values No Values 0.4 No Values No Values

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157 Appendix 7. (Continued) Glucose 6 phosphate isomerase P99078|G6PI Ref No Values No Values 0.4 No Values No Values Protein esaA Q7A7S3|ESA A Ref 0.1 No Values 0.4 0.3 No Values Methicillin resistance mecR1 protein P0A0B0|RAN DOM_MECR R Ref No Values 0 1.2 No Values 1.5 Uncharacterized protein SA1692 P0A0K1|Y169 2 Ref No Values 0 No Values No Values 1.2 Chaperone protein hchA P64313|HCHA Ref No Values No Values 0.5 No Values No Values Clumping factor A Q99VJ4|CLFA Ref No Values 0 No Values No Values 1.2 50S ribosomal protein L10 P99155|RL10 Ref No Values 2.7 0.7 No Values 3 50S ribosomal protein L18 Q7A467|RL18 Ref No Values No Values 2.5 No Values No Values Putative peptidyl prolyl cis trans isomerase Q7A6I1|PPI1 Ref No Values No Values 1.2 No Values No Values Fibrinogen binding protein P68800|FIB Ref No Values No Values 0.1 No Values No Values 1 phosphatidylinositol phosphodiesterase precurosr Q7A888|Q7A8 88 Ref 0 No Values No Values 1 No Values Virulence factor esxA ESXA Ref No Values No Values No Values No Values No Values 50S ribosomal protein L15 P0A0F6|RL15 Ref No Values No Values 0.6 No Values No Values

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158 Appendix 8. Changes in secreted proteins of CA MRSA USA300 compared to HA MRSA USA100 during post exponential phase 3 biological replicates Identified Proteins (109) Accession Number USA100 # 1 USA100 # 2 USA100 # 3 USA300 # 1 USA300 # 2 USA300 # 3 Lipase 1 P65289|LIP1 Ref 2.8 2.6 2.8 1.4 1.3 Putative surface protein SA2285 P61598|PLS Ref 3.9 0.4 3.7 2.3 4.6 Bifunctional autolysin Q99V41|ATL Ref 0.5 1.6 0.2 2.1 2.8 Lipase 2 Q7A7P2|LIP2 Ref 0.6 0.8 2 2.2 1.5 SA0841 protein Q7A6G0|Q7A6 G0 Ref 2.3 1 1.6 1.4 0.7 Penicillin binding protein 2 prime Q7A8C6|Q7A8 C6 Ref 0.9 3.5 0.8 1.5 3.3 Glycerol phosphate lipoteichoic acid synthase Q7A6U1|LTAS Ref 1.2 0.2 0.2 0.3 2 Staphopain B Q7A6A7|SSPB Ref 5 2.5 1.2 3.4 2.3 Thermonuclease Q7A6P2|NUC Ref 3.8 2.7 1.8 4.3 2.5 Alpha Hemolysin Q7A632|Q7A6 32 Ref 2.5 2.5 2.8 0.8 3.2 Probable transglycosylase isaA P99160|ISAA Ref 2.7 1 1.6 2.1 4.6 SA2437 protein Q7A371|Q7A3 71 Ref 1.2 0.7 1.7 0.5 1 SA0620 protein Q7A6Y9|Q7A6 Y9 Ref 2.8 3.1 0.4 4.4 2.8 Glutamyl endopeptidase Q7A6A6|SSPA Ref 4.5 3 0.9 2.9 2 Probable beta lactamase Q7A4X8|Q7A4 X8 Ref No Values 6.9 Reference Missing Reference Missing 6.6 Immunoglobulin G binding protein A P99134|SPA Ref 5.9 4.9 2.7 1 2.3 Enterotoxin type C 3 P0A0L4|ENTC 3 Ref 0.5 0.8 0.1 2.5 1.4 Putative uncharacterized protein SA0359 Q7A7J8|Q7A7J 8 Ref 2.3 2.1 0 3.4 3.2 SA2006 protein Q7A483|Q7A4 83 Ref 2.5 1.3 2.8 1.4 1.1 Staphopain A P65826|SSPP Ref 1.9 1.9 0.8 3.1 0.7 Alkyl hydroperoxide reductase subunit C P99074|AHPC Ref 2 4.3 1.5 5.3 6.5 Putative uncharacterized protein SA0570 Q7A735|Q7A7 35 Ref 9.8 4.3 1.2 10.4 3 Elongation factor Tu P99152|EFTU Ref 1.7 0.6 0.2 0.2 0.6 Glycyl glycine endopeptidase lytM Q7A7T0|LYT M Ref 5.4 1.7 1.2 4.4 0.7

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159 Appendix 8. (Continued) Immunoglobulin binding protein sbi Q99RL2|SBI Ref 2.4 0.3 0.3 2.3 1.1 Truncated beta hemplysin Q99QR7|Q99Q R7 Ref No Values 3.2 Reference Missing No Values 3.3 Uncharacterized leukocidin like protein 1 Q7A4L0|LUKL 1 Ref 4.6 1.4 2.3 3.3 2.4 Enolase P99088|ENO Ref 1.5 0.2 0.6 0.7 1.2 Fructose bisphosphate aldolase P99075|ALF2 Ref 4.2 1.6 2.5 5.8 0.1 Probable transglycosylase sceD Q7A4F2|SCED Ref 3.9 1.6 0.4 3.4 3.2 Serine aspartate repeat containing protein D Q7A780|SDRD Ref 0.8 3.1 3.7 4.8 6.5 SA0587 protein Q7A719|Q7A7 19 Ref 0 0 No Values 1.7 2 SA0914 protein Q99V35|Q99V 35 Ref 5 3.6 0.2 5.4 2.6 60 kDa chaperonin P99083|CH60 Ref 4 0.2 0.2 5 1.1 Glyceraldehyde 3 phosphate dehydrogenase 1 P99136|G3P1 Ref No Values 2.1 1.5 Reference Missing 1.8 Zinc metalloproteinase aureolysin Q7A378|Q7A3 78 Ref 0 0 No Values 1.6 1 Virulence factor esxA Q7A7S4|ESXA Ref 0 0 No Values 0.1 0.1 Serine protease splB Q7A4Y1|SPLB Ref 0.1 1.3 Reference Missing 1.6 0.3 Foldase protein prsA P60748|PRSA Ref 1.3 2.4 0.2 4.2 3.3 50S ribosomal protein L7/L12 P99154|RL7 Ref No Values 4 1.9 No Values 1.4 Iron regulated surface determinant protein A Q7A655|ISDA Ref 0.8 0.3 0.3 0.9 1.2 DNA binding protein HU Q7A5J1|DBH Ref 8.2 3.5 2.2 7.1 1.1 Staphylokinase Q99SU7|SAK Ref 1.2 0.4 1.2 2.9 1.2 Pyruvate dehydrogenase E1 component subunit alpha Q820A6|ODPA Ref 0 0 No Values 2.4 3 Putative uncharacterized protein SA0663 Q7A6V1|Q7A6 V1 Ref 3.1 2.7 1 1.4 2.2 Chaperone protein dnaK P99110|DNAK Ref No Values 0.3 2.7 Reference Missing 1.9 Adenylate kinase P99062|KAD Ref 6.5 1.2 2.2 4.6 1.3 Putative uncharacterized protein SA0908 Q7A6A3|Q7A6 A3 Ref 0.3 1.2 1.7 2.5 2.3

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160 Appendix 8. (Continued) 50S ribosomal protein L30 P0A0G0|RL30 Ref 0 0 No Values 3.7 2.2 Uncharacterized leukocidin like protein 2 Q99SN7|LUKL 2 Ref 3.8 1.8 1.4 2.2 0.2 Dihydrolipoyl dehydrogenase P99084|DLDH Ref No Values 3.8 2.4 Reference Missing 3.9 Phenol soluble modulin alpha 1 peptide P0C7Y7|PSMA 1 Ref 1.3 2.2 2.8 2.9 4.7 Serine protease splF Q7A4Y4|SPLF Ref 0 No Values No Values 1.3 No Values Pyruvate dehydrogenase E1 component subunit beta P99063|ODPB Ref 7.3 8.3 1.9 7 9.4 50S ribosomal protein L17 Q7A469|RL17 Ref 2.7 0.2 1 2.9 1.4 Glutamine synthetase P99095|GLNA Ref No Values 0.4 1.5 Reference Missing 2.5 N acetylmuramoyl L alanine amidase sle1 Q7A7E0|SLE1 Ref No Values 4.5 0.8 Reference Missing Value Missing Alkaline shock protein 23 P99157|ASP23 Ref 1.7 0.4 0.9 2.8 Value Missing Dihydrolipoyllysine residue acetyltransferase component of pyruvate dehydrogenase complex P65636|ODP2 Ref 1.8 2.5 2.8 4.3 2.3 SA2097 protein Q7A418|Q7A4 18 Ref 0 0 No Values 0.5 0.8 Ribosome recycling factor P99130|RRF Ref No Values 0.7 1.2 No Values 2 Elongation factor G P68789|EFG Ref No Values 0.7 1 No Values 1.6 Serine protease splC Q7A4Y2|SPLC Ref 0 No Values No Values 1.6 No Values 50S ribosomal protein L11 P0A0F2|RL11 Ref 2.6 0.5 2 1.1 2.9 Thioredoxin P99122|THIO Ref No Values No Values 0.2 No Values No Values Trigger factor P99080|TIG Ref No Values 2.4 1.5 No Values 1.9 30S ribosomal protein S9 P66646|RS9 Ref No Values 1 1.6 No Values 2 50S ribosomal protein L13 Q7A473|RL13 Ref No Values 1.8 0.4 No Values 0.3 Phosphate acetyltransferase P99092|PTA Ref 3.4 1.4 2.9 3.2 0.2 Acyl carrier protein P0A002|ACP Ref No Values 2.7 2.3 No Values 0.2

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161 Appendix 8. (Continued) Serine aspartate repeat containing protein E Q99W46|SDRE Ref No Values 2 1.7 No Values 1.4 Beta lactamase Q9AC80|Q9AC 80 Ref 1.1 1.2 0.3 1.4 1.3 Cell division protein Q7A620|Q7A6 20 Ref No Values 2.3 1.3 No Values 0.5 Fructose bisphosphate aldolase class 1 P99117|ALF1 Ref No Values 0 No Values No Values 0.6 50S ribosomal protein L14 Q7A463|RL14 Ref No Values 0.1 1.1 No Values 0.3 Phenol soluble modulin alpha 4 peptide P0C824|PSMA 4 Ref 0 0 No Values 1.3 2.2 Staphylococcal secretory antigen ssaA2 Q7A423|SSAA 2 Ref No Values 2.4 0.3 No Values 1 Glycerophosphoryl diester phosphodiesterase Q7A6H7|Q7A6 H7 Ref 0 0 No Values 0.7 0.5 SA2202 protein Q99RL6|Q99R L6 Ref 0 0 No Values 1.6 1.4 50S ribosomal protein L22 Q7A460|RL22 Ref 3.6 No Values 1.7 2.6 No Values Elastin binding protein ebpS Q7A5I6|EBPS Ref 0 0 No Values 2 1.2 Triosephosphate isomerase P99133|TPIS Ref No Values 0.3 2.8 No Values 0.3 P99156|GREA_STA AN P99156|GREA Ref No Values 3.4 2.4 No Values 3.2 Cysteine synthase P63871|CYSK Ref No Values 0.1 2.8 No Values 3.2 Elongation factor Ts P99171|EFTS Ref No Values No Values 2.9 Reference Missing No Values 30S ribosomal protein S6 P99142|RS6 Ref No Values No Values 0.3 No Values No Values Leukotoxin, LukD Q99T54|Q99T5 4 Ref 0 No Values No Values 1.7 No Values Inosine 5' monophosphate dehydrogenase P99106|IMDH Ref 1.8 1.2 1.4 0.2 3.3 Protein grpE P99086|GRPE Ref No Values 0.3 2 No Values 1 50S ribosomal protein L24 P60735|RL24 Ref No Values 0 No Values No Values 1.6 L lactate dehydrogenase 1 P65256|LDH1 Ref 1.4 No Values 1.2 0.6 No Values Superoxide dismutase [Mn/Fe] 1 P99098|SODM 1 Ref No Values No Values 0.9 No Values No Values Transketolase P99161|TKT Ref No Values 0 No Values No Values 2.7 30S ribosomal protein S16 P66440|RS16 Ref 1.8 No Values 1.3 0.2 No Values 10 kDa chaperonin P99104|CH10 Ref No Values 2.9 0.5 No Values 3

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162 Appendix 8. (Continued) SA0295 protein Q7A7Q2|Q7A7 Q2 Ref No Values No Values 0.1 No Values No Values Glucose 6 phosphate isomerase P99078|G6PI Ref No Values No Values 1.5 No Values No Values Protein esaA Q7A7S3|ESAA Ref 0.1 No Values 0.2 1.6 No Values Methicillin resistance mecR1 protein P0A0B0|RAND OM_MECR R Ref No Values 0 2.3 No Values 0.4 Uncharacterized protein SA1692 P0A0K1|Y1692 Ref No Values 0 No Values No Values 0 Chaperone protein hchA P64313|HCHA Ref No Values No Values 0.1 No Values No Values Clumping factor A Q99VJ4|CLFA Ref No Values 0 No Values No Values 0.4 50S ribosomal protein L10 P99155|RL10 Ref No Values 2.7 0.7 No Values 1.8 50S ribosomal protein L18 Q7A467|RL18 Ref No Values No Values 2.2 No Values No Values Putative peptidyl prolyl cis trans isomerase Q7A6I1|PPI1 Ref No Values No Values 1.8 No Values No Values Fibrinogen binding protein P68800|FIB Ref No Values No Values 0.4 No Values No Values 1 phosphatidylinositol phosphodiesterase precurosr Q7A888|Q7A8 88 Ref 0 No Values No Values 1.7 No Values Virulence factor esxA ESXA Ref No Values No Values No Values No Values No Values 50S ribosomal protein L15 P0A0F6|RL15 Ref No Values No Values 0.9 No Values No Values

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163 Appendix 9. Changes in secreted proteins of CA MRSA USA400 compared to HA MRSA USA100 during post exponential phase from 3 biological replicates Identified Proteins (109) Accession Number USA100 # 1 USA100 # 2 USA100 # 3 USA400 # 1 USA400 # 2 USA400 # 3 Lipase 1 P65289|LIP1 Ref 2.8 2.6 0.2 0.1 0.5 Putative surface protein SA2285 P61598|PLS Ref 3.9 0.4 2.9 1.1 3.9 Bifunctional autolysin Q99V41|ATL Ref 0.5 1.6 0.8 1 1.2 Lipase 2 Q7A7P2|LIP2 Ref 0.6 0.8 2 1.4 2.7 SA0841 protein Q7A6G0|Q7A6 G0 Ref 2.3 1 1.2 0 1.2 Penicillin binding protein 2 prime Q7A8C6|Q7A8 C6 Ref 0.9 3.5 0.5 0.3 2.9 Glycerol phosphate lipoteichoic acid synthase Q7A6U1|LTAS Ref 1.2 0.2 0.3 2.5 0.4 Staphopain B Q7A6A7|SSPB Ref 5 2.5 1.6 4.8 0.6 Thermonuclease Q7A6P2|NUC Ref 3.8 2.7 0.8 4.2 3.5 Alpha Hemolysin Q7A632|Q7A63 2 Ref 2.5 2.5 0.8 2.9 3.2 Probable transglycosylase isaA P99160|ISAA Ref 2.7 1 0.8 2.8 1.3 SA2437 protein Q7A371|Q7A37 1 Ref 1.2 0.7 1.7 2.9 2.8 SA0620 protein Q7A6Y9|Q7A6 Y9 Ref 2.8 3.1 0.8 2.6 1.9 Glutamyl endopeptidase Q7A6A6|SSPA Ref 4.5 3 1.7 3.2 0.1 Probable beta lactamase Q7A4X8|Q7A4 X8 Ref No Values 6.9 Reference Missing Reference Missing 6 Immunoglobulin G binding protein A P99134|SPA Ref 5.9 4.9 3.2 1.3 4.6 Enterotoxin type C 3 P0A0L4|ENTC 3 Ref 0.5 0.8 1.1 3.1 1.2 Putative uncharacterized protein SA0359 Q7A7J8|Q7A7J 8 Ref 2.3 2.1 1 3.9 0.7 SA2006 protein Q7A483|Q7A48 3 Ref 2.5 1.3 1.1 2 0.7 Staphopain A P65826|SSPP Ref 1.9 1.9 1.4 3.9 4.1 Alkyl hydroperoxide reductase subunit C P99074|AHPC Ref 2 4.3 0.6 4 4.5 Putative uncharacterized protein SA0570 Q7A735|Q7A73 5 Ref 9.8 4.3 2.7 12 5.5 Elongation factor Tu P99152|EFTU Ref 1.7 0.6 1.4 1.7 1.7

PAGE 176

164 Appendix 9. (Continued) Glycyl glycine endopeptidase lytM Q7A7T0|LYTM Ref 5.4 1.7 0.2 4.9 2.2 Immunoglobulin binding protein sbi Q99RL2|SBI Ref 2.4 0.3 0.7 3.5 0.6 Truncated beta hemplysin Q99QR7|Q99Q R7 Ref No Values 3.2 Reference Missing No Values 3.5 Uncharacterized leukocidin like protein 1 Q7A4L0|LUKL 1 Ref 4.6 1.4 2.3 1 2.8 Enolase P99088|ENO Ref 1.5 0.2 0.3 0.3 0.1 Fructose bisphosphate aldolase P99075|ALF2 Ref 4.2 1.6 1.1 1.2 1 Probable transglycosylase sceD Q7A4F2|SCED Ref 3.9 1.6 0.1 2.2 1.7 Serine aspartate repeat containing protein D Q7A780|SDRD Ref 0.8 3.1 2.4 3.5 5.5 SA0587 protein Q7A719|Q7A71 9 Ref 0 0 No Values 0.1 0.2 SA0914 protein Q99V35|Q99V3 5 Ref 5 3.6 0.2 6 4.1 60 kDa chaperonin P99083|CH60 Ref 4 0.2 0.8 3.7 1 Glyceraldehyde 3 phosphate dehydrogenase 1 P99136|G3P1 Ref No Values 2.1 0.4 No Values 1.9 Zinc metalloproteinase aureolysin Q7A378|Q7A37 8 Ref 0 0 No Values 1.6 1 Virulence factor esxA Q7A7S4|ESXA Ref 0 0 No Values 0.4 0.1 Serine protease splB Q7A4Y1|SPLB Ref 0.1 1.3 Reference Missing 0.3 0.7 Foldase protein prsA P60748|PRSA Ref 1.3 2.4 0.4 1.9 1.8 50S ribosomal protein L7/L12 P99154|RL7 Ref No Values 4 0.6 No Values 3.7 Iron regulated surface determinant protein A Q7A655|ISDA Ref 0.8 0.3 0.4 0.4 0.1 DNA binding protein HU Q7A5J1|DBH Ref 8.2 3.5 0.5 9.4 2.6 Staphylokinase Q99SU7|SAK Ref 1.2 0.4 0.1 2.9 0.4 Pyruvate dehydrogenase E1 component subunit alpha Q820A6|ODPA Ref 0 0 No Values 2.2 0.1 Putative uncharacterized protein SA0663 Q7A6V1|Q7A6 V1 Ref 3.1 2.7 0.2 3 3.5 Chaperone protein dnaK P99110|DNAK Ref No Values 0.3 1.3 Reference Missing 0.5 Adenylate kinase P99062|KAD Ref 6.5 1.2 0.2 5.1 0.8

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165 Appendix 9. (Continued) Putative uncharacterized protein SA0908 Q7A6A3|Q7A6 A3 Ref 0.3 1.2 1.1 0.7 1.2 50S ribosomal protein L30 P0A0G0|RL30 Ref 0 0 No Values 2.2 0.5 Uncharacterized leukocidin like protein 2 Q99SN7|LUKL 2 Ref 3.8 1.8 1.7 2.3 1.9 Dihydrolipoyl dehydrogenase P99084|DLDH Ref No Values 3.8 0.7 No Values 4 Phenol soluble modulin alpha 1 peptide P0C7Y7|PSMA 1 Ref 1.3 2.2 0.8 2.3 3.7 Serine protease splF Q7A4Y4|SPLF Ref 0 No Values No Values 0 No Values Pyruvate dehydrogenase E1 component subunit beta P99063|ODPB Ref 7.3 8.3 0.1 7.4 12 50S ribosomal protein L17 Q7A469|RL17 Ref 2.7 0.2 0.1 2.9 1.6 Glutamine synthetase P99095|GLNA Ref No Values 0.4 1.1 No Values 0.5 N acetylmuramoyl L alanine amidase sle1 Q7A7E0|SLE1 Ref No Values 4.5 0.3 No Values 5.9 Alkaline shock protein 23 P99157|ASP23 Ref 1.7 0.4 0.3 2.1 2.6 Dihydrolipoyllysine residue acetyltransferase component of pyruvate dehydrogenase complex P65636|ODP2 Ref 1.8 2.5 Value Missing 1.6 1.7 SA2097 protein Q7A418|Q7A41 8 Ref 0 0 No Values 0.1 0.1 Ribosome recycling factor P99130|RRF Ref No Values 0.7 0.7 No Values 0.4 Elongation factor G P68789|EFG Ref No Values 0.7 0.4 No Values 1.7 Serine protease splC Q7A4Y2|SPLC Ref 0 No Values No Values 1 No Values 50S ribosomal protein L11 P0A0F2|RL11 Ref 2.6 0.5 1 0.3 0.1 Thioredoxin P99122|THIO Ref No Values No Values 0.3 No Values No Values Trigger factor P99080|TIG Ref No Values 2.4 1.2 No Values 1.4 30S ribosomal protein S9 P66646|RS9 Ref No Values 1 0.5 No Values 0.5 50S ribosomal protein L13 Q7A473|RL13 Ref No Values 1.8 0.9 No Values 2.7 Phosphate acetyltransferase P99092|PTA Ref 3.4 1.4 1 1.8 1 Acyl carrier protein P0A002|ACP Ref No Values 2.7 1.3 No Values 1.5

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166 A ppendix 9. (Continued) Serine aspartate repeat containing protein E Q99W46|SDRE Ref No Values 2 0.9 No Values 2.6 Beta lactamase Q9AC80|Q9AC 80 Ref 1.1 1.2 1.2 1 1.7 Cell division protein Q7A620|Q7A62 0 Ref No Values 2.3 1.3 No Values 2.7 Fructose bisphosphate aldolase class 1 P99117|ALF1 Ref No Values 0 No Values No Values 0.4 50S ribosomal protein L14 Q7A463|RL14 Ref No Values 0.1 1.1 No Values 0.6 Phenol soluble modulin alpha 4 peptide P0C824|PSMA 4 Ref 0 0 No Values 3.2 3 Staphylococcal secretory antigen ssaA2 Q7A423|SSAA 2 Ref No Values 2.4 0.7 No Values 2.3 Glycerophosphoryl diester phosphodiesterase Q7A6H7|Q7A6 H7 Ref 0 0 No Values 2.3 0.3 SA2202 protein Q99RL6|Q99R L6 Ref 0 0 No Values 0.1 0 50S ribosomal protein L22 Q7A460|RL22 Ref 3.6 No Values 0.4 2.5 No Values Elastin binding protein ebpS Q7A5I6|EBPS Ref 0 0 No Values 0.5 0.6 Triosephosphate isomerase P99133|TPIS Ref No Values 0.3 1.1 No Values 0.5 P99156|GREA_STA AN P99156|GREA Ref No Values 3.4 0.3 No Values 3.9 Cysteine synthase P63871|CYSK Ref No Values 0.1 0.1 No Values 3.1 Elongation factor Ts P99171|EFTS Ref No Values No Values 1.5 Reference Missing No Values 30S ribosomal protein S6 P99142|RS6 Ref No Values No Values 0.9 No Values No Values Leukotoxin, LukD Q99T54|Q99T5 4 Ref 0 No Values No Values 0.5 No Values Inosine 5' monophosphate dehydrogenase P99106|IMDH Ref 1.8 1.2 1.4 0.6 1.2 Protein grpE P99086|GRPE Ref No Values 0.3 1.4 No Values 0.2 50S ribosomal protein L24 P60735|RL24 Ref No Values 0 No Values No Values 0.3 L lactate dehydrogenase 1 P65256|LDH1 Ref 1.4 No Values 0.4 0.9 No Values Superoxide dismutase [Mn/Fe] 1 P99098|SODM 1 Ref No Values No Values 1.3 No Values No Values Transketolase P99161|TKT Ref No Values 0 No Values No Values 0.8 30S ribosomal protein S16 P66440|RS16 Ref 1.8 No Values 0.1 1.9 No Values 10 kDa chaperonin P99104|CH10 Ref No Values 2.9 1.5 No Values 3.1

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167 Appendix 9. (Continued) SA0295 protein Q7A7Q2|Q7A7 Q2 Ref No Values No Values 2.2 No Values No Values Glucose 6 phosphate isomerase P99078|G6PI Ref No Values No Values 0.6 No Values No Values Protein esaA Q7A7S3|ESAA Ref 0.1 No Values 0.1 Value Missing No Values Methicillin resistance mecR1 protein P0A0B0|RAND OM_MECR R Ref No Values 0 0.8 No Values 1.4 Uncharacterized protein SA1692 P0A0K1|Y1692 Ref No Values 0 No Values No Values 3.1 Chaperone protein hchA P64313|HCHA Ref No Values No Values 1 No Values No Values Clumping factor A Q99VJ4|CLFA Ref No Values 0 No Values No Values 0.2 50S ribosomal protein L10 P99155|RL10 Ref No Values 2.7 0.4 No Values 3.4 50S ribosomal protein L18 Q7A467|RL18 Ref No Values No Values 0.1 No Values No Values Putative peptidyl prolyl cis trans isomerase Q7A6I1|PPI1 Ref No Values No Values 0.5 No Values No Values Fibrinogen binding protein P68800|FIB Ref No Values No Values 0.6 No Values No Values 1 phosphatidylinositol phosphodiesterase precurosr Q7A888|Q7A88 8 Ref 0 No Values No Values 0.1 No Values Virulence factor esxA ESXA Ref No Values No Values No Values Reference Missing No Values 50S ribosomal protein L15 P0A0F6|RL15 Ref No Values No Values 0.6 No Values No Values

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168 Appendix 10. Changes in secreted proteins of HA MRSA USA200 compared to HA MRSA USA100 during stationary phase from 3 biological replicates Identified Proteins (246) Accession Number USA10 0# 1 USA100 # 2 USA100 # 3 USA200 # 1 USA200 # 2 USA200 # 3 Lipase 2 Q7A7P2|LIP2 Ref 0.4 1.6 0.2 0.7 3.8 Lipase 1 P65289|LIP1 Ref 1 0.8 0.4 2.6 1.5 Alpha Hemolysin Q7A632|Q7A63 2 Ref 0.6 0.2 0.2 1 2.7 Elongation factor Tu P99152|EFTU Ref 1.8 6.7 0.1 2.6 6.7 Enolase P99088|ENO Ref 0.6 6.6 0.4 0.9 6.6 Putative surface protein SA2285 P61598|PLS Ref 4.6 9 4.4 9.8 9 Bifunctional autolysin Q99V41|ATL Ref 0.1 3.1 1.3 2.6 3.1 SA0841 protein Q7A6G0|Q7A6 G0 Ref 1.4 3 2.4 2.1 1 Chaperone protein dnaK P99110|DNAK Ref 3.6 7 0.3 3.9 7 Glyceraldehyde 3 phosphate dehydrogenase 1 P99136|G3P1 Ref 2.5 5.6 0.5 1.5 5.6 DNA binding protein HU Q7A5J1|DBH Ref 1.7 5.1 0.3 6 5.1 Alkyl hydroperoxide reductase subunit C P99074|AHPC Ref 4.7 7 0.9 6.4 7 Inosine 5' monophosphate dehydrogenase P99106|IMDH Ref 0.2 5.8 0.4 0.7 5.8 Immunoglobulin G binding protein A SPA Ref No Values 0 No Values No Values 1.1 Formate -tetrahydrofolate ligase Q7A535|FTHS Ref 1.6 7.6 0.8 3.2 7.6 Dihydrolipoyl dehydrogenase P99084|DLDH Ref 0.7 7.2 0.3 0.3 7.2 50S ribosomal protein L7/L12 P99154|RL7 Ref 0.2 7.4 1.6 0.1 7.4 Pyruvate kinase Q7A559|KPYK Ref 4.6 3.7 1.3 4.5 3.7 SA0587 protein Q7A719|Q7A71 9 Ref 1.3 5 0.1 2.1 5 Phosphate acetyltransferase P99092|PTA Ref 2 6.2 0.6 1.2 6.2 Elongation factor G P68789|EFG Ref 1.5 6.2 0.1 2.1 6.2 Glutamine synthetase P99095|GLNA Ref 2.4 6.7 0.3 2.6 6.7 SA2006 protein Q7A483|Q7A48 3 Ref 0.3 0.8 0.6 2.9 2.4

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16 9 Appendix 10. (Continued) Fructose bisphosphate aldolase P99075|ALF2 Ref 3.8 7.4 Value Missing 3.5 7.4 Glycerol phosphate lipoteichoic acid synthase Q7A6U1|LTAS Ref 1 0.5 0.4 0.3 0.5 Alkaline shock protein 23 P99157|ASP23 Ref 2.4 3.9 0.9 2.5 3.9 Triosephosphate isomerase P99133|TPIS Ref 2 4 1.2 0.8 4 Enterotoxin type C 3 P0A0L4|ENTC 3 Ref 5.3 5.4 0.3 4.7 2.6 Dihydrolipoyllysine residue acetyltransferase component of pyruvate dehydrogenase complex P65636|ODP2 Ref 0.6 5.7 0.7 2.5 5.7 Cysteine synthase P63871|CYSK Ref 0.6 5.1 0.3 1.2 5.1 Elongation factor Ts P99171|EFTS Ref 2.5 5 0.5 3.4 5 Pyruvate dehydrogenase E1 component subunit alpha Q820A6|ODPA Ref 5.5 9.6 0.6 6.8 9.6 Putative uncharacterized protein SA0663 Q7A6V1|Q7A6 V1 Ref 0.1 0.2 0.3 2.6 Value Missing Ornithine aminotransferase 2 P60298|OAT2 Ref 3.2 9.4 0.7 5 9.4 Staphopain B Q7A6A7|SSPB Ref 5.1 3.5 0.4 3.1 2.8 Succinyl CoA ligase [ADP forming] subunit beta P99071|SUCC Ref 1.6 6.2 0.9 4.1 6.2 Phosphocarrier protein HPr P99143|PTHP Ref 1.1 5.6 0.3 2.5 5.6 Putative uncharacterized protein SA0359 Q7A7J8|Q7A7J 8 Ref 5.8 6.1 Value Missing 3.4 6.1 Thermonuclease Q7A6P2|NUC Ref 1.6 0.9 0.5 4.4 0.2 Trigger factor P99080|TIG Ref 1.3 4.9 0.7 0.6 4.9 Aconitate hydratase P99148|ACON Ref 0.1 4.9 0.1 0.7 4.9 Adenylate kinase P99062|KAD Ref 3.1 4.8 0.4 2.6 4.8 Fructose bisphosphate aldolase class 1 P99117|ALF1 Ref 2.3 6.6 1.1 2.5 6.6 50S ribosomal protein L17 Q7A469|RL17 Ref 1.2 3.6 0.1 2.4 3.6 Glutamyl endopeptidase Q7A6A6|SSPA Ref 1.7 3.3 0.6 1.5 1.3 SA0620 protein Q7A6Y9|Q7A6 Y9 Ref 0.1 2.2 1.7 2 3.1 60 kDa chaperonin P99083|CH60 Ref 1.3 5.4 0.7 0.3 5.4 Probable beta lactamase Q7A4X8|Q7A4 X8 Ref 0.8 No Values 4.4 4.4 No Values

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170 Appendix 10. (Continued) Putative uncharacterized protein SA0570 Q7A735|Q7A73 5 Ref 0.1 3.9 Value Missing 2.6 3.9 Alcohol dehydrogenase Q7A742|ADH Ref 0.4 4.8 0.5 1.3 4.8 Catalase Q7A5T2|CATA Ref 3.8 6.9 0.1 5.3 6.9 SA2097 protein Q7A418|Q7A41 8 Ref 1.4 2.7 0.8 1.7 1.4 Penicillin binding protein 2 prime Q7A8C6|Q7A8 C6 Ref 4.4 6.8 0.2 1.1 6.1 Putative uncharacterized protein SAP003 Q9AC87|Q9AC 87 Ref 3.5 6.8 0.9 5.5 6.8 Probable thiol peroxidase P99146|TPX Ref 0.7 6 Reference Missing 0.3 6 30S ribosomal protein S1 Q7A5J0|RS1 Ref 0.9 5.3 Value Missing 2.3 5.3 L lactate dehydrogenase 1 P65256|LDH1 Ref 2.2 1.1 0.5 4 0.8 Phosphoglycerate kinase P99135|PGK Ref 1.6 5.4 0.3 1.2 5.4 Glucose 6 phosphate isomerase P99078|G6PI Ref 1.5 5.4 0 1.3 5.4 Transketolase P99161|TKT Ref 1.9 5.5 3.1 0.7 5.5 Pyruvate dehydrogenase E1 component subunit beta P99063|ODPB Ref No Values 8.1 1.4 Reference Missing 8.1 Glycerophosphoryl diester phosphodiesterase Q7A6H7|Q7A6 H7 Ref 2.9 4.4 Value Missing 1 Value Missing Delta hemolysin P0A0M2|HLD Ref 2.5 No Values 0.4 1.1 No Values Phenol soluble modulin alpha 4 peptide P0C824|PSMA4 Ref 2.3 0.1 0.3 1.3 3.2 Foldase protein prsA P60748|PRSA Ref 3.3 5.7 0.3 1.2 5.1 Glycyl glycine endopeptidase lytM Q7A7T0|LYTM Ref 0.3 0.9 2.3 3.3 5.2 Thioredoxin P99122|THIO Ref 0.2 4.6 0.2 0.7 4.6 SA2437 protein Q7A371|Q7A37 1 Ref 10 8.7 Reference Missing 9.1 Value Missing UPF0477 protein SA0873 Q7A6D4|Y873 Ref 4.7 5.9 1.1 6.9 Value Missing Citrate synthase II Q7A561|Q7A56 1 Ref 5.5 5.8 0.7 7.9 5.8 6 phosphogluconate dehydrogenase, decarboxylating P63334|6PGD Ref 0.9 4.1 0 0.1 Value Missing Superoxide dismutase [Mn/Fe] 1 P99098|SODM1 Ref 4.7 7.7 1.3 3.3 7.7 Phenol soluble modulin alpha 1 peptide P0C7Y7|PSMA 1 Ref 5.3 4.2 0.2 3.8 0.8

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171 Appendix 10. (Continued) Phosphoenolpyruvat e carboxykinase [ATP] P99128|PCKA Ref 0.6 8.4 Value Missing 2.3 8.4 Probable transglycosylase isaA P99160|ISAA Ref 2.4 4.1 0.4 2.1 4.1 Glycyl tRNA synthetase P99129|SYG Ref 0.3 1.7 Value Missing 1.4 1.7 DNA directed RNA polymerase subunit beta' P60285|RPOC Ref 2.4 3.7 1.8 2.4 Value Missing Glycine cleavage system H protein P64214|GCSH Ref 0.9 6.8 0 2.7 Value Missing 50S ribosomal protein L30 P0A0G0|RL30 Ref 0.8 0.7 2.1 0.7 0.7 Cold shock protein cspA Q7A5P3|CSPA Ref No Values 3.9 No Values No Values 3.9 Serine protease splB Q7A4Y1|SPLB Ref 0.2 4.1 0.4 0.2 2.5 DNA directed RNA polymerase subunit beta P60278|RPOB Ref No Values 6.1 0.1 No Values 6.1 2,3 bisphosphoglycerate dependent phosphoglycerate mutase P99153|GPMA Ref No Values 3.4 0.3 Reference Missing 3.4 Dihydrolipoyllysine residue succinyltransferase component of 2 oxoglutarate dehydrogenase complex Q7A5N4|ODO2 Ref 5.2 4.6 1.2 8.3 Value Missing Seryl tRNA synthetase P99178|SYS Ref 6.1 9.3 0.6 8.5 9.3 NAD specific glutamate dehydrogenase Q7A6H8|DHE2 Ref 2.1 3 0.5 3.6 2.8 Succinyl CoA ligase [ADP forming] subunit alpha P99070|SUCD Ref 16.8 25.2 No Values 17.8 25.2 Uncharacterized leukocidin like protein 1 Q7A4L0|LUKL 1 Ref 1.8 1.4 0.4 1.8 1.9 Acyl carrier protein P0A002|ACP Ref 2.5 2.9 0.3 3.8 Value Missing 50S ribosomal protein L21 Q7A583|RL21 Ref 1.8 5.2 Reference Missing 2.9 Value Missing UPF0337 protein SA0772 Q7A6L9|Y772 Ref 2.6 3.3 No Values 4.2 3.3 3 oxoacyl [acyl carrier protein] synthase 2 Q7A6F8|FABF Ref 2.6 3.1 No Values 1.8 3.1 50S ribosomal protein L9 P66318|RL9 Ref 3.2 6.4 Value Missing 3.8 6.4 Beta lactamase Q9AC80|Q9AC 80 Ref 0.3 1.8 1.6 0.2 1.8

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172 Appendix 10. (Continued) Thioredoxin reductase P99101|TRXB Ref No Values 7.7 Value Missing No Values 7.7 Putative dipeptidase SA1572 Q7A522|PEPV L Ref 5.8 9.3 Reference Missing 6 9.3 SA0916 protein Q7A696|Q7A69 6 Ref 3.9 4.7 No Values 6.9 Value Missing Phosphoenolpyruvat e protein phosphotransferase Q99V14|PT1 Ref 0 5.4 Value Missing 1 5.2 30S ribosomal protein S9 P66646|RS9 Ref 2.5 6.4 1 6.1 Value Missing Uncharacterized protein SA1692 P0A0K1|Y1692 Ref 0.3 1.9 0.4 0.9 1.7 50S ribosomal protein L11 P0A0F2|RL11 Ref 0.6 5.9 0.7 2.6 Value Missing Putative universal stress protein SA1532 Q7A551|Y1532 Ref 0.6 2 0.5 0.6 Value Missing 1 pyrroline 5 carboxylate dehydrogenase P99076|ROCA Ref 1 4.6 1 5.2 4.6 Gamma hemolysin component C Q7A3S2|HLGC Ref 5.4 5.2 1.1 5.3 Value Missing Serine aspartate repeat containing protein D Q7A780|SDRD Ref 0 0 No Values 2.7 0 Glutamyl tRNA synthetase P99170|SYE Ref 2.6 7.5 0.9 2.9 7.5 2,3 bisphosphoglycerate independent phosphoglycerate mutase P64270|GPMI Ref 1.2 4.5 0.8 4.8 Value Missing D lactate dehydrogenase P99116|LDHD Ref No Values 0 No Values No Values 0 50S ribosomal protein L10 P99155|RL10 Ref 2.6 5 0.7 3.6 5.1 SA2202 protein Q99RL6|Q99R L6 Ref 0.1 2.5 0.4 0.2 2.4 Gamma hemolysin component A P0A072|HLGA Ref 2.2 No Values 0.8 3.7 No Values 10 kDa chaperonin P99104|CH10 Ref 2.1 5.9 0.4 2 5.9 Dihydroorotase P65906|PYRC Ref 1.6 3.8 0.1 0.7 3.8 UPF0457 protein SA1975.1 Q99S93|Y197A Ref 1.5 5.6 No Values 4.8 Value Missing Ribosome recycling factor P99130|RRF Ref 2.6 4.7 Reference Missing 2.2 3.9 Elongation factor P P99066|EFP Ref 1 5.3 0.3 2 5.3 Uncharacterized leukocidin like protein 2 Q99SN7|LUKL 2 Ref 0.6 No Values 1.7 0.6 No Values UPF0355 protein SA0372 Q7A7I6|UP355 Ref 1.6 4.6 Value Missing 1.3 4.6 Acetate kinase Q99TF2|ACKA Ref 2.6 2.1 1.5 4.2 1.6 Elastin binding protein ebpS Q7A5I6|EBPS Ref 2.6 5 0.2 3.3 5

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173 Appendix 10. (Continued) 30S ribosomal protein S6 P99142|RS6 Ref No Values 2.2 0.8 No Values Value Missing General stress protein 20U Q7A4C8|Q7A4 C8 Ref 0.9 1.4 Value Missing 0.6 Value Missing 50S ribosomal protein L24 P60735|RL24 Ref 2.7 7.6 No Values 3.8 Value Missing Bifunctional protein folD Q7A697|FOLD Ref No Values 0.3 0.1 No Values Value Missing 50S ribosomal protein L22 Q7A460|RL22 Ref 2 4.7 0.4 0.7 3.1 Putative uncharacterized protein SAS040 Q7A5U6|Q7A5 U6 Ref 2.5 8 0.8 1.8 8 Gamma hemolysin component B P0A075|HLGB Ref 1 No Values 0.2 2.1 No Values Putative uncharacterized protein SA0395 Q99WG7|Q99 WG7 Ref No Values No Values 0.7 Reference Missing No Values Putative peptidyl prolyl cis trans isomerase Q7A6I1|PPI1 Ref 2.4 0.1 2.2 6 4.4 50S ribosomal protein L1 Q99W68|RL1 Ref No Values 2.8 0.1 Reference Missing 2.8 Virulence factor esxA ESXA (+1) Ref 0.5 No Values 0.8 0.8 No Values ATP dependent Clp protease ATP binding subunit clpC Q7A797|CLPC Ref 1.9 4.7 2.2 4.8 4.7 SA0914 protein Q99V35|Q99V3 5 Ref 2.4 3.6 0.2 4.8 3.3 30S ribosomal protein S16 P66440|RS16 Ref 1.5 8.5 No Values 1.2 8.5 Translation initiation factor IF 1 P65119|IF1 Ref 5.8 9.1 Reference Missing 9.3 9 DNA directed RNA polymerase subunit alpha P66706|RPOA Ref 1.1 5.9 0.1 2 5.9 Truncated beta hemplysin Q99QR7|Q99Q R7 Ref 19.6 No Values No Values 16 No Values Deoxyribose phosphate aldolase 1 P99102|DEOC1 (+1) Ref 3 4.4 0.1 3.1 4.4 Pyruvate carboxylase Q7A666|Q7A66 6 Ref 0 0 No Values 1.5 0 Uncharacterized protein SA0829 Q7A6H3|Y829 Ref 6.5 No Values No Values 6.8 No Values Probable acetyl CoA acyltransferase Q7A7L2|THLA Ref 2.7 No Values Value Missing 4.1 No Values Protein grpE P99086|GRPE Ref 3.6 4.6 0.4 4.4 Value Missing 50S ribosomal protein L3 P60449|RL3 Ref No Values No Values 0 No Values No Values ATP synthase subunit beta P99112|ATPB Ref 1.6 5.5 0.5 0 5.5

PAGE 186

174 Appendix 10. (Continued) Uncharacterized protein SA0707 Q7A6R6|Y707 Ref 2.5 5.2 0.3 3.9 5.2 Glucose specific phosphotransferase enzyme IIA component P60857|PTGA Ref No Values 0 No Values Reference Missing 0 Transcription elongation factor greA P99156|GREA Ref 2.1 3.6 0.7 2.6 Value Missing SA0758 protein Q7A6M7 Ref 0.3 3.9 2.9 0.5 3.9 Chaperone protein hchA P64313|HCHA Ref 3 No Values 0 5.5 No Values 50S ribosomal protein L5 Q7A465|RL5 Ref 0.6 No Values 1.8 2.4 No Values Serine hydroxymethyltransf erase P99091|GLYA Ref 4 8 0.9 3.3 8 Methionine aminopeptidase AMPM Ref 5.7 9.4 0.3 5.9 9.4 50S ribosomal protein L15 P0A0F6|RL15 Ref 0.9 4.1 0.4 3.6 4.1 ATP dependent Clp protease ATP binding subunit clpL Q7A3F4|CLPL Ref 0.4 7.4 Value Missing 1.8 6.8 UPF0342 protein SA1663 Q7A4V3|Y1663 Ref 2.4 3.6 1.1 0.2 Value Missing SA0759 protein Q7A6M6|Q7A6 M6 Ref 1.8 3.2 No Values 3.4 Value Missing Staphylokinase Q99SU7|SAK Ref 0.8 1.5 Value Missing 0.3 Value Missing Iron regulated surface determinant protein A Q7A655|ISDA Ref No Values 4.7 Value Missing No Values 4.2 Serine aspartate repeat containing protein E Q99W46|SDRE Ref 0 0 No Values 1.8 1.8 Leukotoxin, LukD Q99T54|Q99T5 4 Ref No Values No Values No Values No Values No Values Amidophosphoribos yltransferase P99164|PUR1 Ref No Values No Values 0.3 No Values No Values SA0859 protein Q7A6E5|Q7A6 E5 Ref No Values 6.3 No Values No Values Value Missing Phosphoribosylform ylglycinamidine synthase 1 P99166|PURQ Ref No Values No Values No Values No Values No Values Staphylococcal complement inhibitor Q99SU9|SCIN Ref No Values 1.8 No Values No Values 1 Uncharacterized N acetyltransferase SA1019 Q99UT4|Y1019 Ref 2.5 4.4 0 4 4.4 50S ribosomal protein L27 P66133|RL27 Ref No Values 6.7 0 No Values Value Missing Adenylosuccinate lyase Q7A4Q3|PUR8 Ref 0 0 No Values 1 Value Missing

PAGE 187

175 Appendix 10. (Continued) Naphthoate synthase Q7A6A9|MEN B Ref 8.8 No Values No Values 9.8 No Values Putative uncharacterized protein SA0771 Q7A6M0|Q7A6 M0 Ref 1.3 2.8 Value Missing 1.6 2.8 Xaa Pro dipeptidase Q99TW4|Q99T W4 Ref 5.1 8 No Values 3.7 8 1 phosphatidylinositol phosphodiesterase precurosr Q7A888|Q7A88 8 Ref 0.7 No Values Value Missing 2.5 No Values Probable glycine dehydrogenase [decarboxylating] subunit 1 P64218|GCSPA Ref No Values 0 No Values No Values 0 Putative aldehyde dehydrogenase AldA Q7A825|ALDA Ref No Values 0 No Values No Values 0 L lactate dehydrogenase 2 P99119|LDH2 Ref 1.4 4 Value Missing 3.1 3.8 Formate acetyltransferase Q7A7X6|PFLB Ref 1.2 4.1 1.3 1.8 3.3 Signal transduction protein TRAP Q7A4W3|TRAP Ref 0.6 0 0.2 0.5 0.6 Purine nucleoside phosphorylase Q7A4C9|Q7A4 C9 Ref No Values No Values 1.1 No Values No Values Arginyl tRNA synthetase Q99W05|SYR Ref No Values 7.3 0.8 No Values 7.3 50S ribosomal protein L13 Q7A473|RL13 Ref 1.3 No Values 1 0 No Values Probable transglycosylase sceD Q7A4F2|SCED Ref 0 No Values No Values 2 No Values Organic hydroperoxide resistance protein like Q7A6M9|OHR L Ref 1.8 No Values No Values 0.2 No Values Serine protease splD Q7A4Y3|SPLD Ref No Values No Values Value Missing No Values No Values Putative uncharacterized protein SA1986 Q7A493|Q7A49 3 Ref No Values No Values 0.3 No Values No Values D alanine -poly(phosphoribitol) ligase subunit 2 P0A019|DLTC Ref 2.5 5.9 Value Missing 1.5 Value Missing Immunoglobulin binding protein sbi Q99RL2|SBI Ref 3.3 6.8 Value Missing 4.9 Value Missing Putative uncharacterized protein SA1528 Q7A553|Q7A55 3 Ref 1.7 No Values 0.2 1.9 No Values GMP synthase [glutamine hydrolyzing] P99105|GUAA Ref No Values 5.9 Value Missing No Values Value Missing SA1524 protein Q7A556|Q7A55 6 Ref 1.6 3.1 Value Missing 3.1 3.1

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176 Appendix 10. (Continued) 30S ribosomal protein S11 P66357|RS11 Ref 2.3 7.6 No Values 3.3 Value Missing Putative uncharacterized protein SA0919 Q7A694|Q7A69 4 Ref No Values 4.8 0.2 No Values 3.2 Phenol soluble modulin alpha 3 peptide P0C811|PSMA3 Ref 0.6 No Values 0.4 0.3 No Values Ferritin Q7A4R2|FTN Ref No Values 0 No Values No Values Value Missing Alkyl hydroperoxide reductase subunit F P99118|AHPF Ref No Values 6.2 Value Missing No Values 6.2 Anti sigma B factor antagonist P66838|RSBV Ref No Values 3.2 0.6 No Values 2.3 Nucleoside diphosphate kinase NDK (+1) Ref 3.7 9.6 Value Missing 6.2 9.6 Zinc metalloproteinase aureolysin Q7A378|Q7A37 8 Ref No Values 2.5 Reference Missing Reference Missing 1.7 UPF0173 metal dependent hydrolase SA1529 P99149|Y1529 Ref 14 19.1 No Values 13.9 Value Missing Pyridoxal biosynthesis lyase pdxS P60798|PDXS Ref No Values 6.5 0 No Values 5.9 Alanine dehydrogenase 2 Q99TF4|DHA2 Ref No Values 5.4 0.6 No Values Value Missing Urocanate hydratase P67417|HUTU Ref No Values 0 No Values No Values Value Missing Putative uncharacterized protein SA2309 Q7A3I0|Q7A3I 0 Ref 4.7 3.5 Value Missing Reference Missing Value Missing CTP synthase P99072|PYRG Ref 0.6 5.5 Value Missing 1.2 Value Missing Putative 8 amino 7 oxononanoate synthase/2 amino 3 ketobutyrate coenzyme A ligase P60120|BIKB Ref No Values 23 No Values No Values Value Missing Threonyl tRNA synthetase P67585|SYT Ref No Values No Values 0.6 No Values No Values Probable malate:quinone oxidoreductase 2 P99115|MQO2 Ref 0 0 No Values 2.8 0 Probable branched chain amino acid aminotransferase P99138|ILVE Ref 0 0 No Values 0.1 0.4 Methionyl tRNA synthetase P67579|SYM Ref No Values 0 No Values No Values Value Missing Tryptophanyl tRNA synthetase P67593|SYW Ref 0 No Values No Values 3 No Values Mannitol 1 phosphate 5 dehydrogenase P99140|MTLD Ref 0 No Values No Values 1.8 No Values SA1343 protein Q7A5G2|Q7A5 G2 Ref No Values 0 No Values No Values Value Missing

PAGE 189

177 Appendix 10. (Continued) Staphopain A P65826|SSPP Ref No Values No Values No Values No Values No Values UPF0082 protein SA0624 P67182|Y624 Ref 2.2 5.3 Value Missing 1.9 Value Missing Cell division protein ftsZ P99108|FTSZ Ref 2.5 3.1 0.3 2.3 Value Missing Adenylosuccinate synthetase P99099|PURA Ref No Values 5.1 0.5 No Values 4.8 Histidine ammonia lyase P64416|HUTH Ref No Values 9.6 No Values No Values Value Missing Imidazolonepropiona se P64418|HUTI Ref No Values 0 No Values No Values Value Missing 6 phosphofructokinase P99165|K6PF Ref 3.2 No Values No Values 1.7 No Values Lysyl tRNA synthetase P67610|SYK Ref No Values 1.3 0.9 No Values 0.8 SA1599 protein Q7A501|Q7A50 1 Ref No Values 0 No Values No Values 0.8 SA0231 protein Q7A7W3|Q7A7 W3 Ref No Values 3.4 Value Missing No Values 3.3 Phenylalanyl tRNA synthetase beta chain P67041|SYFB Ref No Values 0 No Values No Values Value Missing P60855|Y370_STAA N P60855|Y370 Ref No Values 0 No Values No Values 0 50S ribosomal protein L23 Q7A459|RL23 Ref No Values No Values 2.3 No Values No Values Putative uncharacterized protein SA0908 Q7A6A3|Q7A6 A3 Ref 3 2.3 0.2 5.1 Value Missing SA1475 protein Q7A581|Q7A58 1 Ref 0 0 No Values 2.3 0.4 Leucyl tRNA synthetase P67513|SYL Ref No Values 6 Value Missing No Values 5.8 Clumping factor A Q99VJ4|CLFA Ref 0 0 No Values 0.9 Value Missing Acetate CoA ligase Q7A3A2|Q7A3 A2 Ref 0 No Values No Values 3.6 No Values Lactonase drp35 RANDOM_DR P35 R Ref 0 No Values No Values 0.3 No Values 50S ribosomal protein L2 P60432|RL2 Ref No Values 0 No Values No Values 0 Glucosamine -fructose 6 phosphate aminotransferase [isomerizing] GLMS Ref No Values No Values No Values No Values No Values Putative uncharacterized protein SA1743 Q7A4N7|Q7A4 N7 Ref No Values 0 No Values No Values 0 Cell division protein Q7A620|Q7A62 0 Ref No Values 0 No Values No Values 0 50S ribosomal protein L18 Q7A467|RL18 Ref No Values 18.9 No Values No Values 18.8 3 hexulose 6 phosphate synthase Q7A774|HPS Ref No Values No Values Reference Missing No Values No Values Acetoin(diacetyl) reductase P99120|BUTA Ref No Values 0 No Values No Values 1.3

PAGE 190

178 Appendix 10. (Continued) SA0022 protein Q99XE9|Q99X E9 Ref 0 No Values No Values 1.9 No Values 3 hydroxy 3 methylglutaryl CoA synthase Q7A3F6|Q7A3 F6 Ref No Values No Values Value Missing No Values No Values 30S ribosomal protein S7 P66616|RS7 Ref 1.5 No Values 1.5 0.1 No Values Uncharacterized lipoprotein SA2158 Q7A3W5|Y215 8 Ref No Values No Values No Values No Values No Values Ribonuclease J 1 Q7A682|RNJ1 Ref No Values No Values Value Missing Reference Missing No Values Trans 2 enoyl ACP reductase Q7A6D8 Ref 0 No Values No Values 2.7 No Values 2 C methyl D erythritol 4 phosphate cytidylyltransferase 2 Q7A7V0|ISPD2 Ref No Values No Values 0.6 No Values No Values 30S ribosomal protein S13 P66388|RS13 Ref No Values No Values No Values No Values No Values 30S ribosomal protein S2 P66544|RS2 Ref No Values 0 No Values No Values 0.2 3 oxoacyl [acyl carrier protein] synthase 3 P99159|FABH Ref No Values No Values No Values No Values No Values

PAGE 191

179 Appendix 11. Changes in secreted proteins of CA MRSA USA300 compared to HA MRSA USA100 during stationary phase from 3 biological replicates Identified Proteins (246) Accession Number USA10 0 # 1 USA100 # 2 USA100 # 3 USA300 # 1 USA300 # 2 USA300 # 3 Lipase 2 Q7A7P2|LIP2 Ref 0.4 1.6 0.6 3 4.8 Lipase 1 P65289|LIP1 Ref 1 0.8 0.6 1.5 2.5 Alpha Hemolysin Q7A632|Q7A63 2 Ref 0.6 0.2 0.6 4.1 4.8 Elongation factor Tu P99152|EFTU Ref 1.8 6.7 0.6 1.4 7 Enolase P99088|ENO Ref 0.6 6.6 0.6 0.3 6.8 Putative surface protein SA2285 P61598|PLS Ref 4.6 9 5.1 9.5 11.5 Bifunctional autolysin Q99V41|ATL Ref 0.1 3.1 0.7 1 2.8 SA0841 protein Q7A6G0|Q7A6 G0 Ref 1.4 3 0.6 0.5 0.3 Chaperone protein dnaK P99110|DNAK Ref 3.6 7 0.6 4.1 7.8 Glyceraldehyde 3 phosphate dehydrogenase 1 P99136|G3P1 Ref 2.5 5.6 0.6 1.8 6.1 DNA binding protein HU Q7A5J1|DBH Ref 1.7 5.1 0.2 3 4.9 Alkyl hydroperoxide reductase subunit C P99074|AHPC Ref 4.7 7 0.6 6.2 6.8 Inosine 5' monophosphate dehydrogenase P99106|IMDH Ref 0.2 5.8 0.6 1.7 5.8 Immunoglobulin G binding protein A SPA Ref No Values 0 No Values No Values 1.7 Formate -tetrahydrofolate ligase Q7A535|FTHS Ref 1.6 7.6 0.5 2.4 8.9 Dihydrolipoyl dehydrogenase P99084|DLDH Ref 0.7 7.2 0.6 0.2 7.5 50S ribosomal protein L7/L12 P99154|RL7 Ref 0.2 7.4 0.6 0.3 7.4 Pyruvate kinase Q7A559|KPYK Ref 4.6 3.7 0.9 3.8 3.6 SA0587 protein Q7A719|Q7A71 9 Ref 1.3 5 0.6 0.6 4.2 Phosphate acetyltransferase P99092|PTA Ref 2 6.2 0 2 7.8 Elongation factor G P68789|EFG Ref 1.5 6.2 0.6 0.4 5.3 Glutamine synthetase P99095|GLNA Ref 2.4 6.7 0.6 1.9 6.7

PAGE 192

180 Appendix 11. (Continued) SA2006 protein Q7A483|Q7A48 3 Ref 0.3 0.8 0.6 3.6 2.5 Fructose bisphosphate aldolase P99075|ALF2 Ref 3.8 7.4 0.6 3.3 10 Glycerol phosphate lipoteichoic acid synthase Q7A6U1|LTAS Ref 1 0.5 0.6 0.5 0.3 Alkaline shock protein 23 P99157|ASP23 Ref 2.4 3.9 0.6 3.5 2.5 Triosephosphate isomerase P99133|TPIS Ref 2 4 0.1 1.4 4.4 Enterotoxin type C 3 P0A0L4|ENTC 3 Ref 5.3 5.4 2 4.5 4.3 Dihydrolipoyllysine residue acetyltransferase component of pyruvate dehydrogenase complex P65636|ODP2 Ref 0.6 5.7 0.4 1.4 6.1 Cysteine synthase P63871|CYSK Ref 0.6 5.1 0.6 0.4 4.3 Elongation factor Ts P99171|EFTS Ref 2.5 5 0.6 3.1 5.2 Pyruvate dehydrogenase E1 component subunit alpha Q820A6|ODPA Ref 5.5 9.6 0.6 6.5 10.4 Putative uncharacterized protein SA0663 Q7A6V1|Q7A6 V1 Ref 0.1 0.2 0.6 3.4 3.6 Ornithine aminotransferase 2 P60298|OAT2 Ref 3.2 9.4 0 4.7 10.3 Staphopain B Q7A6A7|SSPB Ref 5.1 3.5 0.6 3.6 1.4 Succinyl CoA ligase [ADP forming] subunit beta P99071|SUCC Ref 1.6 6.2 0.1 1.5 6.7 Phosphocarrier protein HPr P99143|PTHP Ref 1.1 5.6 1.2 1.9 6 Putative uncharacterized protein SA0359 Q7A7J8|Q7A7J 8 Ref 5.8 6.1 0.6 4.7 5.1 Thermonuclease Q7A6P2|NUC Ref 1.6 0.9 0.6 2.8 1.6 Trigger factor P99080|TIG Ref 1.3 4.9 0.2 2.1 4.7 Aconitate hydratase P99148|ACON Ref 0.1 4.9 0.5 0.5 4.7 Adenylate kinase P99062|KAD Ref 3.1 4.8 0.6 3.7 5.1 Fructose bisphosphate aldolase class 1 P99117|ALF1 Ref 2.3 6.6 0.2 2.4 6.8 50S ribosomal protein L17 Q7A469|RL17 Ref 1.2 3.6 0.6 1.4 3.6 Glutamyl endopeptidase Q7A6A6|SSPA Ref 1.7 3.3 0.6 0.1 1.4 SA0620 protein Q7A6Y9|Q7A6 Y9 Ref 0.1 2.2 1.4 1 0.5

PAGE 193

181 Appendix 11. (Continued) 60 kDa chaperonin P99083|CH60 Ref 1.3 5.4 0.2 0.9 5.6 Probable beta lactamase Q7A4X8|Q7A4 X8 Ref 0.8 No Values 3.7 0.8 No Values Putative uncharacterized protein SA0570 Q7A735|Q7A73 5 Ref 0.1 3.9 0.6 1.5 2.9 Alcohol dehydrogenase Q7A742|ADH Ref 0.4 4.8 0.6 0.6 4.4 Catalase Q7A5T2|CATA Ref 3.8 6.9 Reference Missing 5 7.9 SA2097 protein Q7A418|Q7A41 8 Ref 1.4 2.7 1.1 0.5 2.6 Penicillin binding protein 2 prime Q7A8C6|Q7A8 C6 Ref 4.4 6.8 0.3 4.5 7.5 Putative uncharacterized protein SAP003 Q9AC87|Q9AC 87 Ref 3.5 6.8 0.6 4.8 7 Probable thiol peroxidase P99146|TPX Ref 0.7 6 0.6 0.3 8.5 30S ribosomal protein S1 Q7A5J0|RS1 Ref 0.9 5.3 0.6 1 5 L lactate dehydrogenase 1 P65256|LDH1 Ref 2.2 1.1 0.6 3.8 0.4 Phosphoglycerate kinase P99135|PGK Ref 1.6 5.4 0.6 0.5 5.7 Glucose 6 phosphate isomerase P99078|G6PI Ref 1.5 5.4 0.6 1.4 6 Transketolase P99161|TKT Ref 1.9 5.5 2.2 1 6 Pyruvate dehydrogenase E1 component subunit beta P99063|ODPB Ref No Values 8.1 1.2 Reference Missing 8.6 Glycerophosphoryl diester phosphodiesterase Q7A6H7|Q7A6 H7 Ref 2.9 4.4 0.6 3.1 4.3 Delta hemolysin P0A0M2|HLD Ref 2.5 No Values 0.6 2 No Values Phenol soluble modulin alpha 4 peptide P0C824|PSMA4 Ref 2.3 0.1 0.5 0 2.3 Foldase protein prsA P60748|PRSA Ref 3.3 5.7 0.1 2.9 5.5 Glycyl glycine endopeptidase lytM Q7A7T0|LYTM Ref 0.3 0.9 1.7 0.7 0.9 Thioredoxin P99122|THIO Ref 0.2 4.6 0.6 0.5 5.1 SA2437 protein Q7A371|Q7A37 1 Ref 10 8.7 Reference Missing 10 8.3 UPF0477 protein SA0873 Q7A6D4|Y873 Ref 4.7 5.9 0.5 4.7 6.8 Citrate synthase II Q7A561|Q7A56 1 Ref 5.5 5.8 0 8.5 6.9 6 phosphogluconate dehydrogenase, decarboxylating P63334|6PGD Ref 0.9 4.1 0.5 0.5 4.5 Superoxide dismutase [Mn/Fe] 1 P99098|SODM1 Ref 4.7 7.7 0.6 5 8.1

PAGE 194

182 Appendix 11. (Continued) Phenol soluble modulin alpha 1 peptide P0C7Y7|PSMA 1 Ref 5.3 4.2 0.9 4.1 1.7 Phosphoenolpyruvat e carboxykinase [ATP] P99128|PCKA Ref 0.6 8.4 0.6 1.4 9.4 Probable transglycosylase isaA P99160|ISAA Ref 2.4 4.1 1 3.1 4.8 Glycyl tRNA synthetase P99129|SYG Ref 0.3 1.7 0.6 1.3 1 DNA directed RNA polymerase subunit beta' P60285|RPOC Ref 2.4 3.7 0.6 1.1 5.4 Glycine cleavage system H protein P64214|GCSH Ref 0.9 6.8 0.6 1.5 5 50S ribosomal protein L30 P0A0G0|RL30 Ref 0.8 0.7 4 0 1.2 Cold shock protein cspA Q7A5P3|CSPA Ref No Values 3.9 Reference Missing No Values 3.8 Serine protease splB Q7A4Y1|SPLB Ref 0.2 4.1 0.5 3 1.5 DNA directed RNA polymerase subunit beta P60278|RPOB Ref No Values 6.1 1 No Values 5.8 2,3 bisphosphoglycerate dependent phosphoglycerate mutase P99153|GPMA Ref No Values 3.4 0.6 No Values 2.8 Dihydrolipoyllysine residue succinyltransferase component of 2 oxoglutarate dehydrogenase complex Q7A5N4|ODO2 Ref 5.2 4.6 0 4.9 5.9 Seryl tRNA synthetase P99178|SYS Ref 6.1 9.3 0.5 7.6 9.7 NAD specific glutamate dehydrogenase Q7A6H8|DHE2 Ref 2.1 3 0.6 3.2 3.4 Succinyl CoA ligase [ADP forming] subunit alpha P99070|SUCD Ref 16.8 25.2 No Values 17.5 25.3 Uncharacterized leukocidin like protein 1 Q7A4L0|LUKL 1 Ref 1.8 1.4 0.4 4 3.5 Acyl carrier protein P0A002|ACP Ref 2.5 2.9 0.1 4 3.2 50S ribosomal protein L21 Q7A583|RL21 Ref 1.8 5.2 Reference Missing 2.4 5.9 UPF0337 protein SA0772 Q7A6L9|Y772 Ref 2.6 3.3 Reference Missing 2.9 3.1 3 oxoacyl [acyl carrier protein] synthase 2 Q7A6F8|FABF Ref 2.6 3.1 Reference Missing 2.2 2.5

PAGE 195

183 Appendix 11. (Continued) 50S ribosomal protein L9 P66318|RL9 Ref 3.2 6.4 0.6 4.1 6.5 Beta lactamase Q9AC80|Q9AC 80 Ref 0.3 1.8 0.6 2.4 1.2 Thioredoxin reductase P99101|TRXB Ref No Values 7.7 Value Missing No Values 8.4 Putative dipeptidase SA1572 Q7A522|PEPV L Ref 5.8 9.3 Reference Missing 5.7 9.8 SA0916 protein Q7A696|Q7A69 6 Ref 3.9 4.7 Reference Missing Value Missing Value Missing Phosphoenolpyruvat e protein phosphotransferase Q99V14|PT1 Ref 0 5.4 0.6 1.2 4.4 30S ribosomal protein S9 P66646|RS9 Ref 2.5 6.4 0.5 Value Missing 6.4 Uncharacterized protein SA1692 P0A0K1|Y1692 Ref 0.3 1.9 0.3 1 0.1 50S ribosomal protein L11 P0A0F2|RL11 Ref 0.6 5.9 0.7 2.2 5.8 Putative universal stress protein SA1532 Q7A551|Y1532 Ref 0.6 2 0.5 2.5 1.6 1 pyrroline 5 carboxylate dehydrogenase P99076|ROCA Ref 1 4.6 0.9 2.2 6 Gamma hemolysin component C Q7A3S2|HLGC Ref 5.4 5.2 0.6 2 0.6 Serine aspartate repeat containing protein D Q7A780|SDRD Ref 0 0 No Values 2.8 1.6 Glutamyl tRNA synthetase P99170|SYE Ref 2.6 7.5 0.6 2.9 7.6 2,3 bisphosphoglycerate independent phosphoglycerate mutase P64270|GPMI Ref 1.2 4.5 0.9 1.8 4.8 D lactate dehydrogenase P99116|LDHD Ref No Values 0 No Values No Values 2.2 50S ribosomal protein L10 P99155|RL10 Ref 2.6 5 0.7 3.4 4.8 SA2202 protein Q99RL6|Q99R L6 Ref 0.1 2.5 0.4 0.1 1.5 Gamma hemolysin component A P0A072|HLGA Ref 2.2 No Values 0.6 4.3 No Values 10 kDa chaperonin P99104|CH10 Ref 2.1 5.9 0.2 2.6 6.6 Dihydroorotase P65906|PYRC Ref 1.6 3.8 0.2 2.2 2.9 UPF0457 protein SA1975.1 Q99S93|Y197A Ref 1.5 5.6 Reference Missing 1.7 5.5 Ribosome recycling factor P99130|RRF Ref 2.6 4.7 Reference Missing 3.6 3.9 Elongation factor P P99066|EFP Ref 1 5.3 0.4 2.5 5.7 Uncharacterized leukocidin like protein 2 Q99SN7|LUKL 2 Ref 0.6 No Values 0.5 2.6 No Values

PAGE 196

184 Appendix 11. (Continued) UPF0355 protein SA0372 Q7A7I6|UP355 Ref 1.6 4.6 0.6 0.7 4.1 Acetate kinase Q99TF2|ACKA Ref 2.6 2.1 0.9 4.2 0.1 Elastin binding protein ebpS Q7A5I6|EBPS Ref 2.6 5 0.4 1.7 4.2 30S ribosomal protein S6 P99142|RS6 Ref No Values 2.2 0.6 No Values 1.9 General stress protein 20U Q7A4C8|Q7A4 C8 Ref 0.9 1.4 0.6 1.2 Value Missing 50S ribosomal protein L24 P60735|RL24 Ref 2.7 7.6 Reference Missing 3 7.6 Bifunctional protein folD Q7A697|FOLD Ref No Values 0.3 0.1 No Values 1.4 50S ribosomal protein L22 Q7A460|RL22 Ref 2 4.7 0.6 1 4.1 Putative uncharacterized protein SAS040 Q7A5U6|Q7A5 U6 Ref 2.5 8 0.2 2 8.5 Gamma hemolysin component B P0A075|HLGB Ref 1 No Values 0.6 2.7 No Values Putative uncharacterized protein SA0395 Q99WG7|Q99 WG7 Ref No Values No Values 0.2 Reference Missing No Values Putative peptidyl prolyl cis trans isomerase Q7A6I1|PPI1 Ref 2.4 0.1 0.6 Reference Missing 0.2 50S ribosomal protein L1 Q99W68|RL1 Ref No Values 2.8 0.3 Reference Missing 2.6 Virulence factor esxA ESXA (+1) Ref 0.5 No Values 0.6 1.8 No Values ATP dependent Clp protease ATP binding subunit clpC Q7A797|CLPC Ref 1.9 4.7 2.4 4 4.3 SA0914 protein Q99V35|Q99V3 5 Ref 2.4 3.6 0.6 3.3 2.8 30S ribosomal protein S16 P66440|RS16 Ref 1.5 8.5 Reference Missing 1.4 8.9 Translation initiation factor IF 1 P65119|IF1 Ref 5.8 9.1 Reference Missing 8 8.7 DNA directed RNA polymerase subunit alpha P66706|RPOA Ref 1.1 5.9 0.6 1.8 5.4 Truncated beta hemplysin Q99QR7|Q99Q R7 Ref 19.6 No Values No Values 19.4 No Values Deoxyribose phosphate aldolase 1 P99102|DEOC1 (+1) Ref 3 4.4 0.9 3.6 3.9 Pyruvate carboxylase Q7A666|Q7A66 6 Ref 0 0 No Values 0.5 0.3 Uncharacterized protein SA0829 Q7A6H3|Y829 Ref 6.5 No Values Reference Missing 9.7 No Values Probable acetyl CoA acyltransferase Q7A7L2|THLA Ref 2.7 No Values 0.6 3.2 No Values Protein grpE P99086|GRPE Ref 3.6 4.6 1 5 4.6 50S ribosomal protein L3 P60449|RL3 Ref No Values No Values 0.5 No Values No Values ATP synthase subunit beta P99112|ATPB Ref 1.6 5.5 0.6 0.1 4.9

PAGE 197

185 Appendix 11. (Continued) Uncharacterized protein SA0707 Q7A6R6|Y707 Ref 2.5 5.2 0.6 1.6 5.6 Glucose specific phosphotransferase enzyme IIA component P60857|PTGA Ref No Values 0 No Values Reference Missing 0.4 Transcription elongation factor greA P99156|GREA Ref 2.1 3.6 0.4 1.7 6.2 SA0758 protein Q7A6M7 Ref 0.3 3.9 2.2 1.2 4 Chaperone protein hchA P64313|HCHA Ref 3 No Values 0.6 5 No Values 50S ribosomal protein L5 Q7A465|RL5 Ref 0.6 No Values 0.3 2.5 No Values Serine hydroxymethyltransf erase P99091|GLYA Ref 4 8 0.7 4 8.2 Methionine aminopeptidase AMPM Ref 5.7 9.4 2.9 7 9.5 50S ribosomal protein L15 P0A0F6|RL15 Ref 0.9 4.1 0.6 0.9 3.7 ATP dependent Clp protease ATP binding subunit clpL Q7A3F4|CLPL Ref 0.4 7.4 0.5 0.7 6.7 UPF0342 protein SA1663 Q7A4V3|Y1663 Ref 2.4 3.6 0.6 2.5 4.6 SA0759 protein Q7A6M6|Q7A6 M6 Ref 1.8 3.2 Reference Missing 2.5 3 Staphylokinase Q99SU7|SAK Ref 0.8 1.5 0 2.5 0.4 Iron regulated surface determinant protein A Q7A655|ISDA Ref No Values 4.7 0.2 No Values 3.7 Serine aspartate repeat containing protein E Q99W46|SDRE Ref 0 0 No Values 1.1 2.5 Leukotoxin, LukD Q99T54|Q99T5 4 Ref No Values No Values Reference Missing Reference Missing No Values Amidophosphoribos yltransferase P99164|PUR1 Ref No Values No Values 0.2 No Values No Values SA0859 protein Q7A6E5|Q7A6 E5 Ref No Values 6.3 Reference Missing No Values 8.9 Phosphoribosylform ylglycinamidine synthase 1 P99166|PURQ Ref No Values No Values Reference Missing No Values No Values Staphylococcal complement inhibitor Q99SU9|SCIN Ref No Values 1.8 Reference Missing No Values 0.2 Uncharacterized N acetyltransferase SA1019 Q99UT4|Y1019 Ref 2.5 4.4 1 2.9 4.1 50S ribosomal protein L27 P66133|RL27 Ref No Values 6.7 0.6 No Values 6.8 Adenylosuccinate lyase Q7A4Q3|PUR8 Ref 0 0 No Values 0.2 0.3

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186 Appendix 11. (Continued) Naphthoate synthase Q7A6A9|MEN B Ref 8.8 No Values Reference Missing 9 No Values Putative uncharacterized protein SA0771 Q7A6M0|Q7A6 M0 Ref 1.3 2.8 0.6 1.3 1.8 Xaa Pro dipeptidase Q99TW4|Q99T W4 Ref 5.1 8 Reference Missing 4.2 8.1 1 phosphatidylinositol phosphodiesterase precurosr Q7A888|Q7A88 8 Ref 0.7 No Values 0.4 3.1 No Values Probable glycine dehydrogenase [decarboxylating] subunit 1 P64218|GCSPA Ref No Values 0 No Values No Values 0.9 Putative aldehyde dehydrogenase AldA Q7A825|ALDA Ref No Values 0 No Values No Values 0.7 L lactate dehydrogenase 2 P99119|LDH2 Ref 1.4 4 1.1 3.3 2.7 Formate acetyltransferase Q7A7X6|PFLB Ref 1.2 4.1 0.5 0.3 3.7 Signal transduction protein TRAP Q7A4W3|TRAP Ref 0.6 0 0.2 0.5 0.9 Purine nucleoside phosphorylase Q7A4C9|Q7A4 C9 Ref No Values No Values 0.5 No Values No Values Arginyl tRNA synthetase Q99W05|SYR Ref No Values 7.3 0.8 No Values 7.7 50S ribosomal protein L13 Q7A473|RL13 Ref 1.3 No Values 0.2 0.3 No Values Probable transglycosylase sceD Q7A4F2|SCED Ref 0 No Values No Values 1.3 No Values Organic hydroperoxide resistance protein like Q7A6M9|OHR L Ref 1.8 No Values Reference Missing 1.6 No Values Serine protease splD Q7A4Y3|SPLD Ref No Values No Values 0.6 No Values No Values Putative uncharacterized protein SA1986 Q7A493|Q7A49 3 Ref No Values No Values 2.4 No Values No Values D alanine -poly(phosphoribitol) ligase subunit 2 P0A019|DLTC Ref 2.5 5.9 0.2 1.7 5.3 Immunoglobulin binding protein sbi Q99RL2|SBI Ref 3.3 6.8 0.6 4.9 7.2 Putative uncharacterized protein SA1528 Q7A553|Q7A55 3 Ref 1.7 No Values 0.4 4.2 No Values GMP synthase [glutamine hydrolyzing] P99105|GUAA Ref No Values 5.9 0.6 No Values 6.3 SA1524 protein Q7A556|Q7A55 6 Ref 1.6 3.1 0.6 2.7 4.5

PAGE 199

187 Appendix 11. (Continued) 30S ribosomal protein S11 P66357|RS11 Ref 2.3 7.6 Reference Missing 2.9 8.6 Putative uncharacterized protein SA0919 Q7A694|Q7A69 4 Ref No Values 4.8 0.4 No Values 4.3 Phenol soluble modulin alpha 3 peptide P0C811|PSMA3 Ref 0.6 No Values 0.3 0 No Values Ferritin Q7A4R2|FTN Ref No Values 0 No Values No Values 0.3 Alkyl hydroperoxide reductase subunit F P99118|AHPF Ref No Values 6.2 Value Missing No Values 6.1 Anti sigma B factor antagonist P66838|RSBV Ref No Values 3.2 0.1 No Values 2.9 Nucleoside diphosphate kinase NDK (+1) Ref 3.7 9.6 0.2 5.5 9.8 Zinc metalloproteinase aureolysin Q7A378|Q7A37 8 Ref No Values 2.5 Reference Missing Reference Missing 0.5 UPF0173 metal dependent hydrolase SA1529 P99149|Y1529 Ref 14 19.1 No Values 14.2 19.5 Pyridoxal biosynthesis lyase pdxS P60798|PDXS Ref No Values 6.5 2 No Values 5.9 Alanine dehydrogenase 2 Q99TF4|DHA2 Ref No Values 5.4 1.2 No Values 6.9 Urocanate hydratase P67417|HUTU Ref No Values 0 No Values No Values 0.9 Putative uncharacterized protein SA2309 Q7A3I0|Q7A3I 0 Ref 4.7 3.5 0.5 Reference Missing 3.4 CTP synthase P99072|PYRG Ref 0.6 5.5 0.3 0.6 5.1 Putative 8 amino 7 oxononanoate synthase/2 amino 3 ketobutyrate coenzyme A ligase P60120|BIKB Ref No Values 23 No Values No Values 24.1 Threonyl tRNA synthetase P67585|SYT Ref No Values No Values 0.2 No Values No Values Probable malate:quinone oxidoreductase 2 P99115|MQO2 Ref 0 0 No Values 0.2 0.5 Probable branched chain amino acid aminotransferase P99138|ILVE Ref 0 0 No Values 0.3 0.4 Methionyl tRNA synthetase P67579|SYM Ref No Values 0 No Values No Values 0.1 Tryptophanyl tRNA synthetase P67593|SYW Ref 0 No Values No Values 3.1 No Values Mannitol 1 phosphate 5 dehydrogenase P99140|MTLD Ref 0 No Values No Values 0.7 No Values SA1343 protein Q7A5G2|Q7A5 G2 Ref No Values 0 No Values No Values 1.7

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188 Appendix 11. (Continued) Staphopain A P65826|SSPP Ref No Values No Values No Values No Values No Values UPF0082 protein SA0624 P67182|Y624 Ref 2.2 5.3 0.6 1.8 5 Cell division protein ftsZ P99108|FTSZ Ref 2.5 3.1 0.5 3 3.6 Adenylosuccinate synthetase P99099|PURA Ref No Values 5.1 1.9 No Values 5.2 Histidine ammonia lyase P64416|HUTH Ref No Values 9.6 Reference Missing No Values 9.6 Imidazolonepropiona se P64418|HUTI Ref No Values 0 No Values No Values 1.4 6 phosphofructokinase P99165|K6PF Ref 3.2 No Values Reference Missing 0.7 No Values Lysyl tRNA synthetase P67610|SYK Ref No Values 1.3 1.8 No Values 2.3 SA1599 protein Q7A501|Q7A50 1 Ref No Values 0 No Values No Values 1.3 SA0231 protein Q7A7W3|Q7A7 W3 Ref No Values 3.4 0.2 No Values 3.9 Phenylalanyl tRNA synthetase beta chain P67041|SYFB Ref No Values 0 No Values No Values 1.5 P60855|Y370_STAA N P60855|Y370 Ref No Values 0 No Values No Values 0.7 50S ribosomal protein L23 Q7A459|RL23 Ref No Values No Values 0.5 No Values No Values Putative uncharacterized protein SA0908 Q7A6A3|Q7A6 A3 Ref 3 2.3 1 3.6 2.3 SA1475 protein Q7A581|Q7A58 1 Ref 0 0 No Values 0.7 0.3 Leucyl tRNA synthetase P67513|SYL Ref No Values 6 0.3 No Values 5.8 Clumping factor A Q99VJ4|CLFA Ref 0 0 No Values 0.2 Reference Missing Acetate CoA ligase Q7A3A2|Q7A3 A2 Ref 0 No Values No Values 0.6 No Values Lactonase drp35 RANDOM_DR P35 R Ref 0 No Values No Values 1.5 No Values 50S ribosomal protein L2 P60432|RL2 Ref No Values 0 No Values No Values 0.2 Glucosamine -fructose 6 phosphate aminotransferase [isomerizing] GLMS Ref No Values No Values Reference Missing No Values No Values Putative uncharacterized protein SA1743 Q7A4N7|Q7A4 N7 Ref No Values 0 No Values No Values 0.6 Cell division protein Q7A620|Q7A62 0 Ref No Values 0 No Values Reference Missing 0.5 50S ribosomal protein L18 Q7A467|RL18 Ref No Values 18.9 No Values No Values 18.5 3 hexulose 6 phosphate synthase Q7A774|HPS Ref No Values No Values No Values No Values No Values Acetoin(diacetyl) reductase P99120|BUTA Ref No Values 0 No Values No Values 0.2

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189 Appendix 11. (Continued) SA0022 protein Q99XE9|Q99X E9 Ref 0 No Values No Values 1.2 No Values 3 hydroxy 3 methylglutaryl CoA synthase Q7A3F6|Q7A3 F6 Ref No Values No Values 0.6 No Values No Values 30S ribosomal protein S7 P66616|RS7 Ref 1.5 No Values 0.2 1.2 No Values Uncharacterized lipoprotein SA2158 Q7A3W5|Y215 8 Ref No Values No Values Reference Missing No Values No Values Ribonuclease J 1 Q7A682|RNJ1 Ref No Values No Values 0.5 No Values No Values Trans 2 enoyl ACP reductase Q7A6D8 Ref 0 No Values No Values 0.7 No Values 2 C methyl D erythritol 4 phosphate cytidylyltransferase 2 Q7A7V0|ISPD2 Ref No Values No Values 1.6 No Values No Values 30S ribosomal protein S13 P66388|RS13 Ref No Values No Values Reference Missing No Values No Values 30S ribosomal protein S2 P66544|RS2 Ref No Values 0 No Values No Values 0.2 3 oxoacyl [acyl carrier protein] synthase 3 P99159|FABH Ref No Values No Values No Values No Values No Values

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190 Appendix 12. Changes in secreted proteins of CA MRSA USA400 compared to HA MRSA USA100 during stationary phase from 3 biological replicates Identified Proteins (246) Accession Number USA10 0 # 1 USA100 # 2 USA100 # 3 USA400 # 1 USA400 # 2 USA400 # 3 Lipase 2 Q7A7P2|LIP2 Ref 0.4 1.6 0.7 2.9 3.7 Lipase 1 P65289|LIP1 Ref 1 0.8 1.1 2.5 3.7 Alpha Hemolysin Q7A632|Q7A63 2 Ref 0.6 0.2 0.5 1 2.5 Elongation factor Tu P99152|EFTU Ref 1.8 6.7 0.9 1.7 7.3 Enolase P99088|ENO Ref 0.6 6.6 0.6 0.8 6.9 Putative surface protein SA2285 P61598|PLS Ref 4.6 9 4.2 9.2 9.7 Bifunctional autolysin Q99V41|ATL Ref 0.1 3.1 0.2 1.8 1.8 SA0841 protein Q7A6G0|Q7A6 G0 Ref 1.4 3 1.5 1.5 1 Chaperone protein dnaK P99110|DNAK Ref 3.6 7 0.4 3.6 7.7 Glyceraldehyde 3 phosphate dehydrogenase 1 P99136|G3P1 Ref 2.5 5.6 0.4 2.4 6.3 DNA binding protein HU Q7A5J1|DBH Ref 1.7 5.1 0 2.7 5.8 Alkyl hydroperoxide reductase subunit C P99074|AHPC Ref 4.7 7 1.2 6.4 6 Inosine 5' monophosphate dehydrogenase P99106|IMDH Ref 0.2 5.8 0.6 0.5 6.5 Immunoglobulin G binding protein A SPA Ref No Values 0 No Values No Values Value Missing Formate -tetrahydrofolate ligase Q7A535|FTHS Ref 1.6 7.6 0.5 2.8 8.3 Dihydrolipoyl dehydrogenase P99084|DLDH Ref 0.7 7.2 0.8 0.9 7.1 50S ribosomal protein L7/L12 P99154|RL7 Ref 0.2 7.4 0.4 0.1 7.6 Pyruvate kinase Q7A559|KPYK Ref 4.6 3.7 0.1 4.5 4.2 SA0587 protein Q7A719|Q7A71 9 Ref 1.3 5 0.9 0.8 4.8 Phosphate acetyltransferase P99092|PTA Ref 2 6.2 1.4 2.1 6.9 Elongation factor G P68789|EFG Ref 1.5 6.2 1.2 1.3 6.2 Glutamine synthetase P99095|GLNA Ref 2.4 6.7 0.4 2.4 7.3 SA2006 protein Q7A483|Q7A48 3 Ref 0.3 0.8 1.4 4.1 3.6

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191 Appendix 12. (Continued) Fructose bisphosphate aldolase P99075|ALF2 Ref 3.8 7.4 1.8 3.8 8.1 Glycerol phosphate lipoteichoic acid synthase Q7A6U1|LTAS Ref 1 0.5 0.9 0.1 1.2 Alkaline shock protein 23 P99157|ASP23 Ref 2.4 3.9 2.2 4.1 2.8 Triosephosphate isomerase P99133|TPIS Ref 2 4 0 1.7 4.7 Enterotoxin type C 3 P0A0L4|ENTC 3 Ref 5.3 5.4 4.4 1.2 0.5 Dihydrolipoyllysine residue acetyltransferase component of pyruvate dehydrogenase complex P65636|ODP2 Ref 0.6 5.7 0.7 2.3 6.4 Cysteine synthase P63871|CYSK Ref 0.6 5.1 1.1 0.3 5.1 Elongation factor Ts P99171|EFTS Ref 2.5 5 0.7 2.7 5.7 Pyruvate dehydrogenase E1 component subunit alpha Q820A6|ODPA Ref 5.5 9.6 0.5 7.2 10.3 Putative uncharacterized protein SA0663 Q7A6V1|Q7A6 V1 Ref 0.1 0.2 0.3 4.2 3.2 Ornithine aminotransferase 2 P60298|OAT2 Ref 3.2 9.4 0.9 4.4 9.7 Staphopain B Q7A6A7|SSPB Ref 5.1 3.5 0 3 0.5 Succinyl CoA ligase [ADP forming] subunit beta P99071|SUCC Ref 1.6 6.2 0 2.2 6.6 Phosphocarrier protein HPr P99143|PTHP Ref 1.1 5.6 0.8 1.9 6.3 Putative uncharacterized protein SA0359 Q7A7J8|Q7A7J 8 Ref 5.8 6.1 0.9 2.6 3.5 Thermonuclease Q7A6P2|NUC Ref 1.6 0.9 1.2 2.4 0.7 Trigger factor P99080|TIG Ref 1.3 4.9 0.2 1 5 Aconitate hydratase P99148|ACON Ref 0.1 4.9 0.9 0.8 5.2 Adenylate kinase P99062|KAD Ref 3.1 4.8 0.4 3.3 5.5 Fructose bisphosphate aldolase class 1 P99117|ALF1 Ref 2.3 6.6 0.4 2.3 7.2 50S ribosomal protein L17 Q7A469|RL17 Ref 1.2 3.6 1.4 1.5 3.7 Glutamyl endopeptidase Q7A6A6|SSPA Ref 1.7 3.3 2.7 1.3 1 SA0620 protein Q7A6Y9|Q7A6 Y9 Ref 0.1 2.2 2.2 2.1 2.9 60 kDa chaperonin P99083|CH60 Ref 1.3 5.4 0.9 1.4 5.8 Probable beta lactamase Q7A4X8|Q7A4 X8 Ref 0.8 No Values 2.9 0.4 No Values

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192 Appendix 12. (Continued) Putative uncharacterized protein SA0570 Q7A735|Q7A73 5 Ref 0.1 3.9 0.9 2.9 1.5 Alcohol dehydrogenase Q7A742|ADH Ref 0.4 4.8 0.5 0.7 5.1 Catalase Q7A5T2|CATA Ref 3.8 6.9 Reference Missing 5.3 7.6 SA2097 protein Q7A418|Q7A41 8 Ref 1.4 2.7 0.3 0.1 2.1 Penicillin binding protein 2 prime Q7A8C6|Q7A8 C6 Ref 4.4 6.8 1 3.5 5.8 Putative uncharacterized protein SAP003 Q9AC87|Q9AC 87 Ref 3.5 6.8 0.1 5.3 7.5 Probable thiol peroxidase P99146|TPX Ref 0.7 6 3.1 1.7 6.7 30S ribosomal protein S1 Q7A5J0|RS1 Ref 0.9 5.3 2 1.2 5.7 L lactate dehydrogenase 1 P65256|LDH1 Ref 2.2 1.1 1.5 3.5 0.1 Phosphoglycerate kinase P99135|PGK Ref 1.6 5.4 1.1 0.5 5.3 Glucose 6 phosphate isomerase P99078|G6PI Ref 1.5 5.4 0.9 2.1 5.8 Transketolase P99161|TKT Ref 1.9 5.5 0.6 2.3 5.9 Pyruvate dehydrogenase E1 component subunit beta P99063|ODPB Ref No Values 8.1 0.6 Reference Missing 8.7 Glycerophosphoryl diester phosphodiesterase Q7A6H7|Q7A6 H7 Ref 2.9 4.4 1 2.6 4.1 Delta hemolysin P0A0M2|HLD Ref 2.5 No Values 1.6 0.3 No Values Phenol soluble modulin alpha 4 peptide P0C824|PSMA4 Ref 2.3 0.1 2.4 1.9 4 Foldase protein prsA P60748|PRSA Ref 3.3 5.7 1.7 1.9 4.9 Glycyl glycine endopeptidase lytM Q7A7T0|LYTM Ref 0.3 0.9 0.8 1.9 2.5 Thioredoxin P99122|THIO Ref 0.2 4.6 0.2 0.6 5.4 SA2437 protein Q7A371|Q7A37 1 Ref 10 8.7 Reference Missing 8.2 6.9 UPF0477 protein SA0873 Q7A6D4|Y873 Ref 4.7 5.9 1.3 5.4 5.5 Citrate synthase II Q7A561|Q7A56 1 Ref 5.5 5.8 0.2 8.2 6.5 6 phosphogluconate dehydrogenase, decarboxylating P63334|6PGD Ref 0.9 4.1 0.9 0.5 3.9 Superoxide dismutase [Mn/Fe] 1 P99098|SODM1 Ref 4.7 7.7 0.9 3.9 8 Phenol soluble modulin alpha 1 peptide P0C7Y7|PSMA 1 Ref 5.3 4.2 3.9 1.1 0.2

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193 Appendix 12. (Continued) Phosphoenolpyruvat e carboxykinase [ATP] P99128|PCKA Ref 0.6 8.4 1.1 4.7 9.1 Probable transglycosylase isaA P99160|ISAA Ref 2.4 4.1 0.9 2.9 4.6 Glycyl tRNA synthetase P99129|SYG Ref 0.3 1.7 0.5 3.9 0.8 DNA directed RNA polymerase subunit beta' P60285|RPOC Ref 2.4 3.7 0.2 2.4 4.5 Glycine cleavage system H protein P64214|GCSH Ref 0.9 6.8 0.1 1.2 6.8 50S ribosomal protein L30 P0A0G0|RL30 Ref 0.8 0.7 0.2 0.1 1.4 Cold shock protein cspA Q7A5P3|CSPA Ref No Values 3.9 No Values No Values 4.6 Serine protease splB Q7A4Y1|SPLB Ref 0.2 4.1 0.6 1.5 2.2 DNA directed RNA polymerase subunit beta P60278|RPOB Ref No Values 6.1 0.5 No Values 5.8 2,3 bisphosphoglycerate dependent phosphoglycerate mutase P99153|GPMA Ref No Values 3.4 1.4 No Values 3.9 Dihydrolipoyllysine residue succinyltransferase component of 2 oxoglutarate dehydrogenase complex Q7A5N4|ODO2 Ref 5.2 4.6 0.3 7.1 4.7 Seryl tRNA synthetase P99178|SYS Ref 6.1 9.3 0.6 7.6 9.3 NAD specific glutamate dehydrogenase Q7A6H8|DHE2 Ref 2.1 3 0.5 2.9 3.4 Succinyl CoA ligase [ADP forming] subunit alpha P99070|SUCD Ref 16.8 25.2 No Values 17.9 25.7 Uncharacterized leukocidin like protein 1 Q7A4L0|LUKL 1 Ref 1.8 1.4 1.9 3.9 3.3 Acyl carrier protein P0A002|ACP Ref 2.5 2.9 2 2.5 Value Missing 50S ribosomal protein L21 Q7A583|RL21 Ref 1.8 5.2 Reference Missing 3 5.9 UPF0337 protein SA0772 Q7A6L9|Y772 Ref 2.6 3.3 No Values 2.1 3.9 3 oxoacyl [acyl carrier protein] synthase 2 Q7A6F8|FABF Ref 2.6 3.1 Reference Missing 2.7 2 50S ribosomal protein L9 P66318|RL9 Ref 3.2 6.4 0.4 3.5 6.8 Beta lactamase Q9AC80|Q9AC 80 Ref 0.3 1.8 1.1 2.1 0.6

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194 Appendix 12. (Continued) Thioredoxin reductase P99101|TRXB Ref No Values 7.7 Value Missing No Values 8.5 Putative dipeptidase SA1572 Q7A522|PEPV L Ref 5.8 9.3 Reference Missing 5.1 10 SA0916 protein Q7A696|Q7A69 6 Ref 3.9 4.7 Reference Missing Value Missing 0.9 Phosphoenolpyruvat e protein phosphotransferase Q99V14|PT1 Ref 0 5.4 0.7 0.1 4.4 30S ribosomal protein S9 P66646|RS9 Ref 2.5 6.4 0.9 Value Missing 6.2 Uncharacterized protein SA1692 P0A0K1|Y1692 Ref 0.3 1.9 1.7 1.9 0.7 50S ribosomal protein L11 P0A0F2|RL11 Ref 0.6 5.9 0.5 2.4 5.7 Putative universal stress protein SA1532 Q7A551|Y1532 Ref 0.6 2 1.6 1.4 1.6 1 pyrroline 5 carboxylate dehydrogenase P99076|ROCA Ref 1 4.6 0.3 3.7 5.1 Gamma hemolysin component C Q7A3S2|HLGC Ref 5.4 5.2 0.4 1.7 1.3 Serine aspartate repeat containing protein D Q7A780|SDRD Ref 0 0 No Values 2.8 0.4 Glutamyl tRNA synthetase P99170|SYE Ref 2.6 7.5 2.3 3.4 7.8 2,3 bisphosphoglycerate independent phosphoglycerate mutase P64270|GPMI Ref 1.2 4.5 0.7 Reference Missing 5 D lactate dehydrogenase P99116|LDHD Ref No Values 0 No Values No Values 0.7 50S ribosomal protein L10 P99155|RL10 Ref 2.6 5 1 3.9 4.4 SA2202 protein Q99RL6|Q99R L6 Ref 0.1 2.5 0.4 0.6 1.7 Gamma hemolysin component A P0A072|HLGA Ref 2.2 No Values 0.8 6.3 No Values 10 kDa chaperonin P99104|CH10 Ref 2.1 5.9 0.7 1.6 5.9 Dihydroorotase P65906|PYRC Ref 1.6 3.8 0.4 0.7 4.2 UPF0457 protein SA1975.1 Q99S93|Y197A Ref 1.5 5.6 No Values 1.2 5 Ribosome recycling factor P99130|RRF Ref 2.6 4.7 Reference Missing 2.2 3.7 Elongation factor P P99066|EFP Ref 1 5.3 1.4 1.6 5 Uncharacterized leukocidin like protein 2 Q99SN7|LUKL 2 Ref 0.6 No Values 0.7 3.1 No Values UPF0355 protein SA0372 Q7A7I6|UP355 Ref 1.6 4.6 Reference Missing 0.2 4.1 Acetate kinase Q99TF2|ACKA Ref 2.6 2.1 0 2.9 0.2 Elastin binding protein ebpS Q7A5I6|EBPS Ref 2.6 5 4.4 1.3 2.5

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195 Appendix 12. (Continued) 30S ribosomal protein S6 P99142|RS6 Ref No Values 2.2 2 No Values Value Missing General stress protein 20U Q7A4C8|Q7A4 C8 Ref 0.9 1.4 Value Missing 0.6 2.4 50S ribosomal protein L24 P60735|RL24 Ref 2.7 7.6 Reference Missing 3.6 8 Bifunctional protein folD Q7A697|FOLD Ref No Values 0.3 0.3 No Values Value Missing 50S ribosomal protein L22 Q7A460|RL22 Ref 2 4.7 1.8 0.8 3.7 Putative uncharacterized protein SAS040 Q7A5U6|Q7A5 U6 Ref 2.5 8 0.2 2.3 8.5 Gamma hemolysin component B P0A075|HLGB Ref 1 No Values 0.4 4.5 No Values Putative uncharacterized protein SA0395 Q99WG7|Q99 WG7 Ref No Values No Values 1.2 Reference Missing No Values Putative peptidyl prolyl cis trans isomerase Q7A6I1|PPI1 Ref 2.4 0.1 1.2 6.5 Value Missing 50S ribosomal protein L1 Q99W68|RL1 Ref No Values 2.8 2 Reference Missing 1.6 Virulence factor esxA ESXA (+1) Ref 0.5 No Values 0.2 1.2 No Values ATP dependent Clp protease ATP binding subunit clpC Q7A797|CLPC Ref 1.9 4.7 0.1 5.5 3.7 SA0914 protein Q99V35|Q99V3 5 Ref 2.4 3.6 1.5 4.9 2.1 30S ribosomal protein S16 P66440|RS16 Ref 1.5 8.5 No Values 1.2 9.2 Translation initiation factor IF 1 P65119|IF1 Ref 5.8 9.1 Reference Missing 8 8.2 DNA directed RNA polymerase subunit alpha P66706|RPOA Ref 1.1 5.9 0.2 2.6 4.9 Truncated beta hemplysin Q99QR7|Q99Q R7 Ref 19.6 No Values No Values 19.4 No Values Deoxyribose phosphate aldolase 1 P99102|DEOC1 (+1) Ref 3 4.4 1 2.3 4.1 Pyruvate carboxylase Q7A666|Q7A66 6 Ref 0 0 No Values 1.5 0.7 Uncharacterized protein SA0829 Q7A6H3|Y829 Ref 6.5 No Values Reference Missing 9 No Values Probable acetyl CoA acyltransferase Q7A7L2|THLA Ref 2.7 No Values Reference Missing 2.1 No Values Protein grpE P99086|GRPE Ref 3.6 4.6 1.3 4.6 4.1 50S ribosomal protein L3 P60449|RL3 Ref No Values No Values Value Missing No Values No Values ATP synthase subunit beta P99112|ATPB Ref 1.6 5.5 1.1 1.5 5.4 Uncharacterized protein SA0707 Q7A6R6|Y707 Ref 2.5 5.2 Reference Missing 2.4 5.9

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196 Appendix 12. (Continued) Glucose specific phosphotransferase enzyme IIA component P60857|PTGA Ref No Values 0 No Values Reference Missing 0.2 Transcription elongation factor greA P99156|GREA Ref 2.1 3.6 2 1.7 4.4 SA0758 protein Q7A6M7 Ref 0.3 3.9 0.3 2.4 4.7 Chaperone protein hchA P64313|HCHA Ref 3 No Values 0.5 5.6 No Values 50S ribosomal protein L5 Q7A465|RL5 Ref 0.6 No Values 1.2 2.7 No Values Serine hydroxymethyltransf erase P99091|GLYA Ref 4 8 0.2 4.1 8.8 Methionine aminopeptidase AMPM Ref 5.7 9.4 0.1 6.5 9.3 50S ribosomal protein L15 P0A0F6|RL15 Ref 0.9 4.1 1.5 1.1 2.8 ATP dependent Clp protease ATP binding subunit clpL Q7A3F4|CLPL Ref 0.4 7.4 1.6 1.4 5.4 UPF0342 protein SA1663 Q7A4V3|Y1663 Ref 2.4 3.6 1.3 1.2 4.3 SA0759 protein Q7A6M6|Q7A6 M6 Ref 1.8 3.2 Reference Missing 3.2 2.6 Staphylokinase Q99SU7|SAK Ref 0.8 1.5 0.9 2.9 1.2 Iron regulated surface determinant protein A Q7A655|ISDA Ref No Values 4.7 2 No Values 2.8 Serine aspartate repeat containing protein E Q99W46|SDRE Ref 0 0 No Values 0.3 0.7 Leukotoxin, LukD Q99T54|Q99T5 4 Ref No Values No Values No Values Reference Missing No Values Amidophosphoribos yltransferase P99164|PUR1 Ref No Values No Values 0.2 No Values No Values SA0859 protein Q7A6E5|Q7A6 E5 Ref No Values 6.3 No Values No Values 7.1 Phosphoribosylform ylglycinamidine synthase 1 P99166|PURQ Ref No Values No Values No Values No Values No Values Staphylococcal complement inhibitor Q99SU9|SCIN Ref No Values 1.8 Reference Missing No Values 0 Uncharacterized N acetyltransferase SA1019 Q99UT4|Y1019 Ref 2.5 4.4 1.8 2.5 3.2 50S ribosomal protein L27 P66133|RL27 Ref No Values 6.7 2.7 No Values 6.5 Adenylosuccinate lyase Q7A4Q3|PUR8 Ref 0 0 No Values 0 0.1

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197 Appendix 12. (Continued) Naphthoate synthase Q7A6A9|MEN B Ref 8.8 No Values No Values 11.1 No Values Putative uncharacterized protein SA0771 Q7A6M0|Q7A6 M0 Ref 1.3 2.8 0.9 1.2 1.9 Xaa Pro dipeptidase Q99TW4|Q99T W4 Ref 5.1 8 Reference Missing 3.2 7 1 phosphatidylinositol phosphodiesterase precurosr Q7A888|Q7A88 8 Ref 0.7 No Values 0 2.9 No Values Probable glycine dehydrogenase [decarboxylating] subunit 1 P64218|GCSPA Ref No Values 0 No Values No Values 0.4 Putative aldehyde dehydrogenase AldA Q7A825|ALDA Ref No Values 0 No Values No Values 0.5 L lactate dehydrogenase 2 P99119|LDH2 Ref 1.4 4 0.6 2.5 3.4 Formate acetyltransferase Q7A7X6|PFLB Ref 1.2 4.1 2.1 1 3.4 Signal transduction protein TRAP Q7A4W3|TRAP Ref 0.6 0 2.1 0.3 1.3 Purine nucleoside phosphorylase Q7A4C9|Q7A4 C9 Ref No Values No Values 0.4 No Values No Values Arginyl tRNA synthetase Q99W05|SYR Ref No Values 7.3 1.3 No Values 7.8 50S ribosomal protein L13 Q7A473|RL13 Ref 1.3 No Values 2.4 0.3 No Values Probable transglycosylase sceD Q7A4F2|SCED Ref 0 No Values No Values 0.6 No Values Organic hydroperoxide resistance protein like Q7A6M9|OHR L Ref 1.8 No Values Reference Missing 0.5 No Values Serine protease splD Q7A4Y3|SPLD Ref No Values No Values 0.2 No Values No Values Putative uncharacterized protein SA1986 Q7A493|Q7A49 3 Ref No Values No Values 0.4 No Values No Values D alanine -poly(phosphoribitol) ligase subunit 2 P0A019|DLTC Ref 2.5 5.9 0.6 1.2 6.4 Immunoglobulin binding protein sbi Q99RL2|SBI Ref 3.3 6.8 0.7 2.9 6.7 Putative uncharacterized protein SA1528 Q7A553|Q7A55 3 Ref 1.7 No Values 1 2.3 No Values GMP synthase [glutamine hydrolyzing] P99105|GUAA Ref No Values 5.9 0.4 No Values Value Missing SA1524 protein Q7A556|Q7A55 6 Ref 1.6 3.1 0.1 Value Missing 3.5

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198 Appendix 12. (Continued) 30S ribosomal protein S11 P66357|RS11 Ref 2.3 7.6 Reference Missing 3 8.3 Putative uncharacterized protein SA0919 Q7A694|Q7A69 4 Ref No Values 4.8 1.5 No Values 2.7 Phenol soluble modulin alpha 3 peptide P0C811|PSMA3 Ref 0.6 No Values 3.6 3 No Values Ferritin Q7A4R2|FTN Ref No Values 0 No Values No Values 0.2 Alkyl hydroperoxide reductase subunit F P99118|AHPF Ref No Values 6.2 Value Missing No Values 5.6 Anti sigma B factor antagonist P66838|RSBV Ref No Values 3.2 1.4 No Values 2.1 Nucleoside diphosphate kinase NDK (+1) Ref 3.7 9.6 1.8 5.5 9.4 Zinc metalloproteinase aureolysin Q7A378|Q7A37 8 Ref No Values 2.5 Reference Missing Reference Missing 1 UPF0173 metal dependent hydrolase SA1529 P99149|Y1529 Ref 14 19.1 No Values 13.7 Value Missing Pyridoxal biosynthesis lyase pdxS P60798|PDXS Ref No Values 6.5 0.8 No Values 5.2 Alanine dehydrogenase 2 Q99TF4|DHA2 Ref No Values 5.4 0.1 No Values Value Missing Urocanate hydratase P67417|HUTU Ref No Values 0 No Values No Values 0.2 Putative uncharacterized protein SA2309 Q7A3I0|Q7A3I 0 Ref 4.7 3.5 0.5 Reference Missing Value Missing CTP synthase P99072|PYRG Ref 0.6 5.5 0.7 0.5 4.5 Putative 8 amino 7 oxononanoate synthase/2 amino 3 ketobutyrate coenzyme A ligase P60120|BIKB Ref No Values 23 No Values No Values 23.5 Threonyl tRNA synthetase P67585|SYT Ref No Values No Values 0.1 No Values No Values Probable malate:quinone oxidoreductase 2 P99115|MQO2 Ref 0 0 No Values 0.1 0.7 Probable branched chain amino acid aminotransferase P99138|ILVE Ref 0 0 No Values 0 0.7 Methionyl tRNA synthetase P67579|SYM Ref No Values 0 No Values No Values 0.4 Tryptophanyl tRNA synthetase P67593|SYW Ref 0 No Values No Values Value Missing No Values Mannitol 1 phosphate 5 dehydrogenase P99140|MTLD Ref 0 No Values No Values 0.8 No Values SA1343 protein Q7A5G2|Q7A5 G2 Ref No Values 0 No Values No Values 0.7

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199 Appendix 12. (Continued) Staphopain A P65826|SSPP Ref No Values No Values No Values No Values No Values UPF0082 protein SA0624 P67182|Y624 Ref 2.2 5.3 1.3 1.9 5.4 Cell division protein ftsZ P99108|FTSZ Ref 2.5 3.1 1.9 3.8 Value Missing Adenylosuccinate synthetase P99099|PURA Ref No Values 5.1 0.5 No Values 4.8 Histidine ammonia lyase P64416|HUTH Ref No Values 9.6 Reference Missing No Values 9.8 Imidazolonepropiona se P64418|HUTI Ref No Values 0 No Values No Values 0.7 6 phosphofructokinase P99165|K6PF Ref 3.2 No Values Reference Missing 1.2 No Values Lysyl tRNA synthetase P67610|SYK Ref No Values 1.3 0.4 No Values 3.5 SA1599 protein Q7A501|Q7A50 1 Ref No Values 0 No Values No Values 1.5 SA0231 protein Q7A7W3|Q7A7 W3 Ref No Values 3.4 Value Missing No Values 3.8 Phenylalanyl tRNA synthetase beta chain P67041|SYFB Ref No Values 0 No Values No Values 0.7 P60855|Y370_STAA N P60855|Y370 Ref No Values 0 No Values No Values 0.7 50S ribosomal protein L23 Q7A459|RL23 Ref No Values No Values 1.2 No Values No Values Putative uncharacterized protein SA0908 Q7A6A3|Q7A6 A3 Ref 3 2.3 1.5 5 1.1 SA1475 protein Q7A581|Q7A58 1 Ref 0 0 No Values 3.1 0.7 Leucyl tRNA synthetase P67513|SYL Ref No Values 6 0.9 No Values 6.1 Clumping factor A Q99VJ4|CLFA Ref 0 0 No Values 0.7 Value Missing Acetate CoA ligase Q7A3A2|Q7A3 A2 Ref 0 No Values No Values 0.2 No Values Lactonase drp35 RANDOM_DR P35 R Ref 0 No Values No Values Value Missing No Values 50S ribosomal protein L2 P60432|RL2 Ref No Values 0 No Values No Values 0.1 Glucosamine -fructose 6 phosphate aminotransferase [isomerizing] GLMS Ref No Values No Values No Values No Values No Values Putative uncharacterized protein SA1743 Q7A4N7|Q7A4 N7 Ref No Values 0 No Values No Values 2 Cell division protein Q7A620|Q7A62 0 Ref No Values 0 No Values Reference Missing 0.7 50S ribosomal protein L18 Q7A467|RL18 Ref No Values 18.9 No Values No Values 19.2 3 hexulose 6 phosphate synthase Q7A774|HPS Ref No Values No Values No Values No Values No Values Acetoin(diacetyl) reductase P99120|BUTA Ref No Values 0 No Values No Values 0.5

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200 Appendix 12. (Continued) SA0022 protein Q99XE9|Q99X E9 Ref 0 No Values No Values 1.4 No Values 3 hydroxy 3 methylglutaryl CoA synthase Q7A3F6|Q7A3 F6 Ref No Values No Values 0.5 No Values No Values 30S ribosomal protein S7 P66616|RS7 Ref 1.5 No Values 0.8 0.3 No Values Uncharacterized lipoprotein SA2158 Q7A3W5|Y215 8 Ref No Values No Values No Values No Values No Values Ribonuclease J 1 Q7A682|RNJ1 Ref No Values No Values 0.5 Reference Missing No Values Trans 2 enoyl ACP reductase Q7A6D8 Ref 0 No Values No Values 1.2 No Values 2 C methyl D erythritol 4 phosphate cytidylyltransferase 2 Q7A7V0|ISPD2 Ref No Values No Values 0.7 No Values No Values 30S ribosomal protein S13 P66388|RS13 Ref No Values No Values Reference Missing No Values No Values 30S ribosomal protein S2 P66544|RS2 Ref No Values 0 No Values No Values 0.9 3 oxoacyl [acyl carrier protein] synthase 3 P99159|FABH Ref No Values No Values No Values No Values No Values

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201 Appendix 13. Statistical analyses of the first biological replicate comparing HA MRSA USA100 and HA MRSA USA200 from post exponential phase Ten Most Abundant Proteins Accession Number Averag e Standard Deviation Relative Standard Deviation Standard Error Alpha Hemolysin Q7A632|Q7A632_STAA N 6.97556 2 0.483219009 6.927312411 0.2789866 2 Bifunctional autolysin Q99V41|ATL_STAAN 5.10456 9 0.956585247 18.73978299 0.5522847 5 Glycerol phosphate lipoteichoic acid synthase Q7A6U1|LTAS_STAAN 4.94771 9 0.572118202 11.56327181 0.3303125 9 Lipase 1 P65289|LIP1_STAAN 5.20026 7 0.790831535 15.20751659 0.4565868 Lipase 2 Q7A7P2|LIP2_STAAN 6.21763 0 0.485646794 7.810801323 0.2803883 0 Penicillin binding protein 2' Q7A8C6|Q7A8C6_STAA N 18.7176 8 4.147290327 22.15706589 2.3944391 8 Probable transglycosylase isaA P99160|ISAA_STAAN 1.46850 5 0.299009925 20.36151418 0.1726334 6 Putative surface protein SA2285 P61598|PLS_STAAN 0.15786 1 0.043353443 27.46291637 0.0250301 2 SA0841 protein Q7A6G0|Q7A6G0_STA AN 19.0509 2 3.570102378 18.73978299 2.0611995 6 Thermonuclease Q7A6P2|NUC_STAAN 5.17655 9 0.538252457 10.39787968 0.3107602 0 Least Abundant Protein Enolase P99088|ENO_STAAN 1.42085 9 0.164297809 11.56327181 0. 0948573 8 Relative Standard Deviation Average 15.53919237

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202 Appendix 14. Statistical analyses of the second biological replicate comparing HA MRSA USA100 and HA MRSA USA200 from post exponential phase Ten Most Abundant Proteins Accession Number Averag e Standard Deviation Relative Standard Deviation Standard Error Alpha Hemolysin Q7A632|Q7A632_STAA N 1.55197 9 0.062808912 4.047019914 0.0362627 4 Bifunctional autolysin Q99V41|ATL_STAAN 1.07338 2 0.273251875 25.45708615 0.1577620 4 Glycerol phosphate lipoteichoic acid synthase Q7A6U1|LTAS_STAAN 0.81606 9 0.094364312 11.56327181 0.0544812 6 Lipase 1 P65289|LIP1_STAAN 1.26257 1 0.098616943 7.810801323 0.0569365 1 Lipase 2 Q7A7P2|LIP2_STAAN 1.70502 5 0.139465418 8.179669083 0.0805203 9 Penicillin binding protein 2' Q7A8C6|Q7A8C6_STAA N 3.49856 5 0.40454866 11.56327181 0.2335662 7 Probable transglycosylase isaA P99160|ISAA_STAAN 0.75190 4 0.163402663 21.73185194 0.0943405 7 Putative surface protein SA2285 P61598|PLS_STAAN 0.09079 6 0.00975663 10.74561596 0.0056329 9 SA0841 protein Q7A6G0|Q7A6G0_STAA N 3.32674 0 0.13463385 4.047019914 0.0777308 8 Thermonuclease Q7A6P2|NUC_STAAN 2.16692 8 0.377881349 17.43856716 0.2181698 9 Least Abundant Protein Enolase P99088|ENO_STAAN 1.13177 3 0.17211471 15.20751659 0.0993704 7 Relative Standard Deviation Average 12.52651742

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203 Appendix 15. Statistical analyses of the third biological replicate comparing HA MRSA USA100 and HA MRSA USA200 from post exponential phase Ten Most Abundant Proteins Accession Number Averag e Standard Deviation Relative Standard Deviation Standard Error Alpha Hemolysin Q7A632|Q7A632_STAA N 11.3863 1.574696933 13.82975009 0.9091516 9 Bifunctional autolysin Q99V41|ATL_STAAN 2.31777 0.703685578 30.36035003 0.4062730 5 Glycerol phosphate lipoteichoic acid synthase Q7A6U1|LTAS_STAAN 3.06652 0.58416065 19.04959703 0.3372653 0 Lipase 1 P65289|LIP1_STAAN 6.45108 1.345409444 20.85556249 0.7767725 0 Lipase 2 Q7A7P2|LIP2_STAAN 5.66591 0.392495798 6.927312411 0.2266075 5 Penicillin binding protein 2' Q7A8C6|Q7A8C6_STAA N 8.42533 2.547636686 30.23782481 1.4708787 2 Probable transglycosylase isaA P99160|ISAA_STAAN 1.26059 0.051016689 4.047019914 0.029454 4 9 Putative surface protein SA2285 P61598|PLS_STAAN 0.09355 0.02793254 29.85727152 0.0161268 5 SA0841 protein Q7A6G0|Q7A6G0_STAA N 6.13305 1.168321299 19.04959703 0.6745306 1 Thermonuclease Q7A6P2|NUC_STAAN 4.42601 0.737871913 16.67126423 0.4260105 4 Least Abundant Protein Enolase P99088|ENO_STAAN 0.9137 0.074737667 8.179669083 0.0431498 1 Relative Standard Deviation Average 18.09683806

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204 Appendix 16. Statistical analyses of the first biological replicate comparing HA MRSA USA100 and CA MRSA USA300 from post exponential phase Ten Most Abundant Proteins Accession Number Averag e Standard Deviation Relative Standard Deviation Standard Error Alpha Hemolysin Q7A632|Q7A632_STAA N 20.6408 0.816267 3.954618 0.471272 Bifunctional autolysin Q99V41|ATL_STAAN 3.60024 0.636098 17.66821 0.367252 Glycerol phosphate lipoteichoic acid synthase Q7A6U1|LTAS_STAAN 3.48220 0 0 0 Lipase 1 P65289|LIP1_STAAN 12.7267 1.041007 8.179669 0.601025 Lipase 2 Q7A7P2|LIP2_STAAN 8.29239 1.553977 18.73978 0.897189 Penicillin binding protein 2' Q7A8C6|Q7A8C6_STAA N 5.92752 0.234411 3.954618 0.135337 Probable transglycosylase isaA P99160|ISAA_STAAN 0.89696 0.125036 13.93987 0.072189 Putative surface protein SA2285 P61598|PLS_STAAN 0.21798 0.015101 6.927312 0.008718 SA0841 protein Q7A6G0|Q7A6G0_STAA N 6.13305 1.168321 19.0496 0.674531 Thermonuclease Q7A6P2|NUC_STAAN 10.4798 2.185624 20.85556 1.261871 Least Abundant Protein Enolase P99088|ENO_STAAN 1.10097 0.118306 10.74562 0.068304 Relative Standard Deviation Average 11.27408

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205 Appendix 17. Statistical analyses of the second biological replicate comparing HA MRSA USA100 and CA MRSA USA300 from post exponential phase Ten Most Abundant Proteins Accession Number Averag e Standard Deviation Relative Standard Deviation Standard Error Alpha Hemolysin Q7A632|Q7A632_STAA N 12.4560 1.338483 10.74562 0.772773 Bifunctional autolysin Q99V41|ATL_STAAN 1.15527 0.538799 46.63829 0.311076 Glycerol phosphate lipoteichoic acid synthase Q7A6U1|LTAS_STAAN 3.41005 0.278931 8.179669 0.161041 Lipase 1 P65289|LIP1_STAAN 14.2620 0.577188 4.04702 0.33324 Lipase 2 Q7A7P2|LIP2_STAAN 15.8009 4.717723 29.85727 2.723778 Penicillin binding protein 2' Q7A8C6|Q7A8C6_STA AN 4.40974 1.812254 41.09659 1.046306 Probable transglycosylase isaA P99160|ISAA_STAAN 0.33369 0.063568 19.0496 0.036701 Putative surface protein SA2285 P61598|PLS_STAAN 0.1252 0.008673 6.927312 0.005007 SA0841 protein Q7A6G0|Q7A6G0_STA AN 8.46258 1.44561 17.08238 0.834623 Thermonuclease Q7A6P2|NUC_STAAN 7.36710 1.258477 17.08238 0.726582 Least Abundant Protein Enolase P99088|ENO_STAAN 2.05105 0.160204 7.810801 0.092494 Relative Standard Deviation Average 18.95608

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206 Appendix 18. Statistical analyses of the third biological replicate comparing HA MRSA USA100 and CA MRSA USA300 from post exponential phase Ten Most Abundant Proteins Accession Number Averag e Standard Deviation Relative Standard Deviation Standard Error Alpha Hemolysin Q7A632|Q7A632_STAA N 4.25341 0.924345 21.73185 0.533671 Bifunctional autolysin Q99V41|ATL_STAAN 0.47842 0.037369 7.810801 0.021575 Glycerol phosphate lipoteichoic acid synthase Q7A6U1|LTAS_STAAN 0.41789 0.11444 27.38478 0.066072 Lipase 1 P65289|LIP1_STAAN 2.25452 0.672825 29.84336 0.388456 Lipase 2 Q7A7P2|LIP2_STAAN 1.74389 0.120805 6.927312 0.069747 Penicillin binding protein 2' Q7A8C6|Q7A8C6_STA AN 1.10097 0.118306 10.74562 0.068304 Probable transglycosylase isaA P99160|ISAA_STAAN 0.17882 0.034065 19.0496 0.019667 Putative surface protein SA2285 P61598|PLS_STAAN 0.06506 0.015199 23.35989 0.008775 SA0841 protein Q7A6G0|Q7A6G0_STA AN 1.29216 0.105695 8.179669 0.061023 Thermonuclease Q7A6P2|NUC_STAAN 0.87194 0.060402 6.927312 0.034873 Least Abundant Protein Enolase P99088|ENO_STAAN 0.40873 0.056527 13.82975 0.032636 Relative Standard Deviation Average 15.9809

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207 Appendix 19. Statistical analyses of the first biological replicate comparing HA MRSA USA100 and CA MRSA USA400 from post exponential phase Ten Most Abundant Proteins Accession Number Averag e Standard Deviation Relative Standard Deviation Standard Error Alpha Hemolysin Q7A632|Q7A632_STAA N 1.01446 0.20982 20.68284 0.12114 Bifunctional autolysin Q99V41|ATL_STAAN 2.17455 0.44976 20.68284 0.259669 Glycerol phosphate lipoteichoic acid synthase Q7A6U1|LTAS_STAAN 1.13374 0.193671 17.08238 0.111816 Lipase 1 P65289|LIP1_STAAN 1.05598 0.160589 15.20752 0.092716 Lipase 2 Q7A7P2|LIP2_STAAN 3.41005 0.278931 8.179669 0.161041 Penicillin binding protein 2' Q7A8C6|Q7A8C6_STAA N 1.09741 0.044413 4.04702 0.025642 Probable transglycosylase isaA P99160|ISAA_STAAN 0.58809 0.0238 4.04702 0.013741 Putative surface protein SA2285 P61598|PLS_STAAN 0.13222 0.022588 17.08238 0.013041 SA0841 protein Q7A6G0|Q7A6G0_STAA N 1.48431 0.121412 8.179669 0.070097 Thermonuclease Q7A6P2|NUC_STAAN 1.67714 0.279601 16.67126 0.161428 Least Abundant Protein Enolase P99088|ENO_STAAN 0.93762 0.116176 12.39047 0.067074 Relative Standard Deviation Average 13.11391

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208 Appendix 20. Statistical analyses of the second biological replicate comparing HA MRSA USA100 and CA MRSA USA400 from post exponential phase Ten Most Abundant Proteins Accession Number Averag e Standard Deviation Relative Standard Deviation Standard Error Alpha Hemolysin Q7A632|Q7A632_STAA N 1.26468 0.135898 10.74562 0.078461 Bifunctional autolysin Q99V41|ATL_STAAN 0.85670 0.127044 14.82936 0.073349 Glycerol phosphate lipoteichoic acid synthase Q7A6U1|LTAS_STAAN 3.11402 0.334621 10.74562 0.193193 Lipase 1 P65289|LIP1_STAAN 10.5729 0.732423 6.927312 0.422865 Lipase 2 Q7A7P2|LIP2_STAAN 3.32674 0.134634 4.04702 0.077731 Penicillin binding protein 2' Q7A8C6|Q7A8C6_STAA N 3.28699 1.428436 43.45718 0.824708 Probable transglycosylase isaA P99160|ISAA_STAAN 1.58824 0.062809 3.954618 0.036263 Putative surface protein SA2285 P61598|PLS_STAAN 0.11620 0.027602 23.75351 0.015936 SA0841 protein Q7A6G0|Q7A6G0_STAA N 5.79219 0.234411 4.04702 0.135337 Thermonuclease Q7A6P2|NUC_STAAN 2.04051 0.481623 23.603 0.278065 Least Abundant Protein Enolase P99088|ENO_STAAN 0.56152 0.022206 3.954618 0.012821 Relative Standard Deviation Average 13.64226

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209 Appendix 21. Statistical analyses of the third biological replicate comparing HA MRSA USA100 and CA MRSA USA400 from post exponential phase Ten Most Abundant Proteins Accession Number Averag e Standard Deviation Relative Standard Deviation Standard Error Alpha Hemolysin Q7A632|Q7A632_STAA N 3 .31918 0.860895 25.93696 0.497038 Bifunctional autolysin Q99V41|ATL_STAAN 2.15409 0.266903 12.39047 0.154096 Glycerol phosphate lipoteichoic acid synthase Q7A6U1|LTAS_STAAN 2.10945 0.312818 14.82936 0.180606 Lipase 1 P65289|LIP1_STAAN 11.4446 2.180165 19.0496 1.258719 Lipase 2 Q7A7P2|LIP2_STAAN 5.16867 0.42278 8.179669 0.244092 Penicillin binding protein 2' Q7A8C6|Q7A8C6_STAA N 2.00154 0.555139 27.7356 0.32051 Probable transglycosylase isaA P99160|ISAA_STAAN 1.17618 0.0476 4.04702 0.027482 Putative surface protein SA2285 P61598|PLS_STAAN 0.14702 0.00595 4.04702 0.003435 SA0841 protein Q7A6G0|Q7A6G0_STAA N 4.82990 0.502207 10.39788 0.28995 Thermonuclease Q7A6P2|NUC_STAAN 2.83787 0.759821 26.77427 0.438683 Least Abundant Protein Enolase P99088|ENO_STAAN 1.29414 0.134563 10.39788 0.07769 Relative Standard Deviation Average 14.88961

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210 Appendix 22. Statistical analyses of the first biological replicate comparing HA MRSA USA100 and HA MRSA USA200 from stationary phase Ten Most Abundant Proteins Accession Number Averag e Standard Deviation Relative Standard Deviation Standard Error Alpha Hemolysin Q7A632|Q7A632_STAA N 2.58828 0.269126 10.39788 0.15538 DNA binding protein HU Q7A5J1|DBH_STAAN 0.52736 0.078205 14.82936 0.045151 Elongation factor Tu P99152|EFTU_STAAN 1.00641 0.139185 13.82975 0.080358 Enolase P99088|ENO_STAAN 1.38491 0.113281 8.179669 0.065403 Formate -tetrahydrofolate ligase Q7A535|FTHS_STAAN 0.73816 0.181347 24.5674 0.104701 Glyceraldehyde 3 phosphate dehydrogenase 1 P99136|G3P1_STAAN 2.85928 0.498617 17.43857 0.287877 Lipase 1 P65289|LIP1_STAAN 9.46840 1.319883 13.93987 0.762035 Lipase 2 Q7A7P2|LIP2_STAAN 3.10395 0.125618 4.04702 0.072525 Putative surface protein SA2285 P61598|PLS_STAAN 0.11164 0.038674 34.64102 0.022329 SA0841 protein Q7A6G0|Q7A6G0_STAA N 12.1451 0.841333 6.927312 0.485744 Least Abundant Protein Serine protease splB Q7A4Y1|SPLB_STAAN 2.58010 0.102033 3.954618 0.058909 Relative Standard Deviation Average 13.88659

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211 Appendix 23. Statistical analyses of the second biological replicate comparing HA MRSA USA100 and HA MRSA USA200 from stationary phase Ten Most Abundant Proteins Accession Number Averag e Standard Deviation Relative Standard Deviation Standard Error Alpha Hemolysin Q7A632|Q7A632_STAAN 1.12305 0.044413 3.954618 0.025642 DNA binding protein HU Q7A5J1|DBH_STAAN 1.04784 0.041438 3.954618 0.023924 Elongation factor Tu P99152|EFTU_STAAN 1.15053 0.079701 6.927312 0.046016 Enolase P99088|ENO_STAAN 1.15606 0.159881 13.82975 0.092308 Formate -tetrahydrofolate ligase Q7A535|FTHS_STAAN 1.17999 0.126798 10.74562 0.073207 Glyceraldehyde 3 phosphate dehydrogenase 1 P99136|G3P1_STAAN 1.20366 0.0476 3.954618 0.027482 Lipase 1 P65289|LIP1_STAAN 1.59106 0.426635 26.81436 0.246318 Lipase 2 Q7A7P2|LIP2_STAAN 1.20563 0.098617 8.179669 0.056937 Putative surface protein SA2285 P61598|PLS_STAAN 0.09647 0.021376 22.15707 0.012342 SA0841 protein Q7A6G0|Q7A6G0_STAA N 1.55701 0.16731 10.74562 0.096597 Least Abundant Protein Serine protease splB Q7A4Y1|SPLB_STAAN 1.35545 0.145652 10.74562 0.084092 Relative Standard Deviation Average 11.09171

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212 Appendix 24. Statistical analyses of the third biological replicate comparing HA MRSA USA100 and HA MRSA USA200 from stationary phase Ten Most Abundant Proteins Accession Number Averag e Standard Deviation Relative Standard Deviation Standard Error Alpha Hemolysin Q7A632|Q7A632_STAA N 0.60183 0.0238 3.954618 0.013741 DNA binding protein HU Q7A5J1|DBH_STAAN 0.16219 0.072474 44.68319 0.041843 Elongation factor Tu P99152|EFTU_STAAN 0.07019 0.002776 3.954618 0.001603 Enolase P99088|ENO_STAAN 0.08088 0.00841 10.39788 0.004856 Formate -tetrahydrofolate ligase Q7A535|FTHS_STAAN 0.25040 0.017346 6.927312 0.010015 Glyceraldehyde 3 phosphate dehydrogenase 1 P99136|G3P1_STAAN 0.0599 3 0.006437 10.74562 0.003716 Lipase 1 P65289|LIP1_STAAN 0.49038 0.05099 10.39788 0.029439 Lipase 2 Q7A7P2|LIP2_STAAN 0.58901 0.046006 7.810801 0.026562 Putative surface protein SA2285 P61598|PLS_STAAN 0.05076 0 0 0 SA0841 protein Q7A6G0|Q7A6G0_STAA N 0.3382 8 0.026424 7.810801 0.015256 Least Abundant Protein Serine protease splB Q7A4Y1|SPLB_STAAN 0.32728 0.071125 21.73185 0.041064 Relative Standard Deviation Average 11.67405

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213 Appendix 25. Statistical analyses of the first biological replicate comparing HA MRSA USA100 and CA MRSA USA300 from stationary phase Ten Most Abundant Proteins Accession Number Averag e Standard Deviation Relative Standard Deviation Standard Error Alpha Hemolysin Q7A632|Q7A632_STAA N 15.6923 1.631676 10.39788 0.942049 DNA binding protein HU Q7A5J1|DBH_STAAN 0.77599 0.031404 4.04702 0.018131 Elongation factor Tu P99152|EFTU_STAAN 2.19483 0.088825 4.04702 0.051283 Enolase P99088|ENO_STAAN 1.92932 0.340877 17.66821 0.196805 Formate -tetrahydrofolate ligase Q7A535|FTHS_STAAN 1.71544 0.260877 15.20752 0.150617 Glyceraldehyde 3 phosphate dehydrogenase 1 P99136|G3P1_STAAN 5.61599 1.171247 20.85556 0.67622 Lipase 1 P65289|LIP1_STAAN 5.68343 0.657191 11.56327 0.37943 Lipase 2 Q7A7P2|LIP2_STAAN 7.87275 1.167478 14.82936 0.674044 Putative surface protein SA2285 P61598|PLS_STAAN 0.11324 0.035899 31.69967 0.020726 SA0841 protein Q7A6G0|Q7A6G0_STAA N 5.16021 0.204067 3.954618 0.117818 Least Abundant Protein Serine protease splB Q7A4Y1|SPLB_STAAN 16.7655 0.663015 3.954618 0.382792 Relative Standard Deviation Average 12.56589

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214 Appendix 26. Statistical analyses of the second biological replicate comparing HA MRSA USA100 and CA MRSA USA300 from stationary phase Ten Most Abundant Proteins Accession Number Averag e Standard Deviation Relative Standard Deviation Standar d Error Alpha Hemolysin Q7A632|Q7A632_STAA N 14.2620 0.577188 4.04702 0.33324 DNA binding protein HU Q7A5J1|DBH_STAAN 10.0847 0.408134 4.04702 0.235636 Elongation factor Tu P99152|EFTU_STAAN 16.1745 2.820606 17.43857 1.628478 Enolase P99088|ENO_STAAN 15.0243 2.077825 13.82975 1.199633 Formate -tetrahydrofolate ligase Q7A535|FTHS_STAAN 9.30818 3.193771 34.31142 1.843925 Glyceraldehyde 3 phosphate dehydrogenase 1 P99136|G3P1_STAAN 10.9418 2.050485 18.73978 1.183848 Lipase 1 P65289|LIP1_STAAN 18.4655 2.135221 11.56327 1.23277 Lipase 2 Q7A7P2|LIP2_STAAN 19.6983 0 0 0 Putative surface protein SA2285 P61598|PLS_STAAN 0.46480 0.110408 23.75351 0.063744 SA0841 protein Q7A6G0|Q7A6G0_STA AN 19.6983 0 0 0 Least Abundant Protein Serine protease splB Q7A4Y1|SPLB_STAAN 11.4446 2.180165 19.0496 1.258719 Relative Standard Deviation Average 13.34363

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215 Appendix 27. Statistical analyses of the third biological replicate comparing HA MRSA USA100 and CA MRSA USA300 from stationary phase Ten Most Abundant Proteins Accession Number Averag e Standard Deviation Relative Standard Deviation Standard Error Alpha Hemolysin Q7A632|Q7A632_STAA N 7.15414 0.768757 10.74562 0.443842 DNA binding protein HU Q7A5J1|DBH_STAAN 0.37107 0.030353 8.179669 0.017524 Elongation factor Tu P99152|EFTU_STAAN 0.21798 0.015101 6.927312 0.008718 Enolase P99088|ENO_STAAN 0.30827 0.021355 6.927312 0.01233 Formate -tetrahydrofolate ligase Q7A535|FTHS_STAAN 0.23440 0.029044 12.39047 0.016769 Glyceraldehyde 3 phosphate dehydrogenase 1 P99136|G3P1_STAAN 0.17706 0.012265 6.927312 0.007081 Lipase 1 P65289|LIP1_STAAN 4.01386 1.149492 28.63803 0.663659 Lipase 2 Q7A7P2|LIP2_STAAN 6.71829 1.187002 17.66821 0.685316 Putative surface protein SA2285 P61598|PLS_STAAN 0.06129 0.006374 10.39788 0.00368 SA0841 protein Q7A6G0|Q7A6G0_STAA N 1.95856 0.160204 8.179669 0.092494 Least Abundant Protein Serine protease splB Q7A4Y1|SPLB_STAAN 1.74389 0.120805 6.927312 0.069747 Relative Standard Deviation Average 11.26443

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216 Appendix 28. Statistical analyses of the first biological replicate comparing HA MRSA USA100 and CA MRSA USA400 from stationary phase Ten Most Abundant Proteins Accession Number Averag e Standard Deviation Relative Standard Deviation Standar d Error Alpha Hemolysin Q7A632|Q7A632_STAA N 1.74110 1 0 0 0 DNA binding protein HU Q7A5J1|DBH_STAAN 0.63029 9 0.025508 4.04702 0.014727 Elongation factor Tu P99152|EFTU_STAAN 1.12489 7 0.092013 8.179669 0.053124 Enolase P99088|ENO_STAAN 0.96134 2 0.13401 13.93987 0.077371 Formate -tetrahydrofolate ligase Q7A535|FTHS_STAAN 0.79542 2 0.065063 8.179669 0.037564 Glyceraldehyde 3 phosphate dehydrogenase 1 P99136|G3P1_STAAN 1.38702 5 0.144221 10.39788 0.083266 Lipase 1 P65289|LIP1_STAAN 6.99713 1 0.809097 11.56327 0.467133 Lipase 2 Q7A7P2|LIP2_STAAN 5.66591 7 0.392496 6.927312 0.226608 Putative surface protein SA2285 P61598|PLS_STAAN 0.08485 9 0.023239 27.38478 0.013417 SA0841 protein Q7A6G0|Q7A6G0_STA AN 5.53058 0.218713 3.954618 0.126274 Least Abundant Protein Serine protease splB Q7A4Y1|SPLB_STAAN 4.39652 9 0.343404 7.810801 0.198264 Relative Standard Deviation Average 9.307717

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217 Appendix 29. Statistical analyses of the second biological replicate comparing HA MRSA USA100 and CA MRSA USA400 from stationary phase Ten Most Abundant Proteins Accession Number Averag e Standard Deviation Relative Standard Deviation Standard Error Alpha Hemolysin Q7A632|Q7A632_STAA N 1.41647 0.098124 6.927312 0.056652 DNA binding protein HU Q7A5J1|DBH_STAAN 1.1505 9 0.079701 6.927312 0.046016 Elongation factor Tu P99152|EFTU_STAAN 1.78853 0.192189 10.74562 0.11096 Enolase P99088|ENO_STAAN 1.76009 0.306935 17.43857 0.177209 Formate -tetrahydrofolate ligase Q7A535|FTHS_STAAN 1.49338 0.227107 15.20752 0.13112 Glyceraldehyde 3 phosphate dehydrogenase 1 P99136|G3P1_STAAN 1.66876 0.179319 10.74562 0.10353 Lipase 1 P65289|LIP1_STAAN 2.04784 0.082877 4.04702 0.047849 Lipase 2 Q7A7P2|LIP2_STAAN 1.82740 0.149475 8.179669 0.0863 Putative surface protein SA2285 P61598|PLS_STAAN 0.14046 0.064804 46.13595 0.037415 SA0841 protein Q7A6G0|Q7A6G0_STAA N 2.58010 0.102033 3.954618 0.058909 Least Abundant Protein Serine protease splB Q7A4Y1|SPLB_STAAN 1.87803 0.259728 13.82975 0.149954 Relative Standard Deviation Average 13.10354

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218 Appendix 30. Statistical analyses of the third biological replicate comparing HA MRSA USA100 and CA MRSA USA400 from stationary phase Ten Most Abundant Proteins Accession Number Averag e Standard Deviation Relative Standard Deviation Standar d Error Alpha Hemolysin Q7A632|Q7A632_STAA N 0.72515 0.056641 7.810801 0.032701 DNA binding protein HU Q7A5J1|DBH_STAAN 0.17286 0.067446 39.01742 0.03894 Elongation factor Tu P99152|EFTU_STAAN 0.08880 0.010269 11.56327 0.005929 Enolase P99088|ENO_STAAN 0.12221 0.004833 3.954618 0.00279 Formate -tetrahydrofolate ligase Q7A535|FTHS_STAAN 0.23961 0.025748 10.74562 0.014866 Glyceraldehyde 3 phosphate dehydrogenase 1 P99136|G3P1_STAAN 0.07031 0.018855 26.81436 0.010886 Lipase 1 P65289|LIP1_STAAN 2.60351 0.817272 31.39105 0.471852 Lipase 2 Q7A7P2|LIP2_STAAN 1.70223 0.067317 3.954618 0.038865 Putative surface protein SA2285 P61598|PLS_STAAN 0.05076 0 0 0 SA0841 protein Q7A6G0|Q7A6G0_STA AN 1.35107 0.054678 4.04702 0.031569 Least Abundant Protein Serine protease splB Q7A4Y1|SPLB_STAAN 0.45432 0.196599 43.27277 0.113506 Relative Standard Deviation 16.59741