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DNA polymerase sequence analysis of bacteriophage, podoviridae Reid, Karen E. 2003

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DNA POLYMERASE SEQUENCE ANALYSIS OF BACTERIOPHAGE, PODOVIRIDAE by KAREN E. REED B. Sc., McGill University, 2000 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Earth and Ocean Sciences) We accept this thesis as conforming to the retired standard THE UNIVERSITY OF BRITISH COLUMBIA March 17th 2003 © K a r e n Reid, 2003 In presenting t h i s thesis i n p a r t i a l f u l f i l l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t freely available for reference and study. I further agree that permission for extensive copying of th i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It i s understood that copying or publication of th i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of fczrfh and Qua/) ^CJMl t V i The University of B r i t i s h Columbia Vancouver, Canada ABSTRACT Our oceans are biologically complex systems that incorporate a collection of participants. The most abundant players, which are the bacteria and viruses, are invisible to the naked eye. Seawater typically contains about a million bacteria and 10 million viruses per millilitre. Despite the abundance of viruses in seawater, little is known about their composition or diversity. This dissertation presents the design and application of a new set of PCR primers created to target a subset of bacteriophages. Bacteriophages are the viral fraction that infect and propagate through living bacterial cells. They vary both in terms of their morphology and their genetic constituents. My work focused on the family Podoviridae, one of 3 bacteriophage families that share similar morphological characteristics. The most notable characteristics include an isometric capsid about 60 nm in diameter, and a short, stubby tail. Moreover, although this family has been extensively reported in transmission electron microscopy scans, this study is the first to obtain Podoviridae genetic information from marine viral communities. To have a sense of the level of diversity that this Family constitutes, I designed a set of primers targeting the DNA polymerase gene {pot). The gene is approximately 2.0 kb in length and the primers target a 1.2-1.4 kb region for amplification. Following product verification and method optimization, I turned my attention to analysing environmental samples. Seven environmental samples from the Straight of Georgia, British Columbia, and one sample from the Gulf of Mexico, within the Mississippi River plume, were selected. The amplified products generated from each sample were cloned and 20 clones for each were randomly chosen for genetic comparison. Restriction fragment length polymorphism (RFLP) and sequencing were the methods adopted for analysis. RFLP analysis revealed 29 distinct restriction patterns of DNA pol genetic variation. These patterns indicate that certain samples contain greater genetic diversity than others and also ii f that podovirus communities within a site are more similar than between sites. Sequencing these restriction patterns revealed that at the nucleotide level, 17 sequences were at least 5% different from each other. DNA pol genes from environmental podoviruses are divergent from coliphage T7 and T3, and are more similar to the marine isolates Roseophage SIOl and Cyanophage P60. These latest environmental sequences clustered into four groups, two of which contain no cultured representatives. Moreover, the results indicate that the marine podoviruses share a common ancestor with the coliphage lineage. The work presented in this dissertation is the first study to target and examine the genetic diversity of environmental Podoviridae. iii TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iv LIST OF TABLES vi LIST OF FIGURES vii LIST OF ABBREVIATIONS viii PREFACE ix ACKNOWLEDGMENTS x DEDICATION xi CHAPTER 1. INTRODUCTION - A REVIEW OF SUPPORTING LITERATURE 1 1.1. Viruses and Bacteria in Marine Environments 2 1.2. Studying Marine Viruses 4 1.2.1. Morphological Diversity.. 4 1.2.2. Genetic Diversity and the Molecular Tools Used 5 1.3. Sequenced Marine Viral Genomes 8 1.4. Viral Genes for Primer Design 11 CHAPTER 2. SEQUENCING PODOVIRIDAE DNA POLYMERASE GENES FROM WATER COLUMN AND SEDIMENT SAMPLES 15 2.1 Abstract 16 2.2 Introduction 16 2.3 Materials and Methods 18 iv 2.4 Results 24 2.5 Discussion 30 2.6 Acknowledgments 33 CHAPTER 3. CONCLUSION AND FINAL REMARKS 34 APPENDIX I. VIRAL AND BACTERIAL ENUMERATIONS FROM CANADIAN ARCTIC ICE CORES ; 39 REFERENCES 45 v LIST OF TABLES Table 2.1. Oceanic stations where water samples and sediment samples were collected 29 Table A . l . Viral and bacterial enumerations from Canadian Arctic cores 51 vi LIST OF FIGURES Figure 1.1. Transmission electron micrographs of marine bacteriophages 13 Figure 1.2. An example of DGGE analysis 17 Figure 1.3. Electron micrograph of Roseophage SIOI and Cyanophage P60 19 Figure 1.4. Comparison of genome arrangements between P60, T7, Ye03-12 and SIOI 20 Figure 1.5. PCR analysis of large dsDNA algal virus DNA pol PCR products 22 Figure 1.6. PCR analysis of the g20 gene from Myoviridae cyanophage isolates 23 Figure 2.1. First stage amplification of DNA pol within the Podoviridae family 34 Figure 2.2. RFLP patterns generated from the Mbo II digestion of natural virus DNA pol clones 36 Figure 2.3. A maximum-likelihood trees showing the phylogenetic relationship among the novel marine sequences and the five known Podoviridae DNA pol sequences 37 Figure 2.4. Clonal representation of each sequence from water column and sediment samples 40 Figure A.1. Phylogenetic relationship of the Arctic ice sequence with other Podoviridae DNA pol sequences 53 vn LIST OF ABBREVIATIONS bp Base pairs CCGS Canadian Coast Guard Ships CTD Conductivity temperature and density DGGE Denaturant gradient gel electrophoresis dsDNA Double stranded DNA FITC fluorescein-5-isothicyanate ICTV International committee on taxonomy of viruses kDa Kilo Daltons kPa Kilo Pascals NSERC Natural Sciences and Engineering Research Council of Canada PCR Polymerase chain reaction PFGE Pulsed field gel electrophoresis pol DNA polymerase gene RFLP Restriction fragment length polymorphism TEM Transmission electron microscopy TRFLP Terminal restriction fragment length polymorphism U Unit UV Ultra violet VC Viral concentrate VLP Virus like particles viii P R E F A C E The research performed during my graduate degree was carried out with the intention of eventually publishing the work. In view of this, the core of this dissertation in Chapter 2 presents my major research in publication format. The preceding chapter is an introduction with a literature review covering general information about viral classification and various techniques used in the field of marine molecular virology. The third and final chapter includes some closing remarks and generalized conclusions. Like many graduate student projects, you will learn in this last chapter that my research did not end with the same project as it started with. Considering my time committed and that results were still gathered, these earlier studies are included as a publication note in the Appendix. The text in both Chapter 2 and the Appendix are co-authored by Curtis A. Suttle, my graduate supervisor, and will be reedited and submitted for publication. Al l the research performed was carried out by myself unless otherwise acknowledged. i x A C K N O W L E D G M E N T S There are many people that I have to thank from all different areas of my life; personal, academic and athletic. This dissertation is a tribute to the patience and encouragement displayed for each of them. My deepest appreciation goes to my family and boyfriend whose love and support has been a constant source of strength. Mom, Dad, Pam, Dave and Mark you are my inspirations and I thank you for your belief in me at all times. I would sincerely like to thank Dr. Curtis Suttle for his supervision, guidance, and support throughout my study. Thanks to Dr. Mary Berbee, member of my supervisory committee for her valuable advice and input especially towards the end of my project. I am grateful for the sharing of technical ideas and creative thoughts from those who saw me day-in and day-out in the lab. Thanks to all those who picked me up when there seemed to be nowhere to go, and for the pat on the back when my efforts finally became reality. Sean Brigden, Amy Chan, Jessica Clasen, Alex Culley, Andre Comeau, Cindy Fredrickson, Andrew Lang, Janice Lawrence, Paul Lythgo, Pascale Loret, Nicole McLearn, Alice Ortmann, Jim Rossie, Steve Short and Tanya St. John, you have all shared your boundless enthusiasm for the world of science. Your friendship and encouragement will never be forgotten. A warm thanks to the members of the UBC Rowing team, who have kept me balanced throughout my studies. With their energy and admirable dedication I have learned so much about myself and about life. Finally, I must acknowledge the Natural Sciences and Engineering Research Council of Canada for providing my financial support throughout the better part of my degree. x DEDICATION This thesis is dedicated to my parents for their unfailing encouragement and understanding throughout my academic career. xi CHAPTER 1. INTRODUCTION - A REVIEW OF SUPPORTING LITERATURE l 1.1. Viruses and Bacteria in Marine Environments Although phages were first isolated during the first half of the 20 th century, it was not until 1960 when Spencer reported the first estimates of marine viruses. He calculated the marine viral abundance to be upwards of 10 plaque forming units/ ml, based on the bacterial mixture consisting of forty marine isolates (Spencer, 1960). Considering that most viruses range between 20 to 200 nm in diameter, light microscopy left viruses invisible in marine samples until new methods were adopted. Years later, estimates of total virus abundance by transmission electron microscopy (TEM) (Torrella and Morita, 1979; Bergh et al., 1989; Borsheim et al., 1990; Bratbak et al., 1990b; Hara et al., 1991; Wommack et al., 1992; Weinbauer and Peduzzi, 1994) and subsequently by epifluorescence microscopy (Hennes and Suttle, 1995; Drake et al., 1998; Noble and Fuhrman, 1998; Guixa-Boixereu et al., 1999) revealed counts of approximately 105 to Q 10 virus-like particles (VLPs) per ml. These data sets exposed an opportune field to what is know today as marine virology. Viral morphological observations, such as size and shape, have been collected from the earliest studies to today's observations and complied to build a viral taxonomic classification system. The International Committee on Taxonomy of Viruses (ICTV) classifies viruses into orders, families and genera (Murphy et al., 1995). Typically, the majority of isolated marine bacteriophages are among the order Caudovirales (Maniloff and Ackermann, 1998), the dsDNA tailed viruses (Suttle and Chan, 1993; Proctor, 1997; Lu et al., 2001). The Myoviridae, Podoviridae, and Siphoviridae families fall within this Order and each have distinguishable morphological characteristics (Figure 1.1.). Briefly, myoviruses are characterized by having contractile tails and include genera such as the T4-like phages. Siphoviruses are described as having long, non-contractile tails and include e and Tl-like phages, while the podoviruses have 2 short tails and comprise viruses like the T7-like genus. The work presented in this dissertation will focus on the family Podoviridae. Figure 1.1. Transmission electron micrographs of marine bacteriophages. Scale bars represent 50 nm. a) Family Myoviridae, which possess a contractile tail, b) Family Podoviridae are distinguished by having a short tail, c) Family Siphoviridae, characterized by having a long flexible tail. (Suttle, 2000). It has long been known that prokaryotes make up a large portion of the marine biological 5 7 life. Generally, estimates of 10 to 10 cells/ ml are observed in marine environments (Hobbie et al., 1977; Azam et al., 1983; Bird and Kalff, 1984; Noble and Fuhrman, 1998). Prokaryotes are 90 both abundant, with approximately 10 cells inhabiting our oceans (Whitman et al., 1998), and represent the largest fraction of biomass, contributing about 7 Gt of carbon (Wilhelm and Suttle, 1999). The ocean microorganisms are not only significant in terms of numbers but also with respect to global oxygen production and biogeochemical cycling. As much as 50% of the global oxygen is produced from the marine prokaryotic microorganisms and the small eukaryotes, and 3 they have a leading role in carbon and nutrient cycling in ocean systems (Azam et al., 1983; Fuhrman, 1999). Viruses and bacteria are biologically connected in a microscopic world, with an important influence at the macroscopic level. Together they are responsible for much of the carbon flow through the marine ecosystem (Bratbak et al., 1990b; Fuhrman, 1999; Wilhelm and Suttle, 1999). In order for viruses to sustain their numbers and ultimately their survival, they must infect then subsequently lyse their host. The bacterial mortality due to viral lyses have estimates ranging anywhere from 8 to 66% (Suttle and Chen, 1992; Suttle, 1994; Fuhrman and Noble, 1995). These estimates of viral lysis along with those for protist grazing and other lethal agent such as UV, combine to make up the total bacterial mortality. Once the cells are lysed by viruses, the carbon and nutrients released integrate into a pool of dissolved organic matter and later are reincorporated into the microbial population (Azam et al., 1983; Bratbak et al., 1990b; Wilhelm and Suttle, 1999). The interactions between bacteria and viruses are intriguing as they remain leading contributors to our ocean biological system. 1.2. Studying Marine Viruses 1.2.1. Morphological Diversity As previously discussed, marine viruses represent an enormous abundance, which translates into a large potential for morphological variation. The morphotypes observed are catalogued and classified into their respective families. Even though ocean samples from many locations have been studied, the relative proportions of each bacteriophage morphotype differ with respect to the study (Torrella and Morita, 1979; Bergh et al., 1989; Bratbak et al., 1990a; Hara et al., 1991; Wommack et al., 1992; Borsheim, 1993). In each of these reports however, the 4 TEM observations of seawater revealed that viruses having short tails, non-contractile tails and contractile tails were all represented, covering all the family Caudovirales. In addition, viruses without tails were also observed (Bergh et al., 1989; Bratbak et al., 1990a; Wommack et al., 1992). These tailless-viruses could have been bacteriophages that lost their tails during sample preparation or they could represent another viral family (Bergh et al., 1989; Wommack et al., 1992). Despite the unpredictable representation of the phage morphotypes in natural viral communities observed in TEM, marine phage isolates belong predominantly to the family Myoviridae and the Siphoviridae, whereas Podoviridae isolates are in minority (Waterbury and Valois, 1993; Kellogg et al., 1995; Short and Suttle, 1999; Lu et al , 2001). Such observations are likely a reflection of the selected host culture used for isolation, which does not reflect the natural populations. While morphological classifications correlate to visual observations, the advent of molecular analysis has provided an alternative way of looking at marine viral diversity. 1.2.2. Genetic Diversity and the Molecular Tools Used Over the last 20 years, many molecular challenges have been overcome and a variety of methods have been adapted to gain access to the genetic mysteries enclosed within a virus' capsid. In terms of techniques, molecular methods have included those comparing viruses at a genome level to fingerprinting individual genes and1 randomly cloning marine viral genetic material. PFGE - Whole Genome Separation At the genomic level, one way of characterizing viruses is according to their genome size, via pulsed field gel electrophoresis (PFGE). This method is capable of separating large dsDNA 5 molecules by periodically changing the direction of the charge in the gel. Small sized genomes move rapidly through the gel, while large sized genomes remain at the top of the gel as they have greater difficulty penetrating the agarose matrix. PFGE has been used not only to characterize viral isolates (Hambly et al., 2001) but also to describe the genomic constituents of natural marine viral communities (Wommack et al., 1999a; Wommack et al., 1999b; Steward et al., 2000; Larsen et al., 2001; Fuhrman et al., 2002). Briefly, marine samples show the presence of viruses with genome sizes anywhere from 20 to 300 kb in size, with the majority between 25 to 60 kb. This size range depicts viral communities being composed of a high frequency of bacteriophages and fewer algal viruses. These observations are in concert with what is seen microscopically (Torrella and Morita, 1979; Wommack et al., 1992; Proctor, 1997). Although the PFGE information is a valuable technique for community fingerprinting, it carries limitations such that this method is not very sensitive, in that a substantial amount of DNA is required and secondly genomes sizes alone are not a reliable indicator of viral genetic relatedness. PCR and Associated Methods Several applications have been adopted that depend on PCR. Examples of PCR applications used to study marine viral genetic communities include competitive PCR to estimate the abundance (Fuller et al., 1998), targeting desired sequences for cloning and sequencing (Zhong et al., 2002), and fingerprinting analysis using either restriction fragment length polymorphism (RFLP) (Chen et al., 1996) or denaturing gradient gel electrophoresis (DGGE) (Figure 1.2) (Frederickson et al., submitted; Scanlan and Wilson, 1999; Short and Suttle, 1999; 2002). Details on the latter two will be discussed. 6 Figure 1.2. An example of DGGE analysis. In this gel each lane is the collection of cyanomyovirus g20 genes from natural water samples. The gel separates identical sized fragments (600 bp in this example) based upon their sequence composition. (Frederickson, unpublished) In recent years, DGGE has been used to catalog viral community fingerprints and trace shifts over a temporal and geographic scale (Short and Suttle, 2002). DGGE technology separates PCR amplicons from a single band on an agarose gel, which in theory contains many environmental sequences, on the bases of sequence composition. Disadvantages to this technique include the fact that a single sequence can produce multiple bands, and ultimately overestimate the environments natural diversity (Short and Suttle, 2002). Similar to DGGE, RFLP separates sequences on the basis of their genetic composition. RFLP however, physically cuts a sequence into DNA fragments based on the presence of restriction sites corresponding to a particular restriction enzyme. The work I have performed in this thesis combines cloning along with RFLP analysis. Other work adapting this technique includes the initial genotyping of the DNA polymerase gene of the family Phycodnaviridae (Chen et al., 1996). This method proved to be a quick and effective way of screening samples 7 prior to sequencing. Naturally, the effectiveness of RFLP is dependent on the enzyme chosen to cut the sequence. Typically, this method will underestimate the community's true diversity, which is why we decided to followed-up our RFLP analysis by sequencing the fragments. Shotgun cloning Recently an interesting report was published attempting to investigate the entire viral genetic material from seawater (Breitbart et al., 2002). They did this by randomly shotgun-cloning extracted DNA from the viral size fraction and comparing the sequences acquired to those available in genetic databases. Although most of their sequences did not show similarity to any previously sequenced DNA and only a fraction were related to viral sequences, this is the first report making attempts to look at the entire viral genetic pool (Breitbart et al., 2002). The high amount of overlap with non-viral sequences supports the argument that viral genomes are a result of ongoing gene lateral transfer (Hendrix, 1999; Hendrix et al., 1999; Rohwer and Edwards, 2002). Also, interestingly with respect to the phage fraction, the phage genome with the greatest number of hits was the marine phage Roseophage SIOI, a podovirus genome sequence used in my thesis. 1.3. Sequenced Marine Viral Genomes To date 10,175 viral genomes have been completely sequenced and submitted to GenBank through the National Center for Biotechnology Information. Of these, 83 are genomes from Caudovirales viruses, whereby 13 are myoviruses, 17 are podoviruses and 53 are siphoviruses. Unfortunately for the development of the marine virology field, all but three of these bacteriophage genomes were isolated from non-marine environments. The exceptions 8 include the sequencing of Roseophage SIOI (Rohwer et al., 2000), a lytic virus infecting Roseobacter, and Cyanophage P60 (Chen and Lu, 2002), also a lytic virus infecting the cyanobacterium Synechococcus sp. (Figure 1.3), and the Vibrio parahaemolyticus virus VpV262 (Hardies et al., in press). A l l three fall into the Podoviridae family. Figure 1.3. Electron micrograph of (A) Roseophage SIOI at x 100,000 and (B) Cyanophage P60, scale bar 50 nm. (Chen and Lu, 2002; Rohwer et al., 2000) The sequence information collected from Roseophage SIOI, Cyanophage P60 and VpV262 can were used to compare their genome arrangements and genetic relationships, but the sequence data could also be used for designing new primers or probes for subsequent studies. First, let us look at each these viruses independently. Roseophage SIOI was the first marine bacteriophage genome sequence to be published (Rohwer et al., 2000). SIOI has the greatest genetic relatedness to non-marine podoviruses T7 and T3, particularly with respect to the D N A replication machinery proteins. One interesting aspect that separates SIOI from the Enterobacter phages is that it lacks the RNA polymerase gene, which is an important component to both T7 and T3's life cycle. Rohwer et al. make the suggestion that SIOI is either dependent on the host 9 for RNA transcription or that the RNA polymerase sequence has diverged beyond recognition (Rohwer et al., 2000). Chen and Lu (2002) reported the second complete marine bacteriophage sequence, Cyanophage P60. P60 also belongs to the Podoviridae family. However P60, unlike SIOl, harbour's a RNA polymerase and its DNA machinery is even more closely related to the enterophages T7 and T3 than to marine SIOl (Figure 1.4). Class I i r Class I Class I 2 3 4 5 6 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 34 35 36 37 38 3946 47 ••> ' 1 i i - i 1 1 1 1 1 i i 1 1 i i i i i i ' i i i • ' < • i • • • • i . Cyanophage P60 ,DO> O O C E > . Coliphage T7 Bacteriophage •)>Ye03-12 Roseophage SI01 £ £ 5 > £ > . . \ . . L.<zJ.. Figure 1.4. Comparison of genome arrangements between P60, T7, Ye03-12 and SIOl. Orientation of the arrows indicate the direction of gene transcription. Class I genes are responsible for overcoming host restriction; class II genes are involved in DNA replication metabolism; and class III genes are transcribed as structural genes. (Chen and Lu, 2002) VpV262 is the latest marine bacteriophage genome to be completely sequenced (Hardies et al., in press). Like SIOl and P60, VpV262 also belongs to the T7-like genus within the Podoviridae family. Based on sequence similarity and genome arrangement, VpV262 was found to be most closely related to SIOl, and more distantly related to coliphage T7. Similar to SIOl, it too lacks the RNA polymerase that T7 harbours. However one distinguishing characteristic that 10 separates VpV262 from the previously described podoviruses is that it carries a different replicative module. VpV262 carries replicative machinery which is usually associated with bacteria. This finding alone highlights the challenges associated with capturing the podovirus genetic spectrum with a single gene target, such as the DNA polymerase gene within this replicative module. 1.4. Viral Genes for Primer Design The large amount of lateral gene transfer among phages and the inconsistent presence of many genes among viral isolates (Hendrix et al., 1999; 2000; Rohwer and Edwards, 2002) mean that it is not possible to identify a single gene that can be compared within an entire family let alone be ubiquitous within the order. So although a universal gene may not be possible, studying the diversity of a single gene can still prove to be very valuable. For example, looking at the presence and absence of the viruses that harbour a particular gene and assessing the natural genetic diversity, in terms of time and space, are significant contributions to the field. However, even with the best intentions, the lack of available sequence information adds a considerable challenge to creating specific primers. As of yet only two viral genes have been studied to any extent in marine systems, those being the DNA polymerase gene (pol) from the family of large algal viruses, Phycodnaviridae, and the g20 gene encoding a capsid assembly protein from a subgroup of bacteriophages, the Myoviridae family. Each is discussed briefly. Chen and Suttle (1995a; 1995b) reported on the development of a primer set targeting a -700 bp fragment of the Phycodnaviridae DNA pol gene (Figure 1.5). These viruses harbour a polymerase which has a highly conserved amino acid sequence YGDTDS. A primer set was designed to include this sequence, then these were used to address marine ecological questions 11 concerning viral presence, persistence, distribution and diversity (Chen et al., 1996; Short and Suttle, 1999; 2002). Samples collected from all around the world were analysed and it was found that algal viruses persist in nature over, both temporally and spatially, and related sequences were found to be geographically distributed. 1 2 } 4 5 6 7 8 9 10 11 12 13 Figure 1.5. PCR analysis of large dsDNA algal virus DNA pol PCR products. Lane 1, molecular weight marker (lambda Styl); lanes 2-12, viral isolates infecting Micromonas pusilla and Chlorella-Wko, algae; lane 13, negative control. Fragment sizes are -700 bp. (Chen and Suttle, 1995a) The first publication reporting on a primer set designed for a subset of bacteriophages targeted a gene encoding the capsid assembly protein, g20 (Fuller et al., 1998). More specifically, their primers were designed from three genetically distinct cyanophages within the Myoviridae family. Although this primer set generated a short 165 bp amplicon, this was an important advance for the forthcoming efforts in bacteriophage genetic diversity. Later, two research groups, independent of one another, lengthened the target sequence to capture a -600 bp section of the same gene (Figure 1.6) (Frederickson, unpublished; Zhong et al., 2002). Zhong and his colleagues used this primer set to screen viral concentrates collected from estuarine and 12 oligotrophic oceanic environments (Zhong et al, 2002), obtaining 114 unique g20 sequences demonstrating the high level of genetic diversity in marine myoviruses. Figure 1.6. PCR analysis of the g20 gene from Myoviridae cyanophage isolates. Lane 1, 100 bp ladder; lanes 2-6, viral isolates infecting Synechococcus strains; lane 7, negative control. Fragment sizes are -600 bp. (Frederickson, unpublished) A second study extrapolated from the previous work on g20 to unearth the sequence information surrounding this gene (Hambly et al., 2001). Hambly and her colleagues collected the sequence by primer walking on both sides of the known g20 sequence, to unveil 10 kb of a Synechococcus myovirus genome. This section of DNA matched the entire module that determines the structural components, composing of the gl8 to g23 genes, in the already sequenced T4 genome. They reported on the similarity between these phages' structural components and suggest potential molecular target sites throughout this section of the two Myoviridae genomes. More recently, this information has been used to design a primer set targeting the gene encoding the major head protein, g23, for use in deep oceanic samples (Ortmann, unpublished). To date, no molecular tools have been designed to target the genetic material of the Siphoviridae or Podoviridae. The material in this dissertation will be the first report of a primer set targeting the Podoviridae family. 13 Briefly, my project included designing a primer set to amplify DNA polymerase genes within the T7-like Podoviridae family, and assessing the genetic diversity in marine viral communities. We recognized that this family of viruses were abundant and diverse, based on TEM reports, and saw an opportunity to study them at a genetic level. With the recent sequencing of Roseophage SIOl and Cyanophage P60 genomes, we were able to align the protein sequences of the DNA pol genes of these two marine phages along with T7, T3 and phi-Ye03-12 phages. The primer design was based on consensus regions in the alignment. Since bacterial DNA polymerase genes are selected against in my project, VpV262 was not included in the sequence alignments for this project, but represents the fraction of viruses that are not captured with this primer set. Moreover, in effort to get a glimpse of Podoviridae natural diversity, we used our designed primers on seawater and sediment samples from the Straight of Georgia and the Gulf of Mexico. Each amplicon from the sample was cloned, and then these clones were subjected to RFLP analysis. Based on the restriction patterns generated, selected clones were sequenced, aligned, and their genetic relationship to one another depicted in a phylogenetic tree. The next chapter will cover the major research performed during my thesis and will discuss our findings. 14 CHAPTER 2. SEQUENCING PODOVIRIDAE DNA POLYMERASE GENES F R O M WATER C O L U M N AND SEDIMENT SAMPLES 15 2.1 Abstract Despite the high abundance and ecological significance of viruses in the oceans, we have only begun to explore their diversity. In order to examine the diversity of an important group of bacteriophages, we designed a set of primers targeting of the DNA polymerase {pot) gene of the family Podoviridae. Samples from coastal sediments and water columns produced a PCR-amplified product of about 1.2 kb. Using this approach, the genetic diversity of 7 environmental samples from the Straight of Georgia, British Columbia, Canada and 1 sample from the Gulf of Mexico, within the Mississippi River plume, were analysed. Restriction fragment length polymorphism (RFLP) analysis revealed 29 distinct patterns of genetic variation in DNA pol. RFLP patterns suggest that podovirus communities within a site were more similar than between sites. The sequence data revealed that at the nucleotide level of the 29 different restriction digest patterns, 17 sequences were at least 5% nucleotide different from the others. DNA pol sequences from environmental podoviruses are similar to known marine podoviruses Roseophage SIOl and Cyanophage P60 and to enterophages T7, T3 and phi-Ye-03-12. Our results indicate high genetic diversity within marine podovirus communities and demonstrate that the polymerase genes fall into several groups that are divergent from known bacteriophages. 2.2 Introduction Marine viruses are abundant (Bergh et al., 1989; Borsheim et al., 1990; Hara et al., 1991) and affect community composition by infecting and causing lysis of specific subsets of the microbial community (Hennes et al., 1995; Middelboe et al., 2001). Given that bacteriophages reach their host via diffusion they are most likely to contact bacteria, which are the predominant organisms in the sea (Azam et al., 1983; Fuhrman et al., 1989; Borsheim et al., 1990; Zweifel 16 and Hagstrom, 1995). It has been hypothesized that by causing lysis of numerically dominant host taxa, bacteriophages control species evenness and maintain species richness (Wommack et al., 1999b; Larsen et al., 2001; Middelboe et al., 2001). In addition to affecting community composition, viruses are responsible for significant mortality in planktonic communities (Suttle, 1994) and thereby are important drivers of nutrient and energy cycling (Fuhrman, 1999; Wilhelm and Suttle, 1999). Considering the remarkable abundance of viruses in marine systems (about lxlO 7 / ml) and their important role in nutrient and energy cycling, as well as community composition, viruses represent an important area of study for marine ecology. In theory, all organisms are potential hosts for viral infection, and often more than one virus can infect the same organism. It has been suggested that viruses represent the largest reservoir of genetic diversity on earth (Fuhrman, 1999). A variety of strategies have been applied to look at viral diversity. Morphological observations using transmission electron microscopes (TEM) have frequently been used to characterize marine viruses into morphotypes ( Hara et al., 1991; Borsheim, 1993; Waterbury and Valois, 1993; Lu et al., 2001; Proctor, 1997). Recently, advances in molecular techniques have unveiled new possibilities for studying the genetic variation of viruses in natural viral communities. For example, pulse field gel electrophoresis (PFGE) analysis enables the size fractionation of complete viral genomes (Steward et al., 2000). Wommack et al. (1999a; 1999b) monitored the spatial and temporal changes in viral population diversity, and provided evidence that they influenced the heterotrophic and phytoplankton community dynamics. Also, Kellogg et al. (1995) looked at the genetic diversity of 60 vibriophage isolates by genome restriction digestion and Southern hybridization, and found closely related groups in different oceans. PCR-based techniques have also been used to examine diversity in sub-groups of natural virus communities. For instance, several studies (Frederickson et al., submitted; Fuller et al., 1998; Zhong et al., 2002) have targeted the gene for the capsid assembly protein (g20) of 17 cyanomyovirus demonstrating a high genetic diversity in natural cyanophage communities. Similarly, natural communities of algal viruses belonging to the Phycodnaviridae have been shown to be very diverse and widespread in marine waters based on analysis of DNA pol genes (Chen et al., 1996; Short and Suttle, 1999; 2000; 2002). Recently, genomes of three marine viruses within the Podoviridae were sequenced; Roseophage SIOl (Rohwer et al., 2000), Cyanophage P60 (Chen and Lu, 2002) and Vibriophage VpV262 (Hardies et al., in press). SIOl infects marine Roseobacter SI067 bacterium, while P60 infects a cyanobacterium, Synechecoccus WH7803, and VpV262 infects the bacterium Vibrio parahaemolyticus. The genes required for DNA replications of P60 and SIOl are more similar to the non-marine podoviruses T7, T3 and phi-Ye03-12 than to bacteriophages from other families. The third virus, Vibriophage VpV262 has acquired a bacterial DNA replication gene, but still harbours other similar genes such as the capsid-assembly genes, as well as a common genome arrangement (Hardies et al., in press). In our study, we designed PCR primer based on the DNA pol sequences from T7, T3, phi-Ye03-12, Roseophage SIOl and cyanophage P60. Our goal was to use these primers to amplify viral sequences from marine water and surface sediment samples in order to examine the genetic diversity in uncultured marine podoviruses communities. 2.3 Materials and Methods Water Column and Sediment Collection and Preparation. Water samples were collected at the chlorophyll maximum layers from the inlets adjacent to the Straight of Georgia, British Columbia, Canada, as well as from the north-eastern Gulf of Mexico. GO-FLO bottles mounted on a CTD rosette system were used to collect water samples. Details of the sampling locations are presented in Table 1. For each sample, the natural virus communities were concentrated from ~20 L water to -200 ml via ultrafiltration (Suttle et al., 18 1991; Chen et al., 1996). Briefly, to remove particulate matter, the samples were pressure filtered (<17 kPa) through two 142-mm dia glass-fibre filters (Millipore GC50, nominal pore size 1.2 pm) then through a polyvinylidene difluoride filter (Millipore GVWP, pore size 0.45 pm). The viral size fraction in the filtrate sample was concentrated by ultrafiltration though a 30 kDa molecular weight cut-off spiral cartridge (Amicon S1Y30, Millipore) to produce the final viral concentrates. Viral concentrates were stored at 4 C in the dark. The DNA in the viruses was released by hot/ cold treatment. 200 pi aliquots of the viral concentrates were subject to a hot/cold treatment with 3 cycles of 2 min at 95 C, and 2 min at 4 C in a Hybaid PCR Express thermocycler as previously described (Chen et al., 1996). Finally, a one tenth dilution of the treated viral concentrates was used for the PCR template. Table 2.1. Oceanic stations where water samples and sediment samples were collected Sample Date Collected Type Location Water Depth (m) Latitude Longitude 430 31/07/00 Water column Howe Sound 5 49 27.3'N 123 16.1'W 442 02/08/00 Water column Malaspina Inlet 15 50 04.8'N 124 42.9'W 443 02/08/00 Water column Malaspina Inlet 5 50 04.8'N 124 42.9'W 6B 20/07/01 Water column Gulf of Mexico 25 29 00.0'N 87 17.8'W Mai 1-0 26/07/01 Sediment core Malaspina Inlet 50 50 04.8'N 124 42.9'W Mal4-0 26/07/01 Sediment core Malaspina Inlet 34 49 58.5'N 124 41.1'W Sec 1-0 25/07/01 Sediment core Sechelt Inlet 84 49 43.9'N 123 44.3'W Nanl-0 28/07/01 Sediment core Nanoose Bay 27 49 15.6'N 124 09.6'W Sediment cores were collected using a triple-barrelled gravity corer (Rigosha and Co., Tokyo, Japan) and the sediments processed as described elsewhere (Borsheim, 1993). Briefly, the protocol was as follows. Immediately after retrieval, the sediment-water interface was removed with a wide-bore serological pipette without disrupting the sediment core. Twenty cm 19 of each surface-sediment sample was mixed with 20 ml phosphate-buffered saline and centrifuged at 4000 x g for 5 min at 4 C. The supernatant was filtered through a 47-mm dia glass-fiber filter (Whatman GF/C, nominal pore size 1.2 um) and then through a polyvinylidene difluoride filter (Millipore HVLP, pore size 0.45 um). Following filtration the samples were kept in the dark at 4 C. Prior to DNA extractions the viruses were concentrated by centrifugation at 180,000 x g for 3.5 hrs at 20 C. The viral pellets were allowed to soften overnight at 4 C, and then approximately 100 pi of each pellet was resuspended into 500 pi 50 mM Tris, pH 8.0. DNA extractions were carried out according to Cottrell and Suttle (1991) and diluted one tenth for use as a PCR template. Primer Design and PCR Amplification. Degenerate primers were designed to amplify a DNA polymerase gene fragment from a subset of viruses belonging to the family Podoviridae with the goal of estimating their diversity in seawater samples. The sequences used to design the primers were based on the Family A DNA polymerase amino acid sequence from Roseophage SIOl, cyanophage P60, T7, T3 and phi-Ye03-12 (NC 002519, NC 003390, NC_001604, NC 003298, NC_001271 accession numbers, respectively). The amino acid sequences were collected from GenBank and aligned using Clustal W. Conserved regions were manually selected in the BioEdit software program (version 5.0.9) as primer sites. The forward and reverse primers, Podo-F and Podo-R2, were inferred from their amino acid sequences (5'-GACAC[A/T/C]CT[C/T][A/G]T[A/C/G][A7T/C]TGTC[A/T][A/C]G[A/T][C/T]TG-3') and (5'-[A7C]C[T/G]ACC[A/G]TC[C/T]A[A/G][A/T/G]CC[C/T]TT[A/C]A[T/G]-3'). The primers have degeneracies of 1728 and 768, respectively. The length of the fragments amplified ranged from 1,150 bp in the environmental samples to 1,430 bp for T7. The products were verified through sequencing to confirm the correct target was being amplified. 20 Three pi of either the sediment DNA extract, viral concentrates, or T7 lysate (control) was used as the DNA template in the first-stage PCR amplification reaction mixture (total volume 25 pi). The reagents included Taq DNA polymerase assay buffer [20 mM Tris- HCI (ph 8.4), 50 mM KC1], 2.5 mM MgCl 2 , 160 pM each deoxyribonucleoside triphosphate, 1.2 pM of each Podo-F and Podo-R2 primers and 0.4 U of PLATINUM Taq DNA polymerase (Invitrogen Life Technologies). Negative controls contained all reagents except DNA template. Amplification parameters were as follows: denaturing step at 94 C for 1 min 30 s, followed by 39 cycles of denaturation at 94 C for 45 s, annealing at 56 C for 45 s, and elongation at 72 Cfor 1 min, with a final elongation step of 72 C for 5 min. Amplicons were electrophoresed on a 1.5 % agarose gel in IX TAE (40 mM Tris- acetate, 2 mM EDTA, ph 8.5) at 94 V for 60 min, then stained with ethidium bromide and visualized with a UV transilluminator. A gel image was captured with a Nikon Coolpix 950 digital camera and visualized in Adobe Photoshop 5.0 LE. To increase amplification yield for cloning, a gel plug was excised with a Pasteur pipette and reamplified. First-stage PCR plugs were eluted in 100 pi elution buffer (10 mM Tris- Cl, ph 8.5) and heated for 5 min at 80 C to release the DNA. Six pi of the first stage product plug was added to a second-stage PCR where the conditions were kept the same except amplification was limited to 25 cycles. Five pi of the seconds-stage PCR was electrophoresed to verify the product size. Clone Library Construction and RFLP Analysis. The PCR amplicons from the second-stage PCR were purified with QIAquick PCR purification kit (Qiagen) and ligated into pGEM-T Vector System I (Promega) following the manufacturer's recommendations. The ligation reaction was then used to transform competent Escherichia coli DH5a cells. Positive clones (white colonies) were verified via a colony PCR reaction with the same parameters listed above with the template being a picked colony. Once 20 clones from each sample were confirmed to have the correct insert size, the PCR reaction mix 21 was saved and used for RFLP analysis. The remaining PCR reaction (15 pi) was digested with MboII (New England BioLabs Inc.), a 5 bp recognition-site-cutter, in a reaction containing 1 U/pg of DNA and IX NEBuffer 2 (50 mM NaCl, 10 mM Tris- HCI, 10 mM MgC12, 1 mM DTT, ph 7.9). Reaction mixtures were incubated at 37 C for 1 h and heat inactivated at 65 C for 20 min. A time-course was set up with 1, 2 and 3 h incubations, each resulting in the same restriction patterns, thus confirming complete digestion at 1 h as indicated by the manufacturer. The RFLP products were separated on a 2 % agarose gel in 0.5X TBE (9 mM Tris base, 9 mM boric acid, 2 mM EDTA, ph 8.0) at 115 V for 2 hrs. Based on the restriction pattern, clones were grouped together to reduce the number of sequencing reactions. Also to ensure that sequences producing the identical restriction pattern had the same sequence, 3 clones with the same RFLP patterns were sequenced. Identical RFLP patterns produced the same sequence; this was repeated with 4 different RFLP patterns. Sequencing and Phylogenetic Analysis. In total, 29 RFLP patterns were obtained and at least one clone for each was sequenced (about 1200 bp). DNA was prepared for sequencing either by harvesting the DNA plasmids from the clones using a QIAprep Spin Miniprep Kit (Qiagen), then quantifying the purified plasmid with a GeneQuant DNA spectrophotometer (Pharmacia) or by directly amplifying the fragment using the plasmid's -21M13 and M13R sites on the plasmid and purifying this product with a QIAquick PCR purification kit (Qiagen). Sequence reactions for both methods of DNA preparations were executed using the Big-Dye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems Inc.) according to the manufacturer's recommendations. In order to obtain the entire fragment, each genotype was sequenced with both the -21 Ml3 and M13R primers were used (one reaction generates 700-800 bp of reliable sequence). Following the sequencing reaction, the excess reaction reagents were removed by passing the product through a Centri-Sep 22 spin column (Princeton Separations). Purified sequencing products were then run in an ABI Model 373 Stretch or an ABI Prism 377 automated sequencer at the Nucleic Acid Protein Services at the University of British Columbia. Sequences were handled in BioEdit to construct a single gene sequence from the forward and reverse sequence information. All sequences were aligned using CLUSTAL W and run through a DNA identity matrix whereby sequences with 95% were grouped together for further manipulation. For tree construction, the conserved regions of an alignment were selected and both a maximum-likelihood and a fitch tree were produced. TREE PUZZLE (version 5.0) was used to create the maximum-likelihood tree with the following parameters; 25,000 puzzling steps, the Whelan-Goldman (WAG) substitution model and gamma distributed rates of heterogeneity estimated from the data set. The fitch program was run through PHYLIP (version 3.6), where a final consensus tree was based off a data set of 1000, analogous to the number of bootstraps. Both tree making methods generated trees with parallel topologies. The outgroup sequences include bacterial DNA polymerase I amino acid sequences. They comprised; Nostoc punctiforme (ZP_00107254), Trichodesmium erythraeum (ZP 00072402), Escherichia coli K12 (NP_418300) and Pseudomonas aeruginosa PA01 (NP 254180), which are among the sequences the most similar to this family of podovirus DNA polymerases sequences. Finally, trees were viewed in Tree View (version 1.6.6). Nucleotide Sequence Accession Numbers. The 17 unique sequences obtained in this study were added to the GenBank database. The accession numbers for the sequences SOG W-A, SOG WS-E, SOG W-F, GOM W-O, SOG W-S, SOG WS-N, SOG W-P, SOG S-H, SOG WS-C, GOM W-J, GOM W-M, GOM W-Q, SOG S-R, SOG S-D, SOG S-G, SOG S-L, GOM W-I are AY258449 to AY258461 and AY25863 to AY258466, respectively. 23 2.4 Results A set of degenerate primers (Podo-F and Podo-R2) were designed to target the Podoviridae DNA pol gene with the objective of examining diversity in marine viral communities. These primers amplified the expected 1430 bp product from lysate of the bacteriophage T7 and generated products of ca. 1150-1250 bp (Figure 2.1.) from natural marine virus communities concentrates from seawater samples and surficial sediment samples. Approximately one third of the environmental samples screened produced amplicons. Samples that did not produce products may have contained template concentrations below our detection limit or podoviruses that were not targeted by this primer set. Figure 2.1. First stage amplification of DNA pol within the Podoviridae family. Lanes 1 and 14, 100 bp molecular ladder; lane 2, negative control; lane 3, T7 lysate; lanes 4 to 13, a random selection of viral concentrates collected from the Straight of Georgia in 2000. In total 20 PCR-generated fragments were cloned from each of 8 different environmental samples. Of these 160 cloned fragments, 29 different RFLP patterns were recognized (Figure 2.2.). The frequency of specific RFLP patterns varied among the eight samples, indicating a 24 difference in viral communities at the different sites. For example, based on RFLP patterns there were six distinct genotypes recognized in the Nanoose Bay whereas the Malaspina site-1 sample was dominated by a single genotype (Figure 2.2. D vs. B respectively). At least one clone representing each RFLP patterns was sequenced. Of these sequences, 17 confirmed at least 5 % difference from all others at a nucleotide level. Sequences with 95 % identity to each other were grouped and labelled dependent on the location from which they were collected (SOG = Straight of Georgia; GOM = Gulf of Mexico) and type (W = water column; S = sediment sample) followed by a sequence reference (for example, SOG WS-E). Three sequences did not confirm any sequence similarity to a DNA pol gene. Furthermore, another sequence showed sequence similarity to DNA pol at the nucleotide level (86-99% identity to each other) and recurred in several samples. This sequence however, contained many stop codons at the amino acid level suggesting that these are non-functional prophage sequences that have incorporated mutations with time, yet still maintain a degree of nucleotide identity. Sequences that did not appear to be functional DNA polymerase sequences were excluded from further phylogenetic analysis. 25 B C D E F G H Figure 2.2. RFLP patterns generated from the Mbo II digestion of natural virus D N A pol clones. Marine surface sediment sample from Sechelt Inlet (A), Malaspina Inlet site 1 (B) and 4 (C) and Nanoose Bay (D). Water column samples from Howe Sound (E), Malaspina Inlet sample 442 (F) 26 and 443 (G) and Gulf of Mexico sample 6B (H). Lanes 1 and 22, 100 bp molecular ladder; lanes 2-21, randomly picked clones. The amino acid sequence alignment generated from the 5 known Podoviridae DNA polymerase gene sequences (T7, T3, phi-Ye-03, P60 and SIOI) and sequences obtained in this study showed multiple regions of conservation between them, as well as characteristic regions that define each of the 4 groups. Phylogenetic analysis using both maximum-likelihood and fitch algorithms produced trees with like topologies (Figure 2.3.). Group 1 clusters tightly with cyanophage P60 and Group 2 with Roseophage SIOI, whereas the other groups contain no cultured representatives. A. -I HZ SOG-WS-E SOG-W-F Roseophage SIOI SOG-W-A I Cyanophage P60 1 GOM-W-I SOG-S-D SOG-S-L SOG-S-G phi-Ye-03-12 I" — GOM-W-Q SOG-S-R SOG-W-S GOM-W-0 SOG-WS-N SOG-W-P — SOG-WS-C — SOG-S-H — GOM-W-J GOM-W-M T.erythraeum N.punctiforme _j P.aeruginosa PA01 E.coli K12 27 B. 93/100 56/64 99/99 93/100 98/100 .4 SOG-WS-E SOG-W-F Roseophage SIOI SOG-W-A Group 2 77/81 58/33 81/48 80/87 62/74 61/79 73/48 51/70 99/100 92/100 98/100 Cyanophage P60 GOM-W-I SOG-S-D SOG-S-L SOG-S-G 83/100 92/100 phi-Ye-03-12 T3 77 GOM-W-Q • SOG-S-R SOG-W-S GOM-W-O SOG-WS-N SOG-W-P SOG-WS-C SOG-S-H • GOM-W-J GOM-W-M Group 3 99/100 98/98 0.1 Group 1 Group 4 —— T.erythraeum N.punctiforme — P.aeruginosa PA01 — E.coliK12 Figure 2.3. Maximum-likelihood trees showing the phylogenetic relationship among the novel marine sequences and the five known Podoviridae D N A pol sequences. Known podoviral sequences and outgroup sequences are italicized, while the sequences from this study are in 28 normal text. (A.) the original maximum-likelihood tree as interpreted by TreeView. (B.) an adapted version of the same tree with quartet puzzling support values (maximum-likelihood tree) and bootstrap values (corresponding fitch tree) listed as percentages at the nodes. The scale bar represents 0.1 fixed mutations per amino acid substitution. Sequences were distributed across the tree irrespective of whether the samples were collected from the water column or sediment. Additionally, some sequences recurred more frequently than others. The relative frequency of each sequence (Figure 2.4.) is based on the 20 cloned PCR-fragments from each sample. This assumes that any methodological biases are equally pronounced, considering that all samples were handled in a parallel fashion. Each sample is equally represented as 20 cloned-PCR-fragments were considered from the sites. Within samples collected in British Columbia, 6 sequences occurred in more than one sample, whereas the Gulf of Mexico produced sequences unique only to that location. However, viral communities within the Gulf of Mexico and the coast of British Columbia possess podoviruses with closely related polymerases. 29 I W-A WS-E i W-F : w-o s w-s WS-N i W-P 3 S-H ws-c 4 W-J W-M : W - Q 3 S-R 3 S-B 3 S-D 3 S - G G S-L /I W-I V///////////////////////////, Sechelt Inlet Malaspina -1 Malaspina -4 Nanoose Bay A-a • i •m Z J B ^ ^ ^ ^ ^ Howe Sound 430 Malaspina 442 Malaspina 443 Gulf of Mexico 46 10 20 30 Number of Clones Figure 2.4. Clonal representation of each sequence from (A) water column and (B) sediment samples. Stacked bars identify specific sample distribution for the respective sequence. 2.5 Discussion This is the first report of Podoviridae diversity within natural environments. Based on sequence analysis of DNA polymerase genes these results indicate that podoviruses are diverse and widespread members of natural marine viral communities. The spectrum of sequences collected extends beyond our known cultured representatives clustering into new groups with unknown representation. These results depict the ocean samples as a diverse environment containing podoviruses similar to those infecting heterotrophic bacteria, cyanobacteria and enterobacteria. Our observations demonstrate that the primers designed in this study successfully targeted Family A DNA polymerases specific to Podoviridae. The high degeneracy incorporated 30 into the primers potentially broadened the spectrum of sequences targeted relative to the cultured viruses; however all the sequences collected in our study are monophyletic and clustered with the podovirus Family A polymerases. Moreover, the polymerases from the enterophages are unique compared to the marine sequences as they are consistently 200 bp longer. The additional sequence is distributed throughout the length of the gene and despite this difference they maintain many regions of strong conservation with the marine podoviruses. The complete polymerase gene of phages is approximately 2 kb in length, whereas their hosts' harbour a sequence about 1 kb longer. The fact that the phage sequences are only distantly related to their bacterial host suggests that the podoviruses inherited their pol gene long ago and have simplified their gene to carry but the regions with sequence conservation, perhaps for functional purposes. The majority of Podoviridae genomes that have been sequenced and submitted to GenBank cluster into two distinct groups with respect to their DNA pol, Family A and B, differing with genome sizes and arrangements (Chen and Lu, 2002). The viral genomes of Roseophage SIOI and Cyanophage P60 harbour the Family A DNA pol, as the T7-like phages. This is the subset of podoviruses targeted by the primer set used in this study. Podoviruses harbouring sequences belonging to the Family B or bacterial-like polymerases are among phages not targeted with this primer set. Very recently a new podovirus sequence was submitted to GenBank, a virus infecting Pseudomonas aeruginosa, named PaP3 (accession # NC 004466). The virus has a DNA pol belonging to the Family A, however the polymerase gene contains a 1.6 kb insert. After comparing this virus against our marine sequences, having removed the insert, we found that its closest relative is the SIOI DNA pol. Since viruses have been found to carry large intron inserts into functional genes (Landthaler et al., 2002), this may also be true of this virus also. The marine podovirus polymerase sequences cluster into four distinct groups (Figure 2.3.). Two of the groups were affiliated with cyanophage P60 and Roseophage SIOI, whereas 31 the other two groups have no known cultured representatives. Each of the four groups contains distinct conserved sequences that are not present in any of the other groups. The consensus sequences in the alignments within groups open the potential for future development of specific primers targeting polymerases within each group. Despite the impressive sequence richness observed in this study, the relative proportion of some pol sequences greatly exceeded that of others (Figure 2.4.). For example, SOG W-A from the water column and SOG WS-C extracted from both water column and sediment samples combine to represent about half the entire pool of sequenced pol. This is consistent with there being a high degree of unevenness in podovirus distribution in the environment. Interestingly, we also generally found that viral sequences found within the sediments were solely found within the sediment, and similarly sequences from within the water column tended to be restricted to the water column. For instance, even though four samples were analyzed from Malaspina Inlet (two water column and two from the sediment), sequences extracted from each environment type did not contain a large amount of overlap. These results suggest that the microbes which the viruses infect are also different between the water column and sediment. In this study we report for the first time a phylogenetic derivation of the sequence diversity of natural communities of the bacteriophage family Podoviridae with respect to their DNA pol sequences. We demonstrate that very similar sequences can be found in geographically distant locations (Gulf of Mexico and British Columbia Inlets) and different environment types (water column and sediments). Al l the polymerase sequences fall into a monophyletic group that included the DNA polymerase genes of two known marine podoviruses and three enterophages. Within the marine podovirus DNA pol sequences, four groups were resolved, two with close homology to known marine podoviruses and two with no cultured representatives. Marine phage isolates have typically been characterized as being members of the Myoviridae and Siphoviridae with Podoviridae representatives less frequently isolated (Kellogg 32 et al., 1995; Larsen et al., 2001). Despite this, direct environmental observation via TEM has shown viral proportions to contradict what is observed in our culture collections. Several reports have stated that viruses with short tails (a distinguishing characteristic of podoviruses) are not only abundant, but can predominate in marine viral communities (Bergh et al., 1989; Bratbak et al., 1990a; Hara et al., 1991; Wommack et al., 1992). Since podoviruses are challenging to isolate, the genetic information produced in this report establishes a foundation on which to build our understanding of podovirus diversity in natural communities. 2.6 Acknowledgments We thank Janice Lawrence for providing the extracted DNA from the sediment samples, Cindy Fredrickson for the collection of the Gulf of Mexico sample and the crews of the CCGS Vector for their help in sample collection. This research was supported in part by and NSERC postgraduate scholarship to K. E. Reid and NSERC research grants to C. A. Suttle. 33 CHAPTER 3. CONCLUSION AND FINAL REMARKS 34 The design and proof-of-application for a primer set was the leading accomplishment during my time spent working on my graduate degree. This primer set designed, targets a subgroup of bacteriophages, the Podoviridae family that are naturally present in our environment. They were designed to have degeneracy in their sequence in order to incorporate as wide a range as possible, while limiting the targets to the respective family. In addition to creating molecular tools for PCR technology, I was able to perform RFLP analysis, cloning and sequencing of viral genetic material extracted from marine environments. Samples obtained from both the ocean's water column and sediment were thoroughly analysed. Moreover, the samples included those from the coast of British Columbia within the Straight of Georgia's inlets and the Gulf of Mexico within the Mississippi River plume. The work presented in this dissertation provides a small but positive contribution to the broader research work that examines our limited understanding of bacteriophages inhabiting our world's oceans. Before being able to ask questions concerning viral diversity and their ecological roles in the marine biochemical cycle, basic research such as this is imperative. The underlying motivation throughout my research thesis has been to contribute to the necessary groundwork, providing tools for future ecological questions which are yet to be addressed. Initially, the goal of this project was not to examine the bacteriophage diversity within in the ocean, but rather to obtain genetic information from glacial ice. I had in my possession a small number of ice core samples collected from the Canadian Arctic. At first the idea behind designing a primer set was to capture genetic information of a group of bacteriophages since it had been previously established that the predominant biological lives within glaciers are bacteria (Handfield et al., 1992; Dancer et al., 1997; Karl et a l , 1999; Christner et al., 2000; Skidmore et al., 2000). Once the primers were designed and tested on environmental samples, numerous months of troubleshooting followed to optimize them for this challenging environment. Finally, I suggest that the reason for our disappointing results with the primers are either; 1) there are no 35 podoviruses present; 2) the podovirus abundance is to low to be targeted; or 3) the DNA within the viruses is fragmented such that there is not a continuous stretch of DNA to act as a PCR template. This fragmentation may be caused by the elapsed preservation-time and the extreme conditions. Most likely the reason lies in a combination of the last two suggested explanations. Continuing with our efforts to capture preserved viral genetic material from glacial ice, a second attempt at primer design was undertaken (targeting a shorter -400 bp section of the DNA pol). Unfortunately even though modest results were produced, the primers designed were based on a limited number of known sequences and limited to areas of poor conservation. Given that the scope for successful analysis and meaningful contribution to scientific progress was limited, the project was redirected to what is presented in this dissertation. Even though with greater persistence, this project may have become reality, I felt that it is of equal environmental significance to study bacteriophage diversity within marine environments. Considering we were still able to collect a small number of viral and bacterial counts, I have included this research in the form of a short note (Appendix I), but it was agreed that I would commit this dissertation to the latter part of the research performed during my graduate degree. Following the success in targeting podoviruses from the marine viral communities, I was interested in collecting as much genetic information as possible about this family. To do this, I cloned sequences from the amplicon band and examine the genetic diversity within a sample. From this information, three important conclusions were developed. First, the observation that within the eight samples, the level of diversity varied creating a qualitative impression of the diversity differences between marine samples. This was deduced from the RFLP patterns produced (Figure 2.2. a total of 29 different RFLP patterns). For example the Nanoose Bay sample has a greater number of different RFLP patterns than does the Malaspina site-1 sample. 36 Second, the clones that were sequenced showed substantial genetic diversity. In total 17 new unique sequences confirmed podovirus likeness, which were subsequently clustered into four groups based on their relative relatedness (Figure 2.3.). The phylogenetic tree I produced illustrates that related sequences are widely distributed in nature, both in terms of sample type (water column and sediment) and geographic location (Straight of Georgia and the Gulf of Mexico). This suggests that each viral community is composed of at least a few representatives from the diverse spectrum of bacteriophages. For example, although the sequences from the Gulf of Mexico were not in any way identical to those found in the Straight of Georgia, they covered as great a genetic range over the phylogenetic tree. Third, although the bacteriophage diversity is inspiring, each sequence is not equally represented (Figure 2.4.). I found that some sequences were represented to a greater extent than others. Not only that, but sequences were often restricted to either the water column or the sediment, but not both. Given that viruses infect their respective host, I am suggesting that the bacterial evenness in the marine environment also consistent with these findings. Considering that over a short period of time and with my small contribution of bacteriophage primers, a large amount of information has been gathered. Now a method with the optimized PCR conditions and a library of marine viral sequences exists. This foundation offers the opportunity for new research projects. For example, now that so many viral sequences have been collected from the environment, perhaps a new set of redesigned molecular tools could be created that would capture the potential bacteriophages preserved within glacial ice. Another potential application includes redesigning the primers to target the podocyanophages, a group of viruses infecting cyanobacteria. Cyanobacteria have been acclaimed for producing up to 25% of the ocean's primary production (Waterbury et al., 1986). Should viruses have an effect on controlling the microbial abundance and diversity, being able to target the viral suspects would be an asset. Beyond creating new molecular tools, lies the possibility of applying methods for 37 addressing a wide range of questions. For example, methods such as DGGE or TRFLP (terminal restriction fragment length polymorphism) could be used to examine bacteriophage dynamics over a temporal period. In conclusion, this dissertation provides a detailed explanation of a molecular tool designed to target the Podoviridae family via the DNA pol gene, and also a library of new genetic information about this previously understudied family of bacteriophages. The data and results presented here are utensils for the development of future studies. 38 APPENDIX I. VIRAL AND BACTERIAL ENUMERATIONS F R O M CANADIAN ARCTIC ICE CORES 39 Abstract The analysis of glacial subcores from two Canadian Arctic sites revealed abundances ranging from undetectable to 1.66 x 105 viral-like particles per melted millilitre of water and up to 9.45 x 103 bacteria per millilitre. The cores from both the Agassiz and Devon Island Ice Cap ranged in age from 600 to 120,000 years old. In addition to the counts, a 141 amino acid fragment was amplified from a viral DNA polymerase sequence. The sequence is similar to that of the T7 bacteriophage. This is the first study to report on virus counts from glacial ice. Note Viruses are ubiquitous in nature, they have been observed in environments ranging from deep in marine sediments (Bird et al., 2001; Hewson et al., 2001) to high up in our atmospheric cloud-cover (Castello et al., 1995). Even so, to date there have been no reports on abundances on viruses in glacial ice. Conversely, an accumulation of results demonstrating that bacteria are present in these environments have been reported (Handfield et al., 1992; Abyzov, 1993; 1998; Dancer et al., 1997; Christner et al., 2001; 2000; Skidmore et al., 2000). Not only are the bacterial cells being preserved, but evidence suggests that some of them remain biologically active (Abyzov et al., 1998). In view of the fact that all organisms are potential hosts for viral infection, we hypothesized that since bacteria are present in glacial ice, that viruses should be present as well. In this study we collected samples for viral and bacterial enumeration from ice cores obtained from the Canadian Arctic. Cores from two Canadian Arctic sites were analysed. They include cores drilled from the Agassiz Ice Cap, Ellesmere Island, and the Devon Island Ice Cap. The cores ranged in age from 600 to 120,000 years old (Paterson et al., 1977; Fisher et al., 1995; Zheng et al., 1998). Storage 40 of the ice has been at the Geological Survey of Canada, Ottawa, from the date they were drilled and were kindly provided to us by Roy Koerner. In our laboratory, ice subcores were stored in a -80 C freezer and handled in a -20 C walk-in freezer. Before sampling, approximately 3 ml of 5 pg/ ml FITC (fluorescein-5-isothicyanate) was spread over the outside of each subcore as a measure of experimental contamination. From each core section, a pristine sample was extracted from the center of the core using a succession of increasing sterile, dust free drill-bit sizes. Any sample with trace amounts of FITC (detection limit of 0.1 ng/ ml) was removed from the experiment. A few cubic centimetres of ice were collected, melted and subsequently stained with SYBR Green I. This dye stains all dsDNA and we are able to visualize the bacterial and viral particles under an epifluorescence microscope. Briefly, we filtered between 1 to 1.6 ml of sample through an Anodisc filter (pore size 0.02 pm) followed by staining of SYBR Green I consistent with the protocol of Noble and Furhman (1998). Table A.1. Viral and bacterial enumerations from Canadian Arctic cores Ice Age (ybp) Core Site Viral like particles /ml Bacterial cells /ml 600 Agassiz Ice Cap 1.66 x 105 9.45 x 103 1,100 Agassiz Ice Cap 8.98 x 104 8.27 x 103 5,000 Devon Island Ice Cap <6.0xl03a undetectable b 6,800 Agassiz Ice Cap < 6.0x103 undetectable 8,000 Devon Island Ice Cap <6.0xl03 undetectable 10-11,000 Devon Island Ice Cap 7.68 x 103 undetectable 50-70,000 Devon Island Ice Cap 1.03 x 104 7.38 x 102 120,000 Devon Island Ice Cap <6.0xl03 undetectable a Detection limit equivalent to 1 viral-like particle/ 5 microscope fields at 1,000X b Detection limit is no observable bacterium across all microscope fields 41 Our observations indicate that glacial ice contains as many as 1.66 x 10 viral-like particles/ ml of melted water and 9.45 x 103 bacteria/ ml (Table A.1.)- Furthermore, of the nine cores analysed only four contained values above the detection limit. These observations could be a reflection of the environmental changes during snow deposition over the years or it could be inconsistencies in viral preservation throughout the depth of the ice cap. What is for certain is these abundances are extremely low in comparison to other environments. For example typically our world's oceans harbour approximately 1 x 107 viral-like particles/ ml and 1 xlO 6 bacteria/ ml (reviewed in Fuhrman and Suttle, 1993). After biological counts, those samples with sufficient sample remaining were concentrated 12 times in an ultracentrifuge (4 of the 8 samples) and used in PCR reactions. From one of the samples we PCR amplified viral DNA (AY258467), this being from the 1,100 year old Agassiz subcore. The primers used include Podo-F and Podo-R3, whereby the protocol was the same as described in Chapter 2, with the exception of the reverse primer being Podo-R3 in this experiment. The primer sequences is as follows 5'-GCA(G/C)CI(T/C)G(A/G)TGTTC(A/T)A(G/T)IT(C/G)AAC(A/G)G-3'. The amplified fragment was a 423 bp fragment of the DNA polymerase gene belonging to a Podoviridae bacteriophage. Moreover, it was of sufficient length to confirm its identity and determine that it is more similar to the sequence belonging to the coliphage T7 and T3 than to other bacteriophage sequences extracted from marine environments (Figure A.I.). Our efforts to amplify a longer fragment of the DNA pol gene (1.3 kb) failed, probably due to lesions or partial degradation of the genetic material. Fragmentation of the viral DNA may have been caused by the 1,100 years that the viruses were locked within the ice. 42 T.erythraeum punctiforme P.aeruginosa • E.coliK12 Figure A . l . Phylogenetic relationship of the Arctic ice sequence with other Podoviridae D N A pol sequences. The arrow points to the sequence extracted from the Agassiz Ice core A107, approximately 1,100 years old. Known podoviral sequences and outgroup sequences are italicized, while the sequences from this dissertation are in normal text. The tree is based off 43 maximum-likelihood algorithm and the quartet puzzling support values are shown as percentages at the nodes. The scale bar represents 0.1 fixed mutations per amino acid substitution. Although the presence of viral-like particles does not confirm that the viruses are infectious particles, this study along with that of Castello (1999), where they amplified tomato mosaic tabamovirus from Greenland's ancient glacial ice, is proof that genetic material is being trapped in time. An extension on this study could lead to contributions to our existing knowledge of viral diversity and by following viral genetic material through the depth of a glacial ice, could lead to a stronger understanding of viral evolution. Acknowledgments A special thanks to David Fisher and Roy Koerner from the Geological Survey of Canada for providing the ice subcores. 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