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The molecular analysis of marine algal virus communities Short, Steven Michael 2002

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THE MOLECULAR ANALYSIS OF MARINE ALGAL VIRUS COMMUNITIES by STEVEN MICHAEL SHORT B.Sc, The University of British Columbia, 19-95 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF T H E REQUIREMENTS FOR T H E D E G R E E OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Botany) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December 2002 © Steven Michael Short, 2002 In p r e s e n t i n g this thesis in partial fu l f i lment of the requ i rements fo r an a d v a n c e d d e g r e e at the Univers i ty of Brit ish C o l u m b i a , I agree that t h e Library shal l m a k e it f reely avai lable fo r re fe rence and study. I further agree that p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f this thesis f o r scho la r l y p u r p o s e s m a y b e g r a n t e d b y the h e a d o f m y d e p a r t m e n t o r by his o r her representat ives . It is u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n of this thesis fo r f inancial gain shal l no t b e a l l o w e d w i t h o u t m y w r i t t e n p e r m i s s i o n . D e p a r t m e n t T h e Un ivers i ty o f British C o l u m b i a V a n c o u v e r , C a n a d a D E - 6 (2/88) Abstract Viruses are abundant members of marine microbial communities and important components of marine food webs and geochemical cycles. Previously, PCR was used to amplify D N A polymerase gene fragments from algal viruses belonging to the family Phycodnaviridae. The Phyconaviridae are described by the International Committee on Taxonomy of Viruses as large (genomes > 300 kbp) dsDNA viruses lacking envelopes that infect algae. In order to examine algal virus communities, I developed a denaturing gradient gel electrophoresis (DGGE) protocol to rapidly fingerprint gene fragments amplified from marine algal viruses. This thesis describes the development of this fingerprinting method and its application to the study of marine algal viruses in nature. Initially, PCR and D G G E were used to resolve similar sized products amplified from related but relatively dissimilar virus templates. PCR with degenerate algal-virus-specific primers was used to amplify pol gene fragments from three cultured viruses that infect microalgae and a naturally occurring virus community. Although amplification from all samples resulted in PCR products approximately 700 bp in length, the fragments from cultured viruses focused at different locations in a denaturing gradient gel and several bands were resolved in the natural sample. This study demonstrated that PCR and D G G E could be used to resolve genetically distinct viruses from artificial and natural communities. To determine if pol fragments of similar sequence could be amplified from geographically distant areas, natural algal virus communities were obtained from coastal sites in the Pacific Ocean in British Columbia, Canada, and the Southern Ocean near the Antarctic Peninsula. Genetic fingerprints of algal virus communities were generated using DGGE. D N A polymerase gene fragments were recovered and sequenced from 25 bands extracted from the gradient gel. All 25 sequences fell outside the clusters of known algal viruses, but were within the Phycodnaviridae. In addition, similar virus sequences (>98 % sequence identity) were recovered from British Columbia and Antarctica indicating that closely related viruses occur both in the Southern Ocean and the NE Pacific. The temporal variability of natural algal virus communities and the co-occurring eukaryotic plankton were studied at a single location on a weekly basis over fourteen months. ii The changes in the community were related to physical and biological characteristics of the environment. Comparison of algal virus fingerprints with environmental conditions revealed that, at certain times, changes in algal virus community composition were coincident with changes in tide height, salinity, or chlorophyll a concentration. Overall, algal virus community fingerprints were temporally less variable than eukaryotic community fingerprints. While the algal virus fingerprint patterns were stable throughout most of the study, stable eukaryote fingerprint patterns were observed only during the winter months. It appeared that specific taxa of algal viruses could persist in fluctuating physical and biological environments suggesting that the production of, and mortality from, some algal viruses was constantly occurring. My research has demonstrated that fingerprinting techniques can be used to investigate the geographic and temporal variability of marine algal virus communities. The results show that some marine algal viruses are geographically widespread and that some persist through several seasons. These findings corroborate previous studies showing that viruses are important to the mortality of marine phytoplankton and are therefore ecologically important members of marine food webs. iii Table of Contents Abstract ii Table of Contents iv List of Tables vi List of Figures vii List of Abbreviations and Symbols viii Preface x Acknowledgements xii Dedication xiii Chapter I. Introduction: the Diversity of Algal Virus Communities, the Polymerase Chain Reaction, and Denaturing Gradient Gel Electrophoresis 1 1.1 Summary 2 1.2 Viruses in the Marine Environment 3 1.3 The Polymerase Chain Reaction and the Study of Marine Microbes 7 1.4 The Conserved nature of D N A polymerases 9 1.5 D N A polymerase sequences as genetic markers 11 1.6 PCR amplification of algal virus pol 13 1.7 D N A polymerase genes in the study of marine virus communities 16 1.8 D G G E as a tool for comparing virus communities 19 1.9 Conclusion 23 Chapter II. Denaturing Gradient Gel Electrophoresis Resolves Virus Sequences Amplified with Degenerate Primers 24 2.1 Summary 25 2.2 Introduction 26 2.3 Materials and Methods 28 2.4 Results and Discussion 30 Chapter III. Sequence Analysis of Marine Virus Communities Reveals that Groups of Related Algal Viruses are Widely Distributed in Nature 33 3.1 Summary 34 iv 3.2 Introduction 35 3.3 Materials and Methods 37 3.3.1 Sample collection and preparation 37 3.3.2 PCR and D G G E 38 3.3.3 Sequencing 39 3.3.4 Sequence analysis 40 3.4 Results 42 3.5 Discussion 46 Chapter IV. Temporal Changes in the Composition of Natural Communities of Marine Algal Viruses and Eukaryotes Studied by Denaturing Gradient Gel Electrophoresis 51 4.1 Summary 52 4.2 Introduction 53 4.3 Materials and Methods 55 4.3.1 Sample collection 55 4.3.2 D N A extraction and PCR 56 4.3.3 D G G E and sequencing 57 4.4 Results 61 4.5 Discussion 70 Chapter V. Conclusion 76 References 81 v List of Tables Table 3.1. Sample details 38 Table 3.2. Acronyms, names, and GenBank sources of viruses used in phylogenetic analysis... 41 Table 4.1. Results of database searches with Jericho Pier sequences 64 List of Figures Figure 1.1. Neighbor-joining tree of algal viruses 6 Figure 1.2. Generic map of B-family D N A polymerases 10 Figure 1.3. Relative positions of PCR primers on the DNA polymerase gene 14 Figure 1.4. Phylogenetic tree of 100 bootstrap analyses of D N A polymerase fragments 15 Figure 1.5. Flowchart of PCR primer design 17 Figure 1.6. Gel electrophoresis of amplified algal virus pol fragments 1 22 Figure 2.1. Gel electrophoresis of amplified algal virus pol fragments II 31 Figure 3.1. Map of sample sites 37 Figure 3.2. Gel electrophoresis of PCR fragments amplified in second-stage PCR 43 Figure 3.3. Maximum Likelihood (ML) tree of D N A pol fragments 45 Figure 4.1. Jericho Pier sample site 55 Figure 4.2. Annealing temperature gradient of PCR with AVS primers 61 Figure 4.3. Maximum Likelihood tree of algal viruses from Jericho Pier 63 Figure 4.4. Physical data and AVS fingerprints from Jericho Pier 67 Figure 4.5. Physical data and 18S rDNA fingerprints from Jericho Pier 68 Figure 4.6. Cluster analysis of D G G E fingerprints 69 Figure 5.1. Maximum Likelihood tree of virus pol fragments 80 vii List of Abbreviations and Symbols AVS algal-virus-specific bp base-pair C carbon C O 2 carbon dioxide cpu central processing unit °C degrees Celcius D G G E denaturing gradient gel electrophoresis dia diameter D N A deoxyribonucleic acid dNTP deoxyribonucleoside triphosphate dsDNA double-stranded D N A GHz gigahertz Gt gigatonne h hour kbp kilobase-pair kD kilodalton kPa kilopascal 1 litre M molar mg milligram MHz megahertz min minute ml millilitre mM millimolar mm millimetre ng nanogram PCR polymerase chain reaction PFGE pulsed field gel electrophoresis pmol picomole pol D N A polymerase viii ppt parts per thousand PVDF polyvinylidene fluoride rDNA ribosomal deoxyribonucleic acid RFLP restriction fragment length polymorphism rRNA ribosomal ribonucleic acid s second U units UPGMA unwieghted paired group method with arithmetic means v/v volume per volume w/v weight per volume pg microgram pi microlitre pM micromolar Preface As the title states, this dissertation focuses on the molecular analysis of marine algal virus communities. My interest in this research began when I realized the importance of viruses to the ecology of marine phytoplankton and therefore, global climate change. Although the precession, obliquity, and eccentricity of Earth's orbit may be the principal cause of global glacial and interglacial cycles, the atmospheric concentration of the green house gas C O 2 is also thought to contribute to climate change (88). Since the industrial revolution, fossil fuel combustion has resulted in a dramatic increase in anthropogenic carbon emissions. These emissions have been linked to increased concentrations of atmospheric C O 2 (60). The concentration of atmospheric C O 2 is, in part, controlled by photosynthesis in the ocean; through the "biological pump" phytoplankton convert atmospheric C O 2 into organic carbon that can then be transported to ocean's interior and sediments. The importance of phytoplankton in the global carbon cycle is undisputed. In fact, it has been estimated that approximately half of the global net primary production (ca. 45 Gt C year"1) is due to marine phytoplankton (67). Thus, to understand the ocean's ability to respond to anthropogenic perturbations of the global carbon cycle we must understand the ecology of marine phytoplankton. To have a complete picture of phytoplankton ecology we must examine the viruses that infect them. By contributing to our knowledge of marine algal viruses I hoped my research would further, at least indirectly, our understanding of global climate change. It is interesting to note that from the beginning of my doctoral work in 1996 to the end in 2002 computer processors evolved dramatically. In 1996 the cpu of the computer we used ran at 33 MHz and by the end of my degree I had access to computers with processors running at 2GHz. This is the reason the methods I used for phylogenetic analyses changed. When the phylogenetic analyses for chapter 1 were conducted, the computers required an inordinate amount of time to analyze genetic information using maximum likelihood. At that time we used the neighbor-joining algorithm as it was a quick and therefore, commonly used technique. However, producing trees with accurate branch lengths and support values at nodes was not trivial with the program package I was using. As computer processors evolved it became feasible to analyse sequence data using maximum likelihood. Because it produced multifurcating x dendrograms with accurate branch lengths and node support values, I used a maximum likelihood program to analyze the sequence data in chapter 3 and all subsequent a sequences. As well, during my doctoral research my skills and molecular techniques evolved. Thus, I did not include a single summary of my materials and methods and wrote each chapter as an independent manuscript. The first three chapters of my thesis have been published in peer-reviewed journals. Chapter one was published as: Steven M. Short and Curtis A. Suttle. 1999. Use of the polymerase chain reaction and denaturing gradient gel electrophoresis to study diversity in natural virus communities. Hydrobiologia 401: 19-32. This chapter serves as the introduction to my thesis and in it I review relevant background research and present preliminary data demonstrating the use of molecular methods to study marine algal virus communities in nature. Chapter two was published as: Steven M. Short and Curtis A. Suttle. 2000. Denaturing gradient gel electrophoresis resolves virus sequences amplified with degenerate primers. BioTechniques 28: 20-26. This chapter provides further evidence that the PCR and denaturing gradient gel electrophoresis could be used to study algal virus communities. Chapter three was published as Steven M. Short and Curtis A. Suttle. 2002. Sequence analysis of marine virus communities reveals that groups of related algal viruses are widely distributed in nature. Applied and Environmental Microbiology 68: 1290-1296. This work provided further evidence that some groups of closely related algal viruses were widespread in the world's oceans. I am the primary author of all three of these published manuscripts. As such, I was the author that designed and executed the experiments, and wrote the manuscripts. Curtis A. Suttle was the only other author listed on these manuscripts. He was my graduate supervisor and the principal investigator of the lab in which the research was conducted. As my graduate supervisor, he provided the impetus for all of this work and was the primary editor of the manuscripts. I certify that the preceding statements about authorship are correct. Curtis A. Suttle xi Acknowledgements With my most heartfelt gratitude I want to thank Cindy M . Frederickson for her support, encouragement, and advice through the last years of my doctoral program. For reasons I will not discuss, this period of my life has been extremely difficult. With Cindy's love and care, I found the strength and courage to move forward. I gratefully acknowledge my supervisor Curtis A. Suttle for accepting me as a student and providing the impetus for the work described in this thesis. I also want to thank him for his invaluable advice and comments on my research, presentations and manuscripts. My parents John and Iris Short also deserve my thanks for their encouragement, emotional support, and financial help throughout my academic career. In addition, I would like to acknowledge my supervisory committee P. J. Harrison, F. J. R. Taylor, and W. W. Mohn for their helpful input throughout my graduate research. I would also like to thank Amy Chan for her instruction, Joe Needoba for his moral support and numerous discussions, Wade Jeffrey for taking me on two amazing voyages, and Mary Berbee for her knowledge and help with phylogenetic analyses. I also thank Sean Brigden, Corina Brussaard, Jessica Clasen, Alex Culley, Andre Comeau, Andrew Lang, Janice Lawrence, Pascale Loret, Nicole McLearn, Cydney Nielsen, Alice Ortmann, Karen Reid, Egidio Spinelli, Tanya St. John, Vera Tai, and Derek Yuen for their assistance during my doctoral studies. Finally, I acknowledge the Natural Sciences and Engineering Research Council of Canada for providing me financial support via two postgraduate scholarships and the UBC Department of Graduate Studies for providing a travel award. xii Dedication To Kitty xiii Chapter I. Introduction: the Diversity of Algal Virus Communities, the Polymerase Chain Reaction, and Denaturing Gradient Gel Electrophoresis Published as: Short, S. M. and C. A. Suttle. 1999. Use of the polymerase chain reaction and denaturing gradient gel electrophoresis to study diversity in natural virus communities. Hydrobiologia 401: 19-32. 1 1.1 Summary Viruses are abundant members of marine and freshwater microbial communities, and are important components of aquatic ecology and geochemical cycles. Recent methodological developments have allowed the use of the PCR to examine the diversity of natural communities of viruses without the need for culture. D N A polymerase genes are highly conserved and are therefore suitable targets for PCR analysis of microbes that do not encode rRNA. As natural virus communities include numerous dsDNA viruses, and as many dsDNA algal viruses encode their own D N A polymerase, PCR primers can be designed to amplify fragments of these genes. This approach has been used to examine genetic diversity in natural communities of Phyconaviridae, i.e large (genomes > 300 kbp) dsDNA viruses that infect algae with virions lacking envelopes. Algal-virus-specific (AVS) PCR primers were used to amplify polymerase fragments from natural virus samples, demonstrating the presence of a diverse community of viruses closely related to those that are known to infect phytoplankton. We have modified this approach by using denaturing gradient gel electrophoresis (DGGE) to rapidly analyze PCR products. D G G E will permit rapid and efficient fingerprinting of natural marine viral communities, and allow spatial and temporal differences in viral community structure to be examined. This manuscript provides a brief overview of how PCR and D G G E can be used to examine diversity in natural viral communities drawing on viruses that infect phytoplankton as an example. 2 1.2 Viruses in the Marine Environment Torrella and Morita (121) provided the first evidence that viruses were abundant in seawater, but it was not until a decade later that accurate estimates were obtained of their abundance and ubiquitous distribution in marine and fresh waters (6, 90, 113). Electron and epifluorescence microscopy revealed that abundances of these small (0.02 to 0.2 urn dia.) particles ranged from < 104 to > 108 ml"1, depending on the season and the environment sampled (reviewed in 8, 40, 117). In general, viral abundance and distribution parallels that of other biotic factors such as bacteria, Chlorophyll a, and dissolved and particulate D N A (86, 129). While viruses have an obvious effect on natural populations as a source of mortality, Suttle (109) stated that the greatest impact of viruses on aquatic ecosystems might be through non-steady-state processes such as genetic exchange between microbial populations or through alterations of community composition. In order to replicate, viruses obligately require host cell metabolism. Furthermore, infection of cells by viruses requires direct contact between a virus particle and its host. The probability of successful infection is dependent on the contact rate between viruses and their hosts, and a rapid increase in virus abundance typically occurs only when host cells reach a threshold density (130). As viruses are known to infect cells in a density dependent manner, it follows that viruses potentially have the greatest impact on the most abundant host cells in a community. Lysis of the most abundant host cells may lead to increased diversity through competitive release, or may prevent some species from forming blooms (13, 38, 109, 117). The cosmopolitan phytoplankton Micromonas pusilla may be an example of the latter (26). In this way, viruses may have a direct impact on community composition and species diversity. Viruses may also infect cells through a lysogenic rather than lytic life cycle and may be directly involved in the exchange of genetic material with their hosts. In the lytic pathway of infection, viruses inject their D N A into a host cell. This D N A is then replicated, transcribed, and translated to produce new virus particles. Host cells eventually lyse and new particles are released. In the lysogenic, or latent, life cycle, virus genetic information is not used to replicate new particles. Instead this D N A is archived in the host cell and can remain inactive through many host-cell generations. Eventually, the virus may be induced by an environmental signal, the virus genome is then replicated, and new infectious particles are formed. When hosts maintain 3 viral DNA, host and viral genomes are often intimately associated; this association often results in increased probability of genetic transformation of hosts. To date there is no experimental evidence for widespread lysogeny in marine eukaryotic phytoplankton; however, there is accumulating evidence that lysogeny occurs in many marine prokaryotes (56, 83). Furthermore, Chiura (24) described the process of gene transfer by viruses isolated from marine bacteria. While non-steady-state processes such as competitive release or genetic transformation may be important to phytoplankton communities, there is no direct experimental evidence that virus infection can lead to increased phytoplankton species diversity, or that latent viruses infecting eukaryotic phytoplankton exist. Brown (14) wrote the first comprehensive review of algal viruses. At this time there was substantial evidence, based largely on ultrastructural studies, that viruses infected both prokaryotic and eukaryotic algae, yet few of the viruses were isolated. The first marine algal viruses isolated infected the picoflagellate Micromonas pusilla (70). Currently, lytic viruses infecting at least six taxa of eukaryotic microalgae are established in culture. The first of these to be characterized in detail are a group of viruses that infect freshwater Chlorella-like algae found as symbionts in Hydra viridis and Paramecium bursaria (for review see 124). These viruses have been assigned to a new family, the Phycodnaviridae. The Phyconaviridae are described by the International Committee on Taxonomy of Viruses as large (genomes > 300 kbp) dsDNA viruses lacking envelopes that infect algae. Other cultured Phycodnaviridae infect marine phytoplankton and include viruses that infect the prasinophyte Micromonas pusilla (25, 70), the prymnesiophytes Chrysochromulina spp. (112) and Phaeocystis pouchetii (55), the pelagophyte Aureococcus anophagefferens (73), and the raphidophyte Heterosigma akashiwo (80). With the exception of the A. anophagefferens virus, these viruses share a common morphology; they are large (> 100 nm dia.) polyhedral viruses with double-stranded DNA. An important question that has been difficult to address is the range of phytoplankton species that are infected by viruses. Suttle et al. (113) demonstrated that increasing the abundance of naturally occurring viruses in seawater samples significantly reduced photosynthetic rates. Furthermore, photosynthetic rates of specific subsets of the phytoplankton assemblage were affected by the addition of concentrated marine virus communities (108). While these studies demonstrate that viruses may infect a wide range of marine planktonic algae, determination of the range of phytoplankton species infected has remained elusive. This 4 difficulty stems from the fact that study of the range of organisms infected has relied on culture techniques. Furthermore, studies have shown that the most abundant phytoplankton strains may be resistant to the most abundant viruses (128). If cultured strains of specific phytoplankton species are not representative of a natural community, or if certain strains are not easily cultured, virus isolation with host cultures allows identification of only a small portion of the viruses present in a given sample. Therefore, the diversity of viruses that infect marine phytoplankton may be greatly underestimated. PCR amplification of virus-specific sequences allows the rapid determination of genetic 'diversity in natural samples without the need to purify the organisms in culture. Information on diversity, when combined with data on specific virus isolates can lead to better understanding of the effects of these parasites. For example, DNA polymerase sequences from cultured algal virus strains form distinct clades dependent on the microalgal host infected (23; Figure 1.1). This information permits inferences to be made on the number of host taxa in a natural community; i.e. it is reasonable to hypothesize that virus communities composed of many separate clades of virus sequences infect a larger number of taxa than communities with fewer clades. Further, as more viral polymerase gene sequences are collected, it will be possible to identify specific virus assemblages in nature and infer the host organisms that are infected. Such data should allow a more accurate assessment of the role of marine viruses in regulating algal community structure and primary production. Knowledge of the community structure of algal viruses coupled with data on their abundance and turnover could be used to determine the effect of viruses on the mortality of specific components of the phytoplankton community. A similar approach was used to determine that MpV may be responsible for the mortality of approximately 2.0 to 10.0 % of the M. pusilla population on a daily basis (26) and that cyanophages may remove from 2.0 to 7.0 % of Synechococcus cells daily (111). PCR amplification of viral polymerase genes may also provide a tool for screening of phytoplankton genomic D N A for the presence of lysogens. As mentioned, the algal viruses isolated to date appear to be lytic, and there is no direct evidence of lysogenic viruses that infect eukaryotic phytoplankton. Whether this is a reflection of current methods or a biological reality is unknown. Through molecular techniques, we will be better able to assess algal virus diversity and the existence of latent algal viruses. The following discussion will focus on PCR amplification of D N A polymerase gene sequences as a tool for studying diversity in natural virus 5 Neighbor-joining tree n = 100 o 0.1 76 63 96 100 691— , fi-rV P ° ° l r -94 100 91 OTU2 MpV-PL1 MpV-PB6 MpV-PB7 — MpV-SG1 _ r - O T U 4 ioolMpV-PB8 — OTU3 OTU1 I jMpV-SP2 y i M p V - S P 1 L MpV-GM1 OTU5 100 •CVA-1 — PBCV-1 • NY-2A rCbV-PW1 "iool—CbV-PW3 • HSV-1 communities. Figure 1.1. Neighbor-joining tree of algal viruses The tree shows the phylogenetic relationships among five operational taxonomic units amplified from the Gulf of Mexico and 13 other algal viruses based on sequences of DNA pol gene fragments. Herpes simplex virus type 1 was used as an out-group. The numbers at the nodes indicate bootstrap values (n =100), and branches with values less than 50 have been collapsed. The scale bar represents 0.1 fixed mutation per nucleotide position. Abbreviations are as follows: MpV = Micromonas pusilla virus; CbV = Chrysochrormulina brevifilum virus; CVA-1, PBCV-1, and NY-2A = viruses infecting Chlorella -like algae; HSV-1 = Herpes simplex virus type 1; OTU = operational taxonomic units (modified from, 23). 6 1.3 The Polymerase Chain Reaction and the Study of Marine Microbes Ever since PCR using a thermostable D N A polymerase (Taq) was first described (75, 96), the technique has become widely used in studies ranging from forensics to ecology (50). The widespread use of PCR stems from the fact that it allows the detection and exponential amplification of rare sequences from heterogeneous D N A mixtures. As an example, PCR has permitted the detection of as few as 100 cells against a background of 1011 diverse nontarget organisms (105). Similarly, other studies have demonstrated that PCR, when combined with the use of radiolabeled gene probes, was sensitive enough to detect gene sequences from as few as 1 to 5 bacteria cells in 100 ml environmental water samples (5). For the detection of viral D N A sequences, PCR has been proven to be equally effective. In a study of enteroviruses in water, Kopecka et al. (62) found that PCR was 10 to 103 times more sensitive than cell culture, and was 105 to 107 times more sensitive than direct probe hybridization. A widely utilized PCR-based method for aquatic studies has been the amplification of D N A sequences which encode small subunit rRNA in marine microorganisms. A long-standing dogma in marine microbiology is that most of the bacteria present in a given water sample are uncultivable. Therefore, estimates of population diversity based on cultivable bacteria greatly underestimate the actual diversity present in a sample (42). To circumvent this problem many aquatic microbiologists have used molecular techniques to estimate the diversity of microbes in water samples. For example, Giovannoni et al. (41) used PCR amplified small subunit rRNA sequences to estimate the genetic diversity of bacterioplankton in the Sargasso Sea. Similarly Fuhrman et al. (39) identified hovel archaebacteria using rDNA sequence analysis. Recently, researchers have continued to use these methods for the determination of marine bacterial genetic diversity (35, 114, 139). It is worth noting that molecular techniques have also been used to examine the diversity and distribution of many important primary producers such as the toxic dinoflagellate Alexandrium spp. and prokaryotic cyanobacteria (81, 99). It is apparent that in spite of any shortcomings, PCR has become, and will remain, an important tool for the examination of genetic diversity of organisms from environmental samples. As noted by Giovannoni & Cary (42) molecular techniques such as cloning may potentially involve biases that are not fully understood, however, it was concluded that these biases are much less encumbering than those imposed by cell culture methods. Others have also 7 noted potential problems with molecular techniques used to determine genetic diversity in natural samples. For example Reysenbach et al. (91) noted that PCR primers favored the amplification of rDNA sequences from Saccharomyces cerevisiae in mixtures of D N A from this yeast and an extremely thermophilic archaea species. However, these workers noted that the addition of a denaturant (5 % acetamide) eliminated this primer bias. Further, it has been noted that in some cases the formation of chimeric rDNA may lead to erroneous conclusions about the genetic diversity of certain samples (66). However, these workers concluded that detailed analysis of amplified sequences could prevent spurious results. As is the case for many experimental methods, critical analysis of results is necessary to prevent inaccurate conclusions. Furthermore, when compared to classical culture methods, determination of sample genetic diversity with PCR-based techniques allows the detection of organisms otherwise missed. Therefore, molecular techniques reveal a more accurate picture of species diversity in microbiological studies and will continue to be favored in coming years. However, as viruses do not contain ribosomal RNA sequences, it is necessary to use other conserved genes for phylogenetic inferences. 8 1.4 The Conserved nature of DNA polymerases D N A polymerases are the enzymes responsible for D N A replication and therefore, are essential to all organisms. The structure and function of D N A polymerase enzymes and the genes that code for them have been extensively studied for several decades (for review see 57, 127). A result of these studies has been the discovery, in many D N A polymerases, of regions of highly conserved amino acid sequence. One of the first conserved motifs noted in polymerases was a 14 amino-acid sequence that occurred in polymerases from 15 viruses that infect animals, plants and bacteria (59). The discovery of this conserved region (the amino acid sequence YGDTDS i.e. Asp-Asp motif) in RNA-dependent polymerases of viruses led the authors to suggest that the viruses may be derived from a common ancestor. As more polymerase amino acid sequences were determined, comparisons revealed other homologous segments among several other viruses; namely <p29, Adenoviruses, and Epstein-Barr virus (2). However, the similarity of one of the homologous segments to the Asp-Asp motif described for RNA-dependent polymerases was not noted at this time. It was not until the human DNA-dependent D N A polymerase sequence was determined that the similarities among these motifs were recognized. Furthermore, it was noted that the most highly conserved amino acid sequence was the Asp-Asp motif (137), which has been observed in D N A and RNA-dependent RNA and D N A polymerases of humans, yeast and viruses (e.g. 1, 10, 49, 53, 89). Argos (1) went further and predicted that this amino acid motif would form a strand-loop-strand structure essential to the function of the enzyme. The deduced amino acid sequence of human D N A polymerase a also shared six regions of homology with yeast DNA pol I, bacteriophages T4 and <b29, herpes viruses, vaccinia virus and adenovirus (137). It has also been noted by several researchers that these regions of homology are always in the same linear spatial arrangement (89, 137; 28; Figure 1.2). The discovery of these regions of homology in many polymerases eventually led to the formation of a family of polymerases with similar conserved sequences. Initially this group of polymerases was informally classified as a-like polymerases based on homology to the human D N A polymerase a. However, it was also noted that the a-like polymerases were homologous to E. coli D N A polymerase II (53). Subsequently, based on homology with E. coli D N A polymerase II, this 9 group of D N A dependent D N A polymerases has been classified as B-family D N A polymerases that include polymerases from viruses, bacteria, yeast and higher eukaryotes (52). Furthermore, the B-family D N A polymerases are subdivided into two groups depending on whether they are primed by protein or nucleic acids (10). DNA polymerase 3 kb > o o o X X X CD CD CD catalytic site VI III I V Exonuclease Domain (31- 5') 3 kb dNTP binding site ^1 ^ Polymerase Domain ^ Figure 1.2. Generic map of B-family DNA polymerases Conserved motifs are represented by black boxes. Possible functions are shown for the polymerase domain motifs I, II, III, and V. The most highly conserved amino acid sequence YGDTDS is located in motif I and is a proposed catalytic site for B-family polymerases. dNTP = deoxynucleoside triphosphate (modified from 20). 10 1.5 DNA polymerase sequences as genetic markers As a result of the essential and conserved nature of the a-like polymerases, these enzymes can potentially be used as markers for evolutionary relationships among organisms. It was postulated that because of the high degree of amino-acid sequence conservation, a-like polymerases from both prokaryotic and eukaryotic organisms share a common ancestor and differences are the result of divergent evolution (58, 59). Since this initial hypothesis was formulated, many other workers have used different polymerase gene and amino acid sequences to determine the phylogenetic relationships of these enzymes (e.g. 10, 51, 53). It is apparent that there is considerable utility in the use of polymerase molecular sequences for the determination of phylogenetic relationships of organisms, especially viruses that have genomes of limited complexity when compared to cellular organisms. Phylogenies based on polymerase genes, or any single copy genes, are not always incontrovertible. For example, Meyer et al. (71) noted that phylogenies based on polymerase genes differed slightly from those based on other essential genes. Some workers have also found differences in the homology of different polymerases from two organisms; 45 % and 32 % for Pol 5 and Pol a, respectively, when comparing sequences from Plasmodium falciparum and Saccharomyces cerevisiae (92). Information such as this has led to criticism of single-gene-based taxonomy. For example, Calisher et al. (15) noted several drawbacks to virus phylogenies based on polymerases. For example, they stated that polymerase-based taxonomic schemes are not comprehensive and viruses that do not encode a polymerase will be excluded. In addition, they noted that genes that occur in a wide range of organisms may have moved horizontally between species leading to erroneous taxonomy and recombination events in the evolutionary relationships of some viruses may lead to inaccurate taxonomic relationships. These authors feel that a data driven or "bottom up" strategy of classification, as outlined by the International Committee on Taxonomy of Viruses (ICTV), is the best approach. This "bottom up" strategy involves clustering entities into higher taxa when accumulated data demonstrates relationships between lower taxa (15). While the arguments of Calisher et al. (15) are noteworthy, aspects of them are, as the authors acknowledge, open to debate. As many different viruses lack distinguishing morphological characteristics, such as those that infect unicellular algae, taxonomic schemes 11 must be based on life histories or genetic information. Therefore, if virus and gene relationships are the result of horizontal gene transfer, or recombination between viruses coinfecting the same organism, this is biologically relevant information and is useful for taxonomic inferences. Indeed, coinfection of a single host suggests that two viruses are very similar, at least in host specificity and life history. Calisher et al. (15) state that it is not known with certainty if viruses are monophyletic or polyphyletic in origin and that any taxonomic scheme is contrived. If virus taxonomic schemes are in fact contrived, it is appropriate to base these schemes on biologically relevant information such as host ranges. While polymerase genes may not be appropriate for the determination of phylogenetic relationships of all viruses (i.e. DNA-dependent D N A polymerases are only useful for taxonomy of viruses with D N A genomes) this does not negate the information that can be gained from the study of these genes. When very little information is known for a specific group of viruses, genetic markers such as polymerase genes are a useful tool and sequence homology of these markers can be used to determine preliminary relationships. The initial evidence for Phycodnaviridae-coded a-like D N A polymerases was from a study of viruses that infect a unicellular, exsymbiotic, eukaryotic Chlorella-\ike green alga, strain NC64A (94). This study demonstrated that it was possible to distinguish three different D N A polymerase activities in virus infected Chlorella strain NC64A. The authors also demonstrated that the activities of these enzymes differed between infected and uninfected cells suggesting that the enzymes in virus-infected cells could be host polymerases modified by virus-encoded protein(s), or entirely virus encoded. Further study on the D N A polymerase of two viruses (PBCV-1 and NY-2A) that infect this alga demonstrated that the polymerase gene sequences of these viruses were more homologous to each other than to polymerase sequences of other organisms (43). Interestingly, different regions of the gene had highest homology to different viruses. For example, the amino-acid sequence of the 3'—>5' exonuclease domain had highest identity (23 % identical amino acids) with phages PRD1 and <))29 and herpes simplex virus, while the polymerase domain had highest identity (43 % identical amino acids) with two herpes viruses, HSV and EBV. Nonetheless, these studies of viruses infecting a unicellular algae provided the first evidence that some algal viruses encode their own D N A polymerase and this polymerase is closely related to members of the B-family (a-like) D N A polymerases. 12 1.6 PCR amplification of algal virus pol Following characterization of D N A polymerase genes of viruses infecting a Chlorella-like alga, a D N A polymerase gene from a virus (MpV-SPl) that infects the unicellular alga Micromonas pusilla was sequenced (C. A. Suttle, Pers. Comm.). Analysis of sequences from PBCV-1, NY-2A and MpV-SPl revealed that the DNA polymerase genes of these viruses contain regions both universally conserved in B-family D N A polymerase genes and conserved only among these viruses. This information was used to design, from inferred amino acid sequences, three highly degenerate primers that can be used for amplification of algal virus pol sequences by nested PCR. Two of the primers are complementary to conserved regions of algal virus polymerase genes, and one is complementary to a universally conserved sequence (20, 21). The nested PCR involves two separate PCR reactions using three primers that are complementary to the three regions of the polymerase gene (20, Figure 1.3). The first round of PCR is carried out with the two primers specific to the algal virus polymerase sequence. A second round of PCR is carried out using one of the primers from the first reaction and a third nested pol gene specific primer. This round of PCR is used to confirm the identity of any amplified products from the first reaction (20-22). Following amplification, products are cloned into plasmid vectors and the resulting library can be screened by restriction fragment length polymorphism (RFLP). Unique clones identified in this way can be sequenced, and phylogenetic relationships determined. It was demonstrated that this method allowed the successful amplification of algal virus polymerase sequences from several cultured algal viruses and viruses from natural samples (20, 23, Figure 1.1). Analysis of amino acid sequences deduced from the polymerase genes of algal viruses and 16 other D N A viruses showed that algal viruses clustered as a distinct group (22, Figure 1.4). As well, when compared to data from total viral genome hybridization (27), polymerase sequence analysis produced similar phylogenetic trees thereby demonstrating that D N A pol sequences reflect the whole genome of MpV (22). Analysis of the virus-/w/-based phylogeny indicated that D N A polymerase sequences of the Phycodnaviridae are more closely related to each other than to other dsDNA virus polymerases, suggesting that they share a common ancestor (22). Furthermore, the viruses fall into three clades, which correlate with their hosts. For example, the sequence from the Chlorella virus PBCV-1 is 77 % similar to those from two other 13 Chlorella viruses, NY-2A and CVA-1, while CVA-1 and NY-2A are 64 % similar. Among viruses that infect the members of the Prasinophyceae, including MpV-SPl , the proportion of identical nucleotides for all pairwise combinations ranged from 78 to 99 %. The two viruses that infect members of the Prymnesiophyceae, CbV-PWl and CbV-PW3 shared a high sequence similarity (98 %). Overall, sequence similarities among the D N A polymerase gene fragments of three Chlorella viruses and all Prasinovirus and Prymnesiovirus clones ranged from 49 to 99 %. Furthermore, the Phycodnaviridae clones shared higher sequence similarity to each other than to the most closely related viruses, the Herpesviridae (22). DNA polymerase gene Okb 3kb PCR segment Obp 483 bp 681 bp A V S 1 - ^ Upstream primer (23 mer) <- POL « - A V S 2 Nested primer Downstream primer (17 mer) (23 mer) Figure 1.3. Relative positions of PCR primers on the DNA polymerase gene The upstream and downstream primers (AVS1 and AVS2) are specific for algal viruses, and the nested primer (POL) is universal for B-family DNA polymerases. The amplified segment shown in white is contained within the polymerase domain of the DNA polymerase gene (modified from 20). 14 1 0 0 , , - A C 1 0 0 r 1 0 0 5 0 9 9 1 0 0 f 9 8 f 1 0 0 9 9 | C 5 8 jooT" ioor • B m N P V • H z N P V - L d N P V — MpV-SP1 MpV-PB8 MpV-PL1 CbV-PW1 CbV-PW3 -CVA-1 - PBCV-1 • NY-2A M C M V 1 0 0 — H C M V • G P C M V - E B V HSV - 2 P r V - V Z V — A S F V V a c V • F P V - C b V 0.1 Figure 1.4. Phylogenetic tree of 100 bootstrap analyses of DNA polymerase fragments The neighbor-joining tree was constructed by using 217 of 335 amino acid sites. Virus clones infecting microalgae are indicated by boldface type. The numbers at the nodes indicate bootstrap values, and branches with values less than 75 have been collapsed. The scale bar represents 0.1 fixed mutations per amino acid position. Abbreviations are as follows: AcNPV = Autographa californica nuclear polyhedrosis virus; BmNPV = Bombyx mori nuclear polyhedrosis virus; HzNPV = Helicoverpa zea nuclear polyhedrosis virus; LdNPV = Lymantria dispar nuclear polyhedrosis virus; MpV = Micromonas pusilla virus; CbV = Chrysochormulina brevefilum virus; CVA-1, PBCV-1, and NY-2A = viruses infecting Clorella -like algae; MCMV = Murine cytomegalovirus; HCMV = Human cytomegalovirus; GPCMV = Guinea pig cytomegalovirus; EBV = Epstein-Barr virus; HSV-1 = Herpes simplex virus type 1; HSV-2 = Herpes simplex virus type 2; PrV = Pseudorabies virus; VZV = Varicella-Zoster virus; ASFV = African swine fever virus; VacV = Vaccinia virus; FPV = Fowlpox virus; CbV = Choristoneura biennis poxvirus (modified from 22). 15 1.7 DNA polymerase genes in the study of marine virus communities Many virus genomes contain D N A polymerase genes. For example, marine phages in the family Styloviridae and Myoviridae both encode their own polymerases. Therefore, the approach taken to examine algal virus communities is applicable to other natural virus populations including bacteriophages and cyanophages. The following section will briefly outline the approach to design AVS PCR primers and will summarize the results of specific field studies. The first algal-virus pol genes sequenced were from the viruses NY-2A and PBCV-1. Details of the methods used to obtain these sequences have been previously described (43). This information was combined with information from the D N A polymerase sequence of MpV-SPl to design AVS PCR primers. Obtaining sequence information from MpV-SPl first involved isolating and amplifying cultivable viruses; details of this, as well as purification of MpV genomic. D N A have been previously described (25, 27). Once purified genomic MpV D N A was obtained, restriction digests of the DNA were used in Southern blot hybridizations with a radiolabeled probe according to the protocol of Sambrook et al. (97). The 5' end-labeled probe used to locate the polymerase gene was a degenerate oligonucleotide based on the amino acid sequence YGDTDS (universally conserved among B-family D N A polymerases). Subsequent digestions and subcloning permitted sequencing the entire DNA polymerase gene. Once the sequence was obtained, inferred amino acid sequences were aligned with other D N A pol sequences available in GenBank. Sequences unique to the algal viruses PBCV-1, NY-2A, and MPV-SP1 were used to design two degenerate PCR primers to amplify D N A from these closely related viruses. Furthermore, a degenerate oligonucleotide derived from the amino acid sequence YGDTDS was used as a third primer for nested PCR (21, Figure 1.5). This third primer is of particular importance as it allows confirmation that the desired target sequence, in this case D N A pol, was amplified in the first round of PCR. The motivation for this work was to design PCR primers suitable for the amplification of D N A from eukaryotic phytoplankton (20). However, it should be possible to use the same methods to develop PCR primers specific to other groups of related viruses. 16 Isolat ion of v i rus par t ic les h o Q D N A ext ract ion Micromonas pusilla ce l l l ysa te Res t r i c t i on D iges t of ex t rac ted D N A ^ GATCGGTAC. S o u t h e r n blot hybr id izat ion with un i ve rsa l pol p robe S e q u e n c e c l o n e d f r a g m e n t s I S e q u e n c e a l i g n m e n t a n d ident i f icat ion of P C R p r imers C l o n e ge l -pur i f ied f r a g m e n t into p l a s m i d S u b c l o n e f ragment for s e q u e n c i n g NY-2A PBCV-1 MpV-SP1 HSV-1 I AVS-1 POL AVS-2 I s I I AVS-1 POL AVS-2 I I I I AVS-1 POL AVS-2 I I POL I I Figure 1.5. Flowchart of PCR primer design Chart shows the order of steps taken to clone and sequence the polymerase gene from the Micromonas pusilla virus MpV-SPl. Following sequencing, the pol gene of MpV-SPl was aligned with other known sequences including two Chlorella-like alga viruses (NY -2A and PBCV-1) and Herpes simplex virus type 1 (HSV-1). The primers AVS-1 and AVS -2 are conserved only among algal viruses while the primer Pol is conserved among all B-family DNA polymerases. Use of this PCR method resulted in the amplification of pol sequences from 13 clonal isolates of different viruses. The viruses with amplifiable sequences included 2 viruses that infect an endosymbiotic Chlorella-like alga, 9 viruses that infect M. pusilla, and 2 viruses that infect C. brevifilum. Furthermore, pol sequences were amplified from virus communities concentrated from the Gulf of Mexico, indicating that the PCR method was suitable for the study of natural virus communities (20). Subsequently, PCR with the AVS primers was used to examine the genetic diversity of a concentrated natural virus community collected in the Gulf of Mexico. It was hypothesized that PCR with AVS primers would result in the production, from a single 17 natural virus sample, of several different DNA products of approximately the same size (23). Therefore, amplified pol fragments from a single PCR reaction were cloned to facilitate separation of different sequences. RFLP analysis of cloned fragments revealed that five different sequences were amplified from a single natural virus concentrate. This demonstrated that algal virus polymerase genes, closely related to those of cultured virus strains, could be amplified and sequenced from a natural marine virus sample. Moreover, a single unknown algal virus pol fragment was recovered which did not cluster with any sequences of known marine algal viruses (23). These results indicate that the PCR-based method is useful for examining the genetic diversity of naturally occurring marine viruses. While the primers used in these studies were specific to algal-viruses, it should be possible to use similar methods to study other natural virus communities. As previously mentioned, many marine cyanophages and bacteriophages may encode D N A polymerases and it should be possible to design primers specific for these groups. These molecular techniques allow examination of virus community diversity without the need to isolate or culture specific members of these communities. 18 1.8 DGGE as a tool for comparing virus communities In addition to direct-sequence or RFLP analysis of pol gene fragments, D G G E can be used to examine natural virus communities. This method allows the resolution of D N A fragments of the same size that differ in as little as a single base pair. D G G E has been used to analyze the diversity of complex microbial communities ranging from biofilms in wastewater treatment reactors to hot-spring microbial mats (33, 78, 98). We have begun to use this method to examine the diversity of algal viruses in natural samples. D G G E makes use of the fact that denaturation (separation) of double-stranded (ds) D N A depends on temperature, concentration of denaturant (e.g. urea and/or formamide), and interactions of adjacent base pairs stacked in a helix (79). Therefore, lengths of dsDNA separate under different conditions according to their base composition. Furthermore, as a result of the formation of D N A strands with three ends, partial denaturation inhibits electrophoretic mobility. Resolution of dsDNA samples of the same length, but differing in sequence, occurs by running the D N A in a polyacrylamide gel that has a continuously increasing concentration of denaturant. As each strand that differs in sequence partially denatures at a different concentration of denaturant, migration halts at a different position in the gel. Following gel separation of heterogeneous PCR products, bands can be excised from the gel and sequenced, and phylogenetic relations can be determined. A major advantage of D G G E is that it eliminates cloning PCR products, and offers a rapid means of detecting predominant populations. As the products are separated by electrophoresis, it also eliminates the laborious screening of redundant clones in a library (33). Furthermore, as D G G E is a highly sensitive method for detecting sequence differences, the possibility of mistaking chimeric sequences produced during PCR amplification for sample genetic diversity can be eliminated (78). As a preliminary investigation of the utility of D G G E for the analysis of virus polymerase fragments, several PCR products were analyzed using this technique. Samples of clonal viruses or natural virus communities were amplified using only the outer primer pair (the AVS PCR primers AVS-1 and AVS-2) according to previously established protocols (23). Briefly, colony PCR was performed with stabs from E. coli colonies containing cloned pol fragments from 19 Micromonas pusilla viruses MpV-SPl , MpV-PB5, Chlorella-Xiks, algal viruses PBCV-1 and CVA-1, and Chrysochromulina spp. virus CbV-PW3. Natural communities of viruses that had been concentrated from the Gulf of Mexico (GOM-A1) and British Columbia, Canada, coastal waters (BC-268, BC-271, BC-277, BC-288, BC-308) using ultrafiltration were pelleted by ultracentrifugation and subjected to hot/cold treatments. Samples BC-268, BC-271, and 308 were collected from surface waters at the Jericho Beach pier in the Burrard Inlet while samples BC-277 and BC-288 were collected in the Strait of Georgia. While the amplified products from natural samples are not confirmed algal virus DNA polymerases, negative control experiments have been conducted using D N A templates from a wide range of sources. These have included phage DNA, bacterial DNA, and host algae D N A itself (data not shown). Agarose gel electrophoresis of PCR products was performed by loading 5 pi of clonal samples and 10 pi of natural samples into a gel composed of 1.5 % SeaKem® L E agarose (FMC BioProducts, Rockland, ME) in 0.5 X TBE (45 mM Tris-borate, 1 mM EDTA, pH 8.0). The gel was run at 90 v for 1.5 hours. After electrophoresis, the gel was stained in a 50 pg/ml ethidium bromide solution and examined on a U V transilluminator. Examination of the gel revealed the expected single bands from all clonal viruses, a single 700 bp band from the Gulf of Mexico sample, and two predominant bands of 700 and 550 bp in all samples from British Columbia coastal waters (Figure 1.6a). It is worth noting that the amplicon from the virus PBCV-1 is larger than the other amplicons as the D N A polymerase of this virus contains a 100 bp intron (43). Further analysis of these PCR products was conducted using a 0 to 60 % (100 % denaturant is defined as 7 M urea and 40 % deionized formamide) linear denaturing gradient 6 % polyacrylamide gel, cast with 1.0 X T A E (40 mM Tris-acetate, 2 mM EDTA, pH 8.5). For the clonal virus samples, 10 pi was loaded into wells while 20 pi was loaded for the natural samples. Electrophoresis was carried out for 6 hours at 135 volts using the D-code™ electrophoresis system (Bio-Rad Laboratories, Hercules, CA). The gel was stained in a SyBr® Green I (Molecular Probes, Eugene, OR) solution overnight and was examined on a U V transilluminator. Algal virus PCR products of the same size could be resolved using DGGE. For example, PCR products of cloned virus samples MpV-SPl , MpV-PB5, and CVA-1 cannot be resolved in the agarose gel, yet are clearly separated in the gradient gel (Figure 1.6). Similarly, PCR products from natural samples cannot be resolved by agarose gel electrophoresis, yet several 20 different banding patterns were revealed using D G G E (Figure 1.6b). The bands observed in the sample from the Gulf of Mexico are not present in any of the other samples implying that virus communities in the Gulf of Mexico are genetically different than viral communities of coastal British Columbia. Interestingly, samples BC-268 and BC-271 appear very similar, with the same dominant band. These two samples were collected from the same location but 10 weeks apart, implying that during the time of sample collection (October to January) the genetic composition of algal viruses was relatively stable. However, the other sample from the same location (BC-308) collected in July has a different dominant band demonstrating that the genetic composition of algal virus communities can differ seasonally. Furthermore, samples BC-277 and BC-288 from the Strait of Georgia have very different banding patterns implying that virus communities differ over relatively small spatial scales. The results from this preliminary study using D G G E imply that the genetic structure of algal virus communities differs with time and geographic location and that D G G E is useful for investigating the genetic composition of Phycodnaviridae communities. Recent modifications of the technique have increased its sensitivity and range of application, and will allow further optimization for application of this technique for examination of marine virus communities. For example, it has been demonstrated that attachment of a 40-base-pair G + C rich sequence to one end of the D N A fragments prevents complete denaturation of short amplicons and allows better resolution of very similar sequences. Attachment of this 'GC-clamp' to D N A fragments is easily achieved by incorporation into one of the primers used in PCR. All amplified products from the reaction will have this short sequence incorporated at one end (100, 101). This modification of D G G E has become standard in many applications of this method. Incorporation of a "GC clamp" has been used to examine rDNA sequence diversity in many diverse microbial communities (33, 48, 78, 81, 95). It is worth mentioning that amplified rDNA fragments commonly range in size from 100 to 400 bp necessitating the use of primers with an incorporated G C clamp. Because the amplicons analyzed in this study were all quite long (> 550 bp), incorporation of a GC clamp was not necessary. Even after 6 hours of electrophoresis the amplified fragments remained in the polyacrylamide gel and did not run off. While the majority of studies employing D G G E have been examinations of rDNA sequence diversity, this method is applicable to any PCR-based study of genetic diversity. 21 D G G E is a powerful method for studying diversity in natural virus communities, and will allow rapid examination of viral diversity in different environments. This will permit researchers to obtain "fingerprints" of natural virus communities. Comparisons of fingerprints obtained from different environments will lead to the formulation and testing of hypotheses on the relationship between virus diversity and other environmental parameters. Although much work is necessary before this method will become an optimized tool for studying of natural virus communities, we hope to further demonstrate its usefulness for examining diversity in algal virus communities in the near future. f 4? •» § g < < on o _ O u o ? r < > _- m -t u Zl CO CD CO CD CD TO _ o o o o o _ _ M fO M r o C J —• • ? O ) S N Q3 O < < CO —. S O ) CD TO TO s i z e (bp) 2,072 1,500 100 T J T3 u 55 Q i j o < -o TJ CO f > S-- . m - . - _ . _ j CO CD CO CO CD S "_ O o o o o _ 8 rb io IO ro co — dj Figure 1.6. Gel electrophoresis of amplified algal virus pol fragments I A.) 1.5 % agarose gel of PCR amplified DNA using the algal-virus-specific primers AVS-1 and AVS-2. The marker is a 100 bp ladder (Life Technologies). B.) 6 % polyacrylamide gel with 0 - 60 % linear denaturing gradient loaded with PCR amplified DNA using the AVS primers. Abbreviations are as follows: MpV-SPl and MpV-PB5 = Micromonas pusilla virus strains SP1 and PB5; PBCV-1 and CVA-1 = Chlorella-like algae viruses; GOM-A1 = Gulf of Mexico sample A l ; BC-268, BC-271, BC-277, BC-288, BC-308 = British Columbia, Canada, coastal water samples. See text for further detail. 22 1.9 Conclusion Viruses are currently recognized as numerically and ecologically significant members of aquatic ecosystems (see reviews by: 13, 40, 109, 117). They are also regulators of carbon flow in the marine environment and are probably important controlling agents of bacterial and algal community composition. Currently, very little information on the diversity of natural virus communities, or the range of phytoplankton infected by viruses is available. Molecular techniques such as PCR and D G G E should provide a means for rapid, efficient determination of viral genetic diversity in environmental samples. Data on diversity, when combined with sequence information from cultured viruses, will permit further estimation of the range of phytoplankton species infected by viruses. Furthermore, these molecular techniques permit an alternate means to examine host genomes for latent viruses. Such information is essential to understanding the impact of marine viruses on global primary production and carbon flow. 23 Chapter II. Denaturing Gradient Gel Electrophoresis Resolves Virus Sequences Amplified with Degenerate Primers Published as: Short, S. M. and C. A. Suttle. 2000. Denaturing gradient gel electrophoresis resolves virus sequences amplified with degenerate primers. BioTechniques 28: 20-26. 24 2.1 Summary PCR and D G G E were used to resolve similar sized products amplified from related but relatively dissimilar virus templates. Without using a 'GC-clamp' primer, D G G E was optimized to resolved long (i.e. > 400 bp) PCR products. D N A polymerase gene fragments were amplified from three cultured viruses that infect microalgae and a naturally occurring virus community by PCR with degenerate algal-virus-specific primers. Amplification from all samples resulted in PCR products approximately 700 bp in length. The amplified fragments from cultured viruses focused at different locations in a denaturing gradient gel and several bands were resolved in a denaturing gradient gel loaded with the natural sample. Therefore, it was concluded that incorporation of a 'GC clamp' was unnecessary for the resolution of different sequences amplified from cultured algal viruses or natural algal virus communities. These results provide further evidence that PCR and D G G E can be used to investigate the genetic diversity of algal virus communities in nature. 25 2.2 Introduction D G G E is a versatile technique for studying genetic variation in populations. The technique is based on the separation (melting) of double-stranded (ds) D N A as a function of temperature and concentration of denaturant (i.e. urea and/or formamide) (79). As a result of the formation of molecules with complex structure, partial separation of strands inhibits the electrophoretic mobility of dsDNA. Resolution of dsDNA fragments of the same length that differ in sequence occurs by running the DNA in a polyacrylamide gel that has a continuously increasing concentration of denaturant. Because dsDNA molecules that differ in sequence partially melt at different concentrations of denaturant, rates of migration are inhibited at different positions in a denaturing gradient gel. Furthermore, attachment of a G + C rich sequence (i.e. G C clamp) to one end of the dsDNA fragments that are to be separated increases the sensitivity of DGGE. The G + C rich region creates a high melting temperature domain that allows detection of single base pair substitutions in lower melting domains (101). Attachment of this "GC-clamp" to D N A fragments is easily achieved by incorporation of a GC-rich region into one of the primers used PCR (100, 101). This modification has become standard in most applications of DGGE. Commonly, PCR with non-degenerate primers with a GC-clamp is used to amplify short D N A fragments ranging from 150 to 400 base pairs in length (e.g. 33, 48, 77, 138). While use of a GC-clamp can greatly enhance the sensitivity of DGGE, and allow the detection of single base pair changes, it may not always be advantageous or necessary. In this report we demonstrate that D G G E can be used to resolve longer PCR products without the use of a GC-clamp primer. In addition, PCR with degenerate primers and D G G E with a broad gradient resolved similar sized products amplified from related but relatively dissimilar virus templates. The discovery that viruses are abundant and dynamic components of marine ecosystems, as well as important players in the mortality of microorganisms (40, 109) and in geochemical cycles (e.g. 131) has led to a need to characterize natural virus communities. Recent methodological developments have allowed us to use PCR to examine some groups of viruses that infect unicellular algae. Specifically, the design of algal-virus-specific primers (AVS-1 and AVS-2) that amplify a highly conserved DNA polymerase gene fragment has facilitated examination of natural virus community diversity without the need for culture (19, 21, 23, 102). 26 Initial evidence of the diversity of algal viruses was revealed when five different sequences were obtained from the PCR products of a single environmental sample. The heterogeneity of algal virus sequences amplified in a single PCR reaction was resolved by cloning, restriction fragment length polymorphism (RFLP) analysis, and sequencing (23). While this technique is effective, it is relatively time consuming and cumbersome. As a result we sought alternate methods that would allow direct examination of PCR products amplified from seawater samples to genetically "fingerprint" subsets of natural virus communities. Recently, we modified existing D G G E methods to facilitate rapid analysis of PCR products amplified from seawater samples to follow changes in the genetic diversity of virus communities (102). Here we demonstrate that a mixture of PCR products of the same length that have been amplified using degenerate primers can be clearly resolved by D G G E without using a G C clamp. 27 2.3 Materials and Methods D N A polymerase gene fragments were amplified from three cultured viruses that infect microalgae, and a naturally occurring virus community. Template D N A from the viruses MpV-SP1 and MpV-PJ38, which infect the marine phytoplankton Micromonas pusilla, were prepared as previously outlined (27). Template D N A from the virus CVA-1, which infects the freshwater alga Chlorella sp. (123), was provided by Y. P Zhang and J. L. Van Etten. The target sequences from MpV-SPl and MpV-PB8 share 81 % identity with each other, and 55 % identity with the sequence from CVA-1 (19). The natural virus sample was obtained from Jericho Pier, Vancouver, B.C., Canada, and template DNA was also prepared as previously outlined (23). Target sequences from all templates were amplified using the degenerate (8192-fold and 4096-fold respectively) 23-mer oligonucleotide primers AVS-1 and AVS-2 that are specific for the Phycodnaviridae (21). For reactions with cultured and naturally occurring virus templates, approximately 100 ng of viral D N A was added to 50 pi of a PCR mixture containing Taq D N A polymerase assay buffer (50 mM KC1, 20 mM Tris-HCl, pH 8.4), 1.5 mM MgCl 2 , 0.16 mM of each dNTP, 60 pmol of each primer, and 0.625 U of Taq D N A polymerase (Life Technologies™, Gaithersburg, MD). Two drops of mineral oil were added to each tube. PCR was also performed on a combined template sample containing all the same reagents by adding approximately 50 ng of each cultured virus D N A to the reaction mixture. Negative controls contained all reagents except template. PCR was carried out using a model PTC-150 MiniCycler™ (MJ Research, Watertown, MA) with the following cycle parameters: denaturation at 95°C for 2 min, 30 s, annealing at 50°C for 45 s, and extension at 72°C for 1 min; 29 cycles of denaturation at 95°C for 30 s, annealing at 50°C for 45 s, and extension at 72°C for 1 min; and a final extension at 72°C for 5 min. Following 30 rounds of amplification, 8 pi of the PCR products were electrophoresed in 1.5 % L E agarose (FMC BioProducts, Rockland, ME) in 0.5 X TBE buffer (0.045 M Tris-borate, 1 mM EDTA, pH 8.) at 90 volts for 1 h 20 min. For molecular weight standards, 0.25 pi of 100-bp ladder (Life Technologies) were loaded into a separate lane. The gels were stained with ethidium bromide and examined on a U V transilluminator. 28 D G G E of PCR products was conducted using 0 to 60 % linear denaturing gradient 6 % polyacrylamide gels. Six pi of individual products were loaded into wells, while 18 pi of the combined products were loaded. Electrophoresis of individual and combined products was carried out for 4, 6, and 8 h in a denaturing gradient gel. In another gel, 30 pi of natural products was electrophoresed for 7 h. Both gels were run in 1.0 X T A E (40 mM Tris-base, 20 mM sodium acetate, 1 mM EDTA, pH 8.5) at 135 volts at a constant temperature of 58 °C using the D-Code™ electrophoresis system (Bio-Rad laboratories, Hercules, CA). Gels were stained in a 1.0 X SYBR® Green I (Molecular Probes, Eugene, OR) solution overnight and examined on a U V transilluminator. 29 2.4 Results and Discussion Figure 2.1 A shows that PCR amplification resulted in the production of bands approximately 700 bp in length. The expected size of the PCR products was 692, 683, and 683 bp for the templates CVA-1, MpV-PB8, and MpV-SPl respectively. PCR products in the combined reaction were all resolved by DGGE. Bands corresponding to those in lanes loaded with PCR products from single template reactions can be observed in the lane loaded with PCR products from the combined template reaction (Figure 2. IB). Several distinct bands can be observed in the gel loaded with PCR products from the natural sample (Figure 2.IB). From these results, it is apparent that a broad gradient (0 % to 60 %) of denaturant was adequate for the resolution of these products. After 6 h, the PCR products from cultured viruses had focused in the gel, and longer electrophoretic duration did not result in any further migration of any bands. All of the amplified fragments from cultured viruses focused at different locations in the gradient gel and several bands were resolved in the gel with the natural sample. Therefore, it can be concluded that incorporation of a G C clamp was unnecessary to resolve sequence differences in gene fragments amplified from cultured algal viruses or a natural virus community. As previously shown, amplification of algal virus sequences with the primers AVS-1 and AVS-2 results in the production of approximately 690 base-pair products. It appears that products of this length focus in a denaturing gradient gel without the need of a G C clamp. It could be postulated that as these D N A molecules migrate towards increasing concentrations of denaturant, partial melting of different domains results in D N A conformations that do not migrate in the gel. Once migration of the bands halts, no further denaturation can occur, therefore, the dsDNA does not completely denature. Consequently, no single stranded DNA is produced and the complex structure of the dsDNA allows the bands to focus at specific locations in the gel. 30 Figure 2.1. Gel electrophoresis of amplified algal virus pol fragments II (A) Agarose gel electrophoresis of algal virus samples. Lanes correspond to PCR amplification products from 1) combination of MpV-SPl, MpV-PB8, and CVA-1 templates, 2) MpV-SPl, 3) MpV-PB8, 4) CVA-1, 5) negative control, and 6) Jericho Pier community (negative control not shown). Sizes, in base pairs, are shown to the left of the appropriate bands of the molecular weight marker. (B) DGGE of the same PCR products. Lanes are labeled as above and the duration of electrophoresis is shown above the appropriate lanes. 31 D G G E has become a widely used technique as a result of its flexibility and adaptability for many applications. For most applications, electrophoretic conditions must be determined empirically. While this makes the technique slightly less desirable for new applications, it does imply that this technique lends itself to a certain degree of fine-tuning. As demonstrated by our use of D G G E to resolve differences in algal virus polymerase sequences, the technique is applicable to virtually any study of sequence heterogeneity in related PCR products. PCR combined with D G G E can be used to assess spatial and temporal dynamics in the genetic diversity of algal virus populations amplified with the primers AVS-1 and AVS-2. These results demonstrate that D G G E can be used to rapidly assess the genetic diversity of algal virus communities in nature. 32 Chapter III. Sequence Analysis of Marine Virus Communities Reveals that Groups of Related Algal Viruses are Widely Distributed in Nature Published as: Short, S. M. and C. A. Suttle. 2002. Sequence analysis of marine virus communities reveals that groups of related algal viruses are widely distributed in nature. Applied and Environmental Microbiology 68: 1290-1296 33 3.1 Summary Algal-virus-specific PCR primers were used to amplify D N A polymerase (pol) gene fragments from geographically isolated natural virus communities. Natural algal virus communities were obtained from coastal sites in the Pacific Ocean off British Columbia, Canada and the Southern Ocean near the Antarctic Peninsula. Genetic fingerprints of algal virus communities were generated using DGGE. Sequencing efforts recovered 33 sequences from the gradient gel. Of the 33 sequences examined, 25 encoded a conserved amino-acid motif indicating that the sequences were pol genes fragments. Furthermore, the 25 polymerase sequences were related to pol gene fragments from known algal viruses. In addition, similar virus sequences (> 98 % sequence identity) were recovered from British Columbia and Antarctica. Results from this study demonstrate that D G G E with degenerate primers can be used to qualitatively fingerprint and assess genetic diversity in specific subsets of natural virus communities and that closely related viruses occur in distant geographic locations. D G G E is a powerful tool for genetically fingerprinting natural virus communities and may be used to examine how specific components of virus communities respond to experimental manipulations. 34 3.2 Introduction Renewed interest in oceanic microbial processes stimulated marine virus research during the 1990's. The results of early investigations demonstrated that viruses that infect and lyse marine primary producers are abundant, active components of marine ecosystems (6, 12, 45, 90, 113). Subsequently, it was shown that viruses infecting the marine eukaryotic phytoplankton Micromonas pusilla were widespread, genetically diverse, dynamic, and caused significant mortality in M. pusilla populations (25, 26). These findings inspired the development of the degenerate algal-virus-specific PCR primers AVS1 and AVS2, which amplify a 700 bp fragment of algal virus D N A polymerase (pol) genes (20). To permit unambiguous identification of PCR products amplified with the A V S primers, they were designed to span a short sequence encoding a highly conserved amino acid motif (YGDTDS) found in D N A polymerases (52). Phylogenetic analysis of amplified pol fragments revealed that cultured algal viruses formed a monophyletic group when compared to dsDNA viruses belonging to several families (22). Recently, known algal viruses were assigned the virus family name Phycodnaviridae. Subsequently, the AVS primers were used to amplify unknown algal virus pol fragments from natural virus communities, demonstrating that molecular techniques can be used to study algal virus diversity (23). Denaturing gradient gel electrophoresis (DGGE) is widely used to examine the diversity of PCR products. The technique separates D N A fragments based on sequence rather than length (36, 37), and has been widely used to examine the diversity of gene sequences in complex microbial communities (e.g. 4, 33, 78, 133). Studies have indicated that D G G E analysis of PCR products amplified with AVS-1 and AVS-2 can be used to resolve genetically distinct algal viruses in artificial mixtures, and to examine the diversity of natural algal virus communities (102, 103). In general, it is hypothesized that viruses exert significant control on microbial communities and likely influence host community composition and succession (38, 110, 134). For example, Peduzzi and Weinbauer (87) demonstrated that virus enrichment influenced microbial community dynamics and succession, while Hennes et al. (47) used fluorescently labeled viruses to demonstrate that viruses could control the abundance of susceptible hosts. In 35 addition, recent experiments with Pseudoalteromonas sp. revealed that the growth of virus-resistant cells was correlated to the lysis of sensitive cells (72). Furthermore, the virus-bacteria population dynamics model analyzed by Thingstad predicts that viruses ensure the coexistence of competing bacteria by infecting only the most abundant hosts or "killing the winner" (116). Similarly, the model of virioplankton control of community diversity proposed by Wommack and Colwell (136) predicts that virus community composition should be dynamic and that dominant viruses should be ephemeral. Examination of natural virus communities using pulsed field gel electrophoresis (PFGE) supported these hypotheses. However, it should be noted that PFGE permits determination of genome sizes but not sequence identities of individual viruses. While it seems apparent that viruses influence host community composition, the effect of viruses on phytoplankton communities remains, for the most part, unexamined. A long-term goal of our research is to understand the dynamics and effects of marine phytoplankton viruses. A critical part of this understanding requires examining the genetic composition of natural virus communities. Thus, the purpose of the current study was to use PCR and D G G E to recover and compare unknown algal virus sequences from the natural environment. 36 3.3 Materials and Methods 3.3.1 Sample collection and preparation Natural virus communities were concentrated from several coastal stations in British Columbia, Canada, during the summer of 1999. A coastal Southern Ocean sample was collected near the Antarctic Peninsula from the RV Lawrence M . Gould in 1998. Maps of the sample locations are shown in Figure 3.1. The details of stations sampled and the labels assigned to each sample are listed in Table 3.1. The Southern Ocean and Barkley Sound samples S098 and BSB99 were collected using a submersible pump, the Barkley Sound sample BSA99 was collected with a bucket, and all others were collected using GO-FLO bottles (General Oceanics, Miami, FL) mounted on a rosette. The virus-size-fraction was concentrated using previously established methods (23). Briefly, samples were pressure filtered (< 17 kPa) through 142 mm glass fiber (GC50; nominal pore size, 1.2 pm, Advantec MFS, Dublin, CA) and PVDF filters (GVWP; pore size, 0.45 pm, Millipore, Bedford, MA) connected in series. Remaining particulate material in the filtrates was concentrated using 30 kD cutoff ultrafiltration; Amicon S1Y30 or S10Y30 (Millipore, Bedford, MA) cartridges were used according to manufacturer's recommendations. After processing, concentrated virus samples were stored in the dark at 4 °C until use. 126°W 1 2 4 - W m ° W 8 0 " W 60 W 30°W Figure 3.1. Map of sample sites A) The positions of samples collected from coastal British Columbia, Canada are shown. B) The location of the sample collected in the Southern Ocean near the Antarctic peninsula is shown. Station abbreviations are listed in Table 3.1. 37 Table 3.1. Sample details Sample Date collected Location Latitude Longitude Depth ( m ) Salinity (%,) Temp ( ° C ) Lane no. in gels S098 12/08/1998 Southern Ocean 62° 36' S 58° 15'W 0.5 33.9 -1.0 1 BSA99 06/25/1999 Barkley Sound 4 8 ° 5 1 ' N 125°07 'W 0 25.0 N A 2 BSB99 07/12/1999 Barkley Sound 48° 54' N 125° 01 'W 8.0 35.0 N A 3 MIA99 08/19/1999 Malaspina Inlet 50° 02' N 124° 4 4 ' W 4.0 21.7 18.8 4 HS99 08/22/1999 Howe Sound 49° 27' N 123° 17'W 6.0 17.8 15.0 5 SIA99 08/18/1999 Salmon Inlet 49° 36' N 123° 48' W 15.0 23.4 13.5 6 SIB99 08/18/1999 Salmon Inlet 49° 36' N 123° 48' W 4.0 22.6 15.0 7 MIB99 08/20/1999 Malaspina Inlet 49° 59' N 124°41 'W 22.0 25.4 13.7 8 MIC99 08/20/1999 Malaspina Inlet 49° 59' N 124° 41 'W 5.0 22.2 18.3 9 PSA99 08/21/1999 Pendrell Sound 50° 18 'N 124° 44" W 25.0 28.4 10.1 10 PSB99 08/21/1999 Pendrell Sound 50° 18'N 124° 44' W 19.0 27.7 11.4 11 PSC99 08/21/1999 Pendrell Sound 50° 18'N 124° 44' W 0.5 14.6 22.6 12 3.3.2 PCR and DGGE In order to ensure detection of rare sequences from the environment, PCR of the concentrated virus samples was conducted in two stages. Template DNA for PCR was serially diluted and extracted from virus concentrates using a previously described hot-cold treatment (23). Five microliters of each virus concentrate subsample was then added to a 45 pi first-stage PCR mixture containing Taq DNA polymerase assay buffer (50 mM KC1, 20 mM Tris-HCl, pH 8.4), 1.5 mM MgCh, 0.16 mM of each deoxyribonucleoside triphosphate (dNTP), 60 pmol of each AVS primer (20), and 0.625 U of PLATINUM® Taq D N A polymerase (Invitrogen Life Technologies, Carlsbad, CA). Negative controls contained all reagents except template. PCR was carried out with the following cycle parameters: denaturation at 95 °C for 90 s; 32 cycles of denaturation at 95 °C for 30 s; annealing at 50 °C for 45 s; and extension at 72 °C for 1 min; and a final extension at 72 °C for 5 min. A two microliter subsample from each first-stage reaction was added to a 73 pi second-stage PCR mixture as above except AVS1 and AVS2 were increased to 90 pmol, and 1.0 U of enzyme was used. To minimize the production of PCR 38 artifacts generated from high cycle number, second-stage PCR was limited to 20 cycles. To investigate unexpected PCR products produced in several first-stage reactions, a modified second-stage PCR reaction (SIB99M) was set up using 2 pi of the first-stage reaction from one of the samples (SIB99) as template. Reaction conditions were as described above for second-stage PCR, except the upstream primer AVS1 was used alone. PCR products were electrophoresed in 1.5 % L E agarose (FMC BioProducts, Rockland, ME) in 0.5 X T B E buffer (45 mM Tris-borate, 1 mM EDTA, pH 8.0) at 90 volts for 75 min. The gels were stained with ethidium bromide, examined on an ultraviolet transilluminator, and photographed with a Nikon Coolpix 950 digital camera. Digital images were manipulated using Adobe Photoshop 5.0 LE . D G G E of second-stage PCR products was conducted using 20 to 55 % linear denaturing gradient 6 % polyacrylamide gels. Thirty microliters of products from SIB99M and 50 pi from all other reactions was loaded into wells with 5 pi of 10.0 X loading buffer (50 % v/v glycerol, 0.33 M EDTA, pH 8.0, and 0.08 % w/v bromophenol blue). Electrophoresis was carried out for 15 h in 1.0 X T A E (40 mM Tris-base, 20 mM sodium acetate, 1 mM EDTA, pH 8.5) at 135 volts and a constant temperature of 58 °C using the D-code™ electrophoresis system (Bio-Rad laboratories, Hercules, CA). Gels were stained in a 0.1 X SYBR® Green I (Molecular Probes, Eugene, OR) solution overnight and were examined and photographed as described above for agarose gels. 3.3.3 Sequencing To obtain sequence information from the denaturing gradient gels, individual bands were excised, re-amplified, cloned and sequenced. In total, 31 bands were excised and sequenced from the gradient gel. Excised bands and are referred to by sample abbreviation and position in the gel from top to bottom; e.g., band BSB99-2 refers to the second band from the top of the lane loaded with sample BSB99. After excision, the gel slices were placed in sterile microcentrifuge tubes with 200 pi of 1.0 X T A E and were heated to 95 °C for 5 min to elute the D N A band. One microliter of the resulting eluant was PCR amplified as described above for second stage reactions. D N A fragments amplified from D G G E bands were cloned into the vector pGEM®-5zf(+) (Promega, Madison, Wl) by T A cloning. Subsamples from ligation mixtures were used to transform competent Escherichia coli JM109. To check if multiple sequences could be obtained 39 from one D G G E band, cloned PCR fragments in three colonies from one transformation were sequenced (BSA99-la, BSA99-lb, and BSA99-lc). Plasmid D N A was harvested from the cultures using a QIAprep® spin miniprep kit according to manufacturers recommendations (Qiagen, Valencia, CA). Plasmid D N A (300 to 500 ng) was added to sequencing reactions using AmpliTaq FS® BIGDYE Terminator cycle sequencing chemistry (Applied Biosystems, Foster City, CA) according to manufacturers recommendations. Excess Dye-Terminators were removed from the completed sequencing reactions using CENTRI-SEP™ spin columns (Princeton Separations, Adelphia, NJ), and reactions were run in ABI Model 373 Stretch or ABI Prism 377 automated sequencers (Applied Biosystems, Foster City, CA) at the U B C sequencing facility. 3.3.4 Sequence analysis Sequences obtained from D G G E bands were compared to each other and known virus pol genes available in GenBank (Table 3.2). The GenBank accession numbers for all sequences obtained in this study are listed below. All sequences were edited and translated using BioEdit v 5.0.7 (44). Using the same program, a pairwise D N A identity matrix showing the proportion of identical residues was constructed from D G G E band sequences; only one sequence from any group of sequences with > 98 % identity was included in further analysis. Inferred amino acids of the unknown sequences were aligned with virus pol sequences from GenBank using the multiple sequence alignment program C L U S T A L W and the protein weight matrix B L O S U M (119). The alignment was then edited so that all sequences, with gaps included, were the same length. The alignment of the resulting 337 amino acid positions was used to construct maximum likelihood and neighbor-joining trees using the programs TREE-PUZZLE version 5.0 (107) and PHYLIP version 3.57c (31) respectively. Finally, phylogenetic trees were drawn using the program Tree View (Win32) version 1.6.1 (84). Sequences obtained from Genebank and used in the phylogenetic analysis are listed in table 3.2. The 33 sequences obtained in this study were deposited in the GenBank database. The accession numbers for the sequences S098-1, S098-2, S098-3, S098-4, S098-5, BSA99-1, BSA99-2, BSA99-3, BSA99-4, BSA99-5, BSA99-6, BSA99-7a, BSA99-7b, BSA99-7c, BSA99-8, BSB99-1, BSB99-2, BSB99-4, SIA99-1, MIB99-1, MIB99-2, PSB99-1, PSB99-2, PSB99-3, PSB99-4, PSC99-1, PSC99-2, BSA99-9, BSA99-10, BSB99-3, PSA99-1, PSC99-3, SIB99M-1 are AF405572 through AF405604 respectively. 40 Table 3.2. Acronyms, names, and GenBank sources of viruses used in phylogenetic analysis Acronym Virus names' Accession Nos. Total No. of amino acid residues Amino acids used in phylogenetic analysis Reference M p V - S P l a Micromonas pusilla virus, SP1 U32975 227 0 - 2 2 7 22 M p V - G M l a Micromonas pusilla virus, G M 1 U32977 227 0 - 2 2 7 22 M p V - P B 8 a Micromonas pusilla virus, PB8 U32980 227 0 - 2 2 7 22 M p V - P L l a Micromonas pusilla virus, PL1 U32982 227 0 - 2 2 7 22 M p V - S G l " Micromonas pusilla virus, SGI U32981 227 0 - 2 2 7 22 C b V - P W l ' Chrysochromulina brevifilum virus, PW1 U32983 239 0 - 2 3 9 22 C b V - P W 3 a Chrysochromulina brevifilum virus, PW3 U32984 239 0 - 2 3 9 22 P B C V - 1 " Chlorella strain N C 6 4 A virus, PBCV-1 M86836 913 465 - 699 43 N Y - 2 A a Chlorella strain N C 6 4 A virus, N Y - 2 A M86837 913 465 - 699 43 C V A - l a Chlorella strain Pbi virus, C V A - 1 U32985 230 0 - 2 3 0 22 O T U l a Unidentified phycodnavirus clone, OTU1 U36931 227 0 - 2 2 7 23 0 T U 2 a Unidentified phycodnavirus clone, OTU2 U36932 227 0 - 2 2 7 23 0 T U 3 a Unidentified phycodnavirus clone, OTU3 U36933 227 0 - 2 2 7 23 0TU4" Unidentified phycodnavirus clone, OTU4 U36934 227 0 - 2 2 7 23 0 T U 5 " Unidentified phycodnavirus clone, OTU5 U36935 229 0 - 2 2 9 23 H S V - 1 b Herpes simplex virus 1 (HHV-1) X04771 1235 697-942 64 H S V - 2 b Herpes simplex virus 2 (HHV-2) M16321 1240 702-947 122 H C M V " Human herpesvirus 5 (HHV-5) M14709 1242 697 -966 63 M C M V b Murine cytomegalovirus M73549 1097 604-847 30 E B V b Epstein-Barr virus V01555 1015 564-811 3 A c N P V c Autographa californica nucleopolyhedrovirus ( A c M N P V ) M20744 984 511-729 120 B m N P V c Bombyx mori nucleopolyhedrovirus D16231 988 511-729 18 L d N P V c Lymantria dispar nucleopolyhedrovirus (LdMNPV) D11476 1013 528 - 749 7 A S F V " African swine fever virus X73330 1244 492 -768 69 a. Viruses are members of the family Phycodnaviridae. b. Viruses are members of the family Herpesviridae. c. Viruses are members of the family Baculoviridae. d. Viruses are members of the family Asfarviridae. e. Where it differs from the vernacular acronym used, the current acronym accepted by the ICTV is given. 41 3.4 Results For all samples, different initial template concentrations were required for optimal PCR yield. Nonetheless, agarose gel electrophoresis revealed the presences of well-defined bands in all lanes (Figure 3.2A). All reactions produced the expected 700 bp fragment, yet samples BSA99, MIA99, SIA99, SIB99, MIB99, MIC99, PSA99, PSB99, and PSC99 produced both 700 and 550 bp products (lanes 2, 4, and 6-12). The reaction with primer AVS1 only (SIB99M) produced only the 550 bp product (lane M). D G G E revealed numerous bands in each lane (Figure 3.2B & C). Several bands were present in all lanes while other bands appeared in only a few. For example, the strong band near the bottom of lane 1 can be seen in every lane while the strong bands at the top of lanes 2 and 3 can only be observed in those lanes. As a final example, a single focused band can be observed in lane M , and bands at the same position are present at the same position in lanes 2, 4, and 6-12. Sequence information was recovered from 31 excised bands (Figure 3.2B). Three sequences, BSA99-7a, BSA99-7b, and BSA99-7c, were obtained from clones produced by transformation with sample BSA99-7; while these sequences were not the same, they were approximately 99 % identical (range 98.5 to 99.1). Similarly, two groups of bands at similar positions in different lanes produced nearly identical sequences. Sequences from Bands S098-2 and, BSA99-7a, b, and c were approximately 99 % identical (range 98.9 to 99.5). Bands BSA99-10 and SIB99M-1 produced sequences that were 99.2 % identical. However, gel position and sequence similarity were not related and many similar sequences were recovered from different positions in the gel. A pairwise identity matrix of the 33 sequences recovered from excised bands (data not shown) revealed 26 that formed 7 groups of sequences with > 98 % identity. Sequences with identities > 98 % were grouped as follows: [S098-1, S098-4]; [S098-2, BSA99-6, BSA99-7a, BSA99-7b, BSA99-7c, BSA99-8, BSB99-4]; [BSA99-1, BSA99-3, BSA99-4, BSB99-1]; [BSA99-2, MIB99-1]; [BSA99-9, BSA99-10, BSB99-3, PSA99-1, PSC99-3, SIB99M-1]; [BSB99-2, PSB99-3]; [PSB99-1, PSB99-2, PSB99-4]. The other 7 sequences (S098-3, S098-5, BSA99-5, SIA99-1, MIB99-2, PSC99-1, and PSC99-2) were not > 98 % identical to any other. 42 A L 1 2 3 4 5 6 7 8 9 101112MLN 1500 600 I mm am I MM *Mtf mmmt I J M •3 I I I B 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 M 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 M Figure 3.2. Gel electrophoresis of PCR fragments amplified in second-stage PCR All lane numbers correspond those listed in Table 3.1. Lane M was loaded with the products of the modified reaction (SIB99M) using only the upstream primer AVS1. A) PCR products were electrophoresed in 1.5 % LE agarose in 1 X TAE buffer. Numbers to the left of the gel correspond to the size of standards loaded in lanes L and lane N was loaded with the negative control. B) The lines in panel B specify the position of all bands excised from the gel. C) Lines in panel C indicate the 14 bands, S098-1, S098-2, S098-3, S098-5, BSA99-1, BSA99-2, BSA99-5, BSA99-9, BSB99-2, SIA99-1, MIB99-2, PSB99-1, PSC99-1, and PSC99-2, that produced unique pol-tike sequences (solid lines) and a band BSA99-9 that did not produce a pol-like sequence (broken line). 43 The first sequences in each of the 7 groups and the 7 unique sequences were phylogenetically analyzed. Translation of the 14 D N A sequences revealed, in 12 instances, the presence of the conserved pol motif YGDTDS, and in one case the presence of a very similar sequence HGDTDS (sequence BSA99-5). All of the sequence in the group starting with BSA99-9 did not contain amino acids resembling the conserved motif. As expected, this group of sequences without the pol motif included the band excised from lane M , which was loaded with products from the PCR reaction with the primer AVS1 alone (SIB99M). As stated previously, only 12 of the 14 analyzed sequences coded the pol motif YGDTDS, and one contained a similar sequence, HGDTDS. Not surprisingly, these sequences aligned at this motif with 24 virus pol sequences catalogued in GenBank. An amino acid alignment also revealed extensive sequence conservation among the unknown sequences and pol fragments from viruses belonging to the family Phycodnaviridae. Phylogenetic analysis of aligned amino acids revealed robust relationships between sequences recovered from the gradient gel and other groups of known viruses (Figure 3.3). Both algorithms (maximum likelihood and neighbor joining) used for phylogenetic reconstruction produced identical tree topology at major nodes. The topologies varied only at nodes near branch tips. Furthermore, the Likelihood support and bootstrap values at nodes separating families of viruses were greater than 93 %. All of the sequences recovered with D G G E were closely related to, and clustered among pol sequences of known phycodnaviruses when compared to virus pol sequences from three other families of dsDNA viruses including Asfarviridae, Baculoviridae and Herpesviridae. Several sequences from D G G E (S098-1, S098-2, S098-3, BSA99-2, BSA99-5, SIA99-1, MIB99-2, and PSC99-1) were closely related to pol sequences from the genus Prasinovirus; prasinoviruses (abbreviated MpV) infect the prasinophyte alga Micromonas pusilla. Four sequences (S098-5, BSA99-1, PSB99-1, PSC99-2) formed their own clade yet were also closely related to prasinoviruses and one sequence (BSB99-2) formed a separate branch between prasinoviruses and viruses of the genus Prymnesiovirus (abbreviated CbV). Finally, Baculoviridae and Herpesviridae viruses all clustered with viruses of the same family. 44 96/88 96/97 94/64 96/93 93/100 87/76 • ASFV | A •MCMV - H C M V EBV i - H S V - 1 99/IOO L HSV-2 I CbV-PWl H o.i 98/100 100/ 1 0 0 l - C 5 y _ p W 3 89 / 36" 91 /47 86/74* BSB99-2 ES098-1 S098-2 -S098-3 • BSA99-2 -BSA99-5 SIA99-1 MIB99-2 I-PSC99-1 OTU2 OTU4 HZ. 86/98 70 /NA 62/45 HI / 7 1 1 — 50/NA 84 MpV - SP1 — O T U 1 OTU3 MpV - GM11 MpV - PB8 MpV - PL1 MpV - SGI PSB99-1 PSC99-2 89/69 S098-5 BSA99-1 OTU5 54/100 CVA-1 LdNPV r-AcNPV 99/ ioo L BmNPV 92/99 B PBCV-1 NY2A Figure 3.3. Maximum Likelihood (ML) tree of DNA pol fragments Quartet puzzling support values f o r the ML tree and bootstrap values for a corresponding Neighbor J o i n i n g ( N J ) tree are shown as percentages to the lower left of the appropriate node or as indicated by solid black arrows; NA means the N J tree topology differs at that node. The vertical black lines and letters to the right of the tree indicate virus families as follows: A, Asfarviridae; B, Baculoviridae; H, Herpesviridae; P, Phycodnaviridae. The Phycodnaviridae are also indicated by bold lettering. The scale bar represents the number of amino acid substitutions per residue. 45 3.5 Discussion Three main results were realized during this study. First, PCR and D G G E were used to recover pol sequences from marine algal virus communities. Second, the pol sequences obtained in this study were closely related to known algal viruses. And the final, but perhaps most important result, is that nearly identical sequences were recovered from distant oceans. The following discussion considers PCR and D G G E as tools to genetically fingerprint virus communities, and considers the phylogeny of sequences obtained in this study. PCR reactions produced expected and unexpected products. The unexpected 550 bp products were the result of amplification with the primer AVS1 only, suggesting that they were PCR artifacts or the result of amplification of extant targets in the environment. PCR artifacts are often produced during reactions with mixed templates. For example, chimeric sequences form through recombination of similar PCR products, and therefore, have regions with different phylogenetic affinities. Because of this, many researchers suggest that nearest-neighbor analysis of different domains of suspect sequences can reveal PCR chimeras (61, 93, 126). To determine if any sequences recovered from D G G E were chimeric, phylogenetic trees were constructed from upstream, middle, and downstream regions of all sequences. The topology of phylogenetic trees constructed from the different regions did not differ indicating that the sequences were likely not chimeric (data not shown). To check for the possibility of amplification of other targets, and to determine their identity, the 550 bp sequence SIB99M-1 was compared to sequences in the public databases GenBank, DDBJ, and E M B L using the Basic Local Alignment Search Tool (BLAST) at the National Center for Biological Information web site (http://www.ncbi.nlm.nih.gov). This comparison revealed that the closest relatives of SIB99M were Phycodnaviridae pol sequences; however, only the upstream primer region was related to the Phycodnaviridae sequences and therefore, no conclusions about the identities of the small amplicons were reached. Although the origins and identities of the small PCR products remain unknown, they did not interfere with D G G E analysis of desired PCR products. D G G E facilitated the resolution of heterogeneous D N A fragments amplified from natural environments. However, one must be careful inferring diversity or richness from D G G E fingerprints. This is due to the fact that different sequences can migrate similarly and similar sequences can migrate differently. For example, S098-1 and S098-4 were 98 % identical yet 46 S098-1 migrated one third of the gel length while S098-4 migrated two thirds. On the other hand, S098-3 and BSB99-2 were only 36 % identical yet they migrated the same distance (Figure 3.2B). Nonetheless, the community fingerprints obtained in this study were reproducible; multiple independent PCR reactions of the same sample analyzed with D G G E revealed the same fingerprint (data not shown). Furthermore, because D G G E resolved multiple bands from the PCR products of each reaction, qualitative comparison of PCR products was possible. For example, the banding patterns or 'fingerprints' of samples from Salmon Inlet (SIA99 and SIB99) were identical and easily distinguished from other fingerprints. These samples were collected at the same time and location but different depths suggesting that PCR templates were homogenously distributed with depth. This is reasonable considering the salinity and temperature of Salmon Inlet did not vary greatly with depth, indicating that the water and viruses were well mixed. On the other hand, the fingerprint of pol fragments amplified from surface water at Pendrell Sound (lane 12) is different than the fingerprints from greater depths (lanes 10 and 11). At this location the salinity of surface water was half of that at depth, indicating the water and viruses were stratified with depth. In general, environmental parameters agreed with qualitative lane comparison reinforcing the idea that D G G E can be used to distinguish pol fragments amplified from natural algal virus communities. Preliminary analysis of sequences recovered from D G G E revealed many that were more than 98 % identical. On the other hand, the sequences BSA99-7a, BSA99-7b, and BSA99-7c from 3 different clones of PCR products re-amplified from a single D G G E band were not identical. Although some of the nearly identical sequences may represent strain variation, the methods used in this study cannot distinguish the possibility that some were due to PCR and/or sequencing error. Therefore, only one sequence from each group of sequences with > 98 % identity was included in phylogenetic analyses. To confirm that the sequences from D G G E were pol gene fragments, they were translated and searched for the conserved amino acid sequence corresponding to the putative pol catalytic site, YGDTDS (52). Of the 14 sequences analyzed one (BSA99-9) was not a recognizable pol fragment. This sequence was from the group that included the 550 bp sequence SIB99M-1 mentioned previously. Twelve sequences encoded the pol catalytic motif, and one sequence (BSA99-5) encoded the unusual motif HGDTDS. There are two possible explanations for this sequence variation. First, HGDTDS is not an algal virus pol sequence and was the result of PCR 47 and/or sequencing error. Second, HGDTDS may represent natural sequence variation in B-family pol genes. The amino acids histidine (H) and tyrosine (Y) are both polar and therefore, it is possible that this substitution does not affect pol function. Future studies will support the latter argument if they reveal that this unusual motif is a feature of some algal virus pol genes. On the other hand, if the HGDTDS is not observed again, it is likely this pol motif was an artifact. Phylogenetic inference revealed that the putative pol fragments recovered in this study were closely related to pol genes from the Phycodnaviridae. High likelihood support and bootstrap values indicated that the deep branches of the maximum likelihood tree were robust (Figure 3.3). For example, viruses belonging to the family Baculoviridae branched from the Asfarviridae outgroup with a likelihood support value of 98 %. Similarly, viruses belonging to the closely related families Herpesviridae and Phycodnaviridae branched from the Asfarviridae and Baculoviridae with a support value of 96 %. The Herpesviridae and Phycodnaviridae formed their own clusters with support values of 96 and 93 %, respectively. In addition to having high support values, the phylogeny from this study resembled previously published pol phylogenies (10, 22). Therefore, it is likely that relationships obtained in this study provide accurate relationships of pol genes. Chen and Suttle (22) demonstrated that pol phylogeny reflects the phylogeny of total genomic D N A in Micromonas pusilla viruses (MpV). Interestingly, the results of a previous phylogenetic analysis of phycodnavirus pol sequences based on nucleotide sequences (23) do not agree with the results presented in this study, yet previous phylogenies based on inferred amino acids (22) do. For example, the nucleotide-based phylogeny placed the Chlorella viruses (CVA-1, PBCV-1, NY-2A) more distant from MpV isolates than Chrysochromulina viruses (CbV-PWl, CbV-PW3) (23), whereas in our study CbV isolates were more distant from MpV isolates than Chlorella viruses. An explanation of this minor discrepancy may be that for the sake of reproducibility no eye-refinement was performed on the current amino acid alignment. While the unrefined alignment presented in this paper differed slightly from and may not be as accurate as previous alignments, a strong argument for unrefined alignments can be made because of the inherent irreproducibility of eye-refinement. Nonetheless, these subtle differences do not affect the interpretation of our main result; all pol sequences obtained in this study clustered within the Phycodnaviridae, although outside of established genera. 48 Several sequences obtained in this study were not closely related to any known phycodnaviruses, yet many others (S098-1, S098-2, S098-3, BSA99-2, BSA99-5, SIA99-1, MIB99-2, and PSC99-1) were most closely related to viruses in the genus Prasinovirus (MpV). However, because none of these sequences clustered among MpV, their identity remains unknown. Nonetheless, it is remarkable that sequences from the Southern Ocean were closely related to sequences from coastal British Columbia, in some cases being > 98 % identical, even though the environments were very different. This is the first time nearly identical viruses have been shown to occur in the Southern Ocean as well as in the temperate waters of the North East Pacific. It should be noted that although the samples were collected across the planet, the sample locations were all coastal and may have supported similar algal communities. In addition, the results of this study agree with previous work demonstrating the cosmopolitan nature of some algal viruses and their hosts (25). Five of the analyzed sequences obtained in this study formed two distinct groups within the Phycodnaviridae. These sequences may represent unknown genera within the Phycodnaviridae; further study will be necessary to refine the phylogeny of these unknown sequences. Previously, only one sequence (OTU5) has been recovered from the environment that was not closely related to other prasinoviruses (23). This study is the first to examine sequence diversity of algal viruses in more than one natural sample and provides further evidence that the PCR primers AVS1 and 2 can amplify unknown algal viruses from marine environments. This study demonstrated that PCR and D G G E can be used to recover and identify unknown algal virus pol sequences from the natural environment and that very similar sequences can be recovered from different oceanic provinces across the planet. As more viruses are isolated and cultured from the environment and more sequences become known, we may find viruses that cluster with currently unidentified viruses. This will permit putative identification of uncultured virus sequences. In addition, we demonstrated that D G G E fingerprints could be used for qualitative comparison of virus communities. Therefore, the molecular methods described in this study can be used to relate the genetic composition of marine algal virus communities obtained from different environments. Furthermore, the ability to track changes in virus community composition may lead us closer to understanding the effects of viruses on host community structure. For example, it may be possible to relate changes in host populations to changes in the virus community. This study clearly demonstrates that the genetic composition of algal virus 49 communities can be examined using the molecular techniques presented. Therefore, we are confident our long-term goal to understand the dynamics and effects of marine algal viruses can be obtained, in part, by examining the genetic composition of phytoplankton virus communities. 50 Chapter IV. Temporal Changes in the Composition of Natural Communities of Marine Algal Viruses and Eukaryotes Studied by Denaturing Gradient Gel Electrophoresis 51 4.1 Summary The composition of algal virus communities at a single location was monitored over fourteen months and compared to physical and biological environmental parameters. To examine changes in algal virus and eukaryote communities, PCR and denaturing gradient gel electrophoresis were used to generate genetic fingerprints. For the algal-virus fingerprints, PCR conditions were optimized and previously observed artifacts were eliminated. Sequence analysis of bands extracted from denaturing gradient gels revealed the presence of at least 5 distinct viruses and a diverse community of eukaryotes that included taxa from the viridiplantae, fungi, and metazoa. Comparison of algal virus fingerprints with environmental conditions revealed that, at certain times, changes in algal virus community composition were coincident with changes in tide height, salinity, or chlorophyll a concentration. Overall, algal virus community fingerprints were temporally less variable than eukaryote fingerprints. While the algal virus fingerprint patterns were stable throughout most of the study, stable eukaryote fingerprint patterns were observed only during the winter months. We concluded that specific taxa of algal viruses could persist in fluctuating physical and biological environments suggesting that the production of, and mortality from, some taxa of algal-viruses are constant. This study demonstrated that the presented fingerprinting techniques could be used to investigate temporal changes in algal virus community composition. 52 4.2 Introduction After the discovery that viruses were abundant in the ocean, marine microbiologists quickly recognized their ecological importance (e.g. 13, 109, 117). Recently, it has been proposed that viruses have a significant effect on oceanic energy and material flow (38, 131). An important contribution to the study of marine viruses was the development of PCR techniques that are specific for algal viruses belonging to the Phycodnaviridae (21). Using these culture-independent PCR methods, sequences belonging to unknown taxa of algal viruses were amplified from the Gulf of Mexico demonstrating the utility of this molecular approach (23). Subsequently, we developed a denaturing gradient gel electrophoresis (DGGE) protocol to resolve sequences amplified with the algal-virus-specific (AVS) PCR primers (103). Recently, molecular fingerprinting of algal virus communities facilitated the recovery of algal virus gene sequences from different environments revealing that groups of closely related algal viruses were geographically widespread (104). The results of that study demonstrated that although further refinement of the PCR and D G G E methods was necessary, these methods could be applied to the study of geographic differences in algal virus community composition. The diversity of complex microbial populations was first studied using D G G E when Muyzer et al. obtained molecular fingerprints of 16S rDNA fragments amplified from an aerobically grown bacterial biofilm (78). Since then, D G G E has become a common method to obtain molecular fingerprints and has been used to study microbial diversity in many different contexts. For the most part, D G G E has been applied to the study of bacterial community composition as determined by PCR amplification of 16S rDNA. As examples, D G G E has been used to examine the microbial community diversity of bacterial enrichment cultures (e.g. 54, 98, biostimulated shorelines (82), hot spring microbial mats (34), California estuaries (76), and the Arctic Ocean (32). Recently, PCR and D G G E fingerprinting methods have been developed to examine the diversity of eukaryotic microbes (29, 125). These eukaryote fingerprinting techniques have been applied to investigations of the temporal variability of marine eukaryotes in an enclosure (17) and the spatial variability through a salinity gradient in a solar saltern (16). However, to our knowledge, molecular fingerprinting has not been used to examine temporal changes in marine eukaryotic communities. The primary goal of this study was to determine if there are temporal changes in the 53 genetic fingerprints of natural algal virus communities. Indirect evidence of changes in algal virus community composition was noted when blooms of the alga Emiliania huxleyi were succeeded by an increase in large virus-like particles (11). Furthermore, changes in aquatic viral assemblages have been examined by comparing genome size classes with pulsed field gel electrophoresis (106, 135). However, to date, no direct examination of temporal changes in algal virus community composition has been conducted. A secondary goal of this study was to relate changes in algal virus diversity, as inferred from molecular fingerprints, to changes in eukaryote diversity and the physical environment. The results of this study show that although the temporal changes of algal viruses and eukaryotes were not closely coupled, changes were evident in both communities. More importantly, this study demonstrates that changes in the composition of marine algal virus communities are often coincident with changes in the environment. 54 4.3 Materials and Methods 4.3.1 Sample collection Using a bucket and funnel, forty liter samples were collected weekly from the surface water at Jericho Pier, Vancouver, Canada. A map of the sample site is shown in Figure 4.1. Sample temperature and salinity were measured approximately 15 min after collection and duplicate acetone extracted Chlorophyll a samples were measured fluorometrically using established protocols (85). The virus-size-fraction was concentrated using previously described methods (104). Briefly, samples were pressure filtered (< 17 kPa) through 142 mm dia. glass fiber (GC50; nominal pore size 1.2 pm, Advantec MFS, Dublin, CA) and PVDF filters (GVWP; 0.45 pm pore size, Millipore, Bedford, MA) connected in series. Remaining particulate material in the filtrates was concentrated to approximately 200 ml using a 30 kD-cutoff, Amicon S1Y30 (Millipore) cartridge according to the manufacturer's recommendations. After processing, the concentrated samples were stored at 4 °C in the dark. The glass fiber and PVDF filters were stored at -20 °C in the dark until nucleic acid extraction. 126° W 124° W 122° W Figure 4.1. Jericho Pier sample site The location of the sample collection site at Jericho Pier, Vancouver, British Columbia, Canada is shown. 55 4.3.2 DNA extraction and PCR Nucleic acids were extracted from virus-size-fraction concentrates, glass-fiber filters, and PVDF filters. Thirty-five milliliter subsamples of the virus-size concentrates were centrifuged at 28,500 x g for 3.5 h at 20 °C. Nucleic acids were extracted from the resuspended pellets (100 pi) using an established hot/cold technique (23). D N A was also extracted from aseptically-excised 2 cm 2 portions of the 142 mm dia. filters. Following the protocol of Diez et al. (29), filter subsamples were incubated at 37 °C for 45 minutes in 500 pi lysozyme (final concentration of 1 mg ml"1) and at 65 °C for 1 h after a 500 pi solution of sodium dodecyl sulfate (final concentration, 1 %) and proteinase K (final concentration, 0.2 mg ml"1) was added. D N A was purified from the filter lysates by sequential extractions with equal volumes of phenol, phenol-chloroform-isoamyl alcohol (25:24:1), and finally chloroform-isoamyl alcohol (24:1). The purified D N A was precipitated and washed with ethanol as described (97). Extracted D N A was stored at -20 °C in 10 mM Tris-Cl (pH 8.5) until further use. PCR annealing temperature optimization was necessary to improve the specificity of algal-virus polymerase amplification. Three pi (ca. 0.5 % of the 401 sample) of virus-size extract from a sample (collected on February 1, 2001) that produced non specific PCR products was added to a 47 pi PCR mixture containing Taq DNA polymerase assay buffer (50 mM KC1, 20 mM Tris-HCl, pH 8.4), 1.5 mM MgCl 2 , 0.16 mM of each deoxyribonucleoside triphosphate (dNTP), 10 pmol of the algal-virus-specific primer AVS1 and 30 pmol of AVS2 (20), and 0.625 U of PLATINUM® Taq DNA polymerase (Invitrogen Life Technologies, Carlsbad, CA). The negative control contained all reagents except template and was placed at the lowest annealing temperature. PCR was carried out using a PCR Express thermal cycler (Hybaid Limited, Ashford, UK) with the following cycle parameters: denaturation at 95 °C for 90 s; 35 cycles of denaturation at 95 °C for 45 s; annealing gradient from 42 °C to 56 °C for 45 s; and extension at 72 °C for 45 s with a final 5 min extension. Once the PCR annealing temperature was optimized, algal virus pol was amplified using two separate PCR reactions. First, 3 pi of virus-size extract was added to PCR mixtures as described above. All reaction conditions were as above except the annealing temperature was 45 °C and only 32 cycles were performed. After electrophoresis, a disposable Pasteur pipette was used to excise a single plug from the 700 bp band amplified in each reaction. The plug was 56 placed in 200 pi of sterile 1.0 X T A E (40 mM Tris-base, 20 mM sodium acetate, 1 mM EDTA, pH 8.5) and heated to 65 °C for 20 min to elute DNA. Two pi of the eluant was used in a second stage of PCR to increase product yields for the subsequent D G G E analysis. These reactions were identical to first-stage PCR except the number of cycles was limited to 20 to minimize the production of PCR artifacts generated from high cycle number. Second-stage negative controls were conducted using the eluant of plugs excised from the negative control lane of gels of first-stage PCR. Eukaryotic 18S rDNA fragments were amplified from filter extracts using the previously described eukaryotic 18S rDNA-specific primers EuklA, Euk516r, and Euk516r-GC (29). Five pi of a filter extract were added to a 45 pi PCR mixture containing Taq D N A polymerase assay buffer (50 mM KC1, 20 mM Tris-HCl, pH 8.4), 1.5 mM MgCl 2 , 0.16 mM of each deoxyribonucleoside triphosphate (dNTP), 15 pmol of the 18S primers EuklA and Euk516r, and 0.625 U of PLATINUM® Taq D N A polymerase. PCR was carried out using a Hybaid PCR Express thermal cycler with the following cycle parameters: denaturation at 95 °C for 90 s; 32 cycles of denaturation at 95 °C for 1 min; annealing at 56 °C for 1 min; and extension at 72 °C for 1 min with a final 5 min extension. Like the algal-virus pol reactions, a second PCR reaction was conducted using first-stage gel plugs as templates. In this case, second-stage reactions were used to incorporate a GC clamp onto the PCR products for the subsequent D G G E analysis. All second-stage PCR conditions were the same as first-stage 18S amplification except the downstream primer Euk516r-GC was used and the reaction was limited to twenty cycles. PCR products were electrophoresed in 1.8 % L E agarose (FMC BioProducts, Rockland, ME) in 1.0 X T A E at 90 volts for 75 min. The gels were stained with ethidium bromide, examined on an ultraviolet transilluminator, and photographed with a Nikon Coolpix 950 digital camera. Digital images were inverted and the contrast and brightness was adjusted using Adobe Photoshop 5.0 LE . 4.3.3 DGGE and sequencing Initially, D G G E conditions were optimized for maximum resolution by electrophoresis of several samples in different gradient gels. From the optimization gels, bands were excised, eluted and amplified as described above for the templates of second-stage PCR. For each type of D G G E 57 analysis (AVS or 18S) six bands were selected and pooled for use as standards in subsequent gradient gels. D G G E of second-round AVS PCR products was conducted using 20 to 40 % denaturing gradients cast in a 7 % to 8 % gradient polyacrylamide gels. Approximately 50 pi (range 35 to 60) of the pooled products of 3 separate second-stage reactions was loaded into wells with 5 pi of 10.0 X loading buffer (50 % v/v glycerol, 0.33 M EDTA, pH 8.0, and 0.08 % w/v bromophenol blue). In every gel, 45 pi of the pooled AVS standards was loaded in the wells flanking the samples. Electrophoresis was carried out for 15 h in 1.0 X T A E at 80 volts and a constant temperature of 60 °C using the D-code™ electrophoresis system (Bio-Rad laboratories, Hercules, CA). Similarly, D G G E of the second-round 18S PCR products was conducted using 25 to 47.5 % denaturing gradients cast in 6 % polyacrylamide gels. Approximately 45 pi (range 40 to 50) of the pooled products of 3 separate second-stage reactions was loaded into wells with loading buffer. The amount of D N A loaded for each A V S or 18S sample was standardized by densitometry of agarose gel bands using the program Gel-Pro®Analyzer (Media Cybernetics, Silver Spring, MD). The wells flanking the samples were loaded with 40 pi of the pooled 18S standards. Electrophoresis was carried out for 15 h in 1.0 X T A E at 100 volts and a constant temperature of 59 °C. All gels were stained in a 0.1 X SYBR® Green I (Molecular Probes, Eugene, OR) solution overnight, destained in water for a least 30 min and were examined and photographed as described above for agarose gels. Digital images of the denaturing gradient gels were analyzed using the program GelCompar II (Applied-Maths B V B A , Sint-Martens-Latem, BEL). For each type of D G G E analysis (AVS or 18S), the individual gel images (6 AVS and 12 18S) were normalized using lanes loaded with the appropriate pooled standards. To estimate sample similarities within each fingerprint set (AVS, 0.45 - 1.2 pm 18S, > 1.2 pm 18S) Pearson product moment correlation coefficients were calculated from the densitometry plots of pairs of lanes. The coefficients (similarities) were used to calculate UPGMA dendrograms for each fingerprint set. To create libraries for sequencing, 65 or 138 bands from different gel positions were excised from the AVS or 18S gradient gels, respectively. After excision, the gel slices were placed in sterile microcentrifuge tubes with 200 pi of 1.0 X T A E , heated to 95 °C for 5 min to 58 elute the DNA, and stored at -20 °C until further use. The eluted D N A fragments from ten arbitrarily selected AVS bands were amplified as described above for second-round AVS reactions. Amplified AVS D N A was then cloned using a pGEM®-T Easy kit (Promega, Madison, WI). Plasmid D N A was harvested from transformed Escherichia coli JM109 colonies using a QIAprep® spin miniprep kit according to manufacturers recommendations (Qiagen, Valencia, CA). In addition, the eluted D N A fragments from 19 arbitrarily selected 18S bands were amplified as described for second-round 18S reactions except the downstream primer was Euk516r. Amplified 18S DNA was then used directly in sequencing reactions. A V S plasmid D N A (300 to 500 ng) or 18S amplified D N A (4 pi) and an appropriate oligonucleotide primer (Ml3 forward/reverse for AVS clones and EuklA/516r for 18S PCR products) were added to sequencing reactions using AmpliTaq FS® BIGDYE Terminator cycle sequencing chemistry (Applied Biosystems, Foster City, CA) according to the manufacturer's recommendations. Excess Dye-Terminators were removed from the completed sequencing reactions using CENTRI-SEP™ spin columns (Princeton Separations, Adelphia, NJ), and reactions were run in ABI Model 373 Stretch or ABI Prism 377 automated sequencers (Applied Biosystems, Foster City, CA) at the U B C sequencing facility. Algal-virus pol sequences obtained from D G G E bands were compared to each other and known virus pol genes available in GenBank (accession numbers listed below). All sequences were edited and/or translated using BioEdit v 5.0.7 (44). Inferred amino acids of the unknown sequences were aligned with virus pol sequences from GenBank using the sequence alignment program C L U S T A L X (118) with default settings. The alignment was then edited so that all sequences, with gaps included, were the same length. The alignment of the resulting 340 amino acid positions was used to construct a maximum likelihood tree using the program TREE-PUZZLE version 5.0 (107). Phylogenetic trees were drawn using the program TreeView (Win32) version 1.6.1 (84). The sequences recovered from the 18S gels were compared to nucleotide sequences in GenBank (http://www.ncbi.nlm.nih.gov) using blastn and small subunit rDNA sequences in the Ribosomal Database Project II (http://rdp.cme.msu.edu/html) using Sequence Match (68). All of the sequences obtained in this study were deposited in the GenBank database. The accession numbers assigned to the algal-virus sequences JPavs-22, JPavs-37, JPavs-38, JPavs-42, JPavs-51, JPavs-52, JPavs-53, JPavs-55, JPavs-64, JPavs-65 were AY145089 - AY145098 59 respectively. The accession numbers assigned to the 18S rDNA sequences JPeuk-1, JPeuk-2, JPeuk-4, JPeuk-6, JPeuk-20, JPeuk-27, JPeuk-38, JPeuk-40, JPeuk-51, JPeuk-59, JPeuk-65, JPeuk-71, JPeuk-77, JPeuk-80, JPeuk-84, JPeuk-94, JPeuk-108, JPeuk-113, JPeuk-119 were AY145099 - AY145117 respectively. The accession numbers of virus pol sequences used in the phylogenetic analysis are: ASFV, X73330; BSA99-1, AF405577; BSA99-2, AF405578; BSA99-5, AF405581; BSB99-2, AF405588; CbV-PWl, U32983; CbV-PW3, U32984; CVA-1, U32985; EBV, V01555; EsV-1, AAK14511; FsV, AAB67116; HSV-1, X04771; H C M V , M14709; MIB99-2, AF405592; MpV-GM1, U32977; MpV-PB8, U32980; M p V - P L l , U32982; M p V - S G l , U32981; MpV-SPl , U32975; NY-2A, M86837; OTU1, U36931; OTU2, U36932; OTU3, U36933; OTU4, U36934; OTU5, U36935; PBCV-1, M86836; PSB99-1, AF405593; PSC99-1, AF405597; PSC99-2, AF405598; SIA99-1, AF405590; S098-1, AF405572; S098-2, AF405573; S098-3, AF405574; S098-5, AF405576. 60 4.4 Results Optimization of algal-virus pol PCR successfully eliminated a previously observed 550 bp artifact (104). At annealing temperatures above 48 °C, PCR reactions with the 02/01/01 sample and AVS primers produced 700 and 550 bp bands. The highest yield of the 700 bp band was at the lowest annealing temperature and diminished with increasing temperature, while the highest yield of the 550 bp band was at the highest annealing temperature and disappeared as the temperature decreased to 46 °C (Figure 4.2). Although faint smears were observed above the 700 bp bands in the samples amplified at annealing temperatures from 42 to 52 °C, they were not present when the number of PCR cycles was reduced from 35 to 32 (data not shown). Two-stage AVS PCR using 45 °C annealing temperatures produced only bright 700 bp bands. AVS PCR was also conducted with templates extracted from filters, yet no amplification was achieved. Similarly, second-stage PCR with 18S primers produced only the expected 550 bp DNA fragments (data not shown). Temperature L 42 43 44 46 48 50 52 54 55 56 N Figure 4.2. Annealing temperature gradient of PCR with AVS primers Agarose gel loaded with 15 ul of PCR products from AVS reactions of the template from February 1, 2001. Numbers indicate the annealing temperature in °C. L designates the lane loaded with a 100 bp DNA ladder (Invitrogen, Burlington, CAN) and N designates the lane loaded with the negative control. 61 Ten bands from the AVS fingerprints were identified by sequencing and phylogenetic analysis (Figure 4.3). All ten sequences recovered from the AVS fingerprints encoded the purported pol motif YGDTDS and comparison to other virus pol sequences revealed that their inferred amino acid sequences were closely related to known sequences. Although none of the Jericho Pier sequences were identical, the percentage of identical nucleotides was at least 98 among the sequences JPavs-37, JPavs-42, JPavs-51, JPavs-52. Similarly, the sequences JPavs-64 and JPavs-65 were 99.5 %, and JPavs-22 and JPavs-38 were 98.0 % identical. The remaining sequences (JPavs-53 and JPavs-55) were less than 89 % identical to any other sequence. Furthermore, several sequences were more than 98 % identical to other environmental sequences available in GenBank. For example, the sequences JPavs-64 and 65 were at least 99 % identical to the sequence BSA99-1, and the sequence JPavs-53 was 99 % identical to S098-5. Only one sequence from the A V S fingerprints (JPavs-55) was less than 90 % identical to any known algal virus pol fragment. Nineteen bands excised from the 18S fingerprints were sequenced and compared to sequence databases. When compared to nucleotide sequences in GenBank, all of the sequences recovered from the 18S gradient gels were related to 18S rDNA sequences. The Jericho Pier 18S sequences were related to 18S rDNA sequences from several eukaryote lineages including Alveolata, Fungi, Metazoa, Stramenopiles, and Viridiplantae (Table 4.1). Serial filtration did not effectively fractionate the samples and sequences of some bands from the 0.45 - 1.2 pm 18S fingerprints were closely related to sequences of organisms larger than 1.2 pm in diameter. For example, the 0.45 - 1.2 pm 18S sequences JPeuk2 and JPeuk27 were closely related to the 18S rDNA sequences of a ciliate and a metazoan. 62 H C M V • A S F V 841 H S V - 1 821 89 — F s V E s V - 1 0.1 E B V _ r C b V - P W 1 9 6 « - C b V - PW3 - O T U 5 C V A - 1 P B C V - 1 N Y 2 A B S B 9 9 - 2 J P a v s - 5 5 P S B 9 9 - 1 P S C 9 9 - 2 J P a v s - 6 5 J P a v s - 6 4 B S A 9 9 - 1 S 0 9 8 - 3 B S A 9 9 - 2 :— M I B 9 9 - 2 O T U 2 O T U 4 B S A 9 9 - 5 72 Lr J P a v s - 3 8 63T- J P a v s - 2 2 J P a v s - 5 1 J P a v s - 3 7 J P a v s - 5 2 65 «• J P a v s - 4 2 M p V - S P 1 O T U 1 O T U 3 M p V - P B 8 M M p V - P L 1 M p V - S G 1 M p V - G M 1 S 0 9 8 - 2 P S C 9 9 - 1 S 0 9 8 - 1 S IA99 -1 Figure 4.3. Maximum Likelihood tree of algal viruses from Jericho Pier Maximum Likelihood tree of inferred amino acid sequences of dsDNA viruses with quartet puzzling support values to the lower left of corresponding nodes. Algal virus pol fragments amplified from Jericho Pier are shown in bold. The scale bar indicates the number of amino acid substitutions per position. 63 Table 4.1. Results of database searches with Jericho Pier sequences Sequence Fingerprint NCBI blastn results* RDPII sequence match resultsb Eukaryota; Stramenopiles Eukaryota; Stramenopiles JPeuk-1 0.45 Uncultured Stramenopile B A Q A 2 1 , AF372755 (771) Uncultured Stramenopile OLI51105, AF167414 (0.642) JPeuk-2 0.45 Alveolata; Ciliophora; Spirotrichea Stylonychia mytilus, AJ310499 (622) Alveolata; Ciliophora; Spirotrichea; Stylonychia lemnae, AF164124 (0.588) JPeuk-4 0.45 Fungi; Ascomycota; mitosporic Ascomycota Geosmithia viridis, AB033527 (991) Fungi; Ascomycota; Pezizomycotina Talaromyces flams, M83262 (0.901) JPeuk-6 0.45 Viridiplantae; Streptophyta; Embryophyta Casuarina equisetifolia, U42515 (983) Viridiplantae; Streptophyta; Embryophyta Fagus grandifotia, AF206910 (0.871) Viridiplantae; Chlorophyta; Prasinophyceae Viridiplantae; Chlorophyta; Prasinophyceae JPeuk-20 0.45 Prasinophyte symbiont of radiolarian, AF166380 (922) Prasinophyte symbiont of radiolarian, AF166379 (0.783) JPeuk-27 0.45 Metazoa; Arthropoda; Crustacea Neocalanus tonsus, AF367713 (858) Metazoa; Brachiopoda; Linguliformea Lingula anatine, U08331 (0.483) JPeuk-38 0.45 Viridiplantae; Chlorophyta; Prasinophyceae Mantoniella Antarctica, AB017128 (795) Viridiplantae; Chlorophyta; Prasinophyceae Ostreococcus tauri, Y15814 (0.608) JPeuk-40 0.45 Eukaryota; environmental samples Eukaryote marine clone ME1-1 , AF363153 (932) Viridiplantae; Chlorophyta; Trebouxiophyceae Lobosphaera tirolensis, AB006051 (0.713) JPeuk-51 0.45 Eukaryota; environmental samples Eukaryote marine clone ME1-3 , AF363155 (957) Viridiplantae; Chlorophyta; Trebouxiophyceae Lobosphaera tirolensis, AB006051 (0.716) JPeuk-119 0.45 Eukaryota; environmental samples Eukaryote marine clone ME1-3, AF363155 (660) Viridiplantae; Chlorophyta; Chlorophyceae Coelastrella multistriata, AB012846 (0.487) JPeuk-59 1.2 Stramenopiles; Bacillariophyta; Coscinodiscophyceae; Skeletonema pseudocostatum, X85393 (833) Stramenopiles; Bacillariophyta; Coscinodiscophyceae Skeletonema costatum, M54988 (0.761) JPeuk-65 1.2 Eukaryota; environmental samples Uncultured eukaryote clone LEMD134, AF372806 (321) Alveolata; Apicomplexa; Piroplasmida Theileria sp., AB012202 (0.453) JPeuk-71 1.2 Alveolata; Dinophyceae; Gymnodiniales Amphidinium semilunatum, AF274256 (900) Alveolata; Dinophyceae; Gymnodiniales Karlodinium galatheanum strain KT-77B, AF172712 (0.738) JPeuk-77 1.2 Viridiplantae; Chlorophyta; Trebouxiophyceae Nannochloris sp. R C C 011, AJ131691 (977) Viridiplantae; Chlorophyta; Trebouxiophyceae Nannochloris sp. R C C 011, Al l31691 (0.917) JPeuk-80 1.2 Eukaryota; environmental samples Eukaryote clone OLI11511, AJ402343 (1007) Alveolata; Dinophyceae; Prorocentrales Prorocentrum micans, M14649 (0.603) JPeuk-84 1.2 Alveolata; Ciliophora; Spirotrichea Stylonychia mytilus, AJ310498 (400) Alveolata; Ciliophora; Spirotrichea Halteria grandinella, AF164137 (0.448) JPeuk-94 1.2 Alveolata; Dinophyceae; Gymnodiniales Amphidinium semilunatum, AF274256 (874) Alveolata; Dinophyceae; Gymnodiniales Karlodinium galatheanum, AF172712 (0.756) JPeuk-108 1.2 Fungi; Ascomycota; Pneumocystidomycetes Pneumocystis carinii, X12708 (587) Unidentified eukaryote 18S ribosomal R N A , AJ130849 (0.638) JPeuk-113 1.2 Eukaryota; environmental samples Eukaryote clone OLI11511, AJ402343 (1013) Alveolata; Dinophyceae; Prorocentrales Prorocentrum micans, M14649 (0.571) a. Taxonomy, name, GenBank accession #, and bit score of the highest scoring sequence is shown. b. Taxonomy, name, GenBank accession #, and similarity value of the most similar sequence is shown. 64 The salinity and temperature of the water at Jericho Pier varied seasonally with the highest temperature and lowest salinity occurring during summer and the opposite true of the winter months (Figure 4.4). During winter, temperature and salinity fluctuated less than at other times of the year. With the exception of the winter months, when it was lowest, Chlorophyll a concentrations were variable. Because the local tidal cycle is mixed diurnal, the tide height at Jericho Pier alternated at the times of sample collection. Visual inspection of the composite D G G E images revealed that the banding patterns of the AVS fingerprints (Figure 4.4) appeared less variable than the 18S fingerprints (Figure 4.5). Throughout most of the study period, especially during the months March, April and May, the 18S fingerprint patterns changed from week to week while the AVS patterns were relatively constant. Nonetheless, bands present throughout most of the sample period were observed in all three fingerprints. For example, the bands indicated by an A in the AVS fingerprint can be observed in all but a few samples, and similarly, the bands labeled euk51 and eukll3 were present in the majority of the 0.45 - 1.2 pm 18S and > 1.2 pm 18S samples respectively. On the other hand, some bands were observed in only a few samples. The band avs55 was observed in the January 25th, 2001 fingerprint and was present in every sample until April 4th, 2001; a band at the same position was also present at the beginning of study (March 10th, 2000) and remained until April 7th, 2000. In addition, band avs53 was only present in the February and March 2001 fingerprints, band ' C appeared in only two 0.45 - 1.2 pm 18S fingerprints, and band 'D ' appeared in only one > 1.2 pm 18S fingerprint. Overall, patterns of band appearance and disappearance were observed in all fingerprints. Visual inspection also revealed changing fingerprint patterns coincident with changes in the physical characteristics of samples from Jericho Pier. From the beginning of November to the end of February the salinity was > 25 ppt. while the temperature was < 15 °C and chlorophyll a concentrations were < 3 pg l" 1 . During the same period, the 0.45 - 1.2 pm and the > 1.2 pm 18S fingerprints appeared less variable than at other times of the year; for both size classes, several bands were present in every sample during this period (Figure 4.5). Similarly, the A V S fingerprints remained relatively constant through the winter months, albeit with later timing than the 18S fingerprints; the AVS fingerprints changed little from the middle of December to the middle of March (Figure 4.4). Within the study period, the chlorophyll a concentration was 65 highest on 27 July 2000 at approximately 59 pg l " 1 . High concentrations were also observed on 10 October 2000 (24 pg 1"') and 26 April 2001 (22 pg l"1). The A V S fingerprint pattern on 10 October changed relative to the flanking fingerprints, yet the fingerprints from the 27 July and 26 April samples did not change compared to their flanking fingerprints Cluster analysis of each fingerprint set and inspection of the resulting U P G M A trees revealed that the AVS fingerprints were more similar to one another than were the 18S fingerprints (Figure 4.6). With the exception of the samples collected on 10 March and 19 May 2000, samples from March to May 2000, indicated by white bars in the figure, clustered beside one another in the AVS tree yet were distributed among five separate clusters in each 18S tree. Similarly, with the exception of the fingerprint from 5 October 2000, the fingerprints from mid September to early December, indicated by black bars in the figure, grouped into one cluster in the A V S tree yet were scattered throughout the 18S trees. As a final example, fingerprints from February and March 2001, indicated by grey bars, formed a single group in the AVS tree and were once again distributed throughout the 18S trees. In general, UPGMA analysis of pairwise densitometry correlations illustrated that the AVS fingerprints were more similar to one another, formed fewer clusters, and samples from the same time of year tended to form more similar clusters when compared to either set of 18S fingerprints. 66 o ^ N i - m o i N i f l a n N o ^ N T - i o i i M v i o ^ a i - i o o i - N n ^ o co ^ C ^ C S ^ g ^ g ^ C X J ^ C N J - r - C J O O J O - ^ O T - C O - i - C N ^ O O O C M O C N O - ^ O o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o Figure 4.4. Physical data and AVS fingerprints from Jericho Pier Top: salinity ( o ) , temperature (•), and Chlorophyll a (A) in weekly samples collected at Jericho Pier. Middle: composite image of AVS fingerprints. The callouts on the gel image show the positions and labels of bands sequenced or discussed in the text. Bottom: predicted tidal heights at the time of sample collection are shown. Sample dates are written as YY/MM/DD. 67 o r - i - i n o > c M i o o > n r ^ o ^ - r > » i - m o > C M C 0 o ^ ' c o T - m co f~- c\j ^- co co ^ g c j ^ c j ^ c j ^ c i i ^ w ^ ^ g N g ^ o ^ n i - W T - N o CM o C\J o i - o C O C O ^ 5 Ifi lO CD CD C D S S C O O O O i m O O ^ T ^ T ^ ^ C ^ ^ ^ C\J CVJ CO C O ( ^  ^ LO O O O O O O O O O O O O O O O T - - T - - T - T - T - T - T - 0 0 o o o o o o o o o o o o o o o o o o o o o o o o o o o o o ^ ^ ^ ^ ^ ^ ^ ^ ^ o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o Figure 4.5. Physical data and 18S rDNA fingerprints from Jericho Pier Top: salinity (o), temperature (•), and Chlorophyll a (A) in weekly samples collected at Jericho Pier. Middle: DGGE fingerprints of 18S rDNA amplified from 0.45 to 1.2 um size fraction extracts. Bottom: DGGE fingerprints of 18S fingerprints from the > 1.2 um size fraction extracts. Callouts show the positions and labels of bands sequenced or discussed in the text. Sample dates are written as YY/MM/DD. 68 rEXri N N I - co co in cu r-- T O * - » - co I D o n N O B O c o r o i n s i n c o N V t M S ' - r c e o , e : e o v o i ^ < 0 ' - t o c \ i a CM (O cn m s co rj'-OOJ'-*-OOi-00 CM O CM O CM TT. • " W O N I - W O I C M T - T - O I - O C J O C J CO CM T- O CM OJ CM O r- T- CJ i t t ! ! ; t r r l 5 0 0 , ' 0 < o < o n ^ " N CM CM c o o j c o ^ j - c o o o c B c n o o *- i - * - *- CM ••- co co n v r t r i o m m c o c o c o c o v m m o o o o o o » - o o o o O O O O O O O ^ - O O O O T - O O * - I- y- I- Q I- y- o o o o o o o o o o o o o o o o o o 88880888888 0000 00 8 800 888808 0000008 88808 8888888800 000 1 1 00 07 06 1 1 00 06 08 1 00 08 17 ——i | 00 07 20 ' 01 03 28 —1 LI 010314—' 1 01 04 04 1 01 0411 —1 | 001012 1 | 00 1026 1 CM g> N s n co 0 • - T c CM O o o CM « - CO »— C CO T- i- CM T- T- CO CO CM CI 0 T- 1- 1- 1- o o c T - O Q O Q Q Q O © C 00 0000 000c ^ ^ • M 1 1 "1 J CM CO ' 5 T" CM ( 3 m -r -5 O O C 5 8 S ! 00 06 26 I 1 J 00 05 19 1 Figure 4.6. Cluster analysis of DGGE fingerprints UPGMA trees of sample similarities for the AVS (A), 0.45-1.2 Ltm 18S (B), and > 1.2 (xm 18S (C) fingerprints. The scales to the right of each dendrogram show percent similarity. The resolution of each tree (maximum similarity) was estimated from the minimum similarity of standard lanes (data not shown). The black, gray and white bars below the dates of each dendrogram show the positions of sequences discussed in the text. Sample dates are written as YY/MM/DD. 69 4.5 Discussion The goal of this study was to relate changes in algal virus communities at Jericho Pier over 14 months to changes in eukaryotic diversity, chlorophyll a concentration, temperature, salinity and predicted tidal height. Sequencing efforts revealed at least five ecologically distinct taxa of viruses and several kingdoms of eukaryotes. At certain times, tidal height and salinity changes were coincident with changes in the algal virus community composition. Similarly, blooms of certain phytoplankton were coincident with shifts in algal virus community structure. In general, the composition of the algal virus communities was temporally less variable than that of the eukaryote communities. Before algal virus communities could be monitored using molecular fingerprint techniques, the fidelity of PCR with the primers AVS1 & 2 had to be improved. Established PCR conditions permitted amplification of sequences not related to algal virus pol fragments. In a previous study, we demonstrated that production of these PCR artifacts was due to amplification from the upstream primer AVS1 alone (104). The calculated melting temperature of this primer is approximately 10 °C higher than the downstream primer AVS2 and therefore efforts to increase the stringency of AVS-PCR by increasing the annealing temperature failed. However, when the annealing temperature was reduced the artifact disappeared. It is likely that the increased specificity of the reaction was due to the increased annealing efficiency of AVS2 at lower temperatures. Although AVS PCR with filter extracts was not successful, optimization of the reactions with these templates was not exhaustive. Further efforts may permit the amplification of algal virus pol fragments from nucleic acids extracted from filters. Finally, It is worth noting that every band sequenced in this study was closely related to known algal-virus pol sequences (Figure 4.3), and therefore it is likely that the fingerprints represented algal-virus pol fragments. Many algal virus sequences recovered from Jericho Pier were more than 98 % identical to each other and sequences from other environmental samples. Two sets of sequences (JPavs51 & 52, and JPavs64 & 65) differed by only 3 and 2 nucleotides, respectively. It should be noted that these sequences were recovered from bands at different positions in the fingerprint and sequence differences were due to nucleotides incorporated at sites of primer degeneracy. Therefore, quantitative examination of algal virus richness was not possible with degenerate-primer PCR 70 and DGGE. Nonetheless, when primer positions were excluded, no other sequences recovered from the algal virus were more than 99.8 % identical and it is unlikely that observed differences were due to PCR error alone. Using a Taq error rate of 0.27 x 10"4 misincorporations per bp per cycle (9), on average only 1 base per sequence would be misincorporated during algal-virus pol amplification; this is equal to an identity difference of ca. 0.1 %. Microdiversity, loosely defined as sequence differences less than 3 %, has been observed in other microbial communities and corresponded to physiologically different ecotypes of a single species (74). Microdiversity has also been observed among algal viruses that infect the same host. The major proteins or genomes of the virus isolates MpV-SPl and MpV-SGl are distinct (25), yet their pol fragment sequences, excluding primer sites, are 99 % identical. Currently we do not know if pol sequences that differ by less than 1 % represent ecologically different viruses. Nonetheless, the ten sequences recovered from Jericho Pier resolved onto five separate branches of the maximum likelihood tree (Figure 4.3) and the maximum nucleotide identity between any two sequences on separate branches was ca. 92 %. In comparison the minimum nucleotide identity between any MpV pol fragments was ca. 98 %. Therefore, it is likely that each of the five branches represent distinct virus taxa. All sequences recovered from the 18S fingerprints were related to small subunit rDNA sequences. The eukaryote communities were amplified and fingerprinted from the nucleic acids extracted from ca. 2.5 % of the total filter area; this was equal to a sample volume of approximately one liter. Although the communities were fingerprinted from a relatively small volume, the fingerprint patterns were complex and, through most of the study period, changed from week to week. While the majority of sequences recovered from the 18S fingerprints were related to phytoplankton, three sequences were related to zooplankton, two to fungi and one to a terrestrial plant. It is interesting to note that four of the ten sequences recovered from the 0.45 -1.2 pm 18S fingerprints were related to organisms that should have been retained by the 1.2 pm filter. Nonetheless, sequences related to large phytoplankton, e.g. the diatom Skeletonema and the dinoflagellates Amphidinium or Prorocentrum, were recovered from the 1.2 pm glass fiber filter only. However, because only a limited number of bands were sequenced, the recovered sequences did not quantitatively represent the organisms present. Nonetheless, sequences from the 18S fingerprints demonstrate that several kingdoms of eukaryotes were detected with the described PCR methods. 71 During this study, changes in the virus fingerprints were often related to the physical characteristics of the water at Jericho Pier. The relationship between algal virus community dynamics and the physical environment was apparent when the A V S fingerprints were compared to tide height. At certain times, changes in the tidal height coincided with changes in the A V S fingerprint patterns. The samples from 29 June to 10 August 2000 were collected during alternating high and low tides. At the same time, the position of the top band in each AVS fingerprint alternated coincident with changes in tidal height (Figure 4.4). On the other hand, the fingerprint patterns of virus communities sampled at each low and high tide from May 4th to 6th 2001 did not change (data not shown). Intra-annual variability of the tidal influence on virus communities may be related to other aspects of the physical characteristics of the sample site. During the weeks that tide height changes were coincident with changes in the pattern of successive A V S fingerprints, the salinity was lowest indicating that the Fraser River freshet had peaked; the surface water at Jericho Pier is strongly influenced by freshwater input from the Fraser River (46). During the freshet, freshwater surface flow may have caused more pronounced shifts in phytoplankton and virus community composition at low and high tide than at other times of the year. Another example of the environmental influence on AVS fingerprint patterns was the change in the pattern coincident with one of the three phytoplankton blooms observed during the study; during each bloom the chlorophyll a concentration exceeded 20 pg F 1. These blooms occurred in summer 2000, autumn 2000, and early spring 2001 (Figure 4.4). Examination of Lugol's iodine (85) preserved samples revealed that the summer bloom was a nearly monospecific bloom of the Raphidophyte Heterosigma akashiwo. In contrast, the fall bloom was mixed and dominated by the diatoms Ditylum, Leptocylindrus, and Pseudonitzschia and the dinoflagellates Prorocentrum, Dinophysis and Protoperidinium and the 2001 spring bloom was dominated by the diatoms Skeletonema, Chaetoceros, and Thalassiosira. The fact that the AVS fingerprint did not change during the Heterosigma bloom in the fall or the diatom bloom of the spring was explicable. Although isolates of viruses that infect Heterosigma are established in culture (e.g. 80, 65), we have not successfully amplified pol sequences from these virus suggesting that the AVS primers are not suitable. Furthermore, to our knowledge, no viruses that infect diatoms have been isolated. On the other hand, during the mixed diatom and dinoflagellate bloom of the fall the band-richness of the > 1.2 pm 18S fingerprint was higher than average 72 suggesting that the bloom was a diverse group of phytoplankton. At the same time, the band pattern of the A V S fingerprints changed relative to the fingerprints from surrounding weeks. While these data do not establish a causal relationship between the change in the eukaryote richness and the A V S fingerprint pattern, the coincidence is intriguing; it is likely that the presence of Phycodnaviridae hosts is most probable when eukaryote diversity was high. Also interesting is the fact that recent studies have demonstrated the existence of viruses that infect marine dinoflagellates (e.g. 115, 132) and many were present in the sample from the fall bloom. Because the phytoplankton blooms were not all coincident with AVS-fingerprint changes, it is likely that the viruses detected with the A V S primers infected few of the dominant bloom-forming algae at Jericho Pier. In general, the algal virus fingerprints were less variable than eukaryote fingerprints. From inspection of the composite gel images, it was apparent that the majority of bands in the AVS fingerprints persisted through many successive samples while many bands in the 18S fingerprints changed weekly. While the algal virus fingerprint patterns were stable over most of the year and obvious pattern shifts were rare, the eukaryote patterns were more variable and consistent successive patterns were observed only during the winter months. This contrasting variability of the two communities was not surprising; the viruses detected with the A V S primers all belonged to the Phycodnaviridae (DNA viruses that infect eukaryotic phytoplankton) and therefore, infected only a small subset of eukaryotes. Although the majority of sequences from the 18S fingerprints were related to phytoplankton, several were not. Because only a limited number of bands were sequenced from the 18S fingerprints, the ratios of taxa represented in the sequence data cannot be used to estimate the abundance of different groups of eukaryotes. However, the 18S sequences confirmed the presence of organisms that are not hosts of phycodnaviruses. The presence of non-host organisms may explain the increased complexity of the 18S compared to the A V S fingerprints and the lack of correspondence between virus and eukaryote community dynamics. In the future, development of PCR methods to fingerprint specific groups of phytoplankton, when combined with the virus fingerprinting methods outlined here, will permit comparison of changes in algal virus communities and their hosts. Because the limitations of computerized gel analysis are well documented (e.g. 32), we refrained from detailed comparison of the UPGMA trees. To minimize the possibility that seasonal patterns were due to within gel biases, the order of samples loaded in the gradient gels 73 was arbitrary. In spite of the limitations of fingerprint cluster analysis, gel comparison software was essential to this study; the normalization of individual samples and alignment of common bands within a fingerprint database was not possible without it. Computerized normalization and alignment permitted temporal ordering of fingerprints not run in successive gel lanes. As others have stated, fingerprint approaches can provide useful qualitative information on the spatial and temporal variability of microbial communities (32). Significantly, the fingerprints obtained during this study revealed the temporal variability of both eukaryote and algal virus communities. In all three composite fingerprint sets, bands appeared and disappeared in successive samples and, within each fingerprint set, patterns and variability changed with time. In addition, the lack of coincidence between changes in algal virus and eukaryote community composition was corroborated by cluster analysis of the fingerprints. The minimum similarity of any branches on the AVS tree was higher than the minimum on the 18S trees and, when compared to 18S fingerprints, the AVS fingerprints clustered into groups with higher minimum similarity (Figure 4.6). The cluster analyses also showed that the AVS fingerprints from the same season tended to cluster in larger groups than the 18S fingerprints. There is accumulating evidence that some phytoplankton are infected by viruses not closely related to the Phycodnaviridae (e.g. 65). In addition, our attempts to amplify pol genes from viruses belonging to the Phycodnaviridae (e.g. the viruses EhV, FsV, and EsV-1 that infect Emiliania huxleyi and zoospores of Feldmannia sp. and Ectocarpus siliculosus, respectively) have always failed. Thus, it is apparent that the viruses detected with the AVS primers are only a subset of the Phycodnaviridae. Therefore, the lack of coincidence between changes in algal virus and eukaryote communities at Jericho Pier was not surprising. Although they were not coupled with changes in algal virus community composition, changes were detected in eukaryote communities. Through most of this study, the weekly sampling interval revealed highly variable eukaryote community composition yet relatively stable algal virus community composition. This is the first study demonstrating that specific taxa of algal viruses are temporally stable and can persist in fluctuating physical and biological environments. This suggests that the production of some taxa of algal-viruses is constant and therefore, they are a constant source of mortality to some phytoplankton. Therefore, we can speculate that these viruses influence phytoplankton community structure and may help maintain high host diversity. As well, this study demonstrates that D G G E can be used to follow temporal changes in algal virus community structure. This tool 74 should be useful for further investigations of algal virus community composition and the factors that influence this poorly understood but ecologically important group of viruses. 75 Chapter V. Conclusion The development of a technique to fingerprint gene fragments amplified from algal viruses facilitated investigations of these viruses in nature. Using the PCR and D G G E protocols described in this thesis I was able to study the geographical and temporal diversity of marine algal viruses in nature. Moreover, molecular fingerprinting facilitated the recovery of sequence information from naturally occurring algal viruses. The methods described in this thesis provided new information about the diversity of marine algal viruses and can be applied to further investigation of these ecologically important marine microbes. Moreover, I have demonstrated that D G G E is a powerful technique for studying changes in viral community structure. Using alternative primer sets, and with minor modifications, it should be possible to employ this approach to investigate diversity in a wide range of viral communities. After denaturing gradient gel electrophoresis techniques were optimized for the resolution of pol fragments amplified with degenerate algal-virus-specific primers, algal virus communities were fingerprinted from a variety of samples. These studies demonstrated that qualitative comparison of algal virus communities was possible using PCR and DGGE. Furthermore, fingerprint patterns werereproducible and many PCR reactions and D G G E analyses were repeated with consistent results. The fingerprints of two samples collected from different depths in one British Columbia inlet were identical suggesting that the viruses at this location were homogenously distributed with depth (e.g. lanes 6 and 7, Figure 3.2). At this location the salinity and temperature did not vary with depth, indicating that the water column at this location was well mixed. On the other hand, the fingerprints of samples from different depths in another BC inlet were not similar (e.g. lanes 10 and 12, Figure 3.2). At this location the salinity of surface water was half of that at 19 m, indicating the water and viruses were stratified with depth. It was not surprising that the similarity of algal virus community composition was related to the physical characteristics of the environments sampled. Algal virus communities were also fingerprinted from samples collected weekly at a single location for slightly more than a year (Figure 4.4). Comparison of these fingerprints with environmental conditions revealed that changes in algal virus community composition were often coincident with changes in tidal height, salinity, or chlorophyll a concentration. However, it was surprising to observe that the algal virus fingerprint patterns were relatively stable throughout the study when compared with the fingerprints of eukaryote communities. The eukaryote fingerprint patterns appeared to be stable only during the winter months. It was 77 apparent that specific taxa of algal viruses could persist in fluctuating physical and biological environments suggesting that the production of, and mortality from, some taxa of algal-viruses was constant. PCR and D G G E facilitated the recovery of gene sequences from natural algal virus communities. Remarkably, I found that very similar virus sequences could be recovered from geographically distant locations. For instance, some sequences recovered from the fingerprints of a Southern Ocean sample were closely related to sequences from coastal British Columbia, in some cases being > 98 % identical (Figure 3.3). This was the first time nearly identical viruses have been shown to occur in the Southern Ocean as well as in the temperate waters of the North East Pacific. The results of this study furthered the results of previous work demonstrating the cosmopolitan nature of some algal viruses. Analysis of algal virus sequences obtained during this dissertation revealed that, although MpV-SPI was, in part, used to design the algal virus PCR primers, sequences were amplified from viruses that infect marine phytoplankton other than Micromonas pusilla. To illustrate this point a phylogenetic tree was constructed with all of the pol fragments recovered during the research conducted for this dissertation (Figure 5.1). The unknown algal virus sequences did not cluster with known isolates of MpV. In fact, sequences of prymnesioviruses that infect different genera of phytoplankton (CbV and PgV) were more closely related to each other than any of the unknown sequences were to MpV. It should be noted that the PgV sequences were obtained from viruses that infect the prymnesiophyte Phaeocystis globosa. C. P. D. Brussaard at the Netherlands Institute for Sea Research provided these viral isolates and the sequences will be published collaboratively at a later date. The fact that viruses as similar as CbV and PgV infect different genera of phytoplankton indicate that even closely related viruses infect different phytoplankton. Because the unknown sequences recovered during this dissertation research form many clusters less similar to any other than PgV is to CbV, it is likely that the unknown sequences were amplified from viruses that infect many different genera, and perhaps classes, of phytoplankton. The results from this dissertation have bolstered the hypothesis that the degenerate primers AVS1 and AVS2 can be used to amplify pol gene fragments from a wide range of phytoplankton viruses. Furthermore, by increasing the efficiency and specificity of PCR with the 78 degenerate algal-virus-specific primers, the protocols described in the penultimate chapter of this thesis have increased the utility of this molecular approach to the study of algal virus communities in nature. Moreover, the technique developed to fingerprint PCR amplified algal virus pol fragments can be easily applied to future studies of the ecology and distribution of algal viruses in nature. The molecular fingerprinting technique developed for this dissertation can be applied to study algal virus community dynamics in both natural and artificial systems. Currently, there is little knowledge of the global distribution of members of the Phycodnaviridae. Although closely related viruses of Micromonas pusilla have been observed in the Pacific and Atlantic Oceans, as well as the Mediterranean and Gulf of Mexico (25), it is not known if this group of viruses is present throughout the world's oceans. Sequences obtained from fingerprints of samples collected around the globe will answer questions about the distribution and diversity of algal viruses in different ocean basins. In addition, fingerprints of virus communities in microcosms or mesocosms will permit detailed study of algal virus ecology. For example, it will be possible to determine how nutrient addition or limitation affects algal virus community composition. In addition to providing a new method to study algal virus communities in nature, this dissertation has revealed previously unknown information about Phycodnaviridae diversity and ecology. Thus, this work is a contribution to our knowledge of algal viruses and phytoplankton ecology, and therefore, global biogeochemical cycles. 79 • HSV-1 • EBV FsV EsV-1 I C W - P W 1 " w l — CbV-PW3 • PgV-C (- PgV-D - PgV-3 PgV-4 PgV-7 • PgV-8 0TU5 C NY2A r B S B 9 9 - 2 ~5e1 P S B 9 9 - 3 .61 | - P S C 9 9 - 2 P S B 9 9 - 1 P S B 9 9 - 2 P S B 9 9 - 4 S 0 9 8 - 2 S 0 9 8 - 3 B S A 9 9 - 6 • B S A 9 9 - 7 B S A 9 9 - 8 S I A 9 9 - 1 h P S C 9 9 - 1 J P - 3 7 B S A 9 9 - 2 MpV-PB6 MpV-PB7 MpV-PB8 MpV-GM1 — MpV-SP l — MpV-SP2 MpV-SG1 MpV-PL1 0TU1 0TU3 I t : • M I B 9 9 - 2 0.1 B S A 9 9 - 5 J P - 3 8 s®- J P - 2 2 B S A 9 9 - 3 J P - 6 5 B S A 9 9 - 1 • B S A 9 9 - 4 • J P - 5 5 • S 0 9 8 - 5 " 6 5 U jp-53 B S B 9 9 - 1 J P - 6 4 Figure 5.1. Maximum Likelihood tree of virus pol fragments. Maximum Likelihood tree of inferred amino acid sequences of dsDNA viruses with quartet puzzling support values to the left of corresponding nodes. 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