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The genetic analysis of aquatic cyanophage communities Frederickson, Cindy Marie 2003

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THE GENETIC ANALYSIS OF AQUATIC CYANOPHAGE COMMUNITIES by C I N D Y M A R I E F R E D E R I C K S O N B . S c , The University of Victoria, 1999 A THESIS S U B M I T T E D FN P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E in T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Department of Earth and Ocean Sciences) We accept this thesis as conforming to the required standard „ T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A M a y 2003 © Cindy Marie Frederickson, 2003 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of £flrlj flM Ociqn ZdlCnCe^ The University of British Columbia Vancouver, Canada Date DE-6 (2/88) Abstract Viruses are abundant in the marine environment and are important in terms of carbon and nutrient cycling. Through the specific lysis of host organisms, they play a role in structuring host communities of phytoplankton and bacteria. Phage, viruses that infect bacteria, are the most abundant virus-like particles in seawater, but relatively little is known about their biology, phylogeny and diversity. Previously, sequence analysis revealed a region of similarity among the genomes of three cyanophages belonging to the family Myoviridae and the capsid assembly gene (g20) of coliphage T4. These sequences were used to design two sets of putative cyanophage-specific P C R primers (CPS4GC/CPS5 and CPS4GC/CPS9) . In the first part of the study, natural viral communities were collected from 6 depths in each of 3 inlets in British Columbia, Canada. Cyanophage-specific primers C P S 4 G C and CPS 5 were used to amplify -205 bp (including 40 bp GC-clamp) g20 gene fragments from the samples, and denaturing gradient gel electrophoresis (DGGE) was used to fingerprint the natural cyanophage communities. The results showed that the cyanophage communities differed the most within inlets in which temperature and salinity changed the most with depth. Differences in cyanophage communities sometimes occurred in association with high chlorophyll fluorescence and/or high abundances of infectious cyanophage and Synechococcus cells. In order to obtain more sequence for phylogenetic analysis, a new primer (CPS9) was designed and used with C P S 4 G C to amplify a 595 base pair region of the g20 gene. These primers were used to amplify gene fragments from lakes in British Columbia and Germany, a catfish production pond in Louisiana, a cyanobacterial mat from a high arctic pond, as well as samples from both polar oceans, the Gul f of Mexico, northeast Pacific inlets and the coastal southeast Pacific. D G G E was used to separate the fragments, which were subsequently excised, re-amplified, cloned and sequenced. Phylogenetic analysis demonstrated high variability in the g20 gene sequences and revealed four previously unknown groups of phage, two of which consisted of sequences found only in freshwater. Sequences that were >99% identical in terms of pairwise nucleotide similarity were recovered from both freshwater and marine environments, suggesting that genetically similar phage are globally distributed. M y research has demonstrated that P C R combined with denaturing gradient gel electrophoresis can be used to compare marine cyanophage communities. I have shown that these communities can differ on spatial scales as small as metres and that this is influenced by the physical structure of the water column. A s well , by P C R amplifying the g20 gene from geographically distinct environments, I have shown that this group of phage is global in distribution. I have demonstrated the genetic richness of this group and have potentially identified new groups of freshwater phage. Table of Contents Abstract ii Table of Contents iv List of Tables vii List of Figures viii List of Abbreviations and Symbols ix Preface xi Acknowledgements xi v Dedication xv Chapter I. The Physical Environment Affects Cyanophage Communities in British Columbia Inlets 1 1.1 Summary 2 1.2 Introduction 3 1.3 Materials and Methods 6 1.3.1 Sample collection 6 1.3.2 Concentration of virus communities 6 1.3.3 Abundance of viral particles and Synechococcus spp 7 1.3.4 Estimation of abundance of cyanophages 7 1.3.5 P C R amplification 8 1.3.6 Denaturing gradient gel electrophoresis 9 1.4 Results and Discussion 11 1.4.1 Abundance of viruses, infective cyanophage and Synechococcus spp 11 1.4.2 Temperature and salinity characteristics 13 1.4.3 P C R amplification and reproducibility of denaturing gradient gels 15 1.4.4 Cyanophage D G G E patterns and water column structure 16 1.4.5 Coincidence of high chlorophyll fluorescence with differences in community patterns and high abundances of infective cyanophage and Synechococcus spp 18 1.4.6 Relating sequence richness to the number of bands on the denaturing gradient gel.... 19 iv 1.4.7 Sensitivity of D G G E , 20 1.4.8 Similarity of fingerprints as indicated by U P G M A cluster analysis 21 1.4.9 Conclusion 23 1.5 Acknowledgements 24 Chapter II. Genetic Variation Among Globally Distributed Marine and Freshwater Cyanophages 25 2.1 Summary 26 2.2 Introduction 27 2.3 Materials and Methods 32 2.3.1 Sample collection 32 2.3.2 Concentration of natural virus communities 32 2.3.3 Vi ra l D N A extraction from the cyanobacteria mat 34 2.3.4 P C R 35 2.3.5 Denaturing gradient gel electrophoresis ( D G G E ) 36 2.3.6 D N A sequencing 36 2.3.7 Obtaining sequences from cultured isolates 37 2.3.8 Phylogenetic Analysis 37 2.4 Results 38 2.4.1 The number of unique sequences obtained from the denaturing gradient gels varied between samples 38 2.4.2 Environmental sequences cluster with sequences from cyanophage isolates 38 2.4.3 Phylogenetic analysis reveals four new 'unknown' clusters 40 2.4.4 Variation in sequences obtained from a single location 42 2.4.5 Recovery of similar sequences from different environments 43 2.5 Discussion 44 2.5.1 Most important findings 44 2.5.2 Specificity of cyanophage-specific-primers 44 2.5.3 Separation of large fragments on denaturing gradient gels 44 2.5.4 The Cultured Synechococcus Phage (CSP) phylogenetic group 46 2.5.5 Diversity of phages with g20 genes 47 2.5.6 Are there groups that consist entirely of freshwater phage? 48 v 2.5.7 What is the significance of identical g20 sequences in different environments? 49 2.5.8 Addressing the question of diversity in natural virus communities and the usefulness of identifying 'signature' gene sequences 50 2.5.9 Conclusion 51 2.6 Acknowledgements 52 Chapter III. Summary 53 References 57 v i List of Tables Table 2.1. Details of sample locations 33 List of Figures Figure 1.1. Map of sampling locations 6 Figure 1.2. Abundance of viruses, cyanophage and Synechococcus 12 Figure 1.3. Temperature, salinity and chlorophyll fluorescence profiles 14 Figure 1.4. Agarose gel showing amplification of -200 bp g20 gene fragment 16 Figure 1.5. D G G E of P C R amplified g20 gene fragments from the three inlets 17 Figure 1.6. U P G M A dendrogram showing relatedness of cyanophage communities 22 Figure 2.1. D G G E of P C R amplified g20 gene fragments showing locations of excised and sequenced bands 39 Figure 2.2 Maximum likelihood tree of g20 fragments 41 List of Abbreviations and Symbols bp base-pair c o 2 carbon dioxide °c degrees Celcius C P S cyanophage-specific D G G E denaturing gradient gel electrophoresis dia diameter D N A deoxyribonucleic acid dNTP deoxyribonucleoside triphosphate d s D N A double-stranded D N A h hour k D kilodalton kPa kilopascal 1 litre M molar mg milligram min minute ml millilitre m M millimolar mm millimetre ng nanogram P C R polymerase chain reaction P F G E pulsed field gel electrophoresis pmol picomole ppt parts per thousand P V D F polyvinylidene fluoride s second U units U P G M A unwieghted paired group method with v/v volume per volume w/v weight per volume I X microgram microlitre micromolar Preface The research for my Master of Science degree focussed on the genetic analysis of aquatic cyanophage communities. The impetus for this project stemmed from the recent evolution of research on viruses in the oceans. Approximately 15 years ago, transmission electron microscopy revealed a high abundance of viruses in the marine environment and shortly thereafter, viruses that infect many species of phytoplankton including cyanobacteria were isolated. Phytoplankton form the basis of the food chain in the ocean and are responsible for approximately half of the global net primary production (32). They play a central role in the global carbon cycle via a mechanism known as the 'biological pump'. The biological pump delivers carbon from the atmosphere to the deep sea, where it is concentrated and sequestered for centuries. Because of the importance of marine phytoplankton and the discovery of viruses that infect them, scientists began to question the impact of marine viruses on the cycling of carbon and nutrients in the ocean and their role in structuring communities of phytoplankton and bacteria. Marine viruses affect the abundance and production of natural assemblages of phytoplankton and there is some evidence of viral control of bacteria and phytoplankton community structure. In order to better assess the impact of marine viruses, we must determine the species that are infected by viruses and the variation within groups of viruses that infect a single species. This is not an easy task, as the majority of marine phytoplankton and bacteria have not been cultured. Therefore, our approach has been to use molecular techniques to estimate the genetic richness of virus communities and to elucidate relationships between different viruses. Molecular probes have been used to target conserved genes in marine viruses, such as D N A polymerases. Phylogenetic analysis of conserved gene sequences from cultured isolates has shown, at least in the phytoplankton viruses, that phylogenetic groupings reflect the host species. x i If through further investigation we find this trend in other groups of aquatic viruses, it may be possible to determine types of unknown viruses by P C R amplifying and phylogenetically comparing these target gene sequences from natural environments. A t the beginning of my degree, a conserved region had been discovered in the genomes of three cyanophage isolates that was homologous to the g20 gene of coliphage T4. A primer set had been designed to amplify a 165 bp region of the cyanophage g20 gene and the goal of my project was to use these primers to amplify g20 genes from the coastal waters of British Columbia and to use denaturing gradient gel electrophoresis ( D G G E ) to compare communities of cyanophage. I used these tools to compare communities from six different depths in three British Columbia inlets. I was able to show that the cyanophage community structure reflected the physical structure of the water column and that the communities differed over distances of metres. This work is presented in Chapter I and is published as C. M. Frederickson, S. M. Short and C. A. Suttle. 2003. The physical environment affects cyanophage communities in British Columbia inlets. Microbial Ecology. A t the time of the writing and assembling of this thesis the publication was ' in press'. M y next goal was to obtain sequence information to phylogenetically compare g20 sequences obtained from a wide range of aquatic environments. Because we thought that there may not be enough sequence information in the 165 bp product for phylogenetic analysis, I redesigned the primers to amplify a larger region of the g20 gene. The primer set that I designed included the original upstream primer and a new degenerate downstream primer which together would P C R amplify an approximately 595 bp region of the g20 gene. Wi th these primers, I was able to amplify the fragment from both freshwater and marine environments on a global scale. This was the first amplification of cyanophage g20 genes from freshwater environments of which I am aware. The phylogenetic comparisons demonstrated the genetic richness of x i i this group of cyanophage and showed that genetically similar phage are widely distributed. This work is presented in Chapter II and is titled 'Genetic variation within globally distributed marine and freshwater cyanophage communities'. This work should be submitted for publication in the journal 'Applied and Environmental Microbiology' in the near future. I am the primary author of the first manuscript and w i l l be the primary author of the manuscript that results from Chapter n. I designed and executed the experiments, collected many of the samples and wrote both manuscripts. The second author on the manuscript 'The physical environment affects cyanophage communities in British Columbia inlets', Steven M . Short, devised the sampling scheme, collected and processed the samples, trained me in the realm of molecular biology and provided insightful discussions on the work. There w i l l be no second author on the manuscript 'Genetic variation within globally distributed marine and freshwater cyanophage communities'. The final author on both manuscripts is Curtis A . Suttle, the primary investigator of the laboratory where I conducted my research. He provided the impetus for the work, the workplace and materials and edited the manuscripts. I certify that the preceding statements about authorship are correct. x i i i Acknowledgements The most important person to me during my thesis work was Steven M . Short. Without the mentorship, support and inspiration he provided, I would not have been able to complete this degree. I owe a great deal of gratitude to my supervisor Curtis Suttle, who provided a great place to work with an awesome group of people and who was always supportive and encouraging. M y thanks also go to my parents and family members whose financial and emotional support was much appreciated and to my friends outside of the University of British Columbia who were always there when I needed them. I would like to thank my committee members Rosemary Redfield and B i l l M o h n for their ideas and discussions about my results and A m y Chan for her advice and instruction in the lab. I would like to acknowledge all of the members of the Suttle lab (Sean Brigden, Jessica Clasen, Alex Culley, Andre Comeau, Emma Hambly, Craig Kalnin, Cindy Kam, Andrew Lang, Janice Lawrence, Pascale Loret, Nicole McLearn, Nina Nemcek, Al ice Ortmann, Karen Reid, Tanya St. John, Vera Tai , and Kevin Wen) for making the lab such a great place to work. I would also like to thank the department of Earth and Ocean Sciences for the Robert Rutherford Rae Memorial Scholarship and the travel award that I received. xiv Dedication T o B XV Chapter I. The Physical Environment Affects Cyanophage Communities in British Columbia Inlets Published as: Frederickson, C. M . , Short, S. M . and C. A . Suttle. 2003. The physical environment affects cyanophage communities in British Columbia inlets. Microbial Ecology. In press. 1 1.1 Summary Little is known about the natural distribution of viruses that infect the photosynthetically important group of marine prokaryotes, the cyanobacteria. The current investigation reveals that the structure of cyanophage communities is dependent on water column structure. P C R was used to amplify a fragment of the cyanomyovirus gene (g) 20, which codes for the portal vertex protein. Denaturing gradient gel electrophoresis ( D G G E ) of P C R amplified g20 gene fragments was used to examine variations in cyanophage community structure in three inlets in British Columbia, Canada. Qualitative examination of denaturing gradient gels revealed cyanophage community patterns that reflected the physical structure of the water column as indicated by temperature and salinity. Based on mobility of P C R fragments in the D G G E gels, some cyanophages appeared to be widespread, while others were observed only at specific depths. Cyanophage communities within Salmon Inlet were more related to one another than to communities from either Malaspina Inlet or Pendrell Sound. A s well , surface communities in Malaspina Inlet and Pendrell Sound were different when compared to communities at depth. In the same two locations, distinct differences in community composition were observed in communities that coincided with depths of high chlorophyll fluorescence. The observed community shifts over small distances (only a few metres in depth or inlets separated by less than 100 kilometres) support the idea that cyanophage communities separated by small spatial scales develop independently of each other due to isolation by water column stratification or land mass separation which may ultimately lead to changes in the distribution or composition of the host community. 2 1.2 Introduction Following the initial discovery of abundant marine virus particles in the late 1990's, defining the role of viruses in the world's oceans has become an important field of research. Besides being numerically abundant, viruses are also a diverse and dynamic component of ocean systems that infect heterotrophic and autotrophic prokaryotes, and eukaryotes (2, 4, 39, 52). Photosynthetic and heterotrophic prokaryotes are extremely important, representing an estimated 90% of l iving marine biological carbon (59). Among these, the largest, and most widely distributed group of photosynthetic prokaryotes are cyanobacteria. Members of the cyanobacterial genus Synechococcus are one of the most abundant picoplankton (up to 10 6 ml"1) (58) and are significant primary producers, responsible for 20-95% of carbon fixation in aquatic environments (17, 27, 31). Viruses that infect single strains of Synechococcus are widespread (reviewed in 49) and can reach abundances in excess of 10 5 per ml in coastal seawater (33, 50, 51, 57, 61). The viruses from these communities that are most frequently isolated belong to the family Myoviridae, and exhibit considerable variation in morphology, genetic diversity and host range (50, 57, 61). There is also evidence that marine cyanophages are significant mortality agents of cyanobacteria. A n estimated 1.5-6 percent of Synechococcus cells in natural communities are visibly infected (39) and cyanophage contact rates and decay rates suggest that up to 6.6 percent of Synechococcus cells are lysed per day in coastal and offshore environments (48, 51, 57). Cyanobacterial lysis due to virus infection diverts nutrients and energy from plankton to heterotrophic bacteria, ultimately reducing the transfer of production to higher trophic levels (13, 59). There is also evidence that viruses regulate community diversity of marine bacteria and phytoplankton (24, 35, 54, 64). Due to their high abundance, diversity and activity in marine 3 environments, cyanophage likely influence host community structure and diversity. In addition to influencing host community composition, cyanophage potentially affect the genetic diversity of host communities, through recombination and lysogenic conversion (20, 34, 37). Studies of cyanophage diversity have primarily focused on isolates maintained in the laboratory. Isolates have been characterized in terms of morphology, host range, protein composition, genome size and restriction digest pattern (reviewed in 49). Molecular techniques are currently being used to describe natural cyanophage communities qualitatively and quantitatively to further understand their ecological and genetic role in the marine environment. For example, signal-mediated amplification of R N A technology ( S M A R T ) has been developed to detect cyanophage D N A in mixtures of target and non-target nucleic acids in the laboratory with the goal of simultaneously detecting multiple targets in the environment (18). Recently, a conserved region of three cyanomyoviruses was sequenced and found to be homologous to the T4 g20 gene that encodes a portal vertex protein which functions in capsid assembly (16). P C R primers designed from alignments of the three sequences were used to quantify individual cyanophage isolates in the laboratory using a competitive P C R method (16). Slightly modified P C R primers used with denaturing gradient gel electrophoresis showed that cyanophage communities differed in structure along a transect in the Atlantic Ocean (62). Another set of P C R primers designed to amplify a 592 bp target of the same g20 cyanophage gene were also developed (66). Fragments sequenced from coastal and open ocean environments revealed that cyanophage communities were highly diverse, forming nine clades and that the most closely related sequences were not amplified from the same location. P C R and D G G E have also been used to examine the genetic diversity of algal viruses and phylogenies based on D N A polymerase gene sequences have shown that viruses within the family Phycodnaviridae form separate clades that reflect the family of the host alga (9,42,43). 4 Despite the work done to date, the diversity of cyanophage on scales of meters to kilometers has not been examined in relation to the structure of the water column. The objective of this study was to determine i f the physical environment influences cyanophage communities obtained from three nearby inlets. Cyanophage community structure was compared in coastal waters differing in physical structure (as determined by temperature and salinity profiles) using denaturing gradient gel electrophoresis to separate P C R amplified g20 gene sequences. 5 1.3 Mater ia ls and Methods 1.3.1 Sample Collection Water samples were collected from three inlets (Salmon Inlet, Malaspina Inlet and Pendrell Sound) adjacent to the Strait of Georgia, British Columbia, Canada from August 18-21, 1999 (Fig. 1.1). The depth of the seafloor was 281 m, 30 m and 72 m in Salmon Inlet, Malaspina Inlet and Pendrell Sound respectively. Samples were collected from six depths at each location from the surface to a maximum depth of 25 m using a rosette sampling system fitted with Niskin bottles and a C T D (Seabird Electronics). The C T D was used to measure in situ profiles of temperature, salinity and chlorophyll fluorescence (relative units). Figure 1.1. Map of sampling locations. Water samples were collected from six depths from each of the three inlets (•). 1.3.2 Concentration of Virus Communities Natural virus communities were concentrated from seawater using ultrafiltration as previously described (53). Briefly, between seventeen and twenty liters of seawater were gently 6 filtered through 142-mm diameter, 1.2-um nominal pore-size M F S G C 5 0 glass-fiber filter, (Advantec M F S ) and 0.45 p,m pore-size low-protein binding Durapore membrane filters (Millipore) to remove zooplankton, phytoplankton and most bacteria. The viruses in the filtrate were concentrated to a final volume ranging from 130 to 180 millilitres, using a 30, 000 -MW-cutoff, spiral-wound Amicon S1Y30 ultrafiltration cartridge (Millipore) and were stored in the dark at 4 °C for up to 2.5 years until use. Natural cyanophage communities can be stored for at least a year at 4 °C without significant loss of titer (Rodda and Suttle, unpublished). 1.3.3 Abundance of viral particles and Synechococcus spp. Vira l particles were quantified by epifluorescence microscopy following staining with Yo-Pro-1 (Molecular Probes) (23). Immediately following sampling, two hundred microliters of each seawater sample was diluted with 800 u l of ultrafiltered (<30,000 Da) seawater. The ultrafiltered seawater was prepared using an S1Y30 ultrafiltration cartridge as described above. O f the 1 ml diluted sample, 800 u l was filtered onto a 0.02 \im pore-size filter (Anodisc 25, Whatman), stained for 48 h in the dark at room temperature, and enumerated with an Olympus B X 6 0 epifluorescence microscope using blue-light excitation. Epifluorescence microscopy was used to enumerate Synechococcus spp. using the same Yo-Pro-1 stained virus slides. Autofluorescent cyanobacteria were counted in a minimum of 20 microscope fields and typically 200 cells were counted, except in deeper samples where cells were much less abundant. 1.3.4 Estimation of Abundance of Cyanophages The abundance of cyanophages in the viral concentrates was estimated using a most-probable-number ( M P N ) assay with the cyanobacteria strain D C 2 (=WH7803) (50). M P N assays were conducted 6 to 10 months after sample collection. The concentrations of cyanophages 7 infecting D C 2 were estimated by adding 200 ul of exponentially growing cyanobacteria into each well of a 96-well microtiter plate, followed by 50 u.1 of the viral concentrate which was 10-fold serially diluted in autoclaved artificial seawater media. Each dilution was replicated sixteen times and controls received only artificial seawater media. The plates were incubated under continuous irradiance (20 umol quanta m" 2 s"1) at 22 °C and the wells monitored for 7 to 10 days for evidence of cell lysis. The number of wells in which lysis did or did not occur was scored and the concentration of infectious agents was determined using a computer program written in B A S I C (26). Estimates obtained were converted to ambient abundances assuming that the cyanophages were concentrated from seawater with 100 % efficiency. Given that it is improbable that the viruses would be concentrated with 100% efficiency, these represent minimum estimates. It was assumed that viruses, rather than bacteria were responsible for the lysis of the host Synechococcus. In previous studies the lytic agents purified from viral concentrates by endpoint dilution or plaque assay were always cyanophage (33, 50, 57, 61) and therefore we believe this to be a reasonable assumption. 1.3.5 PCR amplification Virus D N A was extracted by disrupting the virus capsids using a hot-cold method (9). Subsamples (200 ul) were subjected to two cycles of 2 minutes at 0 °C and then 2 minutes at 100 °C and were stored at -20 °C for later use. Twenty ul of 1/10 diluted viral concentrate was added to 50 ul (final volume) P C R mixture containing PLATINUM® Taq D N A polymerase buffer (20 m M T r i s - H C l [pH 8.4], 50 m M KC1), 1.5 m M MgCl2, each deoxynucleoside triphosphate at a concentration of 0.2 m M , 1.0 U PLATINUM® Taq D N A polymerase (Life Technologies) and 20 pmol of each of the 8 cyanophage-specific primers C P S 4 G C and CPS5 (62). P C R amplification was carried out with a Hybaid P C R express™ D N A thermocycler, and reactions were conducted under the following conditions: initial denaturation at 94 °C for 3 min followed by 35 cycles of denaturation at 94 °C (1 min), annealing at 50 °C (1 min) and extension at 72 °C (1 min) and a final extension step of 72 °C for 5 min. Following P C R amplification, reactions were stored at -20 °C until agarose gel electrophoresis. The P C R products (15 ul) were electrophoresed on a 3 % (wt/vol) 3:1 Nusieve® agarose gel ( F M C Bioproducts) in 0.5 X T B E buffer (45 m M Tris-borate, I m M E D T A , p H 8.0) and were visualized on a U V transilluminator with ethidium-bromide staining. To increase D N A yield for D G G E , P C R products were excised from the bands in the . agarose gel and used as template for further P C R amplification. The excised bands were stored in 100 u l of 0.5 X T B E buffer at -20 °C until use. Prior to the second P C R reaction the excised bands were heated to 75 °C for twenty min and vortexed to elute the D N A from the gel plug. One ul of the solution was used as template in a 50 u l P C R reaction and reactions were carried out as outlined above, except that P C R was conducted with twenty cycles of amplification. A subsample of the P C R products (1.5 ul) were electrophoresed on a 2 % (wt/vol) GD3CO B R L Ultrapure® agarose gel (Life Technologies) in 0.5 X T B E buffer (45 m M Tris-borate, 1 m M E D T A , p H 8.0) and were visualized on a U V transilluminator with ethidium-bromide staining. 1.3.6 Denaturing gradient gel electrophoresis D G G E was performed with a DCode™ electrophoresis system (Bio-Rad Laboratories) using a 30 to 42.5 % linear denaturing gradient (100 % denaturant is defined as 7 M urea and 40 % [vol/vol] deionized formamide) 10 % polyacrylamide gel cast with 1 X T A E (40 m M Tris-acetate, 20 m M sodium acetate, 1 m M E D T A , p H 8.5). The D N A concentration of P C R products was determined by densitometric comparison to a L o w D N A Mass® Ladder (Life 9 Technologies) using Gel Pro Analyzer software (MediaCybernetics). Approximately 1 jig of the P C R products were applied to the gel and electrophoresis was performed using 1 X T A E running buffer at 60 °C for 14 h at 135 V . Gels were stained for 3 h with SYBR® Green I nucleic acid stain (Molecular Probes), destained for 1 h in deionized distilled water and visualized with a U V transilluminator. A l l gels were photographed with a Nikon Coolpix 950 digital camera and, contrast and brightness were adjusted using Adobe® Photoshop® 5.0 L E software. Standards for denaturing gradient gel comparisons were made by excising several different bands from the denaturing gradient gel pattern obtained from the separation of the P C R amplified g20 gene fragments from the community obtained from 11 m in Malaspina Inlet. Bands were added to 100 ul of 1 X T A E and heated to 75 °C for 20 min and 2 ul of the solution was used as template for P C R . P C R reactions were pooled and 40 u l was used for each standard lane in the denaturing gradient gels. P C R amplified products from each location were electrophoresed on a separate denaturing gradient gel and thus standard lanes (2 per gel) were run to aid in gel to gel comparison. Corresponding bands in standard lanes were selected using Gel Compar JJ software (Applied Maths) and gel images were adjusted by the program based on the location of selected bands. Densitometric plots of gel lanes were compared using Pearson correlation and were subjected to U P G M A cluster analysis using Gel Compar II software. Standard lanes exhibited similarity values of as low as 80 % and therefore branches on the U P G M A tree were collapsed to this level of similarity. 10 1.4 Results and Discussion 1.4.1 Abundance of viruses, infective cyanophage and Synechococcus spp. Abundances of total viruses, cyanophages infecting Synechococcus strain D C 2 , and Synechococcus spp. were highest in surface waters and decreased with depth (Fig. 1.2). In Malaspina Inlet, total viruses ranged from approximately 4.9 x 10 7ml" 1 at 22 m deep to 1.8 x 10 8 ml" 1 at 5 m deep (Fig. 1.2). Similar estimates of abundance were observed in both Salmon Inlet (1.3 x l f / m l " 1 at 4 m) and Pendrell Sound (1.1 x 1 0 8 m l 4 at 11.6 m). These high abundances are in accordance with those typically observed in productive coastal areas (reviewed in 63). Cyanophages ranged from ca. 10 3 ml" 1 in depths below 10 m in Malaspina Inlet and below 14 m Pendrell Sound to >10 5 ml" 1 at 0.5 m in Salmon Inlet and at 5 and 10 m in Pendrell Sound (Fig. 1.2). These high titers of cyanophages infecting a single strain of Synechococcus spp. (DC-2) are similar to those observed in the shallow coastal waters of the western Gul f of Mexico (50, 51), and those observed in the Gul f Stream (57). Synechococcus spp. were abundant, exceeding 10 5 ml" 1 in waters shallower than 15 m in Malaspina Inlet, at 3.6 m in Salmon Inlet and at 11.6 m in Pendrell Sound, and were similar to the abundances of the cyanophages titered at these depths. It has been argued (57) that the co-occurrence of a high abundance of Synechococcus spp. and cyanophage, indicates that most of the Synechococcus spp. community is resistant to infection. However, the high collision rate between Synechococcus spp. and infectious cyanophage at these abundances (51) means that cells with no resistance could not support the mortality rate that would result from these collision frequencies. Hence, a more likely explanation is that the efficiency of infection is very low requiring many collisions for successful infection (49). Alternatively, the high abundances of infectious cyanophage could result from induction of lysogenized cells (34, 37). 11 Viruses ml"1 x 101 Infective cyanophages (MPN ml"1 x 10s) 0.01 0.1 1 Synechococcus spp. (cells ml"1 x 105) 30 J ' Figure 1.2. Abundance of viruses, cyanophage and Synechococcus Direct counts of virus particles (A), M P N assay of infective cyanophage (B) and counts of Synechococcus spp. (C). Abundance estimates are represented by open circles for Malaspina Inlet, filled square for Salmon Inlet and filled triangles for Pendrell Sound. Symbols with error bars represent samples where abundances were measured in duplicate and the error bar represents the range. No error bars are present for the virus and Synechococcus spp abundance measured in Salmon Inlet and Pendrell Sound because measurements were not duplicated. Error bars are smaller than the width of the symbol if not visible. 12 Cyanophages infecting the Synechococcus strain D C - 2 , although abundant in these inlets, only accounted for up to ca. 0.3 % of the total viral community. In a previous study, during a Synechococcus spp. bloom in the western Gul f of Mexico, cyanophages infecting the same Synechococcus strain, were ca. 10 6 ml" 1 , and represented about 10 % of the viral community as determined by counts of viral particles (reviewed in 49). The cyanophage abundances reported are undoubtedly underestimated because the phages infecting a single strain of Synechococcus w i l l not represent all the infectious cyanophages present. 1.4.2 Temperature and salinity characteristics Temperature and salinity characteristics were used to assess water column structure in the three British Columbia coastal inlets (Fig. 1.3). A l l three inlets exhibited some stratification, but differed in temperature and salinity profiles. In Salmon Inlet, the total variation in temperature and salinity over all sample depths (0.5 m to 15 m) was 4.5 °C and 7.5 ppt. There was a gradient of change in temperature and salinity from approximately 5 m deep to the surface, where the two shallowest samples were collected. The surface sample which was collected from a depth of 0.5 m, was 2 °C warmer and 5.5 ppt less saline then the next deepest sample which was collected at a depth of 4 m. Only minor differences in temperature and salinity (ca. 1 °C and 0.8 ppt) were observed between 5 and 15 m deep where four of the six samples were obtained. In Malaspina Inlet there appeared to be two layers of water differing in both temperature and salinity. The difference in temperature over all depths of sample collection (5 m to 25 m) was only 4.8 °C and the salinity varied by only 3.2 ppt. 13 Figure 1.3. Temperature, salinity and chlorophyll fluorescence profiles Temperature, salinity and chlorophyll fluorescence profiles for Salmon Inlet (A), Malaspina Inlet (B) and Pendrell Sound (C). Salinity (Sal), temperature (Temp) and chlorophyll fluorescence (Fl) profiles are indicated. Depths from which water samples were collected are indicated by a dotted line. The numbers at the right axes correspond to lanes in the gels and labels on the U P G M A dendrogram. 14 The deepest three depths sampled were relatively similar in terms of temperature and salinity varying by only 0.4 ppt and 0.2 °C, while a slightly more pronounced difference in temperature (ca. 1.3 °C) and salinity (ca. 1.8 ppt) occurred in the depths from which the three shallower samples were collected. In contrast, Pendrell Sound profiles of temperature and salinity were broad. There appeared to be an ca. 4 m deep layer of warm (>22 °C) low salinity (<15 ppt) water lying above a water column that exhibited a gradient of change in both temperature and salinity from 5 to 25 m deep. This broad gradient of change may have been the set-up or breakdown of a pycnocline, as the temperatures and salinities below 30 m were fairly homogenous (data not shown). One sample was collected from the wedge of lower salinity, higher temperature water, while the other five samples were collected from the 'confused' portion of the water column (5 to 25 m). Overall, Pendrell Sound exhibited a significant temperature shift of 12.5 °C and a change in salinity of 14 ppt over the depths from which all six samples were collected. 1.4.3 PCR amplification and reproducibility of denaturing gradient gels Although cyanophages represented a fairly small proportion of the total viruses present (Fig. 1.2), P C R amplification using cyanophage-specific primers resulted in the expected -200 bp product from all eighteen samples (Fig. 1.4). The P C R products, when separated by denaturing gradient gel electrophoresis, produced band patterns that could be used to compare communities (Fig. 1.5).These band patterns were reproducible between gels and D G G E analysis of first and second round P C R amplifications showed that bands differed only in the intensity of the products, not the patterns produced (data not shown). There was no evidence that storage of concentrates in the dark at 4 °C affected the preservation of cyanophage. When a concentrated virus community was sub-sampled on two separate occasions more than 18 months apart, P C R amplified D N A resulted in the same D G G E band pattern (data not shown). 15 1 2 3 4 5 6 LI 7 8 9 10 11 12 L2 13 14 IS 16 17 18 i f SB Figure 1.4. Agarose gel showing amplification of -200 bp g20 gene fragment. The fragment is 165 bp plus 40 bp GC-clampp. LI indicates a 100 bp ladder and L2 indicates a low D N A mass ladder with fragment sizes of 2000, 1200, 800, 400, 200 and 100 base pairs. 1.4.4 Cyanophage DGGE patterns and water column structure The similarity of the cyanophage D G G E patterns reflected the differences in temperature and salinity. Similar patterns were observed at most depths within Salmon and Malaspina Inlets, while Pendrell Sound exhibited considerable differences in community composition with depth. In Salmon and Malaspina Inlets, differences were noticeable in the surface samples (lane 6 and 12) where temperature was higher (>18 °C) and salinity was lower (<17 ppt in Salmon Inlet and <23 ppt in Malaspina Inlet). In the Malaspina Inlet surface sample, some bands were present that were not observed at deeper depths and some bands found at greater depths were not present in the surface sample. Similarly in Salmon Inlet, some of the bands found deeper in the water column were not found in the shallowest sample. However, in Pendrell Sound, band patterns were different from the surface to the deepest samples (lanes 13-18), where differences in temperature and salinity were much broader throughout the depths sampled. The variability in the community patterns reflected the somewhat chaotic nature of the water column. Interestingly, in the water above 15 m in Malaspina Inlet, where the three shallow samples were obtained, 16 Salmon Inlet Malaspina Inlet Pendrell Sound S 1 2 3 4 5 6 S S 7 8 9 10 11 12 S S 1314 15 16 17 18 S Figure 1.5. DGGE of PCR amplified g20 gene fragments from the three inlets. Lanes labeled S indicate a standard made up of PCR amplified g20 genes excised from lane 11 of Malaspina Inlet used for comparison between the three gels. The number at the top of the lanes correspond sample numbers also used on the right y-axis in Fig. 1.3 and the U P G M A dendrogram in Fig. 1.6. Numbers on the bottom of the lanes correspond to depths (m) from which the communities were obtained. there was a different temperature and salinity than the water below 17 m where the three deeper samples were obtained, and we did not see a corresponding reflection of this in the D G G E band patterns. This trend of virus community pattern reflecting the physical characteristics of the water column has also been observed in other studies where marine virus communities were compared using similar P C R - D G G E methods. For example, when D G G E was used to separate algal virus D N A pol fragments, PCR-amplified from the same samples in both Salmon Inlet and Pendrell 17 Sound (43). A s well , in the Atlantic Ocean, although the change in temperature with depth was only about 2 °C, D G G E patterns of P C R amplified g20 gene fragments revealed community structures that reflected the thermal stratification (62). Because qualitative examination of D G G E community patterns agrees with the temperature and salinity gradients, we conclude that the physical structure of the water column influences cyanophage community structure. We believe that the differences in the cyanophage communities are most likely the result of differences in the host cyanobacteria communities that are influenced by differences in physical conditions (salinity, temperature and light), and/or because of different biological interactions that occur between physically separated communities. 1.4.5 Coincidence of high chlorophyll fluorescence with differences in community patterns and high abundances of infective cyanophage and Synechococcus spp. Interestingly, samples collected from 5 m in Malaspina Inlet and 10 m in Pendrell Sound, were the least similar to other communities and coincided with high levels of chlorophyll fluorescence (relative units) (Fig. 1.2). In Malaspina Inlet, the community from the depth of highest chlorophyll fluorescence contained unique bands while Pendrell Sound, the community coinciding with high chlorophyll fluorescence had the greatest number of bands in the gel. However, the sample collected from 14 m had an even higher chlorophyll fluorescence, but had fewer bands than the 10 m sample (Fig 1.5, lanes 15 and 16). In Salmon Inlet, because samples were not collected from the depth of the chlorophyll fluorescence peak, community patterns could not be compared. In the Pendrell Sound sample coinciding with higher chlorophyll fluorescence (10 m), a very high titer of cyanophages infecting Synechococcus of ca. 3 x 10 5 ml" 1 (among the highest ever reported) and a high Synechococcus spp. abundance (4.3 x 10 5 ml" 1) was also observed. 18 Other investigators have reported similar findings when detecting cyanophage g20 sequences by P C R amplification. For instance, samples collected from the Atlantic Ocean that had the most D G G E bands were the samples that also had the highest Synechococcus spp. concentration (62). This evidence suggests that there may be a link between the concentration of host organisms and the number and type of genetically different cyanophage that coexist. 1.4.6 Relating sequence richness to the number of bands on the denaturing gradient gel Unfortunately, the number of fragments in a D G G E gel may not represent the actual number of different target sequences amplified. For example, it has been previously reported that sequence analysis of fragments obtained from denaturing gradient gels of algal-virus polymerase P C R products, indicated that in some cases nearly identical sequences migrated to distant locations in the gel, while sequences differing by 70 % migrated to the same position (43). In the same study, a single D G G E band consisted of at least three different polymerase sequences. Relating sequence richness to the number of bands on a D G G E gel may not be as large a problem when using the cyanophage-specific primers, as for the algal virus-specific primers. The algal virus primers A V S 1 and A V S 2 were degenerate, while primers C P S 4 G C and CPS5 used in the present study were not. Degenerate primers are more likely than non-degenerate P C R primers to produce multiple P C R products from a single template. D G G E analysis of most water samples from three British Columbia inlets resulted in the separation of approximately fifteen to twenty visible bands. Assuming the number of D G G E bands is representative of cyanophage genetic richness, then the cyanophage communities in British Columbia coastal waters were more varied than communities in the Atlantic Ocean. Only 2 to 10 bands per sample were detected in the Atlantic (62) compared to 15 to 20 bands per sample (ie lane 16 Fig . 1.5) detected in British Columbia coastal waters when the same primers 19 were used. The higher implied richness may have been due to higher productivity in British Columbia coastal waters than offshore in the Atlantic Ocean, or because there was a Synechococcus bloom (ranging from 4 x 10 5 ml" 1 to 6 x 10 5 ml" 1 in surface samples) in the inlets at the time of sampling, which was greater than the concentrations reported in the Atlantic Ocean which did not exceed 2.1 x 10 4 ml" 1 in 12 out of the 13 samples collected (60). In a previous study in the Gul f of Mexico when Synechococcus spp. abundance exceeded ca. 10 ml" , cyanophage concentrations increased markedly (greater than ca. 10 5 ml" 1 in some cases) (51). ' Similarly in both Salmon Inlet and Pendrell Sound, when Synechococcus spp. concentrations were greater than 10 5 ml" 1 , the highest concentrations of infective cyanophages were observed (ca. 3 x 10 5 ml" 1). The increase in host abundance could have allowed for increased contact rates of hosts and viruses, increasing the production of cyanophages and possibly resulting in an increase in community richness. The number of bands produced in D G G E gels of cyanophage g20 fragments can be regarded as a minimum estimate of cyanophage richness. This results from the restricted subset of cyanomyovirus likely amplified with non-degenerate primers, that other groups of cyanophage (podovirus or siphovirus) D N A wi l l not be amplified with this primer set, and because of the co-migration of fragments of different sequence. Other sequencing based methods have resulted in greater estimates of richness. For example, using the primers CPS1 and CPS8, which also target cyanophage g20 genes, Zhong et al., obtained up to 29 different genotypes from a single sample, and out of 114 g20 homologs recovered, none were identical (66). 1.4.7 Sensitivity of DGGE D G G E appears to be a more sensitive method to estimate diversity when compared to restriction digests of a short region of phage genomes. When over 60 Myoviruses were isolated 20 with a single Vibriophage host, all shared a homologous 1.5 kb region, but only six phage groups were proven to be genetically distinct based on autoradiograms of digested D N A probed with the 1.5 kb fragment (30). Although enumeration of D G G E bands wi l l only represent a minimum estimate of richness, the fingerprints are useful for determining the relatedness of cyanophage communities. 1.4.8 Similarity of fingerprints as indicated by UPGMA cluster analysis U P G M A cluster analysis indicated the similarity of fingerprints among the three gels (Fig 1.6). Communities at all depths in Salmon Inlet (1 through 6) and five out of six communities from Malaspina Inlet (7 through 11) clustered separately from other locations and exhibited a high level of similarity to one another. The differences between locations exists despite the fact that all three inlets are connected to the same body of water, the Strait of Georgia. This implies that community structure is dependent on the environmental conditions of a particular area and that virus communities as near as adjacent inlets are variable. In contrast to the high degree of similarity between different depths in Salmon and Malaspina Inlets, the two surface communities in Pendrell sound (17 and 18) clustered with communities from Malaspina Inlet, while the deepest samples broke into two separate clusters (13-15 and 16) that were not closely related to any other communities. Similarly in Malaspina Inlet, the surface community (12) was located in a separate cluster that was also not related to any other community. 21 % Similarity o o IT) S © o 7 8 9 M 10 11 1 Z 1 1 P 18 1 1 x 1 2 3 s 4 5 6 13 1 1 14 P 15 1 16 - P 12 • M Figure 1.6. UPGMA dendrogram showing relatedness of cyanophage communities. Communities from Malaspina Inlet (M), Pendrell Sound (P) and Salmon Inlet (S) were compared. The top axis represents the similarity. These changes in communities with depth in a single location show that there is variation in cyanophage communities over very small spatial scales. For example, the communities in Pendrell Sound, separated by as few as four metres, exhibited differences in banding pattern. Some cyanophages (represented by a single D G G E band) appeared in all eighteen samples, suggesting that closely related cyanophage were widely distributed within the Strait of Georgia, while some cyanophage (represented by a unique D G G E band) were present in only a single 22 sample. In previous studies closely related viruses have been detected over great distances. For example, similar algal virus pol fragments were recovered from distant oceans (43), genetically related cyanophage were found to be widely distributed in the Sargasso Sea and the Gul f stream (66) and cyanophage originating from different oceans differed by only 8 of 165 bases of the g20 gene fragment (60). Some marine viruses appear to be widespread, while others appear less cosmopolitan. Community shifts over relatively small distances, support the conclusion that virus communities are a dynamic component of marine systems and that these communities develop independently of one another. 1.4.9 Conclusion In conclusion, the composition of natural cyanophage communities was variable depending on both location and depth. These results indicate that the physical structure of the water column influenced the composition of cyanophage communities and the relatedness of communities found at any location. This suggests the physical structure may also have had an effect on host Synechococcus communities, thereby influencing the viral communities infecting them. Cyanophage and cyanobacteria interact in a dynamic manner, and it seems likely the concentration and diversity of host cells influences the diversity of the cyanophages and vice versa. Ultimately, our goal is to understand the complexity of natural cyanophage populations and their role in structuring cyanobacteria populations. This study provides some clues about the influence of physical structure on cyanophage communities and the dynamic nature of communities that are found in close proximity to one another. Further investigation w i l l allow for the comparison of the structure of cyanophage communities from distant oceans and 23 examination of the genetic relatedness of cyanophage from these remote locations. 1.5 Acknowledgements The authors gratefully acknowledge Al ice Ortmann for virus slide preparation, Sean Brigden for providing the cyanobacteria host D C - 2 and Andre Comeau for the preparation of the cruise log. We would also like to thank the crew of the C C G S Vector for their assistance with sample collection and Sara Leckie for her help with Gel Compar II. This study was supported by a grant from the Natural Sciences and Engineering Research Council of Canada ( N S E R C ) to C . A . Suttle. 24 Chapter II. Genetic Variation Among Globally Distributed Marine and Freshwater Cyanophages 25 2.1 Summary Viruses are the most abundant biological entities on the planet and it is estimated that if) there are -10 phage worldwide. Due to the massive volume of the Earth's biosphere that is represented by the oceans, the bulk of viruses may be found here. Despite the abundance and widespread distribution of viruses in the sea, very little is known about their biodiversity and phylogeny. The goal of the present study was to obtain a global perspective on the genetic variation in the g20 gene, which is found within a conserved genetic module in marine cyanophage. Therefore, cyanophage-specific-primers were used to amplify g20 gene fragments from widespread marine and freshwater environments. These primers were used to amplify gene fragments from lakes in British Columbia and Germany, a catfish production pond in Louisiana, a cyanobacterial mat from a high arctic pond, as well as samples from both polar oceans, the Gul f of Mexico , northeast Pacific inlets and the coastal southeast Pacific. The presence of g20 genes in these ecologically and geographically distinct environments demonstrates the global presence of aquatic putative cyanophages. Phylogenetic analysis of fifty-three unique sequences revealed four previously unknown groups of phage, two of which consisted only of freshwater sequences. The discovery of sequences exhibiting >99% sequence identity from geographically widespread locations suggests that genetically similar phages are pathogens of widespread cyanobacteria hosts. 26 2.2 Introduction In the last 15 years, it has become apparent that viruses in aquatic environments are important as agents of mortality of both heterotrophic and photosynthetic microbes (48). They are the most abundant biological entities in freshwater and marine environments, and are approximately 5 tolO-fold more abundant than bacteria (for reviews see 3, 5, 15). Through the specific destruction of host organisms, marine viruses have an effect on host abundance, maintenance of community diversity and the cycling of carbon and nutrients in the sea (59). Furthermore, because every host organism is usually infected by more than one virus strain, it is not unreasonable to believe that marine viruses represent the largest untapped reservoir of genetic diversity in the world. Marine heterotrophic bacteria represent a significant portion of the biological carbon in the ocean and their involvement in the cycling of energy through the microbial loop has been well documented (1). The effect that viruses have on the structure, and therefore function, of bacteria communities is believed to be significant. Based on decay rates and electron microscope analyses, on average, between 10 and 20% of the natural bacterial community is lysed daily to maintain the viral community (39, 48). More specifically, in microcosm experiments, viruses have been shown to control communities of marine heterotrophic bacteria (24) and alter the clonal composition of four strains of marine bacteria from phage sensitive to phage resistant (35). The effect of viruses on primary producers is less certain. Primary producers, including cyanobacteria, form the basis of the food chain, serving as a source of organic carbon for all other marine organisms. The photosynthetic prokaryotes (cyanobacteria) are abundant and widespread and are thought to be responsible for a significant proportion (20-80%) of carbon 27 fixation in aquatic environments (31). Small chrococcoid cyanobacteria of the genus Prochlorococcus and Synechococcus commonly reach abundances exceeding 10 7 cells per liter (11). Enumeration by T E M of visibly infected cells, and viral decay rate experiments suggest that approximately 3 to 10% of the unicellular cyanobacterium Synechococcus spp. are destroyed daily (39, 48, 59). Viruses which infect a single strain of Synechococcus, are widespread and can reach abundances of greater than 10 5 ml" 1 (33, 51, 57). The impact of viruses on the entire photosynthetic community has also been examined. A 20% increase in the virus size fraction can decrease phytoplankton primary production by as much as 50% and removal of viruses from the system could result in an enhancement of photosynthetic rates by about 2% (47). In addition, the association of virus particles with the collapse of blooms of the phytoplankton species Heterosigma akashiwo (36) and Emiliania huxleyi (21) suggests that viruses may be responsible for the termination of some algal blooms. Viruses are thought to increase or maintain the diversity of the communities of organisms that they infect (reviewed in 15, 63). The proposed mechanism is through the selective lysis of only host strains that are present at high concentration and that are fast growing (65). A recently developed mathematical model demonstrates that the interactions between viruses and host cells have the potential to strongly influence the diversity of microbial communities (54). Periodic fluctuations in the abundance of specific viruses in the Chesapeake Bay (65) and the selective regulation of the density of the Vibrio bacteria strain P W F D a by low titers of naturally occurring phages (24) support this hypothesis. Viruses may also alter the genetic make-up of hosts by transferring D N A from one host to another through inter-species transduction (29), but this mechanism is probably less important, because most marine viruses appear to be host specific. To further understand the significance of marine viruses, it is important to determine the types of viruses present and the relationships between viruses in different 28 environments. A number of methods have been used in an effort to understand the diversity of natural virus communities. Transmission electron microscopy has revealed the vast morphological diversity of natural marine virus communities (3), and has shown the presence of all three families of tailed phage as well as large tail-less icosahedral particles. The genetic diversity of viruses is l ikely far greater, because different viruses can be morphologically indistinguishable. Differences in host range or lytic cycle length and the degree of genomic D N A hybridization can distinguish between strains of viruses that are isolated and maintained in culture. Unfortunately, cultured viruses likely represent an insignificant portion of natural viruses because the vast majority (>99%) of marine microbes used to isolate marine viruses remain uncultured. To avoid the inherent biases in culture-based methods, molecular techniques have been developed to estimate the diversity or richness of natural communities of unknown viruses. P C R has been used to target groups of phytoplankton viruses from natural environments (9, 12, 62, 66). PCR-based methods are not quantitative and because there is no gene that is universally present in all viruses, only a subset of the virus community can be targeted with a single P C R primer pair. Pulsed field gel electrophoresis (PFGE) has been used to separate whole 'virus-size' genomes for community comparison (14, 45, 64). Although this technique is quantitative, it is difficult to get adequate separation of similar sized genomes and host identity is unknown. Both PCR-based and P F G E methods are likely missing a large portion of viral diversity because they do not include R N A viruses. Ultimately, the best method to compare marine viruses is through the sequence analysis of whole genomes. Unfortunately, because this technique is both costly and time consuming, only four marine and one freshwater virus have been entirely sequenced to date. These include the freshwater Paramecium bursaria Chlorella algal virus P B C V - 1 (56), and the marine viruses Ectocarpus siliculosus algal virus EsV-1 (10), Roseophage SI01 29 (41), the cyanophage P60 (6) and the Vibriophage V p V 2 6 2 (21). Genomic sequencing of marine viruses w i l l l ikely identify proteins that are conserved among groups of viruses which can be used as genetic markers to identify uncultured representatives in the environment (40). In 1995, the D N A polymerase gene in algal viruses was identified as a possible 'signature gene' that could be used to compare between algal viruses that infect different hosts and to target unknown environmental sequences. The development of the algal-virus-specific primers A V S - 1 and A V S - 2 , allowed for the amplification of a region of the D N A polymerase gene of both cultured and uncultured algal viruses and subsequently the first phylogenetic analysis of marine virus sequences (7). Using A V S - P C R and denaturing gradient gel electrophoresis ( D G G E ) , community 'fingerprints' could be obtained and compared (42,43). These community fingerprints have proven to be useful to observe changes in viral communities over seasons and for the separation of different pol fragments for sequencing (44). The algal virus polymerase sequences obtained from D G G E gels confirmed the existence of uncharacterized groups of viruses within the algal virus family, the Phycodnaviridae, and showed the presence of similar sequences in distant oceans (43). In 1998, southern hybridizations were used to establish regions of similarity in three phages that infect the cyanobacteria Synechococcus (16). These regions, when sequenced, were found to exhibit significant homology to gene (g) 20 from the coliphage T4. The protein product of g20 is involved in attachment of the tail to the capsid and the packaging of D N A into the capsid (25). Conserved regions of the three cyanophages were used to develop primers specific for the amplification of a 165 bp fragment of cyanomyovirus g20 genes. D G G E fingerprints of P C R amplified g20 gene fragments showed that cyanophage communities can differ over spatial scales as small as meters and that they can reflect the structure of the water column (12, 62). In 2002, P C R primers were developed to amplify a larger (-595 bp) region of the 30 cyanophage g20 gene that could be sequenced and used in phylogenetic analyses (66). Phylogenetic analysis of g20 fragments amplified from estuarine and coastal environments of the Sargasso Sea and Gul f Stream revealed 114 different sequences that were separated into nine clades (66). To date, cyanophage g20 gene sequences have only been obtained from one ocean region which includes the Sargasso Sea and the Gul f Stream. The goal of the present study was to assess the 'global' richness of cyanophage g20 genes obtained from widespread marine and freshwater environments and to phylogenetically compare these sequences. P C R primers were designed and used to amplify the -592 bp region of cyanophages belonging to the family Myoviridae from natural samples. Freshwater environments where g20 sequences were obtained included lakes in Canada and Germany, a catfish production pond from Louisiana, and a cyanobacterial mat from a high arctic pond. Marine environments included both polar oceans, the Gul f of Mexico, northeast Pacific inlets and the coastal southeast Pacific. Phylogenetic analysis of sequence information obtained from these 'global' locations revealed very similar sequences in different oceans, sequences >99% identical in both freshwater and marine locations and novel clades of freshwater specific sequences. 31 2.3 Mater ia ls and Methods 2.3.1 Sample collection Samples were collected from both marine and freshwater environments from M a y 1995 to September 2002. Seawater samples were collected from the Gul f of Mexico ( R V F.G. Walton Smith), the Arctic Ocean ( R V Mirai), the Southern Ocean ( R V Lawrence M. Gould), the southeast Pacific (RVD3 Nathaniel B. Palmer), and several inlets in the northeast Pacific ( C C G S Vector). Freshwater samples were collected from two lakes in British Columbia (Chilliwack Lake and Cultus Lake), one lake in Germany (Lake Constance), a commercial catfish production pond (Limco, Inc.) in Crowley, Louisiana, and a shallow pond located on the ice in the high Arctic. Most samples were collected from the euphotic zone where cyanobacteria and therefore cyanophage would be most prevalent. However, one sample was collected from approximately 3000 m in the Chuckchi Sea (Arctic Ocean). Temperatures in the marine environments ranged from below freezing at the poles to 26.8 °C near the equator and salinities ranged from 17.9 to 36.6 ppt. The details of the sites sampled and the labels assigned to each sample are listed in Table 2.1. Ocean samples were collected from the research vessel using Niskin bottles mounted on a rosette. The lake samples were collected from a small boat with a Niskin bottle in both Chil l iwack Lake and Cultus Lake and with a submersible pump in Lake Constance. A small plug was cut from a cyanobacteria mat that was growing in a shallow Arctic pond on melted ice. 2.3.2 Concentration of natural virus communities Two liters of commercial catfish pond water was cleared of solids (cells and mud) by centrifugation at 10,000 rpm for 15 min. Bacteria and remaining cell debris was removed by filtration through 47 mm 0.45 um cellulose acetate filters (Nalgene, Rochester, N . Y . ) . 32 Table 2.1. Details of sample locations Latitude and Sample Origin T .. , Date Collected Depth (m) Tn™? Salinity & Longitude y (°C) (ppt) Marine GOM01 Gulf of Mexico BES02A Beaufort Sea Arctic Ocean CHS02 Chuckchi Sea Arctic Ocean BES02B Beaufort Sea Arctic Ocean ANT98 Antarctic Peninsula Southern Ocean COL00 Coast of Colombia SE Pacific CHI00 Coast of Chile SE Pacific SAI99 Salmon Inlet N E Pacific PES99 Pendrell Sound N E Pacific MAI00 Malaspina Inlet N E Pacific TEAOO Teakerne Arm N E Pacific 27° 30.04' N 88° 24.11'W 72° 30.0' N 151° 20.0' W 73° 30.0' N 157° 00.0' W 70° 12.0' N 137° 0.0' W 61° 11.68' S 54° 36.25' W 3° 00.60' N 82° 37.90' W 29° 00' S 77° 00' W 49° 36.25' N 123° 48.18' W 50° 16.24' N 124° 42.80' W 50° 04.77' N 124° 42.85'W 50° 11.35' N 124° 51.14' W 18-M-2001 14-Sep-2002 9-Sep-2002 26- Sep-2002 3- Nov-1998 4- Aug-2000 27- M-2000 17-Aug-1999 20-Aug-1999 2-Aug-2000 l-Aug-2000 110 35 3000 25 0.5 1-3 1-3 0.5 6.2 1 7 - 1 0 22.5 N A N A N A -0.24 26.8 16.6 16.7 18.3 17.6 18 36.6 N A N A N A 34.1 33.5 34.4 17.9 22.3 25.6 24 Freshwater C U L 0 2 M CUL02H CHL02E CAT02 SPM02 L A C 9 5 A LAC95B Cultus Lake B C , Canada Cultus Lake B C , Canada Chilliwack Lake B C , Canada Catfish pond Crowley, L A , USA Shore Pond Cyano Mat Arctic Lake Constance Germany Lake Constance Germany 49° 03.45' N 121° 59.05' W 49° 03.45' N 121° 59.05'W 49° 04.22' N 121° 25.54' W N A N A N A N A 25-Sep-2002 25-Sep-2002 29-Jun-2002 12-Sept-2002 N A 10-May-1995 16-May-1995 10 18 3 N A N A 3 3 15 7.3 12.7 N A N A N A N A N A N A N A N A N A N A N A 33 The viruses in the filtrate were concentrated to ca. 10 to 15 ml using a 10 kD-cutoff Amicon cartridge (Millipore, Bedford, Mass). In remaining seawater and freshwater samples, the virus size fraction was concentrated using previously established methods as briefly outlined below. Samples were serially filtered through 142 mm dia 1.2 urn (GC50; Advantec M F S , Dublin, C A ) or 0.6 urn (GFF; Whatman, Clifton, NJ) nominal pore size glass-fibre filters followed by a 0.45 um ( G V W P ; Millipore) or 0.2 pjn (Gelman, Pall co., East Hi l l s , N . Y . ) pore size filter to remove organisms and particles larger than viruses. The remaining virus size material was concentrated from ca. 50 to 500 fold using a 10 or 30 kD-cutoff Amicon (S1Y10/S1Y30/S10Y30) cartridge (Millipore), according to the manufacturers recommendations. Subsamples (100 ml) of the virus concentrates from the Arctic Ocean were further concentrated to ca. 500 JLLI using Centricon Plus-20 centrifugal filter devices (Millipore) and stored at -20 °C in the dark. A l l other viral concentrates were stored at 4 °C in the dark until use. 2.3.3 Viral DNA extraction from the cyanobacteria mat D N A was extracted from a 1 cm dia circular plug of cyanobacteria mat (1 mm thick). The plug was incubated at 37 °C for 45 min in 600 p i lysozyme (final concentration of 1 mg ml" 1) and at 37 °C for 1 h after 70 ul of sodium dodecyl sulfate (final concentration, 1 %) and 7 ul proteinase K (final concentration, 0.2 mg ml" 1) was added. After an addition of 90 ul of 10% C T A B in 0.7 M N a C l and 100 u.1 of 5 M N a C l , the mixture was vortexed and incubated at 65 °C for 10 min. D N A was purified from the mat 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 (42). Extracted 34 D N A was stored at -20 °C in 10 m M Tris -Cl (pH 8.5) until further use. 2.3.4 PCR Thirty-five ml subsamples of the viral concentrates from the Gul f of Mexico , the Southern Ocean and the British Columbia lakes were centrifuged at 85,000 x g for 3.5 h and 38-ml subsamples from the S E Pacific samples were centrifuged at 104,000 x g for 3 h. Pelleted viruses were resuspended in 100 p i or 500 p i virus-free water. Nucleic acids were extracted from the resuspended pellets and 100 p i subsamples of the viral concentrates from the N E Pacific Inlets, Lake Constance and the Arctic Ocean using an established hot/cold technique (9). Three j i l of each virus concentrate or extracted D N A was added to a 47-pi P C R mixture containing Taq D N A polymerase assay buffer (50 m M KC1, 20 m M Tr i s -HCl [pH 8.4]), 1.5 m M M g C l 2 , a 0.20 m M concentration of each deoxyribonucleoside triphosphate, 40 pmol each of the cyanophage specific primer CPS4 and CPS9 (5' - S W R A A A T A Y T T I C C R A C R W A K G G A T C - 3 ' ) , and 1.0 U of P L A T I N U M Taq polymerase (Invitrogen Life Technologies, Carlsbad, C A ) . Negative controls contained all reagents and sterile water instead of template. P C R was carried out with the following cycle parameters: denaturation at 94 °C for 90 s, 35 cycles of denaturation at 94 °C for 45 s, annealing at 50 °C for 1 min, extension at 72 °C for 45 s, and a final extension at 72 °C for 5 min. P C R products were electrophoresed in 2% agarose in 0.5X T B E buffer (45 m M Tris-borate, 1 m M E D T A [pH 8.0]) at 100 V for 75 min. Gels were stained with ethidium bromide, visualized on a U V transilluminator, and photographed with a Nikon Coolpix 950 digital camera. The contrast of the digital images was enhances using Adobe Photoshop 5.0 L E . After the P C R products were purified on agarose gels they were used as templates in a second P C R reaction. Using a clean glass pipette, a plug of agarose, containing amplified D N A was removed from each lane. One hundred p i of 0.5X T B E was added to each plug in a sterile microfuge tube and 35 then heated to 65 °C to elute the D N A . Two ul of the eluted D N A was added to a 98-uI P C R mixture and P C R was conducted as described above, except that the number of cycles of amplification was reduced to 25 and a 40 bp G C rich region (GC-clamp) was included at the 5' end of the primer CPS4 so that products could be analyzed by denaturing gradient gel electrophoresis. Amplification in second-stage P C R reactions was confirmed by agarose gel electrophoresis as described above. 2.3.5 Denaturing gradient gel electrophoresis (DGGE) Second stage P C R products (20 to 35 pi) were separated using D G G E . Gels consisted of 20 to 40% linear denaturing gradients (100% denaturant is defined as 7 M urea and 40% deionized formamide) and 7 to 8% polyacrylmide. A l l gels were run for 15 h in I X T A E buffer (40 m M Tris-base, 20 m M sodium acetate, 1 m M E D T A [pH 8.5]) at 80 volts and a constant temperature of 60 °C using the D-code electrophoresis system (Bio-rad laboratories, Hercules, C A ) . Gels were stained in a 0.1X S Y B R Gold (Molecular Probes, Eugene, OR) solution for 3 h and were visualized and photographed as described above. 2.3.6 DNA sequencing Sequences were obtained from bands that were excised from the denaturing gradient gels and then P C R amplified and cloned. Ninety-nine bands were removed from the gels, added to 100 u l I X T A E buffer in sterile microfuge tubes and heated to 95 °C for 10 min. P C R was conducted using 2 ul of the eluted D N A as template under the same conditions as the first-stage P C R , but with 28 cycles of amplification. P C R products were cloned using a p G E M - T Vector System I (Promega, Madison, WI) TA-cloning kit and resulting reactions were used to transform competent E. coli JM109. Colonies were screened for correct inserts by P C R using the 36 cyanophage primers CPS4 and CPS9. Plasmid D N A was harvested from overnight cultures using a QIAprep spin miniprep kit (Qiagen, Valencia, C A ) . Clones were sequenced using Ampl iTaq FS B I G D Y E Terminator cycle sequencing chemistry (Applied Biosystems, Foster City, C A ) and excess Dye-terminators were removed using C E N T R I - S E P spin columns (Princeton Separations, Adelphia, NJ). Reactions were run in A B I Model 373 Stretch or A B I prism 377 automated sequencers (Applied Biosystems). 2.3.7 Obtaining sequences from cultured isolates Sequences from the Synechococcus phages S - P W M 3 and S - P W M 4 were obtained by P C R amplifying g20 gene fragments from filtered culture lysates under the same conditions as the first round P C R reactions of the natural samples. The protocol for cloning and sequencing of the P C R amplified fragments was also identical to the one used for the natural samples. 2.3.8 Phylogenetic Analysis Bioedit (version 5.0.7) was used for sequence editing, translation and generation of pair-wise D N A identity matrices (19). Only one sequence from a single location with >99% identity was used in further analysis. Inferred amino acids of the unknown sequences were aligned with other g20 sequences from GenBank using ClustalX (version 1.81) and the protein weight matrix Gonnet (55). The alignment was used to construct maximum-likelihood and neighbor-joining trees using the programs T R E E - P U Z Z L E (version 5.0) (46) and ClustalX, respectively. Phylogenetic trees were drawn and visualized using Tree View (Win32; version 1.6.1) (38). 37 2.4 Results 2.4.1 The number of unique sequences obtained from the denaturing gradient gels varied between, samples Denaturing gradient gel electrophoresis community patterns resulted in the separation of anywhere between 3 and 18 bands (Fig. 2.1). From six of the eighteen samples ( G O M 0 1 , B E S 0 2 A , A N T 9 8 , C U L 0 2 M , C U L 0 2 H and SPM02), between seven and fourteen bands were excised. Although, each of the six samples varied, only four to eight unique sequences (defined as >99% similarity) were obtained from a single lane. In all of these samples, identical sequences were recovered from multiple bands. A s well , the proportion of unique sequences obtained differed. For example, only five of thirteen bands from the Cultus Lake sample, C U L 0 2 M , were found to be different sequences, while seven of eight bands from the Beaufort Sea sample, B E S 0 2 A , were unique. 2.4.2 Environmental sequences cluster with sequences from cyanophage isolates Phylogenetic analysis revealed that all g20 sequences from culture-maintained Synechococcus phages were found within the group ' C S P ' (=Cultured Synechococcus Phage) (Fig 2.2). The g20 sequences from two cultured Synechococcus phages ( S - P W M 3 and S-PWM4) and 22 of the 53 environmental sequences that were obtained in the present study were also included in this group. None of the 53 sequences were included in any of the 'unknown' groups (A through F) of sequences that were obtained from the northwest Atlantic (Sargasso Sea, Skidaway Estuary, and Gul f Stream). Very few sequences from the three lakes (2 of 15) and the Arctic Ocean (1 of 13) clustered within the C S P group. A relatively higher fraction of the sequences from the Gul f of Mexico (4 of 5), the Southern Ocean (3 of 4), the northeast Pacific 38 .Ci1 ^CF .2 " i C O y a- c +-» — m — ro <n 2 O °-to 1 0 0' c O < Lake Constance r LAC95B "a a Lake Constance < 4jU» LAC9SA * J " " L Shore Pond Mat SPM02 Catfish Pond CAT02 Chilliwack Lake CHL02E Cultus Lake CUL02H as? ?« l v i f e <X>t7s S U V 11 Cultus Lake .1 V 1 ^JMEU CUL02M I^Hl^W Tea kerne Arm TEAOO Malaspina Inlet MAI0O Pendrell Sound PES99 Salmon Inlet SAI99 Coast of Chile CHI00 Coast of Colombia COL00 Antarctic Peninsuia ANT98 Beaufort Sea o a 11 1 l B P a P S P ? BES02B I C h u c k c h i S e a g U k CHS02 Beaufort Sea BES02A GOM01 r r r i l l t Figure 2.1. DGGE of PCR amplified g20 gene fragments showing locations of excised and sequenced bands. Labelled bands were excised from the gel, cloned and then sequenced. Bands in the same lane with the same letter beside them (a, b or c) were >99% nucleotide sequence similarity. From within each lane, only one sequence with >99% nucleotide sequence similarity was included in further analysis. Bands with labels that have an asterix and are in boldface type were included in the phylogenetic analysis and those with 'NS' indicate bands where sequences contained stop codons. inlets (5 of 6), the cyanobacteria mat (4 of 8), the southeast Pacific (2 of 2) and the catfish pond (1 of 1) were closely related to sequences in the CPS group. 2.4.3 Phylogenetic analysis reveals four new 'unknown' clusters Phylogenetic analysis also revealed four new 'unknown' clusters, two of which were entirely composed of sequences from freshwater environments (Fig 2.2). One of the freshwater groups (Group G) included two sequences from Cultus Lake, one sequence from the Arctic cyanobacteria mat, one sequence from Lake Constance and one sequence from Chil l iwack Lake. The other freshwater group (Group J) contained four sequences from Cultus Lake and two sequences from the Arctic cyanobacteria mat. The other two groups consisted of sequences from both freshwater and marine environments. Group 'FT contained five sequences that were obtained from Cultus Lake, the Beaufort Sea and Lake Constance. Group ' K ' contained two sequences from the Chuckchi Sea, and one from the Beaufort Sea. 40 •T4 I LAC95A-1 I AIMT98-20 PGOM01-16 • SPM02-28 •BES02*iSSS • CHS02-10 " CHS02-S —— SE38 I s s « M e : : - . •LAC9SA-4 — BES02A-4 GS273B: •BES02A-3 • BESQ2A-19 • BES02A-17 ?,rrBjwOiicp:s I GS2624 : 6 7 / 1 0 0 W : S S 4 7 Z 3 : : : : : : :7::;-|Siil*>2^::;:;::;H:^ :4:4M|:CUL02^1SH«:¥H::S:::S4S:i:x *'Sr'SPM02-2i mmmm ' *8i|:CULQ2M-i4:o: 6 4 ' C U L 0 2 H - 1 t |CUL02H-19 "LAC95B-12 "CH1.02E-4 ¥ ICUI02H-14 S^ : ? S P M 0 2 - 2 6 :SS i cu i -CUL02H-16 <!4 L •BES02B-28 MARINE GOM01 BES02A/B CHS02 ANT98 COL00 CHIO0 SAI99 PES99 MAI00 TEAOO Gulf of Mexico Beaufort Sea (Arctic Ocean} Chuckchi Sea (Arctic Ocean) Antarctic Peninsula (Southern Ocean) Coast of Colombia (SE Pacific) Coast of Chile (SE Pacific) Salmon Inlet (NE Pacific Coastal Inlet) Pendrell Sound (NE Pacific Coastal Inlet):: Malaspina Inlet (NE Pacific Coastal Inlet) Teakerne Arm (NE Pacific Coastal Inlet) BES02B-27 LAC95B-8 •ANT98-2S • GOM01-15 •CUL02M-17 rzsb>..... SPM2 8al_l BES02A-18 : M 1 SPM02-34 SAI99-44 82IJ-SPM02-36 83" SPMQ2-35 I— ANT98-23 FRESHWATER CUL02M / H Cultus Lake (British Columbia) CHL02E Chilliwack Lake: (British Columbia) CAT02 Catfish Pond SPM02 Shore Pond Mat (Arcticcyanobacteria mat): LAC9SA / B Lake Constance (Germany) •S-BnM1 CAT02-30 ... •: N , . I I I I I I I : S S 4 7 M ' ' : : — - G S 2 7 » 3 ••31Bi-'-: ::-- ::v:''••••••• •SS471S • ANT98-26 73" — PES99-50 GOMOI-IViHSSiH ! l 7 ' 1 0 0 - C O L 0 0 - 3 6 : : : I LAC95B-10 I SPM02A-27 I GOM01-8: GOMOI-'lp: ' "SS<02f .... r P 7 7 ]>P _ p O » S-WHM1 M n s L p - MAI00-2 • flL— PES99-47 CHI00-38 7 7 « 1 3 l — p 8 1 i s ; : P7»;:-x::::;:;.:: S-PWM1 Figure 2.2 Maximum likelihood tree of g20 fragments. Quartet puzzling support values for the maximum-likelihood tree and bootstrap values for the corresponding neighbor-joining tree are shown as percentages to the lower left of the appropriate node. Sequences of cyanophage isolates are indicated in boldface type and sequences in italic type are environmental g20 sequences that were obtained from Genbank (SE=Skidaway Estuary, SS=Sargasso Sea, GS=Gulf Stream). Environmental sequences obtained for the present study are described in the lower left corner and the sequences of the cyanophage isolates obtained for the present study are S-PWM3 and S-PWM4. Clusters A - F and I-III were assigned by Zhong et al. 2001 and clusters G, H , J, K and CSP (^Cultured Synechococcus Phage) were assigned by the authors. Clusters labeled a, b and c indicate where sequences were >99% identical based on nucleotide sequence. A l l clusters were assigned on the basis of phylogenetic relatedness. 2.4.4 Variation in sequences obtained from a single location Genetic variation within the g20 gene sequences from the six locations that were the most intensively sampled was analyzed. There was greater variation in the three freshwater samples when compared to the three marine samples. The pairwise nucleotide similarity in the freshwater samples C U L 0 2 M , C U L 0 2 H , and SPM02 varied from 59.9 to 97.7%, 61.6 to 97.4%, and 59.1 to 99.0%, respectively, and in the marine samples B E S 0 2 A , G O M 0 1 and A N T 9 8 it varied from 56.5 to 86.3%, 61.7 to 88.9 and 60.8 to 79.4%, respectively. Phylogenetic analysis showed that sequences obtained from a single location were divided among two to five different clusters (Fig 2.2). It appeared that the greater the number of distinct sequences obtained, the greater the number of clusters there were representing these sequences. For example, seven unique sequences from the Beaufort Sea sample B E S 0 2 A were divided into 2 clusters ( K and I) plus five sequences that did not cluster with any groups, while eight unique sequences obtained from the cyanobacteria mat sample S P M 0 2 were divided into 5 clusters (a, J, G , I and III c). Five unique sequences were obtained from the Gul f of Mexico sample G O M 0 1 , while four unique sequences were obtained from the Southern Ocean sample A N T 9 8 and the Cultus Lake sample C U L 0 2 H .The sequences from G O M 0 1 and A N T 9 8 were divided among 2 clusters and each had a sequence that did not cluster with any other group and the sequences from C U L 0 2 H were divided among three clusters. 42 2.4.5 Recovery of similar sequences from different environments Identical sequences (>99% identity) were recovered from very different aquatic environments. For example, identical sequences (GOM01-16, SPM02-28, L A C 9 5 A - 1 and ANT98-20) were recovered from the Gul f of Mexico, the Arctic cyanobacteria mat, Lake Constance (Germany) and the Southern Ocean (Fig 2.2, Cluster a). This was also true for the sequences BES02-27 and L A C 9 5 B - 8 , which were recovered from the Beaufort Sea and Lake Constance (Fig 2.2, Cluster b) as well as the sequences COL00-36, L A C 9 5 B - 1 0 and SPM02-27 which were detected in the southeast Pacific, Lake Constance and the Arctic cyanobacteria mat (Fig 2.2, Cluster c). In addition, in 1999 and 2000, sequences that were 87.4% identical (PES99-47 and MAI00-2) were obtained from two northeast Pacific Inlets that are in close proximity to one another. 43 2.5 Discussion 2.5.1 Most important findings Phage g20 gene fragments were amplified from both freshwater and marine locations from pole to pole. The presence of g20 genes in these radically different environments suggests that cyanophages are present in aquatic environments on a global scale. The four most important findings from the present study were the P C R amplification of g20 sequences from remarkably diverse environments, the extent of variation in the g20 sequences obtained, the presence of identical g20 gene fragments in extremely different environments and the first phylogenetic groups of aquatic virus sequences that are defined by their geographic location. 2.5.2 Specificity of cyanophage-specific-primers Consistent with their design, the primers used in the present study amplified a g20 gene fragment from phages belonging to the family Myoviridae that infect Synechococcus spp., but did not amplify g20 from the coliphages T4, RB33 , RB69 and L Z 4 or from other bacteriophage including putative Myoviruses that infect the marine bacterium Vibrio parahaemolyticus, and cyanophage belonging to other families of tailed phage (Podoviridae and Siphoviridae) (data not shown). Other researchers also found there was no amplification when a similar primer pair that amplifies the same g20 fragment in cyanomyoviruses was tested on seven other myoviruses that infect the marine bacteria Alteromonas sp. and Vibrio natriegens (66). These researchers showed their primers amplified g20 gene fragments from the twenty cyanomyophage isolates they tested. P C R using our slightly different g20 primers resulted in positive amplification from three out of the four morphologically distinct cyanomyoviruses I tested. 2.5.3 Separation of large fragments on denaturing gradient gels 44 The original purpose of using denaturing gradient gels was to make comparisons among cyanophage communities, by comparing the D G G E fingerprints generated from P C R amplified g20 gene fragments. This type of analysis assumes that each band in the gel represents a distinct genotype and that identical sequences migrate to the same position in the gel. However, sequences that were identical, or nearly so (>99% similar), migrated to different positions in the gel and, sequences that are different can migrate to the same position. Although the sequences that were >99% similar may have represented different phages with very similar sequences, the possibility that these differences were due to P C R and sequencing error could not be ruled out. In addition, P C R amplification of a single target sequence with degenerate primers can result in several bands in a denaturing gradient gel, giving the false impression that there were as many target sequences in the sample. For example, when P C R amplified g20 gene sequences from cyanophage isolates were separated on denaturing gradient gels, multiple bands were observed (this study, data not shown). D G G E was designed to separate small d s D N A fragments that differed in only one to several base pairs, and therefore clear separation of larger fragments that share from 50 to 100% pairwise nucleotide similarity is more difficult. The presence of a high background haze and smearing was a problem for some of the samples. The lack of clear separation and resolution of bands would have made conclusions drawn from the community comparison analysis less convincing. Thus, the D G G E separation of these larger g20 fragments (-595 bp plus 40 bp G C clamp) must be further optimized before comparisons of cyanophage communities can be made using the D G G E community fingerprints generated using g20 gene fragments amplified with the primers C P S 4 G C and CPS9. Although the D G G E community fingerprints could not be used for community comparisons, they were valuable because they resulted in the separation of g20 fragments that could be excised, cloned and then sequenced. 45 2.5.4 The Cultured Synechococcus Phage (CSP) phylogenetic group The phylogenetic analysis of g20 genes revealed the existence of a group that represented all sequences obtained from cultured Synechococcus phage as well as many of the environmental sequences. The environmental sequences that clustered within the C S P group are likely Synechococcus phages, while the hosts for sequences that cluster outside the group remain unidentified. The majority of sequences from the northeast Pacific Inlets and the Gul f of Mexico fit within this group. This is not surprising, as Synechococcus cells and the phages that infect them are abundant at these locations (12, 50, 51). Because of the presence of cyanobacteria, it was also not surprising that some of the sequences obtained from the Arctic cyanobacteria mat and the catfish production pond were similar to sequences in the C S P group. The two other locations where most sequences clustered within this group were the southeast Pacific and the Southern Ocean. Whether or not Synechococcus phages are common in these locations remains to be determined. On the other hand, almost all sequences obtained from the lakes in British Columbia, Canada and Germany and those obtained from the Arctic did not fit into this group. The presence of Synechococcus phages in these environments is generally unknown, but Synechococcus phages have been isolated from Lake Constance in Germany (C. A . Suttle, pers. comm.) and therefore it is interesting that more of the sequences obtained did not cluster within the C S P group. Perhaps g20 sequences that cluster outside the C S P group represent cyanophages for which there is no cultured representative. It is also known that there are very low abundances of Synechococcus spp. in the Arctic Ocean and therefore the abundances of phages that infect them are also probably very low. Three of the Arctic Ocean sequences were obtained from a depth of approximately 3000 m, where Synechococcus phages i f present, would likely be at low abundances. It is possible that some of these phages infect Synechococcus spp. that are 46 genetically distant from the WH7803-like strains that were used to isolate most of the cultured phages (66) or that they infect hosts that are closely related to Synechococcus spp. such as Prochlorococcus. We also cannot rule out the possibility that these primers amplify g20 gene fragments from phages that do not infect cyanobacteria. It is possible that the sequences were obtained from viruses that are no longer infectious and have remained intact for some time. The isolation and genomic sequencing of additional marine phages w i l l provide answers to these questions. 2.5.5 Diversity of phages with g20 genes O f the 53 g20 sequences obtained from the natural environment in the present study, none clustered with the 'unidentified' groups (A through F) that consist of previously obtained sequences from the Sargasso Sea, Gul f Stream and Skidaway estuary. Twenty-two of them were related'to sequences in the C S P group (which includes previously described clusters I, II and III), nineteen fell within four new groups (G, H , J and K ) while twelve were not related to any group. Either the group of phages that infect cyanobacteria are very diverse, or the sequences outside of the C S P cluster represent a diverse assemblage of phage that do not infect cyanobacteria. The range of phage targeted by these g20 primers is unknown and it is has been demonstrated that they do not even target all of the Myoviruses that infect Synechococcus spp. This being true, then the fraction of all aquatic phage that these primers target is probably miniscule. Despite this primer bias, the sequencing of only a fraction of g20 gene fragments (as represented by D G G E bands) and the sampling of a small number of global niches, we have shown that there is a great deal of genetic variation within natural populations of phage carrying the g20 gene. I believe that the actual diversity of this group of phage must be much greater. It makes sense that the greater the number of unique sequences obtained from an 47 environment, the greater the number of distinct groupings. This pattern carries over on a global scale, where the more sequences we retrieve from aquatic environments, the more unique sequences are obtained that w i l l form new phylogenetic clusters. Eventually we should encounter only redundant sequences, which wi l l allow us to estimate the genetic variation in the gene sequences that we can target with these primers. The current study introduces another four genetically distinct groups of unknown sequences as well as an additional twelve sequences that did not cluster with any of the other groups. This supports our view that we have only uncovered a small fraction of the diversity of this group of phages. It was interesting that the sequences obtained from the three freshwater environments showed a greater divergence in pairwise nucleotide similarity when compared to the three marine environments, but this may be artificial because only six samples were compared. 2.5.6 Are there groups that consist entirely of freshwater phage? O f the four new groups representing unknown phage g20 sequences, two of them were entirely composed of freshwater sequences. These two groups were probably the result of the amplification of g20 genes from closely related phage that infect closely related hosts that may have adapted to and are now confined entirely to freshwater environments. These are the first groups of viruses discovered from aquatic environments that are defined based on geographic location. A l l other groups consisted of a mixture of sequences obtained from many different locations. The lack of geographical separation in phylogenetic groups has been seen in the analysis of gene sequences amplified from other families of marine viruses including Podoviridae (Reid, K and C A . Suttle, unpublished results) and Phycodnaviridae (9, 43, 44). It is entirely possible that sequences from other environments w i l l be very similar to those in these groups of sequences that were obtained exclusively from freshwater environments, but this 48 remains to be seen. 2.5.7 What is the significance of identical g20 sequences in different environments? In three instances in the present study, >99% identical sequences were recovered from very different environments. The most interesting case was for the sequences obtained from the Gul f of Mexico, the Southern Ocean, the Arctic cyanobacteria mat and Lake Constance, Germany because of how different and geographically separated these locations are. Not only were some samples collected from opposite ends of the planet, but in two cases they were collected from entirely land-locked bodies of water. There are three possible scenarios which might explain this: (1) genetically similar phages exist in both environments and have not changed since the separation of these populations (eg the separation of the sea from these lakes) (2) in evolutionarily recent times, phage have had contact through multiple host steps which has allowed for the swapping of a mobile genetic element which includes the g20 gene and (3) there is some mechanism by which phages are transported long distances. These locations likely harbour distinct host communities, as such great distances separate them, but closely related cyanobacteria may exist. It seems reasonable to assume that these sequences are from phage that share closely related hosts. For example, phylogenies inferred from the D N A polymerase gene of large d s D N A viruses (Phycodnaviridae) show that genetically distinct groups of viruses are clearly resolved and correspond to the host taxon they infect (8). Therefore, it may be possible that the g20 gene sequences are from phage that infect closely related hosts and that they have not changed much since separation. It is true that hosts and viruses are continuously co-evolving due to selection pressure for the hosts to develop resistance and for the virus to maintain its ability to infect its host. It is not unreasonable to believe that the majority of the changes in the viruses would be in the tail fiber genes and not the g20 gene, as the tail fibers are the structures 49 that contact the host during infection. The genomes of double-stranded D N A phage are thought to be 'mosaics' that are composed of genetic material from a global phage pool (22). The mosaic nature of these phage is likely due to the sequential assembly of genetic modules when these viruses were created. Recent swapping of these genetic elements has probably not occurred, because genetic exchange through coinfection has not been shown to occur in lytic phages which include this family of phages (Myoviridae). There is a 10 kb genetic module that includes the g20 gene that is conserved between the coliphage T4 and the marine Synechococcus phage S-P M 2 (20), but for gene transfer to occur across such distances would require phage with broad host ranges that are capable of exchanging D N A . 2.5.8 Addressing the question of diversity in natural virus communities and the usefulness of identifying 'signature' gene sequences The importance of both photosynthetic and heterotrophic marine prokaryotes and eukaryotes is established and it is agreed that viruses that infect these organisms w i l l affect their mortality and diversity. To begin to understand the magnitude of the control that viruses exert on their hosts we need to know something about the diversity and structure of natural virus communities. The term 'diversity' implies knowledge of both the number of particular genotypes, as well as the relative abundance of each type. So far, we have no means of enumerating specific groups of viruses or even classifying the viruses into groups. Because most marine viruses infect only a single host species, viruses wi l l l ikely be classified based on the host they infect. To determine whether there are 'signature' gene sequences that w i l l reflect groups of viruses infecting closely related hosts, more sequences need to be obtained from viruses that are established in culture. It is possible that the g20 gene phylogenies w i l l show that groups of cyanophage do not reflect the host taxa and therefore it w i l l not be a useful marker sequence. 50 This may be because the host species (Synechococcus) are closely related and the host ranges of some of these viruses are broad. Some aquatic bacteriophages infect a broad range of hosts (Sphaerotilus natans, Escherichia coli and Pseudomonas aeruginosa) (28). As well , some cyanomyoviruses can infect phycoerythrin- and phycocyanin-dominant strains of Synechococcus; others infect isolates of both Prochlorococcus and Synechococcus (Matt Sullivan pers. comm.). Preliminary data suggests that D N A polymerase sequences may serve such purposes. Phylogenies of PCR-amplified regions of the conserved D N A polymerase gene of phage isolates (Podoviridae) (Reid, K and C A . Suttle, unpublished results) and algal viruses (Phycodnaviridae) (9, 8) have shown that the phylogenetic groupings reflect the host of the viruses. This may also be true of the g20 gene, but this w i l l depend on the identity of the hosts for the majority of the phylogenetic groups. If these 'signature' sequences exist, they may serve as targets for quantitative genetic approaches, such as Q-PCR. Using these methods, we may be able to understand the diversity of aquatic viruses. As the hosts for unknown groups become characterized, we may also be able to obtain an environmental sequence and deduce something about the host of that virus based on its similarity to other known sequences ie. its phylogenetic affiliation. 2.5.9 Conclusion The present study provides insights into the richness of putative cyanomyoviruses with g20 genes. The presence of g20 sequences in wide ranging environments shows that these viruses are extremely widespread and that there may be groups of cyanophage that are exclusively found in one type of environment. Future studies w i l l reveal more information on the diversity of viruses with g20 genes and whether or not phylogenetic groups (based on g20 51 sequences) w i l l reflect the host species or strain of cyanophage. It w i l l be particularly interesting to determine i f g20 genes exist in cyanophages that infect different species of unicellular or filamentous cyanobacteria as well as those that infect Prochlorococcus. 2.6 Acknowledgements I would like to thank the crew and scientists aboard the research vessels R V F.G. Walton Smith, R V Mirai, R V Lawrence M. Gould, RVEB Nathaniel B, Palmer, and the C C G S Vector for their help with sample collection. I would also like to thank Dr. Elizabeth Kutter for supplying the coliphages. I am grateful to Steven W . Wilhe lm for the southeast Pacific viral concentrates, Jessie Clasen for the B . C . lake viral concentrates, A m y Chan for the Lake Constance viral concentrates, Steven M . Short for the Southern Ocean viral concentrate, Vincent Warwick for the Shore Pond cyanobacteria mat, Barry Hurlburt for the catfish production pond viral concentrate and Nina Nemcek for her help with further concentration of samples. Special thanks are reserved for Steven Short for his help with the phylogenetic sequence analysis. This study was supported by a grant from the Natural Sciences and Engineering Research Council of Canada ( N S E R C ) to C. A . Suttle. 52 Chapter III. Summary Before the work for this thesis had begun, very little was known about the diversity of aquatic cyanophage communities. It was previously shown that there are viruses in the marine environment that infect Synechococcus, a small chroococcoid cyanobacterium and that these viruses could be very abundant (>105 ml" 1) (reviewed in 49). Cyanophages were shown to be morphologically diverse, representing all three phage families (Myoviridae, Siphoviridae and Podoviridae), with the majority of cyanophage isolates belonging to the family Myoviridae. It was estimated that they lyse a small percentage of host Synechococcus cells on a daily basis (39) and it was shown that the most abundant Synechococcus spp. are resistant to the most abundant cyanophages present (57). This is thought to be the case in coastal nutrient rich areas where contact rates between host and virus are high and therefore there would be a strong selection for resistance and that sensitive host cells are more prevalent in the open ocean where contact rates are much lower (49). It was also known that viruses that infect freshwater Synechococcus strains were present in Lake Constance, Germany (A. Chan and C A . Suttle, pers. comm.), but no other molecular work on freshwater Synechococcus phage had been done. The discovery of the conserved g20 gene in three cyanomyoviruses facilitated the use of molecular techniques to study natural communities of cyanophage in order to better understand their ecology. The use of g20 primers in the natural environment has been limited and has been restricted to the Atlantic Ocean and Sargasso Sea. Using denaturing gradient gel electrophoresis of P C R amplified g20 fragments it was shown that cyanophage communities differed spatially along a transect in the Atlantic Ocean (60). During the time of my thesis, a manuscript was published that demonstrated the high genetic diversity of marine cyanophage assemblages in the Gul f Stream, Skidaway Estuary and Sargasso Sea (66). Through sequence analysis of a fragment of the g20 gene they showed that up to 29 genotypes encountered in a single sample. 54 M y work has been an extension of the work of others and has also demonstrated spatial differences in cyanophage communities. In addition, I was able to show that these communities could be different over very small spatial scales. These confined areas were separated by landmasses and even by gradients of temperature and salinity. Differences in community fingerprints were observed in relation to differences in relative fluorescence, and the abundances of Synechococcus spp. and infectious cyanophage. The changes in community structure in relation to host cell abundance and relative fluorescence supports the idea that the structure of cyanophage communities is l ikely dictated by interactions with hosts. The interactions of the viruses with their hosts w i l l be affected by the abundance and composition of the host communities within these somewhat isolated regions. M y work also extended the knowledge of cyanophage diversity as inferred by phylogenetic analysis of g20 gene sequences. I used P C R primers to amplify g20 sequences from marine and freshwater environments worldwide. Before my work, g20 fragments had never been P C R amplified from water samples collected outside of the Atlantic Ocean and Sargasso Sea and to my knowledge no one had successfully amplified them from freshwater. I demonstrated that this group of viruses is more diverse than was previously known and that there may be genetically distinct groups that are only present in freshwater environments. I also showed that sequences from areas that were environmentally similar, such as the two polar regions were not more similar to one another than they were to sequences from tropical or temperate regions. In fact, sequences that were greater than 99% similar were found dispersed amongst significantly different environments. In one case, these sequences were obtained from a subtropical marine environment, a polar ocean, a lake in Germany and a freshwater pond in the Arctic. We cannot be sure of the hosts for the majority of the groups that were inferred from the phylogenetic analysis because there are no sequences from isolates that cluster within 55 these groups. Two of the groups of sequences that were >99% similar also fit into this category. We do not know i f these four sequences were amplified from viruses that have the same host or i f their hosts are different. Identification of hosts for these groups wi l l decide whether or not the g20 gene is a good signature sequence that can be used to identify cyanophages. In addition to developing P C R primers that can be used to amplify g20 gene sequences from a wide variety of environments (in fact we have not found an environment where we have failed to amplify these sequences), my work has extended our knowledge of the ecology of this diverse group of phage that infect cyanobacteria. I have shown that there may be segregated populations of hosts and viruses that interact independently of one another. I have also shown that g20 sequences are more geographically widespread and more genetically variable than previously known. More wi l l be revealed about the diversity and ecology of cyanophages with the use of molecular techniques similar to those used in this thesis. 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