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Title:
The use of microarrays in the detection of the gene expression of ribulose- 1,5- bisphosphate carboxylase/oxygenase in the marine environment
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Book
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English
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Bailey, Kathryn Lafaye
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University of South Florida
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Tampa, Fla.
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Subjects / Keywords:
Phytoplankton
CBB Pathway
Mississippi River Plume
RbcL
Pelagophyte
Synechococcus
Dissertations, Academic -- Marine Science -- Masters -- USF   ( lcsh )
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Summary:
ABSTRACT: The Calvin-Benson-Bassham (CBB) pathway is the primary pathway for the entry of inorganic carbon in the biosphere. Autotrophic organisms use this cycle to ultimately convert CO2 into carbohydrates using a key enzyme known as ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). The gene that encodes for the large subunit of RubisCO is rbcL and detection of its expression can be used to determine the autotrophic organisms present in the environment. Recently, microarrays have been used to study functional gene expression from environmental samples such as those obtained from sediments and soil. The purpose of this thesis is to combine microarray technology and rbcL expression analysis to investigate phytoplankton populations in the Mississippi River Plume (MRP). Initially, a macroarray was constructed to determine its capabilities of quantifying gene expression in MRP.PCR amplicons were spotted onto a nylon membrane and labeled transcript RNA was hybridized to each array. Due to the large amount of cross hybridization that was observed, a microarray was used. Microarray analysis revealed large amounts of Synechococcus, pelagophyte and prymnesiophyte expression in the surface waters. Furthermore, there was no chlorophte or Prochlorococcus expression observed in the surface waters. Subsurface microarray data showed high levels of pelagophytes and other Form ID organisms. A significant chlorophyte signal was also observed in the subsurface. This study provides a third level of specificity at which phylogenetic diversity has been sampled in the MRP. Although a limited number of samples were analyzed by microarrays, this technology shows promise and this study was viewed as a pilot for their application.The rbcL probes designed were based upon published sequences from 2003 and we now have a much greater understanding of the diversity of rbcL-containing phytoplanktonic phylotypes. Future studies should employ this knowledge for judicious probe selection.
Thesis:
Thesis (M.S.)--University of South Florida, 2007.
Bibliography:
Includes bibliographical references.
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Mode of access: World Wide Web.
Statement of Responsibility:
by Kathryn Lafaye Bailey.
General Note:
Title from PDF of title page.
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Document formatted into pages; contains 73 pages.

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oclc - 187968261
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ABSTRACT: The Calvin-Benson-Bassham (CBB) pathway is the primary pathway for the entry of inorganic carbon in the biosphere. Autotrophic organisms use this cycle to ultimately convert CO2 into carbohydrates using a key enzyme known as ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). The gene that encodes for the large subunit of RubisCO is rbcL and detection of its expression can be used to determine the autotrophic organisms present in the environment. Recently, microarrays have been used to study functional gene expression from environmental samples such as those obtained from sediments and soil. The purpose of this thesis is to combine microarray technology and rbcL expression analysis to investigate phytoplankton populations in the Mississippi River Plume (MRP). Initially, a macroarray was constructed to determine its capabilities of quantifying gene expression in MRP.PCR amplicons were spotted onto a nylon membrane and labeled transcript RNA was hybridized to each array. Due to the large amount of cross hybridization that was observed, a microarray was used. Microarray analysis revealed large amounts of Synechococcus, pelagophyte and prymnesiophyte expression in the surface waters. Furthermore, there was no chlorophte or Prochlorococcus expression observed in the surface waters. Subsurface microarray data showed high levels of pelagophytes and other Form ID organisms. A significant chlorophyte signal was also observed in the subsurface. This study provides a third level of specificity at which phylogenetic diversity has been sampled in the MRP. Although a limited number of samples were analyzed by microarrays, this technology shows promise and this study was viewed as a pilot for their application.The rbcL probes designed were based upon published sequences from 2003 and we now have a much greater understanding of the diversity of rbcL-containing phytoplanktonic phylotypes. Future studies should employ this knowledge for judicious probe selection.
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PAGE 1

The Use of Microarrays in the Detection of the Gene Expression of Ribulose1,5Bisphosphate Carboxylase/Oxygenase ( RubisCO) in the Marine Environment by Kathryn Lafaye Bailey A thesis submitted in partial fulfillment of the requirements for the degree of Masters of Science College of Marine Science University of South Florida Major Professor: John H. Paul, III, Ph.D. Ashanti J. Pyrtle, Ph.D. Frank Pyrtle, Ph.D. Date of Approval: July 13, 2007 Keywords: Phytoplankton, CBB Pathway, Mississippi River Plume rbcL pelagophyte, Synechococcus Copyright 2007, Kathryn Lafaye Bailey

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Dedication This thesis is dedicated to my mother who has suppo rted me in everything I ever wanted to do in life. Thank you for always being there to give me words of encouragement when I needed them and for the many swift and firm kicks in the behind that kept me going when I felt like giving up. I love you and I live to make you proud of me. I would also like to dedicate this work to the late Mrs. Adrienn e Turner.

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Acknowledgements First and foremost, I want to thank God for His man y blessings. I would also like to thank Him for placing the following people in my li fe, for without them, the completion of this degree would not have been possible: my adv isor, Dr. John H. Paul for allowing me to work with him these past 2 years and for his guidance toward the completion of my research and thesis; Dr. Ashanti Pyrtle for writing the Bridge to the Doctorate Proposal that has funded me for the past two years and also for founding MSPHDS which has enriched my graduate school experience; Mr. Bernard Batson for that email he sent to me on July 26, 2004 that changed my life forever; Amy Long, Jennifer Mobberley, and Michelle Del la Rosa for befriending me when nobody else would; Erica Casper and Dr. Stacey Patterson for always “keeping it real”; Dr. Lauren McDaniel for all of the motherly advice when I could not reach my mother; a nd last but not least, Dr. David John for his patience and friendship.

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i Table of Contents List of Tables iii List of Figures iv Abstract vi Chapter 1Introduction 1 1.1 Primary Production in the Ocean 2 1.2 The CBB Pathway and RuBisCO 4 1.3 Forms and Structure of RuBisCO 8 1.4 RuBisCO in the Marine Environment 9 1.5 Use of Array Technology in the Marine Environm ent 11 1.6 Real Time PCR in the Marine Environment 14 Chapter 2Macroarray Detection of RuBisCO in the M arine Environment 17 Chapter Summary 17 2.1 Introduction 18 2.2 Methods 20 2.2.1 Plasmid Extraction 20 2.2.2 PCR Amplification and Purification 21 2.2.3 Design of preliminary macroarrays 22 2.2.4 Transcript Production 23 2.2.5 Labeling of Transcripts with Biotin 24 2.2.6 Hybridization and Detection 24 2.2.7 Design of Control Arrays 25 2.2.8 Transcription, Hybridization and Detection 26 2.3 Results 27 2.4 Discussion 30 Chapter 3Microarray Detection of RuBisCO in the M ississippi River Plume 32 Chapter Sumary 32 3.1 Introduction 33 3.2 Methods 35 3.2.1 RNA Extraction 35 3.2.2 Transcription 35 3.2.3 Positive Controls 37 3.2.4 Labeling 37 3.2.5 Coupling of dUTPaa-labeled Target to Cy3 D ye 38

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ii 3.2.6 Microarray Construction and Hybridization 38 3.2.7 Washing and Scanning 40 3.3 Results 42 3.3.1 Microarray Performance 42 3.3.2 Tampa Bay 43 3.3.3 Surface Samples 43 3.3.4 Subsurface Samples 44 3.4 Discussion 47 List of References 59 Appendices 64 Appendix A 65

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iii List of Tables Table 2.1 List of clone names, clade and common nam es. 22 Table 3.1 List of cruise samples showing depth, amo unt filtered and type of filter used. 35 Table 3.2 List of transcripts used as positive cont rols for the microarray performance tests. 37

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iv List of Figures Figure 1.1 CBB Pathway. 5 Figure 1.2 Quaternary structure of RubisCO. 8 Figure 1.3 Microarray technology process. 12 Figure 1.4 Comparison of (a) microarray and (b) mac roarray. 13 Figure 2.1 SeaWiFs chlorophyll satellite imagery of the Mississippi River Plume during time of sampling in 2001 overlaid with posi tion of sampling stations. 21 Figure 2.2 Preliminary blot schematic. 23 Figure 2.3 Control blot schematic. 26 Figure 2.4 First successful hybridization experimen t. 27 Figure 2.5 Fully dotted array experiments. 29 Figure 3.1 SeaWiFS ocean color satellite image of t he Mississippi River Plume overlaid with cruise stations. 36 Figure 3.2 Types of array slides. 41 Figure 3.3 Hybridization of clone 3SY1 (HL Prochlor ococcus) to array. 52 Figure 3.4 Hybridization of clone 6SY3 (Synechococc us) to array. 52 Figure 3.5 Hybridization of clones P994GY7 (LL Prochlorococcus ) and P994CH1 (pelagophyte) to array. 53 Figure 3.6 Hybridization of clone 8CH12 (chrysophyt e) and 1CH1 (prymnesiophyte) to array. 53 Figure 3.7 Hybridization of cDNA from water collect ed from Fort Desoto and amplified with Form ID primers. 54

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v Figure 3.8 Hybridization of cDNA from Fort Desoto a nd Station 2G. 54 Figure 3.9 Hybridization of cDNA obtained from Stat ion 5A. 55 Figure 3.10 Hybridization of cDNA obtained from Sta tion 5A. 55 Figure 3.11 Hybridization of cDNA obtained from Sta tion 6A that was amplified with From ID primers. 56 Figure 3.12 Hybridization of cDNA obtained from St ation 6A that was amplified with both Form IA/IB and Form ID primers. 56 Figure 3.13 Hybridization of cDNA obtained from Sta tion 8B. 57 Figure 3.14 Hybridization of cDNA from Station 1B a nd transcript 4SY39 (prasinophyte). 57 Figure 3.15 Hybridization of cDNA obtained from Sta tion 2G. 58 Figure 3.16 Hybridization of cDNA obtained from Sta tion 7X and transcript 6SY3 ( Synechococcus ). 58

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vi The Use of Microarrays in the Detection of the Gene Expression of Ribulose1,5bisphosphate carboxylase/oxygenase (RubisCO) in the Marine Environment Kathryn L. Bailey ABSTRACT The Calvin-Benson-Bassham (CBB) pathway is the prim ary pathway for the entry of inorganic carbon in the biosphere. Autotrophic organisms use this cycle to ultimately convert CO2 into carbohydrates using a key enzyme known as rib ulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). The gene that enco des for the large subunit of RubisCO is rbcL and detection of its expression can be used to dete rmine the autotrophic organisms present in the environment. Recently, mi croarrays have been used to study functional gene expression from environmental sampl es such as those obtained from sediments and soil. The purpose of this thesis is to combine microarray technology and rbcL expression analysis to investigate phytoplankton po pulations in the Mississippi River Plume (MRP). Initially, a macroarray was con structed to determine its capabilities of quantifying gene expression in MRP. PCR amplico ns were spotted onto a nylon membrane and labeled transcript RNA was hybridized to each array. Due to the large amount of cross hybridization that was observed, a microarray was used. Microarray analysis revealed large amounts of Synechococcus pelagophyte and prymnesiophyte expression in the surface waters. Furthermore, the re was no chlorophte or

PAGE 10

vii Prochlorococcus expression observed in the surface waters. Subsur face microarray data showed high levels of pelagophytes and other Form I D organisms. A significant chlorophyte signal was also observed in the subsurf ace. This study provides a third level of specificity at which phylogenetic diversity has been sampled in the MRP. Although a limited number of samples were analyzed by microarr ays, this technology shows promise and this study was viewed as a pilot for their appl ication. The rbcL probes designed were based upon published sequences from 2003 and we now have a much greater understanding of the diversity of rbcL -containing phytoplanktonic phylotypes. Future studies should employ this knowledge for judicious probe selection.

PAGE 11

1 Chapter 1 Introduction Environmental gene expression studies provide a mea ns of determining what biological and biochemical activities are being fac ilitated by microorganisms in the environment. Gene expression is controlled by cell ular responses to changes the cell’s environment (44) and can be broken down into two pr ocesses: transcription and translation. Transcription is a process in which a n enzyme, known as RNA polymerase, makes messenger RNA (mRNA), or an RNA copy, of gene (s) on a DNA strand. In translation, the mRNA is read, or translated, into an amino acid sequence to form protein via the action of ribosomes. The expression, or la ck thereof, of genes in the ocean is dependent upon many environmental factors or stress es. These can include but are not limited to nutrient availability, salinity, pH and temperature. Any modifications to these factors can inhibit the transcription of a gene or increase the amount of expression and thereby affect the production of the protein that i t encodes. The measurement of the expression of discreet gene s in the ocean is in its infancy. In a study of nitrogenase activity in Trichodesmium sp. researchers found that transcription of nifHDK was on a diel pattern (7). The criteria for an en dogenous rhythm are the ability to persist under constant environme ntal conditions, the ability to maintain a

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2 cyclic pattern in different temperatures and the cy cle must occur in a diel period (7, 50). The results showed that transcript production in Tichodesmium sp. maintained a relative constant 24 h cycle in uniform environmental condit ions and with slight temperature change (7). In another study, mRNA expression of a high-affini ty phosphate transporter gene ( TcPHO ) of the prasinophyte, Tetraselmis chui was shown to be correlated to nutrient availability (8). Chung et al. (2003) extracted th e total RNA from a growth-dependent subtracted T. chui cDNA library made from cultures both rich and deple ted of nitrate and phosphate. Real time polymerase chain reaction (PC R) analysis showed that TcPHO mRNA expression in the phosphorous-replete cultures increased during all 4 days of the experiment indicating expression of TcPHO linked to phosphorus availability (8). Cultures replete in nitrogen showed a similar growt h pattern to that of the phosphorusreplete cultures, however, the TcPHO mRNA expression levels of the low-nitrate cultures remained low throughout the course of the experimen t (8). 1.1 Primary Production in the Ocean Primary production is the synthesis and storage of organic molecules during the growth and reproduction of photosynthetic and other autotrophic organisms. Most oceanic primary production occurs in the photic zon e which is located in the upper 200 m of the water column (10). Primary production in th e ocean accounts for nearly half of the earth’s primary production, which ranges from 35 to 65 Gt of carbon annually (10) thus resulting in the ocean acting as a large sink for C O2 (44). Phytoplankton located in the

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3 photic zone use photosynthesis to store energy and release oxygen back into the atmosphere and surrounding waters (27). Marine phy toplankton are responsible for approximately half of Earth’s annual oxygen product ion (44). During photosynthesis, CO2 is taken up by phytoplankton and/or autotrophic ba cteria and incorporated into their cells with oxygen and water as byproducts. The ove rall reaction for photosynthesis is n CO2 + 2 n H2O = (CH2O)n + n O2 + n H2O (24). The factors that control the rates of photosynthesis and thus, primary productivity are t hose that manage photosystems and their rates of reaction, as well as the rates of th e dark reactions (24). The Unites States Geological Survey lists the Missi ssippi River as the second largest river in the United States. The Environmen tal Protection Agency measures it to be 2,302 miles in length from its source, Lake Itas ca in Clearwater Minnesota, to where it empties out into the Gulf of Mexico (43). As the w orld’s 7th largest river in its amount of discharge, an annual average of 10-35 x 103 m3 s-1, the Mississippi River is responsible for approximately 41 % of the drainage of the Unite d States and is responsible for more that 70% of the freshwater input into the Gulf of M exico (1, 42). Much of the drained land consists of farming areas and thus large amoun ts of nitrate and phosphorous, found in fertilizers, are deposited into the northern Gul f of Mexico. Turner and Rabalais (1991) estimated that 44% nitrogen and 28% phosphorous fro m the Mississippi River basin is deposited into the Gulf of Mexico by way of the Mis sissippi and Atchafalaya rivers. Additionally, it has been shown that the Mississip pi River carries 111 4.3 g at NO3-N 1-1 of nitrate and 7.4 0.4 g at P 1-1 pf phosphorous (42).

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4 The increase in the amount of nitrate and phosphoro us deposited into the Gulf has increased the amount of primary production occurrin g in the Mississippi River Plume (MRP) area (1). Studies have shown that primary pr oduction in the plume reaches as high as 8.17 g C m-2 d-1 (32). In a study to asses how the MRP affects the total surfacewater production in the Gulf of Mexico, researchers calculated that the MRP was responsible for approximately 41% of all carbon fix ation occurring in the upper 10 m of the oligotrophic water of the Gulf of Mexico (47). 1.2 The CBB Pathway and RuBisCO There are several pathways utilized by other autotr ophic organisms that allow them to fix CO2, including the tricarboxylic acid pathway and the reductive acetyl-CoA pathway. However the main pathway by which photoau totrophs fix CO2 is the CalvinBenson-Bassham (CBB) pathway or reductive pentose p hosphate pathway (40). The CBB pathway is the fundamental pathway for the move ment of inorganic carbon in the biosphere and is conserved throughout evolution (40 ) (Fig. 1.1). This pathway consists of 13 enzymatic reactions and can be divided into t wo stages. In stage 1, three molecules of ribulose-1, 5-bisphosphate (RuBP) react with CO2, catalyzed by ribulose-1, 5bisphosphate carboxylase/oxygenase (RuBisCO), to gi ve six molecules of 3phosphoglycerate (PGA). One of the six molecules o f PGA is used in the production of carbohydrates. In stage 2 the remaining five molec ules of PGA are converted back into the starting substrate, RuBP through a series of re actions.

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5 Figure 1.1. CBB Pathway. Figure taken from Atomi, 2002. RuBisCO is the key enzyme in the CBB pathway (39, 4 0) because it is the first enzyme, of many, in the pathway and the only enzyme capable of fixing CO2. The slow turnover rate of RuBisCO (1000-2000 mol CO2 fixed/mol enzyme/min) and its poor catalytic activity (40), forces some plants to devo te more than 50% of their protein content by weight to RuBisCO (15, 35) and thus it i s considered to be the most abundant protein on Earth(11, 39, 40) During photosynthes is, phytoplankton capture light energy from the sun. Carbon dioxide is taken up from the atmosphere and fixed via the CBB pathway. Carbohydrates are produced and oxygen is released into the atmosphere and surrounding waters. Carbon is incorporated into th e cells of phytoplankton and cyanobacteria by either direct uptake of CO2 or by the uptake of HCO3 from surrounding waters (19). RuBisCO has two functions: it catalyzes the oxygeno lysis and carboxylation of RuBP (11, 40). CO2 and O2 actively compete with each other for the active si te on

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6 RuBisCO. In the alternate reaction, O2 binds to the active site and is added to RuBP to yield phosphoglycolate which is metabolized in the glycolate pathway (35). This process, known as photorespiration, leads to a 50 % decrease in the overall efficiency of photosynthesis (35). The earliest forms of RuBisCO were not subject to this competition between CO2 and O2 because the atmospheric CO2 concentration was 100 times higher and the O2 concentration was less than 1 % of present day O2 levels, therefore, if CO2 entered the cell diffusively, the active site would be saturated with CO2 (31). As the atmospheric concentrations of both O2 and CO2 changed over geological time, O2 began to aggressively compete with CO2 for the active site (31). As a result of the competition between oxygen and c arbon dioxide, many microorganisms have the ability to concentrate CO2 at the carboxylation site (19). The development of carbon concentrating mechanisms (CCM ) allow for these microorganisms to adapt to changing CO2 levels in the atmospshere and ocean (19) as well as preventing oxygen from binding to the activ e site to form glycolic acid (40). Many species of cyanobacteria contain CCMs that enh ance the efficiency of photosynthesis (3). Marcus et al. (1983) suggested that CCMs were induced by a product from the photorespiration pathway. When mentioning CCMs, one must take carboxysomes into consideration. Carboxysomes are cellular compartments that contain RuBisCO and are bound by a protein membrane (3, 22, 31). The polyhedral shape of these crystalline structures is similar to that of viral particles (3). It is believed that these structures are stores of RuBisCO that act as reserv oirs to protect against photorespiration.

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7 The functioning of CCMs in certain aquatic photoau totrophs is just now becoming understood (4, 14). The bulk of what is k nown about CCM functioning is based on a relatively limited number of organisms i n culture, such as the chlorophyte Chlamydomonas reinhardtii (13, 37) and freshwater cyanobacteria Synechococcus sp. PCC7942 (51) and Synechocystis sp. Strain PCC6803 (23) among others. Such studie s indicate that the components and genetics of CCMs a re quite diverse, and some phytoplankton (i.e. the chrysophytes) appear to lac k a CCM altogether, obtaining inorganic carbon (Ci) merely by diffusion. One component of nearly all CCMs is the enzyme car bonic anhydrase. Carbonic anhydrases (CA) are a broad class of enzymes that c atalyze the reversible conversion of bicarbonate (HCO3 -) to CO2. These may play a role in Ci uptake to the cell, as in the low-CO2-inducible periplasmic CA of C. reinhardtii (37), or in facilitating the enrichment of CO2 concentrations at the site of RuBisCO as for the c arboxysomal CA of cyanobacteria (4). CAs are divided into 5 types (, , and ) with no homology between them as they are thought to be the result o f convergent evolution. One subgroup among the putative CAs with greater c onservation is the CsoS3 carboxysomal shell protein of marine -cyanobacteria, a homologue of which has only recently been shown to have CA activity (36). No ot her putative carboxysomal CA gene is found in these organisms, as found for the -cyanobacteria. Due to the critical role of CCMs in enabling phytoplankton to compete in highly productive coastal or freshwaters where Ci may be reduced, investigation of these mec hanisms is warranted. Because of its relative conservation in sequence, the cyanobacteri al CsoS3 genes represent a good target

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8 for preliminary studies on detecting and quantifyin g the expression of CA genes in the environment and the relationship to changing CO2 concentrations. 1.3 Forms and Structure of RuBisCO RubisCO is found in several forms and is distinguis hed by subunit assembly and its biochemical properties (39, 48). There are fou r forms of Rubisco which include: Form I, Form II, Form III and Form IV. This review will focus on the major forms found in RubisCO containing organisms; Forms I and II (39). Form I consists of eight large (L8) and eight small (S8) subunits with a molecular weight around 550,000 D a (39, 45) and is found primarily in photosynthetic organisms and aer obic chemolithoautotrophs (12, 39, 45) (Fig. 1.2). This hexadecameric structure is the basic and most common of all of the forms and is conserved in many species of bacteria as well as higher plants (40). Form II RubisCO has only a large subunit and is found mainl y in nonsulfur purple photosynthetic bacteria (12, 45) and marine dinoflagellates (26). These organisms usually fix CO2 anaerobically (12). Figure 1.2. Quaternary structure of RubisCO. Image taken from Wikipedia.com (49)

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9 Form I is divided into subgroups denoted as “green” and “red” (12, 39). These subgroups are then further divided into subclasses: “green” is divided into IA and IB while “red” is divided into IC and ID (12, 39). Ru bisCO types from the green subgroup are found in green plants, green algae and cyanobac teria, while those from the red subgroup are found in red algae and purple bacteria (39). The picocyanobacteria Prochlorococcus and some Synechococcus species are the organisms in which most Form IA RubisCO is found (12, 48). Form IB is dominated by all of the green algae and other cyanobacteria (12, 48). Form ID RubisCO is found p rimarily in chromophytic, or nongreen, algae while Form IC has been found in alp haand beta-proteobacteria (12, 48). 1.4 RuBisCO in the Marine Environment The genes that encode for the large and small subun its of RubisCO are denoted as rbcL / cbbL and rbcS / cbbS respectively (2, 40). It is on the large subunit t hat the active site for the carboxylation or oxygenation of RuBP i s located (25, 40). If an organism expresses rbcL then it is assumed that it produces RuBisCO and t herefore fixes CO2 by way of the CBB pathway. The phylogenetic patterns demonstrated by rbcL allow for researchers to identify the organism from which it came without the need for culturing the organism (44). With the use of PCR, one of the first study of rbcL occurrence in a natural phytoplankton community was conducted (28). In thi s study, oligonucleotide primers were designed from sequences of a Synechococcus sp to amplify conserved regions of the rbcL gene (28). These primers were then used in a PCR t o DNA of phytoplankton

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10 samples obtained from a Florida reservoir and algal isolates. Furthermore, extracellular DNA was also amplified thereby indicating that phyt oplankton contribute to the fraction of dissolved DNA in the water column (28). Overall this study was among the first to display the capabilities of PCR amplification to id entify specific genes in natural populations. rbcl transcription has been shown to occur on a diel cyc le. Corredor et al. (2004) conducted a study on RuBisCO transcription and the photosynthetic capacity of phytoplankton. The researchers’ objective was to d etermine whether the amount of mRNA transcription of a phytoplankton community, co uld be related to the biogeochemical cycles in which these communities pl ay an integral role (9). Using the Geochemical Rate-RNA Integration Study, or GRIST, t hey observed that mRNA transcription and carbon fixation occurred on simil ar diel patterns and both reached their peak between 10 a.m. and 1 p.m. (9). In another st udy, researchers observed similar diel patterns in pure cultures of the cyanobacterium Synechococcus and the prymnesiophyte Pavlova gyrans (29). While both organisms were exposed to a 12 h light and 12 h dark cycle, both organisms exhibited slightly different transcription patterns. rbcL transcription levels in Synechococcus peaked around noon and then rapidly disappeared over the next 8 h. On the other hand, P. gyrans’s transcription levels peaked at 4 p.m. and decreased to 66% of it’s maximum level until 8 p.m. thus indicating that chromophytes are capable of fixing carbon later in the day than its cyanobacterium counterpart (29). Furthermore, Wyman et al. (52) e xamined the diel pattern in rbcL transcript abundance in a coccolithophorid. Result s showed that the RuBisCO expression

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11 peaked around sunrise and decreased by an order of magnitude later in the day. This report represents the first study of species-specif ic determinations of RuBisCO expression in a natural population as opposed to th e aforementioned studies which deal with mixed or multiple populations. 1.5 Use of Array Technology in the Marine Environme nt Macroarray and/or microarray technology has been us ed to analyze the function and occurrence of genes in the environment as well as the medical field. A microarray/macroarray works by exploiting the abili ty of a given nucleic acid molecule to bind specifically to, or hybridize to, a DNA templa te with which it shares homology. Arrays can be used to determine the expression of m any genes in an environmental sample in one experiment. Generally, the process e ntails DNA in the form of oligonucleotides or amplicons from polymerase chain reaction (PCR) is spotted onto a glass slide or a nylon membrane. mRNA is then labe led or tagged with a fluorescent dye, either Cy3 or Cy5, and hybridized to the slide or m embrane. Figure 1.3 shows a diagram of this process. The mRNA will bind to the strand of DNA most homologous in sequence to itself. The prefixes macroand microrefer to the size of the spot laid down on either the glass slide or nylon membrane (Fig. 1 .4). Microarray spots must be viewed with high resolution cameras interfaced to special computer software and can hold several hundred of genes. Macroarrays are also a nalyzed with computer software but the spots can be seen with the naked eye and hold m uch fewer spots. Since many

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12 microbial communities are unculturable, array techn ology can be an ideal tool in the identification of these communities. Figure 1.3. Microarry technology process. DNA is extracted from cells and transcribed into mRNA. C ells are labeled with a fluorescent dye and hybridized to a glass slide or memebrane. Array is then scanned to obtain an image and analyzed. There are three general types of arrays. The first and most common, type of array has different genes from a single organism th at are spotted onto the glass slide or membrane in order to represent the genome of that o rganism (44). Arrays of this type are used to determine an organism’s response to environ mental changes. Another type of array utilizes the same gene obtained from differen t organisms. With this method of arraying, one is able to identify the organism from which each gene originated and is often used in diversity studies (44). The third ty pe of array incorporates the use of Isolate mRNA Label mRNA Hybridize to chip/membrane Scan array with appropriate software Scanned image

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13 different functional genes from different organisms to quantify the amount of functional gene expression (44). Figure 1.4. Comparison of (a) microarray and (b) m acroarray. Microarrays can hold more spots than th e macroarray. Macroarray images courtesy Scharf et al 2003. Arrays have been used to determine the diversity of target genes in different environments. Jenkins et al. (2004) used macroarry s to fingerprint the diazotroph communities in the Chesapeake Bay. The results sho wed that Chesapeake Bay is home to a phylogenetically diverse diazotroph community (17). Macroarrays have been used to assess nitrogenase diversity in picoplankton. S teward et al. developed a macroarray to discern the capabilities of such technology to eval uate gene expression in the environment (38). They showed that macroarray resu lts can be easily reproduced and are semiquantitative in assessing gene expression in a mixed sample (38). Microarrays have also been used in microbial ecolog y studies in the identification and monitoring of bacterial communities found in wa stewater and sludge. Loy et al. reports the use of a 16S rRNA oligonucleotide micro array, known as RHC-PhyloChip (probeBase), in the detection of bacteria of the or der Rhodocyclales (21). Many of the a) b)

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14 bacteria of this order are capable of degrading ant hropogenic compounds. The results from this study showed that with the use of this sp ecialized microarray allowed for the detection of Rhodocyclales population that represented less than 1% of the mi crobial community in the sample (21). There are many obstacles that one must consider whe n constructing an array. When dealing with the same gene from different orga nisms, there is high probability that the sequences will be very similar to each other. This problem is not encountered in genomic arrays which target divergent genes from on e organism. Another obstacle researchers may face is that when sampling from het erogeneous populations, probes may have very different melting temperatures ( Tm) due to different sequence lengths (41). To correct for this, Taroncher-Oldenburg et al. (41) c onstructed oligonucleotides of equal length, minimal secondary structure and similar G-C content in the detection and quantification of functional genes found within the nitrogen cycle in the Chesapeake Bay system. The establishment of these parameters allo wed for more uniform conditions during the hybridization process. 1.6 Real Time PCR in the Marine Environment Real time PCR provides a means to observe the PCR w hile it is occurring. Unlike traditional PCR, real time PCR quantifies the amoun t of template produced in each cycle instead of at the end of the reaction. Real time P CR works by detecting the increase of fluorescence of DNA or RNA that has been bound to a fluorescent DNA stain, or to a fluorogenic probe specific for the target PCR produ ct (48). SYBR green is a fluorescent

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15 stain commonly used in real time PCR. The main dis advantage of SYBR green is that it has the tendency to produce false positives. TaqMa n probes are fluorogenically labeled and utilize the 5` exonuclease activity of Taq DNA polymerase to provide a means of examining the amplification of specific PCR product s. Holland et al (1991) were the first to describe thi s process. They developed a method that utilized the 5` 3` exonuclease activity of Thermus aqaticus ( Taq ) DNA polymerase in a PCR that allowed for the quantifica tion of the target during each cycle of the amplification process (16). Thermus aquaticus is a bacterium found in hot pools that has revolutionized the way PCR is conducted today. Taq DNA polymerase replaced the Klenow fragment of E. coli DNA polymerase I PCR because its thermostability eliminates the need to add new DNA polymerase durin g every cycle of amplification. In a study a study to examine rbcL expression in pelagophytes and diatoms, Wawrik et al. (2002), developed a method of detecti ng transcript abundance using real time PCR. A Taq Man probe specific for pelagophytes and diatoms wa s used in real time PCR and allowed for quantification of these groups in particular. Real time PCR data was compared to that obtained from 35Slabeled oligonucleotide hybridization in order t o determine the efficiency of the real time PCR metho d in microbial gene expression studies from environmental samples (48). The result s from the study showed that the mRNA levels detected by both techniques were simila r although hybridization levels were slightly higher (48). Higher hybridization m RNA expression levels were attributed to the predicted bias that hybridization experiment s have towards degraded or partially degraded sequences (48).

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16 The objective of this thesis was to determine what clades or phylogenetic groups are abundant in the primary production occurring in the Mississippi River Plume as determined by rbcL hybridization and real time PCR. I hypothesized t hat microarrays would play an important role in detecting the gene expression of rbcL in the MRP. The goals of my research were to first characterize the phytoplankton community in the MRP using microarray technology. The microarray data w as then compared to RT-PCR and dot-blot hybridization data. Microarray data gathe red for surface waters of the MRP showed high Synechococcus pelagophyte and prymnesiophyte signals, although Synechococcus signals were at times significantly higher than tho se of the pelagopyte and prymnesiophyte probes. Dot-blot hybridization show ed that the chromophytes dominated the surface by as much as 10 times the concentratio n of From IA groups. RT-PCR data from surface water showed that the Form ID organism s were 100 times more abundant than Synechococcus suggesting that the strong signal of the Synechococcus probe on the array was a result of selective PCR amplification.

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17 Chapter 2 Macroarray Detection of RubisCO Expression in Marin e Environment Chapter Summary The Mississippi River is the 7th largest river in the world. It deposits a large amount of nutrients into the Gulf of Mexico (GOM) e levating nutrient levels in the Gulf and stimulating the growth of various phytoplankton species. The elevated nutrient levels in the Gulf causes an increase in the growth of various phytoplankton species. A DNA macroarray was constructed to determine its a bility to quantify gene expression in the Mississippi River Plume (MRP). Ribulose1, 5bisphosphate carboxylase/oxygenase (RuBisCO) is the key enzyme i n the Calvin-Benson-Bassham (CBB) pathway and the expression of the gene which encodes for this enzyme, rbcL, is indicative of carbon fization via the CBB pathway. PCR amplicons made from rbcL plasmid sequences obtained from the Mississippi Riv er Plume were spotted onto a nylon membrane. The rbcl gene was excised from the plasmid, transcribed int o RNA and then hybridized to the arrays. The macroarrays were not able to quantify gene expression in the MRP due to the frequency of cross hybridization of the probe to spots on the array from different phylogenetic groups.

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18 2.1 Introduction Ribulose-1,5-bisphosphate carboxylase/oxygenase (Ru bisCO) is the key enzyme in the Calvin-Benson-Bassham (CBB) pathway of photo synthetic carbon fixation in phytoplankton (40). The CBB pathway is the fundame ntal pathway for the entry of inorganic carbon into the biosphere (40). With reg ards to structure, RuBisCO is found in two major forms: Form I and Form II. Form I consis ts of eight large (L8) and eight small (S8) subunits. Form II consists of two large subunits (L2). The genes that encode for the large and small subunits are denoted as rbcL and rbcS respectively. The active site for carboxylation is found on the large subunit, and as a result, the expression of rbcL in an organism is indicative of carbon fixation via the C BB pathway. Array technology provides a means of simultaneous i dentification and/or expression analysis of thousands of genes. Macroar rays and microarrays can be used in the detection of microbial strains and the presence of functional genes in an environmental sample. What is also unique about ar rays is that the hybridization probetarget relationship is inverted. Traditionally, kn own DNA sequences are labeled with a fluorescent dye (Cy3 or Cy5), and hybridized to unk nown samples on the slide. When using microarray technology, known DNA is spotted o nto the slide and unknown samples are labeled and used as probes. DNA microarrays we re originally developed for the study of nucleic acid sequences but now are commonl y used in gene expression studies (20). Microarrays are ideal for use in the environ ment as most microorganisms are unculturable, they allow the identification and or functionality of genes found in the environment. Roszak and Colwell (1987) shed light on the viable but nonculturable

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19 (VBNC) state in which most marine and estuarine bac teria exist. This term is given to microorganisms that cannot be maintained in a pure culture in the laboratory setting but do have detectable metabolic function (33). Array technology can be used to detect metabolic processes, such as the cycling of nutrien ts, occurring within these microbial communities by examining patterns of gene expressio n. The Mississippi River is responsible for approximat ely 41% of the drainage of the United States and for more that 70 % of the freshwa ter input into the Gulf of Mexico (1, 42). Most of the land through which the Mississipp i River passes is used primarily for agriculture. Phosphorous and nitrogen used in fert ilizers are deposited into the river due to drainage from runoff and groundwater. These nut rients accumulate and are thusly deposited into the Gulf of Mexico. The Mississippi River Plume (MRP) is the dominant source of nutrients to the Gulf. This annual flux of nutrients causes massive phytoplankton blooms at mouth of the river, which e xtends out into the Gulf of Mexico. The purpose of these experiments was to examine fun ctional gene expression of ribulose-1,5-bisphosphate carboxylase/oxygenase in the MRP. The rbcL clades that will be used in this study were Form I type, specificall y, clades IA, IB and ID. In this study, a macroarray designed to detect rbcL transcription was used to characterize the phytoplanktonic community in the oligotrophic Gulf of Mexico. Due to cross hybridization of the target sample to the probes on the arrays, macroarray results were inconclusive.

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20 2.2 Methods 2.2.1 Plasmid extraction Twenty-two clones known to already contain rbcL were selected from the work of Wawrik & Paul (47). These clones were obtained by amplification of the rbcL gene from natural phytoplankton communities of the Gulf of Me xico during the F.G. Walton Smith research cruise in the summer of 2001 (47). Figure 2.1 shows the cruise stations from which all samples were taken in the summer of 2001. Table 2.1 lists the clones that were used in this study as well as their corresponding c lade. Clone names denote the cruise date, station, depth (A-H) from which it was obtain ed and primers used in amplification (Y for Form IA/IB; H for Form ID; and SY to denote when the cyanobacterial reverse primer was used). For example, WS01ST6CH17 is deci phered as Walton Smith, 2001, Station 6, depth C, primer H, clone 17 (47). Clone P994FY27 was taken from Pelican, 1999, Station 4, depth F, primer Y, clone 27 (46). All clones were kept at –80C. Clones were streaked onto LB and Kanamycin plates and plac ed in the incubator at 37C. Overnight cultures were prepared by taking one colo ny of each clone from LB and Kanamycin plates was put into 5 mL of LB broth and 5L of Kanamycin (50 g/L). The cultures were placed in the shaking incubator at 37 C and 200 rpm overnight. Plasmid was extracted from each of the clones using the QIA prep Spin Miniprep Kit (Qiagen) and minipreps of each were sent off for sequencing at t he University of Florida Core Sequencing Facility to ensure they were the correct clones. Upon clone sequence verification, clones were prepared for PCR.

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21 Figure 2.1. SeaWiFs chlorophyll satellite imagery o f the Mississippi River Plume during time of sampli ng in 2001 overlaid with position of sampling stations. 2.2.2 PCR amplification and purification Polymerase chain reaction was performed on each clo ne to amplify the rbcL target sequence. One L of clone plasmid DNA was added to the PCR mixture that contained the primer set corresponding to its phylogenetic gr oup. Form ID clones used 0.5 L of 100 M Form ID fwd (5’GATGATGARAAYATTAACTC -3’) and 0.5 L of 100 M Form ID rev (5’ATTTGDCCACAGTGDATACCA -3’) as the primer set (20 M). Form IA and Form IB used 0.5 L of 100 M Syn fwd (5’CTGAGCGGYAAGAACTAYGG -3’) and 0.5 L of 100 M IA/B rev (5’GGCATRTGCCANACGTGRAT –3’) as the primer set (20 M). In addition to the primer sets, the PCR mixture contained 1 L of 10 mM deoxynucleoside triphosphates (800 M), 5 L thermophilic DNA polymerase 10X reaction buffer, 0. 25 L Taq DNA polymerase and 41.75 L water. Thermocycler conditions consisted of an init ial denaturation at 95C for 2 min, followed by 40 cycl es of 95C for 1 min, 53C for 1 min, and extension at 72C for 1.5 min. There was an ad ditional extension of 72C for 5 min.

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22 Amplicons were purified using the Zymo Kit (Zymo Re search) and quantified using the Hoescht 33258 method (30). Table 2.1. List of clone names, clade and common na mes. 2.2.3 Design of preliminary macroarrays After quantification of PCR amplicons, they were de natured with 10 mM RNAse-free EDTA and 0.4 M NaOH and then heated to 100C for 10 minutes. An equal volume of 2 M ammonium acetate was added to neutralize the DNA mixture. Ammonium acetate acts as a buffer that neutralizes or lowers the pH of NaOH. Amplicons were dotted in duplicate onto a charged nylon membrane using a Bio Rad dot blotter. A diatom (WS01ST6CH1), prymnesiophyte (WS01ST5CH10), chlorop hyte (WS01ST8SY13), chrysophyte (WS01ST1CH4), Synechococcus (WS01ST2SY27) and a prochlorophyte (WS01ST2SY19) were chosen to represent their clades The amplicons were dotted at Clone Name Clade Common Name WS01ST6CH17 ID Diatom WS01ST4CH12 ID Diatom WS01ST4CH36 ID Diatom WS01ST6CH1 ID Diatom WS01ST7CH3 ID Bolidomonas P994CH1 ID Pelagophyte WS01ST7SY24 ID Synechococcus WS01ST8CH5 ID Eustigmatophyte WS01ST6CH33 ID Xanthophyceae WS01ST4CH16 ID Dictophyceae WS01ST8CH15 ID Unknown, deeply rooted chromophyte WS01ST1CH1 ID Prymnesiophyte WS01ST5CH10 ID Prymnesiophyte WS01ST2SY27 IA Synechococcus WS01ST2SY14 IA Synechococcus WS01ST2SY19 IA Prochlorococcus WS01ST3SY5 IA Prochlorococcus WS01ST8SY15 IB Trichodesmium WS01ST3SY4 IB Chlorophyte WS01ST6SY14 IB Chlorophyte P994FY27 IB Prasinophytes WS01ST8SY3 IB Prasinophytes WS01ST6SY8 IB Prasinophytes

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23 different concentrations ranging from 100, 50, 10 a nd 1 ng per dot to determine which concentration is best for hybridization detection ( see Figure 2.2). A total of six membranes were made. Figure 2.2. Preliminary blot schematic. Clones were spotted onto the charged nylon membrane at the concentrations indicated. 2.2.4 Transcript production Restriction digests were performed to linearize pla smid DNA in 40 L reactions prior to in vitro transcription. Digestions were designed s uch that they would be at the 3’ end of the sense orientation of the rbcL gene. The digest was purified by the Zymo DNA Purification Kit or the Promega SV Gel and PCR Puri fication Kit (Promega). The linearized plasmid DNA was then transcribed using t he Riboprobe Combination System (Promega) for 2 h at 37C using either the Sp6 or T 7 RNA polymerase promoter to yield sense transcripts. 100 ng 50 ng 10 ng 1 ng Diatom Prymnes. Chrys. Chloro. Prochloro. Synech. Blank

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24 2.2.5 Labeling of transcripts with biotin An annealing mixture was prepared containing 2.5 g of transcript, 3 L specific primer and RNAse-free water to a total volume of 10 L. The annealing mixture was heated to 70C for 3 min and then cooled to 42C for 2 min. An RT Cocktail consisting of 5X GEAlabeling Buffer for Chemiluminescent Detection ( BN) (SuperArray), Biotin, RNasefree water, RNase inhibitor and reverse transcripta se were combined and heated to 42C for 1 minute before being added to the annealing mi xture. The final mixture was incubated at 42C for 90 min and then denatured at 94C for 5 minutes. Before the labeled transcript was denatured, 1 L of probe was removed and added to 19 L of 1X TAE to give a 20-fold dilution. A 4-fold serial di lution was performed by taking 3 L from the 20-fold dilution and adding it to 9 L of 1X TAE. The remaining serial dilutions were 80-, 320-, 1280and 5120-fold. Thi s process was performed to assess the labeling efficiency of the probe. 2.2.6 Hybridization and detection GEAhyb solution (SuperArray) was warmed to 60C in a water bath. Sheared salmon sperm DNA (SuperArray) was heated to 100C for 5 mi nutes and then immediately placed on ice. Arrays were prehybridized at 60C f or 2 h with 4 mL GEAhyb solution (Super Array) and 40 L sheared salmon sperm DNA (Super Array). Another 4 mL of GEAhyb solution and 40 L sheared salmon sperm DNA were incubated at 60C i n a hybridization oven. After prehybridization, prehyb solution was poured off. Four milliliters of hybridization solution and the entir e volume of labeled transcript were

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25 added to the hybridization tube. Each membrane was probed with a different transcript to ensure that the transcripts only hybridized to a me mber of their corresponding clade. Membranes were placed in the hybridization oven at 60C with continuous rotation for 16 h. After hybridization, membranes were washed t wice at 55C for 15 min with a wash solution containing 2X SSC and 1% SDS with continuo us rotation. Membranes were also washed twice at 55C with a wash solution cont aining 0.1X SSC and 0.5% SDS at 55C for 15 min with continuous rotation. These wa shing steps are a means of increasing the stringency of the array. The last wash was rem oved from the hybridization tubes and 5 mL of GEA Blocking Solution Q (SuperArray) were a dded and tubes were incubated for 40 min with continuous agitation at room temper ature. After discarding GEAblocking Solution Q, 4 mL of Binding Buffer were added to the hybridization tubes. Tubes were incubated for 10 minutes with continuous agitation. Membranes were then washed four times with 8 mL 1X Buffer F (SuperArray ) for 5 minutes each with gentle agitation. Next the membranes were rinsed twice wi th 6 mL Buffer G (SuperArray) for 5 minutes each. Arrays were then treated with CDP-St ar for 2 h in the dark and then exposed to X-ray film for 20 min. 2.2.7 Design of control macroarrys The control arrays were prepared in the same manner as the preliminary arrays, however, the full array was spotted with all 22 clones. Amp licons were dotted in duplicate onto a charged nylon membrane using a BioRad dot blotter. Each of the 22 clones were dotted in duplicate at 50 ng and 10 ng concentrations (Fig 2.3). 2.2.8 Transcription, hybridization and detection

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26 Four control arrays were tested with 4 clones that served as representations for their clades: a prasinophyte (8SY13), a prymnesiophyte (5 CH10), a Synechococcus (2SY4) and a diatom (4CH12). Transcripts were made in the same manner as for the preliminary arrays. Control arrays were hybridized and detecte d in the same manner as the preliminary arrays. Figure 2.3. Control blot schematic. Water was dot ted in each corner to serve as a negative control. Clones were spotted in duplicate on the nylon membrane at 50ng and 10ng concentrations to determine which concentr ation gave the optimum spot density. 50 ng 10 ng 50 ng 10 ng 10 ng 50 ng 10 ng 50 ng H2O P994CH1 1CH4 5SY4 6CH17 6SY3 H2O H2O P994CH1 1CH4 5SY4 6CH17 6SY3 H2O 6CH1 P994FY27 6CH33 2SY4 8CH15 8SY13 6CH1 P994FY27 6CH33 2SY4 8CH15 8SY13 4CH36 2SY19 4CH12 8CH5 8SY15 5CH10 4CH36 2SY19 4CH12 8CH5 8SY15 5CH10 H2O 7SY24 7CH3 8SY3 4CH16 6SY14 H2O H2O 7SY24 7CH3 8SY3 4CH16 6SY14 H2O

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27 2.3 Results The first few hybridization experiments were mostly unsuccessful due to the occurrence of cross hybridization of the transcript to the pro be. However one experiment indicated that the transcripts were hybridizing properly. Th e blot shown in Figure 2.4 was hybridized with an 8SY13 transcript (Form IB) and s trongly hybridized to itself. It also hybridized weakly to both 2SY19 (Form IA) and 2SY27 (Form IA). This hybridization pattern is to be expected since Form IA and Form IB rbcLs are closely related. Figure 2.4. First successful hybridization experi ment. The numbers to the left show the concentrati on of the dots laid down on the membrane. Notation across th e top are the clones spotted onto the membrane: 6CH 1diatom; 8SY13prasinophyte; 1CH1chrysophyte; 2SY 19prochlorophyte,; 1CH4prymnesiophyte; 2SY27Synechococcus This blot was hybridized with a transcript made from 8SY13. Next the full array was spotted containing all 22 clones in the format described in Figure 2.3. Clones 1CH1 nad 2SY27 were replaced wi th 5CH10 and 2SY4, respectively. PCR amplicons were spotted in duplicate in concentr ations of 50 ng and 10 ng. The first array was hybridized with 2.5 g of an 8SY13 probe (Fig. 2.5 a). This experiment indicated cross hybridization occurring between unr elated clones. For example, on one array, a Form IB prasinophyte probe hybridized to a Form ID dictophyceae amplicon and 100 ng 50 ng 10 ng 1 ng Blank 6CH1 8SY13 1CH1 2SY19 1CH4 2SY27

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28 a Form IA Synechococcus amplicon. This had not been a problem in the preli minary arrays; as such the increase in cross hybridization led us to believe that the parameters for hybridization were not stringent enough. In an att empt to correct for this, the hybridization and washing temperature was increased from 60C to 65C. Increasing the hybridization and washing temperature did not preve nt cross hybridization of the probes with dissimilar amplicons. Cross hybridization can be seen in Figures 2.5 b-c, which were hybridized at 65C. Figures 2.5 b-c were hybridize d with a diatom and a Synechococcus sp ., respectively, and when compared to Figure 2.4, i t is evident that cross hybridization has occurred. The diatom (Form ID) probe, hybridiz ed to all Synechococcus dots and to all chlorophyte dots. Additionally, the Synecococcus probe hybridized with a pelagophyte, a dictophyceae, a prymnesiophyte all o f which contain Form ID rbcL

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29 (a) (b) (c) Figure 2.5 Fully dotted array experiments. (a) Fi rst hybridization experiment with the fully dotted array at 60C. This array was probed with a labeled 8SY13 t ranscript (prasinophyte). (b) Array was hybridized with a 4CH12 (diatom) probe at 65C. (c) Array was hybri dized with a 2SY4 ( Synechococcus ) probe at 65C. A B C D E F G H 3 2 5 6 4 8 7 1 10 9 11 12 50 ng 10 ng 50 ng 10 ng 50 ng 10 ng 50 ng 10 ng A B C D E F G H 3 2 5 6 4 8 7 1 10 9 11 12 50 ng 10 ng 50 ng 10 ng 50 ng 10 ng 50 ng 10 ng A B C D E F G H 3 2 5 6 4 8 7 1 10 9 11 12 50 ng 10 ng 50 ng 10 ng 50 ng 10 ng 50 ng 10 ng

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30 2.4 Discussion In this study a macroarray containing clones from t he MRP was hybridized with transcripts from clones also obtained from the MRP. We were faced with difficulties from the onset of the experiment. Initially there was a problem with the labeling step using biotin. During the labeling efficiency step, there would be no spot on the film to indicate that the transcript was labeled with bioti n. In an attempt to correct for this problem, many modifications were made to the protoc ol. We first assumed that the random primers provided by the labeling kit were no t specific enough to our target and thus were not labeling the transcript. Specific pr imers were then used in place of the random primers. When changing the primers did not improve the labeling efficiency, we increased the amount of template by doubling the tr anscription reaction. We believe that the combination of the specific primer and the incr ease in the amount of template contributed to the success of the labeling step. There was a considerable amount of cross hybridizat ion occurring with the macroarrays. With the preliminary arrays there was also some cross hybridization but we believed it to errors made during the dotting of th e macroarrays and thus decided to dot the full array. The non-specific binding of the ta rget was still present; therefore the hybridization temperature was increased from 55C, 60C and 65C, respectively, in separate reactions to increase the stringency of th e array. Increasing the hybridization temperature did not reduce the occurrence of cross hybridization. One theory is that the initial concentration of the probe may have been to o high and thus caused an overload of RNA to the array. During the labeling efficiency s tep, one must judge based on the

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31 intensity of the spots as to what the optimal conce ntration for hybridization should be. A 1:10 dilution of probe 8SY13 was done prior to hybr idization the array yet cross hybridization still occurred. In the next set of e xperiments, approximately 1.25 ng of labeled probe was used for hybridization (Fig 2.5 b -c). Even with half the original concentration of the probes, there was still signif icant cross hybridization with clones from unlike groups. Another possible cause of cros s hybridization of the target could be a result of plasmid sequence overhang on the PCR pr oducts. The rbcL gene is excised from the plasmid by a restriction enzyme and transc ribed into RNA. The PCR products that were spotted onto the array contained the plas mid sequence overhang. The pure transcripts also contain portions of the plasmid se quence which could result in the nonspecific binding of transcripts to spots from di fferent phylogenetic groups.

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32 Chapter 3 Microarray Detection of RuBisCO in the Mississippi River Plume Chapter Summary Here, a DNA microarray was used to detect rbcL expression occurring in the Mississippi River Plume (MRP). Total RNA was ext racted from water samples collected from the MRP in 2005. cDNA was made from total RNA and amplified with rbcL primers in a polymerase chain reaction (PCR). PCR amplicons were labeled and hybridized to microarrays containing known PCR-ampl ified rbcL products obtained from various locations. All microarrays, constructed at Princeton University by the Ward lab, consisted of 70-mer oligonucleotides made from clon es sequences previously obtained from the MRP. Information from the microarrays was compared to real-time PCR (RTPCR) rbcL and dot-blot hybridization data obtained from the plume. Microarray data gathered for surface waters of the MRP showed high Synechococcus pelagophyte and prymnesiophyte signals, although Synechococcus signals were at times significantly higher than those of the pelagopyte and prymnesioph yte probes. Subsurface data showed lesser amounts of Synechococcus and no Prochlorococcus expression. There were also significant signals observed in the chlorophyte pro bes. These results, provide a third layer of the detection of rbcL expression in the MRP.

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33 3.1 Introduction The Calvin-Benson-Bassham cycle is primary the path way for the entry of inorganic carbon to the biosphere, and is the major pathway used by autotrophic organisms such as plants and phytoplankton. Ribulo se-1,5bisphosphate carboxylase oxygenase (RuBisCO) is the enzyme responsible for f ixing CO2 to ribulose bisphosphate. RuBisCO has four forms (I, II, III, and IV) that ca n be distinguished by their assemblage of subunits and their individual biochemical proper ties (39). Form I RuBisCO consists of eight large (L8) and eight small (S8) subunits. It can be further divided into two subgroups of either “green” or “red” (39). Each su bgroup is then divided into subclasses: green is divided into IA and IB which are found in green plants, green algae and cyanobacteria; and red is divided into IC and ID wh ich are found in red algae and purple bacteria (39). Microarray technology offers a new approach to the study of microorganisms by providing a means to identify and examine the funct ionality of many genes simultaneously. Presently, majority of the studies using microarrays have been in the biomedical field, however, the use of microarrays i n environmental studies has begun to increase. The lack of environmental studies is wid ely due to difficulties associated with cultivating microbial communities found in environm ental samples. Nonspecific probe to target binging, temperature variability amongst probes and problems with DNA amplification are the main obstacles that must be a ddressed when working with microarrays (41).

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34 The Mississippi River is responsible for over 41% o f the drainage of the United States and thus deposits a significant amount of nu trients into the Gulf of Mexico (1, 42) forming a plume. These nutrient deposits fuel annu al phytoplankton blooms. Past studies have shown the presence of diverse phytopla nkton groups in the plume (46). In this study, rbcL obtained from the Mississippi River Plume (MRP) wa s extracted and hybridized to a microarray containing known rbcL probes. This data was then compared to real time PCR and dot-blot hybridization data ob tained from the plume.

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35 3.2 Methods 3.2.1 RNA extraction Three-tenths to 20 L of water collected from the Mi ssissippi River Plume in July 2005 were filtered with sterivex filters and 350 L of RLT buffer were added to each filter. Three to nine-hundred mL of water were also filtere d with 0.45 m Durapore filters and which were placed in 2 mL tubes with 750 L RLT buffer and silica or glass beads. Filters were placed in the -80C freezer for storag e. Total RNA was extracted from the filters following the RNeasy Mini Kit (Qiagen) prot ocol. Table 3.1 lists the cruise samples that were used. Figure 3.1 shows the cruis e stations from which these samples were collected. Table 3.1. List of cruise samples showing depth, am ount filtered and type of filter used. Station Depth Water Filtered (L) Filter 8B 55m 15 Sterivex 7X 3m 11.2 Sterivex TB 3m 0.3 Durapore 2G 100m 2 Sterivex 6A 3m 2.6 Sterivex 5A 3m 2.6 Sterivex 1B 60m 15 Sterivex 3.2.2 Transcription cDNA was made using the SuperScript III First-Stran d Synthesis System for RT-PCR (Invitrogen) according to the manufacturer’s protoc ol. Briefly, total RNA up to 5 g, but no less than 1 pg, was combined with 50 ng/uL rando m hexamers and a 10 mM dNTP mixture. Samples were incubated at 65C for 5 minu tes and then placed on ice. cDNA Synthesis mix was prepared using 10X RT buffer, 25 mM MgCl2, 0.1 M DTT, RNaseOut

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36 (40 U/L) and SuperScript III RT (200 U/L) and added to the RNA/primer mixture. This new mixture was then incubated first at 25C f or 10 minutes and then at 50C for 50 minutes followed by incubation at 85C for 5 minute s to terminate the reaction. Samples were placed on ice following incubations and RNAse H reagent was added to each sample followed by incubation at 37C for 20 minute s. cDNA was then quantified using the Quant-iT Pico Green dsDNA Assay Kit (Invitrogen ). cDNA was amplified with PCR using all three rbcL primer sets (Form ID fwd and rev; Form IA/IB fwd a nd rev; Syn fwd, Form IA/IB rev) before labeling. cDNA ranging in c oncentration from 0.1 to 2 ng was added to a mixture containg the forward and reverse primer (100 M each) and PCR Master Mix (Promega). Thermocycler conditions cons isted of an initial denaturation at 95C for 2 min, followed by 40 cycles of 95C for 1 min, 52C for 1 min, and extension at 72C for 1.5 min. There was an additional exten sion of 72C for 5 min. Amplicons were purified using the Zymo Kit and quantified wit h the Quant-iT Pico Green dsDNA Assay Kit (Invitrogen). Figure 3.1. SeaWiFS ocean color satellite image of the Mississippi River Plume overlaid with cruise st ations.

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37 3.2.3 Positive controls Transcripts of clones obtained from the Mississippi River Plume in 2001 (46, 47) were made first by digesting plamids containing the rbcL insert with the appropriate enzymes. Table 3.2 lists the clones that were used as positi ve controls. All digests were purified with the Zymo DNA Purification Kit (Zymo Research). The linearized plasmid DNA was then transcribed using the Riboprobe Combinatio n System (Promega) for 2 h at 37C using either the Sp6 or T7 RNA polymerase promoter to yield sense transcripts. Table 3.2. List of the transcripts used as positive controls for the microarray performance tests. Clone Family RuBisCO Type Reference P994CH1 Pelagophyte ID Wawrik et al., 2003 WS01ST1CH1 Prymnesiophyte ID Wawrik & Paul, 2004 WS01ST8CH12 Chrysophyte ID Wawrik & Paul, 2004 WS01ST3SY1 HL Prochlorococcus IA Wawrik & Paul, 2004 P994GY7 LL Prochlorococcus IA Wawrik et al., 2003 WS01ST6SY3 Synechococcus IA Wawrik & Paul, 2004 WS01ST4SY39 Prasinophyte IB Wawrik & Paul, 2004 3.2.4 Labeling Amplified cDNA was combined in order to minimize wi thin reaction variability and bias. Mixed cDNA was labeled in a random priming labeling reaction using the BioPrime Array CGH Genomic Labeling Module (Invitrogen) with slight modifications. In short, a reaction mixture containing 2.5X random primers (12 5 mM Tris-HCl, template cDNA, 12 mM MgCl2, 25 mM 2-mercaptoethanol, 750 g/mL oligodeoxyribonucleotide primers), 1.2 mM dNTP mix with aminoallyl-dUTP (10 mM dACG mix, 10 mM dTTP, 10 mM dUaa) and Klenow enzyme ( 40 U/L Klenow fragm ent in 100 mM KPO4, 1 mM DTT and 50% glycerol). This mixture will be incuba ted at 37C for 2 h. Labeled product was cleaned with the Qiaquick PCR Purificat ion kit (Qiagen) and quantified with

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38 the Quant-iT Pico Green dsDNA Assay Kit (Invitrogen ) to ensure that the labeling procedure was successful. Following quantificatio n, the labeled product was dried down to a pellet and frozen at -80C. 3.2.5 Coupling of dUTPaa-labeled target to Cy3 dye First, the Cy3 dye pellet was re-suspended in 40 L of dimethyl sulfoxide (DMSO). The dUaa labeled pellet was thawed and re-suspended in 4.5 L of 0.10 M Na2CO3 buffer and incubated at room temperature for 15 min. Next a 4 .5 L aliquot of dye solution was added to the re-suspended pellet and allowed to inc ubate in the dark for 1 hr. Once incubation is complete, 4.5 L 4M hydroxylamine were added to the mixture and al lowed to incubate in the dark for an additional 15 min. Hydroxylamine quenches the coupling reaction of the Cy3 dye with the dUaa label. The m ixture was then purified with the Qiaquick PCR purification kit with minor modificati ons: 25 L of ddH2O were added to each sample before the addition of Buffer PB; 3 L 3M NaOAc was added to ensure low pH of Buffer PB; samples were washed 5 times with B uffer PE; 30 L Buffer EB was added and columns were allowed to sit for 2 min, an d then spun down in a microcentrifuge. The elution step was repeated to yield 60 L of target. Finally, labeled samples were quantified with the Pico Green kit for double stranded DNA and dried down into a pellet. 3.2.6 Microarray construction and hybridization Two types of slides were used in this study (Fig. 3 .2). The first type, referenced as BC008 (Fig 3.2a), consisted of a glass slide and a single gasket. Only one probe can be used with this array. The second slide, referenced as BC009 (Fig 3.2b), is unique in that

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39 there are two gaskets. This allows for the hybridi zation of two probe sets in a single experiment in which a different hybridization solut ion, each with a different Cy3 target, is placed into each gasket and the array slide is p laced onto the coverslip. The DNA laid down on the array were 90-mer oligonucleotides from various organisms in which the first 20 bases were complimentary to the Cy5 refere nce oligonucleotide used in the hybridization solution (Table A-1). All microarray slides were printed at the Microarray Facility at Princeton University Princeton, New Jer sey. This is an ozone free facility due to the sensitivity of Cy5 to the gas. The hybridization protocol for BC009 is different f rom BC008. The hybridization solution consisted of 100 L 2X Hybridization Buffer (Agilient), ( n ) L Cy3-target, ( n ) L Cy5-reference oligonucleotides and distilled wate r to a total of 200 L in a 1.5 mL tube. A total of 4 ng of transcript cDNA and 100 n g of cruise sample cDNA were used in the hybridization solutions. Tubes were mixed and heated to 95C in a wet block with the lid locked for 5 min and then allowed to cool t o room temperature for a minimum of 2 min. The hybridization solution was then applied t o the gasket of the slides. Slides were incubated in a rotating oven overnight at 60C. When using the BC008 slides, 30 mL of prehybridizat ion solution was made containing 7.5 mL of 20X SSC (5X in 30 mL), 3 mL of 10 % SDS(1 % in 30 mL) and 0.3g (1 % in 30 mL) of a blocking reagent in the fo rm of chicken or bovine serum albumen (CSA or BSA). This solution was heated in 5 s intervals until clear and all CSA/BSA is melted. It was then filtered through a 0.45 m filter. Array slides were incubated in this solution for a minimum of 40 min at 64C in the hybridization oven or

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40 warm water bath. After incubation, slides were dip ped in e-pure water 5 times and then rinsed with isopropyl alcohol. Slides were then ce ntrifuged to remove any residual alcohol. Next, parafilm was wrapped around the slides to ser ve as a barrier between the coverslip and the array slide. Slides were then pl aced label side up in the hybridization chamber. The coverslip was secured with more paraf ilm and the slides were placed into the hybridization chamber. Hybridization solution containing 200 ng target DNA, 1 L Cy5 reference oligonucleotide and prehybridization solution to a total of 80 L was pipetted onto the array slide under the coverslip. The hybridization chamber was sealed with black clamps and wrapped in foil to block out any light. Chambers were then placed in the warm water bath or hybridization oven and in cubated overnight. 3.2.7 Washing and Scanning BC009 slides were removed from the rotating oven an d placed in Wash #1(20X SSC, 10% SDS, water) and shaken for 10 min at 100 rpm. The slides were then removed from Wash #1 and placed in Wash #2 (20X SSC, water) for 10 min at 100 rpm. For the final wash, Wash #3 (20X SSC), slides were also shaken fo r 10 min at 100 rpm. BC008 slides use the same reagents, time and rpm’s in the washin g steps but the concentrations are slightly different. Wash #1 contains 1X SSC and 0. 05 % SDS at 55C; Wash #2 contains 0.1X SSC and 0.05 % SDS; and Wash #3 contains 0.1X SSC. All arrays were scanned in the Agilent Scanner (Agilent Technologies) and anal yzed with Gene Pix Software (Molecular Devices).

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41 Figure 3.2. Types of array slides. The first type (a), referred to as BC008, consisted of a glass sli de with one gasket in which only one probe set can be used. Th e second type (b), referred to as BC009, consisted of a glass slide and a coverslip with two gaskets. This allow s for hybridization of a slide with two different p robe sets. (a) Array Coverslip (b) Array Coverslip

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42 3.3 Results cDNA made from cruise samples was sent to the Ward lab in July 2005 and the remaining cDNA was sent in November 2006. Many of the probes on the array consisted of a group of rbcL sequences obtained from GenBank, from which a repr esentative sequence based on similarities in that group, was s elected to characterize all types of that particular group. The probes with accession number s were also obtained from GenBank. Table A.1 lists the probes and the representative s equence that was chosen. All data were normalized by dividing all Cy3/Cy5 ratios for each probe, by the highest ratio in the data set. 3.3.1 Microarray performance Microarray data was arranged into groups: controls, surface samples and subsurface samples. Transcript DNA from clones obtained from the MRP in 2001 (46, 47) were hybridized to microarrays. Figures 3.3-3.6 show the transcripts that were used to test the performance of the microarrays. Clone 3SY1 is a hi gh-light Prochlorococcus (Fig. 3.3); clone 6SY3 is a marine type A Synechococcus (Fig 3.4); clones P994CH1 and P994GY7 are a pelagophyte and low-light Prochlorococcus respectively (Fig. 3.5); and clones 8CH12 and 1CH1 are a chrysophyte and a prymnesiophy te, respectively (Fig. 3.6). All of the clones, with the exception of 8CH12 and P994 GY7, hybridized to the probe whose sequence to which it was most homologous. Clone 8C H12 did not hybridize because there was no similar target on the array. Since P994GY7 did not hybridize specifically to one probe on the array, a pairwised sequence alignment was performed to deter mine how similar the sequences

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43 were to the respective probes to which each hybridi zed. The sequence alignment of P994GY7 showed that it is at least 68% similar to A F381708, an uncultured chlorophyte and to Prochlorococcus marinus The probe with the largest signal was the low li ght Prochlorococcus consensus probe and thus was expected to show a st ronger signal than the other probes. 3.3.2 Tampa Bay Fort Desoto is located at the entrance of Tampa Bay in the southwest corner of Mullet Key. The water samples from Fort Desoto were taken at 3m depth (Table 3.1). There was a large amount of background signal in the Fort Desoto hybridization experiment (Fig. 3.7). This array was hybridized with rbcL product made with Form ID primers, thus only the last eight probes on the far left sho uld have a signal. The signals of the Form IA and Form IB probes were caused by a large b ackground not observed in other samples. Prymnesiophytes were dominant at Fort Des oto, followed by the pelagophytes, the silicoflagellates and the haptophytes. There w ere also significant amounts of Phaeodactylum tricornutm Karenia mikimotoi diatoms and phaeophytes. Figure 3.8 shows another hybridization experiment with cDNA fr om Fort Desoto amplified with Form ID primers. In this experiment there is a hig h signal from the prymnesiophytes. The remaining Form ID probes were below the Cy3/Cy5 ratio cut off. This array was hybridized with MRP Station 2G amplified with Form IA/IB primers, which is discussed later.

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44 3.3.3 Surface samples The surface MRP samples were obtained from Stations 5A and 6A. Water samples from Station 5A were taken at 3m and a total of 3.6 L of water was filtered with Sterivex filters (Table 3.1). Samples collected from Station 6A wer e taken at 3 m depth and 2.6 L were filtered with Sterviex filters (see Table 3.1). Fi gure 3.9 shows the hybridization data from Station 5A for the cDNA that was sent in July 2005 to the Ward lab in Princeton. Figure 3.10 shows the data from Station 5A for the arrays that were hybridized in November 2006. cDNA used in Experiments 5A-1 and 6 A-1 were amplified with only Form ID primers. cDNA used in Experiments 5A-2 and 6A-2 were amplified with both Form ID and Form IA/IB primers. In Experiment 5A-1 pelagophytes are the dominant group followed by diatoms, prymnesiophytes, P. tricornutm haptophytes and silicoflagellates. There are small signals in some of the Form IA and Form IB probes but because only Form ID primers were used, these signa ls are attributed to background. Although the Form ID probes of array 5A-2 appear to have weaker signals than those observed in Station 5A-1, they are proportionate to each other and normalized to the Synechococcus signal. Synechococcus gave the highest signal at Station 5A-2. In array 6A-1, pelagophytes and prymnesiophytes dominated am ongst the Form ID probes (Fig. 3.11). There were also significant signals for the haptophytes and silicoflagellates. P. tricornutum pelagophytes and prymnesiophytes dominated array 6A-2 (Fig. 3.12). There was also a strong signal in the Synechococcus probe. As shown in array Station 5A-2, the Form ID probes in Station 6A-2 were lower than thos e of Station 6A-1, however the

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45 same proportions are observed. Small amount of chl orophytes were present at both Stations 5A-2 and 6A-2 (Figs. 3.12-3.14). 3.3.4 Subsurface samples Subsurface samples were obtained from Stations 8B, 1B, 2G and 7X (Fig. 3.1). In all four stations subsurface stations there is a simila r distribution of Form ID containing organisms. Station 8B is located at the subsurface chlorophyll maximum (SCM) and not in the plume. It is one of only two subsurface st ations in which there is data available for Form ID and Form IA/IB (Fig. 3.13). Water samples collected from Station 8B were obtained at 55 m (Table 3.1). Pelagophytes dominat ed the Form ID probes and there was a significant signal from the prymnesiophytes. Amo ngst the Form IA/IB probes, Synechococcus was the dominant player at Station 8B but there wa s also a significant signal from the prasinophyte, Chlorella, and the un cultivated chlorophyte AF381699. Thus, this subsurface sample was the first to demon strate appreciable hybridization to chlorophyte (green algal) phytoplankton. The SCM of Station 1 (1B) was also sampled (Fig. 3. 14). These samples were taken from a depth of 60 m of which 15 L of water w ere filtered. This array was hybridized with prasinophyte transcript (4SY39) and cDNA obtained from Station 1B. Pelagophytes yielded twice the signal of prymnesiop hytes and there were significant amouns of haptophytes, silicoflagellates, diatoms a nd K. mikimotoi Transcript 4SY39 did not hybridize to any of the chlorophyte/prasino phyte probes due to poor labeling efficiency.

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46 Station 2G is located just outside of the plume at a depth of 100m. cDNA from this station was amplified with both Form ID and Fo rm IA/IB primers, however on different arrays. Figure 3.15 shows the cDNA that was amplified with Form ID prim ers and as expected, only the Form ID probes have signa ls. Pelagophytes are once again the dominant player at Station 2G followed by the prymn esiophytes. There are also significant signals for the haptophytes, silicoflag ellates and diatoms. Figure 3.8 shows cDNA from Station 2G amplified with Form IA/IB prim ers and aforementioned cDNA obtained from Fort Desoto amplified with Form ID pr imers. Synechococcus dominated the Form IA/IB probes. Chlorella and the prasinophytes were significant at Station 2G as well as a few of the other chlorophyte probes, as w as observed for the other subsurface sample, 8B. Station 7X is located outside of the p lume at a depth of 3m (see Fig. 3.16). Pelagophytes dominated among the Form ID probes fol lowed by significant signals from some of the other Form ID probes. This array was a lso hybridized with a Synechococcus transcript and thus explains the strong signal with Synechococcus consensus probe.

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47 3.4 Discussion A microarray was designed to ascertain which clades or phylogenetic groups are most abundant and active in the primary production occur ring in the MRP as determined by rbcL hybridization and real time PCR. Microarrays were designed by the Ward Lab Group at Princeton University, Princeton, NJ. The probes used on the array are made up of a group of sequences taken from GenBank that wer e then assembled into consensus groups. Hybridization experiments were separated i nto three groups: controls, surface and subsurface. With the exception of the prasinophyte and the chr ysophyte, all positive controls hybridized to their respective probes on the array. Initially, we were unaware of what was exactly on the array and a chrysophyte was chos en as a positive control due to its presence in the MRP. However there was no chrysoph yte probe on the array and thus no hybridization signal was observed. The prasinophyt e probe did not label due to poor labeling efficiency of the Cy3 dye the dUTPaa label ed target. cDNA from Fort Desoto was amplified with only Form ID primers, yet there was a signal for every probe on the array including the Form IA and Form IB probes (Figure 3.7). The background levels in this experiment wer e particularly high. Since neither Form IA nor Form IB primers were used to amplify th e cDNA, these signals were ignored and only the Form ID signals were analyzed. These results are in agreement with real time PCR data obtained from Tampa Bay. The re al time PCR assay showed that haptophyte rbcL expression, which includes prymnesiophytes, was sig nificant in Tampa Bay where they were found in concentrations as high as 39 pg L-1. Furthermore,

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48 heterkont rbcL expression, which includes diatoms, pelagophytes, p inguiphytes and some silicoflagellates was found in concentrations as hi gh as 402 pg L-1. Surface stations had high Synechococcus pelagophyte and prymnesiophyte signals. There were no significant signals observe d in the chlorophte or Prochlorococcus probes. We were able to obtain phylogenetic profil es for both Form ID and Form IA/IB groups for Stations 5A and 6A. Dot blot hybridizat ion showed that the chromophytes dominated the surface waters by as much as 10 times the concentration of Form IA groups (18). Furthermore, RT-PCR results from Stat ions 5A and 6A showed that the heterokonts were 100 times more abundant than Synechococcus (18). The strong signal of the Synechococcus probe could be due to selective PCR amplification a nd not due to an abundance of Synechococcus in the plume surface. However, virtually no signal was obtained by PCR or microarray analysis for any Prochlorococcus phylotypes in the surface waters of the MRP. Stations 5A and 6A were located within the plume w here it has been previously shown that diatoms were dominant among the micropla nkton in the plume (18). Microarray analysis revealed an abundance of pelago phytes in the plume. Since the real time PCR assay cannot distinguish between diatoms a nd pelagophytes given their close relationship to each other (46), it is probable tha t the abundance of diatoms previously observed is actually a mixture of diatoms and pelag ophytes. When Station 5A-2 and 6A2 results were compared to Stations 5A and 6A, it w as evident that there was a difference in chromophyte signals on the arrays. However due to the high Synechococcus probe signal, the Form ID probe signals are normalized to the Synechococcus signal and appear

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49 weaker than those observed in arrays 5A-2 and 6A-2. Nonetheless, a similar distribution of organisms is still observed across the two surfa ce stations. Real time PCR assays conducted by John et al. (Subm itted) indicated that diatoms were more than likely responsible for most of the r bcL expression occurring in the MRP. This coincides with microarray analysis which showe d that Form ID organisms, specifically the pelagophytes and prymnesiophytes, were in greater abundance in the surface and subsurface waters. Subsurface samples had lesser amounts of Synechococcus than the surface stations and no prochlorophytes. Pelagophytes were dominant along the SCM. Real time PCR and dot blot hybridization resulted i n significant amounts of heterokonts in the subsurface. Station 8B was the first station at which signals were observed from the Form IB clade. Wawrik et al., (2 003) also found that prasinophytes were high at the SCM and the presence of diatoms. Microarrays detected no chlorophytes in the surfac e but they were found at the SCM. John et al. (Submitted) also found no chlorop hytes in the surface. Station 2G was dominated by Form ID rbcL expression, but there were also significant amounts of Synechococcus and Chlorella rbcL expression. This data coincides with the work of Wawrik et al. (2003) in which a clone library was c onstructed containing a diverse group of phylotypes closely related to green algae. One of the predominant groups among these clones was closely related to Chlorella sp. (46). This finding agrees with the Chlorella probe signal on the microarray (Fig. 3.8). The oth er group discovered was related to Bathycoccus prasinos and was abundant at the SCM (46). A significant p rasinophyte signal was observed in the microarrays at Station 8 B (Figure 3.13) which is located at the

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50 SCM. There was a significant pelagophyte signal at Station 7X. This station was located near the surface where our data shows that pelagoph ytes dominated other surface stations. Previous studies have used flow cytometry and epif luorescence microscopy to study picoplankton community structure in the ocean (5, 6) and thus it is believed that in tropical and subtropical areas, Prochlorococcus dominates the picoplankton. Wawrik et al. (2003) found an abundance of Prochlorococcus in the middle water column of the MRP around 40 m depth. However, no Prochlorococcus probe signals were observed in the subsurface stations on the microarrays. Additi onally there were no Prochlorococcus signals in the surface waters of the MRP which supp orts the work of Wawrik et al., (2003) in which Synechococcus dominated the surface waters. Usually, Synechococ cus dominates where Prochlorococcus is found in lesser amounts and thus could explain why Prochlorococcus was not found in the subsurface. A bundance in Synechococcus has previously been shown to occur at the interface whe re plume waters and water form the Gulf of Mexico mix (47). Furthermore, Prochlorococcus is abundant outside of plume waters in the oligotrophic waters of the Gulf of Me xico. Of the four stations with high to significant signals for the Synechococcus probe, Station 2G was the only one located at the interface where plume waters and blue waters me et. This array design does not provide a thorough repr esentation of the phylogenetic diversity of phytoplankton in the MRP. Figure A-3 in the Appendix shows the phylogenetic tree of the clone library made by Wawr ik et al. (46). Table A.1 lists the probes used on the microarray and their sequences. When the list of probes is compared

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51 to the phylogenetic tree, it is evident that many o f the sequences used in different consensus groups overlap one another on the tree (F igs. A-1 – A-3). The BroadChlr1 consensus group encompasses most of the BroadChlr2 consensus and probe AF381699. Furthermore, BroadChlr1 consensus group contains a few Chlorella species. All of the clones used in the haptophyte consensus were classi fied as prymnesiophytes by Wawrik et al. (2003) and only one prymnesiophyte sequence was used for the prymnesiophyte probe. Figures A-4 – A-6 show updated and more det ailed trees containing the sequences used to make the probe consensus groups, the transc ripts used as controls and their closet relatives. This study provides a third level of specificity a t which phylogenetic diversity has been sampled in the MRP. The first level, dot-blot hybridization is capable of dividing the organisms into their respective clades. Real t ime PCR is the second level and able is to detect specific groups within each clade. Micro arrays take it a step further and are capable of identifying even more organisms within e ach clade. Although a limited number of samples were analyzed by microarrays, thi s technology shows promise and this study was viewed as a pilot for their applicat ion. The rbcL probes designed were based upon published sequences from 2003 and we now have a much greater understanding of the diversity of rbcL -containing phytoplanktonic phylotypes. Future studies should employ this knowledge for judicious probe selection.

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52 3SY1 (HL Pro) 0 0.2 0.4 0.6 0.8 1 1.2 S YN L L PR O HL P R O PROMAR I N U S PR A S CHLORELLA F WC HL R BROADC H LR1 BROADCHLR2 BROADCHLR3 U 3 8 69 5 AF381695 AF3 8 1 6 97 AF381699 AF381703 AF3 8 1 7 08 AF381709 AF381718 HA P TO PR YM S I L I C O P EL A GO PHAEOPHYTE D I A TO MS PHAEODACTYLUM KMIKIMOTOI Cy3/Cy5 Uncultured chlorophytes Form ID Form IB Form IA Figure 3.3. Hybridization of 3SY1 (HL Prochlorococcus ) to array. 6SY3 (Syn)0 0.2 0.4 0.6 0.8 1 1.2 S YN L L PR O HL P R O PROMAR I N U S PR A S CHLORELLA F WC HL R BROADC H LR1 BROADCHLR2 BROADCHLR3 U 3 8 69 5 AF381695 AF3 8 1 6 97 AF381699 AF381703 AF3 8 1 7 08 AF381709 AF381718 HA P TO PR YM S I L I C O P EL A GO PHAEOPHYTE D I A TO MS PHAEODACTYLUM KMIKIMOTOI Cy3/Cy5 Uncultured chlorophytes Form ID Form IB Form IA Figure 3.4. Hybridization of 6SY3 ( Synechococcus ) to array.

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53 P994GY7 (LL Pro) & P994CH1 (Pelag)0 0.2 0.4 0.6 0.8 1 1.2 SYN LL PRO HL PRO PROMARINUS PR A S C HLORE LL A FWCHLR B ROADC H LR1 BROADCHLR2 B R O AD CH LR 3 U3 8 695 AF381695 A F3 81 697 AF3 81 699 AF 3 817 0 3 AF3 8 1708 AF381709 A F3 81 718 HAPTO PRYM SILIC O P EL AG O PHAEOPHYTE DIATOMS P H AEO D AC TY LUM KMIKIMOTO I Cy3/Cy5 Uncultured chlorophytes Form ID Form IB Form IA Figure 3.5. Hybridization of P994GY7 (LL Prochlorococcu s) and P994CH1 (pelagophyte) to array. 8CH12 (Chrys) & 1CH1 (Prym)0.0 0.2 0.4 0.6 0.8 1.0 1.2 SY N L L P RO HL PRO PROMARINUS PRAS CH L O R ELL A F WCHLR BROA D CHLR1 BR O A DCHL R2 BR O A DC HLR3 U3 8 695 AF381 6 9 5 AF 38 16 9 7 AF 38 16 9 9 AF381703 A F 38 17 0 8 A F 38 17 0 9 AF 3 817 1 8 HAPT O PRY M SILICO PELAGO PHAEOPHYTE DI A TOMS PHAEO DA CTY L UM KMIKI M OTOI Cy3/Cy5 Uncultured chlorophytes Form ID Form IB Form IA Figure 3.6. Hybridization of 8CH12 (chrysophyte) an d 1CH1 (prymnesiophyte) to array.

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54 Fort DeSoto0 0.2 0.4 0.6 0.8 1 1.2 S Y N LL P RO HL PRO P ROM A RI N US PRAS CH LO R EL L A F WC HL R BR O ADC HL R1 BR O ADC H LR2 BR O ADC H LR3 U3 8 69 5 AF 3 81695 AF 3 81697 AF38 1 6 99 AF 3 81703 AF38 1 7 08 AF 38 1 7 09 A F 38 1 7 18 HA P TO PRYM S ILICO P EL A GO PHAEOPHYTE DIATOMS PHAEODAC TY LU M K MIKIMOTO I Cy3/Cy5 Uncultured chlorophytes Form IA Form IB Form ID Figure 3.7. Hybridization of cDNA made from water c ollected from Fort Desoto and amplified with Form I D primers. 2G (IA/IB) & Fort Desoto (ID)0 0.2 0.4 0.6 0.8 1 1.2 SYN LL PR O H L P R O P RO M ARINU S P R A S C H LO R E LLA F WC HLR BRO A DC H LR 1 B R O A D CH LR 2 B R O A D CH L R3 U 38695 AF 381 69 5 A F381 69 7 AF38 1 69 9 AF 381 70 3 A F381 70 8 AF38 1 70 9 AF3 81 71 8 H A P TO PRYM SIL I CO P EL A GO PHA EO PHYTE DIATOMS P H A EO D ACTYL U M K M I K I MOTOICy3/Cy5 Form IA Form IB Uncultured chlorophytes Form ID Figure 3.8. Hybridization of cDNA made from water c ollected from Fort Desoto and Station 2G. cDNA obta ined from Station 2G was amplified with Form IA/IB prime rs while cDNA from Fort Desoto was amplified with Form ID primers.

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55 Station 5A-10 0.2 0.4 0.6 0.8 1 1.2 S YN LL PRO HL P R O P RO M AR I NU S P R AS CHLORELLA FWCH L R BR O A D CH LR 1 BR O A D CH LR 2 BR O A D CH L R3 U 3 869 5 A F3 8 16 9 5 A F3 8 16 9 7 AF38169 9 AF38170 3 AF38170 8 A F3 81 7 0 9 A F3 8 17 1 8 H AP T O PRYM SILI C O PELAGO PHA EO PHYTE DI A TO M S PH A EO D AC T YL U M KM I KIMOTOI Cy3/Cy5 Uncultured chlorophytes Form ID Form IB Form IA Figure 3.9. Hybridization of cDNA obtained from Sta tion 5A. cDNA used in this experiment was amplifie d with Form ID primers. Station 5A-2; IA/IB & ID0 0.2 0.4 0.6 0.8 1 1.2 S YN LL PRO HL P R O P RO M AR I NU S P R AS CHLORELLA FWCH L R BR O A D CH LR 1 BR O A D CH LR 2 BR O A D CH L R3 U 3 869 5 A F3 8 16 9 5 A F3 8 16 9 7 AF38169 9 AF38170 3 AF38170 8 A F3 81 7 0 9 A F3 8 17 1 8 H AP T O PRYM SILI C O PELAGO PHA EO PHYTE DI A TO M S PH A EO D AC T YL U M KM I KIMOTOI Cy3/Cy5 Uncultured chlorophytes Form ID Form IB Form IA Figure 3.10. Hybridization of cDNA obtained from St ation 5A. cDNA used in this experiment was amplifie d with Form IA/IB and Form ID primers.

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56 Station 6A-10.0 0.2 0.4 0.6 0.8 1.0 1.2 S YN L L PR O HL P R O PROMAR I N U S PR A S CHLORELLA F WC HL R BROADC H LR1 BROADCHLR2 BROADCHLR3 U 3 8 69 5 AF381695 AF3 8 1 6 97 AF381699 AF381703 AF3 8 1 7 08 AF381709 AF381718 HA P TO PR YM S I L I C O P EL A GO PHAEOPHYTE D I A TO MS PHAEODACTYLUM KMIKIMOTOI Cy3/Cy5 Uncultured chlorophytes Form ID Form IB Form IA Figure 3.11. Hybridization of cDNA obtained from St ation 6A that was amplified with Form ID primers. This cDNA was sent to the Ward Lab at Princeton Universi ty in July 2005. Station 6A-2; IA/IB & ID0 0.2 0.4 0.6 0.8 1 1.2 SYN LL PRO HL PRO P R O M A R I N U S P R AS C H L O R E LL A FWCHLR B R OAD C H LR 1 B R OAD C H LR 2 B R OA DC H LR 3 U 38695 AF381695 AF3816 9 7 A F3 81699 AF381703 AF3817 0 8 A F3 81709 A F 381718 HAPTO P R YM S I L I C O PELAGO PHAEOPHYTE D I A TOMS P H AE O D A C T YLUM K MIKI M O T O I Cy3/Cy5 Uncultured chlorophytes Form ID Form IB Form IA Figure 3.12. Hybridization of cDNA obtained form St ation 6A that was amplified with both Form IA/IB an d Form ID primers. cDNA was sent to the Ward Lab at Princeton University in November 2005.

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57 Station 8B; IA/IB & ID0 0.2 0.4 0.6 0.8 1 1.2 S YN L L PR O HL P R O PROMAR I N U S PR A S CHLORELLA F WC HL R BROADC H LR1 BROADCHLR2 BROADCHLR3 U 3 8 69 5 AF381695 AF3 8 1 6 97 AF381699 AF381703 AF3 8 1 7 08 AF381709 AF381718 HA P TO PR YM S I L I C O P EL A GO PHAEOPHYTE D I A TO MS PHAEODACTYLUM KMIKIMOTOI Cy3/Cy5 Uncultured chlorophytes Form ID Form IB Form IA Figure 3.13. Hybridization of cDNA obtained from St ation 8B. cDNA was amplified with Form IA/IB and F orm ID primers. 4SY39 (pras) & Station 1B (ID)0 0.2 0.4 0.6 0.8 1 1.2 S YN LL PR O H L P R O PROMARINUS PR A S CHLORELLA F W C H L R BR O ADC H L R 1 B ROA DC H L R 2 B ROA D CHLR3 U 3 869 5 AF38 1 695 A F 381697 AF38 1 699 A F 381703 AF3 81 7 0 8 A F3 81 7 09 AF38 1 718 HAPTO PRYM SILICO PELA G O P HA EOP H Y T E DI A T OMS PHAEODACTYLUM K MIK I MOTOI Cy3/Cy5 Uncultured chlorophytes Form ID Form IB Form IA Figure 3.14. Hybridization of cDNA obtained from St ation 1B and transcript 4SY39 (a prasinophyte). cD NA from Station 1B was amplified with Form ID primers.

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58 Station 2G0 0.2 0.4 0.6 0.8 1 1.2 S YN LL PRO HL P R O P RO M AR I NU S P R AS CHLORELLA FWCH L R BR O A D CH LR 1 BR O A D CH LR 2 BR O A D CH L R3 U 3 869 5 A F3 8 16 9 5 A F3 8 16 9 7 AF38169 9 AF38170 3 AF38170 8 A F3 81 7 0 9 A F3 8 17 1 8 H AP T O PRYM SILI C O PELAGO PHA EO PHYTE DI A TO M S PH A EO D AC T YL U M KM I KIMOTOI Cy3/Cy5 Uncultured chlorophytes Form ID Form IB Form IA Figure 3.15. Hybridization of cDNA obtained from St ation 2G. cDNA was amplified with Form ID primers. Station 7X (ID) & 6SY30 0.2 0.4 0.6 0.8 1 1.2 S Y N LL PRO HL PRO P RO M ARINUS PRAS CHLORELLA FWCHLR BROADCHLR1 BRO ADCHLR2 BROADCHLR3 U38 6 95 AF381695 A F 3 81697 A F 3 81699 AF3817 0 3 AF381708 AF381709 AF3 8171 8 HAPTO PRYM SILICO P E L AGO PH AE OPHYTE DIA T O M S P HAE O D ACTYLUM KM IKIM OT O I Cy3/Cy5 Uncultured chlorophytes Form ID Form IB Form IA Figure 3.16 Hybridization of cDNA obtained from S tation 7X and clone 6SY3 (a synechococcus). cDNA f rom Staion 7X was amplified with Form ID primers.

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62 34. Scharf, M. E., D. Wu-Scharf, B. R. Pittendrigh, and G. W. Bennett. 2003. Casteand development-associated gene expression in a lo wer termite. Genome Biol. 4: R62-R62.11. 35. Schneider, G., Y. Lindqvist, and C. I. Branden. 1992. RUBISCO: structure and mechanism. Annu. Rev. Biophys. Biomol. Struct. 21: 119-143. 36. So, A. K. C., G. S. Espie, E. B. Williams, J. M. Sh ively, S. Heinhorst, and G. C. Cannon. 2004. A Novel Evolutionary Lineage of Carbonic Anhy drase ( Class) Is a Component of the Carboxysome Shell. J. Bacter iol. 186: 623-630. 37. Spalding, M. H., K. Van, Y. Wang, and Y. Nakamura. 2002. Acclimation of Chlamydomonas to changing carbon availability. Fun ctional Plant Biology 29: 221-230. 38. Steward, G. F., B. D. Jenkins, B. B. Ward, and J. P Zehr. 2004. Development and Testing of a DNA Macroarray To Assess Nitrogenase (nifH) Gene Diversity. Appl. Environ. Microbiol. 70: 1455-1465. 39. Tabita, F. R. M. 1999. Microbial ribulose 1, 5-bisphosphate carboxyl ase/oxygenase: A different perspective. Photosynthesis Res. 60: 1-28. 40. Tabita, F. R. 1988. Molecular and cellular regulation of autotrop hic carbon dioxide fixation in microorganisms. Microbiol. Rev. 52: 155-189. 41. Taroncher-Oldenburg, G., E. M. Griner, C. A. Franci s, and B. B. Ward. 2003. Oligonucleotide Microarray for the Study of Functi onal Gene Diversity in the Nitrogen Cycle in the Environment. Appl. Environ. Microbiol. 69: 1159-1171. 42. Turner, R. E., and N. N. Rabalais. 1991. Changes in Mississippi River Water Quality This Century. Bioscience 41: 140-147. 43. U.S. Environmental Protection Agency. 2007. Mississippi River Basin and Gulf of Mexico hypoxia: culture and history. [online]: http://www.epa.gov/mbasin/culture.htm 44. Ward, B. B. 2005. MOLECULAR APPROACHES TO MARINE MICROBIAL ECOLOGY AND THE MARINE NITROGEN CYCLE. Annu. Rev. Earth Planet. Sci. 33: 301-333. 45. Watson, G. M., and F. R. Tabita. 1997. Microbial ribulose 1,5-bisphosphate carboxylase/oxygenase: a molecule for phylogenetic and enzymological investigation. FEMS Microbiol. Lett. 146: 13-22. 46. Wawrik, B., J. H. Paul, L. Campbell, D. Griffin, L. Houchin, A. Fuentes-Ortega, and F. Muller-Karger. 2003. Vertical structure of the phytoplankton commu nity

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63 associated with a coastal plume in the Gulf of Mex ico. Mar. Ecol. Prog. Ser. 251: 87–101. 47. Wawrik, B., and J. H. Paul. 2004. Phytoplankton community structure and productivity along the axis of the Mississippi Ri ver plume in oligotrophic Gulf of Mexico waters. Aquat. Microb. Ecol. 35: 185-196. 48. Wawrik, B., J. H. Paul, and F. R. Tabita. 2002. Real-time PCR quantification of rbcL (ribulose-1,5-bisphosphate carboxylase/oxygen ase) mRNA in diatoms and pelagophytes. Appl. Environ. Microbiol. 68: 3771-3779. 49. Wikipedia. 2007. RuBisCO. [online]: http://en.wikipedia.org/wiki/RuBisCO 50. Wilkins, M. B. 1992. Circadian rhythms: their origin and control. New Phytol. 121: 347-375. 51. Woodger, F. J., M. R. Badger, and G. D. Price. 2003. Inorganic Carbon Limitation Induces Transcripts Encoding Components of the CO2 -Concentrating Mechanism in Synechococcus sp. PCC7942 through a Redox-Indep endent Pathway. Plant Physiol. 133: 2069-2080. 52. Wyman, M., J. T. Davies, S. Hodgson, G. A. Tarran, and D. A. Purdie. 2005. Dynamics of Ribulose 1, 5-Bisphosphate Carboxylase /Oxygenase Gene Expression in the Coccolithophorid Coccolithus pel agicus during a Tracer Release Experiment in the Northeast Atlantic {dagg er}. Appl. Environ. Microbiol. 71: 1654-1661.

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64 Appendices

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65 Appendix A Table A-1. rbcL probe list

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66 Appendix A (Continued) Table A-1 (Continued)

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67 Appendix A (Continued) Table A-1 (Continued)

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68 P994CY2 P994EY1 P994DY5F P994FY43 P994EY5 P994FY8 P994AY6 P994AY8 Chlorella ellipsoidea Chlorella like algae Platydorina caudata P994AY1 P994AY2 Chlorarachnion reptans P994AY17 P994AY7 Chlorella like algae Roya anglica Spirotaenia condensata Nitrium digitus Spinach Pentodon pentandrus Zygnema peliosporum Mougeotia sp Cylindrocystis sp mesotaenium cadariorum P994DY2F P994EY8 P994AY15 P994AY4 Sphagnum palustre P994FY24 P994FY29 P994GY9 P974AY1 Bathycoccus prasinos P974HCY2 P994FY27 P974AH9 P994EY4 P9710GY1 P994EY3 P994FY19 P994FY3 Prasinophytes Anabaena sp. Synechococcus PCC7120 P994GY2 Synechococcus PCC6301 P994DY13F Form IB Synechococcus cluster Synechococcus PCC6803 Trichodesmium sp Form IB Hydrogenovibrio marinus Halothiobacillus neapolitanus Nitrobacter vulgaris Prochlorococcus GP2 Synechococcus WH7803 Form IA 100 80 96 94 69 60 58 100 70 61 73 67 99 63 56 98 75 67 92 50 87 52 74 100 100 53 60 70 97 68 0.050 Figure A-1. Phylogenetic tree taken from Wawrik et al., 2003 showing the Form IB clones obtained from the Mississippi River Plume. The probe consensus group s present on the tree have been labeled. Chlorella BroadChlr2 AF381699 Pras. BroadChlr1

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69 Appendix A (Continued) P994AH1 P994BH15 P994DH2 P994BH16 P994GH16 P994CH25 P994EH4 P994BH3 P994BH5 P994EH9 Gymnodinium mikimoitoi P994EH10 P994EH2 P994AH28 P994BH22 P994GH21 Calyptrosphaera sphaeroidea Umbilicosphaera sibogae P994AH7 Chrysochromulina hirta P994DH28 P994AH5 Emiliania huxleyi Gephyrocapsa oceanica P994CH9 P994EH20 Coccolithus pelagicus Pleurochrysis carterae P994DH1 P994HH11 P994HH25 Prymnesiophyta Pavlova salina Gigartina alveata Chondracanthus acicularis Gelidiella acerosa Rhodophyta P994CH2 P994CH1 P994FH18 P994FH2 Pelagomonas calceolata P994FH21 P994HH7 Coccoid pelagophyte Pelagococcus subviridis P994CH26 Pelagophyta Aureoumbra lagunensis Aureococcus anophagefferens Sarcinochrysis marina P994AH8 Botrydium stoloniferum Botrydiopsis intercedens Xanthophyceae P994DH9 Peridinium foleaceum endosymbiont P994GH17 P994GH18 P994GH5 P994FH10 Phaeodactylum tricornutum Rhizosolenia setigera P994FH12 P994GH24 Cylindrotheca sp P994FH16 P994FH1 P994CH3 Skeletonema costatum Thalassiosira nordenskioeldii Detonula confervacea P994FH13 P994CH22 P994HH14 Bacillariophyta Nannochloropsis CCMP531 Nannochloropsis CCMP533 Eustigmatos magna P994EH13 P994EH14 P994EH25 P994BH13 P994DH18 Eustigmatophyceae P994BH8 Chromulina nebulosa P994BH7 P994AH12 P994BH10 Haptophyceae 77 52 99 76 51 67 97 77 50 90 66 63 88 52 93 68 50 51 87 99 61 55 52 85 97 84 64 62 87 65 50 54 98 99 77 0.050 Haptophytes Prymnesiophyte Pelagophytes Silicoflagellates Diatoms Figure A-2. Pylogenetic tree taken from Wawrik et a l., 2003 of the Form ID sequences obtained from the Mississippi River Plume. The probe consensus gr oups present on the tre e have been labeled.

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70 Appendix A (Continued) Prochlorococcus TAK9803 P974AY2 P974DCH6 Prochlorococcus MED4 P994DY19F P974DCH9 P974AH3 P974DCH11 P994DY11F P994DY16F P994DY10F P994BY13 P994DY20F Prochlorococcus SB P994DY17F Prochlorococcus GP2 P994BY2 P994BY1 P994BY7 P974DCH12 P994CY1 P994FY9 P994HH16 Prochlorococcus MIT9313 P994GY8 Prochlorococcus SS120 P994GY23 P994GY20 P994GY7 Vent clone AB038642 Vent clone AB038643 P994GY17 Vent clone AB038641 P994EY27 P994GY1 Prochlorococcus PAC1 Prochlorococcus NATL1 Prochlorococcus NATL2B Vent clone AB038644 Vent clone AB038645 P994FY23 P99SY17 Vent clone AB038646 Synechococcus WH7803 P99SY12 P99SY5 Synechococcus PCC8102 P99SY1 Vent clone AB038633 Thiobacillus ferrioxidans 65 86 64 64 88 52 81 74 98 87 61 97 0.02 High Light Prochlorococcus Form IA Marine A –type 2 Synechococcus Low Light Syn LL Pro HL Pro Figure A-3. Phylogenetic tree taken from Wawrik et al., 2003 showing the Form IA clones found in the Mississippi River Plume. The probe consensus g roups present on the tree have been labeled

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71 Appendix A (Continued) Pseudoneochloris marina Chlorogonium kasakii Percursaria percursa Scenedesmus quadricauda Dunaliella salina P994AY1 4SY39 Tetraselmis marina Tetraselmis suecica Chlorella vulgaris Chlorella ellipsoidea Trebouxia anticipata Myrmecia biatorellae Halimeda opuntia Leptosira erumpens Chlorarachnion reptans P994AY2 P994AY17 P994AY7 P994AY4 P994EY8 Dolichomastix tenuilepis Picocystis salinarum Pedinomonas minor Paulschulzia pseudovolvox Phacotus lenticularis Pteromonas angulosa Volvox globator Platydorina caudata Tetrabaena socialis Yamagishiella unicocca Chlamydomonas reinhardtii Pyramimonas octopus P994CY2 P994DY5F Pyramimonas australis Pyramimonas olivacea Pyramimonas cyclotreta P994FY43 P994FY8 Pyramimonas mantoniae P994AY6 Pyramimonas aureus Nephroselmis olivacea Pseudoscourfieldia marina Pycnococcus provasolii P994AY8 Mantoniella squamata Mamiella sp Micromonas pusilla Bathycoccus prasinos P974HCY2 P994FY27 P974AH9 P994EY4 P994EY3 P994FY19 P994FY29 100 84 53 58 62 100 100 100 99 99 80 89 41 41 58 44 61 89 70 82 60 49 64 18 41 28 74 37 24 65 27 23 27 23 6 43 32 25 26 7 15 2 0 1 21 25 16 7 0 4 4 7 0 7 15 29 85 Figure A-4. Conensus tree of Form IB clone sequence s obtained from the MRP in 1999 and 2001 along with their closest r elatives.

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72 Appendix A (Continued) P994FY29 Synechococcus PCC7002 Synechocystis PCC6803 Crocosphaera watsonii Lyngbya aestuarii PCC 7419 Nostoc punctiforme Synechococcus elongatus Trichodesmium erythraeum IMS101 Trichodesmium thiebautii P994FY23 P994DY11F Prochlorococcus marinus TAK9803 P994DY19F P994DY20F P994CY1 P974AH3 P994BY1 P994DY16F P994BY7 Prochlorococcus marinus SB Prochlorococcus MED4 P994GY1 Prochlorococcus marinus PAC1 Prochlor NATL1 Prochlor NATL2 P994GY17 P994HH16 P994FY9 Prochlorococcus marinus CCMP1375 P994GY7 P994GY8 Prochlorococcus marinus MIT9313 P99SY17 P99SY12 P99SY5 Synechococcus WH7803 Synechococcus WH8102 P99SY1 6SY3 97 66 70 66 97 100 30 30 39 77 44 85 36 67 100 96 63 99 37 76 100 41 96 20 15 17 54 35 35 35 48 37 25 44 78 45 40 Figure A-5. Conensus tree of Form IA clone sequence s obtained from the MRP in 1999 and 2001 along with their closest r elatives.

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73 Appendix A (Continued) 6SY3 Dinophysis fortii Dinophysis tripos P994CH2 P994EH20 Emiliania huxleyi Gephyrocapsa oceanica P994AH5 P994EH10 P994BH22 P994GH21 P994EH9 P994DH2 P994BH15 P994BH3 Calyptrosphaera sphaeroidea Umbilicosphaera sibogae P994AH7 Chrysochromulina hirta Coccolithus pelagicus P994CH25 P994DH1 P994GH16 P994HH11 P994HH25 Pavlova salina P994DH28 Apedinella radians Pseudopedinella elastica P994AH8 Chromulina nebulosa Hibberdia magna Mallomonas asmundae Synura uvella P994BH8 Nannochloropsis CCMP531 Nannochloropsis CCMP533 Eustigmatos magna P994EH25 P994BH13 P994EH14 P994CH1 P994FH2 P994HH7 Pelagomonas calceolata Coccoid pelagophyte Pelagococcus subviridis Aureococcus anophagefferens Heterococcus caespitosus Mischococcus sphaerocephalus Aureoumbra lagunensis Sarcinochrysis marina P994BH7 P994AH12 P994DH9 Peridinium foleaceum endosymbiont P994FH12 P994GH24 Cylindrotheca sp Phaeodactylum tricornutum P994FH10 P994GH17 P994GH5 P994FH16 Rhizosolenia setigera P994FH1 Skeletonema costatum Thalassiosira nordenskioeldii Detonula confervacea P994FH13 P994CH22 97 78 45 99 100 100 97 100 100 95 100 25 73 78 99 56 61 55 100 99 59 49 67 99 40 73 83 41 10 41 6 14 1 16 74 60 38 54 14 67 86 1 81 63 6 13 62 12 48 50 100 43 28 66 15 13 81 17 52 35 12 10 15 22 6 1 8 1 0 0.05 Figure A-6. Conensus tree of Form ID clone sequence s obtained from the MRP in 1999 and 2001 along with their closest relat ives.