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The development of a method to measure microbial gene expression in the marine water-column environment

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Title:
The development of a method to measure microbial gene expression in the marine water-column environment
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xi, 86 leaves : ill. ; 29 cm
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English
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Pichard, Scott L.
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University of South Florida
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Tampa, Florida
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Gene expression   ( lcsh )
Marine phytoplankton   ( lcsh )
Marine bacteria   ( lcsh )
Messenger RNA   ( lcsh )
Dissertations, Academic -- Marine Science -- Masters -- USF   ( fts )

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Thesis (M.S.)--University of South Florida, 1992. Includes bibliographical references (leaves 68-78).

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University of South Florida
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Universtity of South Florida
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aleph - 028934800
oclc - 26617633
usfldc doi - F51-00095
usfldc handle - f51.95
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SFS0040045:00001


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THE DEVELOPMENT OF A METHOD TO MEASURE MICROBIAL GENE EXPRESSION IN THE MARINE WATER-COLUMN ENVIRONMENT by Scott L. Pichard A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science i n the Department of Marine Science in the Un i versity of South Florida May 1992 Major Professor: John H Paul, Ph.D.

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Graduate Council University of South Florida Tampa, Florida CERTIFICATE OF APPROVAL Master's THESIS This is to certify that the Master's Thes i s of Scott L. Pichard with a major in Department of Marine Science has been approved by the Examining Committee on 03/27/92 as satisfactory for the Thesis requirement for the Master of Science degree Thesis Committee: ..x . f1: Major i(rofessor : H. aul, Ph .D. ----'MI;mber: Gabriel A. Varg?. 0

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ACKNOWLEDGEMENTS I wish to thank Sunny Jiang, Marc Frischer, Chris Kellog, Dr. Wade Jeffrey, Jennifer Thurmond, Tamara Pease, and Usa Czares for all their help, guidance and support over this period of my graduate study. I also wish to thank Drs. Gabriel Vargo and Duane Eichler who's technical suggestions, insights into "how it all works .. and statements like, .. RNA is the reason God made DNA .. have been instrumental in the successful development of this method. To the captain and crews of the RN Pelican, RN Cape Hatteras, and the RN Bellows who without them I would have never gone to sea. To my advisor, Dr. John Paul, for his understanding, guidance, teaching me the excitement of science, and for .. all that money .. that kept me going. To Stacie Little who without her great graphics work none of this would ever have been presented. Finally, especially to my mother and father, LaVere and Wilbur Pichard, who if not for them this would not have been poss i ble. iii

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TABLE OF CONTENTS LIST OF TABLES vi LIST OF FIGURES vii ABSTRACT ix CHAPTER 1: INTRODUCTION 1 Models for Gene Expression: a B ioremediation Gene 4 Models for Gene Expression : rbcL, an Environmentally S i gnificant Gene 5 The Need and Use of D i re c t Methods in S tudying M arin e Microbial Ecology 9 De tectio n of M icrobia l Gen e Expression 11 CHAPTER 2 : DEVELOPMENT OF A METHOD FOR DETECTION OF GENE EXPRESSION AS mRNNDNA IN MARINE MICROBIAL COMMUNITIES 14 Introduction 14 Methods and Materi a ls 14 Bacteri a l strains and pl asmids 14 Culture conditions 15 M icrocosm studies 19 RNA extraction method 19 DNA extract io n method 20 Nucle as e d iges t io n of RNA samples, dot blotting, probing and quanititation 21 iv

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Nuclease digestion of DNA samples, dot blotting, probing and quantitation 23 Catechol dioxygenase enzyme assay 23 Results 24 Discussion 34 CHAPTER 3: THE DETECTION OF rbcL GENE EXPRESSION IN NATURAL PHYTOPLANKTON POPULATIONS 40 Introduction 40 Methods and Materials 40 Field sites 40 ANNONA sampling 41 Phytoplankton carbon fixation 41 Biomass/cell count determinations 43 RNA amplification 44 Results 45 Discussion 56 CHAPTER 4 : CONCLUSIONS 66 LITERATURE CITED 68 APPENDICES 79 APPENDIX A: SYNTHESIS OF RNA GENE PROBES 80 APPENDIX B: PROBING PROTOCOL FOR USE WITH RNA GENE PROBES 83 APPENDIX C : SEQUENCES OF DEGENERATE OLIGONUCLEOTIDE PRIMERS FOR AMPLIFICATION OF rbcL 85 v

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LIST OF TABLES 1 Bacterial strains used in this study 16 2. Plasmids used in this study 17 3 Efficiency of RNA extraction form E. coli B cells filtered onto various filter types as determined by spectrophotometry. 25 4. mANA precipitation efficiency. 26 5. DNA determinations of DNA extractions from WJT -1 C (pLV1 013) 27 6. DNA precipitat i on efficiency. 27 vi

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LIST OF FIGURES 1. Maps of plasmid constructs used in thi s study. 18 2 Probing of purified rbcL DNA and RNA with AS and S RNA probes. 29 3 Detection of nptll gene expression in various bacteria in seawater and culture. 31 4. Relative absorbance of expression signals of nptll in E. coli RM1259 32 or Vibrio sp. WJT-1C(pQSR50) from Figure 3. 5. Detection of gene expression in a marine Vibrio containing pLV1013 in culture and in cells resuspended in seawater 33 6. Induction of gene expression in a mar i ne Vibr i o sp. in response to temperature. 35 7. Location of stations sampled for rbcL mANA. 42 8 Diel variations in rbcL mANA levels for Dry Tortugas natural phytoplankton populations and a Synechococcus culture. 46 9 Laser densitometry of the AS probed dot blot in Figure 8 47 1 0. Diel rbcL expression and carbon fixation for natural phytoplankton populations in the Bahamas 48 11. Nearshore-offshore transect of rbcL mANA analysis and re l ated parameters for Gulf of Mexico stations 50 12 Overnight exposures of dotblots of rbcL mANA form 300 m l samples of station 6 seawater. 52 vii

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13 Overnight exposures of dotblots of rbcl DNA from 300 ml samples of station 6 seawater 53 14. Vertical profiles of rbcl mRNA and related parameters for station 6. 54 15. Diel patterns in rbcl mRNA and related parameters 57 1 6 Gel electrophoresis of amplification products of environmental rbcl mRNA. 58 17 Southern analysis of amplification products of environmental rbcl mRNA shown in Figure 16. 59 viii

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THE DEVELOPMENT OF A METHOD TO MEASURE MICROBIAL GENE EXPRESSION IN THE MARINE WATER-COLUMN ENVIRONMENT by Scott L. Pichard An Abstract of a thesis subm i tted in partial fulfillment of the requirements for the degree of Master of Science in the Department of Marine Science in the University of South Florida May 1992 Major Professor: John H Paul, Ph. D ix

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A method has been developed for detecting microbial gene expression in the marine environment that combines the extraction of RNA and DNA with probing of extracts using single-stranded anti sense and sense RNA gene probes. In this study, I have chosen to investigate the expression of the neomycin phosphotransferase II (.QQlli) gene of Tn5, the catechol-2,3-dioxygenase gene, and the ribulose-1,5-bisphosphate carboxylase large subunit (rbcl) gene of Synechococcus sp. PCC6301. The use of Whatman GF/F filters, while enabling large volumes of seawater to be recovered, gave a poor RNA recovery of 14%. Millipore Durapore filters were much better, enabling 89% of the RNA to be recovered Using this same filter, DNA recovery was equally efficient. Lim i ts of detection for .QQ!l! expression in seawater, using GF/F filters were {1. 5 0 .3) x 106 cells of E. coli and (1.0 2 .7) x 106 cells of Vibrio sp mANA analysis was also used to monitor expression of in a marine Vibr i o contain i ng a temperature inducible added to seawater. While, Increases in mANA corresponded to increases in catechol dioxygenase activity, maximum levels mANA were reached before the appearance of enzyme activity. This method was also successful in detecting rbcl expression in natural mar i ne phytoplankton populations. Waters were sampled in the Gulf of Mexico the Dry tortugas, and the Bahamas Levels of rbcl mANA were highest in nearshore environments and decreased in offshore (oligotrophic) waters w i th carbon f ix at i on following the X

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same trend. This suggests a d i rect relationship between carbon fixation and the level of rbcL transcripts Size fractionation experiments of offshore waters indicated that in oligotrophic environments the < 1 I-'m size fraction contained 1 00% of the Synechoccus-like rbcL transcripts yet only 59% of the carbon fixation. Natural populations exhibited d i e l patterns in rbcL expression and carbon fixation During light periods the rbcL mRNNDNA ratio was 4.4 t i mes greater (76 ng mRNNng DNA) than in the dark (17 ng mRNNng DNA). Similarly, chlorophyll specific carbon fixation was elevated in the light. These results ind i cate that diel patterns of carbon fixation may be photoregulated at the level of rbcL transcription not by changes in rbcL gene dose, for natural populations This methodology could be applied to the detect ion of expression of a myriad of ecologically important, conserved gene sequences in the marine environment. Abstract approved : ---"'_ .......... __..... ______ Major Professor: John H Paul, Ph. D Professor, Dept. of Marine Science I I Date of Approval xi

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CHAPTER1 :INTRODUCTION Microorganisms, particularly bacteria, represent the largest portion of biomass in the marine environment and it is through their biochemical activities that much of the biological, chemical, and even physical character of the planet is determined. Autotrophic microorganisms are responsible for photosynthetic carbon fixation, be i ng the primary producers in oceanic ecosystems and producers of most of the oxygen on this planet. Heterotrophic microorganisms remineralize elements and may be significant consumers of this primary production (Fuhrman and Azam, 1982). Up until the 1980's heterotrophic bacteria had been traditionally placed in the role of remineralizers, converting organic carbon, nitrogen, sulfur, and phosphorus into their inorganic forms for use by the phytoplankton. In 1983, Azam et al. suggested the presence of a "microbial loop" whereby this bacterial biomass may be grazed upon by heterotrophic flagellates and thereby returning some energy to the conventional planktonic food chain However, the carbon in these bacteria appear mainly as respired C02 and dissolved organic carbon (Ducklow et al., 1986). In systems dominated by cyanobacterial production, only 6% of the carbon was cycled into higher trophic levels (Hagstrom et al., 1988). Therefore it appeared that direct transfer of bacterial and cyanobacterial production to higher trophic levels was not occuring, and that bacterial production was acting as an energy and carbon "sink". The funct i oning and dynamics

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2 of the microbial loop is not yet completely understood, particularly in light of the recently discovered abundance of viruses (Bergh et al., 1989; Proctor and Fuhrman, 1990). The ecological role that microorganisms play in the mar i ne environment is a direct consequence of their gene-encoded enzyme functions. The information for the structure and biosynthesis of enzymes is contained in the organism's genetic material, the DNA. This information is translated to form macromolecular catalysts in a three step process. In short the DNA strand separates and a RNA polymerase transcribes the genetic code, starting at a element called a promoter, forming a messenger RNA molecule (mANA) Transcription from promoters may be regulated or constitutive (Lewin, 1983) This mANA, containing information, i s then translated by r i bosomes to form a protein This three step process is collectively called gene expression. While the ult i mate source of the information is the DNA the RNA is a necessary i ntermediate in construction of the protein It is the rate of synthesis of and instability of this mANA, its ability to be rapidly turned over, that plays the central role in determ i ning levels of protein synthesis in both prokaryotes and eukaryotes. Each particular message has a unique rate of decay (Kennell, 1986) which is dependant upon secondary structure and the accessibility of target decay sequences This differential stability of mANA regulates whether soluble or particulate hydrogenases are synthesized in Alcaligenes eutrophus (Oelmuller et al., 1990) and controls the relative abundances of plasmid structural gene products in Escherichia coli (Owolabi and Rosen 1990) Albertson et al.{1990) determined functional mANA half-lives by measuring protein synthesis of proteins in a marine Vibrio sp. and found that during starvation the

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3 half-lives of mANAs increased several fold over a 24 hour period. During starvation, the mANA half-lives of some starvation specific proteins were extremely long lived (up to 70 minutes). This half-life is an extreme when compared to mANA half-lives in E. coli. The range of E. coli mANA half-lives is from 30 seconds to 8 minutes at 37C with the half lives being extended at lower temperatures (Kennell, 1986). While rates of mass decay of mANA, reduction in actual amounts of mANA, and functional decay, the ability to be translated, are usually the same, there are some cases where functional inactivation occurs slightly faster than mass decay (Kennell 1986) This decay of mANA is a posttranscriptional mechanism regulating gene expression and occurs by a combination of 3' exonuclease and endonuclease degradation of the mANA (Belasco and Higgins, 1988). Once the mANA has been translated and the protein synthesized, the protein turnover rate may also control gene expression This is accomplished by intracellular protein degradation by generalized and specific proteases. While incorrectly assembled or mutant proteins are degraded in a few minutes, most cellular proteins undergo slow proteolysis. The average protein half-life in E.coli varies from 13 to 60 minutes (Pine, 1980) This rate of turnover may increase as cells respond to nutrient starvation or other conditions of environmental stress Another mechanism of regulation is at the level of gene dosage (gene copies per cell). The majority of gene dosage effects in bacteria appear to be associated with plasmid encoded genes. Nordstrom et al. (1972) found that increased resistance to a variety of antibiotics was directly related to the increase in A-factor copies (2 to 4 fold) per

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4 chromosome. Odakura et al. (1974) found that the hypersynthesis of penicillinase in E. coli was due to duplicated penicillin resistance genes on a plasmid and attributed this to a recombinational event leading to plasmid gene duplication in response to antibiotic selective pressure. Some genes have also been duplicated and transferred from plasmid to chromosome, as for chloramphenicol acetyl transferase genes in E.coli K12 (lyobe et al., 1974). A combination of gene dosage effects were found for several genes in Serratia marcescens (Timmis and Winkler, 1973). A lipase gene was duplicated on the chromosome and a bacteriocin was duplicated on a plasmid in response to changes in culture conditions. Thus, there are a variety of ways a cell can respond to its environment that are collectively termed control of gene expression; however, transcriptional regulation is by far most common. Models for Gene Expression : xviE. a Bioremediation Gene. More than 1 000 new compounds are marketed every year with the total annual world production of synthetic organic chemicals being over 300 million tons. Many of these compounds are recalcitrant and do not degrade rapidly once introduced into the environment (Fewson, 1988) Some compounds are finally broken down in a multistep process by bacteria containing plasmids which encode multioperon degradative genes. One such plasmid, probably the most understood catabolic plasmid, is the TOL plasmid pWWO which encodes the enzymes necessary to degrade toluene (Williams and Murray, 1974; Burlage et al., 1989; Assinder and Williams, 1990). The TOL plas i md is organized

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5 into an upper -pathway and meta-pathway operons. Benzoate and toluates are converted by the meta-pathway into Krebs cycle intermediates which can be used by the cell for growth. The gene, encoding the enzyme catechol 2,3-oxygenase (C230, E.C.1.13 11.2), is located in this meta-pathway and catalyzes the conversion of the aromatic compound catechol to 2-hydroxymuconic semialdehyde (Burlage, 1989). Because of it's ease of assay of enzymatic activity, I have chosen gene as a model bioremediation gene In this project, the gene is plasmid encoded and its expression is under control of the bacteriophage Lambda promoter (pA) and its temperature sensitive repressor (cl857, Winstanley et al., 1989). This construct enables gene expression to be manipulated by temperature changes. Increases in temperature result in increased expression of the gene. Models for Gene Expression : rbcL. an Environmentally Significant Gene. The process of photosynthetic C02 assimilation is for the most part catalyzed by the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco: E.C. 4.1. 1.39) which carboxylates the 5-carbon compound, ribulose-1,5-bisphosphate. This enzyme is responsible for an estimated annual world production of 5x1014 kilograms of organic matter (Knaff, 1989). Unlike which is confined to the prokaryotic world, rubisco is present in a variety of organisms from autotrophic procaryotes to algae and higher plants. The enzyme is of similar mass (560,000 daltons) in all cases to date except in

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6 Rhodospirillum rubrum where there are two forms of the enzyme (Tabita, 1988). The enzyme is a complex of two subunits, one called the large subunit (rbcU and the other the small subunit (rbcS), exisiting in a LaSs configuration. In some cases both the rbcl and rbcS genes are assembled into a single operon existing on either the chromosome (cyanobacteria) or the chloroplast genome (rhodophytes and chromophytes). In other cases the genes are segregated with the rbcl contained on the chloroplast genome and the rbcS contained on the nuclear genome (chlorophyta algae and higher plants). The rbcl gene is highly conserved and shows 85% amino acid sequence similarity between higher plants, chlorophyta algae and cyanobacteria (Shinozaki et al., 1983). However, chromophyte and rhodophyta algae possess divergent sequences. This observation suggests two separate evolutionary lineages for rubisco genes (Valentin and Zetsche, 1989; Valentin and Zetsche, 1990a; Valentin and Zetsche, 1990b; Douglas et al., 1990; Newman and Cattolico, 1990). The photosynthetic bacterium Chromatium vinosum possess two complete sets of rubisco genes, rbcL rbcS and rbcA-rbcB (Viale et al., 1989). These two sets of sequences appear to be identical (Kobayashi et al., 1991) but only rbcA-rbcB is expressed under normal growth conditions (Viale et al., 1990). Both sets appear to be more closely related to cyanobacterial sequences than to chemoautotrophs such as Alcaligenes sp. In Rhodobacter sphaeroides there are two Forms, Form I & Form II, of the rubisco enzyme. Both are structurally and catalytically distinct (Jouanneau and Tabita, 1986), with Form I predominating under conditions where C02 is limiting and Form II under conditions where C02 is saturating.

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7 While the distribution of rubisco genes is diverse, so are the mechanisms of its regulation. The rbcl and rbcS genes appear to be cotranscribed (controlled from a single promoter) in a variety of organisms where the rbcl and rbcS are proximally located on the same genetic element (i.e. procaryotic chromosome and plastid genome). Cotranscription occurs in both the unicellular and multicellular red algae (Valentin and Zetsche, 1989; Valentin and Zetsche, 1990a; Kostrzewa et al., 1990), the cyanobacteria (Nierzwicki-Bauer et al., 1984; Shinozaki and Sugiura, 1985), the chromophyte algae (Douglas et al., 1990; Hwang and Tabita, 1991) and in Cyanophora paradoxa (Starnes et al., 1985). Thus, cotranscription helps to coordinate the synthesis of equimolar amounts of both large and small subunits. In pea plants (Pisum sativum), the coordinated synthesis of both subunit mRNAs is induced by light (Shinozaki et al., 1982; Smith and Ellis, 1981) with both subunits increasing over the light induction period. This induction of rbcl is stimulated by red, violet, and green light wavelengths, yet is inhibited by far-red wavelengths (Sasaki et al., 1988). This suggests that in some higher plants rubisco accumulation is regulated at the transcriptional level or post-transcriptionally by the rate of RNA turnover (Smith and Ellis, 1981). However, for lettuce (Lactuca sativa) and tobacco (Nicotiana tabacum) the production of rbcl mANA did not seem to be regulated (Prioul and Reyss, 1987; Jordan et al., 1989). Furthermore, in Amaranth, accumulation of both subunit transcripts was not coordinated {Berry et al., 1985). However, both subunit mRNAs were lower when plants were in the dark than in the light indicating some form of transcriptional or post transcriptional control of expression.

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8 In unicellular green algae, light promotes the synthesis of both subunit transcripts and seems to exert its effect on rubisco synthesis by modulating the levels of translatable mANA (Steinmuller and Zetsche, 1984; SteinbiB and Zetsche, 1986) Although rubisco seems to be regulated by some form of transcriptional and/or post-transcriptional mechanism, regulation also occurs at the level of the rubisco protein. While post-translational modifications are rare in algae, in the higher plants tobacco and muskmelon the N-terminus of the large subunit of rubisco has been processed by removal of the methionine-1 and serine-2 followed by the acetylation of proline-3 (Houtz et al., 1989). This appears to be necessary to induce a conformational change in the rubisco large subunit which may impact on holoenzyme assembly and activity in vivo Once the rubisco enzyme is assembled, the enzyme is then activated by rubisco activase which carbamylates the epsilon amino group of the Lysine #201 residue on the large subunit near the active site (Portis, Jr., 1990). The rubisco activase may also be regulated by light at the level of mANA synthesis (Rundle and Zielinski, 1991). However, rubisco activase has never been reported in any of the eukaryotic algae or cyanobacteria. Rubisco activase may occur in Synechococcus (Tabita, personal communication) In some plants, rubisco activity is diurnally regulated by the tight binding of a substrate analog inhibitor, 2-carboxyarabinitol-1-phosphate (CA 1 P), to the active site during the dark (Servaites, 1990). CA 1 P is removed from the enzyme upon return to light by a specific chloroplast phosphatase ( Holbrook et al., 1989; Gutteridge and Julien, 1989). This regulation of rubisco by CA 1 P occurs in only a few higher plant species. Finally, it also seems that rbcl gene copy also plays some role in regulating

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9 rubisco expression. In Pisum sativum, the increase in mANA was part i ally due to transcriptional act i vation, yet increases i n the number of large subun i t genes were seen with increased illumination time (Sasaki et al., 1984) This increase i n rbcl DNA was attributed to an increase in chloroplast DNA replication. This relationship between gene dosage and expression has been stud i ed in a chlorophyta alga, Chlamydomonas reinhardtii (Hosler et al., 1989) Levels of total rbcl mANA declined with decreases in the rbcl gene dosage. However, the level of translatable rbcl mANA remained constant suggest i ng that cells accumulate an excess of transcripts and that express i on is regulated by some post-transcriptional mechanism other than gene dosage in th i s organ i sm. It therefore appears that rubisco gene expression may be controlled by a variety of mechanisms in different autotrophs In cyanobacteria, the most abundant type algae in the open ocean, the primary mechanism regu l ating rubisco e x press i on appears to be the synthesis of equal amounts of the rbcl and rbcS gene products from the same operon. Whether this process is constitutive or regulated in response to the light environment requires further study The Need and Use of Direct Methods in Studying Marine Microbial Ecology. Knowledge of the physiology and genetics of m i croorgan i sms i n cu l tures i s not easily extrapolated to understanding the functioning of m i crobes in the env i ronment. A reason for this in part, i s that only 1% or less of the cells from the na t ura l microflora can be isolated using conventional culture techn i ques (Jannasch and Jones 1959). To

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10 overcome this problem, scientists have adapted techniques to directly measure the activity of microbes in their environment. Direct microscopic methods have been developed for counting act i vely growing bacteria (Kogure et al., 1979). Radiometric methods have also been adapted to provide information about rates of autotrophic carbon fixation (Steemann-Nielsen, 1952) and heterotrophic bacterial production in marine surface waters (Fuhrman and Azam, 1982). Within the past few years however, microbial ecologists have adapted new molecular technologies to answer questions on microbial diversity and activity in the environment. These technological breakthroughs have been mainly in identifying novel genotypes or new organisms by use of well characterized genes as targets. The method of DNA hybridization has been applied to detecting a variety of microorganisms in environmental samples. DNA hybridization was successful in screening for bacteria capable of degrading aromatic substrates or detoxifying heavy metals (Sayler et al., 1985; Barkay et al., 1989; Walia, 1990). Hybridization techniques have also been used to detect viral pathogens in estuarine samples (Jiang et al., 1986; Jiang et al., 1987) The advent of the polymerase chain reaction (PCR) and its coupling with a thermostable polymerase (Saiki et al., 1988) to amplify specific gene sequences has revolutionized molecular biology and the field of microbial ecology. The PCR has been used to amplify, clone and sequence 16S rRNA gene sequences from picoplankton assemblages of the Sargasso Sea and the Pacific Ocean revealing a novel genetic cluster (Giovannoni et al., 1990; Schmidt et al., 1991) closely related to the oxygenic phototrophs.

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11 PCR. has also been used to identify the rbcl gene in dissolved and particulate DNA fractions from an aquat i c environment (Paul et al., 1990) Techniques for the amplification of mANA have been recently deve l oped One method of mANA amplification termed AT-PCA, is based upon conversion of the mANA into eDNA using reverse transcriptase and amplificat i on of the eDNA using PCA (Ferre and Garduno, 1989 ; Goblet et al., 1989; Aappolee et al., 1989) Other approaches rely on alternating rounds of eDNA and mANA synthesis (Kwoh et al., 1989; Guatelli et al., 1990) The ATPCA method has been used to amplify mANA sequences specific to Leg i onella pneumophila as a detection method for viable cells in drink i ng water (Bej et al., 1991; Mahbubani et al., 1991 ) Detection of Microbial Gene Expression While the previous methods, excluding mANA amplificat i on, are mainly restricted to detecting the presence or absence of a particular o r ganism or gene, newer methods have been developed to study the act i vity of microorganisms By target ing the expression of spec i fic genes with i n natural populations we can understand the functioning of organisms in relation to their env i ronme nt and their impact on that environment. An antibody spec i fic to the nifH protein of n i trogenase from Trichodesmium sp. (Zehr et al., 1990) was used to investigate the diel var i ation in nitrogenase activity for natural populations of Trichodesmium (Capone et al., 1990) D iel variation in nitrogenase activity was due to nitrogenase synthes i s each morning and it's degradation in late afternoon and

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12 night. Also, bioluminescent reporter plasmids have been used in the monitoring of nah gene activity during naphthalene degradation (King et al., 1990). Recently, a mANA extraction procedure was developed for measuring the abundance of transcripts of the naphthalene catabolic genes of the NAH7 plasmid (Sayler et al., 1989). Tsai et al. (1991) have used a similar direct extraction protocol to obtain mANA transcripts from both nah and mer operons from Pseudomonas aeruginosa seeded into contaminated soils. No gene specific mANA was isolated from natural unseeded soils and no intact transcripts were found for seeded soils. Tsai et al. (1990) have also studied the effect of various mercury compounds on the expression of mer genes in bacteria isolated from the environment. Seventy percent of the methyl mercury resistant strains produced merB transcripts in response to mercury stress, and 90% of the isolates synthesized merA transcripts when exposed to mercury. To increase the detection of sequence divergent mer genes being expressed, DNA gene probing was performed at lowered stringencies, a potential drawback since under reduced stringency non-specific hybridization problems increase. Pichard and Paul (1991) have overcome this problem by using single-stranded RNA probes to detect the expression of a plasmid encoded neomycin phosphotransferase gene in a marine Vibrio sp. and the naturally occurring rbcl gene in natural phytoplankton populations The use of both sense and antisense RNA gene probes enabled detection of mANA abundances and monitoring of non specific hybridization or DNA contamination problems within the RNA sample. However, none of these methods have been able to account for changes in gene expression caused by changes in specific gene copy. With this in mind, I have set

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13 out _to develop a specific method for detection of gene expression that could be normalized to levels of gene dose This method should be efficient at extracting mANA from natural populations and sensitive enough to detect expression in natural populations, where target gene transcripts will be rare. Therefore, the objectives of this study were: 1) To develop a method to extract RNA and DNA from bacteria and phytoplankton and quantitate specific transcripts and genes by use of single-stranded RNA probing. 2) To determine the ability of this method to measure water column microbial gene expression, as mANA per gene dose, of both a model bioremediation gene, and of a gene encoding an ecologically important function, rbcl, In the marine environment. 3) To investigate the use of a mANA amplification technique to increase the sensitivity of detection of microbial gene expression.

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CHAPTER 2 : DEVELOPMENT OF A METHOD FOR DETECTION OF GENE EXPRESSION AS mRNNDNA IN MARINE MICROBIAL COMMUNITIES Introduction 14 Gene expression can be approximated by appearance of phenotyp i c characters Recent developments in RNA extraction procedures have now enabled researchers to detect specific mANA's for ecolog i cally important enzymes from bacteria in soil and sediment environments (Tsai and Olson, 1991; Sayler et al., 1989) While these techniques have been used to estimate relative mANA (gene expression) levels, they have not been able to quant i tate actual levels of gene expression or to account for changes in expression poss i bly due to changes in specif i c gene sequence content within cells (gene dosage) In this part of the study, the development of a method for mANA and DNA extraction is presented The efficiency of the extractions and the quant i tation of gene expression as specific mANA per gene dose is determined for marine bacter ia. Methods and Materials Bacterial strains and p l asmids The strains and plasmids used in this study are listed in Table 1 and Table 2 Two genes were used in laboratory development and testing of this method:

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15 1) the neomycin phosphotransferase gene (.oQlli) of Tn5 which codes for kanamycin and neomycin resistance (Jorgensen et al., 1979) and 2) the catechol dioxygenase gene which codes for the enzyme catechol-2,3-oxygenase The nptll gene was on the broad-host-range plasmid pQSRSO (Meyer et al., 1982) and gene was on the broad-host-range plasmid pLV1013 (Winstanley et al., 1989), a themoregulated expression system. The plasmid pQSRSO was maintained in Escherichia coli RM1259 and in the marine Vibrio sp strain WJT-1C (Frischer et al., 1990). Plasmid pLV1013 was also maintained in the Vibr i o sp. WJT-1 C (Frischer, unpublished results). Culture condit i ons E. coli AM 1259 was grown in L8 broth at 37C in the presence of 50 J..'g/ml of kanamycin and 25 J..'g/ml of streptomycin WJT-1C(pQSR50) and WJT-1C (pLV1 013) were grown on ASWJP plus PY (Paul, 1982) containing 500 J..'g/ml kanamycin and 1 mg/ml streptomycin at 22-24C and 27-40C, respectively. E. coli 8 was grown at 37C in M9 minimal media (Sambrook et al., 1989) All cultures were shaken at 200 RPM. For RNA extraction efficiency calculations total RNA content for a radiation resistant mutant of E. col i 8, E.coli 8/r, was used There were no significant differences in RNA content between these two bacteria (Gilles and Alper, 1960). Synechococcus sp strain PCC 6301 was grown in BG-11 medium {Cote, 1984) at 30 to 60 JJEm-2s-1 {cool white light) and 22 to 25C

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16 Table 1: Bacterial strains used in this study. Strain Characteristic Source E. coli RM1259 Trp(pQSR50) R. Meyer, Uni v .Texas, Austin E coli B J Rose Univ South Fla, Tampa Vibrio sp WJT-1 C(pQSR50) DQ!l! Frischer et al., 1990 V i brio sp. WJT-1 C(pLV1 013) M E Fr ischer Un iv. S. F la. sp. rbcL rbcS Arnerical Type PCC 6301 ATCC 27144 Culture Collection, MD.

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17 Table 2: Plasmids1 used in this study. Plasmid Characteristic Source pQSR50 (R1162::Tn5} lncP-4 Smr Kmr Meyer et al., 1982 pLV1013 lncQ Smr Kmr xyiE under Winstanley et control of Lambda promoter pL al., 1989 and repressor cl857 pGEM3Z pGEM4Z Riboprobe cloning vectors A a nega Cap., with cloning site flanked by Madison, WI. Sp6 and T7 RNA polymerase promoters Apr pNPTII pGEM4Z containing nptll of F rischer et al., Tn5 from pQSR50. Kmr Apr 1990 pEPA53 pUC18 containing s. Cuskey, USEPA, Gulf Breeze,FL pXYL1 pGEM3Z containing from This study pEPA53 Apr pCS751 rbcL, Apr F.R. Tab ita, Ohio State Ur-W.,Cc:i..rrb.s pLC1 pGEM3Z containing rbcL from pCS751,Apr PaJ eta, 1m> 1 Maps of all plasmid constructs are shown in Figure 1

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-----. H I pCS751 p0SR50 ) 4 2kb E 14 4 kb C) l p VGEM4Z VGEM3Z ())) pLV1013 14. 2 K b ., ,.,. p 5 pXYL 1 pNPT I I pLC1 4 .5kb 4 .2kb 4. 7 K b Figure 1. Maps of plasmid constructs used in this study (X)

PAGE 30

19 Microcosm studies For detection of gene expression in E. coli RM1259, Vibrio sp. WJT1C(pQSR50), and Vibrio sp. WJT-1C(pLV1013) in seawater, unfiltered seawater was collected from Bayboro Harbor, St. Petersburg, Fl. Logarithmically growing cells of RM1259 or WJT-1C(pQSR50) were collected by filtrat ion from culture or added to 1 00 ml of seawater at decreasing concentrations and then collected by filtration. Mid-log phase WJT-1C(pLV1013) grown at both 2'flC and 40C were filtered directly or added to 1 00 ml of seawater and then filtered. A 1 0 ml sample was collected for catechol dioxygenase activity and then diethyl pyrocarbonate (DEPC; Sigma Chemical Co., St. Louis, Mo.) was added to 0.1 %, and a second sample (2.0 ml) collected for RNA and DNA determinations. All assays were performed as described below RNA extraction method This extraction method is an adaptation of the single-step method of RNA isolation extraction for mammalian cells (Chomczynski and Sacchi, 1987) Reagents were made in either sterile, disposable labware or glassware that had been baked at 450C for 4 hours All solutions were made with deionized water that had been treated with 0 .1% DEPC (Sam brook et al., 1989). Cells were collected in duplicate by filtration onto Whatman GF/F filters (baked at 450C for 4 hours) or harvested directly by centrifugation Cells were extracted in sterile 2 2 ml bead-beater microcentrifuge tubes (Biospec Products, Bartlesville, OK.). For RNA extraction, 0.5 ml of reagent A (4M guanidinium isothiocyanate,

PAGE 31

20 International Biotechnologies, Inc., New Haven, CT; 0.5% sarcosyl, 25 mM sodium citrate, pH7.0, and 0 1 M 2-mercaptoethanol), 50 J. sodium acetate, pH4 0, 0 5 ml water-saturated phenol, 100 J, chloroform-isoamyl alcohol (49 :1) and 8 scoops (O.Sg) of glass beads (baked at 450C for 4 hours; Biospec Products) were added to the 2.2 ml tube containing cells or the filters to be extracted. Samples were disrupted for 2 min using a micro beadbeater (Biospec products), then cooled on ice for 15 minutes. Samples were then centrifuged for 1 0 minutes at 10 000 x g in a microcentrifuge. The upper aqueous supernatant was removed and the sample was reextracted two more times using reagent A and sodium acetate as described above The supernatants were combined and precipitated with 1 volume of 1 00% isopropanol for 2 hours at -20C. The sample RNA was pelleted by centrifugation for 1 0 minutes as above and then dissolved in 0 .1% DEPC treated-1 mM EDTA, pH7.0. Samples were reprecipitated overnight at -40C by adding 0 1 volume of 2M NaCI, 1 J. glycogen carrier (20 mg/ml, Boehringer Mannheim Biochemicals, Indianapolis, IN) and 2 volumes 100% ethanol. The RNA sample was again centrifuged for 1 0 min and the RNA pellet washed with 1 volume ice-cold 70% ethanol. The RNA pellet was then resuspended in 60 J. DEPC treated 1 mM EDTA, pH7 0 and either directly dot blotted or RNase or DNase digested and then dotted. DNA extraction method A modification of the method of Fuhrman et a1.(1988) was employed for

PAGE 32

21 DNA extraction Cells were collected in duplicate by filtration onto Millipore GS, 0 22 J.'m, filters or by centrifugation, and then placed in sterile 2 2 ml bead beat tubes (Biospec Products). One ml sterile STE, pH8 .0, (Sambrook et al., 1989) was added and the sample stored at -40C until further processing The sample was thawed, 0.1 volume of 10% SDS was added, and the sample placed in boiling water for 2 min Cell debris was removed by a 1 0 min centrifugation and the supernatant recovered The cell debris pellet was reextracted and the supernatants from both extractions combined and precipitated with 0.1 volume 3M NaOAc, pH5.0 and 2 volumes 1 00% ethanol at -20C overnight. The DNA was collected by centrifugation for 1 0 min, and the pellet washed with 1 volume of ice cold 70% ethanol. The DNA was resuspended in sterile de ionized water and either blotted directly or nuclease digested and then blotted Nuclease digestion of RNA samples. dot blott i ng. probing. and guantitation The sample RNA extract was divided into three 20 J.LI aliquots. To one aliquot 2 J.LI2M MgS04 and 4 J.'l RNase A (2300 U/ml Sigma Chemical Co.) was added. To a second aliquot, 4 J.'l RNase-free RQ1-DNase (1 000 U/ml Promega Biotech, Madison, WI), 2 J.LI 2M MgS04 and 2 J .. LI RNasin (placental r i bonuclease inhibitor 40000 U/ml Promega Biotech) were added. All nuclease digestions were done at 37C for 60 min The third aliquot was left untreated The DNase-treated and untreated samples were brought to a final volume of 500 J.LI with DEPC treated 1 mM EDTA, pH7 .0. Two hundred fifty microliter aliquots were then dot-blotted

PAGE 33

22 onto two charged nylon membranes (Zeta-probe, BioRad, Richmond, CA), one for probing with the anti-sense probe and the other for use with the sense probe, using a 96 well DEPC-treated BioDot dot-blot apparatus (BioRad). The membranes were then removed and the RNasetreated samples (kept to a minimal volume) were dot-blotted by hand to avoid RNase contamination of the dot blot apparatus. One membrane was probed with the antisense probe, and the second with the sense probe. A standard curve of target mANA (produced by in vitro synthesis) was also dot blotted at the same time. The RNA dot blot was then baked at 80C for 2 hours under vacuum to fix the sample to the membrane Blots were stored at -20C until probing For production of single-stranded RNA probes of high specific activity the Riboprobe (Promega Corp. ) plasmid construct (containing the gene of interest flanked by Sp6 and T7 RNA polymerase promoters) was linearized with a restriction enzyme that cut downstream of the insert. Using [35S] UTP ( 1289 Ci/mmol; 95% purity; Dupont-New England Nuclear, Wilmington, DE) in the transcription reaction, as in append i x A, and either the T7 or Sp6 RNA polymerase, antisense (AS) or sense (S) radiolabeled RNA probes could be produced (Frischer et al., 1990) as in figure 1 RNA dot blots were hybridized with the AS and S gene probes at 55C and washed at 65C as in appendix B. The probed dot blots were exposed to X-ray film overnight at -40C Dot blots with low signal intensity were either exposed for a longer period or sprayed with an autoradiographic enhancer (EN3Hance : DuPont-New England Nuclear) or both To quantitate the RNA

PAGE 34

23 signals, X-ray films were either scanned by laser densitometry using a LKB Ultrascan XL laser densitometer or the dot blots were scanned directly using a Ambis radioanalytic imaging system (B-counter). In some cases, the dots were cut out and counted by liquid scintillation counting (LSC). The signals were then quantitated by comparison to RNA standards. Nuclease digestion of DNA samples. dot blotting. probing. and quantitation The DNA sample extracts were split into 2 equal volumes One volume was digested with RQ1-DNase as above and the other volume was left untreated. The samples were dot blotted onto charged nylon membrane as described above. Following dot blotting, the DNA dot blot was then placed dot side up onto blot block saturated with 0 5 N NaOH and 1 5 M NaCI for 1 0 min, and then neutralized twice on blot block saturated with 0 5 M Tris-HCI, pH7 5 and 1.5 M NaCI for 5 min The DNA was then fixed to membranes as described for RNA. DNA dot blots were hybridized with AS and S single-stranded RNA gene probes as in appendix B with hybridization at 42C and washing at 65C DNA samples were quantitated by comparison with DNA standards. Catechol dioxvgenase enzyme assay WJT 1C(pLV1013} cells in culture or added to seawater were assayed for catechol dioxygenase activity by the method outlined by Gibson (1971 ) Cells were harvested by filtration using M i llipore GS, 0.22 J.Lm, filters The filters were

PAGE 35

24 placed in acid washed plastic snap cap vials containing 2.0 ml 0 05 M KH2PO 4 / pH7.5, 10% acetone buffer, and placed on ice. Cells were then sonicated on ice for six, 30 sec pulses with 15 sec between pulses. The supernatant was removed, the vials washed with an additional 1 0 ml of buffer, and the combined extracts were centrifuged for 1 0 min One hundred microlilters of the cell-free extract, or a dilution thereof was assayed in a 3 0 ml total volume of phosphate buffer containing 1 00 J.LI 0 .01 M catechol. The absorbance at 375 nm was monitored as a function of time. One unit of enzyme activity was defined as that amount which oxidizes 1 1-Lmole of catechol per min (Gibson 1971). Total protein concentration in the extracts was determined by the method of Lowry et al. (1951) with crystalline bovine serum albumin as the standard. Specific activity was determined as the unit activity per mg protein Results To determine the efficiency of the isolation technique in extracting and recovering RNA, RNA was extracted from E. coli B cells grown in minimal med i a (Table 3). RNA extraction efficiency was directly related to the type of filter used to concentrate cells. The Millipore-Durapore filter, with low binding capacity, was 89% efficient at recovering total RNA based upon a theoretical yield for E. coli 8/r in mid-log growth phase Both the GF/F filter and the Durapore filter were operationally the same in retaining chlorophyll from samples of offshore oligotrophic water 0.081 0 007 J.Lg/1 and 0.085 0.002 J.Lg/1 respectively.

PAGE 36

25 Efficiency of precipitation of target RNA was also determined by precipitating a specific quantity (1 Ong) of target RNA (rbcl) in a total RNA background of 1 J.l.g of transfer RNA (t -RNA). The RNA concentration was succesively decreased for testing different precipitation efficiences at different RNA concentrations. Target RNA was detected by radioactive probing and liquid sci ntillation counting (LSC) of comparable quantities of RNA that had been directly blotted or first precipitated and then blotted. The efficiency of prec i pitation ranged from 80 to 96% with an ave r age of 91% (Table 4). Table 3 : Efficiency of RNA extraction from E. coli 8 cells filtered onto various filter types as determined by spectrophotometry. Filter Avg Avg Measured1 Expected 2 %Recovery Type A2so RNA (JJg) RNA (JJg) Whatman 0.071 1 885 2.86 20.65 14 GF/F 0.020 0 .021 (0.7JJm) Durapore 0.459 1.925 18.38 20 65 89 (0.45JJm) 0 015 0.035 1one A260 unit equals 40 JJg/ml RNA (Sambrook et al., 1989) Samples assayed in 1 ml quartz cuvette 2 expected RNA yield (Ne i dhardt et a17 1990) calculated for 5 0 ml mid-log (A 600 = 0 .5) cells at 7 0 0.56 x 10 cells/mi. DNA extraction efficiency and recovery was also determined by similar methods. Table 5 presents data for DNA recovery from mid-log WJT-1 C(pLV1013) for two different filter types with the extraction performed as described above or

PAGE 37

26 for two different filter types with the extraction performed as described above or with an optional phenol:chloroform extraction as described by Fuhrman et al. (1988) Both the GF/F filter and inclusion of a phenol:chloroform extraction resulted in reduced recovery of DNA. Use of Millipore GS filters resulted in better DNA recoveries than GF/F filters yielding nearly twice the amount of DNA. DNA precipitat i on efficiencies were determined as for RNA. except that calf thymus DNA was used as a carrier instead of t-RNA. DNA recoveries ranged from 78 to 99% with an average recovery of 90% (Table 6). Table 4 : mANA precipitation efficiency Amoun t of CPM1 rbcl mANA (ng) direct 10 268629 27452 1 12705 1688 0.1 576 0.01 125 22 CPM2 precipitate 215553 53697 11724 1203 549 13 120 19 %Recovery 80 92 95 96 avg % rec 91 7 1 RNA was dotted directly onto charged nylon, probed and radioactivity determined by LSC. (CPM = counts per min) 2RNA was precipitated as in RNA extraction method dotted, probed and counts determined by LSC. DNA recoveries were also tested with a cyanobacter i al species Synechococcus sp PCC6301, using a Millipore-Durapore filter instead of a GS filter. DNA extraction yielded an average of 275 ng of DNA for 3.1 x 107 cells i rrespect i ve of the method used to collect the cells (i.e filtration or centrifugat i on). The average DNA content per cell was 8.9 fg DNNcell, consistent with the B i nder

PAGE 38

27 and Chisholm {1990) value of 8.8 fg DNNcell. The latter was determined by flow cytometry and assumes 2 genome equivalents per cell Table 5 : DNA determinat i ons of DNA extractions from WJT-1 C(pLV1 013) 1 Filter Avg DNA extracted2 Modification Type (ng DNA) w/o phenol: GF/F 303 22 GS(0.22 J.m) 594 0 w/ phenol : GF/F 8 0 CHCI3 GS(0 22 J.m) 128 0 1cells were cultured at 27C and mid-log (A600=0.5} cells were f iltered and frozen for 24 hrs before extract i on. 2DNA determ i nations were done in dupl icate using a DNA fluorochrome, Hoechst 33258 and fluorometry (Paul and Meyer, 1982) Table 6 : DNA precipitation efficiency Amount of rbcl DNA (ng) 10 1 0.1 0 .01 CPM1 direct 192090 31493 1159 3517 492 131 112 7 CPM2 precipitate 191366 44777 9064 611 453 59 %Recovery 99 78 92 103 14 92 avg. % rec. 90 9 1 DNA was dotted d i rectly onto charged nylon, probed and radioac t ivity quantitated by LSC 2DNA was precip i tated as in DNA extraction protocol dotted probed and counts determ ined by LSC.

PAGE 39

28 Figure 2 shows the method for differentiating between purified target RNA and DNA (5 ng each) by probing with AS and S gene probes Hybridizat i on to both the AS and S probe was found with purified DNA. RNase-treated samples hybridized to these probes, whereas DNAse treatment resulted in no hybridization Purified RNA hybridized only with the AS probe and was RNase labile. Trace quantities of DNA template in our RNA preparation may have caused the slight hybridization with the S probe. Here RNA can be easily differentiated from DNA by probing with AS and S gene probes. Figure 2 also shows the results of probing of environmental RNA extracts of 25 ml of seawater spiked with rbcl RNA and DNA targets. The same hybridization patterns were found as for RNA and DNA alone indicating that nothing in the extracts interfered with the control nuclease digestions or the hybridization and detection process

PAGE 40

29 DNA RNA E ER ED u D AS R u D s R Figure 2 Probing of purified rbcl DNA and RNA with AS and S RNA probes The dots (5 ng each) i n the row labeled U were undigested, whereas those in rows labeled D and R were digested with DNase and RNase, respectively. An RNA extract from the water co l umn is labeled E, and that extract spiked with rbcl RNA and DNA is labeled ER and ED, respectively.

PAGE 41

30 To determine the capability to recover RNA from cells in seawater, E. coli or Vibrio cells containing nptll were either filtered directly or added to seawater at decreasing concentrations The dot blot of these RNA extracts is presented in Figure 3. Figure 4 shows the results of laser densitometry scanning of the RNA dot blot of this experiment. Hybridization occured only with the AS probe and target RNA was removed by RNase digestion (data not shown). In general a greater amount of hybridization was obtained with Vibrio cells added to seawater than with an equivalent number of E. coli cells. This is reflected in the slopes of the regression lines of absorbance versus cell density (slope = 0 144 for Vibrio sp.; r = 0.91; slope = 0.045 for E. coli; r = 0.96). Figure 5 shows the results of detection of gene expression in Vibrio WJT-1C(pLV1013) in culture and when added to non-sterile seawater. Cells were grown at either the non-permissive (27C) or the permissive ( 40C) temperature to mid-log phase (A600 = 0 .6) and then added to seawater An equivalent number of cells were harvested from both cultures and seawater microcosms for C2,30 activity, RNA, and DNA analysis Cells at 27C had low levels mANA and C2,30 activity while cells at 40C exhibited higher levels of both mANA and C2,30 activity Cells grown at 27C or at 40C had comparable levels DNA suggesting that changes in gene expression levels were not caused by changes in gene dose.

PAGE 42

E .coli Vibrio A B c D E F G H I J K L u D R u D R Figure 3 Detection of nptll gene expression in var i ous bacteria in seawater and culture Columns A and G are cells sampled from culture Columns 8-F and H-L are cells added to seawater at the concentrations shown in Figure 4 AS s (A)

PAGE 43

w (.) z < al a: 0 CJ) al < 3 0 0 --4 5 6 LOG CELL/ ML 32 1 Figure 4. Relative absorbance of expression signals of !1Q1!1 in E. coli RM1259 or Vibrio sp. WJT-1 C (pQSR50) from Figure 3. Cells were either filtered directly from culture (open symbols) or added to and filtered from seawater (solid symbols) Vibrio cells are circles and E. coli are squares.

PAGE 44

DONA .C230 culture seawater cui ture 2 seawater at c 45 0 ... C1. at 30 E ;:) u c( 15 Q. (I) Q C') N 0 33 F i gure 5 Detection express i on in a marine Vibrio containing pLV1013 in culture and in cells resuspended in seawater. Cells were cultured at 27C and then incubated at either 27C or at 40C.

PAGE 45

34 Figure 6 shows expression and C2,30 activ i ty of WJT-1 C cells grown at the nonpermissive temperature and then incubated for 3 hours at the permiss i ve temperature. Levels of mANA was greatest for the first hour at 40C while little or no C2,30 act i vity was measured. Messenger RNA levels then declined over the next 2 hours as C2,30 activity increased The appearance of C2,30 activity lagged beh i nd the appearance mANA by about an hour. DNA rema i ned constant during the first hour at 40C and then began to inc r ease while cells were growing exponentially (data no shown). Because the increase in DNA was less than the cell growth r ate, DNA content per cell was actually decreasing over the incubat i on period This reduction in mANA may be caused by a reduct ion in the number of plasmids contain i ng DNA, although ant i b i ot i c select i on was maintained throughout the experiment. An alternat i ve poss i bility is that the gene was selectively lost from the plasmid (marker segregation) D i scuss ion A number of steps must be performed to efficiently isolate mANA from cells; 1) the cells must be lysed in a solut ion that i nhibits cellular nucleases (RNases are ubi qu i tous) 2) complexes of nuc l eic acids and prote i ns must be d i ssolved as they are a potential loss of mater ial to the aqueous organic i nterface, and 3) to detect gene express i on, RNA must be separated from DNA so as to reduce hybridizat ion fa lse posit i ves because of DNA contaminat ion Successful e xt ract i on of i ntact RNA

PAGE 46

35 < 3 z 0 C) c: < 2 z a: E w >. X 1 C) c: <':1 0 ?'"" -6 1 f4... C230 120 E ... I \ 80 .. a. -/ C) < 60 E E z 4 80 3 a: I ../"" \ E \ < I \ 40 ...,; w u >. / ._ ____ 40 X 2 w C) 20 >. c. / X (J) c: / ":" / C) 0 0 __ _. c ('I') ._ 0 0 0 C\1 (.) 0 1 2 3 4 5 Time (hours) F igure 6 Induction of gene expression in a marine Vibrio sp. in r esponse to temperature. V i brio sp. containing pLV1 013 was grown at 2'f'C and then shifted to 40C at T = 0 (firs t arrow). After three hours cells are returned to 27C (second arrow). The top panel shows the kinetics of mANA accumulation corrected for gene dose The lower panel presents the changes mANA (circles), DNA (triangles) and catechol dioxygenase activity (squares).

PAGE 47

36 is contingent upon the performance of all steps as rapidly as possible. 1 have adapted the method of Chomczynski and Sacchi (1987) which uses the strong protein denaturant guanid i nium isothiocyanate and mechan i cal cell disruption The method takes advantage of solubility differences between RNA and DNA in that at pH 5-6, DNA is selectively retained in the organic phase wh ile RNA rema i ns in the aqueous supernatant (Wallace, 1987). The method also employs a non-ionic detergent to promote the dissolution of lipid membranes and dissociation of protein-nucleic acid complexes and chloroform-isoamyl alcohol which reduces loss of RNA at the organic-aqueous interphase Increases in levels of mANA's may be caused by increased transcriptional activity, increases in specific gene copy, or a combination of both. This assumes that cellular RNase activ i ty remains constant. Therefore I have also developed a method to isolate target DNA from the same sample based on a modification to the method of Fuhrman et al. (1988), which eliminates organic extraction with phenol. This method yields high recoveries, and gave a genomic DNA content for an photoautotrophic picoplankter, Synechococcus sp PCC6301, consistent with literature values (Binder and Chisholm, 1990) It appears that eliminating the phenol extraction of DNA enables 1 00% of the DNA to be recovered. This is important for quantitative studies of changes of absolute copies of DNA. Both the mANA and DNA extraction methods have been combined with single-stranded RNA AS and S gene probing to quantitatively detect gene expression as mANA per gene dose

PAGE 48

37 The utility of this method for detecting cells in the environment based upon specific mANA synthesis was demonstrated using nptll in both E. coli and Vibrio sp. added to seawater The hybridization obtained with RNA extracted from 1 5 x 1 08 E. coli cells was greater than that determined with an equivalent number of cells suspended in seawater. Unlike E. coli, equal hybridization was obtained from Vibrio cells filtered directly from culture or after resuspension in 1 00 ml of seawater Laboratory strains of E. coli are not adapted for growth at salinities of 20 to 35 ppt. Therefore, this added stress may have resulted in an increase in mANA turnover rates as a cellular response to this osmotic stress. The detection limit for both cell types was about 106 cells total (1 04/ml of seawater) using GF/F filters However, other filter types (Durapore) may help to increase the sensitivity of the method by increasing recovery of target mANA. The gene has been used to monitor recombinant bacterial populations in lake water (Morgan et al., 1989; Winstanley et al., 1991 ). By following C2,30 activity, these researchers were able to detect comparable amounts of bacteria (1 04 cells/ml) over a period of 11 days. I have used the ANNONA method, described herein, to monitor xyiE gene expression by WJT-1C(pLV1013) in simulations of the marine environment. Differences in gene expression between induced and uninduced cells at a concentration of i 06 cells/ml have been monitored. Levels mANA were elevated 30 to 70 fold in induced cells over levels in uninduced cells. This increase corresponded to the appearance of C2,30 enzyme activity in the induced cells. A small amount of transcripts were

PAGE 49

38 detected in the uninduced cells. Possibly the lambda promoter and repressor do not function quite as efficiently in Vibrio than in E. coli. WJT-1C was also tested in culture for the appearance of both mANA synthesis and C2,30 enzyme activity over an induction period of 3 hours. mANA was synthesized first followed by the appearance of C2,30 activity coincident with a decline in the mANA level. At shiftdown, the rate of turnover of mANA was only 70% the rate of turnover of enzyme activity suggesting that the mANA was more stable. However, RNA levels had already decreased by half during the 40C incubation while enzyme activity was on the rise prior to shiftdown Therefore, either the rate of mANA turnover increased and/or mANA synthesis per cell was declining due to a reduction in gene dose during the incubation period This separat ion between peaks of gene specific mANA and enzyme activity has been seen with nitrate reductase (NR) for a variety of plants NR mANA accumulated rapidly and then declined while NR activity increased (Hamat et al., 1989; Melzer et al., 1989) Under permissive conditions for expression this reduction in mANA levels may be due to several factors. While the cells are bei ng induced for expression, the temperature shift up may be increasing the instability of the mANA. Thus, the RNA levels decline after a period at 40C because of a shorter turnover time. Temperature elevations of 12C have been shown to decrease mANA pools of an extracellular protease by 50% (McKellar and Cholette, 1987). Over the induction period the cell biomass was increasing exponentially while the gene dose was increasing in a near linear fashion. copy was diluted over this

PAGE 50

39 time because of inefficient plasmid replication or marker segregat i on. S i milar / resu l ts have been seen with other expression systems (Adams and Hatfield, 1984) Expression of galactokinase from efficient promoters, such as Lambda promoters, in transcription fusion vectors has been reduced instead of enhanced as expected This was attributed to a reduction in plasmid replication due to interference from plasmid gene transcript i on (Adams and Hatfield, 1984) While neithe r one of these effects have been determined as the cause of the reduction mRNA levels one or both could be operating to reduce the levels of RNA per cell.

PAGE 51

CHAPTER 3: THE DETECTION OF rbcL GENE EXPRESSION IN NATURAL PHYTOPLANKTON POPULATIONS Introduction 40 Phytoplankton perform the primary function of converting inorganic carbon into organic carbon in the oceans of the world. Without these organisms life in the ocean would not be possible since these organisms supply the carbon upon which higher life forms depend. Therefore it is this process of carbon fixation that has been the subject of intense investigation over the years We now have an understanding of the physiology of photosynthesis and of the enzymes participating in the catalytic steps of the pathway, yet little is known about the regulation of this process and the basis for that regulation In th i s part of the study I utilize the methods developed in the previous section to study the regulation of the synthesis of rbcL mRNA and its relationship to carbon f i xation for natural phytoplankton populations. Methods and Materials Field sites For detection of rbcL gene expression by natural phytoplankton populations several sites were sampled Samples of 2000 ml were filtered thru GF/F filters for a station near Loggerhead Key, Dry Tortugas, FL and Joulters Cay, Bahamas

PAGE 52

41 Samples were also taken at Gulf of Mexico stations shown in figure 7. Various subsurface (1-5 meters) sample volumes were filtered onto Durapore filters depending upon bacterial and phytoplankton densities (eutrophic waters=100ml; oligotrophic waters=300ml) at Gulf of Mexico stations. Samples from Bayboro Harbor and the mouth of Tampa Bay were also filtered onto Whatman GF/F and a low binding filter, Millipore-Durapore, as described below. ANNONA sampling Both RNA and DNA sampling were performed in duplicate as described in the previous chapter (samples were pretreated by addition of DEPC to the sample at 0.1 %) with the exception that the samples were filtered onto sterile autoclaved Millipore-Durapore (polyvinylidene difluoride) 0.45 J.Lm filters. All extraction, probe hybridization, and quantitation was performed as outlined in the previous chapter. After quantitation, RNA dot blots (membranes were not allowed to dry) were stripped of their bound probe by incubation with 50% formamide and 5x SSPE (Sambrook et al., 1989) at 65C for 1 hr The dot blot was then rinsed briefly twice in 2x SSPE. The blot could then be reprobed to detect the expression of a different target gene. Phytoplankton carbon fixation For analysis of photoautotrophic production the method of 14C-C02 incorporation into organic material as outlined by Strickland and Parsons (1968) was followed Light and dark bottle incubations (325 ml sample) were used to determine light dependent rates of carbon fixation Radioactive bicarbonate,

PAGE 53

6 Gulf of Mexico 84 8 3 4 83 Tampa Bay 1 82 42 27 26 Figure 7. Locations of stations samp l ed for rbcl mANA Stat i on 3 (star) was the location of sampling f or a diel study Station 6 (square) was the l ocat i on for sampling rbcl express i on as a funct i on of depth i n the euphot i c zone

PAGE 54

43 [14C]HC03 (53.1 mCi/m mol; Amersham Corporation, Arlington Heights, IL),was added to each light and dark bottle to a final radioactivity of 0.5 J,Ci per mi. A zero point sample was taken by filtration through Millipore GS 0 .22 J,m and both bottles were incubated under constant white light illumination (approx. 40 J.E m-2s-1 ) and relatively constant temperature Duplicate samples were taken at 1 and 2 hrs. Filters were added to glass scintillation vials containing 0 5 ml of 0.5 N HCI and incubated overnight at room temperature Sample radioactivity was determined by dissolving the filter in ethyl acetate, adding 10 ml of a scintillation cocktail (Ecoscint A; National Diagnostics, Manville, IL), and liquid Scintillation counting using a Delta 300 model 6891 liquid scintillation counting system (TM Analytic, Inc., Elk Grove Village, IL). Water samples were titrated by the method of Strickland and Parsons (1968) to determine carbonate alkalinity and total C02 content. The total C02 content and the 14C radioactive counts were used to determine the rate of carbon fixation for each water sample (data provided by John Paul). Biomass/cell count determinations For each station, photosynthetic population size was determined by analysis Samples were filtered in triplicate through Whatman GF/F filters and stored frozen for latter analysis. Chlorophyll was extracted with methanol and the amount of chlorophyll determined fluorometrically (Holm-Hansen and Reimann, 1978). Autofluorescent picoplankton (orange-yellow) and red fluorescing cells were

PAGE 55

44 determined by John Paul and Marc Frischer using the method of Vernet et al. (1990). Cells were filtered in duplicate onto i rgalan black stained 0 22 JJm Nuclepore filters. The filter was then placed onto a slide and a drop of glycerol was placed between the filter and the cover slip, and the slide stored i n the dark at -20C Cells were then enumerated under blue light excitation (450nm) and 400x magnification on an Olympus BH2 epifluoresence microscope RNA amplification rbcl mANA was amplified from RNA extracts from Bayboro Harbor and the Gulf of Mexico using degenerate oligonucleotide primers, D287 and D167 (append i x C), by the method of Becker-Andre and Hahlbrock (1989). Messenger RNA was first converted to DNA using the follow i ng react i on conditions : 50 mM Tris HCI, pH7 .5, 75 mM KCI, 10 mM dithiothreitol, 3 mM MgCI2 100 ng of D287 primer and 0.5 mM each of the deoxynucleotide triphosphates (dNTPs) i n a total volume of 20 JJI. The reaction mix was heated to 65C for 5 min and then cooled to room temperature RNasin (6 units; Promega) and reverse transcriptase (40 units ; Ufe Sciences, Inc., St. Petersburg Fl) were then added, and the reaction continued for 30 min at 42C. The react ion volume was then made to 1 00 JJI by addition of 80 JJI of TE, pH8 0 (Samb r ook et al., 1989). The DNA was then amplified by the polymerase chain reaction For PCR, 3 JJI of DEPC-treated water and 2 J .. of the reverse transcriptase reaction was added to 94.5 JJI of PCR buffer conta i ning 50 mM KCI, 10 mM Tris-HCI pH8 .3, 15 mM MgCI2 0 01% gelat in, 200 JJM each dNTPs, and 1 JJM each of D287 and D167

PAGE 56

45 Reaction mixtures were vortexed and briefly pulse centrifuged. One-half microliter (3 units) of Taq polymerase (Promega) was added, and the reaction was mixed by vortexing and pulsed again. Samples were overlain with 1 00 J .. d of sterile mineral oil and the samples amplified for 39 cycles using the following temperature regime : 1) 1 min at 94C, 2) 2 min at 50C, and 3) 3 min at 72C PCR reactions were then electrophoresed on 1% agarose using a DNA fluorochrome, Hoechst 33258, for visual i zation (DeFiaun and Paul, 1986). The DNA was transferred to charged nylon (Sambrook et al., 1989) and probed with the rbcl gene probe for confirmation of amplification products as in chapter 2 Results Figure 8 shows the results of studies on the extraction of rbcl mANA from a Synechococcus culture and from natural phytoplankton populations of the Dry Tortugas. Extracted RNA from both the cyanobacterial culture and the natural phytoplankton assemblage resulted in material that hybridized to the AS probe and was digestable by RNase. In this natural population the strongest hybridization occurred during the daylight hours and the weakest occurred at night (figure 9). Ught to dark transition hours were variab l e w i th some elevated signals just before the onset of darkness (1900 hr). Similar results were seen for Bahamian surface water phytoplankton populations (Figure 1 0) with the exception that a noon sample had the lowest

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A B C D 19 23 03 07 1 1 15 19 01 07 ...... ... ,. .. ... _, .... . :. # II -Figure 8 Die I variations in rbcl mANA levels for Dry T ortugas natural phytoplankton populations and a Synechococcus culture. Columns A and 8, and C and D, represent l i ght and dark samples of the culture, respectively. Numbers along the top refer to d i e I sampling times for the natural population u 1 0 A .-f-. r; .. ,_ u D A AS s &)

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47 o .sr.A-----------------. AS liil 0.4 1 ... en 0 2 0 1 0 5 0.1 0.6 t-:..r.........J.-.J--.J"-L.........L...L...-.J......L..--L...J._..J.....l__,!__L_.....J._L_L...+ 0 .15 C AS j 1 i e..........._r/j /.""" / l /!.............. 0 5 II 0 1 0.1 0.0..___ ______________ _.....0. 0 19 23 03 07 11 15 19 01 07 TillE OF DAY F i gure 9 Laser densitometry of the AS probed dot blot in F i gure 8. The untreated row is (A), the DNase treated row (B), and the RNase-treated row (C). Each band is the average value of the replicate samples for each time point. (C) Chlorophyll concentration in the wa t er at the time of sampl ing.

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48 0.3 ....J ........... (Q Q 0 1 0 0 1.0 .c ......... 0.5 (.) Cit 0.0 2 z &X: E 1 Q c 0 24 12 24 12 Time of Day F i gure 1 0. Die I rbcl expression and car bon fixation for natural phytop lankton popu lations in the Bahamas. Top panel represents the d iel fluctuat i on in chlorophyll over the sampling period M id dle panel shows the d ial pattern in carbon fixation. Bottom panel shows the diel variation in rbcl mANA levels with the black bars indication periods of dark.

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49 hybridization intensity for rbcL mANA. Rates of carbon fixation exhibited a similar diel trend as the rbcL mRNA. However, surface chlorophyll also followed this sam pattern suggesting that, in this case, carbon fixation and mANA levels were a function of possible diel variations in phytoplankton abundance. Since rubisco is the major carbon fixing enzyme, I was interested in determining if a correlation existed between rbcl mRNA levels and carbon fixation in various marine environments. Surface waters were sampled for rbcl mANA and DNA, chlorophyll carbon fixation, and autofluorescent cell counts (Figure 11) at stations in the Gulf of Mexico (Figure 7) during a nearshore-offshore transect. Picoplankton counts ranged from 6.78 0.21 x 104 to 5.35 1.48 x 103 cells/ml and followed the same general trend observed for chi concentrations Picoplankton were an order of magnitude less abundant in offshore waters than in nearshore waters of the Gulf of Mexico. Red fluorescent cells (presumably eucaryotic picoplankton) ranged from 1 39 0 18 x 1 03 to 38 cells/ml for nea r shore and offshore samples, respectively As with other parameters, carbon fixation was highest in nearshore waters and lowest in offshore waters rbcl mANA levels followed a similar pattern and correlated with carbon fixation rates {r=0.97). Specific levels of carbon fixation (carbon/chi .) and specific levels of rbcL expression (mRNNDNA) correlated at r=0. 74. These RNA dot blots were stripped of bound probe and rehybridized with a divergent rbcl gene probe from the diatom, Cylindrotheca fusiformis (Hwang and Tabita 1989) No signal was obtained using this probe for any of the environments. The same results were

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50 A 9 I 8 I ...J I 7 .._ I I 6 as I PICO ::c I (.) 5 ... I 0 2 I 4 E E ""'-.._ .._ RED. "'3 Q) f/ Q) (.) 1 2 u.,. "0 0 ;; () !/ a. "' j, 1 .. 0 0 .,.... 0 0 6 4 8 3 2 1 STATION# 200 B 40 J::-r 30 ...J .._ < .&: z 20 2 a: 100 E "0 G) ...J )( (.) ; .0 () ... Cl 10 CJ) c ::t 0 0 6 4 8 3 2 1 STATION# Figure 11 Nearshore-offshore transect of rbcL mANA analysis and related parameters for Gulf of Mexico stations. Orange-yellow autofluorescing cell (triangles) and red fluorescing cell (circles) abundances and chlorophyll (squares) concentrations are presented in A. Photosynthetic carbon fixation at a fixed light intensity (circles) and rbcL mANA Levels (squares) are presented in B Normalized parameters of carbon fixation per unit chlorophyll (circles) and rbcL mANA to DNA (squares) are shown in C.

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51 Figure 11. (Continued) c 160 ANA/DNA I \ < 140 I \ z I \ 0 120 I \ _. \ _. I j 0 \ .0 100 I I I 10 as ... I \ I Cl I \ I :2 c:: 80 8 0 I \ < -z I \ I .c a: 60 \ I 6 ...:J E I ) CARIION/0...0 _. 40 4 G) 0 / )( .0 / :;::: ... 20 / 2 Cl I / 0 c:: ., 0 0 0 :::1.. 6 4 8 3 2 1 STATION#

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A) Depth(m) 52 10 30 60 100 u D AS R u D s R I B) 0 1 -1 -2 3 -4 10 10 10 10 10 10 ng Figure 12. Overnight exposures of dot-blots of rbcl mANA from 300 ml samples of station 6 seawater. The vertical profile of rbcl mANA i s shown in A. The rbcl mANA standard curve is in B

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53 A) Depth(m) 10 30 60 100 u AS D B) 1 0 -1 -2 -3 -4 1 0 1 0 1 0 1 0 1 0 1 0 ng Figure 13 Overnight exposures of dot-blots of rbcl DNA from 300 ml samples of station 6 seawater The vertical profile of rbcl DNA is shown in A. The rbcl DNA standard curve is in B

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54 A) 1 0 red cells/ml & B) J.JQ Chla / L 1 0 2 0 0 1 0 2 3 ).JQ C / L h o 10 pica cells/ml 2 4 6 8 10 0 2 0 4 0 6 0 8 1 0 RED 20 20 e -j g I .c 60 .c 60 \ 0. Q) I Q. c Q) I 0 I \ I \ 100 t 100 "---).JQ C /).Jg Chla h C) ng rbcl mANA / L D) 2 4 6 8 or ng rbcl DNA /L o ng rbcl mANA/DNA o 2 4 6 5 15 25 RHA/ONA 20 20 \ 'a_ ......... :--...... ....._ g --.s ---....... .c 60 .c 60 / -a. -a. / Q) Q) 0 0 / / / 100 100 / Figure 14. Vertical profiles of rbcl mANA and related parameters for stat i on 6.

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55 obtained with a gene not expected to be present in these environments 1 also determ i ned the relationship of these same parameters in a vertical profile at the offshore station 6. Dot blots of both rbcL mANA and DNA are shown in Figure 12 and Figure 13. The reduced data is shown in Figure 14. Autofluorescent picoplankton cells were most abundant in surface waters while red cells increased as a function of depth, with a maximum (1.19 0.34 x 1 o3 cells/ml) at the deepest depth sampled (1 00 m). This increase in red cells with depth was reflected in an increase in chi s at 1 00 m relative to surface concentrations. This increase in chlorophyll with depth is also contributed by cellular photoadaptation to decreased light. Phytoplankton at depth tend to have more chlorophyll per cell. Carbon fixation rates were 0 9 J..l.g C/Lhr for 1 0 m waters and exhibited a subsurface peak at 60m that was half of the 1 0 m value. The rbcL DNA values decreased w i th depth to near undetectable levels while mANA values were higher (Figure 14). Carbon fixation per chlorophyll s and rbcL mANNDNA correlated at r=0.61 (Figure 14D} Phytoplankton exhibit diet rhythms in photosynthetic carbon assimilation (Doty and Oguri, 1957) Maximal rates are seen during periods of light and these rates are dramatically reduced or non-existant during dark periods (Harding et al., 1981 ). To investigate the molecular basis for this, 150 L of seawater from station 3 was pumped from 5-1 0 m depth into a decktop incubator and incubated under reduced natural illumination and constant temperature (28C). Samples were collected during light and dark periods for rbcL mANA and DNA, carbon fixation

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56 and chi g (Figure 15). Both carbon fixation and mANA were elevated in the light, 5 times, over the dark period Chi g and DNA biomass remained constant. Specific levels of rbcl gene expression, as mRNNDNA correlated with biomass normalized carbon fixation (r=0.96). This suggests that diel changes in carbon fixation appear to be regulated by changes in transcr i ption of the rbcl genes for open ocean phytoplankton. rbcl mANA amplification was performed as described and gel electrophoresis of amplification products is presented in Figure 16. Samples from the Gulf of Mexico did not amplify while those from Bayboro Harbor did yielding an amplification product of 376 bp specific to a section of rbcl. RNA from a Synechococcus sp PCC 6301 culture also amplified These samples were tested for amplification several weeks later (data not shown) and found not to amplify possibly due to degradation of sample RNA. Amplification products were characterized as being from the rbcl gene by southern hybridization (Figure 17) w i th the Synechococcus rbcl gene probe. D i scussion Phytoplankton are the dominant photosynthet i c organisms in the world s oceans This biomass is composed of diverse assemblages of diatoms, dinoflagellates, cyanobacteria and other phytoplankton with the unicellular cyanobacteria being the most abundant form in oligotrophic systems (ltturiaga and M i tchell, 1986)

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57 -I A 1.0 < ..c z -I ct: -E '0 _.. Q). )( (.) ; j:l 0.5 (.) .... C) :t. -I < B z Q 1.0 -I 0.15 0 -I -_g. :::t .... 0 5 0.1 as C) 1: c: (.) < CARSON/OtA c as z 6 6 1: Q (.) < 4 z. ct: ..c E 2 -. -I u (J Q) j:l )( .... ; 12 24 12 (.) C) Time of Day ::t Figure 15. Diel patterns in rbcl mANA and rel ated parameters. One hundred fifty liters of water was i ncubated on-deck under r educed natural light and constant temperature.

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58 MW 1 2 3 4 5 6 7 8 91011 383bp Figure 16. Gel electrophoresis of amp l ification products of e n v i ronmenta l rbcL mANA. Lane des i gnat i ons are as follows: MW) pBR322 DNA-BstN 1 digest molecular we ight marker 1 )DI water RT-PCR 2) rbcL mANA PCR (no RT}, 3) rbcL mANA RT-PCR 4} 01 water PCR 5} rbcL DNA PCR 6) Station 8 RNA PCR (no RT}, 7) Sta tion 8 RNA RT-PCR 8) Bayboro Harbor RNA PCR, 9) Bayboro harbor RNA RTPCR 1 0) Synechococcus PCC6301 RNA PCR, 11) Synechococcus PCC6301 RNA RT-PCR.

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59 MW 1 2 3 4 5 6 7 8 9 1 0 11 Figure 17 Southern analysis of ampl ifi cat i on products of env i ronmenta l rbcl mANA shown i n F i gure 16 Lane designa t ions a r e as fo ll ows : MW) pBA322 DNA-BstN 1 d i gest molecular we i ght mar k er 1 )DI water RT PCR 2) rbcl mANA PCR (no AT), 3) rbcl mANA RT-PCR, 4) Dl water PCR, 5) rbcl DNA PCR, 6) Stat i on 8 RNA PCR (no AT), 7) Station 8 RNA RT-PCR, 8) Bayboro Harbor RNA PCR, 9) Bayboro harbor RNA RT-PCR, 1 0) Synechococcus PCC6301 RNA PCR, 11) Synechococcus PCC6301 RNA RT-PCR.

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60 However, recent speculation has centered on the underestimation of Trichodesmium abundance in these waters (Carpenter and Roman 1991). The primary function of phytoplankton is the conversion of inorganic carbon to organic carbon through the action of the enzyme rubisco. Yet, little is known about the mechanism of rubisco regulation in these natural populations. Here I have shown that carbon fixation rates directly correlate with rbcL mANA levels in all marine environments tested (oligotrophic, mesotrophic, and eutrophic). Thi s level of mANA may be an underestimation of the total amount of rbcL mANA present in each sample for the following reasons The sample is one of a diverse assemblage which may or may not contain a diversity of rbcL genes The diatom Cylindrotheca fusiform is (Hwang and Tabita 1991) has been shown to contain a diverse rbcL sequence. Diatoms normally dominate in temperate estuaries (Kennish, 1986) and also bloom at certain times of the year in the open ocean (Sargasso Sea spring bloom; Hulburt, E.M. et al., 1960). The rbcL gene probe used in hybridization was from a cyanobacterium, Synechococcus sp. PCC 6301, and thus may only detect cyanobacterial type sequences, thereby underestimating the actual level of rbcL gene expression The < 1 J..Lm open water assemblage at station 6 was responsible for 58 % of the carbon fixation rate, yet contained 1 00% of the hybridizable rbcL mANA. This is further indication that the rbcL probe used in this study may be specific to cyanobacterial and possibly green algal picoplankton forms. Reprobing of blots from this transect with the Cylindrotheca fusiformis rbcL probe yielded no hybridization. This may indicate

PAGE 72

61 that no diatom related sequences were present in the samples or that the diatom probe is unique to Cylindrotheca fusiformis. It is also possibile that during stripping the target mANA was removed from the membrane, although this was not tested This method of stripping and reprobing for multiple targets requires further investigation The sampling method may have also affected the levels of rbcl mANA since non-cyanobacterial a l gae in the sample may have been damaged during collection using a submersible pump. However, the actual level of rbcl mANA may be the upper limit of carbon fixing potential. By only measuring rbcl mANA, researchers may overestimate carbon fixation if actual carbon fixation rates are not measured. The reason for this is as follows. mANA levels are normally in excess of the translation machinery of the cell, the ribosomes, such that the protein synthesis system is saturated (Jensen and Pedersen, 1990). It is this intracellular concentration of ribosomes that best correlates with the rate of protein synthesis. This may explain why decreases in rbcl gene dosage in Chlamydomonas caused a dramatic reduction in rbcl mANA levels yet rubisco synthesis remained unaffected (Hosler et al., 1989) Phytoplankton populations have unique temporal and spatial distributions, both horizontally and in the vertically. In general Synechococcus res i des at high numbers in the mid (50m) to deep(1 OOm) euphotic zone (Li and Wood, 1988) with smaller eucaryotic picoplankton and prochlorophyte types at even deeper depths ( < 1% isolume) In this study, however, autofluorescent cyanobacteria were

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62 present in greatest numbers at 1Om and decreased with depth. The number of red fluorescent cells increased with depth, contributing to the deep chlorophyll maximum. It is interesting that the highest rbcl mANA and DNA levels were found for upper water column populations of possible genetic relatedness to the rbcl gene probe employed. The deeper populations may be genetically distinct Diel fluctuations in phytoplankton carbon fixation rates are not only directly related to the supply of energy from the light harvesting complexes of the cell but may also be due to changes in relative amounts or activity of rubisco. Pichard and Paul (1991) found that natural phytoplankton populations and Synechococcus in culture exhibited diel light/dark variations in the amount of rbcl mANA The Dry Tortugas population appeared to regulate synthesis of rbcl mANA in response to changes in surface illumination. However, this variation could have been due to sampling of diverse phytoplankton assemblages or to the patchy nature of phytoplankton distribution in surface waters. While this explanation has not been ruled out, patchiness was unlikely since surface chlorophyll did not exhibit the same diel trend. Also, I may have sampled patches of phytoplankton that had variable activity. I can not rule out this possibility While we did not quantitate the absolute level of rbcl mANA in Synechococcus or in natural populations from the Dry Tortugas, the amount of hybridization detected in culture (2.2 x 108 cells)was similar to that observed in 2 liters of seawater On the basis of an average chlorophyll content of 18 fg/ cell in Synechococcus, an approximate l y equivalent hybridization signal was obtained for the natural population {0. 72 J.Lg chi E) as for

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63 the culture. However, a lower hybridization signal per unit chlorophyll may be expected for natural populations since these are of an unknown, possibly divergent, genetic homology. In this case, however, the natural population contained only 17 to 33% of the chlorophyll of the culture. This accounts for any differences in biomass content of samples. For surface phytoplankton populations sampled in the Bahamas, a similar trend was seen for both rbcl mANA and carbon fixation, yet this was superimposed upon a diel variation in chlorophyll (biomass). This led to a refinement in diel studies accomplished by capturing a watermass and incubating it on the deck of the research vessel. Open water natural populations incubated in a on-deck incubator, to eliminate patchy sampling, contained higher levels of rbcl mANA corresponding to maximal rates of carbon fixation (light periods). rbcl DNA levels changed little, such that changes in levels of mANA were not caused by gene dose changes. This light stimulation of rbcl transcription has been seen with other algae in culture. In the green algae, Cyanidium caldarium (SteinmOIIer and Zetsche, 1984) and Chlorogonium elongatum (Stein biB and Zetsche, 1986), the synthesis of rubisco protein and its' mANA's is promoted by illumination. Other genes exhibit light regulation of transcription Transcription of psbA, encoding the 01 protein of photosystem II, in Synechococcus occurred only during periods of illumination (Schaffer and Golden, 1989). However this appearance of light stimulation of gene expression does not exclude the possibility that these genes are not regulated by

PAGE 75

64 light but by a different mechanism. Cycles of light and dark have been used to entrain a free running rhythm of photosynthetic oxygen evolution with temporal separation from nitrogen fixation in a marine Synechococcus sp. (Mitsui et al., 1986) A rhythm of expression was found for nitrogen fixation genes in freshwater Synechococcus (Huang et al., 1990). Therefore variations in rbcl expression might be tightly coupled to and regulated by rhythms in cell division of natural populations. This hypothesis is currently under investigation. The fact that rbcl mRNA correlates well with carbon fixation rates does not exclude other enzymes performing equally important functions in regulating carbon fixation. Phosphoenolpyruvate carboxylase (PEP carboxylase) is believed to play a role in phytoplankton carbon ass i milation by the B-carboxylation of phosphoenolpyruvate The maximum possible contribution by PEP carboxylase to total carbon fixation in the light is 25% (Beardall, 1989). However during max i mum rates of photosynthesis, PEP carboxylase contributed only as much as 12.7% of the total photosynthesis (Mortain-Bertrand et al., 1988). In fact, Smith et al. (1983) found that carbon fixation rates correlated with rubisco activity and not with PEP carboxylase activity. Yet, when the physiological state of the phytoplankton is such that rubisco activity is reduced, B-carboxylation reactions may become important. Unlike terrestrial C4-metabo l ism PEP carboxylase appears to play a anaplerotic role in amino acid synthesis while rubisco is the key enzyme necessary for net carbon fixation Finally, variations in levels of such enzymes as phosphoribulokinase, which regenerates the ribulose bisphosphate

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65 substrate, and carbonic anhydrase, wh i ch converts bicarbonate into carbon dioxide (substrate for rubisco), may have a greater effect on carbon fixation rates than PEP carboxylase. Finally, RNA samples that were collected from open ocean populations were not amenable to amplification procedures, possibly due to problems with long term storage of samples. Samples that were freshly isolated from Bayboro Harbor were able to be amplified, yet if these samp l es were stored for several weeks they could not be amplified Thus samples need to be amplif i ed as soon as possible, and if stored for extended periods should possibly be stored as a ethanol precipitate to reduce degradation (Wallace, 1987) These amplification products are being cloned and sequenced to determine their identity and should yield new informat i on on the divers i ty of rub i sco sequences occuring in the marine environment.

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66 CHAPTER 4: CONCLUSIONS The development of this method and the successful demonstration of its potential utility has provided a foundation for future studies on microbial gene expression in the marine water column. The results presented in this study indicate that gene specific mANA levels may be a excellent indicator of microbial activity. When RNA extraction is coupled with current gene amplification technology, this method may enable the detection of a single cell and determine whether that cell is in a metabolically active state. From this study I draw the following conclusions: 1. The use of antisense and sense gene probing enables the detection of gene expression by descrimination of hybridization signals between mANA and DNA target sequences and can account for non-specific hybrid i zation (a problem in environmental hybridizat ions). 2. The mANA detection protocol was able to transcripts that were produced prior to the detection of catechol dioxygenase activity under permissive expression conditions. However, it's ability to monitor gene expression at it s primary level of regulation, transcription and account for variations in expression due to changes in gene copy (gene dose) makes this technology attractive for investigating differences in regulation occuring at transcriptional and post-transcriptionallevels.

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67 3. Rates of carbon fixation appear to be directly related with levels of rbcl mANA for natural phytoplankton populations in both neritic and oceanic surface waters. 4. Diel variations in carbon fixation in natural phytoplankton populations appear to be regulated by control of rbcl transcription and not changes in rbcl gene copy. 5. Vertical profiles suggest that phytoplankton at depth also appear to regulate carbon fixation by controlling levels of rbcl mANA. 6. This method can be successfully applied to measuring the gene expression of any conserved gene in water column microbial populations for which gene probes are available.

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68 LITERATURE CITED Adams, C W and G.W. Hatfield 1984 Effects of promoter strengths and growth conditions on copy number of transcription-fusion vectors. J. Bioi. Chern. 259:7399 7 403. Albertson, N H., T. Nystrom and S Kjelleberg 1990. Funct i onal mRNA half-lives in the marine Vibrio sp. S14 during starvation and recovery J. Gen. Micro. 136:2195-2199 Assinder S J and P .A. Williams. 1990 The TOL plasm ids: determinants of the catabolism of toluene and the xylenes Adv. Mi cro. Physiol. 31: 1-69 Azam, F T Fenchel J.G. F i eld J S Gray, L.A. Meyer-Reil and F Thingstad 1983 The ecological role of water column m i crobes in the sea. Mar. Ecol. Prog Ser 1Q: 257-263 Barkay T., C. Liebert and M G i llman 1989 Hybr i d i zat i on of DNA probes with whole-community genome for detection of that encode microbial responses to pollutants: mer genes and Hg + res i stance. Appl. Environ Microb i al. 55 : 157 4-1577 Beardall, J. 1989. Photosynthesis and photorespiration in marine phytoplankton Aquat. Bot. 34:1 05130 Becker-Andre, M. and K. Hahlbrock 1989 Absolute mRNA quant i f i ca ti on using the polymerase chain react i on (PCR). A novel approach by a PCR aided transcript titration assay (PA TIY) Nucleic. Acid. Res. 17:9437-9446 Bej, A .K., M.H. Mahbubani and R.M. At las. 1991. Detect i on of v i able Leg i onella pneumophila in water by polymerase chain react i on and gene probe methods Appl. Enivron. Microb ial. 57:596-600. Belasco J .G. and C F Higg i ns 1988. Mechanisms of mRNA decay i n bacteria: a perspective. Gene 72: 15-23 Bergh, 0 K.Y. Borsheim, G Bratbak and M Heldal. 1989. High abundance of v i ruses found in aquatic environments Nature (London) 340 : 467-468

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69 Berry, J O., B J. Nikolau, J.P Carr and D .L. Klessig. 1985 Transcriptional and post transcriptional regulation of ribulose 1 ,5-bisphosphate carboxylase gene expression in lightand darkgrown Amaranth cotyledons. Mol. Cell. Bioi. Q:2238-2246 Binder, B.J and S.W. Chisholm. 1990. Relationship between DNA cycle and growth rate in Synechococcus sp. strain PCC 6301. J. Bacterial. 172 : 23132319. Burlage, B.S., S W Hooper and G.S. Sayler. 1989. The TOL (pWWO) catabolic plasmid. Appl. Environ Microbial. 55:1323-1328. Capone, D G J.M O'Neil, J Zehr and E.J. Carpenter. 1990. Basis for diel variation in nitrogenase activity in the marine planktonic cyanobacterium Trichodesmium thiebautii. Appl. Environ. Microbial. 56 : 3532-3536. Carpenter', E J and K. Romans 1991. Major role of the cyanobacterium Trichodesmium in nutrient cycling in the North Atlantic Ocean. Science 254:1356 1358. Chomczynski, P. and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanid i nium thiocyanate-phenol-chloroform extraction Anal. Biochem 162 : 156-159. Cote, R. 1984. ATCC media handbook, 1st. ed. American Type Culture Collection, Rockville, MD DeFiaun, M .F. and J H Paul. 1986 Hoechst 33258 staining of DNA in agarose gel electrophores i s. J. Microbial. Methods. Q : 265-270. Doty, M .S. and M. Oguri. 1957. Evidence for a photosynthetic daily periodicity Limnol. Oceanogr. g:37-40 Douglas, S E., D.G Durnford and C .W. Morden. Nucleotide sequence of the gene for the large subunit of ribulose 1 ,5-bisphosphate carboxylase/oxygenase from Crvptptomonas t : evidence supporting the polyphyletic origin of plastids. J. Phycol. 26:500-508. Ducklow H.W D.A. Purdie, P.J.L. Williams and J.M. Davies 1986 Bacterioplankton: A sink for carbon in a coastal marine plankton community. Science 232:865-867 Ferre, F. and F Garduno 1989 Preparation of crude cell extract suitable for amplification of RNA by the polymerase chain reaction. Nuc. Acid. Res.

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79 APPENDICES

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80 APPENDIX A: SYNTHESIS OF RNA GENE PROBES

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81 1. Digest to completion 2-1 0 J.Lg of the riboprobe plasmid containing the sequence of interest. Use 5-1 0 units of the appropriate restrict i on enzyme per J.Lg DNA at a final DNA concentration of 0.1 J.Lg/ J.LI in a total volume of 50 J.LI for 3 hr to overnight. 2 Check DNA for complete cutting by running 3 J.LI on a 1% agarose gel. If cutting is complete stop reaction by adding 1 /1 0 volume of 0 2M EDT A. 3 Add 1 volume of phenol saturated with STE pH 8 0 and 0 5 volume CHCI3 isoamyl alcohol (24 :1). Vortex 30 sec and centrifuge 1 min Remove the supernatant. 4. Extract a second time as in step 3 5. Add 1 volume CHCI3, vortex 30 sec and cent r ifuge 1 min. Remove the supernatant. 6 Extract a second time as i n step 5. 7. Add 1/1 0 volume 3M NaOAc pH 5 and 250 J.LI 1 00% ethanol and ppt. overnight at -20C 8 Centrifuge the sample remove the supernatant and wash the pellet with 70% ethanol dry and dissolve the pellet in 8 J.LI TE pH 8 0 9 To prepare labeled RNA probe, add 1 J.Lg ( 1 J.LI} linearized template DNA to a 0.5 J.LI t ube and the follow i ng reagents 10 5 J.LI 5X rxn. buffer. 11. 5 J.LI of unlabeled nucleotide m i xture (3 J.LI of each 10 mM stock of ATP CTP, GTP and 3 J.LI of sterile Deionized water) 12 1 J.LI of 120 J.Lm UTP (1 : 83.4 dilution of 10 mM UTP). 13 2 5 J.Ll 100mM OTT. 14. 1 0 J.Ll 35S -UTP. 15. 1 J.LI RNasin and 1 25 J.LI T7 or Sp6 RNA polymerase. 16. Incubate at 37C for 1 hour 17. D i gest DNA template with 1 25 J.LI RQ1DNase at 37C for 10 m i n

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82 18 Perform a phenol chloroform extraction and precipi t ate the aqueous supernatant with 1/10 volume 3M NaOAc pH 5 6 25 J.LI 0 1 mg/ml calf thymus DNA and 2 volumes Ethanol at 80C for 30 m i n 19 Centrifuge for 1 0 min and wash the pellet w i th 70% ethanol. 20 Resuspend the pellet in 1 00 J.LI ster ile STE and 1 J.LI DTI. The labeled RNA is then purified from unincorporated nucleotides by spun column (Sambrook et al., 1989). 21. Add 150 J.LI of hybridization buffer to the probe and use or store at 4C

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83 APPENDIX B : PROBING PROTOCOL FOR USE WITH RNA GENE PROBES

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84 1. Wash filters to be probed in 0.4 M Tris pH 8.0 for 5 min at room temp. 2. Place filters in plastic probe bags, add 1 0-20 ml hybridization solution ( without probe) and seal bags. 3. Pre-hybridize filters for 15 min at 42C for DNA or 55C for RNA. 4. Remove and discard the hybridization solution and add 1 0-20 ml of new hybridization solution containing the RNA gene probe. 5 Hyb(idize filters overnight at 42C for DNA or 55C for RNA. 6 Remove filters from the bags and wash in 2X sse and 1 mM OTT for 5 min at room temp 7 Wash filters in PSE 3X 1 hr at 65C 8. Wash filters in PES 3X 1/2 hr at 65C 9. Allow filters to dry till just damp, mount and expose to x-ray film. Hybridization Solution {200 ml) 50 mi1M pH 7.4 20 ml 2.5M NaCI 0.4 ml O.SM EDTA pH 8 0 1 00 ml deionized formamide 2 0 ml salmon sperm DNA {10 mg/ml denatured) 28.0 ml Deionized water 14 g SDS 2.0 mi1M OTT PSE solution {1 liter) 250 mi1M Na2HP04 pH 7.4 20 g SDS 2 ml O.SM EDTA pH 8.0 PES solution {1 Uter) 40 ml 1M Na2HP04 pH 7.4 10 g SDS 2 ml O.SM EDTA pH 8.0

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APPENDIX C : SEQUENCES OF DEGENERATE OLIGONUCLEOTIDE PRIMERS FOR AMPLIFICATION OF rbcl 85

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86 DESIGN OF PRIMERS A 0167 Syn Thr lle Lys Pro Lys Leu Gly Leu Syn ACG ATC AAA CCA AAA CTC GGT CTG Cotton CGA TAC C.T DEL .. A Crypt G.T T.A .. A Rhodob. ...ATC ... ..G .. G .. G .. G Anab. .. c T.A Chlamy .. A .. T T.A .. T Rhodosp. ...ATC ... ..G .. G .. G .. c Consensuscotton ACG ATC AAA CCA AAA CTC GGT CTG c G T G G G T A G A A T Consensusc T cotton-Anoxygenic PS ACG ATC AAA CCA AAA CTC GGT CTG c G T T T A A A T DESIGN OF PRIMERS B. D286 (3' ) Syn leu His I so His Arg Ala Met Syn CTG CAC ATC CAC GGT GCA ATG Crypt .. T T.A CGT Anab. . c .. G Chlamy. ..A Rhodob. T ... .. T TAT ..G .. T GGC Rhodosp. .. T TAT ..G .G GG. Consensus CTG CAC ATC CAC GGT GCA ATG T T T TAT c G CGT A A G T G C Oxygenic GGT only CTG CAC ATC CAC GCA ATG A T A c T CGT T antisense 3 GAC GTG TAG GTG CCA CGT TAC 5 T AT G A GCA A