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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Many podoviruses have been isolated which infect marine picocyanobacteria, and they may play a potentially important role in regulating the biomass and population composition of picocyanobacteria. However, little is known about the diversity and population dynamics of autochthonous cyanopodoviruses in marine environments. Using a set of newly designed PCR primers which specifically amplify the DNA pol from cyanopodoviruses, a total of 221 DNA pol sequences were retrieved from eight Chesapeake Bay virioplankton communities collected at different times and locations. All DNA pol sequences clustered with the eight known podoviruses that infect different marine picocyanobacteria, and could be divided into at least 10 different subclusters (I-X). The presence of these cyanopodovirus genotypes based on PCR-amplification of DNA pol gene sequences was supported by the existence of similar DNA pol genotypes with metagenome libraries of Chesapeake Bay virioplankton assemblages. The composition of cyanopodoviruses in the Bay also exhibited distinct winter and summer patterns which were likely related to corresponding seasonal changes in the composition of cyanobacterial populations. Our study suggests that diverse and dynamic populations of cyanopodoviruses are present in the estuarine environment. The PCR method developed in this study provides a specific and sensitive tool to explore the abundance, distribution and phylogenetic diversity of cyanopodoviruses in aquatic environments. Linking the dynamics of host and viral populations in the natural environment is critical to broader characterization of the ecological role of virioplankton within microbial communities.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Cyanophages are a class of viruses that infect cyanobacteria. All cyanophages isolated from aquatic environments belong to three recognized families of double-stranded DNA viruses: Myoviridae (T4 like, with contractile tails); Podoviridae (T7 like, with short tails) and Siphoviridae (λ like, with long non-contractile tails) (Suttle, 2000). Myoviruses that infect cyanobacteria (cyanomyoviruses) are commonly isolated from seawater, and they often have a broad host range (Suttle and Chan, 1993; Waterbury and Valois, 1993; Wilson et al., 1993; Lu et al., 2001) extending across the Prochlorococcus, Synechococcus genetic boundary (Sullivan et al., 2003; Weigele et al., 2007). In contrast, cyanophages belonging to the Podoviridae (cyanopodoviruses) are typically highly host specific, and incapable of cross-infecting very closely related host strains (Sullivan et al., 2003; Wang and Chen, 2008). In general, cyanopodoviruses are less commonly isolated from seawater as compared with cyanomyoviruses. Metagenomic analyses have suggested that the cyanopodovirus to cyanomyovirus ratio in metagenome sequence libraries varies with different regions of sea (Angly et al., 2006). For example, this ratio was about 1:1 in the Sargasso Sea, but was roughly 1:10 in the Gulf of Mexico, British Columbia coastal waters and the Arctic Ocean (Angly et al., 2006). Within the estuarine waters of the Chesapeake Bay this ratio was approximately 1:2 (Bench et al., 2007), indicating that cyanopodoviruses could make up a significant portion of cyanophage communities in the Bay. Although not specifically focused on cyanophage, an analysis of phage sequences within the Global Ocean Survey (Rusch et al., 2007) found that podoviruses and siphoviruses exhibited more site-specific distributions compared with myoviruses, and podovirus sequences were most prevalent in temperate, mesotrophic waters (Williamson et al., 2008). This correlation of podoviruses and temperate waters was supported by frequent observations of cyanopodo- and cyanosiphoviruses in the Chesapeake Bay, a temperate estuary (Wang and Chen, 2008).

Despite the general lack of sequence conservation between viral genomes (Hatfull et al., 2006) and their mosaic structure (Hendrix et al., 1999), certain genes appear to be conserved among specific groups of viruses. The examples are the DNA polymerase (DNA pol) gene in algal viruses (Chen and Suttle, 1996; Chen et al., 1996; Short and Suttle, 2003; Short and Short, 2008), the DNA pol among the T7-like phages (Chen and Lu, 2002; Breitbart et al., 2004; Wang and Chen, 2008; Labontéet al., 2009), viral structural genes like g20 and g23 (Fuller et al., 1998; Zhong et al., 2002; Filee et al., 2005), and phage-encoded photosynthesis genes like psbA or psbD in certain cyanophages (Mann et al., 2003; Lindell et al., 2004; Millard et al., 2004; Sullivan et al., 2006). The conservation of viral encoded genes permits the design of PCR primers that can be used to explore the genetic diversity of a specific group of viruses in the natural environment (Labontéet al., 2009; Short et al., 2009). In the past decade, numerous studies have shown that cyanomyoviruses are widely distributed and highly diverse in the aquatic environment (Fuller et al., 1998; Zhong et al., 2002; Marston and Sallee, 2003; Dorigo et al., 2004; Wang and Chen, 2004; Short and Suttle, 2005; Wilhelm et al., 2006; Sullivan et al., 2008). In contrast, little is known about the diversity of cyanopodoviruses and their population dynamics in aquatic environments.

Comparative genomics of podoviruses including cyanopodovirus P60 suggested that the genes responsible for DNA replication (e.g. primase-helicase and DNA pol) in podoviruses are more conserved compared with myoviruses and siphoviruses (Chen and Lu, 2002). Recently, Wang and Chen (2008) reported that the DNA pol gene is conserved among seven T7-like podoviruses infecting a broad range of hosts including marine cluster A and B Synechococcus and marine Prochlorococcus (Wang and Chen, 2008). These marine cyanopodoviruses also had very similar genome and capsid sizes of 45–48 kb and 50–60 nm respectively (Chen and Lu, 2002; Sullivan et al., 2005; Pope et al., 2007; Wang and Chen, 2008). In contrast, cyanomyoviruses and siphoviruses tend to have more variable genome sizes. Cyanopodoviruses isolated from the Chesapeake Bay are highly host specific and have a shorter lysis period compared with cyanomyoviruses and cyanosiphovirues (Wang and Chen, 2008), suggesting that they are able to kill the specific host populations rapidly. The commonly reported observation of narrow host specificity among cyanopodoviruses indicates that host–cyanopodophage relationships may be fundamentally different from that of cyanomyoviruses in the estuarine environment (Wang and Chen, 2008). As a first step towards investigating the unique character of cyanopodovirus–host relationships and the autecology of this viral group in marine environments a set of PCR primers specific for the conserved regions of the T7-like DNA pol gene were developed and tested against eight different viral communities collected at different times of the year from across the Chesapeake Bay.

Results and discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Chesapeake Bay is a temperate estuary that shows strong seasonality. In this study, average surface water temperature varied from c. 3°C in February to 23°C in July 2005 (Table 1). The total picocyanobacterial abundance (mostly Synechococcus) ranged from 890 to 2730 cells ml−1 from the upper to lower bay in winter 2005 (Table 1); whereas, summer abundances were 100–1000 times higher reaching over 1 million cells ml−1 (i.e. Station 804, summer 2004, Table 1). The summer picocyanobacteria also made up a much greater portion of total Chesapeake Bay bacterial communities compared with the winter picocyanobacteria (Table 1). The low picocyanobacterial biomass in winter was also reflected by the low concentration of picophytoplankton Chl. a (< 3 μm) in February 2005. In general, viruses and bacteria were more abundant in summer than winter in the bay. On the spatial scale, salinity increased gradually from the upper bay to lower bay, whereas, nutrients (i.e. nitrate and phosphate) followed the opposite trend (Table 1). The upper bay (Station 858) tended to have relatively lower viral, bacterial and picocyanobacterial abundances compared with the middle and lower bay in both seasons.

Table 1.  Environmental parameters at sampling stations in the Chesapeake Bay.
MeasurementsFebruary 2005February 2004July 2005July 2004
Station 858Station 804Station 707Station 804Station 858Station 804Station 707Station 804
Water temperature (°C)3.12.72.9ND16.827.323.9ND
Salinity (ppt)7.29.618.010.58.113.019.113.2
Ammonium (μM)3.501.111.920.617.551.691.850.91
Nitrate + nitrite (μM)43.635.29.0515.62.290.260.180.60
Phosphate (μM)0.420.220.190.180.500.470.390.27
Picocyanobacteria abundance (103 cells ml−1)0.891.192.730.6596.37984841440
Total chlorophyll a (μg l−1)11.084.985.535.012.394.958.199.0
Chl. a (< 3 μm) (μg l−1)0.700.390.12ND2.283.954.04ND
Viral abundance (×106 viruses ml−1)1.987.198.712.558.4910.311.523.0
Bacterial abundance (×106 cells ml−1)0.741.231.230.654.539.729.557.59

PCR amplification of cyanopodoviruses

The newly designed PCR primers were tested against different Chesapeake Bay viral communities (n > 20) collected during the Microbial Observatory of Virioplankton Ecology (MOVE) cruises. A single PCR amplicon (∼570 bp) was obtained from nearly all tested samples (data not shown). We chose six samples representing the upper, middle and lower bay in winter and summer respectively. In addition, two middle bay samples collected in winter and summer 2004 were also included to examine interannual variation in the diversity of cyanopodovirus DNA pol genes. Eight clone libraries were constructed and approximately 30 clones from each clone library were picked for sequencing. Among the original 239 clones picked for sequencing, 12 clones contained non-translatable sequences, six clones contained chimeric sequences. The remaining 221 sequences were used for further phylogenetic and statistical analyses.

Chesapeake Bay contains diverse cyanopodoviruses

Phylogenetic analysis indicated that Chesapeake Bay virioplankton DNA pol sequences grouped into 10 different subclusters (I to X) (Fig. 1 and Table 2). A subcluster was defined when two or more sequences share less than 0.1 substitutions per amino acid site. These 10 subclusters were all within the larger marine picocyanopodovirus (MPP) cluster that included all eight known cyanopodoviruses – P60, P-SSP7, Syn5, S-CBP1, S-CBP2, S-CBP3, S-CBP4 and S-CBP42 – isolated from diverse species of marine picocyanobacteria (Fig. 1). The MPP cluster can be divided into two large clusters (MPP-A and MPP-B) (Fig. 1), which are correspondent to the two clusters based on the isolated marine cyanopodoviruses (Wang and Chen, 2008). MPP-B contained eight subclusters (I to VIII), while MPP-A only included two subclusters (IX to X). Subclusters II, IV, VI, VII and VIII did not contain a DNA pol sequence from the collection of known cyanopodoviruses and three of these phage – P-SSP7, S-CBP2 and Syn5 – were not contained within a subcluster. The separation into subclusters based on the 90% amino acid identity level is arbitrary. The biological and ecological meanings of these subcluster groupings are still largely unknown.

image

Figure 1. Neighbour-joining phylogenetic tree of DNA pol gene amino acid sequences from clone libraries of environmental samples in the Chesapeake Bay. Numbers at tree branches indicated the bootstrap values (> 50%) from 1000 replicates. The sequences from eight cyanopodovirus isolates are in green, while the summer and winter environmental sequences are labelled in red and blue respectively. The environmental clones were named to reflect the sampling date, sampling site and clone number. For example, ‘CB0204-804-30’ stands for a Chesapeake Bay clone recovered from station 804 in February 2004, and it is the 30th clone in the library. Prefixes for the Synechococcus strains or environmental clones are as follows: CB, Chesapeake Bay; WH, Woods Hole; MED, Mediterranean Sea. The numbers of clones from summer and winter in main phylogenetic clusters were given in parentheses respectively. Bar represents 0.10 substitutions per site.

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Table 2.  Numbers of sequences in phylogenetic subclusters obtained from clone libraries and statistic analysis.
Phylogenetic clustersFebruary 2005February 2004July 2005July 2004
Station 858Station 804Station 707Station 804Station 858Station 804Station 707Station 804
  • a.

    Number of operational taxonomic units (OTUs) determined at 2% DNA sequence divergence.

  • b.

    Estimated abundance-based coverage estimator (ACE) determined at 2% DNA sequence divergence.

  • *

    The ACE value (116) at 2% DNA sequence divergence is not reasonable because the ACE value is 84 and 55 at 1% and 3% DNA sequence divergence respectively.

Subcluster I635917181416
Subcluster II353 1122
Subcluster III   1    
Subcluster IV 1  4210
Subcluster V    1  1
Subcluster VI111  372
Subcluster VII      11
Subcluster VIII171213166231
Subcluster IX 3      
Subcluster X12 11215
Unclustered sequences  1  1  
Total clones (221)2827232730292928
Number of OTUsa1617161522211722
Richness (ACE)b44444557664664*

Overall, the phylogeny of sequences obtained using this new PCR primer set indicated its specificity for amplifying the DNA pol gene from only marine cyanopodoviruses. Moreover, the diversity of DNA pol sequences from the Chesapeake exceeds that from known cyanopodoviruses and the combination of known and environmental sequences indicate that the marine cyanopodoviruses are indeed a highly diverse collection of viruses. The absolute separation of marine cyanopodoviruses from their relatives infecting Proteobacterial hosts points to an ancient divergence between these members of the Podoviridae.

It is intriguing to note that three cyanopodoviruses (S-CBP1, S-CBP3 and S-CBP4), infecting the same host Synechococcus CB0101, belonged to three different DNA pol subclusters (I, III and V) and these three phages only shared c. 80% identity at the amino acid sequence level, and c. 60% identity at the nucleotide sequence level. Fifty-two per cent (116 of 221) of environmental clones in subclusters I-V had close phylogenetic relationship with the three different podoviruses (S-CBP1, S-CBP3 and S-CBP4) that infect a marine cluster B Synechococcus CB0101 (Wang and Chen, 2008) (Fig. 1). Although these environmental clones cluster closely with these phage isolates, it is not necessary that they will infect the same host CB0101. We hypothesized that podoviruses infecting Synechococcus CB0101 and its close relatives undergo a rapid DNA mutation and genomic evolution in the Chesapeake Bay during the summer, and such a genetic variation reflects the continuous arms race between phage and host populations. It is likely that the quick growth of CB0101 (and its mutants) and rapid attacks and replications of their podoviruses in the summer resulted in such a pool of diverse podoviruses. It would be interesting to see how other genes of these podoviruses evolve at the same time. Genome sequencing and comparative genomics on these CB0101 podoviruses will be investigated in the near future. Synechococcus CB0101 is a member of marine cluster B (MC-B) Synechococcus, and MC-B members could be abundant in the upper and middle Bay during the summer, and in the case of the July 2005 samples MC-B Synechococcus comprised 100% and 30% of the picocyanobacterial community in the upper and middle bay respectively (H. Cai et al., unpubl. data). The majority of clones (69%) in subclusters I–V were dominated by the DNA pol sequences recovered from summer samples. The presence of abundant MC-B Synechococcus genotypes and the high production of podoviruses in the summer may explain the dominance of podoviruses in subclusters I-V in the summer.

About 32% (70 of 221) of environmental clones clustered within subcluster VIII that did not contain a sequence from a known cyanopodovirus. Moreover, subcluster VIII was dominated by sequences from winter samples. Interestingly, 55% of winter clones fell within subcluster VIII. Only 7% of Chesapeake environmental clones occurred in subclusters IX and X which contained the two podoviruses infecting oceanic Synechococcus (WH7803, WH7805). Because the primers were designed to amplify the DNA pol from both coastal and oceanic cyanopodoviruses, our results suggest that podoviruses infecting oceanic Synechococcus may not contribute significantly to cyanopodovirus populations in the Bay. The rest of environmental clones (9%) were either in subcluster VI or VII or remained as unclustered due to the lack of close counterparts.

None of the environmental clones were closely related to podovirus P-SSP7, which infects marine Prochlorococcus strain MED4, or podovirus Syn5 which infects marine Synechococcus WH8109 (Fig. 1). Both of these podoviruses carry an integrase gene, and are speculated to be temperate phage (Sullivan et al., 2005; Pope et al., 2007). Nevertheless, P-SSP7 and Syn5 still clustered within the larger MCP cluster suggesting that phage life-cycle characteristics are not necessarily reflected within the phylogenetic placement of cyanopodoviruses based on the DNA pol gene. Both P-SSP7 and Syn5 infect oceanic picocyanobacteria, it would be interesting to see if these types of cyanopodoviruses prevail in the open ocean.

Seasonal and spatial patterns of cyanopodoviruses

Clone libraries constructed from samples collected in February and July, 2004 and 2005, could be readily distinguished into winter and summer classes (Fig. 2). The S-LIBSHUFF analysis allows the comparison of pairwise similarity between samples based on available clone sequences in each library. According to the S-LIBSHUFF analysis, three samples from February 2005 and one sample from February 2004 formed the winter group, while three samples from July 2005 and one sample from July 2004 formed the summer group.

image

Figure 2. Clustering of the different clone libraries based upon ΔCxy values determined from S-LIBSHUFF analysis. The tree was constructed with the unweighted-pair group method using average linkages in MEGA 4.0.

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Most of the DNA pol sequences (72%) fell within only two subclusters (I and VIII) which each contained 88 and 70 clones respectively (Table 2). Subcluster I contained more summer clones (66) than winter clones (23), while subcluster VIII contained more winter clones (59) than summer clones (12). It is quite possible that these trends in the occurrence of DNA pol sequences are indicative of the viral strains comprising seasonal populations of cyanopodoviruses. It is known that Synechococcus CB0101 types thrives in the Chesapeake Bay during the warm season and podovirus S-CBP4, a member of subcluster I, was isolated from CB0101. However, no podoviruses have been isolated from cold-adapted Synechococcus in the Bay. Our recent studies indicate that in the winter the Chesapeake Bay contains a low abundance of distinct groups of Synechococcus (< 103 cells ml−1) that are not found in the summer according to the diversity analysis based on the internal transcribed spacer region (H. Cai et al., unpubl. data). We hypothesize that subcluster VIII represents podoviruses that infect winter populations of Chesapeake Bay picocyanobacteria. Isolation of wintertime Synechococcus phage–host systems might help to substantiate this hypothesis.

Despite the obvious environmental gradients (i.e. salinity and nutrients) which exist across the Bay (Table 1) no trends in the occurrence of specific subclusters was connected with geographic location (Table 2). This is consistent with weak spatial variation of bacterial communities in the Chesapeake Bay (Kan et al., 2007). One possible explanation for the lack of spatial heterogeneity is the relatively long residence time (∼180 days) of Chesapeake Bay water as compared with other estuarine systems (Nixon et al., 1996; Kan et al., 2007).

Occurrence of cyanopodoviral DNA pol within Chesapeake virioplankton metagenomes

When the representative sequences from Fig. 1 were blast searched against the Chesapeake Bay metagenome library (September 2002, Bench et al., 2007), 18 DNA pol-like amino acid sequences were identified at E-value of ≤ 0.001. Subsequently, these 18 sequences were translated and compared with the GenBank non-redundant protein database by blastp (Table S1). Interestingly, 10 of these metagenome sequences had a closest hit with the DNA pol sequences from S-CBP3 or S-CBP1 (Table S1), two podoviruses that infect estuarine Synechococcus CB0101 (Wang and Chen, 2008). This result suggests that cyanopodoviruses were important members of Chesapeake Bay virioplankton assemblages in September 2002.

Despite the fact that the other virioplankton metagenome from Chesapeake station 858 in late October 2004 contained more than twice the number of sequences, no cyanopodoviral-like DNA pol homologues were found (Table S2). It should be noted that the October 2004 metagenomic data set may be biased due to the genome amplification with Genomiphi. The absence of DNA pol homologues likely reflects the low abundance of cyanopodoviruses during fall. Titres of cyanophage are known to co-vary with the abundance of picocyanobacteria (Waterbury and Valois, 1993). Average picocyanobacterial abundance in October 2004 was 2.3 × 104 cells ml−1, c. 10-fold lower than that found in September 2002 samples. Despite low cyanobacterial abundance we were able to detect, via PCR, cyanopodoviruses in winter samples that likely contained very low corresponding titre of cyanopodoviruses. Although more than 200 cyanopodovirus-related DNA pol gene sequences were identified in this study, rarefaction analysis suggests the existence of a much larger diversity (Fig. S2).

Conclusion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We demonstrated that cyanopodoviruses can be specifically detected within environmental samples using a PCR primer set targeting conserved regions of a viral DNA polymerase gene. Phylogenetic analysis of cyanopodoviral-like DNA pol sequences unveiled a great diversity of cyanopodoviruses in the Chesapeake Bay, and showed seasonal changes in the composition of cyanopodoviral subpopulations between winter and summer. In this estuarine ecosystem, cyanopodoviruses could play a critical role in controlling picocyanobacterial populations due to their high host specificity and fast lysis capability. The population structures of picocyanobacteria and their podoviruses in the natural environment can now be co-monitored using different gene markers. These tools will allow us to explore population dynamics of host and phage in nature, which could provide new insights into co-variation and co-evolution of host and phage.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Water sample collection

Water samples were collected from the surface waters of Chesapeake Bay on board the R/V Cape Henlopen during the research cruises for the MOVE project from September 2002 to July 2006 (http://www.virusecology.org/MOVE/Home.html). Viral concentrates (VCs) were prepared on board by ultrafiltration and stored as described previously (Wang and Chen, 2004). Briefly, viral community from each sample was concentrated from 50 l to a final volume of c. 300 ml via ultrafiltration. Six VCs collected from stations 858, 804 and 707 in February and July 2005 were picked to represent viral communities from upper, middle and lower bay (Fig. 3) in winter and summer respectively. In addition, two VCs collected from station 804 (middle bay) in February and July 2004 were also chosen to test whether the seasonal pattern, if present, is repeatable. The physical, chemical and biological parameters of these samples were described in Table 1. Chl. a data were kindly provided by Dr W. Coats at the Smithsonian Environmental Research Center. Viral, bacterial and cyanobacterial abundances were estimated based on the protocols described elsewhere (Kan et al., 2006). Nutrients were analysed in the Chemical Laboratory at the Horn Points Laboratory, University of Maryland Center for Environmental Sciences.

image

Figure 3. The location of sampling sites in the Chesapeake Bay. Samples used in this study were collected in winter and summer of 2004 and 2005.

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PCR primers for cyanopodoviruses

We designed specific PCR primers based on the DNA polymerase gene sequences from seven marine cyanopodoviruses, including P60 (Accession No. AF338467), Syn5 (YP001285436), S-CBP1 (EF535232), S-CBP2 (DQ206830), S-CBP3 (EF535233), S-CBP42 (EF535234) and P-SSP7 (AY939843) (Fig. S1). The 17-mer forward primer CP-DNAP-349F (5′-CCA AAY CTY GCM CAR GT-3′) contains 16× degeneracy. The reverse primer CP-DNAP-533R consists of equal molar amounts of two 18-mer primers 533Ra (5′-CTC GTC RTG SAC RAA SGC-3′) and 533Rb (5′- CTC GTC RTG DAT RAA SGC-3′), and contains 48× degeneracy. These PCR primers only target the partial DNAIgene, and the expected PCR amplicon size is c. 550–600 bp.

DNA extraction and PCR amplification.  Viral particles in the VCs (2 ml) were precipitated with polyethylene glycol 8000 powder (10% w/v in final concentration), centrifuged at 15 000 g in a Beckman JA-21 (c. 15 000 r.p.m) rotor for 1 h. The viral pellets were resuspended in 100–200 μl of SM buffer (10 mM NaCl, 50 mM Tris, 10 mM MgSO4 and 0.1% gelatin) and stored at 4°C. The concentrated VCs (20 μl) were boiled for 5 min to release viruses DNA, which was used as DNA template for PCR. All PCR reactions were performed in 50 μl volume containing 1× reaction buffer with 1.5 mM MgCl2, 100 mM dNTPs, 10 pmol of each primer, 1 unit of Taq DNA polymerase (Promega, Madison, WI) and 2–4 μl boiled viruses DNA solution. The reaction was performed in the PTC-200 Peltier Thermal Cycler (MJ Research, USA) with the following PCR program: denaturing for 3 min at 94°C, 35 cycles of denaturing (30 s at 94°C), annealing (30 s at 50°C) and extension (1 min at 72°C), with a final extension for 10 min at 72°C to facilitate the TA cloning.

Cloning, sequencing and phylogenetic analysis.  PCR products were cloned using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA) by following the manufacturer's instructions. About 30 clones from each clone library were picked for sequence analysis. Clones were sequenced using BigDye terminator chemistry and an ABI 3100 Genetic Analyzer (Applied Biosystems) at the Center of Marine Biotechnology, UMBI. All sequences obtained were carefully checked for chimeric artefacts using the blast (blastn) program (http://www.ncbi.nlm.nih.gov/BLAST), and chimeric sequences were excluded from phylogenetic analysis. The translated sequences were aligned using ClustalX2 (Larkin et al., 2007), and alignment was manually corrected using MEGA 4.0 software (Tamura et al., 2007). Phylogenetic trees of the amino acid sequences were constructed in Mega 4.0 with the default parameters, based on the neighbour-joining algorithm with 1000 bootstrap replicates.

S-LIBSHUFF analyses

S-LIBSHUFF version 1.22 was used to compare libraries statistically (Schloss et al., 2004). It compares more than two libraries at once with the same distance matrix to determine whether the structures of two communities are the same, different, or subsets of one another. The parameter ΔCxy in the S-LIBSHUFF analysis represents the difference in coverage of the two sequence libraries (an increased ΔCxy represents greater dissimilarity between the given communities). The PRODIST program of phylip (http://evolution.genetics.washington.edu/phylip.html) using the Jones-Taylor-Thornton model for amino acid substitution was used to generate the distance matrix analysed by S-LIBSHUFF. Analyses were performed at the web site (http://schloss.micro.umass.edu/software/slibshuff.html). Operational taxonomic units and abundance-based coverage estimator were measured using software DOTUR (http://schloss.micro.umass.edu/software/dotur.html).

GenBank accession numbers

All the DNA pol gene sequences used for the phylogenetic analysis have been deposited in GenBank with accession numbers FJ872594FJ872816.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank the crew of the R/V Cape Henlopen for sample collection. This work was supported by the Grants from the National Science Foundation's Microbial Observatories Program (MCB-0132070, MCB-0238515, MCB-0537041), and the funds from the Xiamen University 111 program to F.C. and 2007CB815904 to N.Z.J.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
  • Angly, F., Felts, B., Breitbart, M., Salamon, P., Edwards, R., Carlson, C., et al. (2006) The marine viromes of four oceanic regions. PLoS Biol 4: e368. doi: 10.1371/journal.pbio.0040368.
  • Bench, S.R., Hanson, T.E., Williamson, K.E., Ghosh, D., Radosovich, M., Wang, K., and Wommack, K.E. (2007) Metagenomic characterization of Chesapeake Bay virioplankton. Appl Environ Microbiol 73: 76297641.
  • Breitbart, M., Miyake, J.H., and Rohwer, F. (2004) Global distribution of nearly identical phage-encoded DNA sequences. FEMS Microb Lett 236: 245252.
  • Chen, F., and Lu, J.R. (2002) Genomic sequence and evolution of marine cyanophage P60: a new insight on lytic and lysogenic phages. Appl Environ Microbiol 68: 25892594.
  • Chen, F., and Suttle, C.A. (1996) Evolutionary relationships among large double-stranded DNA viruses that infect microalgae and other organisms as inferred from DNA polymerase genes. Virology 219: 170178.
  • Chen, F., Short, S.M., and Suttle, C.A. (1996) Genetic diversity in marine algal virus communities as revealed by sequence analysis of DNA polymerase genes. Appl Environ Microbiol 62: 28692874.
  • Dorigo, U., Jacquet, S., and Humbert, J.F. (2004) Cyanophage diversity, inferred from g20 gene analyses, in the largest natural lake in France, Lake Bourget. Appl Environ Microbiol 70: 10171022.
  • Filee, J., Tetart, F., Suttle, C.A., and Krisch, H.M. (2005) Marine T4-type bacteriophages, a ubiquitous component of the dark matter of the biosphere. Proc Natl Acad Sci USA 102: 1247112476.
  • Fuller, N.J., Wilson, W.H., Joint, I.R., and Mann, N.H. (1998) Occurrence of a sequence in marine cyanophages similar to that of T4 g20 and its application to PCR-based detection and quantification techniques. Appl Environ Microbiol 64: 20512060.
  • Hatfull, G.F., Pedulla, M.L., Jacobs-Sera, D., Cichon, P.M., Foley, A., Ford, M.E., et al. (2006) Exploring the mycobacteriophage metaproteome: phage genomics as an educational platform. PLoS Genet 2: e92.
  • Hendrix, R.W., Smith, M.C., Burns, R.N., Ford, M.E., and Hatfull, G.F. (1999) Evolutionary relationships among diverse bacteriophages and prophages: all the world's a phage. Proc Natl Acad Sci USA 96: 21922197.
  • Kan, J., Crump, B., Wang, K., and Chen, F. (2006) Bacterioplankton community in Chesapeake Bay: predictable or random assemblages. Limnol Oceanogr 51: 21572169.
  • Kan, J., Suzuki, M., Wang, K., Evans, S.E., and Chen, F. (2007) High temporal but low spatial heterogeneity of bacterioplankton in the Chesapeake Bay. Appl Environ Microbiol 73: 67766789.
  • Labonté, J., Reid, K.E., and Suttle, C.A. (2009) Phylogenetic analysis indicates evolutionary diversity and environmental segregation of marine podovirus DNA polymerase gene sequences. Appl Environ Microbiol 75: 36343640.
  • Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., et al. (2007) ClustalW2 and ClustalX version 2. Bioinformatics 23: 29472948.
  • Lindell, D., Sullivan, M.B., Johnson, Z.I., Tolonen, A.C., Rohwer, F., and Chisholm, S.W. (2004) Transfer of photosynthesis genes to and from Prochlorococcus viruses. Proc Natl Acad Sci USA 101: 1101311018.
  • Lu, J., Chen, F., and Hodson, R.E. (2001) Distribution, isolation, host specificity, and diversity of cyanophages infecting marine Synechococcus spp. in the Georgia river estuaries. Appl Environ Microbiol 67: 32853290.
  • Mann, N.H., Cook, A., Millard, A., Bailey, S., and Clokie, M. (2003) Bacterial photosynthesis genes in a virus. Nature 424: 741.
  • Marston, M.F., and Sallee, J.L. (2003) Genetic diversity and temporal variation in the cyanophage community infecting marine Synechococcus species in Rhode Island's coastal waters. Appl Environ Microbiol 69: 46394647.
  • Millard, A., Clokie, M.R., Shub, D.A., and Mann, N.H. (2004) Genetic organization of the psbAD region in phages infecting marine Synechococcus strains. Proc Natl Acad Sci USA 101: 1100711012.
  • Nixon, S.W., Ammerman, J.W., Atkinson, L.P., Berounsky, V.M., Billen, G., Boicourt, W.C., et al. (1996) The fate of nitrogen and phosphorus at the land sea margin of the North Atlantic Ocean. Biogeochemistry 35: 141180.
  • Pope, W.H., Weigele, P.R., Chang, J., Pedulla, M.L., Ford, M.E., Houtz, J.M., et al. (2007) Genome sequence, structural proteins, and capsid organization of the cyanophage Syn5: a ‘horned’ bacteriophage of marine Synechococcus. J Mol Biol 368: 966981.
  • Rusch, D.B., Halpern, A.L., Sutton, G., Heidelberg, K.B., Williamson, S., Yooseph, S., et al. (2007) The Sorcerer II global ocean sampling expedition: Northwest Atlantic through Eastern Tropical Pacific. PLoS Biol 5: e77.
  • Schloss, P.D., Larget, B.R., and Handelsman, J. (2004) Integration of microbial ecology and statistics: a test to compare gene libraries. Appl Environ Microbiol 70: 54855492.
  • Short, S.M., and Short, C.M. (2008) Diversity of algal viruses in various North American freshwater environments. Aquat Microb Ecol 51: 1321.
  • Short, S.M., and Suttle, C.A. (2003) Temporal dynamics of natural communities of marine algal viruses and eukaryotes. Aquat Microb Ecol 32: 107119.
  • Short, C.M., and Suttle, C.A. (2005) Nearly identical bacteriophage structural gene sequences are widely distributed in both marine and freshwater environments. Appl Environ Microbiol 71: 480486.
  • Short, S.M., Chen, F., and Wilhelm, S. (2009) MAVE: the construction and analysis of marker gene libraries. In Manual of Aquatic Viral Ecology. Suttle, C.A. et al. (ed.). Limnol Oceanogr Methods (in press).
  • Sullivan, M.B., Waterbury, J.B., and Chisholm, S.W. (2003) Cyanophages infecting the oceanic cyanobacterium Prochlorococcus. Nature 424: 10471051.
  • Sullivan, M.B., Coleman, M.L., Weigele, P., Rohwer, F., and Chisholm, S.W. (2005) Three Prochlorococcus cyanophage genomes: signature features and ecological interpretations. PLoS Biol 3: e144.
  • Sullivan, M.B., Lindell, D., Lee, J.A., Thompson, L.R., Bielawski, J.P., and Chisholm, S.W. (2006) Prevalence and evolution of core photosystem II genes in marine cyanobacterial viruses and their hosts. PLoS Biol 4: e234.
  • Sullivan, M.B., Coleman, M.L., Quinlivan, V., Rosenkrantz, J.E., DeFrancesco, A.S., Tan, G., et al. (2008) Portal protein diversity and phage ecology. Environ Microbiol 10: 28102823.
  • Suttle, C.A. (2000) Cyanophages and their role in the ecology of cyanobacteria, In The Ecology of Cyanobacteria: Their Diversity in Time and Space. Whitton, B.A. and Potts, M. (eds). Boston, MA, USA: Kluwer Academic Publishers, pp. 563589.
  • Suttle, C.A., and Chan, A.M. (1993) Marine cyanophages infecting oceanic and coastal strains of Synechococcus: abundance, morphology, cross-reactivity and growth characteristics. Mar Ecol Prog Ser 92: 99109.
  • Tamura, K., Dudley, J., Nei, M., and Kumar, S. (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24: 15961599.
  • Wang, K., and Chen, F. (2004) Genetic diversity and population dynamics of cyanophage communities in the Chesapeake Bay. Aquat Microb Ecol 34: 105116.
  • Wang, K., and Chen, F. (2008) Prevalence of highly host-specific cyanophages in the estuarine environment. Environ Microbiol 10: 300312.
  • Waterbury, J.B., and Valois, F.W. (1993) Resistance to co-occuring phages enables marine Synechococcus communities to coexist with cyanophages abundant in seawater. Appl Environ Microbiol 59: 33933399.
  • Weigele, P.R., Pope, W.H., Pedulla, M.L., Houtz, J.M., Smith, A.L., Conway, J.F., et al. (2007) Genomic and structural analysis of Syn9, a cyanophage infecting marine Prochlorococcus and Synechococcus. Environ Microbiol 9: 16751695.
  • Wilhelm, S.W., Carberry, M.J., Eldridge, M.L., Poorvin, L., Saxton, M.A., and Doblin, M.A. (2006) Marine and freshwater cyanophages in a Laurentian Great Lake: evidence from infectivity assays and molecular analyses of g20 genes. Appl Environ Microbiol 72: 49574963.
  • Williamson, S.J., Rusch, D.B., Yooseph, S., Halpern, A.L., Heidelberg, K.B., Glass, J.I., et al. (2008) The Sorcerer II global ocean sampling expedition: metagenomic characterization of viruses within aquatic microbial samples. PLoS ONE 3: e1456.
  • Wilson, W.H., Joint, I.R., Carr, N.G., and Mann, N.H. (1993) Isolation and molecular characterization of five marine cyanophages propagated on Synechococcus sp. strain WH7803. Appl Environ Microbiol 59: 37363742.
  • Zhong, Y., Chen, F., Wilhelm, S.W., Poorvin, L., and Hodson, R.E. (2002) Phylogenetic diversity of marine cyanophage isolates and natural virus communities as revealed by sequences of viral capsid assembly protein gene g20. Appl Environ Microbiol 68: 15761584.

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Fig. S1. Nucleotide sequence alignment for design of cyanopodovirus pol gene specific PCR primers. The primer name is numbered according to the corresponding amino acid positions in the reference gene of cyanopodovirus P60. Primers are designed to target the cyanopodoviruses (in red box). For the mixed bases, Y = C/T, M = A/C, R = A/G, S = G/C, D = A/T/G.

Fig. S2. Rarefaction analysis of eight clone libraries based on partial nucleotide sequences of DNA pol gene. The curves were generated using the software DOTUR (http://schloss.micro.umass.edu/software/index.html) at 2% genetic divergence.

Table S1. BLASTP results of translated Chesapeake Bay virioplankton metagenome sequences (18 best hits from the September 2002 metagenome library) against NCBI database.

Table S2. BLASTP results of translated Chesapeake Bay virioplankton metagenome sequences (18 best hits from the GOS MOVE858 metagenome library) against NCBI database.

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