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Investigating the mode of action of a novel _n-sec_-butylthiolated beta-lactam against _staphylococcus aureus_

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Title:
Investigating the mode of action of a novel _n-sec_-butylthiolated beta-lactam against _staphylococcus aureus_
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English
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Prosen, Katherine
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University of South Florida
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MRSA
Antibiotic resistance
Fatty acid synthesis inhibitors
NsBL
Beta-lactams
Dissertations, Academic -- Biology -- Masters -- USF   ( lcsh )
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non-fiction   ( marcgt )

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Abstract:
ABSTRACT: N-sec-butylthioloated beta-lactam (NsBL) is a novel beta-lactam antimicrobial with a mechanism of action proposed to inhibit 3-oxoacyl-acyl carrier protein synthase (ACP) III (FabH), resulting in the inhibition of fatty acid synthesis. It has been suggested that NsβL inhibits FabH indirectly by inactivating coenzyme-A (CoA). CoA is an essential cofactor for numerous proteins involved in glycolysis, the citric acid cycle (TCA), and pyruvate metabolism, in addition to fatty acid biosynthesis. This study aimed to determine the effects of NsBL on a diverse array of laboratory and clinical Staphylococcus aureus isolates by analyzing the mode of resistance in spontaneous and adaptive mutant NsBL-resistant mutants. Phenotypic analysis of the mutants was performed, as well as sequence analysis of fabH; along with comparative proteomic analysis of intracellular proteomes. Our results indicate that NsBL resistance is mediated by drastic changes in the cell wall, oxidative stress response, virulence regulation, and those pathways associated with CoA. It is our conclusion that NsBL has activity towards CoA, resulting in wide-spread effects on metabolism, virulence factor production, stress response, and antimicrobial resistance.
Thesis:
Thesis (MS)--University of South Florida, 2010.
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Includes bibliographical references.
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by Katherine Prosen.
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Investigating the Mode of Action of a Novel N-sec-butylthiolated Beta-lactam Against Staphylococcus aureus by Katherine Rose Prosen A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Cell Biology, Micr obiology, and Molecular Biology College of Arts and Sciences University of South Florida Co-Major Professor: Lindsey Shaw, Ph.D. Co-Major Professor: My Lien Dao, Ph.D. Edward Turos, Ph.D. Daniel Lim, Ph.D. Date of Approval: October 21, 2010 Keywords: MRSA, antibiotic resistance, fatty acid synthesis inhibitors, Ns L, -lactams Copyright 2010, Katherine Prosen

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ACKNOWLEDGEMENTS The author would like to grat efully acknowledge he r committee, Dr. My Lien Dao, Dr. Lindsey Shaw, Dr. Edward Turos, and Dr. Da niel Lim, for their expert advice and support throughout this whole proc ess. Special thanks go to Frances Rivera, Kent Seeley, Jean Horak, and Dr. Stanley Stevens for their assistance with the proteomic analysis. The author would also like to acknowledge Whittn ey Burda for allowing the use of her USA 100 fabH sequence. The assistance of Biplob Bhattacharya was vi tal to the completion of this project, and the authors gives her heartf elt gratitude to him fo r his synthesis of the Ns L antimicrobials used for this study. Further thanks go to Sean McKee, Rachel Raynes, Hamed Mirzaei, the members of the Shaw and Dao Labs, and the CMMB Department for their moral support.

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i TABLE OF CONTENTS List of Tables v List of Figures vii Abstract ix Introduction 1 Staphylococcus aureus 1 Infections Caused by S. aureus 2 Virulence factors 2 Adhesins, Immune Modulation, and Evasion 3 Cytolytic Toxins 4 Staphylococcal Superantigens 5 Clonal Variation of S. aureus 6 Antimicrobial Therapies and Resistance 7 Cell-Wall Synthesis Inhibitors 7 Membrane Disruption 10 DNA Synthesis Inhibition 10 Transcription Inhibition 11 Translation Inhibition 12 Novel Therapeutics 14 Next Generation -lactams 14 Membrane Disruption 15 DNA Synthesis Inhibition 15 Fatty Scid Synthesis Inhibition 16 N -Thiolated -Lactams 17 Materials and Methods 20 Bacterial Strains 20 Culture Media 20 Tryptic Soy Broth (TSB) and Brain-Heart Infusion Broth (BHI) 20 Top Agar 21 Biofilm Media (BIO) 21 Purple Broth 21 Chemically-Defined Media (CDM) 21 Non-Limiting CDM 21 Non-Limiting CDM Plates (CDM-A) 22 Amino Acid-Limiting CDM (AA-CDM) 22 Glucose-Limiting CDM (G-CDM) 22

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ii Phosphate-Limiting CDM (PO4-CDM) 22 Limiting CDM Plates ([L]-CDMA) 22 Metal Ion-Limiting Media (ML) 23 Metal Ion-Limiting Plates (MLA) 23 Milk Plates 23 Blood Plates 24 Sodium Nitrate and Sodi um Nitrite Plates 24 Congo Red Agar (CRA) 24 Maintenance and Growth of Bacterial Cultures 24 Glycerol Stocks 24 Synchronized Cultures 25 Direct Plate Count 25 Determination of Minimum I nhibitory Concentrations 25 Disk Diffusion Assays 26 Fatty Acid 96-Well Assays 26 Antimicrobial Susceptibility of Biofilms 26 Generation of Spontaneous Mutants 27 Calculation of Spontane ous Mutation Frequency 28 Generation of Adaptive Mutants 28 Growth Curve Assays 28 Standard Conditions 28 Nutrient Limiting Conditions 29 Metal Ion-Limiting Conditions 29 Alternative Sugar Fermentation Assay 29 Autolysis Assays 30 Triton X-100 Lysis 30 Penicillin G Lysis 30 Gene Sequencing 30 Solutions 30 TE Buffer 30 50x TAE Buffer 31 DNA Extraction 31 Polymerase Chain Reaction (PCR) Conditions 32 Primers for fabH 32 PCR Sample Preparation 33 Electrophoresis Conditions 33 Gel Extraction 33 Gene sequencing 34 Sequence Analysis 34 Proteomic Analysis 34 Solutions 34 Dissolution Buffer 35 Cytoplasmic Protein Extraction 35 Concentrating Proteins 35 Protein Precipitation 36 Trypsin Digest 36

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iii iTRAQ Labeling 36 Desalt 37 Evaluation of Proteins by Mass Spectrometry 37 Analysis of iTRAQ Data 38 SDS-PAGE 38 Solutions 38 SP Buffer 38 Laemmli Sample Buffer 38 Coomassie Blue Staining Solution 39 Destain Solution 39 Separating Gel 39 Stacking Gel 40 10x Electrophoresis Buffer 40 Extraction of Cell-Wall Associated Proteins 40 Extraction of Secreted Proteins 41 Electrophoresis Conditions 41 Results 42 Activity of Ns L Against a Diverse Array of Laboratory and Clinical Strains of S. aureus 42 Investigation into the Effects of Fatty Acids on the Activity of Ns L 44 Comparison of the Effects of Ns L and Other Antibiotics on S. aureus Biofilms 46 Analysis of Spontaneous Mutation Frequencies for Ns L 48 Analysis of the Effect of Ns L on a Nitrosoguanidine Mutant Library of S. aureus 49 The Adaptation of S. aureus to Prolonged Growth in the Presence of Ns L 50 Phenotypic Characterization of the Ns L-Resistant Mutants 51 Growth Analysis of the Ns L-Resistant Mutants 51 Assessment of the Proteolytic Activity of the Ns L-Resistant Mutants 55 Investigation of Changes in th e Hemolytic Activity of the Ns L-Resistant Mutants 55 Alterations in Capsule Production by the Ns L-Resistant Mutants 56 The Effects of Resistance to Ns L on Virulence Determinant Production and Secretion 57 Evaluation of Electron Transport in the Ns L-Resistant Mutants 59 Alterations in the Ability of the Ns L-Resistant Mutants to Ferment Alternative Carbon Sources 60 Alterations of the Adaptive and Stre ss Resistant Capacities of the Ns LResistant Mutants 61 Exploration of the Effects of Existing Antibiotics on the Ns L-Resistant Mutants 61 Functional Characterization of the Stability of the Cell Envelope of Ns LResistant Mutants 63 Sequence Analysis of fabH in the Ns L-Resistant Mutants 64 Proteomic Analysis of the Ns L-Resistant Mutants 66

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iv Discussion 74 Future Directions 93 References 94 Appendices 106 Appendix A. Chemically-Defined Media 107 Appendix B. Complete Proteomics Data 109 Appendix C. Phyre Results 182

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v LIST OF TABLES Table 1. Overview of relevant antimicrobials and S. aureus resistance. 17 Table 2. Bacterial strains used. 20 Table 3. Minimum inhibitory concen trations for laboratory and clinical S. aureus strains as determined using a microbroth dilution assay. 44 Table 4. MICs for spontaneous Ns L SH1000 mutants. 49 Table 5. MICs for the spontan eous NTG-treated SH1000 mutants. 50 Table 6. MICs of adaptive mutants (AM) and their wild-type (WT) parents. 51 Table 7. Fold changes for antimicrobial ZOIs of SH1000 and its SH-32 spontaneous Ns L-resistant mutant. 62 Table 8. Fold changes for antimicrobi al ZOIs of USA 300 and its adaptive Ns L-resistant mutant. 63 Table 9. Intracellular proteins found to have altered expression at 8 hours in the USA 100 adaptive mutant. 68 Table 10. Intracellular proteins found to have altered expression at 8 hours in the USA 300 adaptive mutant. 68 Table 11. Intracellular proteins found to have altered expression at 8 hours in both the USA 100 and USA 300 adaptive mutants. 70 Table 12. Intracellular proteins found to have altered expression at 15 hours in both the USA 100 and US A 300 adaptive mutants. 73 Table 13. Spontaneous mutation ra tes for various antimicrobials. 77 Table A1. CL1. 107 Table A2. CL2. 107

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vi Table A3. CL3. 107 Table A4. CL4. 107 Table A5. CDM1. 107 Table A6. CDM2. 109 Table A7. CDM3. 109 Table A8. CDM4. 109 Table A9. CDM5. 109 Table A10. Complete list of intracellu lar proteins identified from the USA 100 adaptive mutant at 8 hours. 113 Table A11. Complete list of intracellu lar proteins identified from the USA 300 adaptive mutant at 8 hours. 116 Table A12. Complete list of intracellu lar proteins identified from the USA 100 adaptive mutant at 15 hours. 143 Table A13. Complete list of intracellu lar proteins identified from the USA 300 adaptive mutant at 15 hours. 181

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vii LIST OF FIGURES Figure 1. N-sec-butylthiolated -lactam (Ns L). 18 Figure 2. Disk diffusion assays usi ng NsBL with multiple laboratory and clinical strains of S. aureus 42 Figure 3. Microbroth dilution assay to determine MIC of Ns L using a variety of S. aureus and clinical isolates. 43 Figure 4. Disk diffusion assays compar ing the effects of fatty acids on the activity of NsBL and vancomycin against a variety of laboratory and clinical S. aureus isolates 45 Figure 5. The effects of fatty acids on the antimicrobial activity of Ns L towards S. aureus USA 300 Houston. 46 Figure 6. The effect of selected antimicrobials on S. aureus UAMS-1 biofilms. 47 Figure 7. Quantitative analysis and comparison of antimicrobial-treated biofilms. 48 Figure 8. Spontaneous mutagenesis of S. aureus SH1000 using 7x MIC of Ns L. 49 Figure 9. Spontaneous mutagenesis of an NTG-treated SH1000 mutant library. 50 Figure 10. Growth curve analysis of the Ns L-resistant mutants in both TSB (top) and CDM (bottom). 52 Figure 11. Amino acid-limiting growth curves of the Ns L-resistant mutant strains. 53 Figure 12. Phosphate-limiting growth curves of the Ns L-resistant mutant strains. 54 Figure 13. Metal ion-limiting growth curves for the Ns L-resistant mutant strains. 54

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viii Figure 14. Changes in prot eolytic activity of the Ns L-resistant mutants under aerobic (top) and anaerobi c (bottom) conditions. 55 Figure 15. Alterations in hemolysis of the Ns L-resistant mutant strains. 56 Figure 16. Alterations in capsule production by the Ns L-resistant mutant strains. 57 Figure 17. SDS-PAGE of cell-wall associated virulence determinants for wild-types and their Ns L-resistant mutants. 58 Figure 18. SDS-PAGE of secreted viru lence determinants for wild-types and their Ns L-resistant mutants. 59 Figure 19. Microbroth dilution assay of alternative sugar fermentation by the Ns L-resistant mutants. 61 Figure 20. Disk diffusion assay of cha nges in antimicrobial susceptibility for the SH-32 spontaneous mutant. 62 Figure 21. Disk diffusion assay of cha nges in antimicrobial susceptibility for the USA 300 adaptive mutant. 63 Figure 22. Assays of the stability of the cell envelope of the Ns L-resistant mutants. 64 Figure 23. PCR of fabH genes from parental strains and their Ns L-resistant mutants. 65 Figure 24. FabH amino acid sequence alignment of USA 100 635 wild type (WT) and its Ns L-resistant spontaneous mutant (SM). 65 Figure 25. Diamide 85 Figure 26. Phenotypic changes observe d in the SH-32 spontaneous mutant and proposed mechanisms of those changes. 87 Figure 27. Phenotypic changes obser ved in the USA 100 adaptive mutant (AM) and proposed mechanisms of those changes. 89 Figure 28. Phenotypic changes obser ved in the USA 300 adaptive mutant (AM) and proposed mechanisms of those changes. 91 Figure A1. Results of QuickPhyre analysis of COL (top) and USA 100 635 (bottom) amino acid sequences and predicted structures. 182

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ix ABSTRACT N-sec-butylthioloated -lactam (Ns L) is a novel beta-lactam antimicrobial with a mechanism of action proposed to inhibit 3-oxo acyl-acyl carrier protein synthase (ACP) III (FabH), resulting in the inhibition of fatty acid synthesis. It has been suggested that Ns L inhibits FabH indirectly by inactivating coenzyme-A (CoA). CoA is an essential cofactor for numerous proteins involved in glycolysis, the citric acid cycle (TCA), and pyruvate metabolism, in addition to fatty acid biosynthesis. This study aimed to determine the effects of Ns L on a diverse array of laboratory and clinical Staphylococcus aureus isolates by analyzing the mode of resistance in spontaneous and adaptive mutant Ns L-resistant mutants. Phenotypi c analysis of the mutants was performed, as well as sequence analysis of fabH ; along with comparative proteomic analysis of intracellular proteome s. Our results indicate that Ns L resistance is mediated by drastic changes in the cell wall, oxidative stress re sponse, virulence regulation, and those pathways associated with Co A. It is our conclusion that Ns L has activity towards CoA, resulting in wide-spread effects on me tabolism, virulence factor production, stress response, and antimicrobial resistance.

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INTRODUCTION Staphylococcus aureus Staphylococcus aureus is a Gram-positive coccoid bacterium that is facultatively anaerobic, non-motile, and non-sporeforming. It has a relatively small genome of 2.8 to 2.9 Mb with an approximate G+C content of 32%. S. aureus is part of the normal microflora of the nose, skin, gastrointestinal tract, and genitourinary tract of humans, and colonizes approximately one-thi rd of the US population. It is an opportunistic pathogen, causing disease in immunocompromised indi viduals, such as children, the elderly, HIV/AIDS patients, severe burn vict ims, and intravenous drug users37. Besides being a human pathogen, S. aureus is also known to colonize and infect most other mammals, which can in turn serve as a reservoir for human inoculation. Indeed, there are a number of documented cases wh ere companion animals have been shown to be a source of recurrent huma n infection. Bovine and equine infections are a concern for the agricultural industry, with infections often caused by human-associated strains98. Alternatively, a recent report desc ribes the case of a young, healthy girl infected with the ST398 livestock-associated strain bearing the community acquired MRSA (CA-MRSA)associated Panton-Valentine leukocidin (PVL) toxin. This infection resulted in fatal necrotizing pneumonia, and illust rates the zoonotic potential of S. aureus90. 1

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Infections caused by S. aureus S. aureus is able to cause disease in almost every niche of the human body. It is the major cause of sk in and soft tissue infections (SSTIs) in the United States, causing 76% of all purulent SST Is seen in hospita ls between 2001 and 200376. SSTIs can be uncomplicated (uSSTIs) and typically involv e infection of the epidermis and/or hair follicles, resulting in pimples, boils, abscesses, carbuncles, furuncles, folliculitis, tendonitis, and impeti go. Complicated SSTIs (cSSTIs) stem from uSSTIs and typically exhibit systemic symptoms; for example, S. aureus is also known as a flesh-eating bug due to its ability to cause necrotizing fasciitis. S. aureus can also cause invasive illnesses, including septic arthritis, endocarditis, osteomyelitis, and pneumonia54. S. aureus is also a leading cause of surgical wound infections41 and infections of foreign bodies, such as prosthetic joints, catheters, a nd pacemakers. Foreign body infections can be difficult to treat because they are often caused by biofilms forming on the indwelling device or implant101. The reasons for difficulty in treatment are not well elucidated, but have been attributed to reduced penetration of antibiotics into the aggregate matrix, or via the presence of pe rsister cells, which resist antimicrobial treatment and remain after treatment to re-e stablish the biofilm, leading to recurrent infection. Often, the only eff ective treatment is to remove the implanted or indwelling device altogether61. Virulence factors S. aureus has an impressive array of toxins wh ich are involved in causing illnesses that range from mild to severe. It is differen tiated from other members of its family by its ability to cause disease despite the fact th at 50% of the core genome is common to all 2

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staphylococci10. Indeed, the genomes of individual strains of S. aureus vary by only 22%. These variable accessory regions contai n mobile genetic elem ents (MGE) such as staphylococcal cassette chromosomes (SCC), pa thogenicity islands (PI), bacteriophages ( ), transposons (TN), and plasmids, and ofte n encode virulence factors, toxins, and resistance genes64. Adhesins, immune modulation, and evasion. A factor in the pathogenic success of S. aureus stems in part from its versatile adhesion molecules. Collectively, these are known as the microbial surface components r ecognizing adhesive matrix molecules (MSCRAMM). The host extracellular matrix (ECM) has many components which S. aureus is able to target for the purpose of a dhesion. Collagen, a com ponent of cartilage, is a target for the collagen-binding adhesi n (Cna), which has been implicated in osteomyelitis33. Fibronectin-binding proteins (FnBP) allow for attachment to platelets and, subsequently, activation of clot formation, which has been associated with endocarditis35. S. aureus also binds to fibrinogen, th e precursor to fibrin, through clumping factor A (ClfA), effectively coating th e bacterial cell in fibr inogen and acting as camouflage against phagocytic cells42. Staphylokinase (SAK) binds to plasminogen and is responsible for cl eaving fibrin, allowing S. aureus the opportunity to move to a new site within the host15. Another factor in its succe ss is the presence of several immunomodulatory and immune evasion pr oteins. Surface protein A (SpA) has antiphagocytic activity; it binds the Fc region of immunogl obulin G (IgG), preventing opsonization and, consequently, recognition by phagocytes4. Another IgG-binding protein, the staphylococcal binder of immun oglobulin (Sbi), is secreted and forms 3

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insoluble IgG complexes, reducing opportunities for opsonization. Sbi also has proteolytic activity towards co mplement factor 3 (C3), which is a key component of the complement system and important for the in itial immune response to bacterial infection18. The combination of these surface-associated and secreted proteins affords S. aureus the ability to manipulate their host environment. Cytolytic toxins. A number of staphylococcal secreted toxins have lytic activity towards different cell types present in the host. Generally, they ha ve pore-forming activity which can lead to osmotic disregulation and cell ly sis at high concentrations. Alpha-hemolysin (Hla) is strongly lytic towards rabbit erythr ocytes, but also has activity towards human erythrocytes, monocytes, epithelial cells, and endothelial cells. Hla also has dermonecrotic and neurotoxic activity. Beta-hem olysin (Hlb) displays activity similar to Hla, but is less active in S. aureus isolates causing human diseas e. This is typically due to integration of a prophage within the hlb structural encoding gene Delta toxin (Hld) has a mechanism different from that of either Hla or Hlb, both of which use polymers to form pores in cell membranes; instead, Hld monomers aggregate on a cells surface until they are heavy enough to sink into the host cel l, resulting in holes in the membrane and causing the release of intracellular components and cell death. Gamma-hemolysin is a bicomponent toxin with hemolytic and leukoly tic activity. The leve l of lytic activity depends on the combination of S and F com ponents which make up all staphylococcal bicomponent toxins. The S component s of gamma-hemolysin are encoded by hlgA or hlgC and the single F component is encoded by hlgB Similarly, Panton-Valentine leukocidn (PVL) is a bicomponent toxin, encoded by lukF -PV and lukS -PV, which is 4

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significantly more leukot oxic than gamma-hemolysin14, 29. The phenol soluble modulins (PSM) have been recently described and appe ar to have formed two major classes, PSMs and -PSMs, with the former class having the most leukocytic and inflammatory activity106. Both PVL and PSMare strongly associated with the hypervirulence of CAMRSA infections, as detailed below. These cytolytic toxins have the overall effect of dampening the efficacy of the host immune response by destroying vital immune cells and misdirecting the immune response through the release of pro-inflammatory cytokines from lysed cells. Staphylococcal superantigens. Superantigens are molecules which initiate a nonspecific interaction between the T-cell receptor (TCR) on CD4+ T-cells and the major histocompatbility complex class II (MHC-II) on antigen presenting cells (APC). This interaction leads to a robust im mune response, resulting in a cytokine storm that can lead to multi-system organ failure and death. Different isolates of S. aureus may carry any number of genes encoding superantigens. Toxic shock syndrome toxin 1 (TSST-1) is a bacteriophage-encoded superantigen most ofte n linked to tampon-associated toxic shock syndrome. Scalded skin syndrome, seen al most exclusively in newborns, is caused by exfoliative toxins, which degrade desm oglein 1 and result in burn-like lesions77. A range of staphylococcal enterotoxins (SEA SEG) cause food poisoning, which is often selflimiting and not associated with the more serious affects caused by the other staphylococcal superantigens60, 77. 5

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Clonal variation of S. aureus S. aureus has historically been considered an opportunistic pathogen, infecting primarily the elderly, the hospitalized or otherwise immunocompromised patients. However, recently, so-called CA-MRSA isolates have been causing infection in young, healthy individuals with no history of S. aureus infection or previous contact with the healthcare environment. CA-MRSA infections have been steadily increasing in both the community and hospital settings, while the occurrenc e of hospital-acquired MRSA (HA-MRSA) infections is declining25, 107. CA-MRSA strains are charac terized by enhanced virulence compared to the classical HA-MRSA isolat es. The CA-MRSA strain USA 300 is the major cause of the alteration in epidemiology of MRSA infections in the United States, and now accounts for the majority of SSTIs presenting in emergency rooms53. USA 300 is emerging as a major cause of prosthetic joint infections56 and blood stream infections95 in hospitals, and has been associated with necrotizing pneumonia58 and necrotizing fasciitis63. The necrotizing diseases in particular have been attributed to the presence of the PVL toxin. The association of CA-MRSA virulence with PVL remains controversial, as a number of reports have questioned whether PVL is the mediator of necrotic activity, or merely an indicator of the pres ence of other, as yet undefined, toxins104. Diep, et. al. have suggested that PVL plays a transito ry role in CA-MRSA pathogenesis, being beneficial (to the bacteria) onl y at the onset of infection28. Furthermore, the expression of PSMwas recently shown to be enhanced in CA-MRSA strains, and have been associated with eliciting a st rong immune response in animal models of bacteremia (USA 400) and cSSTIs (USA 300), recr uiting neutrophils to the site of infection, causing cell lysis leading to extensive tissue damage106. 6

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It has recently been suggested that regulation of virulence factors encoded as part of the core genome, as opposed to the acquisiti on of various MGEs encoding toxins or resistance genes (e.g. PVL), is the reason for the increased virulence of CA-MRSA. Li et. al. have shown that agr -regulated virulence factors such as HLA and PSMare significantly upregulated in USA 300 and US A 500 (CA-MRSA isolates) in comparison to USA 100 and USA 200 (HA-MRSA isolates); however, they also indicated that, as staphylococcal regulation is so complex, it is highly lik ely that this process is multifactorial62. Antimicrobial therapies and resistance S. aureus is a remarkably versatile and adap table pathogen, in part due to its multifactorial mode of pathogenesis, but also because it has developed resistance to every therapy introduced in the last 60 years. Here, we discuss the major classes of antimicrobials used for the clinical treatment of S. aureus and the mechanisms of resistance it has developed to cope with ch allenges posed by these various therapies. Cell-wall synthesis inhibitors. With the release of penicillin in the 1940s, previously fatal bacterial infections became treatable, he ralding a new era of infection control. However, within a few short years, strains of S. aureus appeared that were completely resistant to this drug, and it became apparent that controlling bacterial diseases would become more complex than first perceived. Spec ific analysis revealed that resistance was 7

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mediated by penicillinase, which cleaves the active -lactam ring of th is class of drugs, inactivating them entirely19. Methicillin, a derivative of pe nicillin, was introduced onto th e market in 1959 as one of the first semi-synthetic -lactams, and a potential solution to penicillin-resistant strains of bacteria. The reason methicillin was considered to be so important was that its -lactam ring is chemically buried, and is thus protected from access to -lactamases. Unfortunately, the first reported cas e of resistance occurred by 196119 and ushered in the demise of -lactam antibiotics. Methicillin-resistance is mediated by a novel penicillinbinding protein (PBP2a), encoded by the mecA gene. The mecA gene may have been acquired from S. sciuri and incorporated into the staphylococcal chromosome cassette (SCC), resulting in the SCC mec mobile genetic element108. There are currently eight known types of SCCmec, I VIII19. Each type is associated with a different range and level of antimicrobial resistance. SCC mecs I III are associated with HA-MRSA, and SCCmecs IV, V, and VI are associated with CA-MRSA25, 53. In addition to methicillin/ lactam resistance, SCC mec can confer resistance to other antimicrobial classes, such as aminoglycosides, heavy metals, macrolides, and polyketides19. Another commonly used class of -lactams is the cephalosporins, which also inhibit cellwall synthesis by a mechanism similar to the pe nicillins. Cephalospor ins are divided into generations based on the time they were disc overed and their spectrum of activity, with the older generations being the most limite d and the newer generations being the most broadly active. They were first introduced into the clinical environment in 1962, 17 years 8

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after their discovery in 194593. Strains resistant to methicillin are also resistant to cephalosporins, so their therapeutic use is only indicated for known methicillinsusceptible S. aureus (MSSA) strains19. There are currently only a few effective antimic robials available to treat highly resistant MRSA infections. The antibiotic most commonl y used in such situat ions is vancomycin, a glycopeptide that has poor solubility, hi gh toxicity, and can only be administered intravenously. Its mode of action, like -lactams, affects bacterial cell wall synthesis, although via a slightly different mechanism. Reduced susceptibility to vancomycin in clinical isolates was first reported in 1997 in Japan, 39 years after its introduction in 195843. Since this time, so-called vancomycin-intermediate S. aureus (VISA) has become distributed worldwide. They are char acterized by a diminished sensitivity to the drug, requiring increasing concentrations to produce positive therapeu tic outcomes. Fully vancomycin resistant S. aureus (VRSA) was first reported in 2002 and appears to be conferred by acquisition of the vanA resistance gene from vancomycin-resistant Enterococci (VRE). Specifically, a study fr om 2003 describes a diabetes patient coinfected with methicillin-susceptible S. aureus (MSSA) and VRE. The patient was treated with a wide array of antibiotics, including vancomycin, and VRSA cells were isolated before the course of treatment was completed. Sulfamethoxazole-trimethoprim and metronidazole were successful in erad icating the infection, but only after the infection had disseminated to bacteremia and the patients foot was amputated. This was the first time a clinical strain of S. aureus was demonstrated to carry the vanA gene20. Another point for concern has been the ap pearance of heterogenous vancomycin9

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intermediate strains (hVISA). These strains will appear to be susceptible to treatment by vancomycin, but a subset of the ba cterial population (less than 1 in 10-5 10-6) will be resistant. The presence of hVISA is typically associated with repeated MRSA infection and exposure to vancomycin, and is often difficult to detect. Most VISA and hVISA strains do not carry vanA ; instead, they display thickened cell walls and may have altered transcription of regulatory elements44. Membrane disruption. Daptomycin is one of the few novel antimicrobials that targets methicillin-resistant strains of S. aureus and was approved by th e FDA in 2003. It works by disrupting the charge of th e cell envelope, thus weakening membrane stability. The first reported case of reduced susceptibility to daptomycin occurred in 2004 in Boston, Massachusetts69, and resistance often occu rs during a failed course of treatment for an infection previously shown to be daptomycin-susceptible69, 71. The resistance mechanism, whilst not completely understood at this time, has been associated with changes in membrane fluidity and increased positive charge of the bacterial membrane49, 79. DNA synthesis inhibition. Fluoroquinolones such as cipr ofloxacin, levofloxacin, and moxifloxacin inhibit bacterial DNA gyrase a nd topoisomerase IV. Many MRSA strains are resistant to fluoroquinolones, thus, they are infrequently used27; however, many MSSA isolates are still su sceptible. Even so, fluor oquinolones are known to have negative side effects, and are rarely a firstline choice for treatment. As with other therapeutics, resistance can develop during the course of treatment; as such, ciprofloxacin 10

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is often used in combination therapy with rifampicin. Fluoroqui nolone resistance is mediated by the quinolone resistance determin ing regions (QRDR) that are present in the genes encoding the protein targets of fluor oquinolones. Specific mutations leading to resistance have been shown to occur in gyrAB (DNA gyrase) and grlAB (topoisomerase IV), which interferes with quinolone bindi ng whilst still allowing enzymatic function87. The efflux pumps NorA51 and QacBIII78 have also been shown to play a role in decreased susceptibility to these drugs. Trimethoprim-sulfamethoxazole (TMP-SMX), a pyrimidine-sulfonamide combination drug, inhibits folic acid synthesis, an essential precursor for purines and thymine, by competitively binding dihydrofolate reductase (DHFR). TMP-SMX is typically used only for the treatment of MSSA and non-mu ltiply resistant MRSA (nmrMRSA), as multiply-drug resistant MRSA (mdrMR SA) typically demonstrate TMP-SMX resistance34, 91. Resistance is caused by point mutations in the DHFR gene, or by acquisition of a plasmid-encoded accessory DHFR. These accessory DHFRs often have very little sequence homology with othe r prokaryotic DHFRs, rendering them unrecognizable to TMP-SMX24, 47. Transcription inhibition. A common technique used to combat multiple-drug resistant infections is combination drug therapy. Ri fampicin, a transcription inhibitor, is commonly used in combination with ciprofl oxacin, vancomycin, daptomycin, or linezolid for such a purpose. Rifampicin binds to the RNA polymerase subunit, encoded by rpoB interfering with transcription of DNA to RNA. While many strains of MRSA are 11

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resistant to rifampicin5, it can be effective in combination with fusidic acid or vancomycin50, 73. Despite reported successes of va ncomycin-rifampicin therapy, there remains a strong possibility of rifampicin resistance developing during treatment67, 89. Translation inhibition. There are a number of antimicrobial classes available that target different components of protein synthesis. Aminoglycosides (e.g. gentamycin, kanamycin, neomycin) and macrolides (e.g. erythromycin, clarithromycin, azithromycin) are classes that bind to eith er the 30S or 50S subunit of th e ribosome and interfere with translation. These antimicrobia l agents are typically used on ly to treat MSSA infections due to the high prevalence of resistance found in mdrMRSA isolates. Lincosamides (lincomycin and clindamycin) also bind to the 50S ribosomal subunit and inhibit translational initiation, and are also onl y indicated for the treatment of MSSA infections91. The streptogramin B group includes the combination therapy quinupristin/dalfopristin, which work synerg istically to inhib it the elongation of polypeptides. Resistance to any member of the MLS (M acrolides, L incosamides, and S treptrogramin B) group of antibiotics often re sults in cross-resistance to the remaining members, and, specifically, improper use of erythromycin can induce resistance to clindamycin109. Tetracycline antibiotics of the polyketide class target the 30S subunit of ribosomes, blocking translational elongation. Strains of S. aureus often carry the tetracycline resistance genes tetK and tetL encoding efflux pumps, and tetM and tetO encoding ribosomal protection proteins, both of whic h confer high levels of resistance to 12

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tetracycline antibiotics86. A new class of protein synthesi s inhibitors, the glycylcyclines, has been derived from tetracyclines, and includes tigecycline, whic h was approved by the FDA in 2005 to treat MRSA infections. Tigecycline blocks the A-site of the ribosome by binding to the 30S ribosomal subunit with five times greater affinity than tetracycline. The reason for this increased binding affinity is probably due to st ructural differences between tetracycline and tigecycline, with tigecycline forming stronger and more numerous bonds with the ribosome26, 84. In 2004, an initiative was taken to establish the Tigecycline Evaluation and Surveillance Tr ial (TEST) whose purpose was to monitor tigecycline susceptibility and compare it to other clinically relevant dr ugs. As reported in 2008, there has been no change in MSSA or MRSA MICs, making tigecycline an attractive therapeutic option for otherwise untreatable infections30. Linezolid is a synthetic oxazolidinone whic h binds the 23s rRNA component of the 50S ribosomal subunit, and was approved in 2000 to treat multiply-drug resistant infections. Because it is completely synthetic, it was ex pected that there would be no reservoir for resistance. However, the first clinical isolate of linezolid-resistant MRSA was reported in 2001 in Massachusetts102, while the first clinical outbreak, consisting of 12 patients, occurred in 2008 in Madrid, Spain75. Resistance has been associated with the presence of the cfr gene, which is carried on a plasmid, and confers resistance to chloramphenicol, florfenicol, and clindamycin. The mechanism of resistance involves a ltering the target of linezolid by introducing a mutation in to the gene encoding the 23s rRNA75. A specific resistance-inducing mutation, G2576T, in the sa me gene has also been reported to occur 13

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spontaneously in 1 x 10-9 isolates. However, because S. aureus has multiple copies of the 23s rRNA gene, the occurrence of in vivo resistance is still rare88. Novel therapeutics With a remarkable number of antibiotics no longer proving effective in the clinic, it appears troublesome at best to keep pace w ith staphylococcal drug resistance. Very few new antimicrobial classes have been developed in the past 50 years; however, there are a number of new drugs in the pipeline that display improved antimicrobial activity. Next generation -lactams. Two new cephalosporins with broad-spectrum, anti-MRSA activity are being investigated. The firs t, ceftobiprole and its prodrug ceftobiprole medocaril, are being developed by Basilea Pharmaceutica AG and Johnson & Johnson Pharmaceutical Research and Development LLC. Unlike other -lactam antimicrobials, ceftobiprole has a very high affinity for PBP2a, the protein encoded on SCC mec which confers methicillin-resistance, and has a chemical structure more resistant to cleavage by -lactamases. Furthermore, ceftobiprole has also shown good activit y against completely vancomycin-resistant strains111. In vitro experimentation has demonstrated that resistance is rare, although not impossible. To date, Phase III trials for SSTIs and pneumonias, alongside comparison to vancomyc in efficacy, have been completed, with ceftobiprole showing similar cure rate s and patient tolera nce to vancomycin110. Ceftaroline and its prodrug ceftaroline fosam il act by a mechanism similar to ceftobiprole and have been compared favorably to imipenem (a carbapenem) and nitrocefin (a cephalosporin). The spontaneous mutation frequency in MSSA and MRSA is 1 in 10-9 14

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10-10. One Phase III trial has been complete d, studying the usefulness and safety of ceftaroline for the treatment of SSTIs as co mpared to vancomycin, and was found to be equally effective and well tolerated112. Membrane disruption. The majority of drugs available an d in the pipeline today target growing bacteria, with no antim icrobials specifically targeting stationary-phase bacteria currently available. HT61 is a very ear ly-phase, narrow-spect rum compound currently being tested by Helperby Therapeutics for its use as a topical therapeutic in humans. Its mode of action appears to be concentra tion-dependent membrane disruption by rapid depolarization, causing leaking of intracellular contents at low concentrations and cell wall nicking at high concentrations. In vivo HT61-imbued gel completely prevented colonization of mouse skin by stationary-phase S. aureus cultures, as compared to mupirocin, which had no inhibitory effect. HT61 was also shown to be effective in eradicating both MSSA and MRSA in a mouse infection model using log-phase cultures, and was, again, significantly more effectiv e than mupirocin. Interestingly, HT61 is considerably more active towards non-multiply ing cells than actively growing cells, although the precise reason for this has not yet been determined. Significantly, in vitro experiments show that no resistance occurs in either stationaryor log-phase cultures after 50 passages at sub-MIC concentrations45. DNA synthesis inhibition. Iclaprim, like trimethoprim, prevents DNA synthesis by binding DHFR. Unlike TMP, it is active agai nst those strains of MSSA, MRSA, VISA, and VRSA which are TMP resistant93. In vitro resistance has not be en observed thus far, 15

PAGE 27

and the side effects for this drug appear to be negligible, making it a very attractive therapeutic prospect. It also shows, like TMP, synergistic activity with SMX94, and has completed Phase II trials for the treatment of SSTIs57. Fatty acid synthesis inhibition. Currently, triclosan is the only inhibitor of Type II fatty acid synthesis clinically availa ble. It has more than one mode of action, but seems to function by binding to the enoyl-acyl carrier protein (FabI), and prev enting initiation of fatty acid synthesis. Recently, more intere st has emerged in this pathway, as it is believed to be essential to bacterial growth and is distinct from ma mmalian Type I fatty acid synthesis6. Despite the attractiveness of this pathway as a therapeutic target, there has been controversy c oncerning its suitability6, 16, 17. Brinster, et. al. showed that Streptococcus agalactiae a causative agent of neonatal me ningitis, was able to survive the activity of both triclosan and cerulenin (a n inhibitor of b-keto acyl carrier protein synthetase [FabF/B]) when culture medi a was supplemented with long-chain fatty acids17. S. aureus was also demonstrated to survive cerulenin activity in the presence of both short-chain and long-chain fatty acids2, but both of these claims have recently been contested by Balemans, et. al6. Despite this controversy, Merck & Co, Inc. is investigating the potential use of two fatty acid synthesis inhibitors, platensimycin and platencin. Platensimycin inhibits -ketoacyl-[acyl-carrier prot ein (ACP)] synthase II (FabF), while platencin inhibits both FabF and -ketoacyl-[ACP] synthase III (FabH). Both have broad-spectrum in vitro and in vivo activity against Gram-positive pathogens, including MRSA. The rate of resistance for platencin was between 2 x 10-8 and 1 x 10-9 depending on the strain tested and concentration of exposure105. 16

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Mode of Action Antimicrobial Class Date of Release First Reported Date of Resistance Gene/Source of Resistance Mode of Resistance Cell wall synthesis inhibition Penicillin -lactam 1940s 1940s production of a -lactamase penicillinase cleaves -lactam ring, inactivating penicillin Methicillin -lactam 1959 1961 acquisition of mecA PBP2a Vancomycin Glycopeptide 1958 1997 acquisition of vanA altered cell wall prevents antimicrobial binding Membrane disruption Daptomycin Lipopeptide 2003 2004 mutations in mprF or yycG membranes positive charge increases, preventing drug binding DNA synthesis inhibition Ciprofloxacin Fluoroquinolone 1987 1997 mutations in gyrAB or grlAB structure of DNA gyrase is altered, preventing drug binding Transcription inhibition Rifampicin Rifamycin 1989 2004 mutations in rpoB structure of subunit of RNA polymerase is altered, reducing drug binding Translation inhibition Erythromycin Macrolide 1952 1957 acquisition of ermA or ermC structure of 23s rRNA is altered, reducing drug binding Gentamicin Aminoglycoside 1963 1976 acquisition of rmtC enzymatic inactivation of the drug Linezolid Oxazolidinone 2000 2001 acquisition of cfr mutation in gene encoding 23s subunit of 50S ribosome prevents drug binding Tigecycline Glycylcycline 2005 Not yet detected NA NA Table 1. Overview of relevant antimicrobials and S. aureus resistance. Nthiolated -lactams. A novel -lactam, which forms the focus of this study, has been developed, and displays a mode of action unlike any other -lactam previously investigated. N -thiolated lactams have shown str ong activity towards MRSA, Bacillus anthracis and Micrococcus luteus weak activity towards Neisseria gonorrhoeae, 17

PAGE 29

Bacteroides fragilis, and Haemophilus influenza but no activity towards Listeria monocytogenes, Streptococcus pyogenes, S. pn eumoniae, Klebsiella pneumoniae, Vibrio cholerae, Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, or Mycobacterium smegmatis103. Instead of targeting cell wall synthesis, it has been suggested that Ns L interacts with coenzyme A (CoA) by thiolating the active site cysteine residue, and similarly sulfenyl ating the active site cysteine of FabH103, thus both directly and indirectly interferi ng with Type II fatty acid synthesis92. Because of the possible interaction with CoA, other pathways dependent on this intermediate may also be affected by this dr ug. All analogs of N -thiolated lactams are unaffected by lactamases at every concentration tested, an d activity appears to be dependent on both low intracellular concentrations of glutathi one (GSH) and high concentrations of CoA, indicating that N -thiolated lactams may act as a thiolating agent92. Figure 1. N-sec -butylthiolated -lactam (Ns L). This analog has the highest antimicrobial activity of all N L analogs tested. The activity of NsBL against a vari ety of laboratory and clinical S. aureusu isolates was determined, including strains methicillin-, multidrug-, and vancomycin-resistant. Additionally, the aff ects of different in vitro conditions mimicking physiological on the activity of Ns L were explored. In an effort to clarify the mechanism(s) of action of Ns L, we generated mutants resistant to th is drug by two different methods in three 18

PAGE 30

strains. By determining the phenotypic a nd proteomic changes that resulted from the gain of resistance in these isolates, we hope to elucidate the primary target of Ns L. 19

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MATERIALS AND METHODS Bacterial strains Because there is a wide range of clonal variation amongst S. aureus strains, a variety of laboratory and clinical isolates were used in this study (Table 2). Strain Source Significant Characteristics S. epidermidis RP62A NARSA* MRSE clinical isolate S. aureus 8325-4 Simon Foster Defective in rsbU S. aureus RN6390 Mark Smeltzer Derived from 8325-4 S. aureus SH1000 Simon Foster Functional rsbU derivative of 8325-4 S. aureus Newman Mark Smeltzer Laboratory strain S. aureus COL Lab stock Early clinical MRSA isolate S. aureus UAMS-1 Mark Smeltzer Well studied biofilm-former S. aureus USA 100 635 Tampa General Hospital HA-MRSA S. aureus USA 100 Mu50 NARSA VISA S. aureus USA 200 MRSA252 NARSA HA-MRSA S. aureus USA 300 Houston Mark Smeltzer CA-MRSA S. aureus USA 300 LAC Kelly Rice CA-MRSA S. aureus USA 300 FPR3757 Mark Smeltzer CA-MRSA S. aureus USA 400 MW2 NARSA CA-MRSA Table 2. Bacterial strains used. *Network on Antimicro bial Resistance in Staphylococcus aureus Culture media Tryptic soy broth (TSB) and brainheart infusion broth (BHI). Tryptic soy broth (Teknova) and BHI (Bacto) were prepar ed according to the manufacturers specifications. Agar was added at 15 gL-1 as needed for plates. All media was autoclaved for 30 minutes at 121C and 15 psi, unless otherwise noted. 20

PAGE 32

Top Agar. Top agar was prepared by adding 0.7 g agar to 100 mL TSB and autoclaving as described. Biofilm media (BIO). TSB was supplemented with 0.5% de xtrose and 3% NaCl prior to autoclaving. Purple broth. Purple broth was purchased from Fisher Scientific and prepared according to the manufactu rers specifications. Chemically-defined media (CDM). Different types of chemically-defined media were prepared by combining separately prepared lim iting solutions (CL# or CDM#). Refer to the appendix for the complete lists of th e components of the CL and CDM solutions. Non-Limiting CDM. Combine the following solutions: CL1 50% CL2 10% CL3 1% CL4 0.2% CDM4 0.1% CDM5 10% MilliQ dH2O 28.7% 21

PAGE 33

Non-limiting CDM plates (CDM-A). Combine CL1, CL2, CL3, CDM4, and MilliQ dH2O in the proportions described and add 15 gL-1 agar, then autoclave and cool to 55C. Add CL4 and CDM5 in the proportions described. Amino acid-limiting CDM (AA-CDM). Combine the following solutions, then filter sterilize: CDM1 0.07% CDM2 0.02% CDM3 0.05% CDM4 0.001% CDM5 0.1% Glucose-limiting CDM (G-CDM). Reduce the concentration of glucose in CDM5 by ten-fold and follow the recipe for amino acid-limiting CDM. Phosphate-limiting CDM (PO4-CDM). Reduce the concentration of Na2HPO4 and KH2PO4 in CDM1 by five-fold and follow th e recipe for amino-acid limiting CDM. Limiting CDM plat es ([L]-CDMA). Combine CDM1, CDM3, CDM4, and dH2O in the proportions described, and 15 gL-1 agar, then autoclave and cool to 55C. Add CDM2 and CDM5 in the proportions described. Do this for any type of CDM needed. 22

PAGE 34

Metal ion-limiting media (ML). All glassware used for metal ion-limiting solutions was washed in 0.1M HCl overnight, rinsed thoroughly with dH2O, and autoclaved prior to use. All solutions used in metal ion-limiting media were treated with Chelex-100 to remove trace metal ions. Combine the following solutions: CL1 50% CL2 10% CL3 1% CL4 0.2% MilliQ dH2O 38.8% Add 10gL-1 Chelex-100 and stir for 4 hours. Filter sterilize, then add 0.4 mLL-1 sterile 1M MgSO4 in Chelex-treated MilliQ dH2O. Metal ion-limiting plates (MLA). Combine CL1, CL2, CL3, and MilliQ dH2O in the proportions described, and add 10 gL-1 agar, then autoclave and cool to 55C. Add MgSO4 in the proportion described. Do not treat with Chelex-100. Milk plates. Milk plates were prepared by preparing TSA as normal, but with the addition of 2% dry skim milk and autoclavi ng for 10 minutes at 121C and 15 psi. Plates were streaked from an isolated colony on a TSA plate and incubated at 37C overnight. 23

PAGE 35

Blood plates. Remel blood plates containing 5% sheeps blood were purchased from Fisher Scientific. Plates were streaked from an isolated colony on a TSA plate and incubated at 37C overnight. Sodium nitrate and sodium nitrite plates. Sodium nitrite plates were prepared by adding sterile 1M NaNO2 to a final concentration of 1mM NaNO2 or 2M NaNO3 to a final concentration of 20mM NaNO3 to TSA after autoclaving. Plates were streaked from an isolated colony on a TSA plate and incubated in an anaerobic GasPak chamber at 37C overnight. Congo red agar (CRA). Congo red agar was prepared by supplementing TSA with 5% sucrose and 0.8 gL-1 congo red before autoclaving. Maintenance and growth of bacterial cultures Glycerol stocks. Glycerol stocks were pr epared from 1 mL of overnight culture grown in 100 mL TSB in a 250 mL Erlenmeyer flask at 37C with shaking at 250 rpm. The 1 mL aliquot was centrifuged at 13,300 x g for 10 minutes, the supernatant was discarded, and the pellet was resuspended in 1 mL TS B containing 12% glycer ol. Stocks were stored at -80C until needed, when they we re streaked on TSA and incubated at 37C overnight. Stock plates were used for a maxi mum of seven days before new plates were prepared. 24

PAGE 36

Synchronized cultures. Two hundred-fifty mL Erlenmey er flasks containing 100 mL TSB were inoculated with one colony from st ock plates and incubated at 37C overnight with shaking at 250 rpm. A fresh flask was i noculated with 1 mL of the overnight culture and further incubated for 3 hours. Anot her fresh flask was inoculated to ~OD600nm 0.05 and incubated until the desired growth phase was reached. Synchronized cultures were used unless otherwise stated. Direct plate count. Cultures were serially diluted 1:10 in sterile PBS up to a 10-7 dilution. 100 L of the 10-6 and 10-7 dilutions were spread-plated in triplicate and incubated overnight at 37C. Individual co lonies were counted and the colony forming units per milliliter (CFU/mL) was calculated for each plate. The average was taken to give the final CFU/mL. Determination of minimum inhibitory concentrations Minimum inhibitory concentrations (MIC) we re determined using a microbroth 96-well plate method. TSB containing the drug of intere st was prepared in aliquots of at least 1 mL, then 100 L of the antibiotic-containing TSB was added to each well of a sterile 96well flat-bottomed plate (Greiner Bio-One). Cultures grown overnight in 10 mL TSB in a 50 mL Falcon tube were diluted 1:100 befo re adding 10 L to each prepared well, resulting in a final d ilution of 1:1000. At l east three wells containing TSB were left uninoculated to serve as negative controls. Each MIC was performed in triplicate. 25

PAGE 37

Disk diffusion assays Pre-warmed TSA plates were overlaid with 5mL top agar inoculated with 5 uL of overnight culture. The plates were allowed to dry before sterile Whatman paper disks imbued with the chemical stressor of interest were place on the pl ates. Plates were incubated right-side up at 37C overnight. Zones of inhibition were measured in millimeters the next day. After the first round of assays, only those stressors which resulted in a significant difference between the wild-type and mutant strains were repeated. Fatty acid 96-well assays Flat-bottomed 96-well plates were prepared with 200 L per well TSB containing 0.05% bovine serum albumin (BSA, Fisher BioReagents ), 0.1% tween 80 (Fishe r Scientific), or 0.01% oleic acid (Fisher Scientif ic). Tween 80 was prepared as a 10% solution in sterile 5% BSA. Oleic acid was prep ared as a 1% solution in sterile 5% BSA. A range of antibiotic concentrations were tested from sub-MIC up to 100x MIC. Each well was inoculated with an overni ght culture diluted to ~OD600 nm 0.05, and plates were incubated at 37C overnight. Plates were examined for growth in the different media at each antibiotic concentration. To quantify, 100 L was removed from each well and diluted in 900 L diH2O in a cuvette and the absorbance was measured at 600 nm. Antimicrobial susceptibility of biofilms A sterile flat-bottom 96-well plate was treate d with 150 L human plasma per well (20% human plasma in bicarbonate/sodium bicarbonate buffer) and incubated at 4C overnight. 26

PAGE 38

The plasma was gently aspirated from each well, and each prepared well was inoculated with 150 L culture grown overnight in 100 mL TSB-Bio in a 250 mL Erlenmeyer flask. Each culture was arbitrarily standardized to ~OD600 nm 0.05. The plate was incubated at 37C for 24 hours, then the plate was gently rinsed three times with sterile phosphate buffered saline (PBS). 150 L TSB-Bio containing an antibiotic of interest in a range of concentrations was added to each well, and the plate was further incubated at 37C for 24 hours. The plate was gently rinsed three tim es with sterile PBS and each well was fixed with 200 L 100% ethanol. The ethanol was removed and the plate was allowed to air dry. Each well was th en filled with 200 L staining so lution (41% crystal violet w/v, 12% ethanol) and allowed to stain for 2 minutes. The stain was gently aspirated, and each well was washed three times with steril e PBS. The plate was covered and allowed to air dry overnight. The st ain was eluted with 100 L of 100% ethanol for 10 minutes. The absorbance was read using a BioTek S ynergy2 plate reader at 590 nm. Samples were further diluted in 100% ethanol as nece ssary to get a reading within range. Biofilms of interest were compared to biofilms formed by the strains S. aureus 8325-4 (negative control) and S. aureus SH1000 (positive control). Generation of spontaneous mutants TSA plates were supplemented with 5 g/mL Ns L after autoclaving. 1 mL of a 10 mL overnight culture was centrifuged at full speed and all the supernatant except for 100 L was discarded. The pellet was resu spended and spread-plated on the Ns L-TSA plates. At the same time, the CFU/mL of the inoculat ing culture was calculate d (2.3). The plates were incubated ~36 hours at 37C, and a ny colonies were count ed and patched onto 27

PAGE 39

Ns L-TSA plates. The patched plates were incubated at 37 C overnight, and glycerol stocks were made from each patch. Calculation of spontaneous mutation frequency. The spontaneous mutation frequency of each spontaneous mutant was calcul ated us the following equation: ing Where R is the number of resistant coloni es obtained and N is the number of cells screened. Generation of adaptive mutants 4 L of a synchronized culture was used to inoculate a 15 mL Falcon tube containing 2 mL BHI with 0.125 g/mL Ns L (sub-MIC). The Falcon was incubated at 37C for 24 h with shaking. A 4 uL aliquot was used to inoculate a new 15 mL Falcon tube containing 2 mL BHI with 0.25 g/mL Ns L, which was incubated as pr eviously. This process was repeated, doubling the Ns L concentration until no grow th was observed. A 1 mL aliquot from each day was spun down and stored in glycerol at -80C as described. Growth curve assays Standard conditions. Synchronized cultures were used to inoculate 100 mL TSB or CDM in a 250 mL Erlenmeyer flask to ~OD600nm 0.05. For CDM, cells were washed twice with sterile PBS before inoculation. Absorbance readings were taken once every hour for 8 hours. 28

PAGE 40

Nutrient limiting conditions. Synchronized cultures were used to inoculate [L]-CDM to ~OD600nm 0.05 by first measuring the absorbance then calculating the volume needed to reach the desired optical density, then wash ing twice with sterile PBS. Absorbance readings were taken once every hour for 8 hours. Metal ion-limiting conditions. Isolated colonies from TSA were used to streak MLA. Isolated colonies from MLA were used to inoculate 5 mL ML in a 50 mL Falcon tube, which was incubated at 37C for 36 48 hours. An Erlenmeyer fl ask containing 25 mL CL was inoculated to ~OD600 nm 0.005 and in cubated at 37C with shaking. After 12 hours, an identical set of flas ks was set up and incubated at 37C with shaking. Twelve hours from that point is when absorbance m easurements were begun, once an hour for 12 hours, resulting in a total time-point range from 12 hours to 32 hours. Alternative sugar fermentation assay A flat-bottomed 96-well plate was prepared w ith 200 L purple broth per well. Wells were supplemented with 1% of an alternativ e sugar and inoculated with 5 L overnight culture standardized to ~OD600 nm 1.0. Each sugar stock was prepared at a concentration of 100 mg/mL. Alternative sugars used were dextrose, fructose, galactose, D glucosamine, lactose, maltose, mannose, ribose, sucrose, trahalose, raffinose, and xylose. Plates were incubated at 37C overnight, then examined for a change in color from purple to yellow. The color change indicated fermentation of the alternative carbon source. Each assay was performed in triplicate. 29

PAGE 41

Autolysis assays Triton X-100 lysis. Synchronized cultures were used to calculate the volume needed to inoculate a 10 mL solution to ~OD600nm 2.0. The required volume was aliquoted and washed twice with ice cold diH2O, then resuspended in 10 mL sterile 0.05M Tris-HCl (pH 7.6) with or without 0.05% Triton X-100. Absorbance measuremen ts were recorded every 30 minutes for 3 hours. Each assay was performed in triplicate. Penicillin G lysis. Synchronized cultures were used to inoculate 100 mL TSB containing 0.4 g/mL Penicillin G in a 250 mL Erlenmeyer flask to an ~OD600 nm 0.05. Absorbance measurements were recorded every 30 minutes for 5 hours. Each assay was performed in triplicate. Gene sequencing Solutions. All solutions used for gene sequencing were made according to the following formulas: TE buffer Tris-HCl 1 mM EDTA 0.1 mM Adjust pH to 7.5 and autoclave. Store at room temperature. 30

PAGE 42

50x TAE buffer Trisma base 242 g Glacial acetic acid 57.1 mL 0.5 M Na2EDTA, pH 8.0 100 mL Bring up to 1L in diH2O. Dilute 1:10 in diH2O before use. DNA extraction A 5 mL overnight culture was spun down and the pellet was resuspended in 600 L TE buffer in a snap-cap Eppendorf tube. ~0.5 cm of 0.1mm glass beads were added, and the tube was bead beaten for six 10 second intervals. The sample s were spun for 5 minutes at full speed and the supe rnatant was transferred to a ne w snap-cap Eppendorf. 200 L 1.6% sarkosyl and 5 L proteinase K were added, and the samples were incubated at 60C for 60 minutes. 800 L of phenol/chl oroform was added, the samples were vortexed, and spun for 5 minutes at full spee d. The upper aqueous layer was transferred to a 1.5 mL Eppendorf tube, and 500 L 100% isopropanol and 100 L 3M sodium acetate were added and mixed by inversion. The sample wa s incubated at -80C for 30 minutes. The sample was spun at full speed for 5 minutes, the supernatant was discarded, and 500 L 70% ethanol was added. The samp le was again spun at full speed for 5 minutes and the supernatant was discarded. Th e pellet was air-dried at room temperature, gently resuspended in 200 L diH2O, then allowed to fully resuspend at 4C overnight. 31

PAGE 43

Polymerase chain reaction (PCR) conditions. Extracted DNA was prepared in PCR tubes as described and placed in a thermocy cler (DNAEngine, BioRad). The following conditions were used: Initiation: 94C for 2 minutes 30 cycles of o Denaturation: 94C for 1 minute o Annealing: 55C for 30 seconds o Extension: 72C for 1 minute Final elongation: 72C for 7 minutes Hold at 14C Primers for fabH Forward primer: 5-CCT ACC TCT GAC TTG AGT-3 o Melting temperature: 50.1C o GC content: 50% o MW: 5425.6 g/mol Reverse primer: 5-GAC ATC ATT AC C GAT TGG AG-3 o Melting temperature: 50.5C o GC content: 45% o MW: 6141.0 g/mol 32

PAGE 44

PCR sample preparation 12.5 L ExTaq 2 L forward primer 2 L reverse primer 2 L template DNA 6.5 L diH2O Electrophoresis conditions. Gels containing 1% agarose in TAE were prepared depending on the size of the gel tank. E nough TAE buffer was added to completely cover the gel, and 5 10 L of 10 g/mL ethidium bromide was added to the solution. Each well was loaded with the total PCR product, and the gel was run at 110 V and 400 mA for 40 minutes. The gel was placed under ultraviolet light and photographed using the GelDoc-It System to visualize the DNA. Gel extraction. The QIAquick gel extraction kit (Qiage n, California) was used to extract DNA from the agarose gel. Briefly, the ge l was placed on a UV transilluminator and a clean scalpel was used to excise th e DNA bands. The gel was placed in a microcentrifuge tube and 1 mL of Buffer QG was added. The tube was placed in a 50C water bath and occasionally vortexed until the gel was completely dissolved. An equal volume of isopropanol was added to the solu tion. The solution was transferred to a QIAquick spin column in a 2 mL collecti on tube and spun for 1 minute. The flow through was discarded and 5 L Buffer QG wa s added, and the sample was again spun for 1 minute. The flow through was discarde d, and 750 L Buffer PE was added to the 33

PAGE 45

column, allowed to stand for 3 minutes, then spun for 1 minute. The flow through was discarded and the column was spun for 2 minut es, then the spin column was transferred to a clean 1.5 mL Eppendord tube. 50 L diH2O was added to the column, allowed to stand for 1 minute, and spun for 1 minute. Eluted DNA was stored at 4C until needed. Gene sequencing. PCR products extracted from gels were sent to Eurofins MWG Operon (Huntsville, Alabama) for sequencing. Samples containing 2 L of the appropriate primer, 8 L diH2O, and 10 L of the DNA sample were combined in 1.5 mL Eppendorf tubes. A set for both the forward and reverse primers were each prepared in separate Eppendorf tubes. The tubes were double-sealed in parafilm, wrapped in bubble wrap, and shipped overnight via FedEx. Sequence analysis. Sequence massager was used to obtain the complement of reverse primer sequences. Clustal W2 (European Bioinformatics Institue, EMBL-EBI) was used to align the forward and reverse primer sequ ences, resulting in the whole sequenced gene. The same program was used to compare the parent and mutant sequences to determine the presence of any mutati ons at the genetic level. Proteomic analysis Solutions. All solutions used for proteomic analysis were made according to the following formulas: 34

PAGE 46

Dissolution buffer Triethylammonium bicarbonate 500 mM Sodium dodecyl sulfate, ultrapure 0.1% Bring up to volume in HPLC-grade diH2O and store at 4C in the dark. Cytoplasmic protein extraction. A synchronized culture was used to inoculate a 1 L Erlenmeyer flask containing 400 mL TSB to ~OD600 nm 0.05. The flask was incubated until the culture reached the phase of interest The entire culture was spun down at full speed for 10 minutes. The pellet was resusp ended in PBS, transferred to a 50 mL Falcon tube, and washed in PBS three times. The pellet was resuspended in 1 mL dissolution buffer, and 500 L aliquots were transferred to screw-cap tubes, and 0.01mm glass beads were added. The tubes were subjected to vigorous bead beating using a MiniBeadbeater16 (Biospec Products) for four 1 mi nute intervals. The tubes were spun at full speed in a tabletop microcentrifuge for 10 minutes, and the supernatants were combined in an Eppendorf tube. The tubes were spun again, and the supernatant was transferred to another Eppendorf tube. Extracted proteins were stored at -80C until needed. Concentrating proteins. The required concentration for iTRAQ analysis was 5 100 g per 20 L. Millipore concentration tubes we re used to concentrate extracted proteins to the desired concentration as needed, fo llowing the manufacturers recommendations. Protein samples were centrifuged for 30 mi nutes at 13,300 x g for concentration and recovered by centrifuging for 2 minutes at 2000 x g. 35

PAGE 47

Protein precipitation. Concentrated proteins were precipitated by suspending overnight in a 1:10 volume of 100% trichloroacetic acid at 4C. The resulting pellet was washed twice with ice cold 100% et hanol by centrifuging at 8500 x g for 70 minutes. The pellet was allowed to air dry then resuspended by sonication in 1 mL dissolution buffer. The samples were spun for 30 minutes at full speed, the supernatants discarded, and the pellets resuspended in 100 L dissolution buf fer. The Pierce assay was performed following manufacturers recomm endations to determine the final protein concentration. Trypsin digest. The volume of protein sample needed to contain 100 g of protein was calculated and aliquoted to a 1.5 mL Eppendor f tube, and 20 L dissolution buffer and 1 L denaturant was added. The tube was vor texed and 2 L reducing agent was added, and the tube was vortexed again. The tube was incubated at 60C for 1 hour and briefly centrifuged. 1 L cysteine blocking reagen t was added, vortexed to mix, and briefly centrifuged. The tube was incubated at room temperature for 10 minutes. Trypsin was provided with the iTRAQ kit and reconsititute d with 25 L MilliQ water. 10 L was added to each protein sample vortexed, and briefly centrifuged. The tube was incubated at 37C for 16 hours then briefly centrifuge d. The next step was begun immediately. iTRAQ labeling. The iTRAQ reagents required for all the protein samples were allowed to reach room temperature before use, brie fly centrifuged, and 70 L ethanol was added to each tube. The reagents were vortexed, briefly centrifuged, and the contents of one vial was transferred to one protein sample. Samples were vortexed, briefly centrifuged, 36

PAGE 48

and incubated at room temperature for 1 hour. All protein samples were combined into a single fresh 1.5 mL Eppendorf tube, vortexed, and briefly centrifuged. Desalt. A MacroSpin column placed in a 2 mL Eppendorf tube was activated by adding 50 L acetonitrile and centrifuging at 110 x g for 1 minute. The eluent was discarded and the column was dried with a Kim wipe. The column was equi librated by adding 500 L 0.1% formic acid and centrifuged as previo usly. The eluent was discarded and the column was washed with 0.1% formic acid agai n. The labeled protein sample was added to the column and centrif uged as previously, and the eluent was discarded. The MacroSpin column was transferred to a new 2 mL Eppendorf t ube, and the labeled protein sample was eluted by adding 250 L acetonitrile/water (90:10). The sample was spun as previously and the el uent was transferred to a 1.5 mL Eppendorf tube. Samples were dried in a speed vac for ~1 hour and resuspended in 0.1% fo rmic acid to a final concentration of 1 g/L. The desalted, labeled protein sa mple was sonicated for 10 15 minutes, or until completely solubilized, then stored at 4C until needed. Evaluation of proteins by mass spectrometry. The sonicated protein samples were transferred to cylindrical glass tubes and pl aced in the autosampler, which injects 5 L aliquots of the samples into the LTQ Orbitr ap XL (ThermoFinnigan) for analysis. The program used to evaluate the labeled peptid es called for two injections at 180 minutes each. 37

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Analysis of iTRAQ data. Raw iTRAQ data was first run through Mascot. This allowed the identified fractions to be compared to a known database and compiled into a single file. The condensed files were processed by Scaffold Q+ and used to compare parent strains and mutants. SDS-PAGE Solutions. All solutions used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were made according to the following formulas: SP buffer Raffinose 30% MgCl2 20 mM Laemmli sample buffer diH2O 4.0 mL 0.5M Tris, pH 6.8 1.0 mL 100% glycerol 0.8 mL 10% sodium dodecyl sulfate 1.6 mL -mercaptoethanol 0.4 mL Bromophenol blue 0.05% Store at -20C. 38

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Coomassie blue staining solution Methanol 50% Acetic acid 10% Coomassie blue 0.25% Destain solution Methanol 10% Acetic acid 5% Separating gel diH2O 3.35 mL 1.5M Tris-HCl, pH 8.8 2.5 mL 10% SDS 100 L Acrylamide* 4.0 mL 10% APS 50 L TEMED 7 L ProtoGel acrylamide was purchased from Nati onal Diagnostics. It consisted of 30% w/v acrylamide and 0.8% w/v bis-acrylamide. 39

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Stacking gel diH2O 3.05 mL 0.5M Tris-HCl, pH 6.8 1.25 mL 10% SDS 50 L Acrylamide* 650 L 10% APS 25 L TEMED 5 L ProtoGel acrylamide was purchased from Nati onal Diagnostics. It consisted of 30% w/v acrylamide and 0.8% w/v bis-acrylamide. 10x Electrophoresis buffer Glycine 144 gL-1 Tris base 30.3 gL-1 SDS 10 gL-1 Solution was diluted 1:10 in diH2O before use. Extraction of cell-wall associated proteins. The absorbance of overnight cultures were measured at OD600 nm, and the volume calculated to result in an ~OD600nm 50.0 in 100 mL was calculated. The calculated volume was a liquoted and spun down at full speed for 10 minutes. The supernatant was discarded, a nd the pellet was resuspended in 100 L SP buffer. Twenty-five L lysostaphin and 8L protease inhibitor was added to the sample, and the suspension was incubated at 37C fo r 20 minutes. Following incubation, the 40

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samples were spun at full speed for 10 minutes, and the supernatants containing the proteins were collected in an Eppendorf tube to be stored at -20C until needed for SDSPAGE analysis. This protocol was also followed for cultures standardized to ~OD600 nm 100.0 or 150.0. The addition of lysostaphin was increased accordingly. An aliquot of the protein sample was mixed with an equal volum e of laemmli sample buffer and boiled for 5 minutes before loading on a gel. Extraction of secreted proteins. Cultures grown overnight for 10 15 hours were standardized so that all cultures had the same arbitrary absorbance. Cultures were spun down at full speed for 10 minutes, and the supe rnatant containing the proteins was stored at -80C until needed. Upon need, the samp les were thawed on ice, and 1 mL was transferred to a locking-cap Eppendorf t ube. 100 L 100% trichloroacetic acid was added, and the samples were mixed by invers ion and incubated on ice for 30 minutes. The samples were spun at full speed for 5 minutes and washed twice with 500 L acetone. Each time, the pellet was broken with a pipette tip. Following the final wash, the pellet was air-dried before adding 30 L laemmli sample buffer, then boiled for 10 minutes and cooled before loading on a gel. Electrophoresis conditions. BioRads Mini-Protean TETRAce ll system was used to run up to four protein gels at a time. The length of time per run was increased proportionate to the number of gels being run, from ~40 minutes for one gel up to ~2 hours for four gels. The power source was set to 300 V and 30 mA. 41

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RESULTS Activity of Ns L against a diverse array of laboratory and clinical strains of S. aureus S. aureus is a clinically relevant human pa thogen with a high level of clonal variation, resulting in a wide range of antimicrobial resistances. Accordingly, we screened a variety of different laboratory and clinical strains for susceptibility to Ns L using a disk diffusion assay ( Fig. 2 ). The zones of inhibition (ZOI) were found to be comparable for all strains test ed, regardless of their orig in or existing antimicrobial resistance. Ns L was dissolved in dimethylsulfoxide (DMSO). DMSO was not found to have any effect on the growth of S. aureus in this assay (data not shown). Figure 2. Disk diffusion assays using Ns L with multiple laboratory and clinical strains of S. aureus. (A) RN6390; (B) SH1000; (C) Ne wman; (D) COL; (E) UAMS-1; (F) USA100; (G) USA 300 Houston. 42

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In order to quantif y the activity of Ns L, the minimum inhibitory concentration (MIC) was determined for each of these strain s using a 96-well plate microbroth dilution method, which allows for quick and efficient sc reening of multiple samples at a range of concentrations. The MIC is defined as the lo west drug concentration at which there is no visible growth, and was found to be between 0.4 g/mL and 1 g/mL for all strains tested ( Fig. 3; Table 3 ). Figure 3. Microbroth dilution assay to determine MIC of Ns L using a variety of S. aureus and clinical isolates. Circles indicate the lowest concentration with no visible turbidity; ie the MIC. 43

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Strain MIC (g/mL) RN6390 0.5 SH1000 0.75 Newman 0.75 COL 0.75 UAMS-1 1 USA 100 635 1 USA 300 Houston 0.75 Table 3. Minimum inhibitory co ncentrations for laboratory and clinical S. aureus strains as determined using a microbroth dilution assay Investigation into the effects of fatty acids on the activity of Ns L. As a result of these studies, the effect of fatty acids on the activity of Ns L was first tested using a disk diffusion assay. There has been conflicting evidence concerning the effect of fatty acids on the growth of S. aureus and other Gram-positive pathogens in the presence of antimicrobials targeting fatty aci d synthesis. Specifically, it has been suggested that the fatty acids can rescue growth, despit e the presence of antimicrobial agents2, 6, 16, 17. As previous work has indicated that Ns L may target fatty acid synthesis92, 0.01% oleic acid (a long chain unsaturated fa tty acid) or 0.1% Tween 80 (a n oleic acid surrogate) was added to TSA to determine if there was a similar effect on Ns L activity. As a control, Ns L ZOIs were compared to those for vancomyc in (a cell wall synthesis inhibitor). The assay was performed for S. epidermidis RP62A and the S. aureus strains SH1000, Newman, COL, USA 100 Mu50, USA 200 MRSA252, USA 300 Houston, and USA 400 MW2 ( Fig. 4 ). For all strains tested, the Ns L ZOIs were significantly and consistently reduced in the presence of Tween 80. For SH1000, COL, and USA 300 Houston, the vancomycin ZOIs were also reduced in th e presence of Tween 80, but all other strains had consistent ZOIs. 44

PAGE 56

Figure 4. Disk diffusion assays comparing the effects of fatty acids on the activity of Ns L and vancomycin against a variety of laboratory and clinical S. aureus isolates. 45 Quantification of the effects of fatty acids on antimicrobial activity was further evaluated by adding 0.1% Tween 80, 0.01% oleic acid, or 0.5% BSA to TSB and measuring the growth of S. aureus isolates in the presence of Ns L, vancomycin, or triclosan (a commercially used fatty acid synthesis inhib itor). Both Tween 80 and oleic acid were dissolved in BSA, so this was included in the assay as a control. BSA, Tween 80, and oleic acid significantly reduced the MIC for vancomycin, indicating that these additives may work synergistically with vancomcyin to inhibit cell wall synthesis. The MIC for triclosan was increased by Tween 80 20to 50-fold for all strains tested; however, oleic acid did not have significant rescue activity fo r cultures treated with triclosan. The presence of Tween 80 allowed growth in the presence of Ns L concentrations equal to the MIC, but not at the concentrations tested above the MIC. Cultures treated with BSA were also able to grow in the presence of Ns L, but at a level significantly lower than the

PAGE 57

untreated culture. These results were comparab le for all strains tested, suggesting that the diverse isolates tested had similar responses to the presence of fatty acids (only USA 300 data shown: Fig. 5 ). Figure 5. The effects of fatty acids on the antimicrobial activity of Ns L towards S. aureus USA 300 Houston. (A) Ns L; (B) Triclosan; (C) Vancomycin. Comparison of the effects of Ns L and other antibiotics on S. aureus biofilms. S. aureus biofilms are difficult to treat due to a lack of antimicrobial diffusion through the biofilm matrix and because not all of th e cells are in an actively growing state61. The effects of Ns L, triclosan, vancomycin, and tetracycl ine (a protein synthesis inhibitor) on UAMS-1 biofilms were analyzed using a 96-w ell plate method previo usly described by Beenken, et. al. UAMS-1 was chosen as it is a biofilm-forming strain that has previously been well characterized for this aggregate lifestyle11. SH1000 was also used as a positive control, and 8325-4 as a negative control. Biofilms were allowed to establish for 24 46

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hours before fresh media contai ning the relevant antimicrobial was added. Antimicrobial exposure lasted an additional 24 hours before the biofilms were stained with crystal violet for evaluation ( Fig. 6). Figure 6. The effect of selected antimicrobials on S. aureus UAMS-1 biofilms. Biofilms were exposed to the antimicrobials in the concentrations indicated (g/mL) for 24 hours before staining with crystal violet. (A) Ns L; (B) Triclosan; (C) Vancomycin; (D) Tetracycline; (E) Untreated controls. Quantitative analysis was performed by measuring the absorbance of eluted crystal violet at 595 nm in a BioTek Synergy2 96-well plate reader and the percent change from the untreated biofilms was calculated ( Fig. 7 ). Tetracycline and Ns L had the greatest effect on biofilm formation, reducing the absorb ance at 595 nm by approximately 30% and 40%, respectively. Triclosan had only a sl ight effect on biofilms, reducing their 47

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formation by 13 21%. Biofilms were unaff ected by treatment with vancomycin, and had levels of formation comparable to or exceeding the untreated UAMS-1 control. Figure 7. Quantative analysis and comparison of antimicrobial-treated biofilms. Biofilms were stained with crystal violet as described and the absorbance of a 1:100 dilution was measured with a BioTek Synerg y2 plate reader at 590 nm. The percent change as compared to the untreated UAMS-1 biofilms was calculated. p < 0.1 using the Students t-test comparing the antimicrobi al-treated biofilms to the untreated UAMS1 biofilms. Triclosan concentrations were 0, 0.0625, 0.125, 0.25, 0.5, and 1 g/mL. Analysis of spontaneous mutation frequencies for Ns L. Spontaneous mutation leading to resistance is a problem with all clinically available antimicrobials. Knowing the rate of mutation is vital in order to de termine the minimum dosage required to prevent resistance. In addition, it give s a sense of the su itability of a given compound for use in treating infections. As such, TSA wa s supplemented with seven times the Ns L MIC and inoculate with 3 x 109 SH1000 cells in order to dete rmine the spontaneous mutation 48

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49 frequency, calculated as the number of resist ant colonies per cells screened. Four replicates were performed for this assay ( Fig. 8 ), yielding a total of 98 colonies from a total inoculum of 1.38 x 1010 cells yielded a spontaneous mutation rate of 7.1 x 10-9. The Ns L MICs of all colonies obtained were de termined by the microbroth dilution method as described, and found to be 1.5 -fold to 67-fold great er than the parent strain. A total of 26 of the isolated spontaneous mutants had MICs less than 5 g/mL. When this is taken into account, the spontaneous mutation rate becomes 5.2 x 10-9. Two isolates, SH-11 and SH-32, were chosen to use for further experimentation because they had the highest MICs ( Table 4 ). Isolate MIC g/mL Isolate MIC g/mL SH-11 50 SH-60 20 SH-15 X SH-62 20 SH-22 30 SH-64 20 SH-32 30 SH-69 10 SH-38 10 SH-74 10 SH-56 20 SH-75 20 SH-58 10 SH-77 X SH-59 X SH-85 X Figure 8. Spontaneous mutagenesis of S. aureus SH1000 using 7X MIC of Ns L. TSA supplemented with Ns L was inoculated with 1 x 109 SH1000 cells and incubated at 37C for 30 hours. Plates yielded between 11 and 49 colonies. A representative example is shown. Table 4. MICs for spontaneous Ns L SH1000 mutants. These values were determined using the microbroth dilution method, and are shown for randomly selected isolates. Ns L concentrations tested were 0, 10, 20, 30, 40, and 50 g/mL. X indicates no visible growth at all NsL concentrations. The isolates used for further experimentation in this study are highlighted. Analysis of the effect of Ns L on a nitrosoguanidine mutant library of S. aureus We set out to determine if prior DNA mutagene sis would affect the spontaneous mutation rate of S. aureus to Ns L. As such, SH1000 was treated with nitrosoguanidine (NTG), a

PAGE 61

DNA alkylating agent, and the spontaneous mutation frequency was determined as previously described ( Fig. 9; Table 5 ). Twelve colonies were isolated from the 4 x 108 NTG-treated cells plated, yielding a spontaneous mutation rate of 3 x 10-9. However, four of the twelve NTG-treated Ns L-resistant isolates had MICs equal to 1 g/mL, significantly lower than the concentration used to generate them, and changes the spontaneous mutation rate to 2 x 10-8, which is 3.85 times greater than the spontaneous mutation rate of the untreated cells. Figure 9. Spontaneous mutagenesis of an NTG-treated SH1000 mutant library An inoculum of 4 x 108 cells yielded 12 colonies on a 5 g/mL Ns L plate. Isolate MIC g/mL Isolate MIC g/mL NTG-A 10 NTG-G 1 NTG-B 1 NTG-H 10 NTG-C 10 NTG-I 10 NTG-D 10 NTG-J 1 NTG-E 10 NTG-K 10 NTG-F 1 NTG-L 10 Table 5. MICs for the spontaneous NTG-treated SH1000 mutants. The MICs for library mutants were determined using the microbroth dilution method. Ns L concentrations tested were 0, 1, 10, 15, and 20 g/mL. The adaptation of S. aureus to prolonged growth in the presence of Ns L. In contrast to the spontaneous mutant analysis, we also set out to determine th e effect of prolonged Ns L exposure on S. aureus cells. Therefore, S. aureus strains USA 100 and USA 300 were subjected to increasingly higher concentrations of Ns L in order to obtain highly resistant adaptive mutants. Overnight e xposure began at 0.125 g/mL in 2 mL brainheart infusion (BHI) broth. A 4 L aliquot of the culture was transferred to a fresh tube 50

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containing 2 mL BHI with double the Ns L concentration and exposed for 24 hours. This process was repeated, making a glycerol stock of the exposed cells as previously described, and transferring a 4 L aliquot to fresh BHI with double the Ns L concentration each day until no growth was obtained at 64 g/mL. The MICs were confirmed by the microbroth dilution method as previously described ( Table 6 ). Strain MIC (g/mL) USA 100 WT 0.75 USA 100 AM 65 USA 300 WT 0.75 USA 300 AM 55 Table 6. MICs of adaptive mutants (AM) and their wild-type (WT) parents. Phenotypic characterization of the Ns L-resistant mutants. Previous studies on the mode of action of Ns L have indicated that this drug may target FabH, an essential component of fatty acid biosynthesis. The study also speculated that Ns L may have an effect on coenzyme-A (CoA), which is a cof actor for a number of fatty acid synthesis, pyruvate metabolism, and the citric acid cycle (TCA) enzymes92. Thus far, the mode of action of Ns L has not been fully characterized. In an effort to extrapolate the mechanism of activity of Ns L, two of the SH1000 spontaneously generated mutants, SH-11 and SH-32, and the adaptive mutants of USA 100 and USA 300 were evaluated for phenotypic changes from their parental strains. Growth analysis of the Ns L-resistant mutants. Standard growth curves were performed in TSB and chemically-defined me dia (CDM) in order to determine if there were changes in growth of the various mutant strains ( Fig. 10 ). No changes were observed for the SH-11 or SH-32 spontane ous mutants or for the USA 100 adaptive 51

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mutant in TSB. However, the USA 300 adap tive mutant showed decreased levels of growth in TSB in comparison to its parent In CDM, the SH-11 spontaneous mutant showed decreased growth during lag phase in comparison to both SH1000 and the SH-32 spontaneous mutant. The USA 100 adaptive mu tant had increased growth levels during lag and early-exponential phase, but decreased growth during stationary phase. The USA 300 adaptive mutant had increased growth during all phases tested. Figure 10. Growth curve analysis of the Ns L-resistant mutants in both TSB (top) and CDM (bottom). (A, D) SH1000 and its spontaneous mutants; (B, E) USA 100 and its adaptive mutants; (C, F) USA 300 Houston and its adaptive mutants. All assays were performed in triplicate. Nutrient limiting derivatives of CDM were also used to determine if there were defects in response to nutrient starvation in the spontaneous and adaptive mutants. For amino acidlimiting CDM, no significant differences were s een for either spontaneous mutant. The USA 100 adaptive mutant showed increased gr owth levels in early -exponential phase, but 52

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decreased growth in late-e xponential and stationary phase. The USA 300 adaptive mutant showed decreased growth only during exponential phase ( Fig. 11 ). Figure 11. Amino acid-limiting growth curves of the Ns L-resistant mutant strains. (A) SH1000 and its spontaneous mutants; (B) USA 100 and its adaptive mutants; (C) USA 300 Houston and its adaptive mutants. All assays were performed in triplicate. There were no significant differences for any of the mutants when glucose was the limiting factor (data not shown). Likewise, there were no differences for the spontaneous mutants or the USA 100 adaptive mutant when phosphate was limited. However, the USA 300 adaptive mutant showed increased growth during late lag and early -exponential phase, and decreased growth during late-exponential and stationary phase ( Fig. 12 ). 53

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Figure 12. Phosphate-limiting growth curves of the Ns L-resistant mutant strains. (A) SH1000 and its spontaneous mutants; (B) USA 100 and its adaptive mutants; (C) USA 300 Houston and its adaptive mutants. All assays were performed in triplicate. Metal ion-limiting media was also used to analyze growth over a 36 hour period, although the first 12 hours were not assayed as S. aureus has a prolonged lag phase during growth under these conditions. Both sp ontaneous mutants had decreased levels of growth during exponential phase, with SH-32 growth more significantly reduced than SH-11. There was no change for the USA 100 adaptive mutant, while the USA 300 adaptive mutant had higher growth levels dur ing exponential phase than its parent strain ( Fig. 13). Figure 13. Metal ion-limiting growth curves for the Ns L-resistant muta nt strains. (A) SH1000 and its spontaneous mutants; (B) USA 100 and its adaptive mutants; (C) USA 300 Houston and its adaptive mutants. All assays were performed in triplicate. 54

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Assessment of the proteoly tic activity of the Ns L-resistant mutant strains. S. aureus expresses multiple extracellu lar proteases as part of its virulence determinant network. To assess this activity in the Ns L-resistant mutants, these strains were incubated both aerobically and anaerobically ( Fig. 14) on plates cont aining 2% skim milk. SH1000 naturally has very low levels of proteolytic activity, which did not change with its Ns L-resistant mutants (data not show n). Both the USA 100 and USA 300 adaptive mutants displayed reduced aerobic proteolytic activity compared to their parental strains. Only USA 300 retained pr oteolytic activity when grown in an anaerobic chamber, which was not displayed by its adaptive mutant. Figure 14. Changes in proteolytic activity of the Ns L-resistant mutants und er aerobic (top) and anaerobic (bottom) conditions. TSA containing 2% skim milk was inoculated with the parent strains and their respective mutants. A representative example of three experiments is shown. (A, C) USA 100 WT and its adaptive mutant; (B, D) USA 300 WT and its adaptive mutant. Investigation of changes in th e hemolytic activity of the Ns L-resistant mutant strains. One of the major contributions to S. aureus disease comes from its hemolytic activity. To assay these toxins, sheep blood agar was used to compare the hemolytic activity of the parental strains with their respective mutants ( Fig. 15). The SH1000 spontaneous mutants did not s how any apparent change in hemolysis in comparison with the parent (data not shown), while the USA 100 adaptive mu tant had reduced activity. Of 55

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significant interest, the USA 300 adaptive mutant lost all hemolytic activity when assayed using blood agar. Figure 15. Alterations in hemolysis of the Ns L-resistant mutant strains. Wild-types and their mutants were streaked in triplicate on 5% blood agar. A representative example of three experiments is shown. (A) USA 100 and its adaptive mutant; (B) USA 300 and its adaptive mutant. Alterations in capsule production by the Ns L-resistant mutants. S. aureus has the ability to produce a capsule composed of pol ysaccharides which aids in defense from phagocytosis, the hosts primary defense against bacterial inf ection. To assay alterations in the production of capsule by the Ns L-resistant mutants, c ongo red agar (CRA) was inoculated with the wild-types and respective mutant strains ( Fig. 16). SH1000 and its spontaneous mutants displayed colony morp hologies indicative of strong capsule production, i.e. rough, dry, black colonies (dat a not shown). Conversely, while both the USA 100 and USA 300 wild-types displayed capsule production, their adaptive mutants lost the characteristic black pigmentation altogether, indicating that they were not producing capsules. 56

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Figure 16. Alterations in capsule production by the Ns L-resistant mutant strains. CRA was inoculated with each strain and examined fo r colony morphology and cap sule production. (A) USA 100 wild type; (B) USA 100 adaptive mutant; (C) USA 300 wild type; (D) USA 300 adaptive mutant. The effects of resistance to Ns L on virulence determinant production and secretion. S. aureus produces a wide variety of toxins and other secreted proteins associated with virulence. In additi on, MSCRAMMS and other cell-wall associated proteins are involved in invasi on of the host and attachment to various surfaces. Sodium dodecyl sulfate polyacrylamide gel electrophor esis (SDS-PAGE) was used to compare the production of cell-wall asso ciated and secreted proteins produced by the parent and mutant strains. SDS-PAGE of th e cell-wall associated proteins ( Fig. 17 ) shows that SH11 appears to produce slightly in creased levels of these proteins than either the wild-type or SH-32. Both the USA 100 and USA 300 ad aptive mutants produced significantly less 57

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cell-wall associated proteins than their respectiv e parental strains. Particularly notable is a ~45 kDa protein produced by USA 300 wild-typ e which is significan tly diminished in its adaptive mutant. Figure 17. SDS-PAGE of cell-wall associated virulence determina nts for wild-types and their Ns L-resistant mutants. The gel shown here is representative of four experiments. The cell-wall associated proteins were extracted from a culture standardized to OD600nm = 50.0. The white box indicates the ~45 kDa protein expressed in the USA 300 WT but not in the adaptive mutant. SDS-PAGE of secreted proteins ( Fig. 18) showed similar pattern s of expression, with protein levels secreted by SH-11 slightly in creased as compared to the wild-type. The increase in expression of an ~80 kDa protei n was particularly apparent. The proteins secreted by both adaptive mutant strains were significantly decreased compared to their parental strains, particularly an ~80 kDa protein that was also seen over-expressed in SH11, a ~50 kDa protein, and a ~ 35 kDa protein. Interestingly, the levels of two proteins 58

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secreted by both adaptive mutant strains appeared to be less reduced than the rest, seen at ~95 kDa and ~28 kDa. Figure 18. SDS-PAGE of secreted virulen ce determinants for wild-types and their Ns L-resistant mutants. The gel shown here is representative of four experiments. The secreted proteins were extrac ted from a culture standardized to the same absorbance at 600nm. The gray boxes indicates the ~80 kDa protein band, the white box indicates the ~35 kDa protein band, and the black boxes indicate the ~28 kDa and ~95 kDa protein bands. Evaluation of electron transport in the Ns L-resistant mutants. Growth of S. aureus in anoxic environments requires the presence of n itrogen, which serves as a terminal electron acceptor. To assess the phenotype of the Ns L-resistant mutants with regards to electron transport, media contai ning either nitrate or nitrite was inoculated with the wild59

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type strains and their respective Ns L-resistant mutants. These plates were incubated in a GasPak chamber to ensure an anaerobic environment. No significant alterations in growth were observed for any of the mu tant strains tested (data not shown ) Alterations in the ability of the Ns L-resistant mutants to ferment alternative carbon sources. Following from this, we examined the ability of the Ns L-resistant strains to ferment a variety of carbon sources when grow n under anaerobic conditions. Interestingly, S. aureus has the ability to use a variety of different sugars as its carbon source in the absence of glucose. As su ch, purple broth was used, which has a pH indicator that changes the media from purple to yellow when the media becomes acidified as the result of lactate or acetat e production during fermentation. No changes in sugar fermentation were seen for dextrose, fructose, galactose, D -glucosamine, lactose, mannose, sucrose, trehalose, raffinose, or xylose. The spontaneous mutants SH-11 and SH-32 did not show any changes from thei r parent, while both the USA 100 and USA 300 adaptive mutants lost the ability to ferm ent ribose. Further to this, the USA 300 adaptive mutant was also unable to ferment malto se when compared to the parental strain ( Fig. 19). 60

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Figure 19. Microbroth dilution assay of alternative sugar fermentation by the Ns L-resistant mutants. A 96-well plate containing purple broth supplemented with a variety of sugars (at 1%) was inoculated with parent and mutant strains and grown overnight to determine if there were any changes in the ability to ferment alternative carbon sources. Th e plate shown is representative of three experiments. Alterations of the adaptive and stress resistant capacities of the Ns L-resistant mutants. A chemical library containing diverse acids, bases, detergents, disulfides, alcohols, oxidative agen ts, and DNA damaging agents was used to test the spontaneous and adaptive mutants for changes in their stress resistance capabilities. This was assessed via disk diffusion assay, however, no compound s resulted in significant changes in the ZOI for any strains tested (data not shown). Exploration of the effects of existing antibiotics on the Ns L-resistant mutants. Disk diffusions were used to determine changes in ZOI between the pare nt strains and their resistant mutants for a range of antimicrobial compounds. The an timicrobials tested 61

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included representatives of each major class, including inhibitors of DNA synthesis, RNA synthesis, protein synthe sis, fatty acid synthesis, an d cell wall synthesis. Only those antimicrobials that resulted in signi ficant changes in the ZOI are shown. SH-11 and USA 100 did not show any significant cha nges in antimicrobial susceptibility (data not shown), while SH-32 was more resist ant to mupirocin and penicillin G ( Fig. 20; Table 7 ). Interestingly, SH-32 showed signs of heteroresistance to mupirocin, as indicated by the zone of reduced growth around the discrete ZOI. Figure 20. Disk diffusion assay of changes in antimicrobial susceptibility for the SH-32 spontaneous mutant. Mup = Mupirocin; PenG = Penicillin G; Linco = Lincomycin; Kan = Kanamycin Antimicrobial Fold Change from WT Mupirocin -1.77 Penicillin G -2.15 Table 7. Fold change for antimicrobial ZOIs of SH1000 and its SH32 spontaneous Ns L-resistant mutant. Each assay was performed in triplicate. The USA 300 adaptive mutant showed increase d resistance to ampicillin, ciprofloxacin, kanamycin, and erythromycin (Fig. 21; Table 8 ). 62

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Figure 21. Disk diffusion assay of changes in antimicrobial susceptibility for the USA 300 adaptive mutant. Amp = Ampicillin; Cipro = Ciprofloxacin; Rif = Rifampicin; Ery = Erythromycin. Antimicrobial Fold Change Ampicillin -1.81 Erythromycin -1.91 Kanamycin -2.5 Ciprofloxacin Mutant had no zone Table 8. Fold change for antimicrobial ZOIs of USA 300 and its adaptive Ns L-resistant mutant. Each assay was performed in triplicate. Functional characterization of the st ability of the cell envelope of the Ns L-resistant mutants. The integrity of the bacterial cell envelopes for the Ns L-resistant strains was investigated using penicillin-G and Triton X-100 lysis assays ( Fig. 22). For both penicillin-G and Triton X-100, there was no ap parent difference in lysis between SH1000 and its spontaneous mutants. There was also no difference in lysis when USA 100 and its adaptive mutant were treated with Triton X-100. However, the USA 100 adaptive mutant showed a reduced ability to survive prolonge d exposure to penicillin-G, while the USA 300 adaptive mutant appeared to be significa ntly impaired at all timepoints. When 63

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treated with Triton X-100, the USA 300 adap tive mutant showed increased lysis only towards the end of the assay. Figure 22. Assays of the stability of the cell envelope of the NsL-resistant mutants. Penicillin-G lysis (top) was performed over 5 hours in the presence of 0.4 g/mL penicillin G. Triton X-100 lysis (bottom) was performed over 3 hours in the presence of 0.5% Triton X-100. The graphs show the percent survival of each strain. All assays were performed in triplicat e. The dashed lines are unt reated controls. (A, B) SH1000 and its spontaneous mutants; (C, D) USA 100 and its adaptive mutant; (E, F) USA 300 Houston and its adaptive mutant. Sequence analysis of fabH in the Ns L-resistant mutants. Ns L has previously been suggested to inhibit FabH activity92. One of the many ways S. aureus develops resistance to antibiotics is by spontaneously mutating the gene which encodes the target protein(s). Thus, genomic DNA was extracted from each of the mutant and wild-type strains and used to amplify PCR-amplify fabH ( Fig. 23). Sequencing for each strain was attempted, but was unsuccessful, possibly due to problems at the external facility the samples were sent to, or compromised DNA samples (data not shown). 64

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Figure 23. PCR of fabH genes from parental strains and their Ns L-resistant mutants. We were, however, able to obtain sequence data for the fabH gene from an additional USA 100 Ns L-resistant mutant previously generate d in our laboratory (Burda and Shaw, unpublished observation). The amino acid sequences were determined and compared, and no differences in the FabH sequences were found between the wild-type and resistant mutant ( Fig. 24). WT MNVGIKGFGAYAPEKIIDNAYFEQFLDTSDEWISKMTGIKERHWADDDQDTSDLAYEASV MNVGIKGFGAYAPEKIIDNAYFEQFLDTSDEWISKMTGIKERHWADDDQDTSDLAYEASV SM MNVGIKGFGAYAPEKIIDNAYFEQFLDTSDEWISKMTGIKERHWADDDQDTSDLAYEASV WT KAIADAGIQPEDIDMIIVATATGDMPFPTVANMLQERLGTGKVASMDQLAACSGFMYSMI KAIADAGIQPEDIDMIIVATATGDMPFPTVANMLQERLGTGKVASMDQLAACSGFMYSMI SM KAIADAGIQPEDIDMIIVATATGDMPFPTVANMLQERLGTGKVASMDQLAACSGFMYSMI WT TAKQYVQSGDYHNILVVGADKLSKITDLTDRSTAVLFGDGAGAVIIGEVSEGRGIISYEM TAKQYVQSGDYHNILVVGADKLSKITDLTDRSTAVLFGDGAGAVIIGEVSEGRGIISYEM SM TAKQYVQSGDYHNILVVGADKLSKITDLTDRSTAVLFGDGAGAVIIGEVSEGRGIISYEM WT GSDGTGGKHLYLDKDTGKLKMNGREVFKFAVRIMGDASTRVVEKANLTSDDIDLFIPHQA GSDGTGGKHLYLDKDTGKLKMNGREVFKFAVRIMGDASTRVVEKANLTSDDIDLFIPHQA SM GSDGTGGKHLYLDKDTGKLKMNGREVFKFAVRIMGDASTRVVEKANLTSDDIDLFIPHQA WT NIRIMESARERLGISKDKMSVSVNKYGNTSAASIPLSIDQELKNGKLKDDDTIVLVGFGG NIRIMESARERLGISKDKMSVSVNKYGNTSAASIPLSIDQELKNGKLKDDDTIVLVGFGG SM NIRIMESARERLGISKDKMSVSVNKYGNTSAASIPLSIDQELKNGKLKDDDTIVLVGFGG WT GLTWGAMT GLTWGAMT SM GLTWGAMT Figure 24. FabH amino acid sequence alignment of USA 100 635 wild type (WT) and its Ns Lresistant spontaneous mutant (SM). 65

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Proteomic analysis of the Ns L resistant mutants. As a final and global approach to understand the mechanism of action of Ns L, we undertook both comprehensive and quantitative proteomic analysis of th e USA 100 and USA 300 adaptive strains. Differences between the in tracellular proteomes of the USA 100 and USA 300 parent strains and their respective adaptive mutant s were evaluated at 8 hours (post-exponential phase) and 15 hours (stationary phase) using isobaric tagging for re lative and absolute quantitation (iTRAQ) mass spectrometry. For the post-exponential phase cultures, the data for each parental strain and resistant mutant was performed in duplicate, and the average fold cfhange was calculated. At 8 hours, 85 proteins were identified from USA 100 and 66 proteins from USA 300 (see appe ndix for complete lists). Forty of the proteins identified from the USA 100 adaptiv e mutant had expressi on altered by 1.5-fold or greater from the parent stra in, many of which were involv ed in nucleotide synthesis, translation, glycolysis, pyruvate metabolism, and TCA cycle ( Table 9 ). However, only four proteins with a change in expression greater than 1.5-fold were identified from the USA 300 adaptive mutant, which included protei ns involved in fermentation, cell wall degradation, and energy production ( Table 10 ). Of all the protei ns identified, 46 were found to have altered expression in both USA 100 and USA 300, including proteins involved in translati on, glycolysis, pyruvate metabo lism, the TCA cycle, and the oxidative stress response ( Table 11 ). At 15 hours, 796 proteins were identified from USA 100 and 791 proteins from USA 300, 56 of which were found to have significantly altered expression in both strains; however the stationary phase data is purely exploratory, as only one replicate was performed and statistical signi ficance could not be calculated ( Table 12 ). It is worth noting that a num ber of proteins associated with 66

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nucleotide metabolism, transcription, DNA repa ir, amino acid metabolism, translation, peptidoglycan synthesis, cell division, glycol ysis, the TCA cycle, and sugar transport were all significantly decreased in bot h the USA 100 and USA 300 adaptive mutants. Pathway Late Exponential Phase Proteins Identified from USA 100 (40) Accession Number Fold Change from Wild-Type Purine biosynthesis Inosine-5'-monophosphate dehydrogenase (guaB) P99106|IMDH_STAAN -1.5 Phosphoribosylaminoimidazole carboxylase ATPase subunit (purK) Q7A695|PURK_STAAN -2.45 Phosphoribosylformylglycinamidine synthase 1 (purQ) P99166|PURQ_STAAN -2.4 Pyrimidine biosynthesis Aspartate carbamoyltransferase (pyrB) P65618|PYRB_STAAN -2.65 Virulence Determinant Regulation GTP-sensing transcriptional pleiotropic repressor (codY) P63844|CODY_STAAN -2.2 Cysteine metabolism Cysteine synthase (cysK) P63871|CYSK_STAAN -2.05 Nitrogen metabolism Glutamine synthetase (glnA) P99095|GLNA_STAAN -1.5 Electron transport ATP synthase subunit beta (atpD) P99112|ATPB_STAAN -1.7 Energy production L-lactate dehydrogenase 1 (ldhA) P65256|LDH1_STAAN -2.55 NADH dehydrogenase-like protein SA0802 (SA0802) Q7A6J4|Y802_STAAN -2.5 Glycolysis Enolase (eno) P99088|ENO_STAAN -2.15 Glucose-6-phosphate isomerase (pgi) P99078|G6PI_STAAN -1.8 Pyruvate kinase (pyk) Q7A559|KPYK_STAAN -1.6 TCA cycle Aconitate hydratase (acnA) P99148|ACON_STAAN -2.6 Citrate synthase II (citZ) Q7A561|Q7A561_STAAN -2.55 Dihydrolipoyllysine-residue succinyltransferase component of 2oxoglutarate dehydrogenase complex (odhB) Q7A5N4|ODO2_STAAN -2.1 Phosphoglycerate kinase (pgk) P99135|PGK_STAAN -1.7 Probable malate:quinone oxidoreductase 2 (mqo2) P99115|MQO2_STAAN -1.55 Succinyl-CoA ligas e [ADP-forming] subunit alpha (sucD) P99070|SUCD_STAAN -1.55 Pyruvate metabolism Dihydrolipoyl dehydrogenase (pdhD) P99084|DLDH_STAAN -2.25 Phosphate acetyltransferase (pta) P99092|PTA_STAAN -2.25 67

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Pyruvate dehydrogenase E1 component subunit beta (pdhB) P99063|ODPB_STAAN -2.6 PentosePhosphate 6-phosphogluconate dehydrogenase, decarboxylating (gnd) P63334|6PGD_STAAN -2.55 Iron storage Ferritin (ftnA) Q7A4R2|FTN_STAAN -3 Stress response Alkaline shock protein 23 (asp23) P99157|ASP23_STAAN -1.55 Catalase (katA) Q7A5T2|CATA_STAAN -1.95 Chaperone protein (hchA) P64313|HCHA_STAAN -1.65 Putative uncharacterized protein (SA1528) Q7A553|Q7A553_STAAN -2.75 Superoxide dismutase [Mn/Fe] 1 (sodA) P99098|SODM1_STAAN -2.05 Protein Synthesis 30S ribosomal protein S1 (rpsA) Q7A5J0|RS1_STAAN -2.3 30S ribosomal protein S10 (rpsJ) P66334|RS10_STAAN -2.3 30S ribosomal protein S9 (rpsI) P66646|RS9_STAAN -2.1 50S ribosomal protein L11 (rplK) P0A0F2|RL11_STAAN -1.75 50S ribosomal protein L13 (rplM) Q7A473|RL13_STAAN -1.6 50S ribosomal protein L18 (rplR) Q7A467|RL18_STAAN -1.5 Seryl-tRNA synthetase (serS) P99178|SYS_STAAN -1.4 Unknown SA2119 protein (SA2119) Q7A3Z5|Q7A3Z5_STAAN -1.6 DNA-binding protein HU (hup) Q7A5J1|DBH_STAAN -1.85 Table 9. Intracellular proteins found to have alt ered expression at 8 hours in the USA 100 adaptive mutant. Pathway Late Exponential Phase Proteins Identified from USA 300 (4) Accession Number Fold Change from Wild-Type Fermentation Alcohol dehydrogenase (adh) Q2FJ31|ADH_STAA3 -2.3 Energy production Formate acetyltransferase (pflB) Q2FK44|PFLB_STAA3 -2 Cell wall degredation Immunodominant staphylococcal antigen A (isaA) Q2FDT8|ISAA_STAA3 1.45 Unknown Putative uncharacterized protein (SAUSA300_2473) Q2FDX1|Q2FDX1_STAA3 2.5 Table 10. Intracellular proteins fo und to have altered expression at 8 hours in the USA 300 adaptive mutant. Pathway Late Exponential Phase Proteins Identified in Common (46) Fold Change from Wild-Type USA 100 USA 300 Cell division Cell division protein ftsZ (ftsZ) -1.15 0.65 Cysteine metabolism Cysteine synthase (cysK) -2.05 -0.75 DNA replication DNA polymerase III subunit beta (dnaN) -1.4 0.2 Electron transport NADH dehydrogenase-like protein -1.7 -0.15 68

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(SA0802/SAUSA300_0844) Energy production ATP synthase subunit alpha (atpA) -0.8 0.85 ATP synthase subunit beta (atpD) -2.5 0 L-lactate dehydrogen ase 1 (ldhA/ldh1) -2.55 -0.25 Folate synthesis Serine hydroxymethyltransferase (glyA) -0.6 0.65 Glycolysis Enolase (eno) -2.15 -0.5 Glucose-6-phosphate isomerase (pgi) -1.8 -0.7 Glyceraldehyde-3-phosphate dehydrogenase 1 (gapA1) -1.35 0.1 Pyruvate kinase (pyk) -1.6 0.15 Iron storage Ferritin (ftnA) -3 -0.2 Nitrogen metabolism Glutamine synthetase (glnA) -1.5 -0.2 NAD-specific glutamate dehy drogenase (gluD/gudB) -1.6 -0.6 Oxidative stress response Alkyl hydroperoxide reductase subunit C (ahpC) -1.25 0.3 Alkyl hydroperoxide reductase subunit F (ahpF) -1.55 0.5 Catalase (katA) -1.65 -0.25 Pentose-Phosphate 6-phosphogluconate dehydrogen ase, decarboxylating (gnd) -2.55 -0.6 Protein Synthesis 30S ribosomal protein S1 (rpsA) -1.75 -0.35 30S ribosomal protein S2 (rpsB) -0.75 1.05 30S ribosomal protein S5 (rpsE) Value missing 0.4 30S ribosomal protein S9 (rpsI) -2.1 0.15 50S ribosomal protein L1 (rplA) -1.35 0.55 50S ribosomal protein L13 (rplM) -1.5 0.55 50S ribosomal protein L14 (rplN) -1.05 0.4 50S ribosomal protein L2 (rplB) -1.25 0.25 50S ribosomal protein L3 (rplC) N o values 0.6 50S ribosomal protein L6 (rplF) N o values No Values Elongation factor G (fusA) -1.15 0.35 Elongation factor Tu (tuf) -1.3 0.65 Glycyl-tRNA synthetase (glyQS) -0.95 0.25 Stress response Alkaline shock protein 23 (asp23) -1.55 0.3 Purine biosynthesis Inosine-5'-monophosphate dehydrogenase (guaB) -1.5 -0.4 Pyruvate metabolism Dihydrolipoyl dehydrogenase (pdhD/lpdA) -2.25 -0.25 D-lactate dehydrogenase (ldhD/ddh) N o values 0.75 Pyruvate dehydrogenase E1 component subunit alpha (pdhA) N o values 0 Pyruvate dehydrogenase E1 component subunit beta (pdhB) -2.25 -0.35 Formate acetyltransferase (pflB) Value missing -2.3 Regulation GTP-sensing transcriptional pleiotropic repressor (codY) -2.2 0.1 TCA cycle Aconitate hydratase (acnA) -2.55 -1.2 Phosphoglycerate kinase (pgk) -2.6 -0.1 Succinyl-CoA ligase [ADP-forming] subunit alpha (sucD) -1.7 -0.55 69

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Dihydrolipoyllysine-residue su ccinyltransferase component of 2-oxoglutarate dehydrogenase complex (odhB) -1.55 -0.45 Transcription DNA-directed RNA polymerase subunit beta (rpoB) -0.8 0 DNA-directed RNA polymerase subunit beta' (rpoC) -1.2 0.2 Unknown DNA-binding protein HU (hup) -1.85 0 Table 11. Intracellular pr oteins found to have altered expression at 8 hours in both the USA 100 and USA 300 adaptive mutants. Pathway Stationary Phase Proteins Identified in Common (129) Fold Change from Wild-Type USA 100 USA 300 Acetoin catabolism Acetoin(diacetyl) re ductase (butA) -2.3 -2.6 Amino acid metabolism Ornithine carbamoyltransfer ase, catabolic (arcB) -2.7 -2 Arginine metabolism Carbamate kinase 1 (arcC1) -3.3 -2.4 Carbamoyl-phosphate syntha se large chain (carB) -2.6 -2.4 ATP binding Cytidylate kinase (cmk) -2.2 -2.5 ATP synthesis ATP synthase gamma chain (atpG) -2.8 -1.9 ATP synthase subunit alpha (atpA) -2.4 -2.5 ATP synthase subunit b (atpF) -2.8 -2.4 ATP synthase subunit beta (atpD) -2.2 -2.1 ATP synthase subunit delta (atpH) -3.5 -2.6 Cell division Cell division protein (ftsA) -2.1 -1.6 Septation ring formation regulator (ezrA) -1.7 -1.5 GTP-binding protein era homolog (era) -1.9 -1.6 Cell redox homeostasis Alkyl hydroperoxide reductase subunit C (ahpC) -1.5 -1.5 Cell wall degredation; peptidoglycan biosynthesis; cell shape D-alanine--D-alanine ligase (ddl) -1.8 -1.6 DNA repair ATP-dependent DNA helicase (pcrA) -2.1 -2 Protein recA (recA) -1.7 -1.7 DNA replication DNA gyrase subunit B (gyrB) -1.5 -1.7 DNA polymerase III subunit beta (dnaN) -1.9 -1.8 Ribonucleoside-diphosphate reductase (nrdE) -1.8 -2.2 Single-stranded DNA-binding protein (ssb) -1.6 -1.8 DNA replication; stress response Chaperone protein (dnaJ) -1.7 -2.8 Electron transport NADH dehydrogenase-like protein SA0802 (SA0802) -2.6 -2 Probable quinol oxidase subunit 2 (qoxA) -2.4 -3.2 Fatty acid biosynthesis Trans-2-enoyl-ACP reductase (fabI) -2.1 -2 Folic acid metabolism Formate--tetrahydrofolate ligase (fhs) -2.2 -1.9 Formate metabolism Putative formate dehydrogenase (SA2102) -1.6 -1.5 Glutamine metabolism Carbamoyl-phosphate syntha se small chain (carA) -2.3 -1.5 Glycerol Aerobic glycerol-3-phosphate dehydrogenase (glpD) -2.2 -1.7 70

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metabolism Glycolysis 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase (gpmA) -2.3 -2.1 Fructose-bisphosphate aldolase class 1 (fda) -1.7 -1.6 Glyceraldehyde-3-phosphate dehydrogenase 2 (gapA2) -1.5 -2.9 Pyruvate kinase (pyk) -1.9 -1.6 L-lactate dehydrogenase 1 (ldhA) -3 -1.6 L-lactate dehydrogenase 2 (ldhB) -2.7 -2.5 Histidine metabolism Urocanate hydratase (hutU) -2.1 -1.8 Lipoate biosynthesis Lipoyl synthase (lipA) -2 -3.1 Metabolism Putative dipeptidase SA1572 (SA1572) -1.9 -1.5 Pyridoxal biosynthesis lyase (pdxS) -1.8 -2.9 Peptidoglycan biosynthesis Aminoacyltransferase femB (femB) -2.1 -3.1 D-alanine--D-alanine ligase (ddl) -1.8 -1.6 Penicillin binding protein 2 prime (mecA) -2.1 -2.1 UDP-N-acetylmuramoyl-tripeptide--D-alanyl-D-alanine ligase (murF) -1.5 -1.7 MSCRAMMs Elastin-binding protein (ebpS) -1.7 -2 Extracellular matrix protein-binding protein (emp) -2 -1.8 Nucleoside transport Pyrimidine nucleoside transport protein (nupC) -2 -2.2 Nucleotide biosynthesis Ribose-phosphate pyrophosphokinase (prs) -2.8 -1.9 Organic acid metabolism Acetate kinase (ackA) -1.9 -1.6 Phospholipid biosynthesis Glycerol-3-phosphate dehydrogenase [NAD(P)+] (gpsA) -2.3 -2.1 Porphyrin biosynthesis Glutamate-1-semialdehyde 2,1-aminomutase 1 (hemL1) -2.9 -1.9 Glutamate-1-semialdehyde 2,1-aminomutase 2 (hemL2) -1.8 -3.3 Proline biosynthesis 1-pyrroline-5-carboxylate dehydrogenase (rocA) -2.1 -1.7 Ornithine aminotransferase 2 (rocD2) -2.9 -1.7 Pyrroline-5-carboxylat e reductase (proC) -1.6 -1.7 Protein folding ATP-dependent Clp protease ATP-binding subunit (clpX) -2.2 -1.7 Putative peptidyl-prolyl cis-trans isomerase (SA0815) -1.5 -1.7 Protein transport Protein translocase subunit secA 1 (secA1) -2.4 -2.3 Protein-export membrane protein (secF) -2.5 -2.8 Purine biosynthesis Adenylosuccinate synthetase (purA) -1.6 -1.7 Amidophosphoribosyltransferase (purF) -2.3 -2.5 Bifunctional purine biosynthesis protein purH (purH) -2.9 -2.3 Phosphoribosylamine--glycine ligase (purD) -3.1 -2.9 Phosphoribosylaminoimidazole carboxylase ATPase subunit (purK) -2.9 -2.4 Phosphoribosylaminoimidazole-succinocarboxamide synthase (purC) -3.5 -2.7 Phosphoribosylformylglycinamidine synthase 1 (purQ) -2.6 -3.1 Phosphoribosylformylglycinamidine synthase 2 (purL) -3 -3.4 Purine ribonucleoside salvage Hypoxanthine-guanine phosphoribosyltransferase (hpt) -1.8 -1.9 Xanthine phosphoribosyltransferase (xpt) -2.1 -2.1 Pyrimidine Pyrimidine-nucleoside phosphorylase (pdp) -2.9 -1.5 71

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metabolism Uracil phosphoribosy ltransferase (upp) -1.5 -3.4 Uridine kinase (udk) -2.1 -3.2 Pyruvate biosynthesis Pyruvate dehydrogenase E1 component subunit beta (pdhB) -1.8 -1.5 RNA degredation Ribonuclease J 2 (SA1118) -1.8 -2.7 Stress response 10 kDa chaperonin (groS) -1.6 -1.5 ATP-dependent protease ATPase subunit (hslU) -2 -3.2 GTPase obg (obg) -2.6 -2 Stringent response GTP pyrophosphokinase (relA) -2.4 -1.6 Sugar metabolism Acetyl-CoA synthetase (acsA) -1.9 -1.7 Sugar transport PTS system EIIBC component (SA0186) -1.8 -1.5 PTS system glucose-specific EIICBA component (ptsG) -3 -3.6 PTS system glucoside-specific EIICBA component (glcB) -3.2 -1.6 TCA cycle Citrate synthase II (citZ) -2 -1.6 Isocitrate dehydrogenase [NADP] (icd) -1.9 -1.9 Succinate dehydrogenase flavoprotein subunit (sdhA) -2.2 -1.8 Succinate dehydrogenase iron-sulfur protein subunit (sdhB) -1.9 -2 Succinyl-CoA ligase [ADP-forming] subunit alpha (sucD) -1.8 -1.6 Succinyl-CoA ligase [ADP-forming] subunit beta (sucC) -1.5 -1.5 Transcription DNA-directed RNA polymerase subunit beta (rpoB) -1.6 -1.5 DNA-directed RNA polymerase subunit beta' (rpoC) -2 -1.5 Probable DNA-directed R NA polymerase subunit delta (rpoE) -1.9 -1.6 Transcription regulation Serine-protein kinase (rsbW) -2.2 -2.3 Bifunctional protein (pyrR) -3.2 -2.4 Translation Asparaginyl-tRNA synthetase (asnS) -2.5 -1.9 Elongation factor Tu (tuf) -1.8 -1.5 Methionyl-tRNA formyltransferase (fmt) -1.9 -2.3 Prolyl-tRNA synthetase (proS) -1.9 -1.7 Seryl-tRNA synthetase (serS) -3.3 -1.5 Threonyl-tRNA synthetase (thrS) -2.2 -1.5 Translation initiation factor IF-2 (infB) -2.1 -1.9 30S ribosomal protein S11 (rpsK) -2.1 -1.8 30S ribosomal protein S12 (rpsL) -1.9 -1.7 30S ribosomal protein S13 (rpsM) -1.9 -1.6 30S ribosomal protein S14 type Z (rpsZ) -2 -1.8 30S ribosomal protein S3 (rpsC) -1.5 -2.1 30S ribosomal protein S4 (rpsD) -1.5 -1.6 30S ribosomal protein S5 (rpsE) -2.1 -1.5 30S ribosomal protein S6 (rpsF) -1.8 -1.6 50S ribosomal protein L1 (rplA) -2.2 -2 50S ribosomal protein L10 (rplJ) -1.7 -1.6 50S ribosomal protein L13 (rplM) -2.3 -1.6 50S ribosomal protein L14 (rplN) -2.6 -1.6 50S ribosomal protein L19 (rplS) -2.4 -1.8 50S ribosomal protein L2 (rplB) -1.9 -1.7 50S ribosomal protein L20 (rplT) -2.8 -2.1 50S ribosomal protein L3 (rplC) -1.5 -1.9 50S ribosomal protein L4 (rplD) -2.2 -1.9 72

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50S ribosomal protein L5 (rplE) -2.7 -2.5 tRNA modification (Dimethylallyl)adenosine tRNA methylthiotransferase (miaB) -1.5 -1.5 tRNA modification GTPase (mnmE) -1.7 -2.2 Virulence Conserved virulence factor B (cvfB) -1.5 -1.7 Unknown DegV domain-containing protein (SA1258) -2.3 -1.9 GTP-binding protein (engA) -2.5 -1.8 PhoH protein (phoH) -1.9 -1.5 Putative phosphotransferase (SA1392) -2 -1.9 Uncharacterized lipoprotein (SA2158) -3 -1.5 UPF0051 protein (SA0778) -1.9 -1.8 UPF0133 protein (SA0437) -1.5 -1.5 UPF0365 protein SA1402 (SA1402) -1.7 -2 UPF0477 protein (SA0873) -1.9 -1.9 UPF0478 protein SA1560 (SA1560) -1.9 -2 Table 12. Intracellular proteins found to have altered expression at 15 hours in both the USA 100 and USA 300 adaptive mutants. 73

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DISCUSSION Antimicrobial resistance has been an escala ting problem since the advent of clinical antimicrobial use. Every new antibiotic that has been released, including completely synthetic antimicrobials, has rapidly encountered resistan ce in one form or another19, 43, 69, 75, 102. The cost of developing antibiotics is high, especially considering their short lifespan; and is not considered lucrative by pharmaceutical companies. For this reason, the number of new therapies has dwindled, while the number of effective therapeutics continues to decrease. The research presente d here investigates th e mode of action of a novel -lactam antimicrobial, with potent activity against MRSA by analyzing laboratory-generated resistant mutants. A number of diverse la boratory and clinical S. aureus isolates were used in this study because the characteristics of pathogenesis and antimicrobial susceptibility can differ widely depending on the stra in. Importantly, the Ns L MIC was found to be comparable across all strains tested, so the mode of action of this drug does not appear to be affected by existing, intrinsic resistance factors. Previous studies have suggested that Ns L may target 3-oxoacyl-acyl carrier protein syntha se (ACP) III (FabH) either directly or indirectly92, so we set forth to determine if the addition of fatty acids to the culture media would have an effect on the Ns L MICs. 74

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The results of our fatty acid assays with Ns L indicate that fatty acid biosynthesis may not be the sole or primary target of this drug. This is apparent when the effects of fatty acids on Ns L activity are compared to their effects on triclosan and vancomycin activity. The MIC of vancomycin, a cell wall synthesis inhibitor, was reduced by Tween 80. The effect of Tween 80 on the MIC of Ns L was, however, not sign ificant. The MIC of triclosan, an inhibitor of enoyl-ACP reductase (FabI), which catalyzes the final step of fatty acid synthesis, was significantly a ffected by the presence of Tween 80, being increased by at least 20-fold for al l strains tested, from 0.125 g/mL to 6.25 g/mL. It is noteworthy that this dras tic increase in MIC does not ag ree with that published by Balemans, et. al6. They saw that the MIC of triclosan increased from 0.125 g/mL to 2 g/mL in the presence of the same concentr ation of Tween 80 as used in our study, and attributed this to the plasma binding properties of triclosan. They also showed that other known fatty acid synthesis inhibitors behaved similarly6. However, Altenbern showed that cerulenin, an inhibitor of 3-oxoacyl-ACP synthase I/II (FabB/F) which catalyzes the second condensation step of fatty acid synthesi s, lost efficacy in the presence of both saturated and unsaturated fatty acids2. This was confirmed by Br inster, et. al, who used Streptococcus agalactiae as their initial model17, and S. aureus in response to the criticism from Balemans, et. al.16, which is again confirme d by our work. Because Ns L does not follow the rescue pattern established by triclosan and other fatty acid synthesis inhibitors, an enzyme of the fatty acid synt hesis pathway may not be its primary target. Biofilm-mediated infections often lead to chr onic illness due to persistence, and difficulty in treating biofilm-mediated conditions. Currently, a prophylactic view towards 75

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preventing colonization is take n, but there are many limitations, particularly considering only catheter-associated biofilms are targeted by this method. Our study compared the effects of vancomycin, triclosan, and tetracycline to Ns L on biofilms and found that the ability of Ns L to kill established biofilms exceeded that of the other antimicrobials tested, as measured by percent reduction. There are a number of factors that affect the activity of an antimicrobial on the target biofilm, such as the rate of diffusion of the drug through the biofilm61 and the ability of the drug to kill stationary phase bacteria74. Ns L reduced biofilm formation by 43%, which was the greatest observed decrease. Tetracycline was shown in one in vitro experiment to completely reduce metabolic activity in an S. aureus biofilm36 and to have high levels of killing of S. epidermidis biofilms74; we showed here that tetracycline was effective at reducing biofilms by 29%. Both vancomycin and triclosan were ineff ective at killing biof ilms, which has been demonstrated previously in S. aureus32, S. epidermidis74, and Salmonella typhimurium99 to be due to restricted diffusion through the biofilm. The biofilm-killing activity of Ns L may be due to high levels of diffusion through the biofilm matrix, an enhanced ability to kill stationary phase cells, or a combination of both of these factors. It is important, when dealing with antimicrobial agents, to have a sense of the rate at which clinical isolates spontaneously become resistant to the drug. This is particularly important when determining the dose to ad minister to patients. The spontaneous mutation rate for SH1000, a non-resistan t laboratory strain, was 5.2 x 10-9 at 7x the Ns L MIC. Interestingly, resistance o ccurred more frequently at 2 x 10-8 when a nitrosoguanidine (NTG) muta nt library of SH1000 was exposed to the same Ns L 76

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concentration. With that said, these latter isolates had Ns L MICs no greater than 10 g/mL, whereas the spontaneous, untreated SH1000 mutants had Ns L MICs as high as 50 g/mL. The spontaneous mutation rate of Ns L was found to be comparable to a number of clinically releva nt antimicrobials having a wide range of modes of action ( Table 13 ). Rifampicin, for example, is used on ly as a combination therapy due to its high rate of spontaneous mutagenesis resulting in resistance72. Ns L had a spontaneous mutation rate 19to 20-fold lower than rifampic in, indicating that it could conceivably be used in single therapy. Antimicrobial Spontaneous Mutation Rate at 8x MIC References Ceftobiprole 0.9 x 10-10 5.3 x 10-10 13 Ciprofloxacin 3.2 x 10-10 85 Daptomycin 0.8 x 10-10 9.1 x 10-11 13, 55 Levofloxacin 6.4 x 10-10 85 Linezolid 1.3 x 10-9 8.7 x 10-10 13 Minocycline 6.7 x 10-9 9.5 x 10-10 13 Mupirocin 1.5 x 10-8 9.5 x 10-10 13 Ns L (SH1000) 5.2 x 10-9 This study Ns L (MNNG-treated SH1000) 2 x 10-8 This study Quinupristin/Dalfopristin 3.3 x 10-9 8.7 x 10-10 13 Rifampicin 10-7 10-8 72 Teicoplanin 1.6 x 10-8 7.1 x 10-11 55 Telavancin 1 x 10-11 5 x 10-11 55 Table 13. Spontaneous mutation rates for various antimicrobials. The single-step spontaneous mutation rates for various clinically relevant antimicr obials are reported in the literature and compiled here for comparison to Ns L. Ns L spontaneous mutagenesis was performed at 7x MIC. Rifampicin spontaneous mutagenesis was performed at 10x MIC. The spontaneous and adaptive mu tants generated were character ized in order to provide insight into their mechanis m(s) of resistance to Ns L. The rationale for this is that we may be able to extrapolate the mode of action of Ns L, by examining the phenotypic changes of the mutants. Based on previous analysis of the drug92, we would expect to see 77

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changes in those processes invo lving coenzyme-A (CoA), such as glycolysis, the citric acid cycle (TCA), pyruvate me tabolism, and, particularly, fatty acid biosynthesis. Because changes in these pathways would have widespread consequences, we used a global approach to determine phenotypic changes in the Ns L-resistant mutants. The production of virulence factors by S. aureus is a complex and highly regulated process involving multiple regulatory systems which control growth phase-dependent virulence factor production81. No changes were found in virulence factor production for either of the SH1000 spontane ous mutants as determined by growth on blood or milk plates. Conversely, it is interesting to note that SDS-PAGE analysis of SH-11 appears to show an increase in production of cell-wall associated and s ecreted proteins. This may simply be an incongruity in the SDS-PAGE, ra ther than a true phenotypic change, or it may be that SH-11 is producing other virulence factors that were not tested for. On the other hand, virulence determinant producti on in both the USA 100 and USA 300 adaptive mutants was dramatically reduced, as shown by a reduction or loss of proteolytic activity, hemolysis, capsule production, and cell-wall an d secreted protein production. The genes encoding proteases, hemolysins, exotoxins, and capsular polysaccharides are all positively regulated by the central virulence regulator, agr (accessory gene regulator)81, indicating that both the USA 100 and USA 300 adaptive mutants may have developed agr mutations. Because staphylococcal virule nce regulation is so convoluted, there may be additional changes in the adaptive mutant s that resulted in th e observed phenotype, such as repression of, or changes in, the st aphylococcal accessory regulator A (SarA), an activator of agr12. It is also possible that there is repression of, or changes in, SaeRS, 78

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which increases transcription of hemolysins and capsule genes68. Finally, there may be an increase in transcription of the repressor of toxin (Rot), which inhi bits transcription of multiple protease and hemolysin genes48. Interestingly, deletion of s arA was shown to result in increased susceptibility to ciprofloxacin, rifampicin, and vancomycin83 and deletion of sarA and agr together resulted in increased susceptibility to oxacillin, cefoxitin, and imipenem38. To date, changes in sarA or agr have not been associated with increased antimicrobial resistance. These an d other issues will be addressed in more detail in the proteomics section. Amino acid limitation leads to activation of the stringent response in S. aureus, mediated by the synthesis of polyphosphorylated guanosine nucleotid es ((p)ppGpp) by rsh (RelA/SpoT homolog)40. (p)ppGpp induces overexpressi on of genes involved in amino acid synthesis and repression of ribosomal and tRNA genes, allowing the bacteria to survive under less than optimal conditions. Mutants defective in rsh were found to be more susceptible to mupirocin, and not chan ged in susceptibility to vancomycin and ciprofloxacin22, 40. A clinical isolate with a persiste ntly active stringent response was also found to have no changes in susceptibility to vancomycin, teicoplanin, gentamycin, linezolid, daptomycin, tigecycline, rifampicin, or ciprofloxacin39. While neither spontaneous mutant showed changes in growth under amino acid limiting conditions, both adaptive mutants showed changes. The USA 100 adaptive mutant had increased growth levels during lag and early-exponentia l phase, but decreased levels of growth during lateand post-exponentia l phase. The USA 300 adaptive mutant had significantly reduced levels of growth during exponential phase as compared to its parent. While 79

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glucose limitation is also known to induce the stringent response40, none of the strains tested showed changes in growth in glucose limiting media. It has also been suggested that a fully functioning TCA cycle is necessary for effective amino acid catabolism96, which will be further discussed in the proteo mics section. We know from growth curves in tryptic soy broth (T SB) that both adaptive mutants have reduced growth levels during exponential phase, a possible indicator of defects in ener gy production. Furthermore, SarA, proposed above to be repressed, is kno wn to positively regulate expression of amino acid transport and metabolism proteins31; thus, if the adaptive mutants have defects in regulation by SarA, they will have reduced viabil ity under amino acid limiting conditions. Taken together, along with the increased resistance of SH-32 and the USA 300 adaptive mutant to a number of antimicrobials (discussed later), this may indicate that the adaptive mutants are defective in tr ansport of extracellular amino acids, rather than in their overall response to nutrient lim itation as regulated by (p)ppGpp or SarA. Such a deficiency in amino acid utilization ma y lead to weaker pep tidoglycan cross-links, resulting in changes in susceptibility to cell wall-active antimicrobials and autolytic activity97, possibly implying that Ns L has an effect on the integrity of the cell wall. Reduced survivability in response to phosphate limitation has been attributed to a number of factors. Lithgow et. al. have described the role of cysteine synthase65 and FtsH66 in recovery from amino acidand phosphate limitin g-conditions as due to defects in protein synthesis during starvation. Cystei ne synthase is responsible for S. aureus resistance to tellurite, oxidative stress, and disulfides, and is proposed to be important as part of the thiol redox balance in S. aureus65, while FtsH has chaperone-like activities and mediates 80

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resistance to methyl viologen and tellurite66. This is of particul ar interest concerning Ns L, as Revell, et. al. have proposed that this drug may alter the thiol redox balance by reducing CoA, which would prevent it pe rforming its role in this buffer system92. The USA 300 adaptive mutant showed reduced grow th levels in amino acidand phosphatelimiting conditions, and the USA 100 adaptive mu tant had reduced growth levels during late exponential and stationa ry phase in amino acid-limiting conditions, suggesting that cysteine synthase and/or FtsH activity may have been reduced as a result of gaining resistance to Ns L. The loss of the ability to ferment ribose, as seen with both the USA 100 and USA 300 adaptive mutants, has been associated with an increase in fitness under nutrient limiting conditions in Escherichia coli21. This may indicate that gaining resistance to Ns L results in insufficient metabolism or utilizatio n of nutrients, and that the loss of ribose fermentation ameliorates this defect. The Ns L-resistant mutants were subjected to metal ion-limiting conditions by growing them in CL medium, which contains only magnesium as a metal ion source. The spontaneous mutant SH-32 was shown to have decreased fitness during exponential phase under these conditions. The binding and uptake of magnesium from the environment has been shown to be related to the composition of teichoic acids in the bacterial cell wall. Specif ically, increased D-alanine co ntent of teichoic acids in Bacillus subtilis Lactobacillus buchneri and S. aureus resulted in a reduced ability to bind magnesium ions3, 59, 80. Additionally, changes in the D-alanine content of the cell walls 81

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has been associated with increased resistance to -lactam antimicrobials80, 82, which is further addressed below. The oxidative stress response in S. aureus is highly regulated and dependent on the availability of iron, manganese (Mn2+) and zinc. Uptake of Mn2+ is regulated by MntR, which induces expression of supe roxide dismutase (SodA and SodM)52. Recently, it has been shown that SH1000 defective in sarA had increased superoxide dismutase activity in an Mn2+-independent manner7. Based on the increased levels of exponential phase growth in the USA 300 adaptive mu tant, and the obvious lack of Mn2+ ions, it may be that its oxidative stress respons e is increased due to defect s in regulation by SarA. As mentioned previously, SarA deficiencies have been linked to increased susceptibility to a number of antimicrobials38, 83. Additionally, ciprofloxacin and chloramphenicol have been known to induce the oxidative stress response of S. aureus resulting in increased production of superoxide dismuatse1, 9. This is particularly relevant, as the USA 300 adaptive mutant showed a complete loss of susceptibility to ciprofloxacin, and indicates that Ns L may induce a similar response in this strain. Changes in the integrity of the cell wall can l ead to increased autolysis and susceptibility to cell wall-synthesis inhibiting antimicrobial s. While the spontaneous mutants did not exhibit changes in autolysis, SH-32 became 2-fold more resistant to penicillin G than its parent. The mechanism of penicillin G involves inhibiting the cross-linking of peptidoglycan, causing osmotic stress and cell lysi s. As previously mentioned, penicillin G resistance can be correlated to increased D-alanine content of the cell wall82. It is 82

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interesting to note that no changes in autolysi s were observed in this strain, which is often associated with cell wall synthesis-inhi bitor resistance. SH-32 also gained heteroresistance to mupirocin, which has primar ily been associated with the inhibition of isoleucine-tRNA synthetase, but also with inhibiting cell wall synthesis46. This heteroresistance may be similar to that seen with heteroresistance to vancomycin, where only a small subset of the bact erial population is resistant44, indicating that the mutation induced by resistance to Ns L does not confer complete protection from mupirocin in SH-32. While the USA 100 adaptive mutant did no t show any changes in antimicrobial susceptibility, the USA 300 adaptive mutant ga ined resistance to kanamycin, ampicillin, erythromycin, and ciprof loxacin. A study using S. aureus strain COL showed that prolonged exposure to oxacillin resulted in increased resistance to erythromycin, kanamycin, and ciprofloxacin, among others due to increased efflux pump activity70. Considering that this efflux pump activity arose in COL after a similar antimicrobial treatment as used in our study, and that they both had similar antimicrobial resistance profiles, it is possible that the increased resistance of the USA 300 adaptive mutant to Ns L may be due to efflux of these antimicrobials out of the cells. As mentioned before, previous studies have suggested that Ns L may target FabH, the initial enzyme involved in fatty acid biosynthesis92. With this in mind, the fabH gene of a USA 100 spontaneous mutant was sequenced, but found to have no differences from its parent, implying that alte ring FabH did not induce a benefit leading to Ns L-resistance. 83

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Consequently, the sequenced gene was compared to fabH of the COL strain and found to have differences in three distinct amino acids, L40V, D151E, and I276L. Additionally, the USA 100 FabH sequence was found to have the same amino acid differences when compared to Newman, USA 300 Houston, and USA 400 MW2, but was identical to USA 100 Mu50 and USA 200 MRSA252. Comparison of the predicted secondary structures of the amino acid sequences of COL and USA 100 635 using QuickP hyre analysis did not reveal any structural diffe rences in FabH; thus, the stru cture of FabH likely does not have an effect on the res ponse of these strains to Ns L (see appendix for results). Comparative proteomic analysis of the US A 100 and USA 300 wild-type strains, and their respective adaptive muta nts was performed at 8 hours (post-exponentia l phase) in duplicate and a pilot run at 15 hours (stationary phase) in or der to further elucidate the mechanism(s) by which these isol ates gained resistance to Ns L. A change in protein expression of 1.5-fold or more was considered to be significant. During post-exponential phase, the USA 100 adaptive mutant was found to have reduced expression of numerous protei ns involved in nucleotide synthesis, glycolysis, pyruvate metabolism, the TCA cycle, amino acid synthesis, protein synthesis, and stress responses. A specific protein from the TCA cycle, aco nitate hydratase, was reduced by 2.6 fold. Aconitate hydratase has been shown by Somervill e, et. al. to be required for amino acid catabolism, specifically for synthesis of ar ginine, leucine, proline, and histidine96, and this mutant had reduced growth levels under amino acid-limiting conditi ons. This protein was also expressed 2.2-fold less during stationa ry phase. Interestingly, cysteine synthase 84

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(CysK) expression was reduced by 2.05-fold during post-exponential and 1.9-fold during stationary phase. In addition to being invol ved in the synthesis of cysteine, CysK has also been associated with stress response to H2O2, acidic pH, tellurite diamide (Fig. 25), and limited amino acids and phosphate65. Revell, et. al. have proposed that Ns L may act as a sulfenylating agent towards CoA, leading to its inactivation92, similar to the thioloxidating activity of diamide. Specific proteins affected during post-exponential phase that utilize CoA in one form or another are citrate synthase II (2.5-fold reduction), the 2oxoglutarate dehydrogenase co mplex (2.1-fold reduction), and phosphate acetyltransferase (2.25-fold re duction). During stationary phase, the expression of multiple proteins with acetyl-CoA as their final product were significantly reduced, including alcohol-acet aldehyde dehydrogenase (1.7-fo ld reduction), acetyl-CoA synthetase (1.9-fold reduction), format acetyltransferase (2.4-fold reduction), and pyruvate dehydrogenase (3-fold reduction). Notably, while acetyl-CoA is required for initiation of fatty acid synthe sis by FabH, no changes in expr ession of this enzyme were detected at any time point tested. However, AccA, FabF, FabI, and FabZ were all found to have expression reduced between 1.8a nd 2.2-fold during stationary phase. Figure 25. Diamide. Analysis of the USA 300 adaptive mutant at 8 hours resulted in only four proteins with a significant change from the parent; however, analysis at 15 hours resulted in 11 proteins upregulated and 260 downregulat ed proteins changed by 1.5-fold or more. Proteins involved in glycolysis, the TCA cycle, and pyruvate metabolism were found to be decreased during stationary phase, but, unexp ectedly, proteins involved in fatty acid 85

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86 synthesis were not identified as signifi cantly altered. Unlike the USA 100 adaptive mutant or other timepoints tested, the US A 300 adaptive mutant showed a number of proteins overexpressed during st ationary phase. Both forms of superoxide dismutase (SodA and SodM) were upregulated 1.6an d 2.2-fold, respectively, possibly due to deficiencies in regulation by SarA, as discus sed previously. Most interesting in this mutant were the changes in its global regulato rs. CodY and B were detected, but not significantly changed, while Agr (3.8 fold reduc tion), SarR (2.6-fold reduction), and SaeS (2.2-fold reduction) were all significantly reduced. These regulators were previously discussed in reference to the marked decrease in virulence factors detected in this mutant strain. SarR is a repressor of SarA81, expression of which was found to be increased by only 0.6-fold, which may mean that there is another factor involved in regulation of agr The repressor of sarR ( rsr ) was recently described as reducing transcription of both sarR and agr100. This supports the hypothesis that agr was downregulated in this mutant, and that SarA was not playing a significant ro le in virulence factor determination or antimicrobial susceptibility.

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Figure 26. Phenotypic changes observed in SH-32 spontaneous mutant and prop osed mechanisms of those changes. Purple indicates the observed phenotypic change. Arrows with dotted lines pointing to blue indicates the proposed mechanism. 87

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While few changes were observed in the spon taneous mutants used in this study, a key point can be gleaned from analysis of SH32. Based on the increased resistance to penicillin G and the decreased growth in metal ion limiting conditions, resistance to Ns L may have induced changes in the D-alanine content of the cell wall. It is possible that this is the mechanism by which heteroresistance to mupirocin was caused, and that the primary target, isoleucyl-tRNA synthe tase, was not affected by resistance to Ns L. 88

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Figure 27. Phenotypic changes observed in USA 100 adaptive mutant (AM) and proposed mechanisms of those changes. Purple indicates the observed phenotypic change. Arrows with dotted lines pointing to blue indicates the proposed mechanism. Both green and orange indicate proteins observed by proteomic analysis. 89

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Analysis of the comparative proteomics results for the USA 100 adaptive mutant was most insightful and indicates that those pathways reliant on CoA were significantly downregulated. While expression of FabH was not specifically reduced, the majority of proteins involved in fatty acid synthesis we re found to be significan tly reduced, as were glycolysis, pyruvate metabolism, and TCA cycl e enzymes. This supports the conclusions made in previous studies that Ns L has an effect on CoA92. 90

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Figure 28. Phenotypic changes observed in USA 300 adaptive mutant and prop osed mechanisms of those changes. Purple indicates the observed phenotypic change. Arrows with dotted lines pointing to blue indicates the proposed mechanism. Both green and orange indicate proteins observed by proteomic analysis. Green indicates regulators known to affect the indicated phenotype. Both green and orange indicate protei ns observed by proteomic analysis, except for SarA and rsr. Lines with a blunt end indicate downregulation of that protein or gene. 91

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The USA 300 adaptive mutant appears to have developed a number of alterations in its regulatory pathways. SarR expression was found to be reduced, wh ile SarA expression was unaltered. Agr expression was also reduc ed, resulting in abridged expression of various virulence factors. Loss of agr and SarA activity has been shown to cause increased susceptibility to antimicrobials38, 83; however, the USA 300 adaptive mutant displayed increased antimicrobial resistan ce in the disk diffusion assays. These regulatory changes may be secondary to th e expression of a multi-drug efflux pump70, conferring resistance to kanamycin, ampic illin, erythromycin, ciprofloxacin, and, ultimately, Ns L. 92

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FUTURE DIRECTIONS There are a number of experiments that co uld be performed to clarify the precise mechanisms of resistance exhibited by the spontaneous and adaptive mutants. First and foremost, further analysis of the intracellu lar proteome would allow for statistically significant data. SH-11 and SH-32 could also be evalua ted by the same process, particularly as phenotypic anal ysis did not show dramatic changes. It would also be beneficial to perform whol e-genome sequencing of the Ns L-resistant mutants, to further pinpoint genetic changes leading to resistan ce. This would be further confirmed by introducing similar changes into a clean wild-t ype strain and observing if there were the expected changes in resistance to Ns L. A straightforward e xperiment that could be performed is measurement of the efflux activity of the USA 300 adaptive mutant towards Ns L, which would confirm if this was its pr imary mode of resistance, as hypothesized. Complementation of any clean S. aureus strain with a coaABCDE overexpression vector could help determine if Ns L actually targets CoA by monitoring growth for changes in the Ns L MIC as compared to the wild-type strain. 93

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APPENDICES 106

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107 Appendix A. Chemically-defined media. L-glutamic acid 4.44 gL-1 L-histidine 0.88 gL-1 L-aspartic acid 4.44 gL-1 L-valine 0.88 gL-1 L-proline 4.44 gL-1 L-arginine 0.66 gL-1 Glycine 4.44 gL-1 L-cysteine 0.44 gL-1 L-threonine 4.44 gL-1 L-phenylalanine 0.38 gL-1 L-serine 4.44 gL-1 L-tyrosine 0.34 gL-1 L-alanine 4.44 gL-1 L-methionine 0.34 gL-1 L-lysine HCl 1.12 gL-1 L-tryptophan 0.12 gL-1 L-isoleucine 1.12 gL-1 Table A1. CL1. Autoclave and store at -20C. Na2HPO4 70 gL-1 KH2PO4 30 gL-1 Table A2. CL2. Autoclave and store at 4C. Adenine sulfate 2 gL-1 Guanine HCl 2 gL-1 Table A3. CL3. Make up in 0.1M HCl. Autoclave and store at 4C. Pyridoxal 0.4 gL-1 Nicotinic acid 0.2 gL-1 Pyridoxamine HCl 0.4 gL-1 Thiamine HCl 0.2 gL-1 D-pantothenic acid 0.2 gL-1 Biotin 0.01 gL-1 Riboflavin 0.2 gL-1 Table A4. CL4. Add 10g Chelex-100 and stir for 4 hours before filter sterilizing. Store at 4C. L-aspartic acid 2.14 gL-1 L-methionine 1.43 gL-1 L-alanine 1.43 gL-1 L-phenylalanine 1.43 gL-1 L-arginine 1.43 gL-1 L-proline 2.14 gL-1 L-cysteine 0.714 gL-1 L-serine 1.43 gL-1 Glycine 1.43 gL-1 L-threonine 2.14 gL-1 L-glutamic acid 2.14 gL-1 L-tryptophan 1.43 gL-1 L-histidine 1.43 gL-1 L-tyrosine 1.43 gL-1 L-isoleucine 2.14 gL-1 L-valine 2.14 gL-1 L-lysine HCl 1.43 gL-1 Na2HPO4 50 gL-1 L-leucine 2.14 gL-1 KH2PO4 21.43 gL-1 Table A5. CDM1. Autoclave and store at -20C.

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Biotin 0.001 gL-1 Riboflavin 0.02 gL-1 D-panthothenic acid 0.02 gL-1 Nicotinic acid 0.02 gL-1 Pyridoxal 0.04 gL-1 Thiamine HCl 0.02 gL-1 Pyridoxamine HCl 0.04 gL-1 Table A6. CDM2. Filter sterilize and store at 4C. Adenine sulfate 0.4 gL-1 0.4 gL-1 Guanine HCl Table A7. CDM3. Make up in 0.1M HCl. Au toclave and store at 4C. CaCl2 .6H2O 10 gL-1 5 gL-1 MnSO4 FeNH4(SO4)2 .12H2O 6 gL-1 Table A8. CDM4. Make up in 0.1M HCl. Autoclav e and store at room temperature. Glucose 100 gL-1 MgSO4 .7H2O 5g Table A9. CDM 5. Autoclave and store at room temperature. 108

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Appendix B. Complete proteomics data. # Identified Proteins (85) Accession Number Fold Change from WT Average Fold Change 37 SA0295 protein OS=Staphylococcus aureus (strain N315) GN=SA0295 PE=4 SV=1 Q7A7Q2|Q7A7Q2_STAAN -4.2 Value Missing -4.2 40 Ferritin OS=Staphylococcus aureus (strain N315) GN=ftnA PE=1 SV=1 Q7A4R2|FTN_STAAN -2.9 -3.1 -3 64 Chaperone protein hchA OS=Staphylococcus aureus (strain N315) GN=hchA PE=1 SV=1 P64313|HCHA_STAAN -2.9 -2.6 -2.75 65 Aspartate carbamoyltransferase OS=Staphylococcus aureus (strain N315) GN=pyrB PE=1 SV=1 P65618|PYRB_STAAN -2.5 -2.8 -2.65 47 Phosphate acetyltransferase OS=Staphylococcus aureus (strain N315) GN=pta PE=1 SV=1 P99092|PTA_STAAN -2.7 -2.5 -2.6 73 Phosphoglycerate kinase OS=Staphylococcus aureus (strain N315) GN=pgk PE=1 SV=1 P99135|PGK_STAAN -2.6 -2.6 -2.6 8 L-lactate dehydrogenase 1 OS=Staphylococcus aureus (strain N315) GN=ldhA PE=1 SV=1 P65256|LDH1_STAAN -2.5 -2.6 -2.55 20 Aconitate hydratase OS=Staphylococcus aureus (strain N315) GN=acnA PE=1 SV=1 P99148|ACON_STAAN -2.7 -2.4 -2.55 29 6-phosphogluconate dehydrogenase, decarboxylating OS=Staphylococcus aureus (strain N315) GN=gnd PE=1 SV=1 P63334|6PGD_STAAN -2.6 -2.5 -2.55 23 ATP synthase subunit beta OS=Staphylococcus aureus (strain N315) GN=atpD PE=1 SV=1 P99112|ATPB_STAAN -2.6 -2.4 -2.5 32 Phosphoribosylformylglycinamidine synthase 1 OS=Staphylococcus aureus (strain N315) GN=purQ PE=1 SV=1 P99166|PURQ_STAAN -2.3 -2.6 -2.45 79 Phosphoribosylaminoimidazole carboxylase ATPase subunit OS=Staphylococcus aureus (strain N315) GN=purK PE=1 SV=1 Q7A695|PURK_STAAN -2.3 -2.5 -2.4 52 50S ribosomal protein L18 OS=Staphylococcus aureus (strain N315) GN=rplR PE=1 SV=1 Q7A467|RL18_STAAN -2.3 -2.3 -2.3 74 Seryl-tRNA synthetase OS=Staphylococcus aureus (strain N315) GN=serS PE=1 SV=1 P99178|SYS_STAAN -2.3 -2.3 -2.3 4 Dihydrolipoyl dehydrogenase OS=Staphylococcus aureus (strain N315) GN=pdhD PE=1 SV=1 P99084|DLDH_STAAN -2.2 -2.3 -2.25 5 Pyruvate dehydrogenase E1 component subunit beta OS=Staphylococcus aureus (strain N315) GN=pdhB PE=1 SV=1 P99063|ODPB_STAAN -2.1 -2.4 -2.25 61 GTP-sensing transcriptional pleiotropic repressor codY OS=Staphylococcus aureus (strain N315) GN=codY PE=1 SV=1 P63844|CODY_STAAN -2.2 -2.2 -2.2 15 Enolase OS=Staphylococcus aureus (strain N315) GN=eno PE=1 SV=1 P99088|ENO_STAAN -2 -2.3 -2.15 25 30S ribosomal protein S9 OS=Staphylococcus aureus (strain N315) GN=rpsI PE=1 SV=1 P66646|RS9_STAAN -2 -2.2 -2.1 109

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76 Citrate synthase II OS=Staphylococcus aureus (strain N315) GN=citZ PE=1 SV=1 Q7A561|Q7A561_STAAN -2.2 -2 -2.1 62 Cysteine synthase OS=Staphylococcus aureus (strain N315) GN=cysK PE=1 SV=1 P63871|CYSK_STAAN -2.1 -2 -2.05 75 Putative uncharacterized protein SA1528 OS=Staphylococcus aureus (strain N315) GN=SA1528 PE=4 SV=1 Q7A553|Q7A553_STAAN -2.1 -2 -2.05 70 Superoxide dismutase [Mn/Fe] 1 OS=Staphylococcus aureus (strain N315) GN=sodA PE=1 SV=1 P99098|SODM1_STAAN -1.8 -2.1 -1.95 45 DNA-binding protein HU OS=Staphylococcus aureus (strain N315) GN=hup PE=1 SV=1 Q7A5J1|DBH_STAAN -1.8 -1.9 -1.85 24 Glucose-6-phosphate isomerase OS=Staphylococcus aureus (strain N315) GN=pgi PE=1 SV=1 P99078|G6PI_STAAN -1.7 -1.9 -1.8 28 30S ribosomal protein S1 OS=Staphylococcus aureus (strain N315) GN=rpsA PE=1 SV=1 Q7A5J0|RS1_STAAN -1.7 -1.8 -1.75 18 Succinyl-CoA ligase [ADP-forming] subunit alpha OS=Staphylococcus aureus (strain N315) GN=sucD PE=1 SV=1 P99070|SUCD_STAAN -1.8 -1.6 -1.7 82 NADH dehydrogenase-like protein SA0802 OS=Staphylococcus aureus (strain N315) GN=SA0802 PE=1 SV=1 Q7A6J4|Y802_STAAN -1.7 -1.7 -1.7 17 Catalase OS=Staphylococcus aureus (strain N315) GN=katA PE=1 SV=2 Q7A5T2|CATA_STAAN -1.4 -1.9 -1.65 35 Pyruvate kinase OS=Staphylococcus aureus (strain N315) GN=pyk PE=1 SV=1 Q7A559|KPYK_STAAN -1.6 -1.6 -1.6 43 SA2119 protein OS=Staphylococcus aureus (strain N315) GN=SA2119 PE=1 SV=1 Q7A3Z5|Q7A3Z5_STAAN -1.6 -1.6 -1.6 66 30S ribosomal protein S10 OS=Staphylococcus aureus (strain N315) GN=rpsJ PE=1 SV=1 P66334|RS10_STAAN -1.5 -1.7 -1.6 81 NAD-specific glutamate dehydrogenase OS=Staphylococcus aureus (strain N315) GN=gluD PE=1 SV=1 Q7A6H8|DHE2_STAAN -1.8 -1.4 -1.6 6 Probable malate:quinone oxidoreductase 2 OS=Staphylococcus aureus (strain N315) GN=mqo2 PE=1 SV=1 P99115|MQO2_STAAN -1.6 -1.5 -1.55 9 Alkaline shock protein 23 OS=Staphylococcus aureus (strain N315) GN=asp23 PE=1 SV=1 P99157|ASP23_STAAN -1.5 -1.6 -1.55 57 Dihydrolipoyllysine-residue succinyltransferase component of 2oxoglutarate dehydrogenase complex OS=Staphylococcus aureus (strain N315) GN=odhB PE=1 SV=1 Q7A5N4|ODO2_STAAN -1.6 -1.5 -1.55 50 Alkyl hydroperoxide reductase subunit F OS=Staphylococcus aureus (strain N315) GN=ahpF PE=1 SV=1 P99118|AHPF_STAAN -1.7 -1.4 -1.55 10 Inosine-5'-monophosphate dehydrogenase OS=Staphylococcus aureus (strain N315) GN=guaB PE=1 SV=1 P99106|IMDH_STAAN -1.5 -1.5 -1.5 19 Glutamine synthetase OS=Staphylococcus aureus (strain N315) GN=glnA PE=1 SV=1 P99095|GLNA_STAAN -1.5 -1.5 -1.5 21 50S ribosomal protein L13 OS=Staphylococcus aureus (strain N315) GN=rplM PE=1 SV=1 Q7A473|RL13_STAAN -1.4 -1.6 -1.5 110

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34 Q7A3N7|RANDOM_Q7A3N7_STAAN-R Q7A3N7|RANDOM_Q7A3N7_STAAN-R (+2) -1.4 -1.4 -1.4 48 DNA polymerase III subunit beta OS=Staphylococcus aureus (strain N315) GN=dnaN PE=1 SV=1 P99103|DPO3B_STAAN -1.4 -1.4 -1.4 58 NADPH-dependent oxidoreductase OS=Staphylococcus aureus (strain N315) GN=nfrA PE=3 SV=1 Q7A7J0|RANDOM_NFRA_STAAN -R -1.4 -1.4 -1.4 59 50S ribosomal protein L11 OS=Staphylococcus aureus (strain N315) GN=rplK PE=1 SV=2 P0A0F2|RL11_STAAN -1.3 -1.5 -1.4 2 Glyceraldehyde-3-phosph ate dehydrogenase 1 OS=Staphylococcus aureus (strain N315) GN=gapA1 PE=1 SV=1 P99136|G3P1_STAAN -1.4 -1.3 -1.35 13 50S ribosomal protein L1 OS=Staphylococcus aureus (strain N315) GN=rplA PE=1 SV=1 Q99W68|RL1_STAAN -1.3 -1.4 -1.35 1 Elongation factor Tu OS=Staphylococcus aureus (strain N315) GN=tuf PE=1 SV=1 P99152|EFTU_STAAN -1.3 -1.3 -1.3 22 50S ribosomal protein L22 OS=Staphylococcus aureus (strain N315) GN=rplV PE=1 SV=1 Q7A460|RL22_STAAN -1.4 -1.2 -1.3 53 Adenylosuccinate lyase OS=Staphylococcus aureus (strain N315) GN=purB PE=1 SV=1 Q7A4Q3|PUR8_STAAN -1.2 -1.4 -1.3 7 Alkyl hydroperoxide reductase subunit C OS=Staphylococcus aureus (strain N315) GN=ahpC PE=1 SV=1 P99074|AHPC_STAAN -1.3 -1.2 -1.25 11 50S ribosomal protein L2 OS=Staphylococcus aureus (strain N315) GN=rplB PE=1 SV=1 P60432|RL2_STAAN -1.2 -1.3 -1.25 69 D-alanine aminotransferase OS=Staphylococcus aureus (strain N315) GN=dat PE=1 SV=1 P99090|DAAA_STAAN -1.3 -1.2 -1.25 42 DNA-directed RNA polymerase subunit beta' OS=Staphylococcus aureus (strain N315) GN=rpoC PE=1 SV=1 P60285|RPOC_STAAN -1.3 -1.1 -1.2 77 SA1224 protein OS=Staphylococcus aureus (strain N315) GN=SA1224 PE=4 SV=1 Q7A5Q0|Q7A5Q0_STAAN -1.2 -1.2 -1.2 3 Elongation factor G OS=Staphylococcus aureus (strain N315) GN=fusA PE=1 SV=2 P68789|EFG_STAAN -1.2 -1.1 -1.15 12 Cell division protein ftsZ OS=Staphylococcus aureus (strain N315) GN=ftsZ PE=1 SV=1 P99108|FTSZ_STAAN -1.1 -1.2 -1.15 36 50S ribosomal protein L21 OS=Staphylococcus aureus (strain N315) GN=rplU PE=1 SV=1 Q7A583|RL21_STAAN -1 -1.1 -1.05 51 50S ribosomal protein L14 OS=Staphylococcus aureus (strain N315) GN=rplN PE=1 SV=1 Q7A463|RL14_STAAN -1 -1.1 -1.05 72 Glycyl-tRNA synthetase OS=Staphylococcus aureus (strain N315) GN=glyQS PE=1 SV=1 P99129|SYG_STAAN -0.9 -1 -0.95 33 Aspartyl/glutamyl-tRNA(Asn/Gln) amidotransferase subunit B OS=Staphylococcus aureus (strain N315) GN=gatB PE=1 SV=1 P99169|GATB_STAAN -1 -0.8 -0.9 49 ATP synthase subunit alpha OS=Staphylococcus aureus (strain N315) GN=atpA PE=1 SV=1 P99111|ATPA_STAAN -0.8 -0.8 -0.8 111

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56 DNA-directed RNA polymerase subunit beta OS=Staphylococcus aureus (strain N315) GN=rpoB PE=1 SV=1 P60278|RPOB_STAAN -0.8 -0.8 -0.8 16 30S ribosomal protein S2 OS=Staphylococcus aureus (strain N315) GN=rpsB PE=1 SV=1 P66544|RS2_STAAN -0.8 -0.7 -0.75 30 50S ribosomal protein L19 OS=Staphylococcus aureus (strain N315) GN=rplS PE=1 SV=1 P66083|RL19_STAAN -0.7 -0.7 -0.7 60 Cell division protein ft sA OS=Staphylococcus aureus (strain N315) GN=ftsA PE=1 SV=1 P63765|FTSA_STAAN -0.6 -0.8 -0.7 14 Serine hydroxymethyltransferase OS=Staphylococcus aureus (strain N315) GN=glyA PE=1 SV=1 P99091|GLYA_STAAN -0.7 -0.5 -0.6 26 Bifunctional autolysin OS=Staphylococcus aureus (strain N315) GN=atl PE=1 SV=1 Q99V41|ATL_STAAN -0.4 -0.4 -0.4 27 Uncharacterized protein SA0829 OS=Staphylococcus aureus (strain N315) GN=SA0829 PE=1 SV=1 Q7A6H3|Y829_STAAN No Values No Values 31 D-lactate dehydrogenase OS=Staphylococcus aureus (strain N315) GN=ldhD PE=1 SV=1 P99116|LDHD_STAAN No Values No Values 38 Q7A7W3|Q7A7W3_STAAN Q7A7W3|Q7A7W3_STAAN Value Missing Value Missing 39 ATP-dependent Clp protease ATP-binding subunit clpL OS=Staphylococcus aureus (strain N315) GN=clpL PE=1 SV=1 Q7A3F4|CLPL_STAAN No Values No Values 41 Phosphoribosylaminoimidazolesuccinocarboxamide synthase OS=Staphylococcus aureus (strain N315) GN=purC PE=1 SV=1 P99064|PUR7_STAAN No Values No Values 44 50S ribosomal protein L3 OS=Staphylococcus aureus (strain N315) GN=rplC PE=1 SV=1 P60449|RL3_STAAN No Values No Values 46 50S ribosomal protein L6 OS=Staphylococcus aureus (strain N315) GN=rplF PE=1 SV=1 Q7A466|RL6_STAAN No Values No Values 54 Aerobic glycerol-3-phos phate dehydrogenase OS=Staphylococcus aureus (strain N315) GN=glpD PE=1 SV=2 Q7A5V7|GLPD_STAAN No Values No Values 55 2-oxoglutarate dehydrogenase E1 component OS=Staphylococcus aureus (strain N315) GN=odhA PE=1 SV=1 Q99U74|ODO1_STAAN No Values No Values 63 (3R)-hydroxymyristoyl-[acyl-carrier-protein] dehydratase OS=Staphylococcus aureus (strain N315) GN=fabZ PE=3 SV=1 P64108|FABZ_STAAN No Values No Values 67 30S ribosomal protein S5 OS=Staphylococcus aureus (strain N315) GN=rpsE PE=1 SV=1 P66579|RS5_STAAN Value Missing Value Missing 68 1-pyrroline-5-carboxylate dehydrogenase OS=Staphylococcus aureus (strain N315) GN=rocA PE=1 SV=1 P99076|ROCA_STAAN No Values No Values 71 P99119|LDH2_STAAN P99119|LDH2_STAAN No Values No Values 78 Ribonuclease J 2 OS=Staphylococcus aureus (strain N315) GN=SA1118 PE=1 SV=1 Q7A5X6|RNJ2_STAAN No Values No Values 80 Naphthoate synthase OS=Staphylococcus aureus (strain N315) GN=menB PE=1 SV=1 Q7A6A9|MENB_STAAN No Values No Values 112

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83 Formate acetyltransferase OS=Staphylococcus aureus (strain N315) GN=pflB PE=1 SV=1 Q7A7X6|PFLB_STAAN Value Missing Value Missing 84 Pyruvate dehydrogenase E1 component subunit alpha OS=Staphylococcus aureus (strain N315) GN=pdhA PE=1 SV=1 Q820A6|ODPA_STAAN No Values No Values 85 ATP-dependent Clp protease ATP-binding subunit clpC OS=Staphylococcus aureus (strain N315) GN=clpC PE=1 SV=1 Q7A797|RANDOM_CLPC_STAAN-R Value Missing Value Missing Table A10. Complete list of intracellular proteins identified from the USA 100 adaptive mutant at 8 hours. # Identified Proteins (66) Accession Number Fold Change from WT Average Fold Change 54 Formate acetyltransferase OS=Staphylococcus aureus (strain USA300) GN=pflB PE=3 SV=1 Q2FK44|PFLB_STAA3 -2 -2.6 -2.3 18 Alcohol dehydrogenase OS=Staphylococcus aureus (strain USA300) GN=adh PE=3 SV=1 Q2FJ31|ADH_STAA3 -2.1 -1.9 -2 24 Aconitate hydratase OS=Staphylococcus aureus (strain USA300) GN=acnA PE=4 SV=1 Q2FH85|Q2FH85_STAA3 -1.3 -1.1 -1.2 9 Formate--tetrahydrofolate ligase OS=Staphylococcus aureus (strain USA300) GN=fhs PE=3 SV=1 Q2FG06|FTHS_STAA3 -1.2 -1.1 -1.15 36 Putative universal stress protein SAUSA300_1656 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1656 PE=3 SV=1 Q2FG28|Y1656_STAA3 -1.2 -1.1 -1.15 65 Pyruvate carboxylase OS=Staphylococcus aureus (strain USA300) GN=pyc PE=4 SV=1 Q2FHW6|Q2FHW6_STAA3 -1 -0.8 -0.9 52 Cysteine synthase OS=Staphylococcus aureus (strain USA300) GN=cysK PE=3 SV=1 Q2FJC8|Q2FJC8_STAA3 -0.9 -0.6 -0.75 42 Glucose-6-phosphate isomerase OS=Staphylococcus aureus (strain USA300) GN=pgi PE=3 SV=1 Q2FIB3|G6PI_STAA3 -0.7 -0.7 -0.7 56 Phosphoenolpyruvate carboxykinase [ATP] OS=Staphylococcus aureus (strain USA300) GN=pckA PE=3 SV=1 Q2FFV5|PCKA_STAA3 -0.9 -0.5 -0.7 40 Succinyl-CoA ligase [ADP-forming] subunit beta OS=Staphylococcus aureus (strain USA300) GN=sucC PE=3 SV=1 Q2FHJ3|SUCC_STAA3 -0.7 -0.6 -0.65 50 6-phosphogluconate dehydrogenase, decarboxylating OS=Staphylococcus aureus (strain USA300) GN=gnd PE=3 SV=1 Q2FGM3|Q2FGM3_STAA3 -0.7 -0.5 -0.6 62 NAD-specific glutamate dehydrogenase OS=Staphylococcus aureus (strain USA300) GN=gudB PE=3 SV=1 Q2FIB7|Q2FIB7_STAA3 -0.7 -0.5 -0.6 32 Succinyl-CoA ligase [ADP-forming] subunit alpha OS=Staphylococcus aureus (strain USA300) GN=sucD PE=3 SV=1 Q2FHJ2|Q2FHJ2_STAA3 -0.6 -0.5 -0.55 8 Enolase OS=Staphylococcus aureus (strain USA300) GN=eno PE=3 SV=1 Q2FIL7|ENO_STAA3 -0.6 -0.4 -0.5 51 Dihydrolipoamide acetyltransferase OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0995 PE=3 SV=1 Q2FHY5|Q2FHY5_STAA3 0.2 -1.1 -0.45 113

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26 Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex OS=Staphylococcus aureus (strain USA300) GN=odhB PE=3 SV=1 Q2FH26|ODO2_STAA3 -0.6 -0.3 -0.45 6 Inosine-5'-monophosphate dehydrogenase OS=Staphylococcus aureus (strain USA300) GN=guaB PE=3 SV=1 Q2FJM6|IMDH_STAA3 -0.5 -0.3 -0.4 5 Pyruvate dehydrogenase E1 component, beta subunit OS=Staphylococcus aureus (strain USA300) GN=pdhB PE=4 SV=1 Q2FHY6|Q2FHY6_STAA3 -0.4 -0.3 -0.35 43 30S ribosomal protein S1 OS=Staphylococcus aureus (strain USA300) GN=rpsA PE=4 SV=1 Q2FGW6|Q2FGW6_STAA3 -0.4 -0.3 -0.35 3 Dihydrolipoyl dehydrogenase OS=Staphylococcus aureus (strain USA300) GN=lpdA PE=3 SV=1 Q2FHY4|Q2FHY4_STAA3 -0.3 -0.2 -0.25 14 L-lactate dehydrogenase 1 OS=Staphylococcus aureus (strain USA300) GN=ldh1 PE=3 SV=2 Q2FK29|LDH1_STAA3 -0.3 -0.2 -0.25 23 Catalase OS=Staphylococcus aureus (strain USA300) GN=katA PE=3 SV=1 Q2FH99|CATA_STAA3 -0.3 -0.2 -0.25 16 Malate:quinone-oxidoreduc tase OS=Staphylococcus aureus (strain USA300) GN=mqo PE=3 SV=1 Q2FDQ3|Q2FDQ3_STAA3 -0.3 -0.1 -0.2 25 Glutamine synthetase OS=Staphylococcus aureus (strain USA300) GN=glnA PE=3 SV=1 Q2FHD0|Q2FHD0_STAA3 -0.3 -0.1 -0.2 30 Ferritin OS=Staphylococcus aureus (strain USA300) GN=ftnA PE=3 SV=1 Q2FFK2|FTN_STAA3 -0.3 -0.1 -0.2 47 NADH dehydrogenase-like protein SAUSA300_0844 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0844 PE=3 SV=1 Q2FID4|Y844_STAA3 -0.3 0 -0.15 13 Phosphoglycerate kinase OS=Staphylococcus aureus (strain USA300) GN=pgk PE=3 SV=1 Q2FIM0|PGK_STAA3 -0.2 0 -0.1 46 Bifunctional protein folD OS=Staphylococcus aureus (strain USA300) GN=folD PE=3 SV=1 Q2FI15|FOLD_STAA3 -0.2 0 -0.1 66 Isocitrate dehydrogenase [NADP] OS=Staphylococcus aureus (strain USA300) GN=icd PE=3 SV=1 Q2FG43|Q2FG43_STAA3 -0.1 0 -0.05 39 ATP synthase subunit beta OS=Staphylococcus aureus (strain USA300) GN=atpD PE=3 SV=1 Q2FF24|ATPB_STAA3 0 0 0 20 Pyruvate dehydrogenase E1 component, alpha subunit OS=Staphylococcus aureus (strain USA300) GN=pdhA PE=4 SV=1 Q2FHY7|Q2FHY7_STAA3 -0.1 0.1 0 33 DNA-binding protein HU OS=Staphylococcus aureus (strain USA300) GN=hup PE=3 SV=1 Q2FGW9|Q2FGW9_STAA3 -0.1 0.1 0 34 DNA-directed RNA polymerase subunit beta OS=Staphylococcus aureus (strain USA300) GN=rpoB PE=3 SV=2 Q2FJ98|RPOB_STAA3 -0.1 0.1 0 64 Phosphoenolpyruvate-prote in phosphotransferase OS=Staphylococcus aureus (strain USA300) GN=ptsI PE=3 SV=1 Q2FHZ6|Q2FHZ6_STAA3 -0.1 0.3 0.1 4 Glyceraldehyde-3-phosphat e dehydrogenase, type I OS=Staphylococcus aureus (strain USA300) GN=gap PE=3 SV=1 Q2FIM1|Q2FIM1_STAA3 0 0.2 0.1 38 GTP-sensing transcriptional pleiotropic repressor codY OS=Staphylococcus aureus (strain USA300) GN=codY PE=3 SV=1 Q2FHI3|CODY_STAA3 0 0.2 0.1 114

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12 Pyruvate kinase OS=Staphylococcus aureus (strain USA300) GN=pyk PE=3 SV=1 Q2FG40|KPYK_STAA3 0.1 0.2 0.15 63 30S ribosomal protein S9 OS=Staphylococcus aureus (strain USA300) GN=rpsI PE=3 SV=1 Q2FES2|RS9_STAA3 0.1 0.2 0.15 11 DNA-directed RNA polymerase subunit beta' OS=Staphylococcus aureus (strain USA300) GN=rpoC PE=3 SV=2 Q2FJ97|RPOC_STAA3 0.1 0.3 0.2 60 DNA polymerase III, beta subunit OS=Staphylococcus aureus (strain USA300) GN=dnaN PE=3 SV=1 Q2FKQ4|Q2FKQ4_STAA3 0.1 0.3 0.2 28 50S ribosomal protein L2 OS=Staphylococcus aureus (strain USA300) GN=rplB PE=3 SV=1 Q2FEP2|RL2_STAA3 0.3 0.2 0.25 37 Glycyl-tRNA synthetase OS=Staphylococcus aureus (strain USA300) GN=glyQS PE=3 SV=1 Q2FGF8|SYG_STAA3 0.2 0.3 0.25 22 Alkyl hydroperoxide reductase subunit C OS=Staphylococcus aureus (strain USA300) GN=ahpC PE=3 SV=1 Q2FJN4|AHPC_STAA3 0.3 0.3 0.3 7 Alkaline shock protein 23 OS=Staphylococcus aureus (strain USA300) GN=asp23 PE=3 SV=1 Q2FEV0|ASP23_STAA3 0.2 0.4 0.3 2 Elongation factor G OS=Staphylococcus aureus (strain USA300) GN=fusA PE=3 SV=3 Q2FJ93|EFG_STAA3 0.3 0.4 0.35 48 50S ribosomal protein L14 OS=Staphylococcus aureus (strain USA300) GN=rplN PE=3 SV=1 Q2FEP9|RL14_STAA3 0.4 0.4 0.4 55 UPF0457 protein SAUSA300_2132 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2132 PE=3 SV=1 Q2FEV9|Y2132_STAA3 0.4 0.4 0.4 31 30S ribosomal protein S5 OS=Staphylococcus aureus (strain USA300) GN=rpsE PE=3 SV=1 Q2FEQ6|RS5_STAA3 0.3 0.5 0.4 19 Transketolase OS=Staphylo coccus aureus (strain USA300) GN=tkt PE=4 SV=1 Q2FH92|Q2FH92_STAA3 0.4 0.5 0.45 53 Alkyl hydroperoxide reductase, subunit F OS=Staphylococcus aureus (strain USA300) GN=ahpF PE=3 SV=1 Q2FJN5|Q2FJN5_STAA3 0.4 0.6 0.5 21 50S ribosomal protein L1 OS=Staphylococcus aureus (strain USA300) GN=rplA PE=3 SV=1 Q2FJA2|RL1_STAA3 0.5 0.6 0.55 57 50S ribosomal protein L13 OS=Staphylococcus aureus (strain USA300) GN=rplM PE=3 SV=1 Q2FES1|RL13_STAA3 0.4 0.7 0.55 27 50S ribosomal protein L3 OS=Staphylococcus aureus (strain USA300) GN=rplC PE=3 SV=1 Q2FEN9|RL3_STAA3 0.5 0.7 0.6 1 Elongation factor Tu OS=Staphylococcus aureus (strain USA300) GN=tuf PE=3 SV=1 Q2FJ92|EFTU_STAA3 0.6 0.7 0.65 15 Cell division protein ftsZ OS=Staphylococcus aureus (strain USA300) GN=ftsZ PE=3 SV=1 Q2FHQ1|Q2FHQ1_STAA3 0.6 0.7 0.65 17 Serine hydroxymethyltransferase OS=Staphylococcus aureus (strain USA300) GN=glyA PE=3 SV=1 Q2FF15|GLYA_STAA3 0.6 0.7 0.65 35 D-lactate dehydrogenase OS=Staphylococcus aureus (strain USA300) GN=ddh PE=3 SV=1 Q2FDY1|Q2FDY1_STAA3 0.6 0.9 0.75 115

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49 ATP synthase subunit alpha OS=Staphylococcus aureus (strain USA300) GN=atpA PE=3 SV=1 Q2FF22|ATPA_STAA3 0.7 1 0.85 10 Chaperone protein dnaK OS=Staphylococcus aureus (strain USA300) GN=dnaK PE=2 SV=1 Q2FGE3|DNAK_STAA3 1 1.1 1.05 29 30S ribosomal protein S2 OS=Staphylococcus aureus (strain USA300) GN=rpsB PE=3 SV=1 Q2FHI2|RS2_STAA3 1 1.1 1.05 61 Probable transglycosylase isaA OS=Staphylococcus aureus (strain USA300) GN=isaA PE=3 SV=1 Q2FDT8|ISAA_STAA3 1.2 1.7 1.45 41 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2473 PE=4 SV=1 Q2FDX1|Q2FDX1_STAA3 2.2 2.8 2.5 44 50S ribosomal protein L6 OS=Staphylococcus aureus (strain USA300) GN=rplF PE=3 SV=1 Q2FEQ4|RL6_STAA3 No Values No Values 45 Glucosamine--fructose-6-phosphate aminotransferase (Isomerizing) OS=Staphylococcus aureus (strain USA300) GN=glmS PE=3 SV=1 Q2FEX8|Q2FEX8_STAA3 No Values No Values 59 Transcription termination factor NusA OS=Staphylococcus aureus (strain USA300) GN=nusA PE=4 SV=1 Q2FHH2|Q2FHH2_STAA3 No Values No Values 58 Glyceraldehyde-3-phosphat e dehydrogenase, type I OS=Staphylococcus aureus (strain USA300) GN=gap PE=3 SV=1 Q2FG50|Q2FG50_STAA3 Value Missing Value Missing Table A11. Complete list of intracellular proteins identified from the USA 300 adaptive mutant at 8 hours. # Identified Proteins (796) Accession Number Fold Change from WT 213 Probable molybdate-binding protein OS=S taphylococcus aureus (strain N315) GN=modA PE=4 SV=1 Q99RZ3|Q99RZ3_ STAAN -4.4 30 Lipase 2 (lip2) Q7A7P2|LIP2_STA AN -3.7 161 Phosphoribosylaminoimidazole-succino carboxamide synthase OS=Staphylococcus aureus (strain N315) GN=purC PE=1 SV=1 P99064|PUR7_STA AN -3.5 290 ATP synthase subunit delta OS=Staphyloco ccus aureus (strain N315) GN=atpH PE=1 SV=1 P99109|ATPD_ST AAN -3.5 251 Seryl-tRNA synthetase OS=Staphylococcus aureus (strain N315) GN=serS PE=1 SV=1 P99178|SYS_STA AN -3.3 318 SA0916 protein OS=Staphylococcus aure us (strain N315) GN=SA0916 PE=1 SV=1 Q7A696|Q7A696_ STAAN -3.3 493 Carbamate kinase 1 OS=S taphylococcus aureus (strain N315) GN=arcC1 PE=1 SV=1 Q7A627|ARCC1_S TAAN -3.3 33 Immunoglobulin-binding protein (sbi) Q99RL2|SBI_STA AN -3.2 248 Bifunctional protein pyrR OS=Staphylococcu s aureus (strain N315) GN=pyrR PE=1 SV=1 P65944|PYRR_ST AAN -3.2 577 PTS system glucoside-specific EIICBA component OS=Staphylococcus aureus (strain N315) GN=glcB PE=1 SV=1 Q7A3G4|PTU3C_S TAAN -3.2 103 Ferritin OS=Staphylococcus aureus (strain N315) GN=ftnA PE=1 SV=1 Q7A4R2|FTN_STA AN -3.1 114 Phosphoribosylamine--glycine ligase OS=Staphylococcus aureus (strain N315) GN=purD PE=1 SV=1 P65896|PUR2_STA AN -3.1 167 Deoxyribose-phosphate aldolase 2 OS=Staphylococcus aureus (strain N315) GN=deoC2 PE=1 SV=1 P99174|DEOC2_S TAAN -3.1 116

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386 SA0537 protein OS=Staphylococcus aure us (strain N315) GN=SA0537 PE=1 SV=1 Q7A765|Q7A765_ STAAN -3.1 413 UvrABC system protein A OS=Staphylococcus aureus (strain N315) GN=uvrA PE=1 SV=1 P63383|UVRA_ST AAN -3.1 32 Phosphoribosylformylglycinamidine synthase 2 (purL) P65901|PURL_ST AAN -3 49 Dihydrolipoyllysine-residue acetyltransferas e component of pyr uvate dehydrogenase complex (pdhC) P65636|ODP2_ST AAN -3 106 L-lactate dehydrogenase 1 OS=Staphyloco ccus aureus (strain N315) GN=ldhA PE=1 SV=1 P65256|LDH1_ST AAN -3 199 PTS system glucose-specific EIICBA component OS=Staphylococcus aureus (strain N315) GN=ptsG PE=1 SV=1 Q7A807|PTG3C_S TAAN -3 282 SA0243 protein OS=Staphylococcus aure us (strain N315) GN=SA0243 PE=4 SV=1 Q7A7V2|Q7A7V2_ STAAN -3 320 Putative uncharacterized protein SA2101 OS=Staphylococcus aureus (strain N315) GN=SA2101 PE=4 SV=1 Q7A414|Q7A414_ STAAN -3 457 Uncharacterized lipoprotein SA2158 OS=S taphylococcus aureus (strain N315) GN=SA2158 PE=1 SV=1 Q7A3W5|Y2158_S TAAN -3 84 Bifunctional purine biosynthesis protein purH (purH) P67544|PUR9_STA AN -2.9 100 Ornithine aminotransferase 2 OS=Staphylo coccus aureus (strain N315) GN=rocD2 PE=1 SV=1 P60298|OAT2_ST AAN -2.9 120 Pyrimidine-nucleoside phosphorylase OS=Staphylococcus aureus (strain N315) GN=pdp PE=1 SV=2 Q7A4D0|PDP_STA AN -2.9 198 Phosphoribosylaminoimidazole carboxylase ATPase subunit OS=Staphylococcus aureus (strain N315) GN=purK PE=1 SV=1 Q7A695|PURK_ST AAN -2.9 264 Probable malate:quinone oxidor eductase 1 OS=Staphylococcus aureus (strain N315) GN=mqo1 PE=1 SV=1 P65422|MQO1_ST AAN -2.9 266 Glutamate-1-semialdehyde 2,1-aminomutase 1 OS=Staphylococcus aureus (strain N315) GN=hemL1 PE=1 SV=1 P99096|GSA1_ST AAN -2.9 324 SA0248 protein OS=Staphylococcus aure us (strain N315) GN=SA0248 PE=4 SV=1 Q7A7U7|Q7A7U7_ STAAN -2.9 419 Tyrosyl-tRNA synthetase OS=Staphylococcu s aureus (strain N315) GN=tyrS PE=1 SV=1 Q7A537|SYY_STA AN -2.9 9 Probable malate:quinone oxidoreductase 2 (mqo2) P99115|MQO2_ST AAN -2.8 16 Dihydrolipoyl dehydrogenase (pdhD) P99084|DLDH_ST AAN -2.8 34 SA0587 protein (SA0587) Q7A719|Q7A719_ STAAN -2.8 73 Immunodominant staphylococcal antigen B (isaB) Q7A377|ISAB_ST AAN -2.8 127 Ribose-phosphate pyrophosphokina se OS=Staphylococcus aureus (strain N315) GN=prs PE=1 SV=1 P65237|KPRS_ST AAN -2.8 221 ATP synthase subunit b OS=Staphylococcus aureus (strain N315) GN=atpF PE=1 SV=1 Q7A4E7|ATPF_ST AAN -2.8 253 ATP synthase gamma chain OS=Staphyloco ccus aureus (strain N315) GN=atpG PE=1 SV=1 Q7A4E8|ATPG_ST AAN -2.8 274 SA2302 protein OS=Staphylococcus aure us (strain N315) GN=SA2302 PE=4 SV=1 Q7A3I7|Q7A3I7_S TAAN -2.8 286 50S ribosomal protein L20 OS=Staphylococcus aureus (strain N315) GN=rplT PE=1 SV=1 P66108|RL20_STA AN -2.8 480 Argininosuccinate synthase OS=Staphylococcus aureus (strain N315) GN=argG PE=3 SV=1 P63645|ASSY_ST AAN -2.8 35 50S ribosomal protein L5 (rplE) Q7A465|RL5_STA AN -2.7 56 SA0295 protein (SA0295) Q7A7Q2|Q7A7Q2_ STAAN -2.7 145 L-lactate dehydrogenase 2 OS=Staphylococcus aure us (strain N315) GN=ldhB PE=1 P99119|LDH2_ST -2.7 117

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SV=1 AAN 307 Putative uncharacterized protein SA0618 OS=Staphylococcus aureus (strain N315) GN=SA0618 PE=4 SV=1 Q7A6Z0|Q7A6Z0_ STAAN -2.7 326 Putative uncharacterized protein SAS081 OS=Staphylococcus aureus (strain N315) GN=SA2054.1 PE=4 SV=1 Q7A450|Q7A450_ STAAN -2.7 387 DNA topoisomerase 4 subunit A OS=Staphylococcus aureus (strain N315) GN=parC PE=1 SV=1 Q93KF4|PARC_ST AAN -2.7 394 Putative uncharacterized protein SA2171 OS=Staphylococcus aureus (strain N315) GN=SA2171 PE=4 SV=1 Q7A3V3|Q7A3V3_ STAAN -2.7 406 D-alanine--poly(phosphoribitol) ligase subunit 2 OS=Staphylococcus aureus (strain N315) GN=dltC PE=3 SV=1 P0A019|DLTC_ST AAN -2.7 462 Ornithine carbamoyltransferas e, catabolic OS=Staphylococcus aureus (strain N315) GN=arcB PE=1 SV=1 P65602|OTCC_ST AAN -2.7 570 Putative uncharacterized protein SA1649 OS=Staphylococcus aureus (strain N315) GN=SA1649 PE=4 SV=1 Q7A4W5|Q7A4W5 _STAAN -2.7 632 Branched-chain alpha-keto acid dehydrogena se E1 OS=Staphylococcus aureus (strain N315) GN=bfmBAA PE=4 SV=1 Q7A5F7|Q7A5F7_ STAAN -2.7 38 Carbamoyl-phosphate synthase large chain (carB) P63740|CARB_ST AAN -2.6 71 NADH dehydrogenase-lik e protein SA0802 (SA0802) Q7A6J4|Y802_ST AAN -2.6 146 50S ribosomal protein L14 OS=Staphyloco ccus aureus (strain N315) GN=rplN PE=1 SV=1 Q7A463|RL14_ST AAN -2.6 162 SA1224 protein OS=Staphylococcus aure us (strain N315) GN=SA1224 PE=4 SV=1 Q7A5Q0|Q7A5Q0_ STAAN -2.6 183 GTPase obg OS=Staphylococcus aureus (strain N315) GN=obg PE=1 SV=1 Q7A584|OBG_ST AAN -2.6 197 2,3-bisphosphoglycerate-independent phos phoglycerate mutase OS=Staphylococcus aureus (strain N315) GN=gpmI PE=1 SV=1 P64270|GPMI_ST AAN -2.6 334 SA0589 protein OS=Staphylococcus aure us (strain N315) GN=SA0589 PE=4 SV=1 Q7A717|Q7A717_ STAAN -2.6 347 Glycerol kinase OS=Staphylococcus au reus (strain N315) GN=glpK PE=1 SV=1 P99113|GLPK_ST AAN -2.6 441 SA1606 protein OS=Staphylococcus aure us (strain N315) GN=SA1606 PE=1 SV=1 Q7A4Z8|Q7A4Z8_ STAAN -2.6 455 Phosphoribosylformylglycinamidine syntha se 1 OS=Staphylococcus aureus (strain N315) GN=purQ PE=1 SV=1 P99166|PURQ_ST AAN -2.6 612 Putative uncharacterized protein SA0637 OS=Staphylococcus aureus (strain N315) GN=SA0637 PE=4 SV=1 Q7A6X4|Q7A6X4_ STAAN -2.6 729 SA0682 protein OS=Staphylococcus aure us (strain N315) GN=SA0682 PE=3 SV=1 Q7A6T5|Q7A6T5_ STAAN -2.6 44 D-lactate dehydrogenase (ldhD) P99116|LDHD_ST AAN -2.5 94 Asparaginyl-tRNA synthetase OS=Staphyloco ccus aureus (strain N315) GN=asnS PE=1 SV=1 P67572|SYN_STA AN -2.5 279 GTP-binding protein engA OS=Staphylococcu s aureus (strain N315) GN=engA PE=1 SV=1 P64060|ENGA_ST AAN -2.5 364 SA1989 protein OS=Staphylococcus aure us (strain N315) GN=SA1989 PE=1 SV=1 Q7A492|Q7A492_ STAAN -2.5 415 SA1749 protein OS=Staphylococcus aure us (strain N315) GN=SA1749 PE=4 SV=1 Q7A4N0|Q7A4N0_ STAAN -2.5 469 Kanamycin nucleotidyltransferase OS=Staphy lococcus aureus (strain N315) GN=aadD PE=4 SV=1 Q7A8D0|Q7A8D0_ STAAN -2.5 477 Protein-export membrane protein SecDF OS=Staphylococcus aureus (strain N315) GN=secF PE=4 SV=1 Q7A586|Q7A586_ STAAN -2.5 617 UPF0297 protein SA1445 OS=Staphylococcus aureus (strain N315) GN=SA1445 PE=1 SV=1 P60359|Y1445_ST AAN -2.5 667 Putative uncharacterized protein SA0814 OS=Staphylococcus aureus (strain N315) GN=SA0814 PE=4 SV=1 Q7A6I2|Q7A6I2_S TAAN -2.5 118

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6 Formate acetyltransferase (pflB) Q7A7X6|PFLB_ST AAN -2.4 53 Protein translocase subunit secA 1 (secA1) Q7A6R5|SECA1_S TAAN -2.4 54 ATP synthase subunit alpha (atpA) P99111|ATPA_ST AAN -2.4 66 Probable quinol oxidase subunit 2 (qoxA) Q7A698|QOX2_ST AAN -2.4 116 50S ribosomal protein L19 OS=Staphyloco ccus aureus (strain N315) GN=rplS PE=1 SV=1 P66083|RL19_STA AN -2.4 139 Phosphate acetyltransferase OS=Staphyloco ccus aureus (strain N315) GN=pta PE=1 SV=1 P99092|PTA_STA AN -2.4 340 Putative uncharacterized protein SA0933 OS=Staphylococcus aureus (strain N315) GN=SA0933 PE=4 SV=1 Q7A687|Q7A687_ STAAN -2.4 356 GTP pyrophosphokinase OS=Staphylococcus aureus (strain N315) GN=relA PE=1 SV=2 Q99TL8|RELA_ST AAN -2.4 450 Virulence factor esxA OS=Staphylococcus aureus (strain N315) GN=esxA PE=1 SV=1 ESXA_STAAN (+1) -2.4 573 Molybdopterin synthase sulfur carrier subunit OS=Staphylococcus aureus (strain N315) GN=moaD PE=1 SV=1 Q7A441|MOAD_S TAAN -2.4 631 SA2201 protein OS=Staphylococcus aure us (strain N315) GN=SA2201 PE=3 SV=1 Q7A3S5|Q7A3S5_ STAAN -2.4 82 Fructose-bisphosphate aldolase (fba) P99075|ALF2_STA AN -2.3 83 PBP2 (pbp2) Q7A5K8|Q7A5K8_ STAAN -2.3 96 50S ribosomal protein L13 OS=Staphylococcus aureus (strain N315) GN=rplM PE=1 SV=1 Q7A473|RL13_ST AAN -2.3 137 2,3-bisphosphoglycerate-dependent phos phoglycerate mutase OS=Staphylococcus aureus (strain N315) GN=gpmA PE=1 SV=1 P99153|GPMA_ST AAN -2.3 140 Probable catabolite control protein A OS=Staphylococcus aureus (strain N315) GN=ccpA PE=1 SV=1 P99175|CCPA_ST AAN -2.3 142 N-acetylmuramoyl-L-alanine amidase sle1 OS=Staphylococcus aureus (strain N315) GN=sle1 PE=1 SV=1 Q7A7E0|SLE1_ST AAN -2.3 220 Acetoin(diacetyl) reductase OS=Staphyloco ccus aureus (strain N315) GN=butA PE=1 SV=1 P99120|BUTA_ST AAN -2.3 223 Ffh protein OS=Staphylococcus aureus (strain N315) GN=ffh PE=4 SV=1 Q7A5Z0|Q7A5Z0_ STAAN -2.3 332 DNA polymerase I OS=Staphylococcus aureus (strain N315) GN=polA PE=3 SV=1 Q7A565|Q7A565_ STAAN -2.3 363 DegV domain-containing protein SA1258 OS =Staphylococcus aureus (strain N315) GN=SA1258 PE=1 SV=1 P67371|Y1258_ST AAN -2.3 384 Putative uncharacterized protein SA1933 OS=Staphylococcus aureus (strain N315) GN=SA1933 PE=4 SV=1 Q7A4D3|Q7A4D3_ STAAN -2.3 393 Carbamoyl-phosphate synthase small chain OS=Staphylococcus aureus (strain N315) GN=carA PE=1 SV=1 P99147|CARA_ST AAN -2.3 420 50S ribosomal protein L24 OS=Staphyloco ccus aureus (strain N315) GN=rplX PE=1 SV=1 P60735|RL24_STA AN -2.3 491 Amidophosphoribosyltransferase OS=Staphylococcus aureus (strain N315) GN=purF PE=1 SV=1 P99164|PUR1_STA AN -2.3 506 Glycerol-3-phosphate dehydrogenase [NAD( P)+] OS=Staphylococcus aureus (strain N315) GN=gpsA PE=1 SV=1 P64191|GPDA_ST AAN -2.3 621 Putative uncharacterized protein SA1168 OS=Staphylococcus aureus (strain N315) GN=SA1168 PE=4 SV=1 Q7A5T3|Q7A5T3_ STAAN -2.3 785 Ribonuclease 3 OS=Staphylococcus aure us (strain N315) GN=rnc PE=1 SV=1 P66668|RNC_STA AN -2.3 18 ATP synthase subunit beta (atpD) P99112|ATPB_ST AAN -2.2 40 Aconitate hydratase (acnA) P99148|ACON_ST -2.2 119

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AAN 51 50S ribosomal protein L1 (rplA) Q99W68|RL1_STA AN -2.2 69 50S ribosomal protein L6 (rplF) Q7A466|RL6_STA AN -2.2 77 Glucose-6-phosphate isomerase (pgi) P99078|G6PI_STA AN -2.2 92 Succinate dehydrogenase flavoprotein subun it OS=Staphylococcus aureus (strain N315) GN=sdhA PE=4 SV=1 Q7A642|Q7A642_ STAAN -2.2 104 Formate--tetrahydrofolate ligase OS=Staphylococcus aureus (strain N315) GN=fhs PE=1 SV=1 Q7A535|FTHS_ST AAN -2.2 105 Aerobic glycerol-3-phosphate dehydrogenase OS=Staphylococcus aureus (strain N315) GN=glpD PE=1 SV=2 Q7A5V7|GLPD_S TAAN -2.2 115 Threonyl-tRNA synthetase OS=Staphylococcu s aureus (strain N315) GN=thrS PE=1 SV=1 P67585|SYT_STA AN -2.2 130 50S ribosomal protein L22 OS=Staphyloco ccus aureus (strain N315) GN=rplV PE=1 SV=1 Q7A460|RL22_ST AAN -2.2 141 NAD-specific glutamate dehydrogenase OS=Staphylococcus aureus (strain N315) GN=gluD PE=1 SV=1 Q7A6H8|DHE2_ST AAN -2.2 156 Transcription termination-antitermination factor OS=Staphylococcus aureus (strain N315) GN=nusA PE=4 SV=1 Q7A5Y2|Q7A5Y2_ STAAN -2.2 157 50S ribosomal protein L4 OS=Staphylococcu s aureus (strain N315) GN=rplD PE=1 SV=1 P61059|RL4_STA AN -2.2 180 Adenylosuccinate lyase OS=Staphylococcu s aureus (strain N315) GN=purB PE=1 SV=1 Q7A4Q3|PUR8_ST AAN -2.2 196 SA1599 protein OS=Staphylococcus aure us (strain N315) GN=SA1599 PE=4 SV=1 Q7A501|Q7A501_ STAAN -2.2 205 HTH-type transcriptional regulator mgrA OS=Staphylococcus aureus (strain N315) GN=mgrA PE=1 SV=3 Q7A6X2|MGRA_S TAAN -2.2 230 UPF0355 protein SA0372 OS=Staphylococcus aureus (strain N315) GN=SA0372 PE=1 SV=1 Q7A7I6|UP355_ST AAN -2.2 247 ATP-dependent Clp protease ATP-binding su bunit clpX OS=Staphylococcus aureus (strain N315) GN=clpX PE=1 SV=1 P63790|CLPX_ST AAN -2.2 268 Putative uncharacterized protein SA0633 OS=Staphylococcus aureus (strain N315) GN=SA0633 PE=4 SV=1 Q7A6X7|Q7A6X7_ STAAN -2.2 283 Putative uncharacterized protein SA0919 OS=Staphylococcus aureus (strain N315) GN=SA0919 PE=4 SV=1 Q7A694|Q7A694_ STAAN -2.2 305 Acetyl-coenzyme A carboxylase carboxyl tran sferase subunit alpha OS=Staphylococcus aureus (strain N315) GN=accA PE=1 SV=1 Q7A558|ACCA_ST AAN -2.2 333 Putative uncharacterized protein SA1331 OS=Staphylococcus aureus (strain N315) GN=SA1331 PE=1 SV=1 Q7A5H1|Q7A5H1_ STAAN -2.2 362 Serine-protein kinase rsbW OS=Staphyloco ccus aureus (strain N315) GN=rsbW PE=1 SV=1 P0A0H7|RSBW_S TAAN -2.2 411 SA1585 protein OS=Staphylococcus aure us (strain N315) GN=SA1585 PE=4 SV=1 Q7A512|Q7A512_ STAAN -2.2 454 Cytidylate kinase OS=Staphylococcus aureus (strain N315) GN=cmk PE=3 SV=1 P63806|KCY_STA AN -2.2 481 Uncharacterized peptidase SA1530 OS=Staphylococcus aureus (strain N315) GN=SA1530 PE=1 SV=2 Q7A552|Y1530_ST AAN -2.2 519 UPF0173 metal-dependent hydrolase SA1529 OS =Staphylococcus aureus (strain N315) GN=SA1529 PE=1 SV=1 P99149|Y1529_ST AAN -2.2 536 SA0658 protein OS=Staphylococcus aure us (strain N315) GN=SA0658 PE=4 SV=1 Q7A6V6|Q7A6V6_ STAAN -2.2 598 SA1450 protein OS=Staphylococcus aure us (strain N315) GN=SA1450 PE=4 SV=1 Q7A595|Q7A595_ STAAN -2.2 691 UvrABC system protein B OS=Staphylococcus aureus (strain N315) GN=uvrB PE=1 SV=1 P67425|UVRB_ST AAN -2.2 718 (3R)-hydroxymyristoyl-[acyl-carrier-prote in] dehydratase OS=Staphylococcus aureus (strain N315) GN=fabZ PE=3 SV=1 P64108|FABZ_ST AAN -2.2 120

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7 Penicillin binding pr otein 2 prime (mecA) Q7A8C6|Q7A8C6_ STAAN -2.1 39 1-pyrroline-5-carboxylate dehydrogenase (rocA) P99076|ROCA_ST AAN -2.1 50 30S ribosomal protein S5 (rpsE) P66579|RS5_STAA N -2.1 65 Transketolase (tkt) P99161|TKT_STA AN -2.1 68 30S ribosomal protein S11 (rpsK) P66357|RS11_STA AN -2.1 86 Cell division protein (ftsA) P63765|FTSA_STA AN -2.1 88 Translation initiation factor IF-2 (infB) P65134|IF2_STAA N -2.1 147 Naphthoate synthase OS=Staphylococcus au reus (strain N315) GN=menB PE=1 SV=1 Q7A6A9|MENB_S TAAN -2.1 159 Uncharacterized protein SA1069 OS=Staphy lococcus aureus (strain N315) GN=SA1069 PE=1 SV=1 Q7A5Z4|Y1069_S TAAN -2.1 160 Copper chaperone copZ OS=Staphylococcus aureus (strain N315) GN=copZ PE=1 SV=1 Q7A3E5|COPZ_ST AAN -2.1 172 SA0859 protein OS=Staphylococcus aure us (strain N315) GN=SA0859 PE=1 SV=1 Q7A6E5|Q7A6E5_ STAAN -2.1 176 6-phosphofructokinase OS=Staphylococcus au reus (strain N315) GN=pfkA PE=1 SV=1 P99165|K6PF_STA AN -2.1 217 50S ribosomal protein L21 OS=Staphyloco ccus aureus (strain N315) GN=rplU PE=1 SV=1 Q7A583|RL21_ST AAN -2.1 225 Xanthine phosphoribosyltransferase OS=Sta phylococcus aureus (strain N315) GN=xpt PE=1 SV=1 Q7A7I5|XPT_STA AN -2.1 276 Trans-2-enoyl-ACP reductase OS=Staphyloco ccus aureus (strain N315) GN=fabI PE=4 SV=1 Q7A6D8|Q7A6D8_ STAAN -2.1 299 30S ribosomal protein S18 OS=Staphylococcu s aureus (strain N315) GN=rpsR PE=1 SV=1 P66468|RS18_STA AN -2.1 304 Urocanate hydratase OS=Staphylococcus aureus (strain N315) GN=hutU PE=1 SV=1 P67417|HUTU_ST AAN -2.1 350 SA1969 protein OS=Staphylococcus aure us (strain N315) GN=SA1969 PE=4 SV=1 Q7A4A8|Q7A4A8_ STAAN -2.1 369 30S ribosomal protein S17 OS=Staphylococcu s aureus (strain N315) GN=rpsQ PE=1 SV=1 Q7A462|RS17_ST AAN -2.1 379 Putative uncharacterized protein SA2309 OS=Staphylococcus aureus (strain N315) GN=SA2309 PE=4 SV=1 Q7A3I0|Q7A3I0_S TAAN -2.1 381 Aminoacyltransferase femB OS=Staphylococcus aureus (strain N315) GN=femB PE=1 SV=1 P0A0A7|FEMB_ST AAN -2.1 400 SA0884 protein OS=Staphylococcus aure us (strain N315) GN=SA0884 PE=4 SV=1 Q7A6C5|Q7A6C5_ STAAN -2.1 404 Anti-sigma-B factor antagonist OS=Staphylococcus aureus (strain N315) GN=rsbV PE=1 SV=1 P66838|RSBV_ST AAN -2.1 432 SA1054 protein OS=Staphylococcus aure us (strain N315) GN=SA1054 PE=4 SV=1 Q7A605|Q7A605_ STAAN -2.1 436 Putative uncharacterized protein SAS053 OS=Staphylococcus aureus (strain N315) GN=SA1670.1 PE=4 SV=1 Q7A4U6|Q7A4U6_ STAAN -2.1 488 ATP-dependent DNA helicase pcrA OS=Staphy lococcus aureus (strain N315) GN=pcrA PE=1 SV=1 P64319|PCRA_ST AAN -2.1 496 Putative uncharacterized protein SA1534 OS=Staphylococcus aureus (strain N315) GN=SA1534 PE=4 SV=1 Q7A550|Q7A550_ STAAN -2.1 503 Putative uncharacterized protein SA2247 OS=Staphylococcus aureus (strain N315) GN=SA2247 PE=4 SV=1 Q7A3N9|Q7A3N9_ STAAN -2.1 563 Probable ribokinase OS=Staphylococcus aureus (strain N315) GN=rbsK PE=3 SV=1 Q7A7T7|Q7A7T7_ STAAN -2.1 640 Putative uncharacterized protein SA1455 OS=Staphy lococcus aureus (strain N315) Q7A590|Q7A590_ -2.1 121

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GN=SA1455 PE=4 SV=1 STAAN 663 Uridine kinase OS=Staphylococcus au reus (strain N315) GN=udk PE=1 SV=1 P67411|URK_STA AN -2.1 698 P0A0I2|SECE_STAAN P0A0I2|SECE_ST AAN -2.1 26 DNA-directed RNA polymerase subunit beta' (rpoC) P60285|RPOC_ST AAN -2 45 Putative universal stress protein SA1532 (SA1532) Q7A551|Y1532_ST AAN -2 62 ATP-dependent Clp protease ATP-binding subunit (clpL) Q7A3F4|CLPL_ST AAN -2 64 Catalase (katA) Q7A5T2|CATA_S TAAN -2 107 UPF0342 protein SA1663 OS=Staphylococcus aureus (strain N315) GN=SA1663 PE=1 SV=1 Q7A4V3|Y1663_S TAAN -2 164 SA0351 protein OS=Staphylococcus aure us (strain N315) GN=SA0351 PE=4 SV=1 Q7A7K4|Q7A7K4_ STAAN -2 170 Putative uncharacterized protein SA1607 OS=Staphylococcus aureus (strain N315) GN=SA1607 PE=4 SV=1 Q7A4Z7|Q7A4Z7_ STAAN -2 178 UPF0356 protein SA0941 OS=Staphylococcus aureus (strain N315) GN=SA0941 PE=3 SV=2 Q99V08|Y941_ST AAN -2 208 SA2119 protein OS=Staphylococcus aureus (strai n N315) GN=SA2119 PE=1 SV=1 Q7A3Z5_STAAN -2 210 Citrate synthase II OS=Staphylococcus aureus (strain N315) GN=citZ PE=1 SV=1 Q7A561|Q7A561_ STAAN -2 249 30S ribosomal protein S10 OS=Staphyloco ccus aureus (strain N315) GN=rpsJ PE=1 SV=1 P66334|RS10_STA AN -2 252 Putative 2-hydroxyacid dehydrogenase SA 2098 OS=Staphylococcus aureus (strain N315) GN=SA2098 PE=1 SV=1 Q7A417|Y2098_ST AAN -2 261 Extracellular matrix protein-binding prot ein emp OS=Staphylococcus aureus (strain N315) GN=emp PE=1 SV=1 Q7A6P4|EMP_ST AAN -2 267 SA1524 protein OS=Staphylococcus aure us (strain N315) GN=SA1524 PE=3 SV=1 Q7A556|Q7A556_ STAAN -2 308 Uncharacterized oxidoreductase SA2266 OS =Staphylococcus aureus (strain N315) GN=SA2266 PE=1 SV=1 Q7A3L9|Y2266_S TAAN -2 309 ATP-dependent protease ATPase subunit HslU OS=Staphylococcus aureus (strain N315) GN=hslU PE=1 SV=1 P63797|HSLU_ST AAN -2 313 Putative phosphotransferase SA1392 OS=Sta phylococcus aureus (strain N315) GN=SA1392 PE=3 SV=1 P67200|Y1392_ST AAN -2 316 Uncharacterized epimerase/dehydratase SA0511 OS=Staphylococcus aureus (strain N315) GN=SA0511 PE=1 SV=1 Q7A788|Y511_ST AAN -2 342 Uncharacterized N-acetyltransferase SA1019 OS=Staphylococcus aureus (strain N315) GN=SA1019 PE=1 SV=1 Q99UT4|Y1019_S TAAN -2 408 Lipoyl synthase OS=Staphylococcus aureus (strain N315) GN=lipA PE=1 SV=1 P65286|LIPA_STA AN -2 486 Putative uncharacterized protein SA0319 OS=Staphylococcus aureus (strain N315) GN=SA0319 PE=4 SV=1 Q7A7N3|Q7A7N3_ STAAN -2 495 Glutathione peroxidase ho molog BsaA OS=Staphylococcus aureus (strain N315) GN=bsaA PE=1 SV=1 P99097|BSAA_ST AAN -2 509 Putative uncharacterized protein SA2143 OS=Staphylococcus aureus (strain N315) GN=SA2143 PE=4 SV=1 Q7A3X9|Q7A3X9_ STAAN -2 560 Pyrimidine nucleoside transport protein OS=Staphylococcus aureus (strain N315) GN=nupC PE=4 SV=1 Q7A7A0|Q7A7A0_ STAAN -2 594 30S ribosomal protein S14 type Z OS=Staphylococcus aureus (strain N315) GN=rpsZ PE=3 SV=1 P66412|RS14Z_ST AAN -2 603 SA1155 protein OS=Staphylococcus aure us (strain N315) GN=SA1155 PE=4 SV=1 Q7A5U5|Q7A5U5_ STAAN -2 645 UPF0039 protein SA0906 OS=Staphylococcus aureus (strain N315) GN=SA0906 PE=3 SV=1 P0A0M7|Y906_ST AAN -2 122

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671 SA2166 protein OS=Staphylococcus aure us (strain N315) GN=SA2166 PE=4 SV=1 Q7A3V7|Q7A3V7_ STAAN -2 673 SA0572 protein OS=Staphylococcus aure us (strain N315) GN=SA0572 PE=4 SV=1 Q7A733|Q7A733_ STAAN -2 730 Putative uncharacterized protein SA0626 OS=Staphylococcus aureus (strain N315) GN=SA0626 PE=4 SV=1 Q7A6Y4|Q7A6Y4_ STAAN -2 12 Pyruvate kinase (pyk) Q7A559|KPYK_ST AAN -1.9 23 Cysteine synthase (cysK) P63871|CYSK_ST AAN -1.9 36 Phosphoglycerate kinase (pgk) P99135|PGK_STA AN -1.9 47 Pyruvate dehydrogenase E1 component subunit alpha (pdhA) Q820A6|ODPA_ST AAN -1.9 59 50S ribosomal protein L2 (rplB) P60432|RL2_STA AN -1.9 60 Acetate kinase (ackA) Q99TF2|ACKA_ST AAN -1.9 67 UPF0478 protein SA1560 (SA1560) Q7A531|Y1560_ST AAN -1.9 78 Isocitrate dehydrogenase [NADP] (icd) P99167|IDH_STA AN -1.9 80 DNA polymerase III subunit beta (dnaN) P99103|DPO3B_ST AAN -1.9 85 Putative aldehyde dehy drogenase SA1924 (SA1924) Q7A4D8|ALD1_ST AAN -1.9 93 30S ribosomal protein S7 OS=Staphylococcus aureus (strain N315) GN=rpsG PE=1 SV=1 P66616|RS7_STAA N -1.9 126 6-phosphogluconate dehy drogenase, decarboxylating OS=Sta phylococcus aureus (strain N315) GN=gnd PE=1 SV=1 P63334|6PGD_ST AAN -1.9 128 30S ribosomal protein S13 OS=Staphylococcu s aureus (strain N315) GN=rpsM PE=1 SV=1 P66388|RS13_STA AN -1.9 134 Prolyl-tRNA synthetase OS=Staphylococcus aureus (strain N315) GN=proS PE=1 SV=1 Q7A5Y3|SYP_STA AN -1.9 138 30S ribosomal protein S12 OS=Staphylococcu s aureus (strain N315) GN=rpsL PE=1 SV=1 P0A0G8|RS12_ST AAN -1.9 144 30S ribosomal protein S8 OS=Staphylococcus aureus (strain N315) GN=rpsH PE=1 SV=1 P66630|RS8_STAA N -1.9 151 Phosphoglucosamine mutase OS=Staphylococcu s aureus (strain N315) GN=glmM PE=1 SV=1 P99087|GLMM_ST AAN -1.9 171 Putative dipeptidase SA1572 OS=Staphyloco ccus aureus (strain N315) GN=SA1572 PE=1 SV=1 Q7A522|PEPVL_S TAAN -1.9 174 Putative aldehyde dehydrogenase AldA OS=Staphylococcus aureus (strain N315) GN=aldA PE=1 SV=1 Q7A825|ALDA_ST AAN -1.9 188 SA0959 protein OS=Staphylococcus aure us (strain N315) GN=SA0959 PE=4 SV=1 Q7A671|Q7A671_ STAAN -1.9 204 UPF0477 protein SA0873 OS=Staphylococcus aureus (strain N315) GN=SA0873 PE=1 SV=1 Q7A6D4|Y873_ST AAN -1.9 209 50S ribosomal protein L23 OS=Staphyloco ccus aureus (strain N315) GN=rplW PE=1 SV=1 Q7A459|RL23_ST AAN -1.9 214 Succinate dehydrogenase iron-sulfur protein subunit OS=Staphylococcus aureus (strain N315) GN=sdhB PE=4 SV=1 Q99UV7|Q99UV7_ STAAN -1.9 228 Probable thiol peroxidase OS=Staphyloco ccus aureus (strain N315) GN=tpx PE=1 SV=1 P99146|TPX_STA AN -1.9 291 SA1387 protein OS=Staphylococcus aure us (strain N315) GN=SA1387 PE=4 SV=1 Q7A5D2|Q7A5D2_ STAAN -1.9 294 Glucokinase OS=Staphylococcus aureus (strain N315) GN=glcK PE=4 SV=1 Q7A5D8|Q7A5D8_ STAAN -1.9 306 UPF0051 protein SA0778 OS=Staphylococcus aureus (strain N315) GN=SA0778 PE=1 Q7A6L4|Y778_ST -1.9 123

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SV=1 AAN 322 Transcriptional regulatory protein srrA OS=Staphylococcus aureus (strain N315) GN=srrA PE=1 SV=1 Q7A5H6|SRRA_S TAAN -1.9 345 GTP-binding protein era homolog OS=Staphylococcus aureus (strain N315) GN=era PE=1 SV=1 P64085|ERA_STA AN -1.9 360 SA1548 protein OS=Staphylococcus aure us (strain N315) GN=SA1548 PE=4 SV=1 Q7A539|Q7A539_ STAAN -1.9 377 SA0317 protein OS=Staphylococcus aure us (strain N315) GN=SA0317 PE=4 SV=1 Q7A7N5|Q7A7N5_ STAAN -1.9 378 SA2202 protein OS=Staphylococcus aure us (strain N315) GN=SA2202 PE=4 SV=1 Q99RL6|Q99RL6_ STAAN -1.9 391 Methionyl-tRNA formyltransferase OS=Staphylococcus aureus (strain N315) GN=fmt PE=1 SV=1 P99127|FMT_STA AN -1.9 392 Mannitol-1-phosphate 5-dehydrogenase OS =Staphylococcus aureus (strain N315) GN=mtlD PE=1 SV=1 P99140|MTLD_ST AAN -1.9 399 PhoH protein OS=Staphylococcus aure us (strain N315) GN=phoH PE=4 SV=1 Q7A5C7|Q7A5C7_ STAAN -1.9 412 Acetyl-CoA synthetase OS=Staphylococcus aureus (strain N315) GN=acsA PE=4 SV=1 Q99TD1|Q99TD1_ STAAN -1.9 466 Fructose specific permease OS=Staphyloco ccus aureus (strain N315) GN=fruA PE=4 SV=1 Q7A6V9|Q7A6V9_ STAAN -1.9 475 UDP-N-acetylglucosamine 1-carboxyvinyltransferase 2 OS=Staphylococcus aureus (strain N315) GN=murA2 PE=1 SV=1 P65457|MURA2_S TAAN -1.9 485 Putative uncharacterized protein SA1242 OS=Staphylococcus aureus (strain N315) GN=SA1242 PE=4 SV=1 Q7A5N6|Q7A5N6_ STAAN -1.9 568 Probable DNA-directed RNA polymerase s ubunit delta OS=Staphylococcus aureus (strain N315) GN=rpoE PE=1 SV=1 P66715|RPOE_ST AAN -1.9 627 50S ribosomal protein L32 OS=Staphylococcus aureus (strain N315) GN=rpmF PE=1 SV=1 P66210|RL32_STA AN -1.9 629 Dihydrofolate reductase OS=Staphylococcu s aureus (strain N315) GN=folA PE=1 SV=2 P99079|DYR_STA AN -1.9 647 50S ribosomal protein L9 OS=Staphylococcu s aureus (strain N315) GN=rplI PE=1 SV=1 P66318|RL9_STA AN -1.9 660 Membrane-associated protein tcaA OS=Staphylococcus aureus (strain N315) GN=tcaA PE=2 SV=1 Q7A3X6|TCAA_S TAAN -1.9 672 Putative uncharacterized protein SA1068 OS=Staphylococcus aureus (strain N315) GN=SA1068 PE=4 SV=1 Q7A5Z5|Q7A5Z5_ STAAN -1.9 687 Putative uncharacterized protein SA1086 OS=Staphylococcus aureus (strain N315) GN=SA1086 PE=4 SV=1 Q7A5Y9|Q7A5Y9_ STAAN -1.9 767 Putative uncharacterized protein SA1062 OS=Staphylococcus aureus (strain N315) GN=SA1062 PE=4 SV=1 Q7A5Z9|Q7A5Z9_ STAAN -1.9 1 Elongation factor Tu (tuf) P99152|EFTU_ST AAN -1.8 2 Bifunctional autolysin (atl) Q99V41|ATL_STA AN -1.8 29 Pyruvate dehydrogenase E1 component subunit beta (pdhB) P99063|ODPB_ST AAN -1.8 48 Aspartyl/glutamyl-tRNA(Asn/Gln) amidotransferase subunit B (gatB) P99169|GATB_ST AAN -1.8 58 Glutamyl-tRNA(Gln) amidot ransferase subunit A (gatA) P63489|GATA_ST AAN -1.8 75 Ribonucleoside-diphosphate reductase (nrdE) Q7A6T2|Q7A6T2_ STAAN -1.8 122 SA1745 protein OS=Staphylococcus aure us (strain N315) GN=SA1745 PE=4 SV=1 Q7A4N5|Q7A4N5_ STAAN -1.8 129 DNA-directed RNA polymerase subunit alpha OS=Staphylococcus aureus (strain N315) GN=rpoA PE=1 SV=1 P66706|RPOA_ST AAN -1.8 148 Pyridoxal biosynthesis lyase pdxS OS=Staphylococcus aureus (strain N315) GN=pdxS PE=1 SV=1 P60798|PDXS_ST AAN -1.8 124

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166 Succinyl-CoA ligase [ADP-forming] subunit alpha OS=Staphylococcus aureus (strain N315) GN=sucD PE=1 SV=1 P99070|SUCD_ST AAN -1.8 179 Glycine cleavage system H protein OS=Sta phylococcus aureus (strain N315) GN=gcvH PE=1 SV=1 P64214|GCSH_ST AAN -1.8 219 D-alanine aminotransferase OS=Staphyloco ccus aureus (strain N315) GN=dat PE=1 SV=1 P99090|DAAA_ST AAN -1.8 229 3-oxoacyl-[acyl-carrier-protein] synthase 2 OS=Staphylococcus aureus (strain N315) GN=fabF PE=1 SV=1 Q7A6F8|FABF_ST AAN -1.8 233 Putative 8-amino-7-oxononanoate synthase/2 -amino-3-ketobutyrate coenzyme A ligase OS=Staphylococcus aureus (strain N315) GN=SA0508 PE=1 SV=1 P60120|BIKB_STA AN -1.8 235 D-alanine--poly(phosphoribitol) ligase subunit 1 OS=Staphylococcus aureus (strain N315) GN=dltA PE=1 SV=1 P99107|DLTA_ST AAN -1.8 241 30S ribosomal protein S6 OS=Staphylococcus aureus (strain N315) GN=rpsF PE=1 SV=1 P99142|RS6_STAA N -1.8 245 Uridylate kinase OS=Staphylococcus au reus (strain N315) GN=pyrH PE=1 SV=1 P65936|PYRH_ST AAN -1.8 257 Chaperone protein hchA OS=Staphylococcu s aureus (strain N315) GN=hchA PE=1 SV=1 P64313|HCHA_ST AAN -1.8 280 CTP synthase OS=Staphylococcus aure us (strain N315) GN=pyrG PE=1 SV=1 P99072|PYRG_ST AAN -1.8 285 D-alanine--D-alanine ligase OS=Staphyloco ccus aureus (strain N315) GN=ddl PE=1 SV=1 P63892|DDL_STA AN -1.8 293 Putative NAD(P)H nitroreductase SA2311 OS =Staphylococcus aureus (strain N315) GN=SA2311 PE=1 SV=1 Q7A3H8|Y2311_S TAAN -1.8 300 30S ribosomal protein S9 OS=Staphylococcu s aureus (strain N315) GN=rpsI PE=1 SV=1 P66646|RS9_STAA N -1.8 302 50S ribosomal protein L31 type B OS=Staphylococcus aureus (strain N315) GN=rpmE2 PE=1 SV=1 P66196|RL31B_ST AAN -1.8 311 Imidazolonepropionase OS=Staphylococcus aureus (strain N315) GN=hutI PE=1 SV=1 P64418|HUTI_STA AN -1.8 329 50S ribosomal protein L29 OS=Staphyloco ccus aureus (strain N315) GN=rpmC PE=1 SV=1 P66173|RL29_STA AN -1.8 358 Putative uncharacterized protein SA1657 OS=Staphylococcus aureus (strain N315) GN=SA1657 PE=4 SV=1 Q7A4V9|Q7A4V9_ STAAN -1.8 361 PTS system EIIBC component SA0186 OS=Staphylococcus aureus (strain N315) GN=SA0186 PE=1 SV=1 Q7A804|PTXBC_S TAAN -1.8 366 Ribonuclease J 2 OS=Staphylococcus aureus (strain N315) GN=SA1118 PE=1 SV=1 Q7A5X6|RNJ2_ST AAN -1.8 383 Hypoxanthine-guanine phosphoribosyltransferase OS=Staphylococcus aureus (strain N315) GN=hpt PE=1 SV=1 P99085|HPRT_ST AAN -1.8 398 30S ribosomal protein S16 OS=Staphylococcu s aureus (strain N315) GN=rpsP PE=1 SV=1 P66440|RS16_STA AN -1.8 459 Glutamate-1-semialdehyde 2,1-aminomutase 2 OS=Staphylococcus aureus (strain N315) GN=hemL2 PE=1 SV=1 Q7A4T5|GSA2_ST AAN -1.8 473 Leucyl-tRNA synthetase OS=Staphylococcu s aureus (strain N315) GN=leuS PE=1 SV=1 P67513|SYL_STA AN -1.8 499 30S ribosomal protein S20 OS=Staphylococcu s aureus (strain N315) GN=rpsT PE=1 SV=1 Q7A5C0|RS20_ST AAN -1.8 624 Response regulator protein vraR OS=Staphy lococcus aureus (strain N315) GN=vraR PE=1 SV=1 Q7A4R9|VRAR_S TAAN -1.8 652 Protein nagD homolog OS=Staphylococcus aureus (strain N315) GN=nagD PE=3 SV=1 Q7A6K4|NAGD_S TAAN -1.8 701 Putative uncharacterized protein SA1453 OS=Staphylococcus aureus (strain N315) GN=SA1453 PE=4 SV=1 Q7A592|Q7A592_ STAAN -1.8 17 Protein map (map) P69775|MAP1_ST AAN -1.7 24 Fructose-bisphosphate aldolase class 1 (fda) P99117|ALF1_STA AN -1.7 42 UPF0365 protein SA1402 (SA1402) Q7A5C5|Y1402_S -1.7 125

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TAAN 57 Uncharacterized protein SA0829 (SA0829) Q7A6H3|Y829_ST AAN -1.7 79 Glutamine synthetase (glnA) P99095|GLNA_ST AAN -1.7 98 60 kDa chaperonin OS=Staphylococcus au reus (strain N315) GN=groL PE=1 SV=1 P99083|CH60_STA AN -1.7 99 Triosephosphate isomerase OS=Staphylococcu s aureus (strain N315) GN=tpiA PE=1 SV=1 P99133|TPIS_STA AN -1.7 101 50S ribosomal protein L10 OS=Staphyloco ccus aureus (strain N315) GN=rplJ PE=1 SV=1 P99155|RL10_STA AN -1.7 113 Septation ring formation regulator ezrA OS=Staphylococcus aureus (strain N315) GN=ezrA PE=1 SV=1 P64003|EZRA_ST AAN -1.7 119 GTP-sensing transcriptional pleiotropic repressor codY OS=Staphylococcus aureus (strain N315) GN=codY PE=1 SV=1 P63844|CODY_ST AAN -1.7 143 Protein recA OS=Staphylococcus aure us (strain N315) GN=recA PE=1 SV=1 P68844|RECA_ST AAN -1.7 149 50S ribosomal protein L7/L12 OS=Staphyloco ccus aureus (strain N315) GN=rplL PE=1 SV=1 P99154|RL7_STA AN -1.7 152 Putative uncharacterized protein SA1986 OS=Staphylococcus aureus (strain N315) GN=SA1986 PE=4 SV=1 Q7A493|Q7A493_ STAAN -1.7 193 Putative uncharacterized protein SA0359 OS=Staphylococcus aureus (strain N315) GN=SA0359 PE=4 SV=1 Q7A7J8|Q7A7J8_S TAAN -1.7 200 Putative uncharacterized protein SA1528 OS=Staphylococcus aureus (strain N315) GN=SA1528 PE=4 SV=1 Q7A553|Q7A553_ STAAN -1.7 232 Transcription antitermination protein nusG OS=Staphylococcus aureus (strain N315) GN=nusG PE=1 SV=1 P0A096|NUSG_ST AAN -1.7 236 Glycerol phosphate lipoteichoic acid synthase OS=Staphylococcus aureus (strain N315) GN=ltaS PE=1 SV=1 Q7A6U1|LTAS_ST AAN -1.7 254 Elastin-binding protein ebpS OS=Staphyloco ccus aureus (strain N315) GN=ebpS PE=1 SV=3 Q7A5I6|EBPS_ST AAN -1.7 263 UDP-N-acetylmuramoylalanine--D-glutamate ligase OS=Staphylococcus aureus (strain N315) GN=murD PE=1 SV=1 P0A090|MURD_S TAAN -1.7 270 Arginyl-tRNA synthetase OS=Staphylococcu s aureus (strain N315) GN=argS PE=1 SV=1 Q99W05|SYR_ST AAN -1.7 272 Orotidine 5'-phosphate decarboxylase OS=Staphylococcus aureus (strain N315) GN=pyrF PE=1 SV=1 P99145|PYRF_ST AAN -1.7 277 Alcohol-acetaldehyde dehydrogenase OS=Staphylococcus aureus (strain N315) GN=adhE PE=4 SV=1 Q7A843|Q7A843_ STAAN -1.7 317 50S ribosomal protein L17 OS=Staphyloco ccus aureus (strain N315) GN=rplQ PE=1 SV=1 Q7A469|RL17_ST AAN -1.7 390 Translation initiation factor IF-3 OS=Sta phylococcus aureus (strain N315) GN=infC PE=1 SV=1 P65140|IF3_STAA N -1.7 395 Putative uncharacterized protein SA1946 OS=Staphylococcus aureus (strain N315) GN=SA1946 PE=1 SV=1 Q7A4C4|Q7A4C4_ STAAN -1.7 421 SA1565 protein OS=Staphylococcus aure us (strain N315) GN=SA1565 PE=4 SV=1 Q7A527|Q7A527_ STAAN -1.7 433 Cystathionine gamma-syntha se OS=Staphylococcus aureus (strain N315) GN=metB PE=3 SV=1 Q99WE2|Q99WE2 _STAAN -1.7 437 Phosphate acyltransferase OS=Staphylococcu s aureus (strain N315) GN=plsX PE=1 SV=1 P65739|PLSX_STA AN -1.7 446 Putative uncharacterized protein SA1840 OS=Staphylococcus aureus (strain N315) GN=SA1840 PE=1 SV=1 Q7A4J0|Q7A4J0_S TAAN -1.7 500 Ribosomal RNA small subunit methyltransferase H OS=Staphylococcus aureus (strain N315) GN=rsmH PE=1 SV=1 P60392|RSMH_ST AAN -1.7 514 Putative uncharacterized protein SA0635 OS=Staphylococcus aureus (strain N315) GN=SA0635 PE=4 SV=1 Q7A6X5|Q7A6X5_ STAAN -1.7 555 Putative uncharacterized protein SA1464 OS=Staphylococcus aureus (strain N315) GN=SA1464 PE=4 SV=1 Q7A585|Q7A585_ STAAN -1.7 126

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579 SA0675 protein OS=Staphylococcus aure us (strain N315) GN=SA0675 PE=4 SV=1 Q7A6U0|Q7A6U0_ STAAN -1.7 628 tRNA modification GTPase mnmE OS=Staphylococcus aureus (strain N315) GN=mnmE PE=3 SV=1 P66972|MNME_ST AAN -1.7 669 Chaperone protein dnaJ OS=Staphylococcus aureus (strain N315) GN=dnaJ PE=1 SV=1 P63971|DNAJ_ST AAN -1.7 11 Cell division protein ftsZ (ftsZ) P99108|FTSZ_STA AN -1.6 27 Staphylococcal secretory antigen (ssaA2) Q7A423|SSAA2_S TAAN -1.6 72 DNA-directed RNA polymerase subunit beta (rpoB) P60278|RPOB_ST AAN -1.6 90 Trigger factor OS=Staphylococcus aureus (strain N315) GN=tig PE=1 SV=1 P99080|TIG_STAA N -1.6 121 Putative formate dehydrogenase SA2102 OS =Staphylococcus aureus (strain N315) GN=SA2102 PE=1 SV=1 Q99RW4|FDHL_S TAAN -1.6 175 Adenylosuccinate synthetase OS=Staphyloco ccus aureus (strain N315) GN=purA PE=1 SV=1 P99099|PURA_ST AAN -1.6 181 Putative uncharacterized protein SA1263 OS=Staphylococcus aureus (strain N315) GN=SA1263 PE=4 SV=1 Q7A5M5|Q7A5M5 _STAAN -1.6 182 UDP-N-acetylglucosamine 1-carboxyvinyltransferase 1 OS=Staphylococcus aureus (strain N315) GN=murA1 PE=1 SV=1 P84058|MURA1_S TAAN -1.6 239 Putative uncharacterized protein SA1743 OS=Staphylococcus aureus (strain N315) GN=SA1743 PE=1 SV=1 Q7A4N7|Q7A4N7_ STAAN -1.6 242 Chaperone protein clpB OS=Staphylococcus aureus (strain N315) GN=clpB PE=1 SV=1 Q7A6G6|CLPB_ST AAN -1.6 281 Thioredoxin reductase OS=Staphylococcus au reus (strain N315) GN=trxB PE=1 SV=1 P99101|TRXB_ST AAN -1.6 298 DNA gyrase subunit A OS=Staphylococcus aureus (strain N315) GN=gyrA PE=1 SV=1 Q99XG5|GYRA_S TAAN -1.6 314 Ribosome-recycling factor OS=Staphylococcus aureus (strain N315) GN=frr PE=1 SV=1 P99130|RRF_STA AN -1.6 328 Putative septation protein spoVG OS=Staphy lococcus aureus (strain N315) GN=spoVG PE=1 SV=2 Q7A7B5|SP5G_ST AAN -1.6 365 Aspartate semialdehyde dehydrogenase OS=Staphylococcus aureus (strain N315) GN=asd PE=3 SV=1 Q7A5P8|Q7A5P8_ STAAN -1.6 397 Branched-chain alpha-keto acid dehydrogena se E2 OS=Staphylococcus aureus (strain N315) GN=bmfBB PE=3 SV=1 Q7A5F9|Q7A5F9_ STAAN -1.6 414 10 kDa chaperonin OS=Staphylococcus aureus (strain N315) GN=groS PE=1 SV=1 P99104|CH10_STA AN -1.6 470 Putative uncharacterized protein SAS056 OS=Staphylococcus aureus (strain N315) GN=SA1738.1 PE=4 SV=1 Q7A4P2|Q7A4P2_ STAAN -1.6 483 Pyrroline-5-carboxylate reductase OS=Staphy lococcus aureus (strain N315) GN=proC PE=1 SV=1 Q7A5G8|P5CR_ST AAN -1.6 511 Arginase OS=Staphylococcus aureus (strain N315) GN=arg PE=1 SV=1 P60088|ARGI_STA AN -1.6 539 SA2056 protein OS=Staphylococcus aure us (strain N315) GN=SA2056 PE=4 SV=1 Q7A448|Q7A448_ STAAN -1.6 540 Menaquinone biosynthesis methyltransferase ubiE OS=Staphylococcus aureus (strain N315) GN=ubiE PE=1 SV=1 P67062|UBIE_STA AN -1.6 554 50S ribosomal protein L28 OS=Staphyloco ccus aureus (strain N315) GN=rpmB PE=3 SV=1 P66153|RL28_STA AN -1.6 585 Uncharacterized protein SA1737 OS=Staphy lococcus aureus (strain N315) GN=SA1737 PE=1 SV=1 Q7A4P4|Y1737_ST AAN -1.6 593 Single-stranded DNA-bindi ng protein OS=Staphylococcus aureus (strain N315) GN=ssb PE=4 SV=1 Q7A7K2_STAAN (+1) -1.6 680 Ribosome-binding factor A OS=Staphylococcu s aureus (strain N315) GN=rbfA PE=1 SV=1 P65967|RBFA_ST AAN -1.6 3 Elongation factor G (fusA) P68789|EFG_STA -1.5 127

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AN 10 Inosine-5'-monophosphate dehydrogenase (guaB) P99106|IMDH_ST AAN -1.5 19 Enolase (eno) P99088|ENO_STA AN -1.5 25 30S ribosomal protein S2 (rpsB) P66544|RS2_STAA N -1.5 28 30S ribosomal protein S3 (rpsC) P66553|RS3_STAA N -1.5 55 Alkyl hydroperoxide reductase subunit C (ahpC) P99074|AHPC_ST AAN -1.5 74 Phosphocarrier protein HPr (ptsH) P99143|PTHP_STA AN -1.5 76 GMP synthase [glutamine-hydrolyzing] (guaA) P99105|GUAA_ST AAN -1.5 91 Lysyl-tRNA synthetase OS=Staphylococcus aureus (strain N315) GN=lysS PE=1 SV=1 P67610|SYK_STA AN -1.5 102 Probable branched-chain-amino-acid aminot ransferase OS=Staphylococcus aureus (strain N315) GN=ilvE PE=1 SV=1 P99138|ILVE_STA AN -1.5 108 50S ribosomal protein L3 OS=Staphylococcu s aureus (strain N315) GN=rplC PE=1 SV=1 P60449|RL3_STA AN -1.5 125 Bifunctional protein folD OS=Staphylococcus aureus (strain N315) GN=folD PE=1 SV=1 Q7A697|FOLD_ST AAN -1.5 133 Succinyl-CoA ligase [ADP-forming] subunit beta OS=Staphylococcus aureus (strain N315) GN=sucC PE=1 SV=1 P99071|SUCC_ST AAN -1.5 165 UPF0133 protein SA0437 OS=Staphylococcus aureus (strain N315) GN=SA0437 PE=1 SV=1 P99126|Y437_STA AN -1.5 186 S-adenosylmethionine synthase OS=Staphy lococcus aureus (strain N315) GN=metK PE=1 SV=1 P66767|METK_ST AAN -1.5 195 50S ribosomal protein L18 OS=Staphylococcus aureus (strain N315) GN=rplR PE=1 SV=1 Q7A467|RL18_ST AAN -1.5 202 Glyceraldehyde-3-phosphate de hydrogenase 2 OS=Staphylococcus aureus (strain N315) GN=gapA2 PE=1 SV=1 P99067|G3P2_STA AN -1.5 215 Glutamyl-tRNA synthetase OS=Staphylococcu s aureus (strain N315) GN=gltX PE=1 SV=1 P99170|SYE_STA AN -1.5 222 Putative uncharacterized protein SA1661 OS=Staphylococcus aureus (strain N315) GN=SA1661 PE=4 SV=1 Q7A4V5|Q7A4V5_ STAAN -1.5 224 Putative peptidyl-prolyl cis-trans isomerase OS=Staphylococcus aureus (strain N315) GN=SA0815 PE=1 SV=1 Q7A6I1|PPI1_STA AN -1.5 238 Aspartyl-tRNA synthetase OS=Staphylococcus aureus (strain N315) GN=aspS PE=1 SV=1 P67015|SYD_STA AN -1.5 250 Uracil phosphoribosyltransferase OS=Staphylococcus aureus (strain N315) GN=upp PE=1 SV=1 P67396|UPP_STA AN -1.5 258 DNA gyrase subunit B OS=Staphylococcus aureus (strain N315) GN=gyrB PE=1 SV=2 P66937|GYRB_ST AAN -1.5 287 30S ribosomal protein S4 OS=Staphylococcus aureus (strain N315) GN=rpsD PE=1 SV=1 P66563|RS4_STAA N -1.5 327 Putative uncharacterized protein SA1668 OS=Staphylococcus aureus (strain N315) GN=SA1668 PE=4 SV=1 Q7A4U8|Q7A4U8_ STAAN -1.5 335 UDP-N-acetylmuramoyl-L-alanyl-D-glutam ate--L-lysine ligase OS=Staphylococcus aureus (strain N315) GN=murE PE=1 SV=1 P65480|MURE_ST AAN -1.5 346 Peptide methionine sulfoxide reductase msrA 2 OS=Staphylococcus aureus (strain N315) GN=msrA2 PE=1 SV=1 P65446|MSRA2_S TAAN -1.5 352 SA1243 protein OS=Staphylococcus aure us (strain N315) GN=SA1243 PE=4 SV=1 Q7A5N5|Q7A5N5_ STAAN -1.5 374 SA0758 protein OS=Staphylococcus aure us (strain N315) GN=SA0758 PE=4 SV=1 Q7A6M7|Q7A6M7 _STAAN -1.5 401 Mannitol-specific phosphotransferase enzyme IIA component OS=Staphylococcus aureus (strain N315) GN=mtlF PE=3 SV=2 P0A0D8|PTMA_S TAAN -1.5 128

PAGE 140

403 Dephospho-CoA kinase OS=Staphylococcus aureus (strain N315) GN=coaE PE=1 SV=1 P63831|COAE_ST AAN -1.5 428 UDP-N-acetylmuramoyl-tripeptide--D-ala nyl-D-alanine ligase OS=Staphylococcus aureus (strain N315) GN=murF PE=3 SV=1 Q7A4F9|Q7A4F9_ STAAN -1.5 449 (Dimethylallyl)adenosine tRNA methylthiotr ansferase miaB OS=Staphylococcus aureus (strain N315) GN=miaB PE=1 SV=1 Q7A5W3|MIAB_S TAAN -1.5 508 RNA polymerase sigma factor rpoD OS=Staphylococcus aureus (strain N315) GN=rpoD PE=1 SV=1 Q99TT5|RPOD_ST AAN -1.5 517 Conserved virulence factor B OS=Staphyloco ccus aureus (strain N315) GN=cvfB PE=1 SV=1 Q7A5Q1|CVFB_S TAAN -1.5 557 UPF0312 protein SA2479 OS=Staphylococcus aureus (strain N315) GN=SA2479 PE=1 SV=1 Q7A339|Y2479_ST AAN -1.5 699 GMP reductase OS=Staphylococcus aure us (strain N315) GN=guaC PE=1 SV=1 P60563|GUAC_ST AAN -1.5 761 Methicillin resistance mecR1 protein OS=Staphylococcus aureus (strain N315) GN=mecR1 PE=1 SV=1 P0A0B0|MECR_S TAAN -1.5 769 SA0777 protein OS=Staphylococcus aure us (strain N315) GN=SA0777 PE=4 SV=1 Q7A6L5|Q7A6L5_ STAAN -1.5 21 Glyceraldehyde-3-phosphat e dehydrogenase 1 (gapA1) P99136|G3P1_STA AN -1.4 109 Phosphoenolpyruvate-protein phosphotransfe rase OS=Staphylococcus aureus (strain N315) GN=ptsI PE=1 SV=1 Q99V14|PT1_STA AN -1.4 110 Putative uncharacterized protein SA1698 OS=Staphylococcus aureus (strain N315) GN=SA1698 PE=4 SV=1 Q7A4S0|Q7A4S0_ STAAN -1.4 118 Putative uncharacterized protein SA0775 OS=Staphylococcus aureus (strain N315) GN=SA0775 PE=4 SV=1 Q7A6L6|Q7A6L6_ STAAN -1.4 154 Serine hydroxymethyltransferase OS=Staphylococcus aureus (strain N315) GN=glyA PE=1 SV=1 P99091|GLYA_ST AAN -1.4 191 SA1549 protein OS=Staphylococcus aure us (strain N315) GN=SA1549 PE=4 SV=1 Q7A538|Q7A538_ STAAN -1.4 237 50S ribosomal protein L30 OS=Staphyloco ccus aureus (strain N315) GN=rpmD PE=1 SV=1 RL30_STAAN (+1) -1.4 262 50S ribosomal protein L16 OS=Staphyloco ccus aureus (strain N315) GN=rplP PE=1 SV=1 Q7A461|RL16_ST AAN -1.4 265 Alanyl-tRNA synthetase OS=Staphylococcus aureus (strain N315) GN=alaS PE=1 SV=1 P67011|SYA_STA AN -1.4 292 Aspartyl/glutamyl-tRNA(Asn/Gln) amidot ransferase subunit C OS=Staphylococcus aureus (strain N315) GN=gatC PE=1 SV=1 P68808|GATC_ST AAN -1.4 315 Signal transduction protein TRAP OS=Staphy lococcus aureus (strain N315) GN=traP PE=1 SV=1 Q7A4W3|TRAP_S TAAN -1.4 319 Acetate-CoA ligase OS=Staphylococcus aureus (strain N315) GN=SA2402 PE=4 SV=1 Q7A3A2|Q7A3A2_ STAAN -1.4 343 DNA-directed RNA polymerase subunit omeg a OS=Staphylococcus aureus (strain N315) GN=rpoZ PE=1 SV=1 P66726|RPOZ_ST AAN -1.4 388 Putative uncharacterized protein SAS040 OS=Staphylococcus aureus (strain N315) GN=SA1154.1 PE=4 SV=1 Q7A5U6|Q7A5U6_ STAAN -1.4 418 UPF0637 protein SA0957 OS=Staphylococcus aureus (strain N315) GN=SA0957 PE=1 SV=1 Q99UZ6|Y957_ST AAN -1.4 447 Branched-chain alpha-keto acid dehydrogena se E1 OS=Staphylococcus aureus (strain N315) GN=bfmBAB PE=4 SV=1 Q7A5F8|Q7A5F8_ STAAN -1.4 489 Lipase 1 OS=Staphylococcus aureus (strain N315) GN=lip1 PE=3 SV=2 P65289|LIP1_STA AN -1.4 492 Organic hydroperoxide resistance protein-lik e OS=Staphylococcus aureus (strain N315) GN=SA0755 PE=1 SV=1 Q7A6M9|OHRL_S TAAN -1.4 501 Methionine aminopeptidase OS=Staphylococcu s aureus (strain N315) GN=map PE=1 SV=1 P99121|AMPM_ST AAN -1.4 43 Glycyl-tRNA synthetase (glyQS) P99129|SYG_STA AN -1.3 87 UPF0457 protein SA1975.1 (SA 1975.1) Q99S93|Y197A_ST -1.3 129

PAGE 141

AAN 95 Cell-division protein OS=Staphylococcus au reus (strain N315) GN=ftsH PE=4 SV=1 Q7A7A5|Q7A7A5_ STAAN -1.3 117 Putative uncharacterized protein SAS030 OS=Staphylococcus aureus (strain N315) GN=SA0930.1 PE=4 SV=1 Q7A690|Q7A690_ STAAN -1.3 123 Putative uncharacterized protein SA1671 OS=Staphylococcus aureus (strain N315) GN=SA1671 PE=1 SV=1 Q7A4U5|Q7A4U5_ STAAN -1.3 132 Foldase protein prsA OS=Staphylococcus aureus (strain N315) GN=prsA PE=1 SV=1 P60748|PRSA_ST AAN -1.3 155 Alkyl hydroperoxide reductase subunit F OS=Staphylococcus aureus (strain N315) GN=ahpF PE=1 SV=1 P99118|AHPF_ST AAN -1.3 168 Uncharacterized hydrolase SA2367 OS=Sta phylococcus aureus (strain N315) GN=SA2367 PE=1 SV=1 Q7A3C4|Y2367_S TAAN -1.3 190 Translation initiation factor IF-1 OS=Sta phylococcus aureus (strain N315) GN=infA PE=1 SV=1 P65119|IF1_STAA N -1.3 194 SA2095 protein OS=Staphylococcus aure us (strain N315) GN=SA2095 PE=4 SV=1 Q7A420|Q7A420_ STAAN -1.3 203 3-oxoacyl-[acyl-carrier-protein] reductase OS=Staphylococcus aureus (strain N315) GN=fabG PE=1 SV=1 P99093|FABG_ST AAN -1.3 227 Adenylate kinase OS=Staphylococcus aureus (strain N315) GN=adk PE=1 SV=1 P99062|KAD_STA AN -1.3 255 Dihydrolipoyllysine-residue succinyltrans ferase component of 2-oxoglutarate dehydrogenase complex OS=Staphylococcu s aureus (strain N315) GN=odhB PE=1 SV=1 Q7A5N4|ODO2_S TAAN -1.3 338 Probable uridylyltransferase SA1974 OS=S taphylococcus aureus (strain N315) GN=SA1974 PE=1 SV=1 Q7A4A4|URTF_ST AAN -1.3 373 Elongation factor P OS=Staphylococcus aureus (strain N315) GN=efp PE=1 SV=1 P99066|EFP_STAA N -1.3 382 Staphopain A OS=Staphylococcus aureus (strain N315) GN=sspP PE=3 SV=1 P65826|SSPP_STA AN -1.3 402 HTH-type transcriptional regulator sarR OS=Staphylococcus aureus (strain N315) GN=sarR PE=1 SV=3 Q7A425|SARR_ST AAN -1.3 435 50S ribosomal protein L33 1 OS=Staphylococcus aureus (strain N315) GN=rpmG1 PE=3 SV=1 P66228|RL331_ST AAN -1.3 440 Hit-like protein involved in cell-cycle re gulation OS=Staphylococcus aureus (strain N315) GN=hit PE=4 SV=1 Q7A4W0|Q7A4W0 _STAAN -1.3 505 Putative uncharacterized protein SAS027 OS=Staphylococcus aureus (strain N315) GN=SA0884.1 PE=4 SV=1 Q7A6C4|Q7A6C4_ STAAN -1.3 556 Cold shock protein cspB OS=Staphylococcus aureus (strain N315) GN=cspB PE=3 SV=1 Q7A326|Q7A326_ STAAN -1.3 633 Uncharacterized protein SA1186 OS=Staphy lococcus aureus (strain N315) GN=SA1186 PE=1 SV=1 Q7A5S4|Y1186_ST AAN -1.3 5 DNA-binding protein HU (hup) Q7A5J1|DBH_STA AN -1.2 14 30S ribosomal protein S1 (rpsA) Q7A5J0|RS1_STA AN -1.2 37 Thioredoxin (trxA) P99122|THIO_STA AN -1.2 81 SA0774 protein (SA0774) Q7A6L7|Q7A6L7_ STAAN -1.2 124 Polyribonucleotide nucleotidyltransferase OS=Staphylococcus aureus (strain N315) GN=pnp PE=1 SV=1 Q7A5X7|PNP_STA AN -1.2 177 2-oxoglutarate dehydrogenase E1 component OS=Staphylococcus aureus (strain N315) GN=odhA PE=1 SV=1 Q99U74|ODO1_ST AAN -1.2 207 Methionyl-tRNA synthetase OS=Staphyloco ccus aureus (strain N315) GN=metG PE=1 SV=1 P67579|SYM_STA AN -1.2 297 Regulatory protein msrR OS=Staphylococcus aureus (strain N315) GN=msrR PE=1 SV=1 Q99Q02|MSRR_ST AAN -1.2 130

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351 Glucose-6-phosphate 1-dehydrogenase OS =Staphylococcus aureus (strain N315) GN=SA1336 PE=3 SV=1 Q7A5G7|Q7A5G7_ STAAN -1.2 375 NH(3)-dependent NAD(+) synthetase OS =Staphylococcus aureus (strain N315) GN=nadE PE=1 SV=1 P99150|NADE_ST AAN -1.2 430 Peptide methionine sulfoxide reductase ms rB OS=Staphylococcus aureus (strain N315) GN=msrB PE=1 SV=1 P99065|MSRB_ST AAN -1.2 443 Redox-sensing transcriptional repressor re x OS=Staphylococcus aureus (strain N315) GN=rex PE=3 SV=1 P60386|REX_STA AN -1.2 543 Chromosome segregation SMC protein OS=Staphylococcus aureus (strain N315) GN=smc PE=4 SV=1 Q7A5Z2|Q7A5Z2_ STAAN -1.2 136 Putative uncharacterized protein SA0570 OS=Staphylococcus aureus (strain N315) GN=SA0570 PE=4 SV=1 Q7A735|Q7A735_ STAAN -1.1 295 Malonyl CoA-acyl carrier protein transacylase OS=Staphylococcus aureus (strain N315) GN=fabD PE=1 SV=1 Q7A5Z3|FABD_ST AAN -1.1 301 Transcription elongation factor greA OS =Staphylococcus aureus (strain N315) GN=greA PE=1 SV=1 P99156|GREA_ST AAN -1.1 344 30S ribosomal protein S19 OS=Staphylococcu s aureus (strain N315) GN=rpsS PE=1 SV=1 P66494|RS19_STA AN -1.1 348 Probable cysteine desulfurase OS=Staphyloco ccus aureus (strain N315) GN=csd PE=1 SV=1 P99177|CSD_STA AN -1.1 359 S-ribosylhomocysteine lyase OS=Staphyloco ccus aureus (strain N315) GN=luxS PE=1 SV=1 P65330|LUXS_ST AAN -1.1 368 Probable glycine dehydrogena se [decarboxylating] subunit 2 OS=Staphylococcus aureus (strain N315) GN=gcvPB PE=1 SV=1 P99168|GCSPB_ST AAN -1.1 464 SA1035 protein OS=Staphylococcus aure us (strain N315) GN=SA1035 PE=1 SV=1 Q7A612|Q7A612_ STAAN -1.1 465 Putative uncharacterized protein SA2262 OS=Staphylococcus aureus (strain N315) GN=SA2262 PE=4 SV=1 Q7A3M3|Q7A3M3 _STAAN -1.1 526 Low molecular weight protein-tyrosinephosphatase ptpA OS=Staphylococcus aureus (strain N315) GN=ptpA PE=1 SV=1 Q7A4S1|PTPA_ST AAN -1.1 550 Probable transglycosylase sceD OS=Staphylococcus aureus (strain N315) GN=sceD PE=2 SV=1 Q7A4F2|SCED_ST AAN -1.1 578 OppF protein OS=Staphylococcus aure us (strain N315) GN=oppF PE=4 SV=1 Q7A6F3|Q7A6F3_ STAAN -1.1 618 3-phosphoshikimate 1-carboxyvinyltransfera se OS=Staphylococcus aureus (strain N315) GN=aroA PE=1 SV=1 P63585|AROA_ST AAN -1.1 620 SA1475 protein OS=Staphylococcus aure us (strain N315) GN=SA1475 PE=4 SV=1 Q7A581|Q7A581_ STAAN -1.1 646 Aminomethyltransferase OS=Staphylococcus aureus (strain N315) GN=gcvT PE=1 SV=1 P64225|GCST_ST AAN -1.1 4 UPF0337 protein SA0772 (SA0772) Q7A6L9|Y772_ST AAN -1 41 Acyl carrier protein (acpP) P0A002|ACP_STA AN -1 46 Uncharacterized leukocid in-like protein 2 (SA1813) Q99SN7|LUKL2_S TAAN -1 89 Probable manganese-dependent inorganic pyrophosphatase (ppaC) P65753|PPAC_ST AAN -1 185 50S ribosomal protein L11 OS=Staphyloco ccus aureus (strain N315) GN=rplK PE=1 SV=2 P0A0F2|RL11_ST AAN -1 442 Homoserine dehydrogenase OS=Staphyloco ccus aureus (strain N315) GN=dhoM PE=3 SV=1 Q7A5T6|Q7A5T6_ STAAN -1 467 Cysteinyl-tRNA synthetase OS=Staphylococcu s aureus (strain N315) GN=cysS PE=1 SV=1 Q99W73|SYC_ST AAN -1 504 Bla regulator protein blaR1 OS=Staphyloco ccus aureus (strain N315) GN=blaR1 PE=4 SV=1 Q9AC79|Q9AC79_ STAAN -1 523 FMN-dependent NADPH-azoreductase OS=S taphylococcus aureus (strain N315) GN=azo1 PE=1 SV=1 Q7A782|AZO1_ST AAN -1 548 tRNA uridine 5-carboxymethylaminomethyl modification enzyme mnmG P64230|MNMG_S -1 131

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OS=Staphylococcus aureus (strain N315) GN=mnmG PE=1 SV=1 TAAN 613 UPF0447 protein SA0544 OS=Staphylococcus aureus (strain N315) GN=SA0544 PE=1 SV=1 Q7A759|Y544_ST AAN -1 716 Putative trmH family tRNA/rRNA methyltransferase OS=Staphylococcus aureus (strain N315) GN=SA0490 PE=1 SV=1 Q7A794|TRMHL_ STAAN -1 746 UPF0154 protein SA1178 OS=Staphylococcus aureus (strain N315) GN=SA1178 PE=1 SV=1 P67291|Y1178_ST AAN -1 750 SA0605 protein OS=Staphylococcus aure us (strain N315) GN=SA0605 PE=4 SV=1 Q7A701|Q7A701_ STAAN -1 8 Alkaline shock protein 23 (asp23) P99157|ASP23_ST AAN -0.9 111 50S ribosomal protein L27 OS=Staphyloco ccus aureus (strain N315) GN=rpmA PE=1 SV=1 P66133|RL27_STA AN -0.9 112 SA2327 protein OS=Staphylococcus aure us (strain N315) GN=SA2327 PE=4 SV=1 Q7A3G3|Q7A3G3_ STAAN -0.9 163 ATP-dependent Clp protease ATP-binding su bunit clpC OS=Staphylococcus aureus (strain N315) GN=clpC PE=1 SV=1 Q7A797|CLPC_ST AAN -0.9 192 3-hexulose-6-phosphate synthase OS=S taphylococcus aureus (strain N315) GN=SA0528 PE=1 SV=1 Q7A774|HPS_STA AN -0.9 246 Valyl-tRNA synthetase OS=Staphylococcus aureus (strain N315) GN=valS PE=1 SV=1 Q99TJ8|SYV_STA AN -0.9 310 Probable glycine dehydrogena se [decarboxylating] subunit 1 OS=Staphylococcus aureus (strain N315) GN=gcvPA PE=1 SV=1 P64218|GCSPA_S TAAN -0.9 380 Ribonucleoside-diphosphate reductase minor subunit OS=Staphylococcus aureus (strain N315) GN=nrdF PE=4 SV=1 Q7A6T1|Q7A6T1_ STAAN -0.9 490 SA1558 protein OS=Staphylococcus aure us (strain N315) GN=SA1558 PE=4 SV=1 Q7A533|Q7A533_ STAAN -0.9 522 Probable ctpA-like serine protease OS =Staphylococcus aureus (strain N315) GN=SA1253 PE=1 SV=1 Q7A5M9|CTPAL_ STAAN -0.9 527 Putative uncharacterized protein SA0936 OS=Staphylococcus aureus (strain N315) GN=SA0936 PE=4 SV=1 Q7A686|Q7A686_ STAAN -0.9 600 Putative uncharacterized protein SA1293 OS=Staphylococcus aureus (strain N315) GN=SA1293 PE=4 SV=1 Q7A5J9|Q7A5J9_S TAAN -0.9 766 Threonine synthase OS=Staphylococcus au reus (strain N315) GN=thrC PE=4 SV=1 Q7A5T5|Q7A5T5_ STAAN -0.9 97 Phosphoenolpyruvate carboxykinase [ATP] OS=Staphylococcus aureus (strain N315) GN=pckA PE=1 SV=1 P99128|PCKA_ST AAN -0.8 189 2-C-methyl-D-erythritol 4-phosphate cytidyl yltransferase 2 OS=Staphylococcus aureus (strain N315) GN=ispD2 PE=1 SV=1 Q7A7V0|ISPD2_S TAAN -0.8 405 Cold-shock protein C OS=Staphylococcus aureus (strain N315) GN=cspC PE=3 SV=1 Q7A6P1|Q7A6P1_ STAAN -0.8 422 Glucose-specific phosphotransferase enzyme IIA component OS=Staphylococcus aureus (strain N315) GN=crr PE=1 SV=1 P60857|PTGA_ST AAN -0.8 478 ATP-binding cassette transporter A OS=Staphylococcus aureus (strain N315) GN=SA0599 PE=4 SV=1 Q7A708|Q7A708_ STAAN -0.8 623 Iron-regulated ABC transporter siderophor e-binding protein SirA OS=Staphylococcus aureus (strain N315) GN=sirA PE=4 SV=1 Q7A869|Q7A869_ STAAN -0.8 135 Transcriptional regulator sarA OS=Staphylo coccus aureus (strain N315) GN=sarA PE=1 SV=3 Q7A732|SARA_ST AAN -0.7 206 General stress protein 20U OS=Staphyloco ccus aureus (strain N315) GN=dps PE=3 SV=1 Q7A4C8|Q7A4C8_ STAAN -0.7 212 Uncharacterized protein SA0707 OS=Staphy lococcus aureus (strain N315) GN=SA0707 PE=1 SV=1 Q7A6R6|Y707_ST AAN -0.7 303 UPF0082 protein SA0624 OS=Staphylococcus aureus (strain N315) GN=SA0624 PE=1 SV=1 P67182|Y624_STA AN -0.7 532 Penicillin-binding protein 1 OS=Staphyloco ccus aureus (strain N315) GN=pbpA PE=4 SV=1 Q7A619|Q7A619_ STAAN -0.7 602 Putative uncharacterized protein SA1593 OS=Staphylococcus aureus (strain N315) GN=SA1593 PE=4 SV=1 Q99T94|Q99T94_S TAAN -0.7 132

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711 Putative uncharacterized protein SA2221 OS=Staphylococcus aureus (strain N315) GN=SA2221 PE=4 SV=1 Q7A3R1|Q7A3R1_ STAAN -0.7 712 Aminopeptidase ampS OS=Staphylococcus aureus (strain N315) GN=ampS PE=4 SV=1 Q7A4S3|Q7A4S3_ STAAN -0.7 717 Na(+)/H(+) antiporter subunit E1 OS=Staphylococcus aureus (strain N315) GN=mnhE1 PE=1 SV=1 P60689|MNHE1_S TAAN -0.7 153 Isoleucyl-tRNA synthetase OS=Staphyloco ccus aureus (strain N315) GN=ileS PE=1 SV=1 P67509|SYI_STAA N -0.6 158 Putative uncharacterized protein SA1573 OS=Staphylococcus aureus (strain N315) GN=SA1573 PE=4 SV=1 Q7A521|Q7A521_ STAAN -0.6 169 Probable DEAD-box ATP-dependent RNA helicase SA1885 OS=Staphylococcus aureus (strain N315) GN=SA1885 PE=1 SV=1 Q7A4G0|Y1885_S TAAN -0.6 289 Superoxide dismutase [Mn/ Fe] 1 OS=Staphylococcus aureus (strain N315) GN=sodA PE=1 SV=1 P99098|SODM1_S TAAN -0.6 367 SA0691 protein OS=Staphylococcus aure us (strain N315) GN=SA0691 PE=4 SV=1 Q7A6S7|Q7A6S7_ STAAN -0.6 448 Epimerase family protein SA0724 OS=S taphylococcus aureus (strain N315) GN=SA0724 PE=1 SV=1 Q7A6Q5|Y724_ST AAN -0.6 476 SA1563 protein OS=Staphylococcus aure us (strain N315) GN=SA1563 PE=1 SV=1 Q7A529|Q7A529_ STAAN -0.6 705 Putative uncharacterized protein SA0711 OS=Staphylococcus aureus (strain N315) GN=SA0711 PE=4 SV=1 Q7A6R2|Q7A6R2_ STAAN -0.6 187 Cell cycle protein gpsB OS=Staphylococcus aureus (strain N315) GN=gpsB PE=1 SV=1 Q7A5L1|GPSB_ST AAN -0.5 284 Superoxide dismutase [Mn/ Fe] 2 OS=Staphylococcus aureus (strain N315) GN=sodM PE=1 SV=1 P66831|SODM2_S TAAN -0.5 479 Aminoacyltransferase femA OS=Staphyloco ccus aureus (strain N315) GN=femA PE=1 SV=1 Q7A5R3|FEMA_S TAAN -0.5 482 HTH-type transcriptional regulator sarS OS=Staphylococcus aureus (strain N315) GN=sarS PE=1 SV=1 Q7A872|SARS_ST AAN -0.5 582 Putative uncharacterized protein SA0695 OS=Staphylococcus aureus (strain N315) GN=SA0695 PE=4 SV=1 Q7A6S4|Q7A6S4_ STAAN -0.5 625 SA0422 protein OS=Staphylococcus aure us (strain N315) GN=SA0422 PE=4 SV=1 Q7A7E1|Q7A7E1_ STAAN -0.5 715 Peptide deformylase OS=Staphylococcus aureus (strain N315) GN=def PE=1 SV=1 P99077|DEF_STA AN -0.5 271 FMN-dependent NADH-azoreductase OS=S taphylococcus aureus (strain N315) GN=azoR PE=1 SV=1 Q99X11|AZOR_ST AAN -0.4 552 Lytic regulatory protein truncated with T n554 OS=Staphylococcus aureus (strain N315) GN=truncated-SA PE=4 SV=1 Q99SB2|Q99SB2_ STAAN -0.4 553 Clumping factor A OS=Staphylococcus au reus (strain N315) GN=clfA PE=1 SV=1 Q99VJ4|CLFA_ST AAN -0.4 20 Chaperone protein dnaK (dnaK) P99110|DNAK_ST AAN -0.3 321 Hydroxamate siderophore binding lipoprotein OS=Staphylococcus aureus (strain N315) GN=fhuD2 PE=4 SV=1 Q7A433|Q7A433_ STAAN -0.3 664 Methylenetetrahydrofolate--tRNA-(uracil-5-)-methyltransferase trmFO OS=Staphylococcus aureus (strain N315) GN=trmFO PE=1 SV=1 P64235|TRMFO_S TAAN -0.3 752 Putative uncharacterized protein SA2267 OS=Staphylococcus aureus (strain N315) GN=SA2267 PE=4 SV=1 Q99RF4|Q99RF4_ STAAN -0.3 765 Putative uncharacterized protein SA1957 OS=Staphylococcus aureus (strain N315) GN=SA1957 PE=4 SV=1 Q7A4B6|Q7A4B6_ STAAN -0.2 31 SA2437 protein (SA2437) Q7A371|Q7A371_ STAAN -0.1 216 SA1979 protein OS=Staphylococcus aure us (strain N315) GN=SA1979 PE=1 SV=1 Q7A499|Q7A499_ STAAN -0.1 288 2',3'-cyclic-nucleotide 2'-phosphodiesterase OS=Staphylococcus aureus (strain N315) GN=cvfA PE=1 SV=1 P67278|CNPD_ST AAN -0.1 444 Anti repressor OS=Staphylococcus aureus (strai n N315) GN=SA1801 PE=4 SV=1 Q99SP8|Q99SP8_S -0.1 133

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TAAN 545 Putative uncharacterized protein SA1274 OS=Staphylococcus aureus (strain N315) GN=SA1274 PE=4 SV=1 Q7A5L6|Q7A5L6_ STAAN -0.1 22 Probable transglycosylase isaA (isaA) P99160|ISAA_STA AN 0 735 Heme-degrading monooxygenase isdI OS=Staphylococcus aureus (strain N315) GN=isdI PE=1 SV=1 Q7A827|ISDI_STA AN 0 275 UPF0435 protein SA1696 OS=Staphylococcus aureus (strain N315) GN=SA1696 PE=1 SV=1 Q7A4S2|Y1696_ST AAN 0.1 575 Putative uncharacterized protein SA0943 OS=Staphylococcus aureus (strain N315) GN=SA0943 PE=4 SV=1 Q7A681|Q7A681_ STAAN 0.1 708 SA1343 protein OS=Staphylococcus aure us (strain N315) GN=SA1343 PE=4 SV=1 Q7A5G2|Q7A5G2_ STAAN 0.1 325 6,7-dimethyl-8-ribityllumazine synthase OS=Staphylococcus aureus (strain N315) GN=ribH PE=1 SV=1 P99141|RISB_STA AN 0.2 15 Elongation factor Ts (tsf) P99171|EFTS_STA AN 0.4 218 50S ribosomal protein L25 OS=Staphyloco ccus aureus (strain N315) GN=rplY PE=1 SV=1 Q7A7B3|RL25_ST AAN 0.4 52 Putative uncharacterized protein SAS049 (SA1451.1) Q7A594|Q7A594_ STAAN 0.5 63 Putative uncharacterized protein SA2323 (SA2323) Q7A3G7|Q7A3G7_ STAAN 0.5 259 UPF0337 protein SA1452 OS=Staphylococcus aureus (strain N315) GN=SA1452 PE=1 SV=1 Q7A593|Y1452_ST AAN 0.7 355 GTP cyclohydrolase folE2 OS=Staphylococcus aureus (strain N315) GN=folE2 PE=1 SV=1 Q7A777|GCH4_ST AAN 0.8 547 Delta-hemolysin OS=Staphylococcus au reus (strain N315) GN=hld PE=1 SV=2 P0A0M2|HLD_ST AAN 0.8 240 SA0246 protein OS=Staphylococcus aure us (strain N315) GN=SA0246 PE=4 SV=1 Q7A7U9|Q7A7U9_ STAAN No Values 312 UDP-N-acetylmuramate--L-alanine ligase OS=Staphylococcus aureus (strain N315) GN=murC PE=1 SV=1 P65475|MURC_ST AAN No Values 353 Pseudouridine synthase OS=Staphylococcus aureus (strain N315) GN=SA1040 PE=3 SV=1 Q7A610|Q7A610_ STAAN No Values 385 Probable cytosol aminopeptidase OS=Staphy lococcus aureus (strain N315) GN=ampA PE=4 SV=1 Q7A6J3|Q7A6J3_S TAAN No Values 396 Aldehyde dehydrogenase OS=Staphylococcu s aureus (strain N315) GN=aldH PE=4 SV=1 Q7A4P5|Q7A4P5_ STAAN No Values 407 Fumarate hydratase class II OS=Staphyloc occus aureus (strain N315) GN=fumC PE=1 SV=1 P64173|FUMC_ST AAN No Values 416 Putative uncharacterized protein SA0908 OS=Staphylococcus aureus (strain N315) GN=SA0908 PE=4 SV=1 Q7A6A3|Q7A6A3_ STAAN No Values 424 Bleomycin resistance protein OS=Staphylococcus aureus (strain N315) GN=ble PE=1 SV=2 Q7A8D1|BLE_ST AAN No Values 425 Protein grpE OS=Staphylococcus aure us (strain N315) GN=grpE PE=1 SV=1 P99086|GRPE_ST AAN No Values 434 Putative uncharacterized protein SAS010 OS=Staphylococcus aureus (strain N315) GN=SA0359.1 PE=4 SV=1 Q7A7J7|Q7A7J7_S TAAN No Values 453 Exodeoxyribonuclease 7 small subunit OS=Staphylococcus aureus (strain N315) GN=xseB PE=3 SV=1 P67461|EX7S_STA AN No Values 456 Serine-aspartate repeat-containing protein D OS=Staphylococcus aureus (strain N315) GN=sdrD PE=1 SV=1 Q7A780|SDRD_ST AAN No Values 460 Putative uncharacterized protein SA1277 OS=Staphylococcus aureus (strain N315) GN=SA1277 PE=4 SV=1 Q7A5L3|Q7A5L3_ STAAN No Values 468 50S ribosomal protein L15 OS=Staphyloco ccus aureus (strain N315) GN=rplO PE=1 SV=1 P0A0F6|RL15_ST AAN No Values 471 30S ribosomal protein S15 OS=Staphylococcu s aureus (strain N315) GN=rpsO PE=1 SV=1 Q7A5X8|RS15_ST AAN No Values 134

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497 Methionine import ATP-binding protein Me tN 2 OS=Staphylococcus aureus (strain N315) GN=metN2 PE=1 SV=1 Q7A6M2|METN2_ STAAN No Values 498 Putative uncharacterized protein SA1363 OS=Staphylococcus aureus (strain N315) GN=SA1363 PE=4 SV=1 Q7A5E8|Q7A5E8_ STAAN No Values 502 SA0182 protein OS=Staphylococcus aure us (strain N315) GN=SA0182 PE=1 SV=1 Q7A808|Q7A808_ STAAN No Values 510 SA1559 protein OS=Staphylococcus aure us (strain N315) GN=SA1559 PE=4 SV=1 Q7A532|Q7A532_ STAAN No Values 513 Protein kinase OS=Staphylococcus aureus (strain N315) GN=SA1063 PE=4 SV=1 Q7A5Z8|Q7A5Z8_ STAAN No Values 516 SA2140 protein OS=Staphylococcus aure us (strain N315) GN=SA2140 PE=4 SV=1 Q7A3Y2|Q7A3Y2_ STAAN No Values 518 3-methyl-2-oxobutanoate hydroxymethyltransferase OS=Staphylococcus aureus (strain N315) GN=panB PE=1 SV=1 P65656|PANB_ST AAN No Values 525 ATP synthase epsilon chain OS=Staphylococcus aureus (strain N315) GN=atpC PE=1 SV=1 P63665|ATPE_ST AAN No Values 528 SA2346 protein OS=Staphylococcus aure us (strain N315) GN=SA2346 PE=4 SV=1 Q7A3E4|Q7A3E4_ STAAN No Values 529 Uncharacterized protein SA0370 OS=Staphy lococcus aureus (strain N315) GN=SA0370 PE=1 SV=1 P60855|Y370_STA AN No Values 531 Coenzyme A disulfide reductase OS=Sta phylococcus aureus (strain N315) GN=cdr PE=1 SV=3 Q7A6H1|CDR_ST AAN No Values 533 Molybdopterin molybdenumtransferase OS=Staphylococcus aureus (strain N315) GN=moeA PE=1 SV=1 P99139|MOEA_ST AAN No Values 535 Putative uncharacterized protein SA1031 OS=Staphylococcus aureus (strain N315) GN=SA1031 PE=3 SV=1 Q7A616|Q7A616_ STAAN No Values 537 SA1121 protein OS=Staphylococcus aure us (strain N315) GN=SA1121 PE=3 SV=1 Q7A5X4|Q7A5X4_ STAAN No Values 538 Putative uncharacterized protein SA2160 OS=Staphylococcus aureus (strain N315) GN=SA2160 PE=4 SV=1 Q7A3W3|Q7A3W3 _STAAN No Values 541 Adenine phosphoribosyltransferase OS=Staphylococcus aureus (strain N315) GN=apt PE=1 SV=1 P68779|APT_STA AN No Values 542 SA1311 protein OS=Staphylococcus aure us (strain N315) GN=SA1311 PE=4 SV=1 Q7A5I7|Q7A5I7_S TAAN No Values 546 SA2434 protein OS=Staphylococcus aure us (strain N315) GN=SA2434 PE=4 SV=1 Q7A374|Q7A374_ STAAN No Values 558 SA2332 protein OS=Staphylococcus aure us (strain N315) GN=SA2332 PE=4 SV=1 Q7A3F8|Q7A3F8_ STAAN No Values 561 2,3,4,5-tetrahydropyridine-2,6-dicarboxyla te N-acetyltransfer ase OS=Staphylococcus aureus (strain N315) GN=dapH PE=3 SV=1 Q7A5P7|DAPH_ST AAN No Values 566 TelA-like protein SA1238 OS=Staphylococcu s aureus (strain N315) GN=SA1238 PE=1 SV=1 P60108|TELL_STA AN No Values 567 Putative uncharacterized protein SA2374 OS=Staphylococcus aureus (strain N315) GN=SA2374 PE=4 SV=1 Q7A3B7|Q7A3B7_ STAAN No Values 571 Putative uncharacterized protein SA0721 OS=Staphylococcus aureus (strain N315) GN=SA0721 PE=4 SV=1 Q7A6Q7|Q7A6Q7_ STAAN No Values 576 Chorismate synthase OS=Staphylococcus aureus (strain N315) GN=aroC PE=3 SV=1 P63614|AROC_ST AAN No Values 580 PurR protein OS=Staphylococcus aureus (strain N315) GN=purR PE=4 SV=1 Q7A7B7|Q7A7B7_ STAAN No Values 583 DNA topoisomerase 1 OS=Staphylococcus au reus (strain N315) GN=topA PE=1 SV=1 Q7A5Y5|TOP1_ST AAN No Values 584 SA2200 protein OS=Staphylococcus aure us (strain N315) GN=SA2200 PE=4 SV=1 Q7A3S6|Q7A3S6_ STAAN No Values 590 SA2490 protein OS=Staphylococcus aure us (strain N315) GN=SA2490 PE=1 SV=1 Q7A331|Q7A331_ STAAN No Values 591 Putative uncharacterized protein SA1454 OS=Staphylococcus aureus (strain N315) GN=SA1454 PE=4 SV=1 Q7A591|Q7A591_ STAAN No Values 596 Nucleoside-triphosphatase OS=Staphylococcus aureus (strain N315) GN=SA0998 PE=1 P99094|NTPA_ST No Values 135

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SV=1 AAN 597 Purine nucleoside phosphorylase OS=Staphy lococcus aureus (strain N315) GN=deoD PE=1 SV=1 Q7A4C9|Q7A4C9_ STAAN No Values 599 UPF0473 protein SA1443 OS=Staphylococcus aureus (strain N315) GN=SA1443 PE=1 SV=1 Q7A598|Y1443_ST AAN No Values 601 Glycolytic operon regulator OS=Staphyloco ccus aureus (strain N315) GN=gapR PE=4 SV=1 Q7A6Q3|Q7A6Q3_ STAAN No Values 608 Putative uncharacterized protein SA1405 OS=Staphylococcus aureus (strain N315) GN=SA1405 PE=4 SV=1 Q7A5C4|Q7A5C4_ STAAN No Values 609 Putative uncharacterized protein SA1451 OS=Staphylococcus aureus (strain N315) GN=SA1451 PE=4 SV=1 Q99TM6|Q99TM6 _STAAN No Values 610 Sensor protein srrB OS=Staphylococcus aureus (strain N315) GN=srrB PE=1 SV=2 Q7A5H7|SRRB_ST AAN No Values 611 Phosphoglucomutase OS=Staphylococcus aureus (str ain N315) GN=pgcA PE=1 SV=2 PGCA_STAAN No Values 619 Putative uncharacterized protein SAS054 OS=Staphylococcus aureus (strain N315) GN=SA1692.1 PE=4 SV=1 Q7A4S5|Q7A4S5_ STAAN No Values 626 SA0247 protein OS=Staphylococcus aure us (strain N315) GN=SA0247 PE=4 SV=1 Q7A7U8|Q7A7U8_ STAAN No Values 630 SA2244 protein OS=Staphylococcus aure us (strain N315) GN=SA2244 PE=4 SV=1 Q7A3P2|Q7A3P2_ STAAN No Values 636 UPF0349 protein SA0800 OS=Staphylococcus aureus (strain N315) GN=SA0800 PE=3 SV=1 Q7A6J6|Y800_ST AAN No Values 638 Glycine betaine transporter OS=Staphyloc occus aureus (strain N315) GN=opuD PE=4 SV=1 Q99UC9|Q99UC9_ STAAN No Values 639 Glutamate racemase OS=Staphylococcus aureus (strain N315) GN=murI PE=1 SV=1 P63638|MURI_ST AAN No Values 641 Putative uncharacterized protein SA1795 OS=Staphylococcus aureus (strain N315) GN=SA1795 PE=4 SV=1 Q99SQ6|Q99SQ6_ STAAN No Values 642 DNA ligase OS=Staphylococcus aureus (strain N315) GN=ligA PE=3 SV=1 Q7A4Q5|DNLJ_ST AAN No Values 643 SA2498 protein OS=Staphylococcus aure us (strain N315) GN=SA2498 PE=4 SV=1 Q7A321|Q7A321_ STAAN No Values 644 SA0817 protein OS=Staphylococcus aure us (strain N315) GN=SA0817 PE=1 SV=1 Q7A6H9|Q7A6H9_ STAAN No Values 650 SA1566 protein OS=Staphylococcus aure us (strain N315) GN=SA1566 PE=4 SV=1 Q7A526|Q7A526_ STAAN No Values 651 Alpha-D-1,4-glucosidase OS=Staphylococcus aureus (strain N315) GN=malA PE=1 SV=1 Q7A5G5|Q7A5G5_ STAAN No Values 653 Putative uncharacterized protein SA0696 OS=Staphylococcus aureus (strain N315) GN=SA0696 PE=1 SV=1 Q7A6S3|Q7A6S3_ STAAN No Values 654 Putative uncharacterized protein SA0529 OS=Staphylococcus aureus (strain N315) GN=SA0529 PE=4 SV=1 Q7A773|Q7A773_ STAAN No Values 655 SA0165 protein OS=Staphylococcus aure us (strain N315) GN=SA0165 PE=4 SV=1 Q7A821|Q7A821_ STAAN No Values 657 Alanine dehydrogenase 2 OS=Staphylococcu s aureus (strain N315) GN=ald2 PE=1 SV=1 Q99TF4|DHA2_ST AAN No Values 658 SA1216 protein OS=Staphylococcus aure us (strain N315) GN=SA1216 PE=4 SV=1 Q7A5Q5|Q7A5Q5_ STAAN No Values 659 UTP--glucose-1-phosphate uridylyltransferas e OS=Staphylococcus aureus (strain N315) GN=gtaB PE=1 SV=1 Q7A3J9|GTAB_ST AAN No Values 668 Glucosamine--fructose-6-phosphate aminotra nsferase [isomerizing] OS=Staphylococcus aureus (strain N315) GN=glmS PE=1 SV=2 GLMS_STAAN No Values 676 Bifunctional protein glmU OS=Staphylococcu s aureus (strain N315) GN=glmU PE=1 SV=1 Q7A7B4|GLMU_S TAAN No Values 677 SA0229 protein OS=Staphylococcus aure us (strain N315) GN=SA0229 PE=4 SV=1 Q7A7W6|Q7A7W6 _STAAN No Values 678 PTS enzyme II, phosphoenolpyruvate-depende nt, trehalose-specific OS=Staphylococcus aureus (strain N315) GN=treP PE=4 SV=1 Q7A7D1|Q7A7D1_ STAAN No Values 136

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681 SA0557 protein OS=Staphylococcus aure us (strain N315) GN=SA0557 PE=4 SV=1 Q7A747|Q7A747_ STAAN No Values 683 SA2170 protein OS=Staphylococcus aure us (strain N315) GN=SA2170 PE=4 SV=1 Q7A3V4|Q7A3V4_ STAAN No Values 684 Transcription-repair-coupling factor OS=Sta phylococcus aureus (strain N315) GN=mfd PE=1 SV=1 Q7A7B2|MFD_ST AAN No Values 688 SA0677 protein OS=Staphylococcus aure us (strain N315) GN=SA0677 PE=4 SV=1 Q7A6T8|Q7A6T8_ STAAN No Values 689 SA2248 protein OS=Staphylococcus aure us (strain N315) GN=SA2248 PE=4 SV=1 Q7A3N8|Q7A3N8_ STAAN No Values 690 UDP-N-acetylglucosamine 2-epimerase OS=Staphylococcus aureus (strain N315) GN=mnaA PE=3 SV=1 Q7A4E3|Q7A4E3_ STAAN No Values 692 SA2240 protein OS=Staphylococcus aure us (strain N315) GN=SA2240 PE=3 SV=1 Q7A3P6|Q7A3P6_ STAAN No Values 693 Putative uncharacterized protein SA1966 OS=Staphylococcus aureus (strain N315) GN=SA1966 PE=4 SV=1 Q7A4B0|Q7A4B0_ STAAN No Values 694 Transcriptional repressor nrdR OS=Staphy lococcus aureus (strain N315) GN=nrdR PE=3 SV=1 P67316|NRDR_ST AAN No Values 695 Uroporphyrinogen decarboxylase OS=Staphylococcus aureus (strain N315) GN=hemE PE=1 SV=1 P67420|DCUP_ST AAN No Values 696 SA0902 protein OS=Staphylococcus aure us (strain N315) GN=SA0902 PE=3 SV=1 Q7A6A5|Q7A6A5_ STAAN No Values 697 SA2099 protein OS=Staphylococcus aure us (strain N315) GN=SA2099 PE=4 SV=1 Q7A416|Q7A416_ STAAN No Values 702 Pseudouridine synthase OS=Staphylococcus aureus (strain N315) GN=rluB PE=3 SV=1 Q7A5H5|Q7A5H5_ STAAN No Values 707 Putative uncharacterized protein SA0013 OS=Staphylococcus aureus (strain N315) GN=SA0013 PE=4 SV=1 Q7A8E3|Q7A8E3_ STAAN No Values 709 Putative uncharacterized protein SAS032 OS=Staphylococcus aureus (strain N315) GN=SA0959.1 PE=4 SV=1 Q7A670|Q7A670_ STAAN No Values 710 Formimidoylglutamase OS=Staphylococcus aureus (strain N315) GN=hutG PE=1 SV=1 P99158|HUTG_ST AAN No Values 713 Probable GTP-binding protein engB OS=S taphylococcus aureus (strain N315) GN=engB PE=1 SV=1 P64071|ENGB_ST AAN No Values 714 Putative uncharacterized protein SA2298 OS=Staphylococcus aureus (strain N315) GN=SA2298 PE=4 SV=1 Q7A3J0|Q7A3J0_S TAAN No Values 719 Delta-aminolevulinic acid dehydratase OS=Staphylococcus aureus (strain N315) GN=hemB PE=1 SV=1 P64334|HEM2_ST AAN No Values 721 Serine acetyltransferase OS=Staphylococcus aureus (strain N315) GN=cysE PE=1 SV=1 P67765|CYSE_ST AAN No Values 723 SA2436 protein OS=Staphylococcus aure us (strain N315) GN=SA2436 PE=4 SV=1 Q7A372|Q7A372_ STAAN No Values 724 Diacylglycerol kinase OS=Staphylococcus aureus (strain N315) GN=dagK PE=3 SV=1 Q7A4Q8|DAGK_S TAAN No Values 725 Putative uncharacterized protein SA1546 OS=Staphylococcus aureus (strain N315) GN=SA1546 PE=4 SV=1 Q7A541|Q7A541_ STAAN No Values 726 SA1291 protein OS=Staphylococcus aure us (strain N315) GN=SA1291 PE=4 SV=1 Q7A5K1|Q7A5K1_ STAAN No Values 727 Putative uncharacterized protein SA1017 OS=Staphylococcus aureus (strain N315) GN=SA1017 PE=4 SV=1 Q7A623|Q7A623_ STAAN No Values 731 SA0590 protein OS=Staphylococcus aure us (strain N315) GN=SA0590 PE=4 SV=1 Q7A716|Q7A716_ STAAN No Values 732 Protein esaA OS=Staphylococcus aureus (strain N315) GN=esaA PE=3 SV=1 Q7A7S3|ESAA_ST AAN No Values 733 SA0270 protein OS=Staphylococcus aure us (strain N315) GN=SA0270 PE=4 SV=1 Q7A7S5_STAAN (+1) No Values 734 Ribonuclease R OS=Staphylococcus aureus (strain N315) GN=rnr PE=4 SV=1 Q99VK1|Q99VK1_ STAAN No Values 736 Putative uncharacterized protein SA0447 OS=Staphyloco ccus aureus (strain N315) Q7A7C1|Q7A7C1_ No Values 137

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GN=SA0447 PE=4 SV=1 STAAN 737 UPF0403 protein SA1261 OS=Staphylococcus aureus (strain N315) GN=SA1261 PE=1 SV=1 Q7A5M6|Y1261_S TAAN No Values 738 Uncharacterized lipoprotein SA2273 OS=S taphylococcus aureus (strain N315) GN=SA2273 PE=1 SV=1 Q7A3L3|Y2273_S TAAN No Values 739 DNA polymerase III polC-type OS=Staphylo coccus aureus (strain N315) GN=polC PE=1 SV=1 P63982|DPO3_ST AAN No Values 741 HmrA protein OS=Staphylococcus aure us (strain N315) GN=hmrA PE=4 SV=1 Q99Q45|Q99Q45_ STAAN No Values 742 Putative uncharacterized protein SA2238 OS=Staphylococcus aureus (strain N315) GN=SA2238 PE=4 SV=1 Q99RI2|Q99RI2_S TAAN No Values 743 Queuine tRNA-ribosyltransfer ase OS=Staphylococcus aure us (strain N315) GN=tgt PE=1 SV=1 P66905|TGT_STA AN No Values 744 Streptomycin 3''-adenylyltran sferase OS=Staphylococcus au reus (strain N315) GN=ant1 PE=1 SV=1 P0A0D1|S3AD_ST AAN No Values 745 5-methyltetrahydropteroy ltriglutamate--homocyste ine methyltransferase OS=Staphylococcus aureus (str ain N315) GN=metE PE=3 SV=1 P65343|METE_ST AAN No Values 748 Glycine betaine/carnitine/choline ABC tr ansporter opuCC OS=Staphylococcus aureus (strain N315) GN=opuCC PE=4 SV=1 Q7A3Q0|Q7A3Q0_ STAAN No Values 749 Putative uncharacterized protein SA2224 OS=Staphylococcus aureus (strain N315) GN=SA2224 PE=4 SV=1 Q7A3Q8|Q7A3Q8_ STAAN No Values 751 SA0530 protein OS=Staphylococcus aure us (strain N315) GN=SA0530 PE=4 SV=1 Q7A772|Q7A772_ STAAN No Values 753 Putative uncharacterized protein SA1403 OS=Staphylococcus aureus (strain N315) GN=SA1403 PE=4 SV=1 Q99TS2|Q99TS2_S TAAN No Values 756 Ribosomal RNA small subunit methyltransferase G OS=Staphylococcus aureus (strain N315) GN=rsmG PE=3 SV=1 P64240|RSMG_ST AAN No Values 757 Acetyl-coenzyme A carboxylase carboxyl tran sferase subunit beta OS=Staphylococcus aureus (strain N315) GN=accD PE=1 SV=1 Q7A557|ACCD_ST AAN No Values 758 Porphobilinogen deaminase OS=Staphylococcus aureus (strain N315) GN=hemC PE=1 SV=1 P64341|HEM3_ST AAN No Values 760 Peptide methionine sulfoxide reductase msrA 1 OS=Staphylococcus aureus (strain N315) GN=msrA1 PE=1 SV=1 P0A082|MSRA1_S TAAN No Values 762 Glycine betaine aldehyde de hydrogenase gbsA OS=Staphylococcus aureus (strain N315) GN=gbsA PE=4 SV=1 Q7A399|Q7A399_ STAAN No Values 763 Preprotein translocase subunit secY OS=Sta phylococcus aureus (strain N315) GN=secY PE=1 SV=1 Q7A468|SECY_ST AAN No Values 764 Putative uncharacterized protein SA1985 OS=Staphylococcus aureus (strain N315) GN=SA1985 PE=4 SV=1 Q7A494|Q7A494_ STAAN No Values 768 Putative uncharacterized protein SA1018 OS=Staphylococcus aureus (strain N315) GN=SA1018 PE=4 SV=1 Q7A622|Q7A622_ STAAN No Values 772 Serine-aspartate repeat-containing protein E OS=Staphylococcus aureus (strain N315) GN=sdrE PE=1 SV=1 Q99W46|SDRE_ST AAN No Values 773 Peptidase T OS=Staphylococcus aureus (strain N315) GN=pepT PE=3 SV=1 P65806|PEPT_STA AN No Values 775 DNA topoisomerase 3 OS=Staphylococcus aureus (strain N315) GN=topB PE=3 SV=1 Q7A455|TOP3_ST AAN No Values 776 Ferredoxin--NADP reductase OS=Staphylococcus aureus (strain N315) GN=SA2162 PE=1 SV=2 Q7A3W1|FENR_S TAAN No Values 777 SA0849 protein OS=Staphylococcus aure us (strain N315) GN=SA0849 PE=4 SV=1 Q7A6F2|Q7A6F2_ STAAN No Values 779 SA2194 protein OS=Staphylococcus aure us (strain N315) GN=SA2194 PE=3 SV=1 Q7A3T2|RANDO M_Q7A3T2_STAA N-R No Values 780 Putative uncharacterized protein SA0673 OS=Staphylococcus aureus (strain N315) GN=SA0673 PE=4 SV=1 Q7A6U2|Q7A6U2_ STAAN No Values 781 Guanylate kinase OS=Staphylococcus aureus (strain N315) GN=gmk PE=1 SV=1 P99176|KGUA_ST AAN No Values 138

PAGE 150

782 Glycerol uptake facilitator OS=Staphylococcu s aureus (strain N315) GN=glpF PE=3 SV=1 Q7A5V8|Q7A5V8_ STAAN No Values 783 Putative uncharacterized protein SA0960 OS=Staphylococcus aureus (strain N315) GN=SA0960 PE=4 SV=1 Q7A669|Q7A669_ STAAN No Values 784 Putative uncharacterized protein SA1742 OS=Staphylococcus aureus (strain N315) GN=SA1742 PE=4 SV=1 Q7A4N8|Q7A4N8_ STAAN No Values 786 MutS2 protein OS=Staphylococcus aureus (strain N315) GN=mutS2 PE=1 SV=1 P65496|MUTS2_S TAAN No Values 787 SA2324 protein OS=Staphylococcus aure us (strain N315) GN=SA2324 PE=4 SV=1 Q7A3G6|Q7A3G6_ STAAN No Values 788 SA1562 protein OS=Staphylococcus aure us (strain N315) GN=SA1562 PE=4 SV=1 Q7A530|Q7A530_ STAAN No Values 789 2-succinyl-5-enolpyruvyl-6-hydroxy-3-c yclohexene-1-carboxylate synthase OS=Staphylococcus aureus (str ain N315) GN=menD PE=3 SV=1 Q7A6B1|MEND_S TAAN No Values 790 Sensor protein kinase walK OS=Staphyloco ccus aureus (strain N315) GN=walK PE=1 SV=1 Q7A8E0|WALK_S TAAN No Values 792 Thymidylate synthase OS=Staphylococcus aureus (strain N315) GN=thyA PE=3 SV=1 P67047|TYSY_ST AAN No Values 793 UPF0124 protein SA1030 OS=Staphylococcus aureus (strain N315) GN=SA1030 PE=3 SV=1 Q7A617|Y1030_ST AAN No Values 794 DNA polymerase III gamma and tau subunits OS=Staphylococcus aureus (strain N315) GN=dnaX PE=4 SV=1 Q7A7C7|Q7A7C7_ STAAN No Values 795 Dehydrosqualene desaturase OS=Staphyloco ccus aureus (strain N315) GN=crtN PE=1 SV=1 Q7A3E2|CRTN_ST AAN No Values 796 Pantothenate synthetase OS=Staphylococcu s aureus (strain N315) GN=panC PE=1 SV=1 P65659|PANC_ST AAN No Values 354 Putative uncharacterized protein SA0771 OS=Staphylococcus aureus (strain N315) GN=SA0771 PE=4 SV=1 Q7A6M0|Q7A6M0 _STAAN Reference Missing 569 SA2422 protein OS=Staphylococcus aure us (strain N315) GN=SA2422 PE=4 SV=1 Q7A383|Q7A383_ STAAN Reference Missing 662 Primosomal protein OS=Staphylococcus aureus (strain N315) GN=dnaI PE=4 SV=1 Q7A568|Q7A568_ STAAN Reference Missing 13 Cold shock protein (cspA) Q7A5P3|CSPA_ST AAN Value Missing 61 Glucosamine--fructose-6-phosphate am inotransferase [isomerizing] (glmS) P64228|GLMS_ST AAN Value Missing 70 Uncharacterized leukocid in-like protein 1 (SA1812) Q7A4L0|LUKL1_S TAAN Value Missing Value Missing 131 Immunoglobulin G-binding protein A OS=Staphy lococcus aureus (strain N315) GN=spa PE=1 SV=1 SPA_STAAN 150 Phosphoribosylformylglycinamidine cycloligase OS=Staphylococcus aureus (strain N315) GN=purM PE=1 SV=1 P99163|PUR5_STA AN Value Missing 173 Alcohol dehydrogenase OS=Staphylococcus aureus (strain N315) GN=adh PE=1 SV=1 Q7A742|ADH_ST AAN Value Missing 184 Pyruvate carboxylase OS=Staphylococcus aureus (strain N315) GN=pycA PE=4 SV=1 Q7A666|Q7A666_ STAAN Value Missing 201 Threonine dehydratase catabolic OS=Staphylococcus aureus (strain N315) GN=tdcB PE=1 SV=1 Q7A5L8|THD2_ST AAN Value Missing 211 Alpha-Hemolysin OS=Staphylococcus au reus (strain N315) GN=SA1007 PE=4 SV=1 Q7A632|Q7A632_ STAAN Value Missing 226 SA0231 protein OS=Staphylococcus aure us (strain N315) GN=SA0231 PE=1 SV=1 Q7A7W3|Q7A7W3 _STAAN Value Missing 231 Probable quinol oxidase subunit 1 OS=Staphylococcus aureus (strain N315) GN=qoxB PE=1 SV=1 Q7A699|QOX1_ST AAN Value Missing 234 UDP-N-acetylglucosamine--N-acetylmuramyl-(pentapeptide) pyrophosphorylundecaprenol N-acetylglucosamine transferase OS=Staphylococcus aureus (strain N315) GN=murG PE=1 SV=1 P65482|MURG_ST AAN Value Missing 243 Poly D-alanine transfer protein OS=Staphylococcus aureus (strain N315) GN=dltD PE=4 SV=1 Q7A6K0|Q7A6K0_ STAAN Value Missing 139

PAGE 151

244 Transcriptional regulatory protein walR OS=Staphylococcus aureus (strain N315) GN=walR PE=1 SV=2 Q7A8E1|WALR_S TAAN Value Missing 256 SA0759 protein OS=Staphylococcus aure us (strain N315) GN=SA0759 PE=4 SV=1 Q7A6M6|Q7A6M6 _STAAN Value Missing 260 SA1000 protein OS=Staphylococcus aure us (strain N315) GN=SA1000 PE=4 SV=1 Q7A639|Q7A639_ STAAN Value Missing 269 Transcription termination factor Rho OS=S taphylococcus aureus (strain N315) GN=rho PE=4 SV=1 Q99SD7|Q99SD7_ STAAN Value Missing 273 3-hydroxy-3-methylglutaryl CoA synthase OS=Staphylococcus aureus (strain N315) GN=mvaS PE=1 SV=1 Q7A3F6|Q7A3F6_ STAAN Value Missing 278 Accessory gene regulator protein A OS=Sta phylococcus aureus (strain N315) GN=agrA PE=1 SV=1 P0A0I5|AGRA_ST AAN Value Missing 296 Putative uncharacterized protein SA0663 OS=Staphylococcus aureus (strain N315) GN=SA0663 PE=4 SV=1 Q7A6V1|Q7A6V1_ STAAN Value Missing 323 SA0969 protein OS=Staphylococcus aure us (strain N315) GN=SA0969 PE=4 SV=1 Q7A661|Q7A661_ STAAN Value Missing 330 UDP-N-acetylenolpyruvoylglucosamine reduc tase OS=Staphylococcus aureus (strain N315) GN=murB PE=1 SV=1 P65463|MURB_ST AAN Value Missing 331 Aminoacyltransferase femX OS=Staphyloco ccus aureus (strain N315) GN=femX PE=1 SV=1 Q7A447|FEMX_ST AAN Value Missing 336 Dihydroorotase OS=Staphylococcus aureus (strain N315) GN=pyrC PE=1 SV=1 P65906|PYRC_ST AAN Value Missing 337 Chromosomal replication initiator protein dnaA OS=Staphylococcus aureus (strain N315) GN=dnaA PE=1 SV=1 P68866|DNAA_ST AAN Value Missing 339 Putative uncharacterized protein SA1868 OS=Staphylococcus aureus (strain N315) GN=SA1868 PE=4 SV=1 Q7A4H1|Q7A4H1_ STAAN Value Missing 341 Putative uncharacterized protein SA0606 OS=Staphylococcus aureus (strain N315) GN=SA0606 PE=1 SV=1 Q7A700|Q7A700_ STAAN Value Missing 349 SA2375 protein OS=Staphylococcus aure us (strain N315) GN=SA2375 PE=3 SV=1 Q7A3B6|Q7A3B6_ STAAN Value Missing Value Missing 357 Nucleoside diphosphate kinase OS=Staphylo coccus aureus (strain N315) GN=ndk PE=1 SV=1 NDK_STAAN 370 Putative uncharacterized protein SA0517 OS=Staphylococcus aureus (strain N315) GN=SA0517 PE=1 SV=1 Q7A783|Q7A783_ STAAN Value Missing 371 Probable acetyl-CoA acyltransferase OS=Staphylococcus aureus (strain N315) GN=SA0342 PE=1 SV=1 Q7A7L2|THLA_ST AAN Value Missing 372 Ferrochelatase OS=Staphylococcus aureus (strain N315) GN=hemH PE=1 SV=1 P64125|HEMH_ST AAN Value Missing 376 Alpha-acetolactate synthase OS=Staphylococcu s aureus (strain N315) GN=alsS PE=4 SV=1 Q7A481|Q7A481_ STAAN Value Missing 389 Protein fmtA OS=Staphylococcus aureus (strain N315) GN=fmtA PE=1 SV=1 Q7A6A2|FMTA_S TAAN Value Missing 409 Phosphopentomutase OS=Staphylococcus au reus (strain N315) GN=deoB PE=1 SV=1 P99100|DEOB_ST AAN Value Missing 410 SigmaB regulation protein RsbU OS=Staphylococcus aureus (strain N315) GN=rsbU PE=4 SV=1 Q7A4H0|Q7A4H0_ STAAN Value Missing 417 Xaa-Pro dipeptidase OS=Staphylococcus aureus (strain N315) GN=SA1360 PE=3 SV=1 Q99TW4|Q99TW4 _STAAN Value Missing 423 ATP-dependent Clp protease proteolytic subunit OS=Staphylococcus aureus (strain N315) GN=clpP PE=1 SV=1 P99089|CLPP_STA AN Value Missing 426 SA1626 protein OS=Staphylococcus aure us (strain N315) GN=SA1626 PE=4 SV=1 Q7A4Y5|Q7A4Y5_ STAAN (+1) Value Missing 427 Ribonuclease J 1 OS=Staphylococcus aureus (strain N315) GN=SA0940 PE=1 SV=1 Q7A682|RNJ1_ST AAN Value Missing 429 D-3-phosphoglycerate dehydrogenase OS=Staphylococcus aureus (strain N315) GN=serA PE=4 SV=1 Q7A542|Q7A542_ STAAN Value Missing 431 Phosphoribosylglycinamide formyltransferas e OS=Staphylococcus aureus (strain N315) GN=purN PE=1 SV=1 P99162|PUR3_STA AN Value Missing 438 Ornithine carbamoyltransferase OS=Staphylococcus aureus (strain N315) GN=argF P99073|OTC_STA Value 140

PAGE 152

PE=1 SV=1 AN Missing 439 Alanine dehydrogenase 1 OS=Staphylococcu s aureus (strain N315) GN=ald1 PE=1 SV=1 P99151|DHA1_ST AAN Value Missing 445 Peptide chain release factor 3 OS=Staphylo coccus aureus (strain N315) GN=prfC PE=1 SV=1 Q99V72|RF3_STA AN Value Missing 451 DNA repair protein OS=Staphylococcus au reus (strain N315) GN=recN PE=4 SV=1 Q7A5F5|Q7A5F5_ STAAN Value Missing 452 HTH-type transcriptional regulator rot OS =Staphylococcus aureus (strain N315) GN=rot PE=1 SV=2 Q7A514|ROT_STA AN Value Missing 458 Glycine betaine/carnitine/choline ABC tr ansporter opuCA OS=Staphylococcus aureus (strain N315) GN=opuCA PE=4 SV=1 Q7A3P8|Q7A3P8_ STAAN Value Missing 461 Putative uncharacterized protein SA1161 OS=Staphylococcus aureus (strain N315) GN=SA1161 PE=4 SV=1 Q7A5T9|Q7A5T9_ STAAN Value Missing 463 SA2297 protein OS=Staphylococcus aure us (strain N315) GN=SA2297 PE=4 SV=1 Q7A3J1_STAAN (+1) Value Missing 472 tRNA pseudouridine synthase B OS=Staphylococcus aureus (strain N315) GN=truB PE=1 SV=1 P65855|TRUB_ST AAN Value Missing 474 Alkaline phosphatase synthesis transcriptional regulatory protein OS=Staphylococcus aureus (strain N315) GN=phoP PE=4 SV=1 Q7A562|Q7A562_ STAAN Value Missing 484 Putative uncharacterized protein SA1364 OS=Staphylococcus aureus (strain N315) GN=SA1364 PE=4 SV=1 Q7A5E7|Q7A5E7_ STAAN Value Missing 487 SA2156 protein OS=Staphylococcus aure us (strain N315) GN=SA2156 PE=4 SV=1 Q7A3W7|Q7A3W7 _STAAN Value Missing 494 HPr kinase/phosphorylase OS=Staphyloco ccus aureus (strain N315) GN=hprK PE=1 SV=1 P60701|HPRK_ST AAN Value Missing 507 DNA topoisomerase 4 subunit B OS=Staphylococcus aureus (strain N315) GN=parE PE=1 SV=1 P66939|PARE_ST AAN Value Missing 512 Arginine deiminase OS=Staphylococcus au reus (strain N315) GN=arcA PE=1 SV=1 P63554|ARCA_ST AAN Value Missing 515 Histidine protein kinase saeS OS=Staphylo coccus aureus (strain N315) GN=saeS PE=1 SV=1 Q7A6V4|SAES_ST AAN Value Missing 520 Accessory gene regulator C OS=Staphyloco ccus aureus (strain N315) GN=agrC PE=4 SV=1 Q7A4I6|Q7A4I6_S TAAN Value Missing 521 Putative uncharacterized protein SA1423 OS=Staphylococcus aureus (strain N315) GN=SA1423 PE=4 SV=1 Q7A5B3|Q7A5B3_ STAAN Value Missing 524 Glutamine amidotransferase subunit pdxT OS=Staphylococcus aureus (strain N315) GN=pdxT PE=1 SV=1 Q7A7A1|PDXT_S TAAN Value Missing 530 SA1708 protein OS=Staphylococcus aure us (strain N315) GN=SA1708 PE=4 SV=1 Q7A4R3|Q7A4R3_ STAAN Value Missing 534 Protoporphyrinogen oxidase OS=Staphylococcus aureus (strain N315) GN=hemY PE=4 SV=1 Q7A4W4|Q7A4W4 _STAAN Value Missing Value Missing 544 Putative uncharacterized protein SA1662 OS=Staphylococcus aureus (strain N315) GN=SA1662 PE=4 SV=1 Q7A4V4_STAAN 549 Phenylalanyl-tRNA synthetase beta chai n OS=Staphylococcus aureus (strain N315) GN=pheT PE=1 SV=1 P67041|SYFB_ST AAN Value Missing 551 SA0791 protein OS=Staphylococcus aure us (strain N315) GN=SA0791 PE=4 SV=1 Q7A6K3|Q7A6K3_ STAAN Value Missing 559 SA2278 protein OS=Staphylococcus aure us (strain N315) GN=SA2278 PE=4 SV=1 Q7A3K8|Q7A3K8_ STAAN Value Missing 562 Cmp-binding-factor 1 OS=Staphylococcus aureus (strain N315) GN=cbf1 PE=4 SV=1 Q7A4V6|Q7A4V6_ STAAN Value Missing 564 UPF0316 protein SA1727 OS=Staphylococcus aureus (strain N315) GN=SA1727 PE=1 SV=1 P61544|Y1727_ST AAN Value Missing 565 Putative uncharacterized protein SA1426 OS=Staphylococcus aureus (strain N315) GN=SA1426 PE=4 SV=1 Q7A5B1|Q7A5B1_ STAAN Value Missing 572 Penicillin binding protein 4 OS=Staphyloco ccus aureus (strain N315) GN=pbp4 PE=3 SV=1 Q7A709|Q7A709_ STAAN Value Missing 574 Putative uncharacterized protein SA0513 OS=Staphylococcus aureus (strain N315) GN=SA0513 PE=4 SV=1 Q7A787|Q7A787_ STAAN Value Missing 141

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581 tRNA-specific 2-thiouridylase mnmA OS=Staphylococcus aureus (strain N315) GN=mnmA PE=1 SV=1 Q99TM8|MNMA_ STAAN Value Missing 586 Thymidylate kinase OS=Staphylococcus aureus (strain N315) GN=tmk PE=3 SV=1 P65249|KTHY_ST AAN Value Missing 587 GTP-binding protein lepA OS=Staphylococcu s aureus (strain N315) GN=lepA PE=1 SV=1 P65272|LEPA_ST AAN Value Missing 588 DNA mismatch repair protein mutL OS=Staphylococcus aureus (strain N315) GN=mutL PE=1 SV=1 P65492|MUTL_ST AAN Value Missing 589 Aspartate carbamoyltransferase OS=Staphylococcus aureus (strain N315) GN=pyrB PE=1 SV=1 P65618|PYRB_ST AAN Value Missing 592 Penicillin-binding protein 3 OS=Staphyloco ccus aureus (strain N315) GN=pbp3 PE=4 SV=1 Q7A5D5|Q7A5D5_ STAAN Value Missing 595 SA0357 protein OS=Staphylococcus aure us (strain N315) GN=SA0357 PE=4 SV=1 Q7A7K0|Q7A7K0_ STAAN Value Missing 604 Fibrinogen-binding protein OS=Staphyloco ccus aureus (strain N315) GN=fib PE=1 SV=1 P68800|FIB_STAA N Value Missing 605 4,4'-diaponeurosporene oxida se OS=Staphylococcus aureus (strain N315) GN=crtP PE=1 SV=1 Q7A3D9|CRTP_ST AAN Value Missing 606 SA2243 protein OS=Staphylococcus aure us (strain N315) GN=SA2243 PE=4 SV=1 Q7A3P3_STAAN (+1) Value Missing 607 SA2103 protein OS=Staphylococcus aure us (strain N315) GN=SA2103 PE=4 SV=1 Q7A413|Q7A413_ STAAN Value Missing 614 Membrane protein oxaA OS=Staphylococcus aureus (strain N315) GN=oxaA PE=1 SV=1 P65629|OXAA_ST AAN Value Missing 615 SA1329 protein OS=Staphylococcus aure us (strain N315) GN=SA1329 PE=4 SV=1 Q7A5H3|Q7A5H3_ STAAN Value Missing 616 Competence-damage inducible protein cinA OS=Staphylococcus aureus (strain N315) GN=cinA PE=4 SV=1 Q7A5W9|Q7A5W9 _STAAN Value Missing 622 Putative uncharacterized protein SA0449 OS=Staphylococcus aureus (strain N315) GN=SA0449 PE=4 SV=1 Q7A7C0|Q7A7C0_ STAAN Value Missing 634 OppD protein OS=Staphylococcus aure us (strain N315) GN=oppD PE=4 SV=1 Q7A6F4|Q7A6F4_ STAAN Value Missing 635 Putative uncharacterized protein SA0832 OS=Staphylococcus aureus (strain N315) GN=SA0832 PE=4 SV=1 Q7A6H0|Q7A6H0_ STAAN Value Missing 637 Transcriptional regulator ctsR OS=Staphylo coccus aureus (strain N315) GN=ctsR PE=1 SV=1 Q7A799|CTSR_ST AAN Value Missing 648 Ribose-5-phosphate isomerase A OS=Staphylococcus aureus (strain N315) GN=rpiA PE=3 SV=1 P66695|RPIA_STA AN Value Missing 649 Putative uncharacterized protein SA2090 OS=Staphylococcus aureus (strain N315) GN=SA2090 PE=4 SV=1 Q7A424|Q7A424_ STAAN Value Missing 656 SA2132 protein OS=Staphylococcus aure us (strain N315) GN=SA2132 PE=4 SV=1 Q99RT2|Q99RT2_ STAAN Value Missing 661 2-dehydropantoate 2-reducta se OS=Staphylococcus aureus (strain N315) GN=SA2232 PE=3 SV=1 Q7A3Q3|Q7A3Q3_ STAAN Value Missing 665 Putative uncharacterized protein SA0184 OS=Staphylococcus aureus (strain N315) GN=SA0184 PE=4 SV=1 Q7A806|Q7A806_ STAAN Value Missing 666 Gamma-hemolysin component B OS=Staphylococcus aureus (strain N315) GN=hlgB PE=1 SV=1 P0A075|HLGB_ST AAN Value Missing 670 Mannose-6-phosphate isomerase OS=Staphylococcus aureus (strain N315) GN=pmi PE=3 SV=1 Q7A373|Q7A373_ STAAN Value Missing 674 SA0566 protein OS=Staphylococcus aure us (strain N315) GN=SA0566 PE=4 SV=1 Q7A739|Q7A739_ STAAN Value Missing 675 Putative uncharacterized protein SA0561 OS=Staphylococcus aureus (strain N315) GN=SA0561 PE=4 SV=1 Q7A743|Q7A743_ STAAN Value Missing 679 Putative uncharacterized protein SA0954 OS=Staphylococcus aureus (strain N315) GN=SA0954 PE=4 SV=1 Q7A675|Q7A675_ STAAN Value Missing 682 Peptide chain release factor 1 OS=Staphylo coccus aureus (strain N315) GN=prfA PE=3 SV=1 P66019|RF1_STAA N Value Missing 685 Putative uncharacterized protein SA0739 OS=Staphy lococcus aureus (strain N 315) Q7A6P8|Q7A6P8_ Value 142

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GN=SA0739 PE=4 SV=1 STAAN Missing Putative zinc metalloprotease SA1105 OS=S taphylococcus aureus (strain N315) GN=SA1105 PE=1 SV=1 P63333|Y1105_ST AAN Value Missing 686 Putative uncharacterized protein SA1942 OS=Staphylococcus aureus (strain N315) GN=SA1942 PE=4 SV=1 Q7A4C7|Q7A4C7_ STAAN Value Missing 700 SA0958 protein OS=Staphylococcus aure us (strain N315) GN=SA0958 PE=4 SV=1 Q7A673|Q7A673_ STAAN Value Missing 703 SA0734 protein OS=Staphylococcus aure us (strain N315) GN=SA0734 PE=4 SV=1 Q7A6Q0|Q7A6Q0_ STAAN Value Missing 704 Putative uncharacterized protein SA0296 OS=Staphylococcus aureus (strain N315) GN=SA0296 PE=4 SV=1 Q7A7Q1|Q7A7Q1_ STAAN Value Missing 706 Undecaprenyl-diphosphatase OS=Staphylococcu s aureus (strain N315) GN=uppP PE=3 SV=1 P67391|UPPP_STA AN Value Missing 720 Carbamate kinase 2 OS=S taphylococcus aureus (strain N315) GN=arcC2 PE=1 SV=1 P99069|ARCC2_S TAAN Value Missing 722 SA0895 protein OS=Staphylococcus aure us (strain N315) GN=SA0895 PE=4 SV=1 Q7A6B2|Q7A6B2_ STAAN Value Missing 728 Urease accessory protein ureG OS=Staphylo coccus aureus (strain N315) GN=ureG PE=1 SV=1 Q7A427|UREG_ST AAN Value Missing 740 Orotate phosphoribosyltransferase OS=Staphylococcus aureus (strain N315) GN=pyrE PE=1 SV=1 PYRE_STAAN (+1) Value Missing 747 Sulfite reductase flavoprotein OS=Staphy lococcus aureus (strain N315) GN=SA2413 PE=3 SV=1 Q7A392|Q7A392_ STAAN Value Missing 754 SA1699 protein OS=Staphylococcus aure us (strain N315) GN=SA1699 PE=4 SV=1 Q99SZ8|Q99SZ8_S TAAN Value Missing 755 Putative uncharacterized protein SA1672 OS=Staphylococcus aureus (strain N315) GN=SA1672 PE=4 SV=1 Q7A4U4|Q7A4U4_ STAAN Value Missing 759 Putative uncharacterized protein SA0601 OS=Staphylococcus aureus (strain N315) GN=SA0601 PE=3 SV=1 Q7A705|Q7A705_ STAAN Value Missing 770 NADPH-dependent oxidoreductase OS=Staphylo coccus aureus (strain N315) GN=nfrA PE=3 SV=1 Q7A7J0|NFRA_ST AAN Value Missing 771 Putative uncharacterized protein SA1526 OS=Staphylococcus aureus (strain N315) GN=SA1526 PE=4 SV=1 Q7A555|Q7A555_ STAAN Value Missing 774 Value Missing 30S ribosomal protein S5 OS=Staphylococcu s aureus (strain N315) GN=rpsE PE=1 SV=1 RS5_STAAN 778 791 Dihydrolipoyl dehydrogenase OS=Staphylo coccus aureus (strain N315) GN=SA1349 PE=3 SV=1 Q7A5F6|Q7A5F6_ STAAN Value Missing Table A12. Complete list of intracellular proteins identified from the USA 100 adaptive mutant at 15 hours. # Identified Proteins (791) Accession Number Fold Change from WT 47 Uncharacterized leukocid in-like protein 2 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1975 PE=3 SV=1 Q2FFA2|LUKL2_STAA3 -4.4 67 Uncharacterized leukocid in-like protein 1 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1974 PE=3 SV=1 Q2FFA3|LUKL1_STAA3 -4.4 429 Glycosyl transferase, group 1 family protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0939 PE=4 SV=1 Q2FI41|Q2FI41_STAA3 -4.4 428 Proline dehydrogenase OS=Staphylococcus aureus (strain USA300) GN=putA PE=4 SV=1 Q2FFX4|Q2FFX4_STAA3 -4.3 671 Hydrolase, alpha/beta hydrolase fold family OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0604 PE=4 SV=1 Q2FJ21|Q2FJ21_STAA3 -4.1 143

PAGE 155

338 Dihydroxyacetone kinase, DhaL s ubunit OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0637 PE=4 SV=1 Q2FIY8|Q2FIY8_STAA3 -4 521 Accessory gene regulator protei n C OS=Staphylococcus aureus (strain USA300) GN=agrC PE=4 SV=1 Q2FF86|Q2FF86_STAA3 -3.8 205 PTS system glucose-specific EIICBA component OS=Staphylococcus aureus (strain USA300) GN=ptsG PE=3 SV=1 Q2FK73|PTG3C_STAA3 -3.6 436 Alanine dehydrogenase 1 OS=Staphylococcus aureus (strain USA300) GN=ald1 PE=3 SV=1 Q2FH00|DHA1_STAA3 -3.6 552 UPF0316 protein SAUSA300_1892 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1892 PE=3 SV=1 Q2FFI4|Y1892_STAA3 -3.6 587 Putative staphylococcal enterot oxin OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0370 PE=4 SV=1 Q2FJP4|Q2FJP4_STAA3 -3.5 31 Phosphoribosylformylglycinamidin e synthase 2 OS=Staphylococcus aureus (strain USA300) GN=purL PE=3 SV=1 Q2FI09|PURL_STAA3 -3.4 244 Uracil phosphoribosyltransferase OS=Staphylococcus aureus (strain USA300) GN=upp PE=3 SV=1 Q2FF16|UPP_STAA3 -3.4 579 GTP-binding protein lepA OS=Staphylococcus aureus (strain USA300) GN=lepA PE=3 SV=1 Q2FGD9|LEPA_STAA3 -3.4 269 Hydroxymethylglutaryl-CoA synthase OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2484 PE=4 SV=1 Q2FDW0|Q2FDW0_STAA3 -3.3 392 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2328 PE=4 SV=1 Q2FEB5|Q2FEB5_STAA3 -3.3 453 Glutamate-1-semialdehyde 2,1-am inomutase 2 OS=Staphylococcus aureus (strain USA300) GN=hemL2 PE=3 SV=1 Q2FFN1|GSA2_STAA3 -3.3 8 Formate acetyltransferase OS=S taphylococcus aureus (strain USA300) GN=pflB PE=3 SV=1 Q2FK44|PFLB_STAA3 -3.2 65 Probable quinol oxidase subunit 2 OS=Staphylococcus aureus (strain USA300) GN=qoxA PE=3 SV=1 Q2FI17|QOX2_STAA3 -3.2 305 ATP-dependent hsl protease ATP-binding subunit hslU OS=Staphylococcus aureus (strain USA300) GN=hslU PE=3 SV=1 Q2FHI4|HSLU_STAA3 -3.2 319 Accessory gene regulator protein A OS=Staphylococcus aureus (strain USA300) GN=agrA PE=4 SV=1 Q2FF85|Q2FF85_STAA3 -3.2 534 AcrB/AcrD/AcrF family protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2213 PE=4 SV=1 Q2FEN0|Q2FEN0_STAA3 -3.2 662 Uridine kinase OS=Staphylococcu s aureus (strain USA300) GN=udk PE=3 SV=1 Q2FGB5|URK_STAA3 -3.2 146 Phosphoribosylformylglycinamidine cyclo-ligase OS=Staphylococcus aureus (strain USA300) GN=purM PE=3 SV=1 Q2FI07|PUR5_STAA3 -3.1 278 Putative teichoic acid biosynthesis protein B OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0247 PE=4 SV=1 Q2FK17|Q2FK17_STAA3 -3.1 297 Putative lipoprotein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0693 PE=4 SV=1 Q2FIT2|Q2FIT2_STAA3 -3.1 144

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327 Aminoacyltransferase femX OS=S taphylococcus aureus (strain USA300) GN=femX PE=3 SV=1 Q2FEM9|FEMX_STAA3 -3.1 339 Chromosomal replication initiator protein dnaA OS=Staphylococcus aureus (strain USA300) GN=dnaA PE=3 SV=1 Q2FKQ5|DNAA_STAA3 -3.1 361 Hydrolase, haloacid dehalogenase -like family OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0544 PE=4 SV=1 Q2FJ81|Q2FJ81_STAA3 -3.1 411 Lipoyl synthase OS=Staphylococcus aureus (strain USA300) GN=lipA PE=3 SV=1 Q2FIE9|LIPA_STAA3 -3.1 61 Glucosamine--fructose-6-phosphate aminotransferase (Isomerizing) OS=Staphylococcus aureus (strain USA300) GN=glmS PE=3 SV=1 Q2FEX8|Q2FEX8_STAA3 -3 171 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1729 PE=4 SV=1 Q2FFV7|Q2FFV7_STAA3 -3 116 Phosphoribosylamine--glycine li gase OS=Staphylococcus aureus (strain USA300) GN=purD PE=3 SV=1 Q2FI04|Q2FI04_STAA3 -2.9 148 Pyridoxal biosynthesis lyase pdxS OS=Staphylococcus aureus (strain USA300) GN=pdxS PE=3 SV=1 Q2FJC1|PDXS_STAA3 -2.9 200 Glyceraldehyde-3-phosphat e dehydrogenase, type I OS=Staphylococcus aureus (strai n USA300) GN=gap PE=3 SV=1 Q2FG50|Q2FG50_STAA3 -2.9 333 ABC transporter ATP-binding protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0620 PE=4 SV=1 Q2FJ05|Q2FJ05_STAA3 -2.9 442 DNA repair protein RecN OS=Staphylococcus aureus (strain USA300) GN=recN PE=4 SV=1 Q2FGL4|Q2FGL4_STAA3 -2.9 228 Probable quinol oxidase subunit 1 OS=Staphylococcus aureus (strain USA300) GN=qoxB PE=3 SV=1 Q2FI18|QOX1_STAA3 -2.8 270 Alcohol dehydrogenase, iron-cont aining OS=Staphylococcus aureus (strain USA300) GN=adhE PE=4 SV=1 Q2FKB2|Q2FKB2_STAA3 -2.8 452 Oxidoreductase, aldo/keto reduc tase family OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0688 PE=4 SV=1 Q2FIT7|Q2FIT7_STAA3 -2.8 471 Protein-export membrane protein SecF OS=Staphylococcus aureus (strain USA300) GN=secF PE=4 SV=1 Q2FG90|Q2FG90_STAA3 -2.8 668 Chaperone protein dnaJ OS=Staphylococcus aureus (strain USA300) GN=dnaJ PE=3 SV=1 Q2FGE4|DNAJ_STAA3 -2.8 159 Phosphoribosylaminoimidazole -succinocarboxamide synthase OS=Staphylococcus aureus (strain USA300) GN=purC PE=3 SV=1 Q2FI12|PUR7_STAA3 -2.7 271 ABC transporter, ATP-binding protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2453 PE=4 SV=1 Q2FDZ1|Q2FDZ1_STAA3 -2.7 357 Ribonuclease J 2 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1168 PE=3 SV=2 Q2FHG3|RNJ2_STAA3 -2.7 225 Acetoin(Diacetyl) reductase OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0129 PE=3 SV=1 Q2FKD2|Q2FKD2_STAA3 -2.6 232 ATP synthase subunit delta OS=Staphylococcus aureus (strain USA300) GN=atpH PE=3 SV=1 Q2FF21|ATPD_STAA3 -2.6 145

PAGE 157

276 UPF0637 protein SAUSA300_1006 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1006 PE=3 SV=1 Q2FHX4|Y1006_STAA3 -2.6 341 Dihydroorotate dehydrogenase OS =Staphylococcus aureus (strain USA300) GN=pyrD PE=3 SV=1 Q2FDR8|Q2FDR8_STAA3 -2.6 359 Glycosyl transferase, group 2 family protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0252 PE=4 SV=1 Q2FK12|Q2FK12_STAA3 -2.6 383 Staphopain A OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1890 PE=4 SV=1 Q2FFI6|Q2FFI6_STAA3 -2.6 404 HTH-type transcriptional regulator sarR OS=Staphylococcus aureus (strain USA300) GN=sarR PE=3 SV=3 Q2FEJ8|SARR_STAA3 -2.6 752 Dihydroxyacetone kinase, DhaK subunit OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0636 PE=4 SV=1 Q2FIY9|Q2FIY9_STAA3 -2.6 759 RNA methyltransferase OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1805 PE=4 SV=1 Q2FFP0|Q2FFP0_STAA3 -2.6 788 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2378 PE=4 SV=1 Q2FE65|RANDOM_Q2FE65_STAA3R -2.6 15 Cold shock protein cspA OS=Staphylococcus aureus (strain USA300) GN=cspA PE=3 SV=1 Q2FH36|CSPA_STAA3 -2.5 35 50S ribosomal protein L5 OS=Staphylococcus aureus (strain USA300) GN=rplE PE=3 SV=1 Q2FEQ1|RL5_STAA3 -2.5 55 ATP synthase subunit alpha OS=Staphylococcus aureus (strain USA300) GN=atpA PE=3 SV=1 Q2FF22|ATPA_STAA3 -2.5 154 L-lactate dehydrogenase 2 OS=Staphylococcus aureus (strain USA300) GN=ldh2 PE=3 SV=1 Q2FDQ7|LDH2_STAA3 -2.5 235 UDP-N-acetylglucosamine--N-acet ylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acet ylglucosamine transferase OS=Staphylococcus aureus (strain USA300) GN=murG PE=3 SV=1 Q2FH20|MURG_STAA3 -2.5 330 Oxidoreductase, aldo/keto reduc tase family OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1450 PE=4 SV=1 Q2FGN2|Q2FGN2_STAA3 -2.5 387 Phosphomethylpyrimidine kinase OS=Staphylococcus aureus (strain USA300) GN=thiD PE=4 SV=1 Q2FJ63|Q2FJ63_STAA3 -2.5 426 Amidophosphoribosyltransferase OS=Staphylococcus aureus (strain USA300) GN=purF PE=4 SV=1 Q2FI08|Q2FI08_STAA3 -2.5 450 Cytidylate kinase OS=Staphyloco ccus aureus (strain USA300) GN=cmk PE=4 SV=1 Q2FGW4|Q2FGW4_STAA3 -2.5 516 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0278 PE=4 SV=1 Q2FJY6|Q2FJY6_STAA3 -2.5 582 Aspartate carbamoyltransferase OS =Staphylococcus aureus (strain USA300) GN=pyrB PE=3 SV=1 Q2FHN8|PYRB_STAA3 -2.5 603 Membrane protein oxaA OS=Staphylo coccus aureus (strain USA300) GN=oxaA PE=3 SV=1 Q2FF36|OXAA_STAA3 -2.5 32 ABC transporter, substrate-binding protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0618 PE=3 SV=1 Q2FJ07|Q2FJ07_STAA3 -2.4 146

PAGE 158

39 Carbamoyl-phosphate synthase large chain OS=Staphylococcus aureus (strain USA300) GN=carB PE=3 SV=1 Q2FHN5|CARB_STAA3 -2.4 196 Phosphoribosylaminoimidazole carboxylase, ATPase subunit OS=Staphylococcus aureus (strain USA300) GN=purK PE=4 SV=1 Q2FI13|Q2FI13_STAA3 -2.4 217 ATP synthase subunit b OS=Staphylococcus aureus (strain USA300) GN=atpF PE=3 SV=1 Q2FF20|ATPF_STAA3 -2.4 249 Bifunctional protein pyrR OS=Staphylococcus aureus (strain USA300) GN=pyrR PE=3 SV=1 Q2FHP0|PYRR_STAA3 -2.4 334 Dihydroorotase OS=Staphylococcus aureus (strain USA300) GN=pyrC PE=3 SV=1 Q2FHN7|Q2FHN7_STAA3 -2.4 440 Glycine betaine/carnitine/cholin e ABC transporter ATP-binding protein OS=Staphylococcus aureus (strain USA300) GN=opuCa PE=4 SV=1 Q2FE50|Q2FE50_STAA3 -2.4 486 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0331 PE=4 SV=1 Q2FJT3|Q2FJT3_STAA3 -2.4 496 Carbamate kinase 1 OS=Staphyloco ccus aureus (strain USA300) GN=arcC1 PE=3 SV=1 Q2FHR7|ARCC1_STAA3 -2.4 559 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1780 PE=4 SV=1 Q2FFR5|Q2FFR5_STAA3 -2.4 699 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1583 PE=4 SV=1 Q2FGA0|Q2FGA0_STAA3 -2.4 53 Protein translocase subunit secA 1 OS=Staphylococcus aureus (strain USA300) GN=secA1 PE=3 SV=1 Q2FIN8|SECA1_STAA3 -2.3 89 Bifunctional purine biosynthesis protein purH OS=Staphylococcus aureus (strain USA300) GN=purH PE=3 SV=1 Q2FI05|PUR9_STAA3 -2.3 138 Catabolite control protein A OS=Staphylococcus aureus (strain USA300) GN=ccpA PE=4 SV=1 Q2FG02|Q2FG02_STAA3 -2.3 188 Putative serine protease HtrA OS =Staphylococcus aureus (strain USA300) GN=SAUSA300_1674 PE=4 SV=1 Q2FG10|Q2FG10_STAA3 -2.3 354 Serine-protein kinase rsbW OS=Staphylococcus aureus (strain USA300) GN=rsbW PE=3 SV=1 Q2FF59|RSBW_STAA3 -2.3 399 Methionyl-tRNA formyltransferase OS=Staphylococcus aureus (strain USA300) GN=fmt PE=3 SV=1 Q2FHM2|FMT_STAA3 -2.3 696 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2093 PE=4 SV=1 Q2FEY9|Q2FEY9_STAA3 -2.3 71 Ribonucleoside-diphosphate reductase OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0716 PE=3 SV=1 Q2FIQ9|Q2FIQ9_STAA3 -2.2 336 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0982 PE=4 SV=1 Q2FHZ8|Q2FHZ8_STAA3 -2.2 390 Fmt protein OS=Staphylococcus aureus (strain USA300) GN=fmt PE=4 SV=1 Q2FI21|Q2FI21_STAA3 -2.2 523 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1554 PE=4 SV=1 Q2FGC9|Q2FGC9_STAA3 -2.2 147

PAGE 159

548 Pyrimidine nucleoside transport protein OS=Staphylococcus aureus (strain USA300) GN=nupC PE=4 SV=1 Q2FJB9|Q2FJB9_STAA3 -2.2 557 ABC transporter, permease pr otein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2358 PE=3 SV=1 Q2FE85|Q2FE85_STAA3 -2.2 621 tRNA modification GTPase mnmE OS=Staphylococcus aureus (strain USA300) GN=mnmE PE=3 SV=1 Q2FDE8|MNME_STAA3 -2.2 737 Heme-degrading monooxygenase isdI OS=Staphylococcus aureus (strain USA300) GN=isdI PE=3 SV=1 Q2FK96|ISDI_STAA3 -2.2 9 Penicillin-binding protein 2' OS=S taphylococcus aureus (strain USA300) GN=mecA PE=4 SV=1 Q2FKM6|Q2FKM6_STAA3 -2.1 19 ATP synthase subunit beta OS=Staphylococcus aureus (strain USA300) GN=atpD PE=3 SV=1 Q2FF24|ATPB_STAA3 -2.1 29 30S ribosomal protein S3 OS=Staphylococcus aureus (strain USA300) GN=rpsC PE=3 SV=1 Q2FEP5|RS3_STAA3 -2.1 136 2,3-bisphosphoglycerate-depende nt phosphoglycerate mutase OS=Staphylococcus aureus (strain USA300) GN=gpmA PE=3 SV=1 Q2FE81|GPMA_STAA3 -2.1 160 Alcohol dehydrogenase OS=Staphylococcus aureus (strain USA300) GN=adh PE=3 SV=1 Q2FJ31|ADH_STAA3 -2.1 223 Xanthine phosphoribosyltransferase OS=Staphylococcus aureus (strain USA300) GN=xpt PE=3 SV=1 Q2FJM8|XPT_STAA3 -2.1 230 Putative pyridoxal phosphate-dependent acyltransferase OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_0535 PE=3 SV=1 Q2FJ90|Q2FJ90_STAA3 -2.1 251 Putative arsenate reductase OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0790 PE=4 SV=1 Q2FII8|Q2FII8_STAA3 -2.1 282 50S ribosomal protein L20 OS=Staphylococcus aureus (strain USA300) GN=rplT PE=3 SV=1 Q2FG58|RL20_STAA3 -2.1 315 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2212 PE=4 SV=1 Q2FEN1|Q2FEN1_STAA3 -2.1 371 ATP-binding protein, Mrp/Nbp35 fa mily OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2125 PE=4 SV=1 Q2FEW6|Q2FEW6_STAA3 -2.1 403 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1223 PE=4 SV=1 Q2FHA8|Q2FHA8_STAA3 -2.1 412 Phosphopentomutase OS=Staphylococcus aureus (strain USA300) GN=deoB PE=3 SV=1 Q2FKC0|DEOB_STAA3 -2.1 504 Putative lipoprotein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2403 PE=4 SV=1 Q2FE40|Q2FE40_STAA3 -2.1 506 3-phosphoshikimate 1-carboxyvinylt ransferase OS=Staphylococcus aureus (strain USA300) GN=aroA PE=3 SV=1 Q2FGX6|AROA_STAA3 -2.1 508 Glycerol-3-phosphate de hydrogenase [NAD(P)+] OS=Staphylococcus aureus (strain USA300) GN=gpsA PE=3 SV=1 Q2FGW8|GPDA_STAA3 -2.1 729 Amino acid/peptide transporter (Peptide:H+ symporter) OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_0712 PE=3 SV=1 Q2FIR3|Q2FIR3_STAA3 -2.1 148

PAGE 160

11 Malate:quinone-oxidoreductase OS =Staphylococcus aureus (strain USA300) GN=mqo PE=3 SV=1 Q2FDQ3|Q2FDQ3_STAA3 -2 42 UPF0365 protein SAUSA300_1533 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1533 PE=3 SV=1 Q2FGF0|Y1533_STAA3 -2 46 Putative universal stress protein SAUSA300_1656 OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_1656 PE=3 SV=1 Q2FG28|Y1656_STAA3 -2 48 50S ribosomal protein L1 OS=Staphylococcus aureus (strain USA300) GN=rplA PE=3 SV=1 Q2FJA2|RL1_STAA3 -2 68 UPF0478 protein SAUSA300_1685 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1685 PE=3 SV=1 Q2FFZ9|Y1685_STAA3 -2 73 NADH dehydrogenase-like protein SAUSA300_0844 OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_0844 PE=3 SV=1 Q2FID4|Y844_STAA3 -2 179 GTPase obg OS=Staphylococcus aureus (strain USA300) GN=obg PE=3 SV=1 Q2FG83|OBG_STAA3 -2 209 Succinate dehydrogenase, iron-sulfur protein OS=Staphylococcus aureus (strain USA300) GN=sdhB PE=4 SV=1 Q2FHT2|Q2FHT2_STAA3 -2 262 Transcription termination factor Rho OS=Staphylococcus aureus (strain USA300) GN=rho PE=4 SV=1 Q2FF07|Q2FF07_STAA3 -2 285 Elastin-binding protein ebpS OS=Staphylococcus aureus (strain USA300) GN=ebpS PE=3 SV=3 Q2FGW1|EBPS_STAA3 -2 301 Trans-2-enoyl-ACP reductase OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0912 PE=4 SV=1 Q2FI66|Q2FI66_STAA3 -2 316 Putative AMP-binding enzyme OS=S taphylococcus aureus (strain USA300) GN=SAUSA300_2542 PE=4 SV=1 Q2FDQ2|Q2FDQ2_STAA3 -2 317 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2257 PE=4 SV=1 Q2FEI6|Q2FEI6_STAA3 -2 344 Glucose-6-phosphate 1-dehydrogenase OS=Staphylococcus aureus (strain USA300) GN=zwf PE=3 SV=1 Q2FGM8|Q2FGM8_STAA3 -2 437 Ornithine carbamoyltransferase OS =Staphylococcus aureus (strain USA300) GN=argF PE=3 SV=1 Q2FHR8|Q2FHR8_STAA3 -2 485 Hydrolase, MutT/nudix family OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2432 PE=4 SV=1 Q2FE12|Q2FE12_STAA3 -2 490 ATP-dependent DNA helicase, Pc rA OS=Staphylococcus aureus (strain USA300) GN=pcrA PE=4 SV=1 Q2FFJ0|Q2FFJ0_STAA3 -2 491 Mur ligase family protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1873 PE=4 SV=1 Q2FFK3|Q2FFK3_STAA3 -2 517 Histidine protein kinase saeS OS=Staphylococcus aureus (strain USA300) GN=saeS PE=3 SV=1 Q2FIT5|SAES_STAA3 -2 641 Putative lipoprotein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0769 PE=4 SV=1 Q2FIK9|Q2FIK9_STAA3 -2 670 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1118 PE=4 SV=1 Q2FHL3|Q2FHL3_STAA3 -2 149

PAGE 161

750 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1240 PE=4 SV=1 Q2FH91|Q2FH91_STAA3 -2 37 Triacylglycerol lipase OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0320 PE=4 SV=1 Q2FJU4|Q2FJU4_STAA3 -1.9 78 Isocitrate dehydrogenase [NADP] OS =Staphylococcus aureus (strain USA300) GN=icd PE=3 SV=1 Q2FG43|Q2FG43_STAA3 -1.9 91 Translation initiation factor IF-2 OS=Staphylococcus aureus (strain USA300) GN=infB PE=3 SV=1 Q2FHG9|IF2_STAA3 -1.9 94 Asparaginyl-tRNA synthetase OS =Staphylococcus aureus (strain USA300) GN=asnS PE=3 SV=1 Q2FGY6|SYN_STAA3 -1.9 103 Formate--tetrahydrofolate ligase OS=Staphylococcus aureus (strain USA300) GN=fhs PE=3 SV=1 Q2FG06|FTHS_STAA3 -1.9 109 50S ribosomal protein L3 OS=Staphylococcus aureus (strain USA300) GN=rplC PE=3 SV=1 Q2FEN9|RL3_STAA3 -1.9 130 Ribose-phosphate pyrophosphokinase OS=Staphylococcus aureus (strain USA300) GN=prs PE=3 SV=1 Q2FJE1|Q2FJE1_STAA3 -1.9 155 50S ribosomal protein L4 OS=Staphylococcus aureus (strain USA300) GN=rplD PE=3 SV=1 Q2FEP0|RL4_STAA3 -1.9 156 Uncharacterized protein SAUSA300_1119 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1119 PE=4 SV=1 Q2FHL2|Y1119_STAA3 -1.9 210 UPF0477 protein SAUSA300_0916 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0916 PE=3 SV=1 Q2FI62|Y916_STAA3 -1.9 264 Glutamate-1-semialdehyde 2,1-am inomutase 1 OS=Staphylococcus aureus (strain USA300) GN=hemL1 PE=3 SV=1 Q2FG69|GSA1_STAA3 -1.9 273 ATP synthase gamma chain OS=Staphylococcus aureus (strain USA300) GN=atpG PE=3 SV=1 Q2FF23|Q2FF23_STAA3 -1.9 300 Threonine dehydratase catabolic OS=Staphylococcus aureus (strain USA300) GN=tdcB PE=3 SV=1 Q2FH01|THD2_STAA3 -1.9 311 Putative phosphotransferase SAUSA300_1523 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1523 PE=3 SV=1 Q2FGG0|Y1523_STAA3 -1.9 355 DegV family protein OS=Staphylo coccus aureus (strain USA300) GN=SAUSA300_1318 PE=4 SV=1 Q2FH13|Q2FH13_STAA3 -1.9 388 Hypoxanthine phosphoribosyltransferase OS=Staphylococcus aureus (strain USA300) GN=hpt PE=4 SV=1 Q2FJD1|Q2FJD1_STAA3 -1.9 497 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1658 PE=4 SV=1 Q2FG26|Q2FG26_STAA3 -1.9 551 Ribokinase OS=Staphylococcus aureus (strain USA300) GN=rbsK PE=3 SV=1 Q2FK02|Q2FK02_STAA3 -1.9 567 tRNA-specific 2-thiouridylase mn mA OS=Staphylococcus aureus (strain USA300) GN=mnmA PE=3 SV=2 Q2FGA5|MNMA_STAA3 -1.9 666 Mannose-6-phosphate isomerase, class I OS=Staphylococcus aureus (strain USA300) GN=manA PE=3 SV=1 Q2FDL7|Q2FDL7_STAA3 -1.9 150

PAGE 162

697 Delta-hemolysin OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1988 PE=4 SV=1 Q2FF89|Q2FF89_STAA3 -1.9 751 Orotate phosphoribosyltransferase OS=Staphylococcus aureus (strain USA300) GN=pyrE PE=3 SV=1 Q2FHN3|Q2FHN3_STAA3 -1.9 70 30S ribosomal protein S11 OS=Staphylococcus aureus (strain USA300) GN=rpsK PE=3 SV=1 Q2FER4|RS11_STAA3 -1.8 81 DNA polymerase III, beta subunit OS=Staphylococcus aureus (strain USA300) GN=dnaN PE=3 SV=1 Q2FKQ4|Q2FKQ4_STAA3 -1.8 92 Succinate dehydrogenase, flavoprotein subunit OS=Staphylococcus aureus (strain USA300) GN=sdhA PE=4 SV=1 Q2FHT3|Q2FHT3_STAA3 -1.8 125 50S ribosomal protein L19 OS=Staphylococcus aureus (strain USA300) GN=rplS PE=3 SV=1 Q2FHJ7|RL19_STAA3 -1.8 167 ABC transporter, ATP-binding protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1285 PE=4 SV=1 Q2FH46|Q2FH46_STAA3 -1.8 204 Transcriptional regulator, MarR family OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0672 PE=4 SV=1 Q2FIV3|Q2FIV3_STAA3 -1.8 216 Oxidoreductase, short chain de hydrogenase/reductase family OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_2275 PE=4 SV=1 Q2FEG8|Q2FEG8_STAA3 -1.8 220 Signal recognition particle protein OS=Staphylococcus aureus (strain USA300) GN=ffh PE=4 SV=1 Q2FHK1|Q2FHK1_STAA3 -1.8 237 D-alanine-activating enzyme/D-ala nine-D-alanyl, dltD protein OS=Staphylococcus aureus (strain USA300) GN=dltD PE=4 SV=1 Q2FIE0|Q2FIE0_STAA3 -1.8 247 Extracellular matrix protein-bindi ng protein emp OS=Staphylococcus aureus (strain USA300) GN=emp PE=3 SV=1 Q2FIK4|EMP_STAA3 -1.8 292 Putative NADP-dependent malic en zyme OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1648 PE=3 SV=1 Q2FG36|Q2FG36_STAA3 -1.8 294 GTP-binding protein engA OS=Staphylococcus aureus (strain USA300) GN=engA PE=3 SV=1 Q2FGW7|ENGA_STAA3 -1.8 304 Urocanate hydratase OS=Staphylococcus aureus (strain USA300) GN=hutU PE=4 SV=1 Q2FEG5|Q2FEG5_STAA3 -1.8 306 UPF0051 protein SAUSA300_0822 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0822 PE=3 SV=1 Q2FIF6|Y822_STAA3 -1.8 340 DNA-directed RNA polymerase subunit omega OS=Staphylococcus aureus (strain USA300) GN=rpoZ PE=3 SV=1 Q2FHM8|RPOZ_STAA3 -1.8 367 Putative thioredoxin OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0789 PE=4 SV=1 Q2FII9|Q2FII9_STAA3 -1.8 568 Oligopeptide ABC transporter, ATP-binding protein OS=Staphylococcus aureus (strain USA300) GN=oppF PE=4 SV=1 Q2FI88|Q2FI88_STAA3 -1.8 585 Single-stranded DNA-binding protei n OS=Staphylococcus aureus (strain USA300) GN=ssb PE=4 SV=1 Q2FJP7|Q2FJP7_STAA3 -1.8 586 30S ribosomal protein S14 type Z OS=Staphylococcus aureus (strain USA300) GN=rpsZ PE=3 SV=2 Q2FEQ2|RS14Z_STAA3 -1.8 151

PAGE 163

590 Aminotransferase, class V OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1579 PE=3 SV=1 Q2FGA4|Q2FGA4_STAA3 -1.8 766 Threonine synthase OS=Staphylo coccus aureus (strain USA300) GN=thrC PE=3 SV=1 Q2FHA4|Q2FHA4_STAA3 -1.8 40 1-pyrroline-5-carboxylate dehydroge nase OS=Staphylococcus aureus (strain USA300) GN=rocA PE=3 SV=1 Q2FDV3|ROCA_STAA3 -1.7 62 50S ribosomal protein L2 OS=Staphylococcus aureus (strain USA300) GN=rplB PE=3 SV=1 Q2FEP2|RL2_STAA3 -1.7 104 Aerobic glycerol-3-phosphate de hydrogenase OS=Staphylococcus aureus (strain USA300) GN=glpD PE=3 SV=1 Q2FHD8|GLPD_STAA3 -1.7 105 Ornithine aminotransferase OS=Staphylococcus aureus (strain USA300) GN=rocD PE=3 SV=1 Q2FIB8|Q2FIB8_STAA3 -1.7 133 Prolyl-tRNA synthetase OS=Staphylococcus aureus (strain USA300) GN=proS PE=3 SV=1 Q2FHH5|SYP_STAA3 -1.7 137 30S ribosomal protein S12 OS=Staphylococcus aureus (strain USA300) GN=rpsL PE=3 SV=1 Q2FJ95|RS12_STAA3 -1.7 150 Phosphoglucosamine mutase OS=Staphylococcus aureus (strain USA300) GN=glmM PE=3 SV=1 Q2FEX1|GLMM_STAA3 -1.7 152 Protein recA OS=Staphylococcus aureus (strain USA300) GN=recA PE=3 SV=1 Q2FHF3|Q2FHF3_STAA3 -1.7 181 Adenylosuccinate synthetase OS =Staphylococcus aureus (strain USA300) GN=purA PE=3 SV=1 Q2FKN9|Q2FKN9_STAA3 -1.7 187 Translation initiation factor IF-1 OS=Staphylococcus aureus (strain USA300) GN=infA PE=3 SV=1 Q2FER1|IF1_STAA3 -1.7 221 Putative peptidyl-prolyl cis-tr ans isomerase OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0857 PE=3 SV=1 Q2FIC1|PPI1_STAA3 -1.7 255 DNA gyrase subunit B OS=Staphylococcus aureus (strain USA300) GN=gyrB PE=3 SV=3 Q2FKQ1|GYRB_STAA3 -1.7 256 ATP-dependent Clp protease ATP-binding subunit clpX OS=Staphylococcus aureus (strain USA300) GN=clpX PE=3 SV=1 Q2FG62|CLPX_STAA3 -1.7 261 Malate:quinone-oxidoreductase OS =Staphylococcus aureus (strain USA300) GN=mqo PE=3 SV=1 Q2FED1|Q2FED1_STAA3 -1.7 382 UDP-N-acetylmuramoyl-tripeptid e--D-alanyl-D-alanine ligase OS=Staphylococcus aureus (strain USA300) GN=murF PE=3 SV=1 Q2FF44|Q2FF44_STAA3 -1.7 384 2-oxoisovalerate dehydrogenase, E2 component, dihydrolipoamide acetyltransferase OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1464 PE=3 SV=1 Q2FGL8|Q2FGL8_STAA3 -1.7 435 Acetyl-coenzyme A synthetase OS =Staphylococcus aureus (strain USA300) GN=acsA PE=4 SV=1 Q2FG05|Q2FG05_STAA3 -1.7 482 Pyrroline-5-carboxylate reductase OS=Staphylococcus aureus (strain USA300) GN=proC PE=4 SV=1 Q2FGN0|Q2FGN0_STAA3 -1.7 519 Conserved virulence factor B OS=Staphylococcus aureus (strain USA300) GN=cvfB PE=3 SV=1 Q2FH47|CVFB_STAA3 -1.7 152

PAGE 164

560 Chromosome segregation protein SMC OS=Staphylococcus aureus (strain USA300) GN=smc PE=4 SV=1 Q2FHK4|Q2FHK4_STAA3 -1.7 574 Uncharacterized protein SAUSA300_1902 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1902 PE=3 SV=1 Q2FFH4|Y1902_STAA3 -1.7 625 2-oxoisovalerate dehydrogenase, E1 component, alpha subunit OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_1466 PE=4 SV=1 Q2FGL6|Q2FGL6_STAA3 -1.7 719 RNA methyltransferase, TrmH family, group 3 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0517 PE=4 SV=1 Q2FJA8|Q2FJA8_STAA3 -1.7 727 Na(+)/H(+) antiporter subunit E1 OS=Staphylococcus aureus (strain USA300) GN=mnhE1 PE=3 SV=1 Q2FIC7|MNHE1_STAA3 -1.7 731 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0657 PE=4 SV=1 Q2FIW8|Q2FIW8_STAA3 -1.7 14 Pyruvate kinase OS=Staphylococcus aureus (strain USA300) GN=pyk PE=3 SV=1 Q2FG40|KPYK_STAA3 -1.6 26 Fructose-bisphosphate aldolase class 1 OS=Staphylococcus aureus (strain USA300) GN=fda PE=3 SV=1 Q2FDQ4|ALF1_STAA3 -1.6 63 Acetate kinase OS=Staphyloco ccus aureus (strain USA300) GN=ackA PE=3 SV=1 Q2FG27|ACKA_STAA3 -1.6 88 Cell division protein ftsA OS=Staphylococcus aureus (strain USA300) GN=ftsA PE=4 SV=1 Q2FHQ2|Q2FHQ2_STAA3 -1.6 96 50S ribosomal protein L13 OS=Staphylococcus aureus (strain USA300) GN=rplM PE=3 SV=1 Q2FES1|RL13_STAA3 -1.6 101 50S ribosomal protein L10 OS=Staphylococcus aureus (strain USA300) GN=rplJ PE=3 SV=1 Q2FJA1|RL10_STAA3 -1.6 108 L-lactate dehydrogenase 1 OS=Staphylococcus aureus (strain USA300) GN=ldh1 PE=3 SV=2 Q2FK29|LDH1_STAA3 -1.6 131 30S ribosomal protein S13 OS=Staphylococcus aureus (strain USA300) GN=rpsM PE=3 SV=1 Q2FER3|RS13_STAA3 -1.6 142 50S ribosomal protein L14 OS=Staphylococcus aureus (strain USA300) GN=rplN PE=3 SV=1 Q2FEP9|RL14_STAA3 -1.6 168 Succinyl-CoA ligase [ADP-forming] subunit alpha OS=Staphylococcus aureus (strain USA300) GN=sucD PE=3 SV=1 Q2FHJ2|Q2FHJ2_STAA3 -1.6 184 GTP-binding protein OS=Staphylococcus aureus (strain USA300) GN=typA PE=4 SV=1 Q2FHX1|Q2FHX1_STAA3 -1.6 208 Citrate synthase II OS=Staphylococcus aureus (strain USA300) GN=gltA PE=3 SV=1 Q2FG42|Q2FG42_STAA3 -1.6 241 30S ribosomal protein S6 OS=Staphylococcus aureus (strain USA300) GN=rpsF PE=3 SV=1 Q2FJP8|RS6_STAA3 -1.6 274 30S ribosomal protein S4 OS=Staphylococcus aureus (strain USA300) GN=rpsD PE=3 SV=1 Q2FG18|RS4_STAA3 -1.6 281 D-alanine--D-alanine ligase OS=S taphylococcus aureus (strain USA300) GN=ddl PE=3 SV=1 Q2FF43|DDL_STAA3 -1.6 153

PAGE 165

307 UPF0082 protein SAUSA300_0655 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0655 PE=3 SV=1 Q2FIX0|Y655_STAA3 -1.6 308 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0649 PE=4 SV=1 Q2FIX6|Q2FIX6_STAA3 -1.6 342 GTP pyrophosphokinase OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1590 PE=3 SV=1 Q2FG93|Q2FG93_STAA3 -1.6 343 GTP-binding protein era homolog OS=Staphylococcus aureus (strain USA300) GN=era PE=3 SV=1 Q2FGF6|ERA_STAA3 -1.6 381 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2085 PE=4 SV=1 Q2FEZ7|Q2FEZ7_STAA3 -1.6 427 D-3-phosphoglycerate dehydrogenase OS=Staphylococcus aureus (strain USA300) GN=serA PE=3 SV=1 Q2FG14|Q2FG14_STAA3 -1.6 542 D-isomer specific 2-hydroxyacid dehydrogenase OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0834 PE=3 SV=1 Q2FIE4|Q2FIE4_STAA3 -1.6 558 Probable DNA-directed RNA polymerase subunit delta OS=Staphylococcus aureus (strain USA300) GN=rpoE PE=3 SV=1 Q2FF00|RPOE_STAA3 -1.6 644 PTS system glucoside-specific EIICBA component OS=Staphylococcus aureus (strain USA300) GN=glcB PE=3 SV=1 Q2FDW8|PTU3C_STAA3 -1.6 682 Putative membrane-associated zinc metalloprotease OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_1155 PE=3 SV=1 Q2FHH6|Q2FHH6_STAA3 -1.6 1 Elongation factor Tu OS=Staphylococcus aureus (strain USA300) GN=tuf PE=3 SV=1 Q2FJ92|EFTU_STAA3 -1.5 27 DNA-directed RNA polymerase subunit beta' OS=Staphylococcus aureus (strain USA300) GN=rpoC PE=3 SV=2 Q2FJ97|RPOC_STAA3 -1.5 30 Pyruvate dehydrogenase E1 component, beta subunit OS=Staphylococcus aureus (strain USA300) GN=pdhB PE=4 SV=1 Q2FHY6|Q2FHY6_STAA3 -1.5 54 30S ribosomal protein S5 OS=Staphylococcus aureus (strain USA300) GN=rpsE PE=3 SV=1 Q2FEQ6|RS5_STAA3 -1.5 58 5'-nucleotidase, lipoprotein e(P4) family OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0307 PE=4 SV=1 Q2FJV7|Q2FJV7_STAA3 -1.5 72 DNA-directed RNA polymerase subunit beta OS=Staphylococcus aureus (strain USA300) GN=rpoB PE=3 SV=2 Q2FJ98|RPOB_STAA3 -1.5 98 Phosphoenolpyruvate carboxykina se [ATP] OS=Staphylococcus aureus (strain USA300) GN=pckA PE=3 SV=1 Q2FFV5|PCKA_STAA3 -1.5 110 Threonyl-tRNA synthetase OS=Staphylococcus aureus (strain USA300) GN=thrS PE=3 SV=1 Q2FG54|SYT_STAA3 -1.5 114 Putative formate dehydrogenase SAUSA300_2258 OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_2258 PE=3 SV=1 Q2FEI5|FDHL_STAA3 -1.5 115 Septation ring formation regulator ezrA OS=Staphylococcus aureus (strain USA300) GN=ezrA PE=3 SV=1 Q2FG20|EZRA_STAA3 -1.5 118 Pyrimidine-nucleoside phosphorylase OS=Staphylococcus aureus (strain USA300) GN=pdp PE=3 SV=2 Q2FEZ3|PDP_STAA3 -1.5 154

PAGE 166

124 Succinyl-CoA ligase [ADP-forming] subunit beta OS=Staphylococcus aureus (strain USA300) GN=sucC PE=3 SV=1 Q2FHJ3|SUCC_STAA3 -1.5 161 Alkyl hydroperoxide reductase, subunit F OS=Staphylococcus aureus (strain USA300) GN=ahpF PE=3 SV=1 Q2FJN5|Q2FJN5_STAA3 -1.5 162 Putative dipeptidase SAUSA300_1697 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1697 PE=3 SV=1 Q2FFY7|PEPVL_STAA3 -1.5 164 GTP-binding protein YchF OS=Staphylococcus aureus (strain USA300) GN=ychF PE=4 SV=1 Q2FJQ0|Q2FJQ0_STAA3 -1.5 165 UPF0133 protein SAUSA300_0453 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0453 PE=3 SV=1 Q2FJG3|Y453_STAA3 -1.5 180 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1323 PE=4 SV=1 Q2FH08|Q2FH08_STAA3 -1.5 183 Pyruvate carboxylase OS=Staphylococcus aureus (strain USA300) GN=pyc PE=4 SV=1 Q2FHW6|Q2FHW6_STAA3 -1.5 252 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0385 PE=4 SV=1 Q2FJM9|Q2FJM9_STAA3 -1.5 254 Seryl-tRNA synthetase OS=Staphy lococcus aureus (strain USA300) GN=serS PE=3 SV=1 Q2FKP7|SYS_STAA3 -1.5 260 50S ribosomal protein L16 OS=Staphylococcus aureus (strain USA300) GN=rplP PE=3 SV=1 Q2FEP6|RL16_STAA3 -1.5 320 Staphylococcal respiratory response protein, SrrA OS=Staphylococcus aureus (strain USA300) GN=srrA PE=4 SV=1 Q2FGP0|Q2FGP0_STAA3 -1.5 321 Glycerophosphoryl diester phosphodi esterase family protein OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_1020 PE=4 SV=1 Q2FHW0|Q2FHW0_STAA3 -1.5 351 PTS system EIIBC component SAUSA300_0194 OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_0194 PE=3 SV=1 Q2FK70|PTXBC_STAA3 -1.5 377 Nucleoside diphosphate kinase OS =Staphylococcus aureus (strain USA300) GN=ndk PE=3 SV=2 Q2FGX3|NDK_STAA3 -1.5 400 Carbamoyl-phosphate synthase, small subunit OS=Staphylococcus aureus (strain USA300) GN=carA PE=3 SV=1 Q2FHN6|Q2FHN6_STAA3 -1.5 402 Phosphate starvation-induced protein, PhoH family OS=Staphylococcus aureus (strain USA300) GN=phoH PE=4 SV=1 Q2FGF2|Q2FGF2_STAA3 -1.5 409 Oxidoreductase, aldo/keto reduc tase family OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1728 PE=4 SV=1 Q2FFV8|Q2FFV8_STAA3 -1.5 413 10 kDa chaperonin OS=Staphylococcus aureus (strain USA300) GN=groS PE=3 SV=1 Q2FF94|CH10_STAA3 -1.5 425 Ribonuclease J 1 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0989 PE=3 SV=1 Q2FHZ1|RNJ1_STAA3 -1.5 441 (Dimethylallyl)adenosine tRNA methylthiotransferase miaB OS=Staphylococcus aureus (strain USA300) GN=miaB PE=3 SV=1 Q2FHE6|MIAB_STAA3 -1.5 565 HAD-superfamily hydrolase, subfamily IA, variant 1 OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_0540 PE=4 SV=1 Q2FJ85|Q2FJ85_STAA3 -1.5 155

PAGE 167

575 Uncharacterized lipoprotein S AUSA300_2315 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2315 PE=4 SV=1 Q2FEC8|Y2315_STAA3 -1.5 601 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0668 PE=4 SV=1 Q2FIV7|Q2FIV7_STAA3 -1.5 637 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1585 PE=4 SV=1 Q2FG98|Q2FG98_STAA3 -1.5 639 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1082 PE=3 SV=1 Q2FHP9|Q2FHP9_STAA3 -1.5 12 Inosine-5'-monophosphate dehydr ogenase OS=Staphylococcus aureus (strain USA300) GN=guaB PE=3 SV=1 Q2FJM6|IMDH_STAA3 -1.4 41 Aconitate hydratase OS=Staphylococcus aureus (strain USA300) GN=acnA PE=4 SV=1 Q2FH85|Q2FH85_STAA3 -1.4 50 Aspartyl/glutamyl-tRNA(Asn/Gln) amidotransferase subunit B OS=Staphylococcus aureus (strain USA300) GN=gatB PE=3 SV=1 Q2FFJ6|GATB_STAA3 -1.4 69 50S ribosomal protein L6 OS=Staphylococcus aureus (strain USA300) GN=rplF PE=3 SV=1 Q2FEQ4|RL6_STAA3 -1.4 74 Immunodominant staphylococcal antigen B OS=Staphylococcus aureus (strain USA300) GN=isaB PE=3 SV=1 Q2FDM1|ISAB_STAA3 -1.4 174 UPF0356 protein SAUSA300_0990 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0990 PE=3 SV=1 Q2FHZ0|Y990_STAA3 -1.4 191 Dehydrogenase family protein OS =Staphylococcus aureus (strain USA300) GN=SAUSA300_2251 PE=4 SV=1 Q2FEJ2|Q2FEJ2_STAA3 -1.4 234 Aspartyl-tRNA synthetase OS=Staphylococcus aureus (strain USA300) GN=aspS PE=3 SV=1 Q2FG97|SYD_STAA3 -1.4 246 Uridylate kinase OS=Staphylococcus aureus (strain USA300) GN=pyrH PE=3 SV=1 Q2FHI0|PYRH_STAA3 -1.4 259 Chaperone protein hchA OS=Staphy lococcus aureus (strain USA300) GN=hchA PE=3 SV=1 Q2FJ89|HCHA_STAA3 -1.4 272 CTP synthase OS=Staphylococcus aureus (strain USA300) GN=pyrG PE=3 SV=1 Q2FF01|PYRG_STAA3 -1.4 288 DNA gyrase subunit A OS=Staphylococcus aureus (strain USA300) GN=gyrA PE=3 SV=1 Q2FKQ0|GYRA_STAA3 -1.4 322 Uncharacterized epimerase/dehydratase SAUSA300_0538 OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_0538 PE=3 SV=1 Q2FJ87|Y538_STAA3 -1.4 360 30S ribosomal protein S17 OS=Staphylococcus aureus (strain USA300) GN=rpsQ PE=3 SV=1 Q2FEP8|RS17_STAA3 -1.4 373 Glycerol kinase OS=Staphyloco ccus aureus (strain USA300) GN=glpK PE=3 SV=1 Q2FHD9|GLPK_STAA3 -1.4 378 Lipoate-protein ligase A family protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0930 PE=4 SV=1 Q2FI50|Q2FI50_STAA3 -1.4 391 Cold shock protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0777 PE=3 SV=1 Q2FIK1|Q2FIK1_STAA3 -1.4 156

PAGE 168

420 Phosphopantothenoylcysteine d ecarboxylase/phosphopantothenate-cysteine ligase OS=Staphylococcus aureus (strain USA300) GN=coaBC PE=4 SV=1 Q2FHM7|Q2FHM7_STAA3 -1.4 472 Phosphate acyltransferase OS=Staphylococcus aureus (strain USA300) GN=plsX PE=3 SV=1 Q2FHK9|PLSX_STAA3 -1.4 473 ABC transporter, ATP-binding protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0630 PE=4 SV=1 Q2FIZ5|Q2FIZ5_STAA3 -1.4 475 Methicillin resistance protein FemA OS=Staphylococcus aureus (strain USA300) GN=femA PE=4 SV=1 Q2FH62|Q2FH62_STAA3 -1.4 505 Uncharacterized oxidoreductase SAUSA300_2422 OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_2422 PE=3 SV=1 Q2FE21|Y2422_STAA3 -1.4 515 Decarboxylase family protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0666 PE=4 SV=1 Q2FIV9|Q2FIV9_STAA3 -1.4 578 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1793 PE=4 SV=1 Q2FFQ2|Q2FFQ2_STAA3 -1.4 669 Menaquinone biosynthesis methyltransferase ubiE OS=Staphylococcus aureus (strain USA300) GN=ubiE PE=4 SV=1 Q2FGX1|Q2FGX1_STAA3 -1.4 687 UvrABC system protein B OS=S taphylococcus aureus (strain USA300) GN=uvrB PE=3 SV=1 Q2FIN5|Q2FIN5_STAA3 -1.4 25 30S ribosomal protein S2 OS=Staphylococcus aureus (strain USA300) GN=rpsB PE=3 SV=1 Q2FHI2|RS2_STAA3 -1.3 33 Immunoglobulin-binding protein sbi OS=Staphylococcus aureus (strain USA300) GN=sbi PE=3 SV=1 Q2FE79|SBI_STAA3 -1.3 49 Pyruvate dehydrogenase E1 component, alpha subunit OS=Staphylococcus aureus (strain USA300) GN=pdhA PE=4 SV=1 Q2FHY7|Q2FHY7_STAA3 -1.3 60 ATP-dependent Clp protease ATP-binding subunit clpL OS=Staphylococcus aureus (strain USA300) GN=clpL PE=3 SV=1 Q2FDV8|CLPL_STAA3 -1.3 95 30S ribosomal protein S7 OS=Staphylococcus aureus (strain USA300) GN=rpsG PE=3 SV=2 Q2FJ94|RS7_STAA3 -1.3 112 50S ribosomal protein L27 OS=Staphylococcus aureus (strain USA300) GN=rpmA PE=3 SV=1 Q2FG82|RL27_STAA3 -1.3 121 Bifunctional protein folD OS=S taphylococcus aureus (strain USA300) GN=folD PE=3 SV=1 Q2FI15|FOLD_STAA3 -1.3 126 50S ribosomal protein L22 OS=Staphylococcus aureus (strain USA300) GN=rplV PE=3 SV=1 Q2FEP4|RL22_STAA3 -1.3 140 Phosphate acetyltransferase OS=Staphylococcus aureus (strain USA300) GN=pta PE=4 SV=1 Q2FJ55|Q2FJ55_STAA3 -1.3 153 Transcription termination factor NusA OS=Staphylococcus aureus (strain USA300) GN=nusA PE=4 SV=1 Q2FHH2|Q2FHH2_STAA3 -1.3 186 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase OS=Staphylococcus aureus (strain USA300) GN=ispD PE=3 SV=1 Q2FK15|ISPD_STAA3 -1.3 195 Immunoglobulin G binding protein A OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0113 PE=3 SV=1 Q2FKE8|Q2FKE8_STAA3 -1.3 157

PAGE 169

243 30S ribosomal protein S10 OS=Staphylococcus aureus (strain USA300) GN=rpsJ PE=3 SV=1 Q2FEN8|RS10_STAA3 -1.3 332 UDP-N-acetylenolpyruvoylglucosamine reductase OS=Staphylococcus aureus (strain USA300) GN=murB PE=3 SV=1 Q2FIQ3|MURB_STAA3 -1.3 350 1-acyl-sn-glycerol-3-phosphate acy ltransferases OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1673 PE=4 SV=1 Q2FG11|Q2FG11_STAA3 -1.3 352 Phosphoribosylaminoimidazole carboxylase, catalytic subunit OS=Staphylococcus aureus (strain USA300) GN=purE PE=4 SV=1 Q2FI14|Q2FI14_STAA3 -1.3 379 Ribonucleoside-diphosphate reductase, beta subunit OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_0717 PE=4 SV=1 Q2FIQ8|Q2FIQ8_STAA3 -1.3 380 Phosphoribosylglycinamide formyltransferase OS=Staphylococcus aureus (strain USA300) GN=purN PE=4 SV=1 Q2FI06|Q2FI06_STAA3 -1.3 385 Methicillin resistance protein FemB OS=Staphylococcus aureus (strain USA300) GN=femB PE=4 SV=1 Q2FH61|Q2FH61_STAA3 -1.3 408 Sigma-B regulation protein OS=Staphylococcus aureus (strain USA300) GN=rsbU PE=4 SV=1 Q2FF57|Q2FF57_STAA3 -1.3 627 Uncharacterized protein SAUSA300_1248 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1248 PE=3 SV=1 Q2FH83|Y1248_STAA3 -1.3 658 50S ribosomal protein L9 OS=Staphylococcus aureus (strain USA300) GN=rplI PE=3 SV=1 Q2FKP1|RL9_STAA3 -1.3 660 2-dehydropantoate 2-re ductase OS=Staphylococcus aureus (strain USA300) GN=panE PE=3 SV=1 Q2FE55|Q2FE55_STAA3 -1.3 20 Enolase OS=Staphylococcus aureus (strain USA300) GN=eno PE=3 SV=1 Q2FIL7|ENO_STAA3 -1.2 122 ABC transporter, ATP-binding protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1911 PE=4 SV=1 Q2FFG5|Q2FFG5_STAA3 -1.2 123 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1804 PE=4 SV=1 Q2FFP1|Q2FFP1_STAA3 -1.2 139 NAD-specific glutamate dehydrogena se OS=Staphylococcus aureus (strain USA300) GN=gudB PE=3 SV=1 Q2FIB7|Q2FIB7_STAA3 -1.2 145 Isoleucyl-tRNA synthetase OS=Staphylococcus aureus (strain USA300) GN=ileS PE=3 SV=1 Q2FHP4|SYI_STAA3 -1.2 170 Probable DEAD-box ATP-dependent RNA helicase SAUSA300_2037 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2037 PE=3 SV=1 Q2FF45|Y2037_STAA3 -1.2 175 Adenylosuccinate lyase OS=Staphylococcus aureus (strain USA300) GN=purB PE=3 SV=1 Q2FFI7|PUR8_STAA3 -1.2 182 S-adenosylmethionine synthase OS=Staphylococcus aureus (strain USA300) GN=metK PE=3 SV=1 Q2FFV6|METK_STAA3 -1.2 194 2,3-bisphosphoglycerate-indepe ndent phosphoglycerate mutase OS=Staphylococcus aureus (strain USA300) GN=gpmI PE=3 SV=1 Q2FIL8|Q2FIL8_STAA3 -1.2 218 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1792 PE=4 SV=1 Q2FFQ3|Q2FFQ3_STAA3 -1.2 158

PAGE 170

233 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1909 PE=4 SV=1 Q2FFG7|Q2FFG7_STAA3 -1.2 267 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0664 PE=4 SV=1 Q2FIW1|Q2FIW1_STAA3 -1.2 298 30S ribosomal protein S9 OS=Staphylococcus aureus (strain USA300) GN=rpsI PE=3 SV=1 Q2FES2|RS9_STAA3 -1.2 329 DNA polymerase I OS=Staphylococcus aureus (strain USA300) GN=polA PE=3 SV=1 Q2FG47|Q2FG47_STAA3 -1.2 348 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1788 PE=4 SV=1 Q2FFQ7|Q2FFQ7_STAA3 -1.2 375 Putative oxidoreductase OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0329 PE=4 SV=1 Q2FJT5|Q2FJT5_STAA3 -1.2 456 Ornithine carbamoyltransferase OS =Staphylococcus aureus (strain USA300) GN=arcB PE=3 SV=1 Q2FDM5|Q2FDM5_STAA3 -1.2 469 UDP-N-acetylglucosamine 1-carboxyvinyltransferase OS=Staphylococcus aureus (strain USA300) GN=murA PE=3 SV=1 Q2FF04|Q2FF04_STAA3 -1.2 476 S-adenosyl-L-methionine-depende nt methyltransferase mraW OS=Staphylococcus aureus (strain USA300) GN=mraW PE=3 SV=2 Q2FHQ8|MRAW_STAA3 -1.2 483 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1495 PE=4 SV=1 Q2FGI8|Q2FGI8_STAA3 -1.2 511 Multidrug resistance protein A, drug resistance transporter OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_2299 PE=4 SV=1 Q2FEE4|Q2FEE4_STAA3 -1.2 522 UPF0173 metal-dependent hydrolase SAUSA300_1653 OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_1653 PE=3 SV=1 Q2FG31|Y1653_STAA3 -1.2 531 Penicillin-binding protein 1 OS=S taphylococcus aureus (strain USA300) GN=pbpA PE=4 SV=1 Q2FHQ6|Q2FHQ6_STAA3 -1.2 589 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1720 PE=4 SV=1 Q2FFW5|Q2FFW5_STAA3 -1.2 610 UPF0297 protein SAUSA300_1574 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1574 PE=3 SV=1 Q2FGA9|Y1574_STAA3 -1.2 617 DNA-binding response regulator OS=Staphylococcus aureus (strain USA300) GN=vraR PE=4 SV=1 Q2FFL1|Q2FFL1_STAA3 -1.2 654 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0956 PE=4 SV=1 Q2FI24|Q2FI24_STAA3 -1.2 18 Dihydrolipoyl dehydrogenase OS =Staphylococcus aureus (strain USA300) GN=lpdA PE=3 SV=1 Q2FHY4|Q2FHY4_STAA3 -1.1 56 Glutamyl-tRNA(Gln) ami dotransferase subunit A OS=Staphylococcus aureus (strain USA300) GN=gatA PE=3 SV=1 Q2FFJ5|GATA_STAA3 -1.1 100 Triosephosphate isomerase OS=Staphylococcus aureus (strain USA300) GN=tpiA PE=3 SV=1 Q2FIL9|TPIS_STAA3 -1.1 102 Ferritin OS=Staphylococcus aureus (strain USA300) GN=ftnA PE=3 SV=1 Q2FFK2|FTN_STAA3 -1.1 159

PAGE 171

111 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1863 PE=4 SV=1 Q2FFL3|Q2FFL3_STAA3 -1.1 143 30S ribosomal protein S8 OS=Staphylococcus aureus (strain USA300) GN=rpsH PE=3 SV=1 Q2FEQ3|RS8_STAA3 -1.1 149 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2144 PE=4 SV=1 Q2FEU8|Q2FEU8_STAA3 -1.1 202 50S ribosomal protein L23 OS=Staphylococcus aureus (strain USA300) GN=rplW PE=3 SV=1 Q2FEP1|RL23_STAA3 -1.1 213 50S ribosomal protein L21 OS=Staphylococcus aureus (strain USA300) GN=rplU PE=3 SV=1 Q2FG80|RL21_STAA3 -1.1 248 Putative 2-hydroxyacid dehydrogenase SAUSA300_2254 OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_2254 PE=3 SV=1 Q2FEI9|Y2254_STAA3 -1.1 386 DNA topoisomerase IV, subunit A OS=Staphylococcus aureus (strain USA300) GN=parC PE=4 SV=1 Q2FH80|Q2FH80_STAA3 -1.1 394 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2097 PE=4 SV=1 Q2FEY5|Q2FEY5_STAA3 -1.1 414 Aminotransferase OS=Staphyloco ccus aureus (strain USA300) GN=SAUSA300_1916 PE=4 SV=1 Q2FFG0|Q2FFG0_STAA3 -1.1 459 2-oxoisovalerate dehydrogenase E1 component, beta subunit OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_1465 PE=4 SV=1 Q2FGL7|Q2FGL7_STAA3 -1.1 502 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1494 PE=4 SV=1 Q2FGI9|Q2FGI9_STAA3 -1.1 509 RNA polymerase sigma factor OS=Staphylococcus aureus (strain USA300) GN=rpoD PE=3 SV=1 Q2FGG2|Q2FGG2_STAA3 -1.1 545 Preprotein translocase, YajC subunit OS=Staphylococcus aureus (strain USA300) GN=yajC PE=4 SV=1 Q2FG89|Q2FG89_STAA3 -1.1 594 Cardiolipin synthetase OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1216 PE=4 SV=1 Q2FHB5|Q2FHB5_STAA3 -1.1 602 UPF0447 protein SAUSA300_0569 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0569 PE=3 SV=1 Q2FJ56|Y569_STAA3 -1.1 714 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1230 PE=4 SV=1 Q2FHA1|Q2FHA1_STAA3 -1.1 17 30S ribosomal protein S1 OS=Staphylococcus aureus (strain USA300) GN=rpsA PE=4 SV=1 Q2FGW6|Q2FGW6_STAA3 -1 59 Uncharacterized protein SAUSA300_0871 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0871 PE=3 SV=1 Q2FIA7|Y871_STAA3 -1 77 GMP synthase [glutamine-hydrolyzing] OS=Staphylococcus aureus (strain USA300) GN=guaA PE=3 SV=1 Q2FJM5|GUAA_STAA3 -1 85 Putative aldehyde dehy drogenase SAUSA300_2076 OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_2076 PE=3 SV=1 Q2FF06|ALD1_STAA3 -1 87 Probable manganese-dependent inorganic pyrophosphatase OS=Staphylococcus aureus (strain USA300) GN=ppaC PE=3 SV=1 Q2FFH6|PPAC_STAA3 -1 160

PAGE 172

93 Lysyl-tRNA synthetase OS=Staphy lococcus aureus (strain USA300) GN=lysS PE=3 SV=1 Q2FJC3|SYK_STAA3 -1 120 Polyribonucleotide nucleotidyltransf erase OS=Staphylococcus aureus (strain USA300) GN=pnp PE=3 SV=1 Q2FHG4|PNP_STAA3 -1 127 DNA-directed RNA polymerase subunit alpha OS=Staphylococcus aureus (strain USA300) GN=rpoA PE=3 SV=1 Q2FER5|RPOA_STAA3 -1 147 50S ribosomal protein L7/L12 OS=Staphylococcus aureus (strain USA300) GN=rplL PE=3 SV=1 Q2FJA0|RL7_STAA3 -1 163 ATP-dependent Clp protease ATP-binding subunit clpC OS=Staphylococcus aureus (strain USA300) GN=clpC PE=3 SV=1 Q2FJB5|CLPC_STAA3 -1 169 Oligoendopeptidase F OS=Staphylococcus aureus (strain USA300) GN=pepF PE=4 SV=1 Q2FI76|Q2FI76_STAA3 -1 199 6-phosphofructokinase OS=Staphylococcus aureus (strain USA300) GN=pfkA PE=3 SV=1 Q2FG39|Q2FG39_STAA3 -1 253 Putative flavohemoprotein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0234 PE=3 SV=1 Q2FK30|Q2FK30_STAA3 -1 279 Phosphoribosylformylglycinamidin e synthase OS=Staphylococcus aureus (strain USA300) GN=purS PE=4 SV=1 Q2FI11|Q2FI11_STAA3 -1 283 ATP-dependent RNA helicase, DEAD/DEAH box family OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_1518 PE=4 SV=1 Q2FGG5|Q2FGG5_STAA3 -1 345 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1304 PE=4 SV=1 Q2FH27|Q2FH27_STAA3 -1 432 50S ribosomal protein L33 2 OS=Staphylococcus aureus (strain USA300) GN=rpmG2 PE=3 SV=1 Q2FGH2|RL332_STAA3 -1 467 tRNA pseudouridine synthase B OS=Staphylococcus aureus (strain USA300) GN=truB PE=3 SV=1 Q2FHG7|TRUB_STAA3 -1 638 ABC transporter, ATP-binding protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0704 PE=4 SV=1 Q2FIS1|Q2FIS1_STAA3 -1 656 Protein nagD homolog OS=Staphylo coccus aureus (strain USA300) GN=nagD PE=3 SV=1 Q2FIE5|NAGD_STAA3 -1 667 Transporter, CorA family OS=Staphylococcus aureus (strain USA300) GN=cobI PE=4 SV=1 Q2FEC0|Q2FEC0_STAA3 -1 717 Peptide deformylase OS=Staphylococcus aureus (strain USA300) GN=def PE=3 SV=1 Q2FHY9|Q2FHY9_STAA3 -1 5 Elongation factor G OS=Staphylococcus aureus (strain USA300) GN=fusA PE=3 SV=3 Q2FJ93|EFG_STAA3 -0.9 13 Cell division protein ftsZ OS=S taphylococcus aureus (strain USA300) GN=ftsZ PE=3 SV=1 Q2FHQ1|Q2FHQ1_STAA3 -0.9 64 Catalase OS=Staphylococcus au reus (strain USA300) GN=katA PE=3 SV=1 Q2FH99|CATA_STAA3 -0.9 83 Penicillin binding protein 2 OS=S taphylococcus aureus (strain USA300) GN=pbp2 PE=4 SV=1 Q2FGZ0|Q2FGZ0_STAA3 -0.9 161

PAGE 173

106 Branched-chain-amino-acid aminotransferase OS=Staphylococcus aureus (strain USA300) GN=ilvE PE=3 SV=1 Q2FJ86|Q2FJ86_STAA3 -0.9 129 Phosphoenolpyruvate-prote in phosphotransferase OS=Staphylococcus aureus (strain USA300) GN=ptsI PE=3 SV=1 Q2FHZ6|Q2FHZ6_STAA3 -0.9 178 UDP-N-acetylglucosamine 1-carboxyvinyltransferase 1 OS=Staphylococcus aureus (strain USA300) GN=murA PE=3 SV=1 Q2FF27|Q2FF27_STAA3 -0.9 198 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1652 PE=4 SV=1 Q2FG32|Q2FG32_STAA3 -0.9 203 3-oxoacyl-(Acyl-carrier-protein) reductase OS=Staphylococcus aureus (strain USA300) GN=fabG PE=3 SV=1 Q2FHK7|Q2FHK7_STAA3 -0.9 219 D-alanine aminotransferase OS=Staphylococcus aureus (strain USA300) GN=dat PE=3 SV=1 Q2FFY8|Q2FFY8_STAA3 -0.9 222 Methionyl-tRNA synthetase OS =Staphylococcus aureus (strain USA300) GN=metS PE=3 SV=1 Q2FJF2|Q2FJF2_STAA3 -0.9 258 Arginyl-tRNA synthetase OS=Staphylococcus aureus (strain USA300) GN=argS PE=3 SV=1 Q2FJ29|SYR_STAA3 -0.9 275 Orotidine 5'-phosphate decarboxylase OS=Staphylococcus aureus (strain USA300) GN=pyrF PE=3 SV=1 Q2FHN4|PYRF_STAA3 -0.9 290 Putative NAD(P)H nitroreductase SAUSA300_2462 OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_2462 PE=3 SV=1 Q2FDY2|Y2462_STAA3 -0.9 325 Ribosomal large subunit pseudouridine synthase, RluD subfamily OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_1800 PE=4 SV=1 Q2FFP5|Q2FFP5_STAA3 -0.9 326 Putative septation protein spoVG OS=Staphylococcus aureus (strain USA300) GN=spoVG PE=3 SV=1 Q2FJE4|SP5G_STAA3 -0.9 405 Dephospho-CoA kinase OS=Staphylo coccus aureus (strain USA300) GN=coaE PE=3 SV=1 Q2FG49|COAE_STAA3 -0.9 433 Redox-sensing transcriptional re pressor rex OS=Staphylococcus aureus (strain USA300) GN=rex PE=3 SV=1 Q2FF78|REX_STAA3 -0.9 434 HIT family protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1787 PE=4 SV=1 Q2FFQ8|Q2FFQ8_STAA3 -0.9 609 Rod shape-determining protein MreC OS=Staphylococcus aureus (strain USA300) GN=mreC PE=4 SV=1 Q2FG78|Q2FG78_STAA3 -0.9 3 Autolysin OS=Staphylococcus aureus (strain USA300) GN=atl PE=4 SV=1 Q2FI25|Q2FI25_STAA3 -0.8 24 Cysteine synthase OS=Staphyloco ccus aureus (strain USA300) GN=cysK PE=3 SV=1 Q2FJC8|Q2FJC8_STAA3 -0.8 57 Alkyl hydroperoxide reductase subunit C OS=Staphylococcus aureus (strain USA300) GN=ahpC PE=3 SV=1 Q2FJN4|AHPC_STAA3 -0.8 82 FeS assembly ATPase SufC OS=Staphylococcus aureus (strain USA300) GN=sufC PE=4 SV=1 Q2FIG0|Q2FIG0_STAA3 -0.8 99 60 kDa chaperonin OS=Staphylococcus aureus (strain USA300) GN=groL PE=3 SV=1 Q2FF95|CH60_STAA3 -0.8 162

PAGE 174

135 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0602 PE=4 SV=1 Q2FJ23|Q2FJ23_STAA3 -0.8 177 Glycine cleavage system H prot ein OS=Staphylococcus aureus (strain USA300) GN=gcvH PE=3 SV=1 Q2FII7|GCSH_STAA3 -0.8 231 Transcription antitermination protein nusG OS=Staphylococcus aureus (strain USA300) GN=nusG PE=3 SV=1 Q2FJA4|Q2FJA4_STAA3 -0.8 295 Peptide methionine sulfoxide reductase regulator MsrR OS=Staphylococcus aureus (strain USA300) GN=msrR PE=4 SV=1 Q2FH74|Q2FH74_STAA3 -0.8 417 Tyrosyl-tRNA synthetase OS=Staphylococcus aureus (strain USA300) GN=tyrS PE=3 SV=1 Q2FG09|SYY_STAA3 -0.8 451 Phosphoribosylformylglycinamidin e synthase 1 OS=Staphylococcus aureus (strain USA300) GN=purQ PE=3 SV=1 Q2FI10|PURQ_STAA3 -0.8 470 Phenylalanyl-tRNA synthetase (Beta subunit) OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1688 PE=4 SV=1 Q2FFZ6|Q2FFZ6_STAA3 -0.8 480 Uncharacterized pe ptidase SAUSA300_1654 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1654 PE=3 SV=1 Q2FG30|Y1654_STAA3 -0.8 538 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1333 PE=4 SV=1 Q2FGZ8|Q2FGZ8_STAA3 -0.8 787 Dihydrolipoyl dehydrogenase OS =Staphylococcus aureus (strain USA300) GN=lpdA PE=3 SV=1 Q2FGL5|Q2FGL5_STAA3 -0.8 43 Glycyl-tRNA synthetase OS=Staphylococcus aureus (strain USA300) GN=glyQS PE=3 SV=1 Q2FGF8|SYG_STAA3 -0.7 76 Transketolase OS=Staphylococcus aureus (strain USA300) GN=tkt PE=4 SV=1 Q2FH92|Q2FH92_STAA3 -0.7 97 Putative cell division protein FtsH OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0489 PE=4 SV=1 Q2FJD0|Q2FJD0_STAA3 -0.7 117 FeS assembly protein SufD OS=Staphylococcus aureus (strain USA300) GN=sufD PE=4 SV=1 Q2FIF9|Q2FIF9_STAA3 -0.7 119 GTP-sensing transcriptional pleiotropic repressor codY OS=Staphylococcus aureus (strain USA300) GN=codY PE=3 SV=1 Q2FHI3|CODY_STAA3 -0.7 128 6-phosphogluconate dehydr ogenase, decarboxylating OS=Staphylococcus aureus (strain USA300) GN=gnd PE=3 SV=1 Q2FGM3|Q2FGM3_STAA3 -0.7 158 2-oxoglutarate dehydrogenase E1 component OS=Staphylococcus aureus (strain USA300) GN=odhA PE=3 SV=1 Q2FH25|ODO1_STAA3 -0.7 193 Transaldolase OS=Staphylococcu s aureus (strain USA300) GN=SAUSA300_1725 PE=3 SV=1 Q2FFW0|Q2FFW0_STAA3 -0.7 211 Uncharacterized protein SAUSA300_0736 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0736 PE=3 SV=1 Q2FIN9|Y736_STAA3 -0.7 238 50S ribosomal protein L30 OS=Staphylococcus aureus (strain USA300) GN=rpmD PE=3 SV=1 Q2FEQ7|RL30_STAA3 -0.7 245 Valyl-tRNA synthetase OS=Staphylo coccus aureus (strain USA300) GN=valS PE=3 SV=1 Q2FG72|SYV_STAA3 -0.7 163

PAGE 175

265 Alanyl-tRNA synthetase OS=Staphylococcus aureus (strain USA300) GN=alaS PE=3 SV=1 Q2FGA8|SYA_STAA3 -0.7 335 Acetyltransferase, GNAT family OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1070 PE=4 SV=1 Q2FHR1|Q2FHR1_STAA3 -0.7 353 Alcohol dehydrogenase, zinc-cont aining OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2147 PE=4 SV=1 Q2FEU5|Q2FEU5_STAA3 -0.7 356 Aspartate semialdehyde dehydrogenase OS=Staphylococcus aureus (strain USA300) GN=asd PE=3 SV=1 Q2FH44|Q2FH44_STAA3 -0.7 376 Acetyltransferase family protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2460 PE=4 SV=1 Q2FDY4|Q2FDY4_STAA3 -0.7 422 ATP-dependent Clp protease proteolytic subunit OS=Staphylococcus aureus (strain USA300) GN=clpP PE=3 SV=1 Q2FIM5|CLPP_STAA3 -0.7 500 Epimerase family protein SAUSA300_0753 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0753 PE=3 SV=1 Q2FIM4|Y753_STAA3 -0.7 563 Molybdopterin converting factor, subunit 1 OS=Staphylococcus aureus (strain USA300) GN=moaD PE=4 SV=1 Q2FEM2|Q2FEM2_STAA3 -0.7 631 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0874 PE=4 SV=1 Q2FIA4|Q2FIA4_STAA3 -0.7 701 GMP reductase OS=Staphylococcus aureus (strain USA300) GN=guaC PE=3 SV=1 Q2FH96|GUAC_STAA3 -0.7 79 Glucose-6-phosphate isomerase OS=Staphylococcus aureus (strain USA300) GN=pgi PE=3 SV=1 Q2FIB3|G6PI_STAA3 -0.6 86 UPF0457 protein SAUSA300_2132 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2132 PE=3 SV=1 Q2FEV9|Y2132_STAA3 -0.6 166 Uncharacterized hydrolase SAUSA300_2518 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2518 PE=3 SV=1 Q2FDS6|Y2518_STAA3 -0.6 197 Putative lipoprotein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0372 PE=4 SV=1 Q2FJP2|Q2FJP2_STAA3 -0.6 224 Alcohol dehydrogenase, zinc-cont aining OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0250 PE=4 SV=1 Q2FK14|Q2FK14_STAA3 -0.6 236 Chaperone clpB OS=Staphylococcus aureus (strain USA300) GN=clpB PE=3 SV=1 Q2FIA1|Q2FIA1_STAA3 -0.6 250 Fibrinogen-binding protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1052 PE=4 SV=1 Q2FHS8|Q2FHS8_STAA3 -0.6 287 2',3'-cyclic-nucleotide 2'-phosphodiesterase OS=Staphylococcus aureus (strain USA300) GN=cvfA PE=3 SV=1 Q2FHF2|CNPD_STAA3 -0.6 364 30S ribosomal protein S19 OS=Staphylococcus aureus (strain USA300) GN=rpsS PE=3 SV=1 Q2FEP3|RS19_STAA3 -0.6 370 Probable uridylyltransferase SAUSA300_2130 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2130 PE=3 SV=1 Q2FEW1|URTF_STAA3 -0.6 444 PTS system, mannitol specific IIA component OS=Staphylococcus aureus (strain USA300) GN=mtlA PE=4 SV=1 Q2FEX5|Q2FEX5_STAA3 -0.6 164

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460 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2418 PE=4 SV=1 Q2FE25|Q2FE25_STAA3 -0.6 498 Glutathione peroxidase OS=Staphy lococcus aureus (strain USA300) GN=SAUSA300_1197 PE=3 SV=1 Q2FHD4|Q2FHD4_STAA3 -0.6 566 Putative lipoprotein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0992 PE=4 SV=1 Q2FHY8|Q2FHY8_STAA3 -0.6 583 Bifunctional protein glmU OS=S taphylococcus aureus (strain USA300) GN=glmU PE=3 SV=1 Q2FJE2|GLMU_STAA3 -0.6 592 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1351 PE=4 SV=1 Q2FGY0|Q2FGY0_STAA3 -0.6 606 Competence/damage-inducible pr otein cinA OS=Staphylococcus aureus (strain USA300) GN=cinA PE=4 SV=1 Q2FHF4|Q2FHF4_STAA3 -0.6 36 Phosphoglycerate kinase OS=Staphylococcus aureus (strain USA300) GN=pgk PE=3 SV=1 Q2FIM0|PGK_STAA3 -0.5 51 Dihydrolipoamide acetyltransfer ase OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0995 PE=3 SV=1 Q2FHY5|Q2FHY5_STAA3 -0.5 90 Trigger factor OS=Staphylococcus aureus (strain USA300) GN=tig PE=3 SV=1 Q2FG61|TIG_STAA3 -0.5 289 Aspartyl/glutamyl-tRNA(Asn/Gln) amidotransferase subunit C OS=Staphylococcus aureus (strain USA300) GN=gatC PE=3 SV=1 Q2FFJ4|GATC_STAA3 -0.5 415 Excinuclease ABC, A subunit OS=Staphylococcus aureus (strain USA300) GN=uvrA PE=3 SV=1 Q2FIN4|Q2FIN4_STAA3 -0.5 431 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0373 PE=4 SV=1 Q2FJP1|Q2FJP1_STAA3 -0.5 443 Cystathionine gamma-synthase OS =Staphylococcus aureus (strain USA300) GN=metB PE=3 SV=1 Q2FJI1|Q2FJI1_STAA3 -0.5 477 Lytic regulatory protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2100 PE=4 SV=1 Q2FEY2|Q2FEY2_STAA3 -0.5 501 30S ribosomal protein S20 OS=Staphylococcus aureus (strain USA300) GN=rpsT PE=3 SV=1 Q2FGD8|RS20_STAA3 -0.5 754 Sulfite reductase flavoprotein OS =Staphylococcus aureus (strain USA300) GN=SAUSA300_2554 PE=3 SV=1 Q2FDP0|Q2FDP0_STAA3 -0.5 22 Glyceraldehyde-3-phosphat e dehydrogenase, type I OS=Staphylococcus aureus (strai n USA300) GN=gap PE=3 SV=1 Q2FIM1|Q2FIM1_STAA3 -0.4 132 Foldase protein prsA OS=Staphylococcus aureus (strain USA300) GN=prsA PE=3 SV=1 Q2FFQ5|PRSA_STAA3 -0.4 151 Serine hydroxymethyltransferase OS=Staphylococcus aureus (strain USA300) GN=glyA PE=3 SV=1 Q2FF15|GLYA_STAA3 -0.4 214 Glutamyl-tRNA synthetase OS=Staphylococcus aureus (strain USA300) GN=gltX PE=3 SV=1 Q2FJB2|SYE_STAA3 -0.4 229 D-alanine--poly(phosphoribitol) ligase subunit 1 OS=Staphylococcus aureus (strain USA300) GN=dltA PE=3 SV=1 Q2FIE3|DLTA_STAA3 -0.4 165

PAGE 177

239 3-oxoacyl-(Acyl-carrier-protein) synthase II OS=Staphylococcus aureus (strain USA300) GN=fabF PE=3 SV=1 Q2FI92|Q2FI92_STAA3 -0.4 240 Glycerol phosphate lipoteichoic aci d synthase OS=Staphylococcus aureus (strain USA300) GN=ltaS PE=3 SV=1 Q2FIS2|LTAS_STAA3 -0.4 277 Thioredoxin reductase OS=Staphylococcus aureus (strain USA300) GN=trxB PE=3 SV=1 Q2FIM9|Q2FIM9_STAA3 -0.4 313 Ribosome-recycling factor OS=Staphylococcus aureus (strain USA300) GN=frr PE=3 SV=1 Q2FHH9|RRF_STAA3 -0.4 318 Iron compound ABC transporter, iron compound-binding protein OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_2235 PE=4 SV=1 Q2FEK8|Q2FEK8_STAA3 -0.4 323 50S ribosomal protein L29 OS=Staphylococcus aureus (strain USA300) GN=rpmC PE=4 SV=1 Q2FEP7|Q2FEP7_STAA3 -0.4 337 UDP-N-acetylmuramoyl-L-alanyl-D-glutamate--L-lysine ligase OS=Staphylococcus aureus (strain USA300) GN=murE PE=3 SV=1 Q2FI59|MURE_STAA3 -0.4 421 Putative thioredoxin OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1690 PE=4 SV=1 Q2FFZ4|Q2FFZ4_STAA3 -0.4 535 Nitroreductase family protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1986 PE=4 SV=1 Q2FF91|Q2FF91_STAA3 -0.4 544 50S ribosomal protein L28 OS=Staphylococcus aureus (strain USA300) GN=rpmB PE=3 SV=1 Q2FHL4|RL28_STAA3 -0.4 596 Putative transcriptional regulator OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2259 PE=4 SV=1 Q2FEI4|Q2FEI4_STAA3 -0.4 599 Fibrinogen-binding protein OS=Staphylococcus aureus (strain USA300) GN=efb PE=4 SV=1 Q2FHS5|Q2FHS5_STAA3 -0.4 607 Arginine deiminase OS=Staphylococcus aureus (strain USA300) GN=arcA PE=3 SV=1 Q2FDM4|Q2FDM4_STAA3 -0.4 44 Acyl carrier protein OS=Staphylococcus aureus (strain USA300) GN=acpP PE=3 SV=1 Q2FHK6|ACP_STAA3 -0.3 189 Cell cycle protein gpsB OS=Staphylococcus aureus (strain USA300) GN=gpsB PE=3 SV=1 Q2FGZ4|GPSB_STAA3 -0.3 445 HTH-type transcriptional regulator rot OS=Staphylococcus aureus (strain USA300) GN=rot PE=3 SV=1 Q2FFX6|Q2FFX6_STAA3 -0.3 448 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1803 PE=4 SV=1 Q2FFP2|Q2FFP2_STAA3 -0.3 464 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1904 PE=4 SV=1 Q2FFH2|Q2FFH2_STAA3 -0.3 546 Cold shock protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2639 PE=3 SV=1 Q2FDF5|Q2FDF5_STAA3 -0.3 550 Cmp-binding-factor 1 OS=Staphylococcus aureus (strain USA300) GN=cbf1 PE=4 SV=1 Q2FFQ4|Q2FFQ4_STAA3 -0.3 564 N utilization substance protei n B homolog OS=Staphylococcus aureus (strain USA300) GN=nusB PE=3 SV=1 Q2FGK9|NUSB_STAA3 -0.3 166

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626 Dihydrofolate reductase OS=Staphy lococcus aureus (strain USA300) GN=folA PE=3 SV=1 Q2FH12|Q2FH12_STAA3 -0.3 80 Glutamine synthetase OS=Staphylo coccus aureus (strain USA300) GN=glnA PE=3 SV=1 Q2FHD0|Q2FHD0_STAA3 -0.2 144 Naphthoate synthase OS=Staphylococcus aureus (strain USA300) GN=menB PE=4 SV=1 Q2FI32|Q2FI32_STAA3 -0.2 173 Putative aldehyde dehydrogenase AldA OS=Staphylococcus aureus (strain USA300) GN=aldA PE=3 SV=1 Q2FK94|ALDA_STAA3 -0.2 190 3-hexulose-6-phosphate synthase OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0555 PE=3 SV=1 Q2FJ70|HPS_STAA3 -0.2 192 50S ribosomal protein L18 OS=Staphylococcus aureus (strain USA300) GN=rplR PE=3 SV=1 Q2FEQ5|RL18_STAA3 -0.2 299 Transcription elongation factor gr eA OS=Staphylococcus aureus (strain USA300) GN=greA PE=3 SV=1 Q2FGB6|GREA_STAA3 -0.2 302 30S ribosomal protein S18 OS=Staphylococcus aureus (strain USA300) GN=rpsR PE=3 SV=1 Q2FJP6|RS18_STAA3 -0.2 312 Probable glycine dehydrogenase [decarboxylating] subunit 1 OS=Staphylococcus aureus (strai n USA300) GN=gcvPA PE=3 SV=1 Q2FGI6|GCSPA_STAA3 -0.2 314 50S ribosomal protein L17 OS=Staphylococcus aureus (strain USA300) GN=rplQ PE=3 SV=1 Q2FER6|RL17_STAA3 -0.2 347 ABC transporter, substrate-binding protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0798 PE=4 SV=1 Q2FII0|Q2FII0_STAA3 -0.2 368 Amino acid ABC transporter, amino acid-binding protein OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_2359 PE=4 SV=1 Q2FE84|Q2FE84_STAA3 -0.2 424 Homoserine dehydrogenase OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1226 PE=3 SV=1 Q2FHA5|Q2FHA5_STAA3 -0.2 430 Peptide methionine sulfoxide re ductase msrB OS=Staphylococcus aureus (strain USA300) GN=msrB PE=3 SV=1 Q2FH15|MSRB_STAA3 -0.2 447 PTS system, glucose-specific IIA component OS=Staphylococcus aureus (strain USA300) GN=crr PE=4 SV=1 Q2FH16|Q2FH16_STAA3 -0.2 479 Argininosuccinate synthase OS=Staphylococcus aureus (strain USA300) GN=argG PE=3 SV=1 Q2FIB4|ASSY_STAA3 -0.2 484 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1303 PE=4 SV=1 Q2FH28|Q2FH28_STAA3 -0.2 495 Organic hydroperoxide resistance protein-like OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0786 PE=3 SV=1 Q2FIJ2|OHRL_STAA3 -0.2 52 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1581 PE=4 SV=1 Q2FGA2|Q2FGA2_STAA3 -0.1 113 Pyruvate oxidase OS=Staphyloco ccus aureus (strain USA300) GN=cidC PE=3 SV=1 Q2FDW7|Q2FDW7_STAA3 -0.1 207 Molybdenum ABC transporter, mo lybdenum-binding protein ModA OS=Staphylococcus aureus (strain USA300) GN=modA PE=4 SV=1 Q2FEL3|Q2FEL3_STAA3 -0.1 167

PAGE 179

212 Iron compound ABC transporter, iron compound-binding protein OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_2136 PE=4 SV=1 Q2FEV5|Q2FEV5_STAA3 -0.1 366 Elongation factor P OS=Staphylococcus aureus (strain USA300) GN=efp PE=3 SV=1 Q2FGJ3|EFP_STAA3 -0.1 406 Anti-sigma factor antagonist OS=Staphylococcus aureus (strain USA300) GN=rsbV PE=3 SV=1 Q2FF58|Q2FF58_STAA3 -0.1 461 Fructose specific permease OS=Staphylococcus aureus (strain USA300) GN=fruA PE=4 SV=1 Q2FIU0|Q2FIU0_STAA3 -0.1 466 Leucyl-tRNA synthetase OS=Staphylococcus aureus (strain USA300) GN=leuS PE=3 SV=1 Q2FFY0|SYL_STAA3 -0.1 487 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0985 PE=4 SV=1 Q2FHZ5|Q2FHZ5_STAA3 -0.1 562 Penicillin-binding protein 4 OS=S taphylococcus aureus (strain USA300) GN=pbp4 PE=3 SV=1 Q2FIZ6|Q2FIZ6_STAA3 -0.1 629 50S ribosomal protein L32 OS=Staphylococcus aureus (strain USA300) GN=rpmF PE=3 SV=1 Q2FHV3|RL32_STAA3 -0.1 7 DNA-binding protein HU OS=Staphy lococcus aureus (strain USA300) GN=hup PE=3 SV=1 Q2FGW9|Q2FGW9_STAA3 0 84 Fructose bisphosphate aldolase OS=Staphylococcus aureus (strain USA300) GN=fba PE=4 SV=1 Q2FF03|Q2FF03_STAA3 0 226 Adenylate kinase OS=Staphylococcus aureus (strain USA300) GN=adk PE=3 SV=1 Q2FER0|Q2FER0_STAA3 0 303 50S ribosomal protein L31 type B OS=Staphylococcus aureus (strain USA300) GN=rpmE2 PE=3 SV=1 Q2FF08|RL31B_STAA3 0 309 Signal transduction protein TRAP OS=Staphylococcus aureus (strain USA300) GN=traP PE=3 SV=1 Q2FFR1|TRAP_STAA3 0 481 Staphylococcal accessory regulat or OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0114 PE=4 SV=1 Q2FKE7|Q2FKE7_STAA3 0 541 Phenylalanyl-tRNA synthetase beta chain OS=Staphylococcus aureus (strain USA300) GN=pheT PE=3 SV=1 Q2FHU2|SYFB_STAA3 0 580 Penicillin-binding protein 3 OS=S taphylococcus aureus (strain USA300) GN=pbp3 PE=4 SV=1 Q2FGH1|Q2FGH1_STAA3 0 615 Iron compound ABC transporter, iron compound-binding protein SirA OS=Staphylococcus aureus (strain USA300) GN=sirA PE=4 SV=1 Q2FKE4|Q2FKE4_STAA3 0 711 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2378 PE=4 SV=1 Q2FE65|Q2FE65_STAA3 0 6 UPF0337 protein SAUSA300_0816 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0816 PE=3 SV=1 Q2FIG2|Y816_STAA3 0.1 21 Chaperone protein dnaK OS=Staphylococcus aureus (strain USA300) GN=dnaK PE=2 SV=1 Q2FGE3|DNAK_STAA3 0.1 107 UPF0342 protein SAUSA300_1795 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1795 PE=3 SV=1 Q2FFQ0|Y1795_STAA3 0.1 168

PAGE 180

157 Copper chaperone copZ OS=Staphylococcus aureus (strain USA300) GN=copZ PE=3 SV=1 Q2FDU9|COPZ_STAA3 0.1 227 Thiol peroxidase OS=Staphyloco ccus aureus (strain USA300) GN=tpx PE=4 SV=1 Q2FG25|Q2FG25_STAA3 0.1 268 FMN-dependent NADH-azoreductase OS=Staphylococcus aureus (strain USA300) GN=azoR PE=3 SV=1 Q2FK58|AZOR_STAA3 0.1 293 Glucokinase OS=Staphylococcus au reus (strain USA300) GN=glk PE=4 SV=1 Q2FGH6|Q2FGH6_STAA3 0.1 419 50S ribosomal protein L24 OS=Staphylococcus aureus (strain USA300) GN=rplX PE=3 SV=1 Q2FEQ0|RL24_STAA3 0.1 458 Putative cell-division initiation prot ein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1086 PE=4 SV=1 Q2FHP5|Q2FHP5_STAA3 0.1 647 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2246 PE=4 SV=1 Q2FEJ7|Q2FEJ7_STAA3 0.1 722 (3R)-hydroxymyristoyl-[acyl-carrier-protein] dehydratase OS=Staphylococcus aureus (strain USA300) GN=fabZ PE=3 SV=1 Q2FF28|FABZ_STAA3 0.1 34 N-acetylmuramoyl-L-alanine amidase domain protein OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_2579 PE=4 SV=1 Q2FDL5|Q2FDL5_STAA3 0.2 185 50S ribosomal protein L11 OS=Staphylococcus aureus (strain USA300) GN=rplK PE=3 SV=3 Q2FJA3|RL11_STAA3 0.2 215 50S ribosomal protein L25 OS=Staphylococcus aureus (strain USA300) GN=rplY PE=3 SV=1 Q2FJE0|RL25_STAA3 0.2 389 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1215 PE=4 SV=1 Q2FHB6|Q2FHB6_STAA3 0.2 492 Chorismate mutase/phospho-2-de hydro-3-deoxyheptonate aldolase OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_1683 PE=4 SV=1 Q2FG01|Q2FG01_STAA3 0.2 507 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0931 PE=4 SV=1 Q2FI49|Q2FI49_STAA3 0.2 683 Putative GTP-binding protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1136 PE=4 SV=1 Q2FHJ5|Q2FHJ5_STAA3 0.2 771 NADPH-dependent oxidoreductase OS=Staphylococcus aureus (strain USA300) GN=nfrA PE=3 SV=1 Q2FJN3|NFRA_STAA3 0.2 10 Alkaline shock protein 23 OS=Staphylococcus aureus (strain USA300) GN=asp23 PE=3 SV=1 Q2FEV0|ASP23_STAA3 0.3 172 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1698 PE=4 SV=1 Q2FFY6|Q2FFY6_STAA3 0.3 286 Dihydrolipoyllysine-residue succiny ltransferase component of 2oxoglutarate dehydrogenase comple x OS=Staphylococcus aureus (strain USA300) GN=odhB PE=3 SV=1 Q2FH26|ODO2_STAA3 0.3 401 30S ribosomal protein S16 OS=Staphylococcus aureus (strain USA300) GN=rpsP PE=3 SV=1 Q2FHK0|RS16_STAA3 0.3 768 SUF system FeS assembly protein, NifU family OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0821 PE=4 SV=1 Q2FIF7|Q2FIF7_STAA3 0.3 169

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28 Secretory antigen SsaA OS=Staphy lococcus aureus (strain USA300) GN=ssaA PE=4 SV=1 Q2FEJ4|Q2FEJ4_STAA3 0.4 141 N-acetylmuramoyl-L-alanine amidase sle1 OS=Staphylococcus aureus (strain USA300) GN=sle1 PE=3 SV=1 Q2FJH7|SLE1_STAA3 0.4 324 6,7-dimethyl-8-ribityllumazine synthase OS=Staphylococcus aureus (strain USA300) GN=ribH PE=3 SV=1 Q2FFX3|RISB_STAA3 0.4 393 Mannitol-1-phosphate 5-dehydrogenase OS=Staphylococcus aureus (strain USA300) GN=mtlD PE=3 SV=1 Q2FEX4|MTLD_STAA3 0.4 398 Translation initiation factor IF-3 OS=Staphylococcus aureus (strain USA300) GN=infC PE=3 SV=1 Q2FG56|IF3_STAA3 0.4 407 D-alanine--poly(phosphoribitol) ligase subunit 2 OS=Staphylococcus aureus (strain USA300) GN=dltC PE=3 SV=1 Q2FIE1|DLTC_STAA3 0.4 489 Triacylglycerol lipase OS=Staphylococcus aureus (strain USA300) GN=lip PE=4 SV=1 Q2FDJ1|Q2FDJ1_STAA3 0.4 618 NLPA lipoprotein OS=Staphylococc us aureus (strain USA300) GN=SAUSA300_0437 PE=4 SV=1 Q2FJH8|Q2FJH8_STAA3 0.4 38 Thioredoxin OS=Staphylococcus aureus (strain USA300) GN=trxA PE=3 SV=1 Q2FHT6|THIO_STAA3 0.5 396 NH(3)-dependent NAD(+) syntheta se OS=Staphylococcus aureus (strain USA300) GN=nadE PE=3 SV=1 Q2FFI3|NADE_STAA3 0.5 571 Putative lipoprotein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0724 PE=4 SV=1 Q2FIQ1|Q2FIQ1_STAA3 0.5 75 Phosphocarrier protein HPr OS=Staphylococcus aureus (strain USA300) GN=ptsH PE=4 SV=1 Q2FHZ7|Q2FHZ7_STAA3 0.6 134 Transcriptional regulator sarA OS=Staphylococcus aureus (strain USA300) GN=sarA PE=3 SV=1 Q2FJ20|SARA_STAA3 0.6 310 Acetyl-coenzyme A carboxylase carboxyl transferase subunit alpha OS=Staphylococcus aureus (strai n USA300) GN=accA PE=1 SV=1 Q2FG38|ACCA_STAA3 0.6 624 Probable transglycosylase sceD OS=Staphylococcus aureus (strain USA300) GN=sceD PE=2 SV=1 Q2FF31|SCED_STAA3 0.6 296 Malonyl CoA-acyl carrier protein transacylase OS=Staphylococcus aureus (strain USA300) GN=fabD PE=4 SV=1 Q2FHK8|Q2FHK8_STAA3 0.7 540 tRNA uridine 5-carboxymethylaminomethyl modification enzyme mnmG OS=Staphylococcus aureus (strain USA300) GN=mnmG PE=3 SV=2 Q2FDE9|MNMG_STAA3 0.7 372 Methionine-S-sulfoxide reductase OS=Staphylococcus aureus (strain USA300) GN=msrA PE=3 SV=1 Q2FH14|Q2FH14_STAA3 0.8 503 Methionine aminopeptidase OS=Staphylococcus aureus (strain USA300) GN=map PE=3 SV=1 Q2FFK7|Q2FFK7_STAA3 0.8 619 Clumping factor A OS=Staphyloco ccus aureus (strain USA300) GN=clfA PE=3 SV=1 Q2FIK6|Q2FIK6_STAA3 0.8 23 Probable transglycosylase isaA OS=Staphylococcus aureus (strain USA300) GN=isaA PE=3 SV=1 Q2FDT8|ISAA_STAA3 0.9 170

PAGE 182

45 D-lactate dehydrogenase OS=Staphylococcus aureus (strain USA300) GN=ddh PE=3 SV=1 Q2FDY1|Q2FDY1_STAA3 0.9 263 UPF0435 protein SAUSA300_1861 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1861 PE=3 SV=2 Q2FFL5|Y1861_STAA3 0.9 349 S-ribosylhomocysteine lyase OS =Staphylococcus aureus (strain USA300) GN=luxS PE=3 SV=1 Q2FEZ4|LUXS_STAA3 0.9 478 Carboxyl-terminal protease OS=Staphylococcus aureus (strain USA300) GN=ctpA PE=3 SV=1 Q2FH18|Q2FH18_STAA3 0.9 659 Membrane-associated protein tcaA OS=Staphylococcus aureus (strain USA300) GN=tcaA PE=3 SV=1 Q2FEE1|TCAA_STAA3 0.9 358 Transferrin receptor OS=Staphylo coccus aureus (strain USA300) GN=SAUSA300_0721 PE=4 SV=1 Q2FIQ4|Q2FIQ4_STAA3 1 418 Probable glycine dehydrogenase [decarboxylating] subunit 2 OS=Staphylococcus aureus (strai n USA300) GN=gcvPB PE=3 SV=1 Q2FGI7|GCSPB_STAA3 1 525 Glutamine amidotransferase subunit pdxT OS=Staphylococcus aureus (strain USA300) GN=pdxT PE=3 SV=1 Q2FJC0|PDXT_STAA3 1 713 Aminopeptidase PepS OS=Staphylococcus aureus (strain USA300) GN=pepS PE=4 SV=1 Q2FFL6|Q2FFL6_STAA3 1 66 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2473 PE=4 SV=1 Q2FDX1|Q2FDX1_STAA3 1.1 266 UDP-N-acetylmuramoylalanin e--D-glutamate ligase OS=Staphylococcus aureus (strain USA300) GN=murD PE=3 SV=1 Q2FHQ4|MURD_STAA3 1.1 206 General stress protein 20U OS=Staphylococcus aureus (strain USA300) GN=dps PE=3 SV=1 Q2FEZ0|Q2FEZ0_STAA3 1.3 363 Imidazolonepropionase OS=Staphylococcus aureus (strain USA300) GN=hutI PE=3 SV=1 Q2FEG6|HUTI_STAA3 1.5 16 Elongation factor Ts OS=Staphylococcus aureus (strain USA300) GN=tsf PE=3 SV=1 Q2FHI1|EFTS_STAA3 1.6 284 Superoxide dismutase [Mn/Fe] 1 OS=Staphylococcus aureus (strain USA300) GN=sodA PE=3 SV=1 Q2FGH0|SODM1_STAA3 1.6 513 Arginase OS=Staphylococcus aureus (strain USA300) GN=rocF PE=3 SV=1 Q2FEW8|Q2FEW8_STAA3 1.7 374 GTP cyclohydrolase folE2 OS=Staphylococcus aureus (strain USA300) GN=folE2 PE=3 SV=1 Q2FJ74|GCH4_STAA3 1.8 462 Cysteinyl-tRNA synthetase OS=Staphylococcus aureus (strain USA300) GN=cysS PE=3 SV=1 Q2FJB0|SYC_STAA3 1.8 2 Antibacterial protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1067 PE=4 SV=1 Q2FHR4|Q2FHR4_STAA3 2.2 280 Superoxide dismutase [Mn/Fe] 2 OS=Staphylococcus aureus (strain USA300) GN=sodM PE=3 SV=1 Q2FKC6|SODM2_STAA3 2.2 4 Antibacterial protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1068 PE=4 SV=1 Q2FHR3|Q2FHR3_STAA3 2.7 171

PAGE 183

257 UPF0337 protein SAUSA300_1582 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1582 PE=3 SV=1 Q2FGA1|Y1582_STAA3 2.7 438 Phi77 ORF014-like protein, phage anti-repressor protein OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_1966 PE=4 SV=1 Q2FFB1|Q2FFB1_STAA3 2.9 176 Deoxyribose-phosphate aldolase OS=Staphylococcus aureus (strain USA300) GN=deoC PE=3 SV=1 Q2FKC1|Q2FKC1_STAA3 No Values 291 UDP-N-acetylmuramate--L-alanine ligase OS=Staphylococcus aureus (strain USA300) GN=murC PE=3 SV=1 Q2FFZ8|MURC_STAA3 No Values 331 Pseudouridine synthase OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1090 PE=3 SV=1 Q2FHP1|Q2FHP1_STAA3 No Values 397 Fumarate hydratase, class II OS=Staphylococcus aureus (strain USA300) GN=fumC PE=4 SV=1 Q2FFP4|Q2FFP4_STAA3 No Values 410 Cytosol aminopeptidase OS=Staphylococcus aureus (strain USA300) GN=ampA PE=3 SV=1 Q2FID3|Q2FID3_STAA3 No Values 446 Exodeoxyribonuclease 7 small subunit OS=Staphylococcus aureus (strain USA300) GN=xseB PE=3 SV=1 Q2FGL1|EX7S_STAA3 No Values 449 Protein grpE OS=Staphylococcus aureus (strain USA300) GN=grpE PE=3 SV=1 Q2FGE2|GRPE_STAA3 No Values 454 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1336 PE=4 SV=1 Q2FGZ5|Q2FGZ5_STAA3 No Values 463 50S ribosomal protein L15 OS=Staphylococcus aureus (strain USA300) GN=rplO PE=3 SV=1 Q2FEQ8|RL15_STAA3 No Values 465 30S ribosomal protein S15 OS=Staphylococcus aureus (strain USA300) GN=rpsO PE=3 SV=1 Q2FHG5|RS15_STAA3 No Values 474 Serine-aspartate repeat-containing protein D OS=Staphylococcus aureus (strain USA300) GN=sdrD PE=3 SV=1 Q2FJ78|SDRD_STAA3 No Values 488 L-lactate permease OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2313 PE=4 SV=1 Q2FED0|Q2FED0_STAA3 No Values 494 Putative zinc-binding dehydrogena se OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2317 PE=4 SV=1 Q2FEC6|Q2FEC6_STAA3 No Values 512 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1684 PE=4 SV=1 Q2FG00|Q2FG00_STAA3 No Values 518 Esterase-like protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2296 PE=4 SV=1 Q2FEE7|Q2FEE7_STAA3 No Values 520 3-methyl-2-oxobutanoate hydroxymethyltransferase OS=Staphylococcus aureus (strain USA300) GN=panB PE=3 SV=2 Q2FDR0|PANB_STAA3 No Values 526 ATP synthase epsilon chain OS=Staphylococcus aureus (strain USA300) GN=atpC PE=3 SV=1 Q2FF25|ATPE_STAA3 No Values 528 D-isomer specific 2-hydroxyacid dehydrogenase family protein OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_2496 PE=3 SV=1 Q2FDU8|Q2FDU8_STAA3 No Values 529 Coenzyme A disulfide reductase OS=Staphylococcus aureus (strain USA300) GN=cdr PE=3 SV=1 Q2FIA5|CDR_STAA3 No Values 172

PAGE 184

530 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0383 PE=4 SV=1 Q2FJN1|Q2FJN1_STAA3 No Values 532 Molybdopterin biosynthesis protein A OS=Staphylococcus aureus (strain USA300) GN=moeA PE=4 SV=1 Q2FEL9|Q2FEL9_STAA3 No Values 533 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1171 PE=3 SV=1 Q2FHG0|Q2FHG0_STAA3 No Values 536 Adenine phosphoribosyltransferase OS=Staphylococcus aureus (strain USA300) GN=apt PE=3 SV=1 Q2FG92|APT_STAA3 No Values 543 Methionine import ATP-binding pr otein MetN 2 OS=Staphylococcus aureus (strain USA300) GN=metN2 PE=3 SV=1 Q2FII2|METN2_STAA3 No Values 547 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2482 PE=4 SV=1 Q2FDW2|Q2FDW2_STAA3 No Values 549 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-acetyltransferase OS=Staphylococcus aureus (strain USA300) GN=dapH PE=3 SV=1 Q2FH41|DAPH_STAA3 No Values 554 TelA-like protein SAUSA300_1299 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1299 PE=3 SV=1 Q2FH32|TELL_STAA3 No Values 561 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0749 PE=4 SV=1 Q2FIM7|Q2FIM7_STAA3 No Values 572 DNA topoisomerase 1 OS=Staphylococcus aureus (strain USA300) GN=topA PE=3 SV=2 Q2FHI8|TOP1_STAA3 No Values 573 ABC transporter, ATP-binding protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2357 PE=4 SV=1 Q2FE86|Q2FE86_STAA3 No Values 576 Putative N-acetyltransferase OS=S taphylococcus aureus (strain USA300) GN=SAUSA300_2631 PE=3 SV=1 Q2FDG3|Q2FDG3_STAA3 No Values 581 DNA mismatch repair protein mutL OS=Staphylococcus aureus (strain USA300) GN=mutL PE=3 SV=1 Q2FHE2|MUTL_STAA3 No Values 588 Purine nucleoside phosphorylase OS=Staphylococcus aureus (strain USA300) GN=deoD PE=3 SV=1 Q2FEZ1|Q2FEZ1_STAA3 No Values 591 UPF0473 protein SAUSA300_1572 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1572 PE=3 SV=1 Q2FGB1|Y1572_STAA3 No Values 593 Glycolytic operon regulator OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0755 PE=4 SV=1 Q2FIM2|Q2FIM2_STAA3 No Values 598 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1536 PE=4 SV=1 Q2FGE7|Q2FGE7_STAA3 No Values 600 Sensor protein OS=Staphylococcus aureus (strain USA300) GN=srrB PE=3 SV=1 Q2FGP1|Q2FGP1_STAA3 No Values 614 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0173 PE=4 SV=1 Q2FK91|Q2FK91_STAA3 No Values 616 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1180 PE=4 SV=1 Q2FHF1|Q2FHF1_STAA3 No Values 620 Putative teichoic acid biosynth esis protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0251 PE=4 SV=1 Q2FK13|Q2FK13_STAA3 No Values 173

PAGE 185

628 Glycine betaine transporter opuD OS=Staphylococcus aureus (strain USA300) GN=opuD PE=4 SV=1 Q2FH86|Q2FH86_STAA3 No Values 632 UPF0349 protein SAUSA300_0842 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0842 PE=3 SV=1 Q2FID6|Y842_STAA3 No Values 635 Glutamate racemase OS=Staphylococcus aureus (strain USA300) GN=murI PE=3 SV=1 Q2FHT1|MURI_STAA3 No Values 636 PhiPVL ORF41-like protein OS =Staphylococcus aureus (strain USA300) GN=SAUSA300_1961 PE=4 SV=1 Q2FFB6|Q2FFB6_STAA3 No Values 640 DNA ligase OS=Staphylococcus aureus (strain USA300) GN=ligA PE=3 SV=1 Q2FFJ1|DNLJ_STAA3 No Values 643 Putative chromosome partioning protein, ParB family OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_2643 PE=4 SV=1 Q2FDF1|Q2FDF1_STAA3 No Values 648 Glutamyl-aminopeptidase OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1691 PE=4 SV=1 Q2FFZ3|Q2FFZ3_STAA3 No Values 650 Bacterial luciferase family protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1580 PE=4 SV=1 Q2FGA3|Q2FGA3_STAA3 No Values 652 Alpha glucosidase OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1456 PE=3 SV=1 Q2FGM6|Q2FGM6_STAA3 No Values 653 Oligoendopeptidase F OS=Staphylococcus aureus (strain USA300) GN=pepF PE=4 SV=1 Q2FH53|Q2FH53_STAA3 No Values 674 ABC transporter, substrate-binding protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0231 PE=4 SV=1 Q2FK33|Q2FK33_STAA3 No Values 675 PTS system, trehalose-specific IIBC component OS=Staphylococcus aureus (strain USA300) GN=treP PE=4 SV=1 Q2FJG8|Q2FJG8_STAA3 No Values 679 Transcription-repair-coupling fact or OS=Staphylococcus aureus (strain USA300) GN=mfd PE=3 SV=1 Q2FJD8|MFD_STAA3 No Values 680 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2327 PE=4 SV=1 Q2FEB6|Q2FEB6_STAA3 No Values 681 Phosphoglucomutase OS=Staphylococcus aureus (strain USA300) GN=pgcA PE=3 SV=2 Q2FE11|PGCA_STAA3 No Values 684 Putative osmoprotectant ABC transporter, ATP-binding protein OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_0706 PE=4 SV=1 Q2FIR9|Q2FIR9_STAA3 No Values 685 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2404 PE=4 SV=1 Q2FE39|Q2FE39_STAA3 No Values 686 UDP-N-acetylglucosamine 2-epimerase OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2065 PE=3 SV=1 Q2FF17|Q2FF17_STAA3 No Values 688 Para-nitrobenzyl esterase OS=S taphylococcus aureus (strain USA300) GN=pnbA PE=3 SV=1 Q2FE47|Q2FE47_STAA3 No Values 689 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2112 PE=4 SV=1 Q2FEX0|Q2FEX0_STAA3 No Values 690 Transcriptional repressor nrdR OS=Staphylococcus aureus (strain USA300) GN=nrdR PE=3 SV=1 Q2FG51|NRDR_STAA3 No Values 174

PAGE 186

691 UTP--glucose-1-phosphate uridyly ltransferase OS=Staphylococcus aureus (strain USA300) GN=gtaB PE=3 SV=2 Q2FE05|GTAB_STAA3 No Values 692 Uroporphyrinogen decarboxylase OS=S taphylococcus aureus (strain USA300) GN=hemE PE=3 SV=1 Q2FFR2|DCUP_STAA3 No Values 693 Aminotransferase, class I OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0952 PE=3 SV=1 Q2FI28|Q2FI28_STAA3 No Values 694 Indole-3-pyruvate decar boxylase OS=Staphylococcus aureus (strain USA300) GN=ipdC PE=3 SV=1 Q2FK74|Q2FK74_STAA3 No Values 695 Monooxygenase family protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2255 PE=4 SV=1 Q2FEI8|Q2FEI8_STAA3 No Values 703 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0814 PE=4 SV=1 Q2FIG4|Q2FIG4_STAA3 No Values 704 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0740 PE=4 SV=1 Q2FIN6|Q2FIN6_STAA3 No Values 706 DHH subfamily 1 protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0014 PE=4 SV=1 Q2FKP2|Q2FKP2_STAA3 No Values 707 Acetyltransferase, GNAT family OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0451 PE=4 SV=1 Q2FJG5|Q2FJG5_STAA3 No Values 709 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1010 PE=4 SV=1 Q2FHX0|Q2FHX0_STAA3 No Values 710 Acetyl-CoA carboxylase, biotin carboxylase OS=Staphylococcus aureus (strain USA300) GN=accC PE=4 SV=1 Q2FGC0|Q2FGC0_STAA3 No Values 712 Formimidoylglutamase OS=Staphylococcus aureus (strain USA300) GN=hutG PE=3 SV=1 Q2FEG2|HUTG_STAA3 No Values 715 Probable GTP-binding protein engB OS=Staphylococcus aureus (strain USA300) GN=engB PE=3 SV=1 Q2FG63|ENGB_STAA3 No Values 716 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2447 PE=4 SV=1 Q2FDZ7|Q2FDZ7_STAA3 No Values 718 NADH-dependent flavin oxidoreductase OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0859 PE=4 SV=1 Q2FIB9|Q2FIB9_STAA3 No Values 720 Putative phage infection protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2578 PE=4 SV=1 Q2FDL6|Q2FDL6_STAA3 No Values 723 Diacylglycerol kinase OS=Staphylo coccus aureus (strain USA300) GN=dagK PE=3 SV=1 Q2FFJ7|DAGK_STAA3 No Values 724 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1671 PE=4 SV=1 Q2FG13|Q2FG13_STAA3 No Values 725 Delta-aminolevulinic acid dehydratase OS=Staphylococcus aureus (strain USA300) GN=hemB PE=3 SV=1 Q2FG68|Q2FG68_STAA3 No Values 726 Glycosyl transferase, group 1 family protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1349 PE=4 SV=1 Q2FGY2|Q2FGY2_STAA3 No Values 728 Ribonuclease R OS=Staphylococcus aureus (strain USA300) GN=rnr PE=3 SV=1 Q2FIL3|Q2FIL3_STAA3 No Values 175

PAGE 187

732 Iron-dependent repressor OS=S taphylococcus aureus (strain USA300) GN=SAUSA300_0621 PE=4 SV=1 Q2FJ04|Q2FJ04_STAA3 No Values 733 SIS domain protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0556 PE=4 SV=1 Q2FJ69|Q2FJ69_STAA3 No Values 734 Serine acetyltransferase OS=Staphy lococcus aureus (strain USA300) GN=cysE PE=4 SV=1 Q2FJB1|Q2FJB1_STAA3 No Values 735 Putative membrane protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0279 PE=4 SV=1 Q2FJY5|Q2FJY5_STAA3 No Values 738 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0466 PE=4 SV=1 Q2FJF3|Q2FJF3_STAA3 No Values 739 UPF0403 protein SAUSA300_1321 OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1321 PE=3 SV=1 Q2FH10|Y1321_STAA3 No Values 741 DNA polymerase III polC-type OS=Staphylococcus aureus (strain USA300) GN=polC PE=3 SV=1 Q2FHH4|DPO3_STAA3 No Values 742 Putative peptidase OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2087 PE=4 SV=1 Q2FEZ5|Q2FEZ5_STAA3 No Values 743 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2394 PE=4 SV=1 Q2FE49|Q2FE49_STAA3 No Values 744 Queuine tRNA-ribosyltransferase OS =Staphylococcus aureus (strain USA300) GN=tgt PE=3 SV=1 Q2FG88|TGT_STAA3 No Values 745 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2527 PE=4 SV=1 Q2FDR7|Q2FDR7_STAA3 No Values 746 Glycine betaine/carnitine/choline ABC transporter OS=Staphylococcus aureus (strain USA300) GN=opuCc PE=4 SV=1 Q2FE52|Q2FE52_STAA3 No Values 747 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2381 PE=4 SV=1 Q2FE62|Q2FE62_STAA3 No Values 748 tRNA (guanine-N(7)-)-methyltransferase OS=Staphylococcus aureus (strain USA300) GN=trmB PE=3 SV=1 Q2FFZ0|TRMB_STAA3 No Values 749 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1534 PE=4 SV=1 Q2FGE9|Q2FGE9_STAA3 No Values 753 HAD-superfamily hydrolase, subfamily IA, variant 1 OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_0557 PE=4 SV=1 Q2FJ68|Q2FJ68_STAA3 No Values 755 Ribosomal RNA small subun it methyltransferase G OS=Staphylococcus aureus (strain USA300) GN=rsmG PE=3 SV=1 Q2FDF0|RSMG_STAA3 No Values 757 Acetyl-coenzyme A carboxylase carboxyl transferase subunit beta OS=Staphylococcus aureus (strai n USA300) GN=accD PE=3 SV=1 Q2FG37|ACCD_STAA3 No Values 758 Porphobilinogen deaminase OS=Staphylococcus aureus (strain USA300) GN=hemC PE=3 SV=1 Q2FG66|HEM3_STAA3 No Values 760 Thymidylate synthase OS=Staphylococcus aureus (strain USA300) GN=thyA PE=3 SV=1 Q2FH11|TYSY_STAA3 No Values 761 Glycine betaine aldehyde dehydr ogenase OS=Staphylococcus aureus (strain USA300) GN=betB PE=3 SV=1 Q2FDP8|Q2FDP8_STAA3 No Values 176

PAGE 188

762 Preprotein translocase subunit s ecY OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2184 PE=3 SV=1 Q2FEQ9|Q2FEQ9_STAA3 No Values 763 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2143 PE=4 SV=1 Q2FEU9|Q2FEU9_STAA3 No Values 765 Peptide methionine sulfoxide re ductase MsrA OS=Staphylococcus aureus (strain USA300) GN=msrA PE=3 SV=1 Q2FH75|Q2FH75_STAA3 No Values 767 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1069 PE=4 SV=1 Q2FHR2|Q2FHR2_STAA3 No Values 770 Serine-aspartate repeat-containing protein E OS=Staphylococcus aureus (strain USA300) GN=sdrE PE=3 SV=1 Q2FJ77|SDRE_STAA3 No Values 772 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1650 PE=4 SV=1 Q2FG34|Q2FG34_STAA3 No Values 773 Peptidase T OS=Staphylococcus au reus (strain USA300) GN=pepT PE=3 SV=1 Q2FIP8|PEPT_STAA3 No Values 774 DNA topoisomerase 3 OS=Staphylococcus aureus (strain USA300) GN=topB PE=3 SV=1 Q2FEN5|TOP3_STAA3 No Values 775 Oligopeptide ABC transporter, substrate-binding protein OS=Staphylococcus aureus (strain USA300) GN=oppA PE=4 SV=1 Q2FI87|Q2FI87_STAA3 No Values 776 Putative lipoprotein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0079 PE=4 SV=1 Q2FKI1|Q2FKI1_STAA3 No Values 777 Ferredoxin--NADP reductase OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2319 PE=3 SV=1 Q2FEC4|FENR_STAA3 No Values 778 Urea amidolyase-related protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0702 PE=4 SV=1 Q2FIS3|Q2FIS3_STAA3 No Values 779 Guanylate kinase OS=Staphylococcus aureus (strain USA300) GN=gmk PE=3 SV=1 Q2FHM9|KGUA_STAA3 No Values 780 Glycerol uptake facilitator OS=S taphylococcus aureus (strain USA300) GN=glpF PE=3 SV=1 Q2FHE0|Q2FHE0_STAA3 No Values 781 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1011 PE=4 SV=1 Q2FHW9|Q2FHW9_STAA3 No Values 782 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1908 PE=4 SV=1 Q2FFG8|Q2FFG8_STAA3 No Values 783 FtsK/SpoIIIE family protein OS =Staphylococcus aureus (strain USA300) GN=SAUSA300_1687 PE=4 SV=1 Q2FFZ7|Q2FFZ7_STAA3 No Values 784 MutS2 protein OS=Staphylococcus aureus (strain USA300) GN=mutS2 PE=3 SV=1 Q2FHT7|MUTS2_STAA3 No Values 785 2-succinyl-5-enolpyruvyl-6-hydr oxy-3-cyclohexene-1-carboxylate synthase OS=Staphylococcus au reus (strain USA300) GN=menD PE=3 SV=1 Q2FI34|MEND_STAA3 No Values 786 Sensor protein kinase walK OS=Staphylococcus aureus (strain USA300) GN=walK PE=3 SV=1 Q2FKN7|WALK_STAA3 No Values 789 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1081 PE=4 SV=1 Q2FHQ0|Q2FHQ0_STAA3 No Values 177

PAGE 189

790 DNA polymerase III, gamma and tau subunits OS=Staphylococcus aureus (strain USA300) GN=dnaX PE=4 SV=1 Q2FJG4|Q2FJG4_STAA3 No Values 791 Pantothenate syntheta se OS=Staphylococcus aureus (strain USA300) GN=panC PE=3 SV=1 Q2FDR1|PANC_STAA3 No Values Reference Missing 346 Cysteine desulfurases, SufS subfamily subfamily OS=Staphylococcus aureus (strain USA300) GN=sufS PE=3 SV=1 Q2FIF8|Q2FIF8_STAA3 Reference Missing 395 Aldehyde dehydrogenase OS=Staphylococcus aureus (strain USA300) GN=aldA2 PE=3 SV=1 Q2FFH5|Q2FFH5_STAA3 Reference Missing 455 Transcriptional regulator OS=S taphylococcus aureus (strain USA300) GN=SAUSA300_0958 PE=4 SV=1 Q2FI22|Q2FI22_STAA3 Reference Missing 499 HPr kinase/phosphorylase OS=Staphylococcus aureus (strain USA300) GN=hprK PE=3 SV=1 Q2FIN3|HPRK_STAA3 Reference Missing 524 FMN-dependent NADPH-azoreducta se OS=Staphylococcus aureus (strain USA300) GN=azo1 PE=3 SV=1 Q2FJ80|AZO1_STAA3 Reference Missing 605 Transcriptional regulator, Fur fa mily OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1448 PE=4 SV=1 Q2FGN4|Q2FGN4_STAA3 630 Oligopeptide ABC transporter, ATP-binding protein OS=Staphylococcus aureus (strain USA300) GN=oppD PE=4 SV=1 Q2FI89|Q2FI89_STAA3 Reference Missing 663 Methylenetetrahydrofolate--tRNA-(uracil-5-)-methyltransferase trmFO OS=Staphylococcus aureus (strain USA300) GN=trmFO PE=3 SV=1 Q2FHI7|TRMFO_STAA3 Reference Missing 672 Putative iron compound ABC transporter, iron compound-binding protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0598 PE=4 SV=1 Q2FJ27|Q2FJ27_STAA3 Reference Missing Reference Missing 677 Ribosome-binding factor A OS=Staphylococcus aureus (strain USA300) GN=rbfA PE=3 SV=1 Q2FHG8|RBFA_STAA3 Reference Missing 708 Peptidase, M20/M25/M40 family OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1460 PE=4 SV=1 Q2FGM2|Q2FGM2_STAA3 Reference Missing 730 Undecaprenyl-diphosphatase OS=S taphylococcus aureus (strain USA300) GN=uppP PE=3 SV=1 Q2FIV6|UPPP_STAA3 Value Missing 201 Alpha-hemolysin OS=Staphylococc us aureus (strain USA300) GN=SAUSA300_1058 PE=4 SV=1 Q2FHS2|Q2FHS2_STAA3 Value Missing 242 Transcriptional regulatory protein walR OS=Staphylococcus aureus (strain USA300) GN=walR PE=3 SV=1 Q2FKN8|WALR_STAA3 Value Missing 328 S1 RNA binding domain protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2021 PE=4 SV=1 Q2FF61|Q2FF61_STAA3 Value Missing 362 Probable acetyl-CoA acyltransferase OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0355 PE=3 SV=1 Q2FJQ9|THLA_STAA3 Value Missing 365 Ferrochelatase OS=Staphylococcus aureus (strain USA300) GN=hemH PE=3 SV=1 Q2FFR3|Q2FFR3_STAA3 Value Missing 369 Alpha-acetolactate synthase OS=S taphylococcus aureus (strain USA300) GN=alsS PE=3 SV=1 Q2FES6|Q2FES6_STAA3 416 Proline dipeptidase OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1491 PE=3 SV=1 Q2FGJ2|Q2FGJ2_STAA3 Value Missing 178

PAGE 190

Value Missing 423 Type I restriction-modi fication system, M subunit OS=Staphylococcus aureus (strain USA300) GN=hsdM PE=4 SV=1 Q2FFT5|Q2FFT5_STAA3 (+1) Value Missing 439 Peptide chain release factor 3 OS=Staphylococcus aureus (strain USA300) GN=prfC PE=3 SV=1 Q2FI57|RF3_STAA3 Value Missing 457 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2446 PE=4 SV=1 Q2FDZ8|Q2FDZ8_STAA3 468 Alkaline phosphatase synthesis tran scriptional regulatory protein PhoP OS=Staphylococcus aureus (strain USA300) GN=phoP PE=4 SV=1 Q2FG44|Q2FG44_STAA3 Value Missing Value Missing 493 Chorismate synthase OS=Staphylococcus aureus (strain USA300) GN=aroC PE=3 SV=1 Q2FGX4|AROC_STAA3 Value Missing 510 DNA topoisomerase IV, subunit B OS=Staphylococcus aureus (strain USA300) GN=parE PE=3 SV=1 Q2FH81|Q2FH81_STAA3 Value Missing 514 Protein kinase OS=Staphylococcus aureus (strain USA300) GN=pknB PE=4 SV=1 Q2FHL8|Q2FHL8_STAA3 Value Missing 527 Low molecular weight protein-tyrosine-phosphatase ptpA OS=Staphylococcus aureus (strain USA300) GN=ptpA PE=3 SV=1 Q2FFL4|PTPA_STAA3 Value Missing 537 Pyridine nucleotide-disulfide oxidoreductase OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1369 PE=4 SV=1 Q2FGW2|Q2FGW2_STAA3 539 Phosphotransferase system, fr uctose-specific IIABC component OS=Staphylococcus aureus (s train USA300) GN=SAUSA300_2576 PE=4 SV=1 Q2FDL8|Q2FDL8_STAA3 Value Missing Value Missing 553 Hydrolase, HAD-superfamily, s ubfamily IIIA OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1557 PE=4 SV=1 Q2FGC6|Q2FGC6_STAA3 Value Missing 555 Panton-Valentine leukocidin, LukF-PV OS=Staphylococcus aureus (strain USA300) GN=lukF-PV PE=4 SV=1 Q2FGV0|Q2FGV0_STAA3 Value Missing 556 Tributyrin esterase OS=Staphylococcus aureus (strain USA300) GN=estA PE=4 SV=1 Q2FDN0|Q2FDN0_STAA3 Value Missing 569 Aldo/keto reductase family pr otein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0589 PE=4 SV=1 Q2FJ36|Q2FJ36_STAA3 Value Missing 570 Pur operon repressor OS=Staphylococcus aureus (strain USA300) GN=purR PE=4 SV=1 Q2FJE6|Q2FJE6_STAA3 Value Missing 577 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2525 PE=4 SV=1 Q2FDR9|Q2FDR9_STAA3 Value Missing 584 Thymidylate kinase OS=Staphylo coccus aureus (strain USA300) GN=tmk PE=3 SV=1 Q2FJG0|KTHY_STAA3 Value Missing 595 4,4'-diaponeurosporene oxidase OS =Staphylococcus aureus (strain USA300) GN=crtP PE=3 SV=1 Q2FDU3|CRTP_STAA3 Value Missing 597 Protoporphyrinogen oxidase OS=Staphylococcus aureus (strain USA300) GN=hemG PE=4 SV=1 Q2FFR4|Q2FFR4_STAA3 Value Missing 604 ABC transporter, ATP-binding protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2399 PE=4 SV=1 Q2FE44|Q2FE44_STAA3 608 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1857 PE=4 SV=1 Q2FFL9|Q2FFL9_STAA3 Value Missing 179

PAGE 191

Value Missing 611 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0812 PE=4 SV=1 Q2FIG6|Q2FIG6_STAA3 Value Missing 612 Hydrolase, TatD family OS=Staphy lococcus aureus (strain USA300) GN=SAUSA300_0468 PE=4 SV=1 Q2FJF1|Q2FJF1_STAA3 613 5-methyltetrahydropteroyltr iglutamate--homocysteine methyltransferase OS=Staphylococcus aureus (strain USA300) GN=metE PE=3 SV=1 Q2FJQ7|METE_STAA3 Value Missing Value Missing 622 Hydroxymethylglutaryl-CoA reductase OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2483 PE=3 SV=1 Q2FDW1|Q2FDW1_STAA3 Value Missing 623 Glutamyl-aminopeptidase OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2400 PE=4 SV=1 Q2FE43|Q2FE43_STAA3 Value Missing 633 Carboxylesterase OS=Staphylococcus aureus (strain USA300) GN=est PE=4 SV=1 Q2FIL4|Q2FIL4_STAA3 Value Missing 634 Transcriptional regulator ctsR OS=Staphylococcus aureus (strain USA300) GN=ctsR PE=3 SV=1 Q2FJB8|CTSR_STAA3 Value Missing 642 Universal stress protein family OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0067 PE=4 SV=1 Q2FKJ2|Q2FKJ2_STAA3 Value Missing 645 ABC transporter, ATP-binding protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2288 PE=4 SV=1 Q2FEF5|Q2FEF5_STAA3 Value Missing 646 Ribose 5-phosphate isomerase A OS=Staphylococcus aureus (strain USA300) GN=rpiA PE=3 SV=1 Q2FEG0|Q2FEG0_STAA3 Value Missing 649 Alanine dehydrogenase 2 OS=Staphylococcus aureus (strain USA300) GN=ald2 PE=3 SV=1 Q2FG29|DHA2_STAA3 Value Missing 651 Aminomethyltransferase OS=Staphylococcus aureus (strain USA300) GN=gcvT PE=3 SV=1 Q2FGI5|GCST_STAA3 Value Missing 655 Isochorismate synthase family pr otein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0945 PE=4 SV=1 Q2FI35|Q2FI35_STAA3 Value Missing 657 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0725 PE=4 SV=1 Q2FIQ0|Q2FIQ0_STAA3 Value Missing 661 Primosomal protein DnaI OS=Staphylococcus aureus (strain USA300) GN=dnaI PE=4 SV=1 Q2FG53|Q2FG53_STAA3 Value Missing 664 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0192 PE=4 SV=1 Q2FK72|Q2FK72_STAA3 Value Missing 665 Gamma-hemolysin component B OS=Staphylococcus aureus (strain USA300) GN=hlgB PE=4 SV=1 Q2FE76|Q2FE76_STAA3 Value Missing 673 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0593 PE=4 SV=1 Q2FJ32|Q2FJ32_STAA3 Value Missing 676 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1003 PE=4 SV=1 Q2FHX7|Q2FHX7_STAA3 Value Missing 678 Peptide chain release factor 1 OS=Staphylococcus aureus (strain USA300) GN=prfA PE=3 SV=1 Q2FF10|RF1_STAA3 698 ATPase, AAA family OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1584 PE=4 SV=1 Q2FG99|Q2FG99_STAA3 Value Missing 180

PAGE 192

Value Missing 700 Pseudouridine synthase OS=Staphylococcus aureus (strain USA300) GN=rluB PE=3 SV=1 Q2FGN9|Q2FGN9_STAA3 Value Missing 702 Inositol monophosphatase family pr otein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1007 PE=4 SV=1 Q2FHX3|Q2FHX3_STAA3 Value Missing 705 ABC transporter, permease pr otein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0308 PE=4 SV=1 Q2FJV6|Q2FJV6_STAA3 Value Missing 721 Carbamate kinase 2 OS=Staphyloco ccus aureus (strain USA300) GN=arcC2 PE=3 SV=1 Q2FDM7|ARCC2_STAA3 Value Missing 736 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0042 PE=4 SV=1 Q2FKL6|Q2FKL6_STAA3 Value Missing 740 Urease accessory protein ureG OS=Staphylococcus aureus (strain USA300) GN=ureG PE=3 SV=1 Q2FEK0|UREG_STAA3 Value Missing 756 Putative membrane protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_1864 PE=3 SV=1 Q2FFL2|Q2FFL2_STAA3 Value Missing 764 Haloacid dehalogenase-like hydr olase OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_2102 PE=4 SV=1 Q2FEY0|Q2FEY0_STAA3 769 Putative uncharacterized protein OS=Staphylococcus aureus (strain USA300) GN=SAUSA300_0632 PE=3 SV=1 Q2FIZ3|Q2FIZ3_STAA3 Value Missing Table A13. Complete list of intracellular proteins identified from the USA 300 adaptive mutant at 15 hours. 181

PAGE 193

182 Appendix C. Phyre results. Figure A1. Results of QuickPhyre analysis of COL (top) and USA 100 635 (bottom) amino acid sequences and predicted structures. Red = predicted -helix. Blue = predicted -pleated sheet.


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Prosen, Katherine.
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Investigating the mode of action of a novel _n-sec_-butylthiolated beta-lactam against _staphylococcus aureus_
h [electronic resource] /
by Katherine Prosen.
260
[Tampa, Fla] :
b University of South Florida,
2010.
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Thesis (MS)--University of South Florida, 2010.
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Includes bibliographical references.
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Text (Electronic thesis) in PDF format.
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Mode of access: World Wide Web.
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ABSTRACT: N-sec-butylthioloated beta-lactam (NsBL) is a novel beta-lactam antimicrobial with a mechanism of action proposed to inhibit 3-oxoacyl-acyl carrier protein synthase (ACP) III (FabH), resulting in the inhibition of fatty acid synthesis. It has been suggested that NsβL inhibits FabH indirectly by inactivating coenzyme-A (CoA). CoA is an essential cofactor for numerous proteins involved in glycolysis, the citric acid cycle (TCA), and pyruvate metabolism, in addition to fatty acid biosynthesis. This study aimed to determine the effects of NsBL on a diverse array of laboratory and clinical Staphylococcus aureus isolates by analyzing the mode of resistance in spontaneous and adaptive mutant NsBL-resistant mutants. Phenotypic analysis of the mutants was performed, as well as sequence analysis of fabH; along with comparative proteomic analysis of intracellular proteomes. Our results indicate that NsBL resistance is mediated by drastic changes in the cell wall, oxidative stress response, virulence regulation, and those pathways associated with CoA. It is our conclusion that NsBL has activity towards CoA, resulting in wide-spread effects on metabolism, virulence factor production, stress response, and antimicrobial resistance.
590
Advisor: Lindsey Shaw, Ph.D.
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MRSA
Antibiotic resistance
Fatty acid synthesis inhibitors
NsBL
Beta-lactams
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Dissertations, Academic
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x Biology
Masters.
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t USF Electronic Theses and Dissertations.
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