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Appl Environ Microbiol. Sep 2008; 74(17): 5317–5324.
Published online Jun 27, 2008. doi:  10.1128/AEM.02480-07
PMCID: PMC2546643

Phylogenetic Diversity of Sequences of Cyanophage Photosynthetic Gene psbA in Marine and Freshwaters[down-pointing small open triangle]

Abstract

Many cyanophage isolates which infect the marine cyanobacteria Synechococcus spp. and Prochlorococcus spp. contain a gene homologous to psbA, which codes for the D1 protein involved in photosynthesis. In the present study, cyanophage psbA gene fragments were readily amplified from freshwater and marine samples, confirming their widespread occurrence in aquatic communities. Phylogenetic analyses demonstrated that sequences from freshwaters have an evolutionary history that is distinct from that of their marine counterparts. Similarly, sequences from cyanophages infecting Prochlorococcus and Synechococcus spp. were readily discriminated, as were sequences from podoviruses and myoviruses. Viral psbA sequences from the same geographic origins clustered within different clades. For example, cyanophage psbA sequences from the Arctic Ocean fell within the Synechococcus as well as Prochlorococcus phage groups. Moreover, as psbA sequences are not confined to a single family of phages, they provide an additional genetic marker that can be used to explore the diversity and evolutionary history of cyanophages in aquatic environments.

There are approximately 10 to 100 million viruses per ml in marine and fresh waters, often exceeding prokaryotic abundance more than 10-fold. As significant agents of mortality, viruses facilitate nutrient cycling, influence bacterial and algal biodiversity, and mediate microbial mortality and genetic transfer (21, 22, 29). In the oceans, a significant proportion of cyanobacteria of the genus Synechococcus are visibly infected by viruses (13). In fact, cyanophages infecting this genus can be readily isolated from seawater (23, 25, 28, 30), where they can reach abundances in excess of 105 ml−1 (19, 24). Their titers fluctuate with temperature, salinity, and host abundance (23, 24, 30), and it is estimated that viral lysis removes from <1 to several percent of Synechococcus cells each day (3, 13, 24, 28).

Several non-culture-based methods have been used to assess the genetic diversity of cyanophages in nature. Although no gene is universally conserved in cyanophages, there are genes found within specific groups that can be used as PCR targets. For example, a homologue to a gene (g20) that encodes a portal vertex protein in phage T4 has been targeted in a number of studies (16, 27, 33). Although these results indicated that cyanophage diversity is very high in seawater, the primers target only cyanomyoviruses and likely other myoviruses that do not infect cyanobacteria (16).

Recently, genes homologous to psbA and psbD that code for the D1 and D2 proteins involved in oxygenic photosynthesis were discovered in cyanophages infecting Prochlorococcus (5) and Synechococcus (7, 10) spp., a finding that potentially provides another marker with which to investigate the evolution and molecular diversity of cyanophages. Viral encoded psbA was found in 88% of the cyanophage genomes surveyed by Sullivan et al. (17), including those of all 32 cyanomyoviruses and 5 Prochlorococcus podoviruses, but it was not found in Prochlorococcus siphoviruses or Synechococcus podoviruses. Subsequently, Wang and Chen (27) found the gene in podoviruses infecting Synechococcus spp. Phylogenetic analysis clustered psbA sequences into two groups infecting Prochlorococcus spp. (podoviruses and myoviruses) and a group infecting Synechococcus spp. (myoviruses). As psbA is not restricted to a specific viral family and can be amplified from environmental samples (17, 32), it provides an additional marker with which to examine the diversity and evolutionary history of cyanophages in nature.

The present study assessed the phylogenetic diversity of cyanophage psbA gene sequences in marine and fresh waters with the goal of determining whether sequences clustered based on the environment from which they were obtained. Previously, environmental cyanophage psbA sequences had been obtained from the Red Sea, Mediterranean Sea, Norwegian coast, and near Hawaii (14, 15, 17, 32). The present study examined environmental sequences from the Arctic Ocean, the Gulf of Mexico, the northeast Pacific Ocean, and lakes in North America and Europe. Phylogenetic analysis demonstrated that viral psbA sequences from freshwaters have an evolutionary history that is distinct from that of their marine counterparts, and the tree architecture was consistent with viral psbA sequences clustering based on the phage family and the hosts they infect.

MATERIALS AND METHODS

Sample collection.

Samples were collected from fresh and marine waters during May 1995 to July 2004. Seawater samples were collected from the Arctic Ocean (RV Mirai), the Gulf of Mexico (RV F. G. Walton Smith), and several inlets in the northeast Pacific (CCGS Vector). Freshwater samples were collected from lakes 227 and 240 in the Experimental Lakes Area, Ontario, Canada, and from Lake Constance, Germany. Details on the stations and samples are listed in Table Table1.1. The Lake Constance sample (LAC95) was a composite containing three virus communities that were collected using a submersible pump, while the Jericho Pier, lake 227, and lake 240 samples were collected with a bucket or by submersing a carboy. All others were collected from a research vessel by use of Niskin bottles mounted on a rosette. In all cases, viruses were concentrated from the samples by tangential flow filtration as outlined below.

TABLE 1.
Details of sampling locations

Concentration of natural virus communities.

To remove organisms and particles larger than viruses, water samples were filtered through a nominally 1.2-μm (GC50; Advantec MFS, Dublin, CA)- or 0.7-μm (GFF; Whatman, Clifton, NJ)-pore-size glass-fiber filter, followed by a 0.45-μm (GVWP; Millipore, Bedford, MA)- or 0.2-μm (Gelman Sciences Inc., East Hills, NY)-pore-size filter. The remaining virus-size material was concentrated ca. 50- to 400-fold by use of a 1-kDa- or 30-kDa-cutoff ultrafiltration cartridge (S1Y10/S1Y30/S10Y30) (Millipore) as described previously (26). Viral concentrates were stored at 4°C in the dark until analysis.

DNA extraction and amplification of psbA.

Subsamples (250 μl) of the viral concentrates were filtered through a 0.22-μm-pore-size filter (PDVF; Millipore) to remove cells and treated with DNase to remove free DNA. Nucleic acids were extracted using a Powersoil DNA kit (Qiagen, Mississauga, Ontario, Canada).

Extracted DNA (2 μl) was added to a 48-μl PCR mixture containing Platinum Taq DNA polymerase assay buffer (50 mM KCl, 20 mM Tris-HCl, pH 8.4), 5.0 mM MgCl2, 200 μM deoxyribonucleoside triphosphate, the primers (17) Pro-psbA-1F (5′-AACATCATYTCWGGTGCWGT-3′) and Pro-psbA-1R (5′-TCGTGCATTACTTCCATACC-3′) (0.25 μM each), and 2.0 U of Platinum Taq DNA polymerase (Invitrogen, Carlsbad CA). Platinum Taq was chosen over DNA polymerases because of its lower error rate. Negative controls contained all of the reagents, but sterile water was used as the template. PCR was carried out as follows: denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 52°C for 1 min, extension at 72°C for 1.5 min, and a final extension at 72°C for 10 min (28). The ~650-bp amplification products were subjected to electrophoresis using 1.5% agarose-0.5× Tris-borate-EDTA buffer (45 mM Tris-borate, 1 mM EDTA [pH 8.0]) at 100 V for 60 min. Gels were stained with ethidium bromide and visualized under conditions of UV illumination. By use of a clean glass pipette, a plug of agarose containing amplified DNA was removed from each lane. The plug was added to 100 μl of 0.5× Tris-borate-EDTA buffer and heated at 65°C for 60 min to elute the DNA. The eluted DNA (2 μl) was added to a 48-μl PCR mixture, and the PCR was conducted as described above, except that the number of cycles was decreased to 30. Amplification in the second-round PCR was confirmed by agarose gel electrophoresis in the manner described above.

DGGE.

Second-round amplification products (75 μl) were separated using denaturing gradient gel electrophoresis (DGGE). Gels with a 10 to 30% linear denaturing gradient (where a 100% denaturing gradient results from 7 M urea and 40% deionized formamide) and 6 to 8% polyacrylamide were run for 20 h using 1× Tris-acetate-EDTA buffer (40 mM Tris base, 20 mM sodium acetate, 1 mM EDTA [pH 8.5]) at 120 V and 60°C in a D-code electrophoresis system (Bio-Rad Laboratories, Hercules, CA). The gels were stained in 0.1× SYBR green solution (Invitrogen) for 3 h and visualized and photographed with an Alpha Imager 3400 system.

By use of a sterile pipette, plugs were removed from 48 DGGE bands. Each plug was added to 100 μl of 1× Tris-acetate-EDTA buffer and heated at 95°C for 15 min to elute the DNA. PCR was conducted using 2 μl of the eluted DNA as the template under the same conditions as the second-round PCR described above. Products were purified with a MinElute PCR cleanup kit (Qiagen). The purified products were cloned with a TOPO TA cloning kit (Invitrogen) as described by the manufacturer. Plasmid DNA harvested from overnight cultures was added to a 24-μl PCR mixture containing Platinum Taq DNA polymerase assay buffer, deoxyribonucleoside triphosphate, MgCl2 (all in the quantities specified above), primers T3 (5′-ATTAACCCTCACTAAAGGGA-3′) and T7 (5′-TAATACGACTCACTATAGGG-3′) (0.5 μM each), and 1.0 U of Platinum Taq DNA polymerase (Invitrogen). After electrophoresis, the remaining PCR products were purified using a minelute cleanup kit (Qiagen) and sequenced using Applied Biosystems BigDye version 3.1 Terminator chemistry. Sequencing services were provided by University of British Columbia Nucleic Acid and Protein Service Facility.

Phylogenetic analysis.

A total of 45 environmental viral psbA sequences were manually edited in BioEdit (Table (Table1;1; GenBank accession numbers EU258956 to EU258999). The 249 other psbA sequences were retrieved from GenBank and included sequences from marine and freshwater cyanobacteria (GenBank accession numbers AY119759, AY599033, BX548174, DQ158145 to DQ1581557, DQ158160 to DQ158163, DQ473676 to DQ473678, DQ473681, DQ473683 to DQ473685, DQ473687, DQ473688, NC005072, NC005042, NC010475, U18090, U21331, X13547, X15514, and Y00885) and cyanophages (GenBank accession numbers AJ629075, AJ629221, DQ473648 to DQ473666, DQ473668 to DQ473673, DQ206826, DQ206828, and EU256551 to EU256570) and environmental samples from Hawaii (17) (GenBank accession numbers DQ473720 to DQ473827), Norway (14) (GenBank accession numbers DQ787237 to DQ787256), the Mediterranean Sea (15) (GenBank accession numbers DQ401466 to DQ401504), and the Red Sea (32) (GenBank accession numbers AY713428 to AY713432). Cyanophage psbA sequences were ascribed to families based on the original descriptions (5, 9, 19, 17, 27).The sequences were aligned with CLUSTALX and refined by eye with BioEdit. Amino-acid alignments were used as the basis for the manual alignment of the nucleotide sequences.

Phylogenetic trees were constructed using PAUP 4.0b8 software and the neighbor-joining (NJ) and maximum-parsimony (MP) methods. MP (100 replicates) was used to reconstruct the tree topology. Heuristic searches were performed with 100 (MP) random addition sequence replicates and using the tree-bisection and reconnection branch-swapping algorithms. Bootstrap analyses of 1,000 (NJ) and 100 (MP) resamplings were carried out to generate confidence estimates for the inferred topologies. Eukaryotic psbA gene sequences from Heterosigma akashiwo and H. carterae were used as outgroups for the phylogenetic analysis.

RESULTS

Synechococcus spp. and their cyanophages formed distinct monophyletic groups according to the results of analysis by either the NJ (not shown) or the MP method, whereas Prochlorococcus and viral psbA sequences clustered together (Fig. (Fig.1;1; see also Fig. S1 in the supplemental material). Prochlorococcus psbA sequences clustered according to ecotype (low light versus high light). Although bootstrap support for many of the groups was weak, the tree was generally congruent with the results for phage families and host range; hence, it is likely that the overall architecture of the tree is largely correct. These analyses resolved five groups of marine cyanophage isolate and environmental psbA sequences.

FIG. 1.
Phylogenetic tree of psbA determined by MP using PAUP version 4.0b8. The percent bootstrap support (n = 100) is indicated by the color of the filled circles at the nodes: black, >85%; gray, 75 to 84%, light blue, 74 to ...

Mar1-SM (Marine1 Synechococcus myoviruses) is a weakly supported (<50%) group that includes several more strongly supported clades containing sequences from myoviruses infecting Synechococcus spp. and from 30 environmental isolates from the Norwegian coast, the northeastern Pacific Ocean, the Arctic Ocean, and the Mediterranean Sea. The fact that nearly all of the clusters of sequences from myovirus isolates infecting marine Synechococcus spp. fall into the Mar1-SM group, even though there is little statistical support for the idea that these clusters belong in the same clade, provides additional evidence that Mar1 is a valid group. Therefore, it is a reasonable to hypothesize that most environmental sequences in this cluster also are from myoviruses infecting Synechococcus. A group containing a single sequence from a myovirus infecting Prochlorococcus spp. also fell within this clade, but given its poor bootstrap support, it is likely misplaced.

Mar2-SP (Marine2 Synechococcus podoviruses) is a small group with moderate (79%) bootstrap support that includes two psbA sequences from podoviruses infecting marine Synechococcus spp. and two environmental sequences from the Red Sea. Two sequences reportedly from myoviruses infecting Synechococcus spp. (9) form a clade that is a sister to the Mar2-SP clade, although with little (<50%) support. However, these viruses were from cultures containing podoviruses and myoviruses (9); hence, the origin of the sequences is unclear.

Mar4-PP (Marine4 Prochlorococcus podoviruses) is a broad group that contains a series of clades with various degrees of bootstrap support that encompass 4 psbA sequences from podoviruses infecting Prochlorococcus spp. and 46 environmental sequences from near Hawaii, the Gulf of Mexico, the Arctic Ocean, and the Sargasso Sea.

Finally, Mar6-PM (Marine6 Prochlorococcus myoviruses) is a weakly (<50%) supported group that comprises psbA sequences from nine Prochlorococcus myoviruses and two Synechococcus myoviruses as well as sequences from 23 environmental isolates from near Hawaii, the Arctic Ocean, and the Mediterranean Sea. Most of these sequences fall into two relatively well-supported clades, Mar6a-PM (<50%) and Mar6b-PM (76%), although sequences from an isolate (P-SSM4) and a Hawaiian sample fell outside the clusters with significant bootstrap support.

The phylogenetic reconstruction resolved five other groups that consist entirely of environmental sequences, Mar3 (Marine3), Mar5 (Marine5), Mar7 (Marine7), FW1 (Freshwater1), and FW2 (Freshwater2). Only Mar3, Mar5, FW2, and most of FW1 have strong (>85%) bootstrap support. Although clades within Mar7 were well supported, the overall architecture of the group was not; hence, the group may not remain stable as more sequences become available. Mar3 consists of 15 sequences from the Arctic Ocean and Mediterranean Sea, while Mar5 comprises 18 sequences from the coast of Hawaii. The 27 sequences included in Mar7 are from the Gulf of Mexico, the northeastern Pacific, the Arctic Ocean, and the Sargasso Sea. FW1 and FW2 are mainly comprised of sequences from lakes. FW1 contains seven sequences from lakes 240 and 227, one sequence from Jericho Pier, and two sequences from the Mediterranean. FW2 includes five sequences from Lake Constance and one from lake 240.

The GC contents of psbA sequences, as well as the variable triplet peptides associated with the D1 protein motif R/K ETTXXXSQ/H (15), were analyzed for groups comprised only of marine environmental sequences (Fig. (Fig.2).2). The percentages of GC content ranged from 44.5 to 45.9 (x = 45.2) for Mar3, 41.3 to 43.3 (x = 42.6) for Mar5, and 45.4 to 49.3 (x = 47.2) for Mar7. In addition, most variable triplet motifs identified in these groups are virus-like. Mar3 primarily contained virus-like triplets (GLE, GLT, and DNE), with two exceptions (SLE and ETE), while the triplet motifs in Mar5 (ENE and EVE) were exclusively virus-like. Similarly, Mar7 triplets EQV, ETV, EQE, ENV, EEV, and EVE were virus-like, with the exception of one sequence (EAE) that is mainly found in Synechococcus spp. Among the six virus-like triplets identified in this cluster, two of them (ETV and ENV) appeared to be unique to viruses (15).

FIG. 2.
Phylogenetic relationship, percent GC content, and the triplet sequences from the D1 protein motifs of partial psbA genes from environmental samples clustering in the three unknown marine groups (Mar3, Mar5, and Mar7). The MP tree was constructed with ...

DISCUSSION

Previous observations revealing that many cyanophages contain psbA, which encodes one of the core proteins required for photosynthesis (5, 7, 10, 17, 27), raise many questions about the diversity and evolutionary history of this gene in viruses. The results of the present study suggest that a number of previously unknown evolutionary groups of psbA sequences are likely phage associated, thereby considerably expanding the known sequence space of these genes in nature. Furthermore, the fact that evolutionary groups of psbA sequences appear to be associated with specific families of phages and their hosts, as well as with the environments in which they occur, implies that psbA sequences can be useful markers for examining the environmental diversity of cyanophages. These results and their implications are considered below.

Phylogenetic reconstruction showed discrete evolutionary groups of psbA sequences from cyanobacteria and cyanophages and of sequences from marine and freshwaters. In similarity to previous findings (17), psbA sequences from Synechococcus spp., from low-light and high-light Prochlorococcus spp., and from the phage families that infect them fell into distinct clades, implying that each has its own evolutionary history, with limited genetic exchange among the clades. Consequently, environmental sequences that fall into clades with those of representative cyanophage isolates can be used to infer the identity of the virus family (myoviruses versus podoviruses) as well as of the host (Synechococcus or Prochlorococcus spp.) that the viruses infect. Moreover, freshwater and marine phage psbA sequences could be discriminated. Although bootstrap support values are weak for many clades (e.g., Mar1-SM, FW-1, Mar2-SP, Mar6b-PM, and Mar7), suggesting that the tree topology is not stable, much of the tree architecture is logically consistent in that sequences from families of viruses that infect the same host species are generally grouped together, as are sequences from freshwaters. Other groups (e.g., Mar3, Mar4-PP, Mar5, Mar6a-PM, and FW2) have good bootstrap support. Moreover, the basic tree topology is supported by the results of both NJ and MP analyses, and phages and their hosts are placed into logical groups, as is consistent with the results of other studies (4, 17). For example, with one exception, psbA sequences from marine cyanophage isolates that infect Synechococcus spp. fell into the Mar1-SM and Mar2-SP groups. For phages infecting marine Synechococcus spp., most psbA sequences belonging to myoviruses fall within Mar1-SM whereas those from podoviruses fell within Mar2-SP. Nonetheless, the weak bootstrap support at some nodes suggests that the tree topology may change as more psbA sequences become available. Ultimately, the isolation of representative taxa may provide additional support for the overall topology of the tree.

Phages S-SSM1 and S-ShM1 are exceptional, as they are myoviruses infecting Synechococcus spp. but that fall within the group of myoviruses (Mar6-PM) infecting Prochlorococcus spp. Although these isolates appear to have a relatively restricted host range (17), some cyanomyoviruses infect both Prochlorococcus and Synechococcus spp. (18). This infection pattern could lead to lateral gene transfer among relatively distantly related cyanophages during coinfection (17). Similarly, the psbA sequence for the Prochlorococcus myovirus (P-SSM1) falls among those associated with Synechococcus myoviruses. This phage has a broad host range and infects low- and high-light ecotypes of Prochlorococcus spp. (17), and, based on phylogenetic analysis of psbA, it likely infects Synechococcus spp. as well.

Many environmental psbA sequences from the northeastern Pacific and the coast of Norway clustered with those from Synechococcus phages (Fig. (Fig.1)1) and had percent GC content consistent with that found in Synechococcus phages (data not shown). Synechococcus spp. are important phototrophs in these regions, and titers of cyanophages infecting Synechococcus spp. can be high (8, 11, 24). Moreover, sequences homologous to those of cyanomyovirus structural genes have been found at these locations (1, 16). Most environmental sequences within Mar1-SM fell into well-supported clades that were distinct from clades encompassing sequences from isolates, indicating that isolates do not currently capture the diversity of phage psbA sequences found in nature.

psbA sequences from Prochlorococcus podoviruses and myoviruses also formed two distinct groups (Mar4-PP and Mar6-PM, respectively), within which many environmental sequences were dispersed. Most of the environmental sequences were from samples collected near Hawaii, where Prochlorococcus spp. are abundant (12) and where viruses infecting Prochlorococcus spp. would also be expected to be present. Sequences were also recovered, however, from samples collected in the Mediterranean, Gulf of Mexico, and Arctic Ocean.

The results of this study have revealed several previously unknown evolutionary groups of putative cyanophage psbA genes that have no cultured representatives and demonstrate that the genetic richness of psbA is considerably greater than previously recognized. Two supported clades (FW1 and FW2) were composed of sequences from freshwaters except for three sequences in FW1, including one sequence that originated from Jericho Pier, an estuarine environment that is heavily influenced by freshwater inflow (19 ppt) from the Fraser River. These results strongly suggest that psbA in freshwater cyanophages has an evolutionary history that is distinct from that of its marine counterparts. This idea is supported by observations (16) that some clades of myovirus structural gene (g20) sequences were restricted to freshwaters. The results differed, however, in that there were also clades in which g20 sequences from marine and fresh waters cooccurred.

Both MP (Fig. (Fig.1)1) and NJ (not shown) analyses revealed three other exclusively marine and previously unknown groups of psbA sequences. Mar3 and Mar5 are well-supported groups that are likely derived from Prochlorococcus phages. Mar3 is most closely related to and branches with Mar4-PP, a group containing Prochlorococcus podoviruses. Similarly, Mar5 is well supported and is part of a larger group that includes Mar6-PM, a clade that encompasses sequences from Prochlorococcus myoviruses as well as sequences from Prochlorococcus host cells. Although there are no representive isolates within Mar5, these sequences are likely to be viral. First, the sequences come from samples that were filtered through 0.2-μm pores to remove host cells (17). Further evidence is derived from nucleotide linguistics based on the distribution of predicted amino acids within a variable triplet region within the D1 protein motif (R/K ETTXXXS Q/H) (15). The variable triplet sequences found in Prochlorococcus strains are ETE and GLE. In the case of Mar5, the variable triplet sequences were EVE and ENE, the virus-like sequences (Fig. (Fig.2).2). Overall, the evidence is persuasive that sequences within Mar5 are of phage origin.

The final group, Mar7, is comprised entirely of sequences collected from the Arctic to the Mediterranean and is revealed by both NJ (data not shown) and MP (Fig. (Fig.1)1) analyses. Although bootstrap support for Mar7 is low, within the clade there are many well-supported groups. Low bootstrap support, and phylogenetic analyses which place the branch point of Mar7 subordinate to psbA sequences from Prochlorococcus and its phages but superior to sequences from Synechococcus and its phages, makes the origin of the Mar7 sequences difficult to ascertain based on phylogenetic analysis. If nodes with little support are collapsed, Mar7 becomes unresolved with other poorly supported clades in Mar1-SM; thus, Mar7 may represent previously unknown diversity of psbA sequences within Synechococcus phages.

Nonetheless, there is good evidence that the psbA sequences that make up Mar7 are viral. First, the samples were filtered through 0.22-μm pores to remove host cells and were DNase treated to remove free DNA before the psbA sequences were amplified. Second, the average percentages of GC content of the sequences ranged from 46.2% to 49.2%, which is in the range for Synechococcus phages but is considerably higher than is found for Prochlorococcus spp. and their phages (15, 32). The distribution of predicted amino acids within a variable triplet region within the D1 protein motif (15) also supports the viral hypothesis (Fig. (Fig.2).2). The variable triplet sequences EQV, ETV, EQE, ENV, EEV, and EVE are characteristic of viruses, while the triplets ETV and ENV have been found only in viruses (15). In total, the evidence provides strong support for the idea that the sequences within the Mar7 group are from phages.

It was surprising that psbA sequences from the Arctic Ocean were clustering within several groups of cyanophage sequences (Mar1-SM, Mar6-PM, and Mar5). These results appear to provide persuasive evidence that cyanomyoviruses infecting Synechococcus spp., as well as viruses related to those that infect Prochlorococcus spp., are present in the Arctic Ocean. Prochlorococcus spp. are thought to be restricted to oligotrophic waters between 40°N and 40°S, where water temperatures are above 10°C (6). These results suggest the existence of an unknown Arctic relative of Prochlorococcus spp., although it is possible that the sequences originate from cyanophages that infect Synechococcus spp. For example, psbA sequences from the S-ShM1 and S-SSM1 myoviruses that infect Synechococcus spp. are more similar to sequences from myoviruses that infect Prochlorococcus spp. (17). A final possibility is that cyanophages are advected from temperate waters far to the south.

In order to understand the control that viruses exert on their hosts, we need to know which viruses infect which hosts. Since most viruses are host specific, knowledge of the structure of the viral community provides insights into the host communities that are infected (20). There are no universally conserved marker genes for viruses; consequently, signature genes that identify groups of viruses represent an important tool in the quest to understand the impact of viruses on microbial communities.

Previous studies have used structural gene sequences to examine cyanomyovirus richness (2, 14, 16, 31, 33), but primer specificity has been a concern (16). In contrast, phage psbA genes are clearly from viruses that infect cyanobacteria and are not restricted to a specific phage family. Moreover, the sequences provide insights into the genera of the organisms that are infected. psbA appears to occur in all cyanomyoviruses and Prochlorococcus cyanopodoviruses (17), as well as in some Synechococcus podoviruses (27), but has not been found in siphoviruses infecting Prochlorococcus and Synechococcus spp. Unlike structural gene sequences such as g20 (16, 33) and g23 (1), psbA targets more than one phage family and distinguishes between most phages infecting Prochlorococcus and Synechococcus spp. However, psbA is not a perfect marker; it is not found in all cyanophages, in some cases host and viral sequences can cluster together, and the broad host range of some cyanophages can confound some interpretations. Nonetheless, more than any other marker, psbA has the potential to reveal greater insights into the structure, evolutionary history, and genetic richness of cyanophages communities in marine and fresh waters and of the hosts that they infect.

Supplementary Material

[Supplemental material]

Acknowledgments

We are grateful to M. T. Maldonado for discussions, to S. W. Graham for providing valuable insights into the analysis and interpretation of the results, to J. P. Payet for helping with the figure editing, and to J. L. Clasen for helpful comments on the manuscript and for providing samples from the Experimental Lakes Area lakes. A. M. Chan, S. M. Short, C. M. Short, and A. C. Ortmann collected and processed samples from Lake Constance, Jericho Pier, the Arctic Ocean, and the Strait of Georgia, respectively.

This work was supported by an NSERC Discovery Grant to C.A.S.

Footnotes

[down-pointing small open triangle]Published ahead of print on 27 June 2008.

Supplemental material for this article may be found at http://aem.asm.org/.

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