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Environ Microbiol. Nov 2010; 12(11): 3035–3056.
PMCID: PMC3037559

Genomic analysis of oceanic cyanobacterial myoviruses compared with T4-like myoviruses from diverse hosts and environments

Abstract

T4-like myoviruses are ubiquitous, and their genes are among the most abundant documented in ocean systems. Here we compare 26 T4-like genomes, including 10 from non-cyanobacterial myoviruses, and 16 from marine cyanobacterial myoviruses (cyanophages) isolated on diverse Prochlorococcus or Synechococcus hosts. A core genome of 38 virion construction and DNA replication genes was observed in all 26 genomes, with 32 and 25 additional genes shared among the non-cyanophage and cyanophage subsets, respectively. These hierarchical cores are highly syntenic across the genomes, and sampled to saturation. The 25 cyanophage core genes include six previously described genes with putative functions (psbA, mazG, phoH, hsp20, hli03, cobS), a hypothetical protein with a potential phytanoyl-CoA dioxygenase domain, two virion structural genes, and 16 hypothetical genes. Beyond previously described cyanophage-encoded photosynthesis and phosphate stress genes, we observed core genes that may play a role in nitrogen metabolism during infection through modulation of 2-oxoglutarate. Patterns among non-core genes that may drive niche diversification revealed that phosphorus-related gene content reflects source waters rather than host strain used for isolation, and that carbon metabolism genes appear associated with putative mobile elements. As well, phages isolated on Synechococcus had higher genome-wide %G+C and often contained different gene subsets (e.g. petE, zwf, gnd, prnA, cpeT) than those isolated on Prochlorococcus. However, no clear diagnostic genes emerged to distinguish these phage groups, suggesting blurred boundaries possibly due to cross-infection. Finally, genome-wide comparisons of both diverse and closely related, co-isolated genomes provide a locus-to-locus variability metric that will prove valuable for interpreting metagenomic data sets.

Introduction

T4-like phages

Double-stranded DNA bacteriophages (Caudovirales) are the primary viral types observed in marine systems. Myoviruses (contractile-tailed phages) predominate among these, as determined by viral metagenomic surveys (Breitbart et al., 2002; 2004; Angly et al., 2006; Williamson et al., 2008) and in culture experiments (Suttle and Chan, 1993; Waterbury and Valois, 1993; Wilson et al., 1993; Lu et al., 2001; Marston and Sallee, 2003; Sullivan et al., 2003). Myoviruses also dominated the viral signal in microbial-fraction metagenomic data sets from Hawaii (DeLong et al., 2006) and from the surface waters sampled in the Global Ocean Survey (GOS; Rusch et al., 2007; Yooseph et al., 2007), the latter of which reports that five of the six most abundant GOS proteins were attributed to T4-like myoviruses (Yooseph et al., 2007). The viral signal in these microbial metagenomes is thought to represent infecting viruses captured inside infected host cells, suggesting that T4-like phages are both numerically abundant and actively infectious (DeLong et al., 2006).

The canonical Escherichia coli bacteriophageT4 has a well-characterized infection cycle, genome and transcriptome (Luke et al., 2002; Miller et al., 2003a). A watershed of papers has defined the ‘core’ genes representative of the growing family of known T4-like phages. Relatively early work (Hambly et al., 2001) first noted that the ocean cyanobacterial T4-like virus S-PM2 had a module of capsid gene sequences similar to those of phage T4 – isolated using E. coli from sewage – suggesting that at least portions of these phage genomes might be shared across distantly related phages. Subsequent work (Desplats et al., 2002) expanded these observations, using a larger fraction of an E. coli T4-like phage genome (RB49) to show that the general virion structural components and the DNA replication apparatus were also conserved across T4-like phages. Whole-genome comparison followed that compared the archetype T4 phage with marine T4-like vibriophage KVP40 (Miller et al., 2003b), and T4-like coliphage JS98 (Chibani-Chennoufi et al., 2004); these studies showed that the ‘T4 core’ genes encode structural proteins to produce virus particles, as well as the metabolic machinery required for infection of the host.

As new genomes became available, further whole-genome comparisons refined our understanding of the T4 core (e.g. phages T4, RB49 and Aeh1 share 90 genes, Comeau et al., 2007) and shifted the focus to characterizing the flexible genome of T4-like phages (Nolan et al., 2006). These flexible genes encode proteins that interact with the host cell, e.g. tail fibres and internal scaffolding proteins, or likely offer other niche-defining functions such as base modification and differential complements of tRNAs (Comeau et al., 2007). Most of these genes are thought to represent ancient lateral transfer events, as 90% of them exhibited early/middle promoter control similar to that seen for the corresponding T4 core genes (Nolan et al., 2006).

Cyanobacterial T4-like phages

Ocean microbes drive globally important biogeochemical cycles, including carbon, oxygen, nitrogen and sulfur cycles (Arrigo, 2005; Howard et al., 2006; Karl, 2007), and the enormous numbers of ocean viruses (typically > 107 ml−1, or approximately 10 for every microbial cell) drive the evolution of microbial processes through host mortality (Fuhrman, 1999; Wommack and Colwell, 2000; Weinbauer, 2004; Suttle, 2005), horizontal gene transfer (Paul, 1999; Miller, 2001) and the modulation of host metabolism (Breitbart et al., 2007). Among marine microbes, the picocyanobacteria Prochlorococcus and Synechococcus are highly abundant (Waterbury et al., 1979; 1986; Partensky et al., 1999), and some estimates suggest that they account for as much as one-third of oceanic primary production (Li, 1994; 1995;). These two genera are commonly present at 105 cells ml−1 and usually co-occur: Prochlorococcus is numerically dominant in the vast, low-nutrient open oceans (Partensky et al., 1999; Johnson et al., 2006; Coleman and Chisholm, 2007), while Synechococcus dominates in coastal waters (Waterbury et al., 1979; 1986;).

In previous studies, four Prochlorococcus and Synechococcus T4-like cyanophage genomes were found to share up to 45 genes (out of ~150 total) with the non-cyanophages (Mann et al., 2005; Sullivan et al., 2005; Weigele et al., 2007, but also see Millard et al., 2009 and Note added in proof). In addition, these studies revealed the power of phage–host co-evolution in the context of ocean-basin scale ecological settings. For example, cyanophage genomes were found to contain ‘host’ genes involved in central host metabolism and photosynthesis (Mann et al., 2003; Lindell et al., 2004; Millard et al., 2004; 2009; Mann et al., 2005; Sullivan et al., 2005; Weigele et al., 2007), and these genes are expressed during phage infection (Lindell et al., 2005; 2007; Clokie et al., 2006). Further, the viral version of these host genes dominates the GOS surface ocean microbial-fraction metagenomes, e.g. 60% of the identifiable psbA genes were viral (Sharon et al., 2007). The distributions of these host photosynthetic genes among phage types appear driven by the physiology of the phage (e.g. host range for psbD and lytic cycle length for psbA, Sullivan et al., 2006). In fact cyanophages may be among the drivers of photosystem evolution as portions of the ‘host’ genes carried on cyanophages are able to recombine back into the host gene pool (Zeidner et al., 2005; Sullivan et al., 2006).

In contrast to the near-ubiquity of the core photosystem II psbA gene present in cyanophage genomes, other ‘host’ genes are sporadically distributed among cyanophage genomes but also may impact phage fitness. On the one hand, T4-like viral contigs assembled from marine metagenomes contain up to seven clustered photosystem I genes thought to form an intact monomeric PSI complex to funnel reducing power from electron transport chains to PSI-related functions during infection (Sharon et al., 2009). Interestingly, such PSI genes have yet to be identified in any genome from a cyanophage isolate (Chen et al., 2002; Mann et al., 2005; Sullivan et al., 2005; Weigele et al., 2007; Millard et al., 2009). On the other hand, the functional role of cyanophage-encoded phycobilin synthesis genes (pcyA and pebS) remains a mystery (Dammeyer et al., 2008). In this case, despite the fact that Prochlorococcus hosts lack intact phycobilisomes and that these cyanophage-encoded genes are highly divergent relative to host copies, they are expressed in vivo during infection and are functional in vitro (Dammeyer et al., 2008). It is likely that these and other sporadically distributed genes serve specific niche-defining roles for phages' adaptation to their particular hosts and environments that will reveal themselves as more genome and physiology data become available.

Here we expand the T4-like cyanophage database, nearly doubling the number of T4-like phage genomes by adding 12 new ocean cyanophage genomes to the previous four (Table 1). We use this augmented database to explore the ecology and evolution of T4-like cyanophages through an analysis of the genomes of 16 marine cyanophages compared with 10 non-cyanophage T4-like genomes from the Tulane Genome Sequencing Project (http://phage.bioc.tulane.edu/). The cyanophages were isolated from 15 different habitats over a period of 16 years, using 10 different host strains (four Prochlorococcus and six Synechococcus), while the non-cyanophages were isolated over decades using at least seven different source waters and six different hosts. Thus, these conditions optimize the potential for revealing diversity across the 26 phage isolates (Table 1) examined in this study. With this data set, we asked the following questions: What gene sets are shared and not shared among various hierarchical groupings of T4-like phages, and how do these genes inform our understanding of T4-like cyanophage and non-cyanophage biology? What mechanisms likely drive differential and sporadic distribution of non-shared genes among the cyanophages?

Table 1
General features of the T4-like genomes and isolates

Results and discussion

General features of the 16 cyanophage genomes

All available annotation information for the 16 cyanophage genomes is provided in a detailed overview figure (Fig. 1). With two exceptions, cyanophage genome sizes ranged from 174 to 196 kb (summarized in Table 1, details provided in Table S1), as commonly observed previously for non-cyanophages (Miller et al., 2003a,b; Nolan et al., 2006; Petrov et al., 2006). The exceptional cyanophages were S-SSM7 (232 kb) and P-SSM2 (252 kb), which contained large lipopolysaccharide gene clusters (Fig. 1, discussed below) that accounted for about 72–85% of the expanded genome size. Cyanophage genome size was correlated with the number of predicted open reading frames (ORFs) (R2 = 0.743), and there was no apparent relationship between the genome size and the genus of the host on which it was isolated (Fig. S1).

Fig. 1
Overview of 16 cyanophage genome annotations. Each drawn box represents a predicted open reading frame (ORF) with forward strand ORFs above and reverse strand ORFs below. ORFs are colour-coded as per the legend in the figure, while colour-coded lines ...

While significant variation in genome-wide %G+C exists among the non-cyanophages (Table 1), even for those isolated on the same host, we note that this metric is less variable among the cyanophage genomes (Table 1). As well, the average genome-wide %G+C content of phages isolated on Prochlorococcus (37.2 ± 1.0%) is significantly different (P ≤ 0.0001) from that of phages isolated on Synechococcus (40.1 ± 1.0%). Such cyanophage variability may reflect host range-constrained swapping of genetic material followed by subsequent genome-wide amelioration of the new genes in the phage genome. For example, Synechococcus cells have higher %G+C genomes than Prochlorococcus (Kettler et al., 2007; Dufresne et al., 2008) and even high %G+C material from Synechococcus hosts would ameliorate once in the phage genome towards the overall lower %G+C of phage genomes. In contrast, Prochlorococcus phage %G+Cs are often closer to that of their host genomes, so the impact of such genome-wide amelioration pressures are minimal compared with that seen in Synechococcus. Such observations in cyanophage-encoded core photosynthesis genes proved diagnostic for tracing intragenic recombination events among cyanophage genomes (Zeidner et al., 2005; Sullivan et al., 2006). That one cyanophage, S-PM2, deviates from the general pattern may hold clues regarding the host range of this particular phage (also see below).

Gene distributions among hierarchical groupings of the genomes

In preparation for analyses of gene content and order in the different genomes, we clustered orthologous genes into T4 Gene Clusters (‘T4-GCs’; see Experimental procedures), and used these to define core gene sets common to hierarchical groupings of the genomes (Fig. 2A, see discussion below). A total of 6798 predicted genes in the 26 genomes clustered into 892 T4-GCs, with 1873 genes remaining as singletons.

Fig. 2
T4-like gene set relatedness representations.

Gene presence/absence network analysis

To examine how similar the genomes are to each other with respect to the presence or absence of each T4-GC, we represented the presence/absence table as a network (Fig. 2B), which links T4-GCs to the genomes in which they are found. Genomes with many T4-GCs in common appear in close proximity due to the many connections that they share. The resulting network shows clustering of the cyanophage (blue diamonds, Fig. 2B) separate from non-cyanophage (red diamonds, Fig. 2B) T4-like genomes by this metric. Core genes shared by all 26 genomes connect the two groups of phage and are highlighted as the central purple cluster (Fig. 2B).

Core and pan-genomes

To explore the features of the core and pan-genomes of the cyanophage and non-cyanophage subsets given the number of genomes sequenced, we identified the shared and unshared gene sets of all possible combinations of choosing k genomes (k = 1 to n) from n sequenced genomes (Fig. 3). The core genes shared within the two groups (discussed in detail below) levelled off quickly as new genomes were added to the analysis, suggesting that this small sample size of diverse T4-like phages is adequate for determining the core. As expected, the total number of unique genes identified (the pan-genome) steadily increased with the number of available genomes in both cases. The size of the pan-genome reached 1388 and 1445 genes for the cyanophages and non-cyanophages respectively (Fig. 3A and B). The rate of increase of both pan-genomes as more genomes are added to the analysis is far from saturated, indicating the existence of a much larger and diverse gene pool than has been captured by the 26 sequenced genomes. Interestingly, however, the cyanophage pan-genome showed a slower rate of increase (Fig. 3A) than that of the non-cyanophages (Fig. 3B).

Fig. 3
The core and pan-genomes of the (A) cyanophage and (B) non-cyanophage groups, where the core and pan-genomes are represented by square and triangles respectively. The core and pan-genomes were analysed for k genomes from cyanophages (n = 16) or non-cyanophages ...

The T4 core, shared by all 26 T4-like phage genomes

Thirty-eight genes were common to all 26 genomes (Fig. 2A, Table S2), while also maintaining remarkable synteny (Fig. S2). The only exceptions to the synteny included a large inversion among the cyanophages relative to the non-cyanophages, and a few notable smaller-scale breaks in synteny likely due to mobile element activity (see Genome evolution in the cyanophages). Of the 38 genes shared by all the genomes, 27 form sequence-based orthologous groups (T4-GCs; see Experimental procedures), while the remaining 11 display enough sequence divergence that these functional homologues are placed into multiple T4-GCs. While the number of core genes decreased as more T4-like phage genomes were added to these analyses (Miller et al., 2003b; Mann et al., 2005; Sullivan et al., 2005; Comeau et al., 2007; Weigele et al., 2007; Millard et al., 2009), it appears that we have now adequately defined the core (Fig. 3) and that these T4 core functions involve appropriating host metabolic machinery, replicating the viral genome during infection, and building the viral particles.

Nearly ‘T4 core’ genes

Beyond the T4 core genes are a handful of noteworthy nearly core genes, i.e. those present in at least 22 genomes across the 26 T4-like phage genomes. An analysis of the patterns of their distributions makes these genes potentially useful targets for experimental functional identification, or indicators of novel functions in particular groups of isolates. This set of genes includes the gp51 baseplate hub assembly catalyst (missing only in Aeh1, but note that cyanophage gp51 are only ~20% of the length of non-cyanophage gp51, Fig. S3), nucleotide metabolism and recombination/repair genes uvsX, uvsY (both missing in the same three phages – 44RR, PHG25, PHG31), and the gp59 loader of gp41 helicase (found in 22 of 26 T4-like phages).

The non-cyanophage core, shared by all 10 non-cyanophage genomes

In addition to the 38 genesshared by all the genomes, the non-cyanophage genomes shared an additional 32 non-cyanophage core genes (Fig. 2A, Table S3), giving this group a shared core of 70 genes down from the most recent estimate of 90 core genes shared among three non-cyanophage T4-like genomes (Comeau et al., 2007). All but six of the 32 non-cyanophage core genes have been functionally annotated in coliphage T4 (Miller et al., 2003a), and the larger proteins such as structural proteins gp7, gp10 and gp12 were so divergent as to be comprised of up to nine T4-GC clusters (Table S3). Many of these additional non-cyanophage core genes encode functions involved in ‘host specialized’ viral structure (e.g. tail fibres) and DNA replication machinery. We expect that experiments targeting functional annotation of shared hypothetical proteins in the cyanophages will reveal that many of these host specific functions exist in the cyanophages, but as divergent gene copies. In contrast, other genes, such as nrdD and nrdH genes, are likely only relevant to the specific habitat of some of these non-cyanophages (e.g. anaerobic sewage).

The cyanophage core, shared by all 16 cyanophage genomes

Twenty-five genes were shared by all 16 cyanophages (Fig. 2A, Table S4), in addition to the 38 that form the T4 core, for a total of 63 genes shared across the cyanophages which now appears to be a stable shared gene set among the T4-like cyanophages (Fig. 3A). All but one of these 25 cyanophage core genes was absent from the non-cyanophages (Table S4). This exception is the phoH gene that was found in only one of the other genomes – the marine vibriophage KVP40 – and may represent an adaptation valuable both for infection of cyanobacteria, but also more generally of marine hosts (e.g. marine vibrios) rather than a cyanophage-specific function. However, some do appear cyanophage-specific, such as the previously described cobalamin biosynthesis protein (cobS), or photosynthesis proteins for the central photosystem II reaction centre protein (psbA) and highlight-inducible proteins (hli) (Mann et al., 2005; Sullivan et al., 2005; Weigele et al., 2007). Other cyanophage core genes include proteins that likely encode basic phage functions, such as a heat shock family protein (hsp20) that might be important for scaffolding during maturation of the capsid, and two experimentally determined virion structural proteins (T4-GCs 15, 190). In addition, the cyanophage core includes phosphate stress-induced protein (phoH), pyrophosphatase (mazG), and dioxygenase proteins (T4-GCs 101, 155 with similarity to PFAM PF05721) that are discussed in greater detail below. The remaining genes encode hypothetical proteins of unknown function. An understanding of the functions of these proteins, combined with a deeper understanding of the PhoH and MazG proteins (discussed below) should further elucidate the nature of cyanophage–host interactions.

Notable cyanophage core and nearly cyanophage core genes

The cyanophage core gene mazG has received a lot of recent attention. In E. coli, MazG appears to be a regulator of nutrient stress and programmed cell death (Magnusson et al., 2005; Gross et al., 2006; Lee et al., 2008), as its dNTP pyrophosphatase activity acts on the signalling nucleotide guanosine tetraphosphate (ppGpp) to regulate up to one-third of E. coli genome (Traxler et al., 2008). In cyanophages, MazG is also thought to act as a global transcriptional regulator through modulation of ppGpp levels, which may extend the period of cell survival under the stress of phage infection (Clokie and Mann, 2006; Weigele et al., 2007). However, MazG enzymes are highly specific for non-canonical NTPs, suggesting that identifying their substrates likely requires solving crystal structures along with activity and binding assays for each new enzyme (Galperin et al., 2006). Thus the cyanophage MazG substrate should be cautiously interpreted.

Regardless of function, the mazG gene has a notable distribution among T4-like cyanophages. Recently, it was found by PCR screens to be present in nine out of 17 cyanophage myovirus isolates (Bryan et al., 2008). In contrast, all 16 of our cyanophage myovirus genomes contained this gene. While this difference could be real, it likely reflects the limitations of PCR screening, which can only reveal the presence of a gene whose sequence is known (Millard et al., 2004; 2009;). Consistent with this interpretation, Bryan and colleagues (2008) observed > 99% identity among their sequenced mazG PCR products obtained from geographically diverse isolates, while the mazG sequences of our genomes showed marked sequence divergence (Fig. S4). Nonetheless, in agreement with Bryan and colleagues (2008), our analyses also suggest that mazG arose from outside the cyanobacteria (Fig. S4), as opposed to most other ‘host’ genes in cyanophages which originate from their host strains (Sullivan et al., 2005; Williamson et al., 2008), and is most closely related to the genes from Choloroflexus.

Finally, in addition to the core mazG gene, nine genes are nearly cyanophage core genes as they are found in 15 of the 16 cyanophages, missing only in the anomalous S-PM2 phage (see below).

Genome variability of two co-isolated cyanophages

To explore genomic diversity among spatially coexisting phages capable of infecting the same host, we included in this sequencing project two phages isolated from the same water sample on the same host strain (Fig. 4A). These two cyanophages, P-HM1 and P-HM2, are highly syntenic and share 200 of 241 and 242 genes, respectively, whose protein sequences are on average 83% identical (Fig. 4A). In contrast, pairwise genome comparisons showed that among the non-co-isolated cyanophages, the genomes share as much as 77–80% of their genes with average identity 72–75% (Fig. 4B) or at the least 22–33% of their genes, with only 48–49% average identity (Fig. 4C).

Fig. 4
Whole-genome pairwise comparisons across the bounds of the cyano T4 phage genome diversity are examined here. In all three panels, two genomes are compared where lines between the genomes connect homologues, coloured ORFs indicate genes that are unique ...

Further comparison of the two co-isolated phage genomes (Fig. 4A) showed that, while the protein identity of orthologues shared between P-HM1 and P-HM2 averaged 83%, there was an enormous range (21–100%) in this value. On the one hand, orthologue identities could be quite low (21–32%) and include hypothetical proteins and even proteins that are part of the cyanophage core such as CoA-dioxygenase and Hsp20. On the other hand, orthologue identities could be quite high (100%) for other cyanophage core proteins such as Hli03, gp55, as well as for non-conserved hypothetical proteins such as T4-GCs 429, 542 and 559, which are found only in a subset of Prochlorococcus phages. The non-shared proteins, predominately hypotheticals, were notably clustered into distinct regions of the genomes (Fig. 4A) akin to cyanobacterial genomic ‘islands’ (sensuColeman et al., 2006). In addition to hypotheticals, the non-shared gene set did include some annotation (Table S5): a purine biosynthesis gene (purM) and plastoquinol terminal oxidase (PTOX, described further below) are unique to P-HM1, while a pair of endonucleases and a Kelch-repeat-containing protein are unique to P-HM2. In addition, peptidase genes were present in syntenic genomic locations in both phages (Fig. 4A) even though their sequences had diverged to the point of forming separate gene clusters (T4-GCs 573, 452). These phages also contain 70 genes found in both phages, but not in any of the other sequenced cyanophages. These 70 genes encode an S8 peptidase (T4-GC518), glycine dehyrogenase (T4-GC540), two asparaginyl beta-hydroxylases (T4-GCs 536, 546), an acyl carrier protein (ACP, T4-GC457) and its synthetase (ACPS, T4-GC500), a terminal quinol oxidase (T4-GC555), taurine catabolism dioxygenase (T4-GC447), and hypotheticals. That genes encoding these proteins were found only in these two co-isolated MED4-infecting phages might provide clues to requirements for infection of Prochlorococcus MED4 in these Hawaii Ocean waters.

The cyanophage-exclusive, but not universal, gene set

We identified 143 genes that occurred in four or more of the 16 cyanophage genomes, but were absent from all of the non-cyanophage genomes (summarized in Table 2). Ninety-six of these encode hypothetical proteins, but others encode a diversity of photosynthesis (psbD, petE, petF, PTOX, pebS), phosphate stress (pstS), carbon metabolism (talC, CP12) and virion structural (24 genes) proteins, the functions of which are consistent with our notion of a cyanophage lifestyle. Some of these are discussed further below.

Table 2
Summary of the 143 ‘non-core’ genes that are enriched in cyanophages (found in > 3 genomes), but are absent from non-cyanophages

The Synechococcus-enriched gene set

We found no genes that were universal and exclusive to the 10 cyanophages isolated on Synechococcus. However, there were 48 genes that occurred in three or more of this phage set, and occurred in no others (Table S6). Notably, these genes clustered in four ‘hot-spot’ regions of the genomes: (i) near gp5 with tRNAs, (ii) with small genes between gp46 and gp25, (iii) between gp16 and gp17 (previously identified by Millard et al., 2009) and (iv) near psbA, again commonly with numerous tRNAs (Fig. 1). Although 42 of these 48 genes encode hypothetical proteins, two are involved in carbon metabolism (zwf, gnd– discussed below), three had PFAM domains that suggested function (PA14 carbohydrate-binding domain, DUF1583, and SAICAR synthetase purine biosynthesis), and one is a virion structural protein (T4-GC969; see Experimentally identified cyanophage structural proteins).

The Prochlocococcus T4 core and enriched gene set

Two genes were universal and exclusive to cyanophages isolated on Prochlorococcus (Table S7). These Prochlorococcus T4 core genes encode a possible photosystem II PsbN (Pfam domain PF02468, T4-GC163, no functional role has yet been determined for PsbN), and a hypothetical (T4-GC285). As well, there were 16 genes that occurred in three or more of this phage set, and occurred in no others (Table S7). These clustered in ‘hot-spot’ genome regions homologous to those described above for the Synechococcus-enriched genes (Fig. 1), and include genes encoding a highlight-inducible protein (T4-GC436), a phycocyanobilin biosynthesis protein (pcyA, T4-GC413) and 14 hypothetical proteins. Finally, two hypothetical proteins were universal among the six Prochlorococcus phages, but not exclusive to them (T4-GC082 also found in S-SSM7 and S-SSM5; T4-GC224 also found in S-SSM7).

The odd cyanophage out

Synechococcus cyanophage S-PM2 appears quite distinct from the 15 other cyanophages. First, its %G+C content is similar to that of a Prochlorococcus phage (Table 1). Second, S-PM2 lacks nine nearly cyanophage core genes that are found in all of the 15 other cyanophages, and two genes found in 14 of the 15 other cyanophages. In contrast, only one other cyanophage (P-SSM2) is missing even a single gene (T4-GC424) that is ubiquitous among the other 15 cyanophages. Among the genes ‘missing’ in S-PM2 are eight hypothetical genes, an endonuclease and two carbon metabolic proteins (transaldolase and CP12 = T4-GCs 63, 337). Finally, S-PM2 contains only seven of the 45 ‘Synechococcus-enriched’ phage genes, whereas, other than Synechococcus phage S-SSM7 (containing only two), the rest of the cyanophage genomes contained 18–27 (average = 23) of the 45 Synechococcus phage-enriched genes. Given the data set at hand, we cannot identify any variables that might explain why this particular phage is so different from the others.

Sporadically distributed ‘host’ genes – a link to cyanobacterial phage – host ecology and evolution

In contrast to the syntenic, widely distributed sets of genes described above, a number of genes exhibit more sporadic distributions across the cyanophage genomes (Table 3), and these are likely driving niche differentiation of cyanophage–host systems (Lindell et al., 2004; Coleman et al., 2006). Here we highlight a few of these genes, the putative functions of which can be readily connected to known variables in cyanobacterial and cyanophage ecology.

Table 3
Summary of cyanobacterial specific sporadically distributed genes among 16 T4-like cyanophages

Phosphorus utilization genes

Phosphorus often limits productivity in oligotrophic marine systems, and cyanophages have been shown to contain the phosphate stress gene, pstS (Sullivan et al., 2005), which shuttles phosphate from the outer to the inner membrane in cyanobacteria. Two Prochlorococcus T4-like phages isolated from the Sargasso Sea have been shown to encode the gene, while it was not found in two Synechococcus T4-like phages from coastal waters (Mann et al., 2005; Sullivan et al., 2005; Weigele et al., 2007, but also see Note added in proof). This raises the question of whether pstS distribution is driven by host strain, source waters, or both. Here we observed that homologues of the pstS gene were found in nine of the 16 cyanophages (Table 3). While the nine phages were isolated on six different Prochlorococcus and Synechococcus host strains, all originated from low-nutrient waters, where phosphorus is likely in short supply. Thus it appears that the source waters used for phage isolation are more important than host strain for predicting the presence or absence of pstS in the phage genome – a relationship that has been observed in metagenomic analyses of surface ocean samples (Williamson et al., 2008). In addition to the gene itself, we also identified transcriptional regulatory machinery flanking all nine pstS genes, including promoters (Fig. 5, Fig. S5) and terminators (Fig. 5). No single regulatory solution was apparent across the genomes. Interestingly, two of the phages (S-SM1, S-SM2) contained phoA, which encodes an alkaline phosphatase, next to pstS (Fig. 5). If functional, this could facilitate access to organic phosphorus.

Fig. 5
Close-up genome representation of the phosphate genes cluster from cyanophages. Genomic features are as described in Fig. 1. To orient the reader to the genome location of the cluster being portrayed, a box is drawn in a reference genome for each or a ...

Homologues of phoH, a gene that belongs to the phosphate regulon in E. coli and encodes a putative ATPase, were found in all 16 cyanophages as well as the marine T4-like vibriophage KVP40 (Miller et al., 2003b). This gene is absent from some other non-T4-like marine cyanophages [e.g. podoviruses P-SSP7 (Sullivan et al., 2005) and P60 (Chen et al., 2002), siphovirus P-SS2 (Sullivan et al., 2009)], but present in other marine phages, i.e. the distant T7-like roseophage SIO1 (Rohwer et al., 2000); thus clear patterns are not evident. We had previously described (Sullivan et al., 2005) such phage-encoded phoH genes as apparent parts of a multi-gene family with divergent functions from phospholipid metabolism and RNA modification (COG1702 phoH genes) to fatty acid beta-oxidation (COG1875 phoH genes) (Kazakov et al., 2003); indeed the function of the phoH gene, particularly in cyanobacteria, remains unclear. For example, under phosphate stress, the gene has been shown to be upregulated in E. coli (Wanner, 1996) and Corynebacterium glutamicum (Ishige et al., 2003), downregulated in Synechococcus WH8102 (Tetu et al., 2009), and unaffected in at least two Prochlorococcus strains (Martiny et al., 2006). The uniform presence of the gene in the T4-like cyanophages, combined with this mosaic of other patterns of distribution and expression, is intriguing.

Carbon metabolism genes

The distribution of carbon metabolism genes among the cyanophage genomes (Table 3) suggests that many have co-opted critical enzymes to access reducing power from glucose via the pentose phosphate pathway (PPP). All but S-PM2 (Mann et al., 2005) have the transaldolase gene (talC), thought to be important in mobilizing stored carbon through the PPP, and observed previously in three T4-like cyanophage genomes (Sullivan et al., 2005; Weigele et al., 2007). These phages also carry the gene that encodes CP12, a cyanobacterial regulatory protein that inhibits several Calvin cycle enzymes, promoting carbon flux through the PPP at night (Tamoi et al., 2005). We recently identified a homologue of CP12 in Prochlorococcus, whose identity was strengthened by a diel expression pattern consistent with this function (Zinser et al., 2009). This led to the identification and analysis of cp12 in these phage genomes, with the diel expression patterns of PPP genes (Zinser et al., 2009) informing their possible role in cyanophages (L.R. Thompson et al., in preparation). In addition to carrying talC and cp12, eight Synechococcus cyanophages encode two other pentose phosphate pathway enzymes, of varying sequence conservation (see below), which generate NADPH: zwf, a glucose-6-phosphate dehydrogenase, and gnd, a 6-phosphogluconate dehydrogenase. The existence of as many as four PPP genes in some phages suggests that this pathway is critical to cyanophage infection. We suggest that this may be due either to increased reducing power stored in carbon substrates or to the production of ribulose-5-phosphate which may alleviate bottlenecks in nucleotide metabolism.

Nitrogen metabolism genes

A well-known cyanobacterial response to nitrogen stress is the degradation of phycobilisomes through the activity of the non-bleaching protein NblA. While the nblA gene has been observed in a freshwater cyanophages (Yoshida et al., 2008), this gene has not been found in marine cyanobacteria and has not been observed among marine cyanophage. Here we propose cyanophage involvement in host nitrogen metabolism that likely involves a response to intracellular levels of 2-oxoglutarate (2OG) in the host. Ammonium, the preferred nitrogen source for cyanobacteria, is assimilated through incorporation into a 2OG carbon skeleton. Ammonia limitation thus results in 2OG accumulation in the cell, which serves as an indicator of nitrogen status (Irmler et al., 1997; Forchhammer, 1999; Muro-Pastor et al., 2001). DNA binding of the global nitrogen regulator, NtcA, is 2OG-dependent such that NtcA is inactive when 2OG is limiting and the cell has excess available nitrogen, whereas the opposite is true under nitrogen stress conditions (Schwartz and Forchhammer, 2005).

Three features of the cyanophage genomes suggest that they modulate 2OG levels to stimulate NtcA activity as needed to promote phage gene expression (Fig. 6). First, all 16 genomes contain numerous NtcA binding sites (1–16 per genome; average = 8.9), which apparently promote a diversity of both T4 phage and cyanophage functions (Fig. 1). Second, 14 of the 16 genomes contain numerous 2OG-FeII oxygenase superfamily proteins (Table 3). Third, all 16 cyanophages contain at least one and often numerous hypothetical proteins with possible phytanoyl-CoA-dioxygenase domains, (Table S4), which may act on 2OG, in this case as oxidoreductases.

Fig. 6
Proposed role of 2-oxoglutarate (2OG) during cyanophage infection.

Photosynthesis-related genes

Cyanophage-encoded phycobilin biosynthesis genes have previously been shown to be expressed during infection (pebS) and functional in vitro (pcyA, pebS, ho1; Dammeyer et al., 2008). These genes, pcyA, pebS, ho1, occur in three, four and four of the 16 cyanophage genomes respectively (Table 3). As well, the cpeT gene previously observed in S-PM2, S-RSM4 and Syn9 (Mann et al., 2005; Weigele et al., 2007; Millard et al., 2009) is found in 12 of the 16 cyanophage genomes examined here (Table 3). Notably, the cpeT gene in marine cyanobacteria is part of a phycoerythrin cpeESTR operon, so the role of the cyanophage-encoded copy remains unresolved given the lack of cpeESR.

Sporadically distributed among the cyanophage genomes are two electron transport genes, petE and PTOX, which encode proteins that commonly co-occur with the carbon metabolism genes (zwf and gnd, described above) as part of a hypothesized mobile gene cassette (Fig. 7) and likely prevent electrons from backing up and damaging photosynthetic reaction centres. The petE gene encodes plastocyanin, and has previously been described in cyanophages (Sullivan et al., 2005; Weigele et al., 2007; Millard et al., 2009). PTOX proteins are normally associated with carotenoid desaturation (Kuntz, 2004), but in cyanophages are hypothesized to help maintain balanced pools of ATP and NADPH in infected host cells (Weigele et al., 2007; Millard et al., 2009). Consistent with this hypothesis, a marine Synechococcus was shown recently to use PTOX-related oxidases to shunt off excess inter-photosystem electrons to oxygen rather than to PSI (Bailey et al., 2008), which would significantly impact ATP/NADPH pools. This alternate electron flow was thought to be particularly important under Fe-limiting conditions when PSI/PSII reaction centre ratios drop (Bailey et al., 2008). Consistent with this observation, PTOX genes are abundant in open ocean surface water microbial metagenomes (McDonald and Vanlerberghe, 2005), and are found in many surface water oligotrophic Prochlorococcus (AS9601, MIT9301, MIT9215, MIT9312, MED4, NATL1A, NATL2A) and Synechococcus (BL101, WH8102, CC9902) isolates (data not shown), although lacking in their less Fe-limited counterparts from deeper or coastal waters (e.g. LL Prochlorococcus and SynCC9605).

Fig. 7
Close-up genome representation of the carbon metabolic gene cluster from cyanophage genomes. Genomic features are as described in Fig. 1, and genome location orientation is as described for Fig. 5.

Experimentally identified cyanophage structural proteins

To maximize our ability to annotate cyanophage structural proteins, we analysed the proteome of S-SM1 experimentally, and detected multiple peptides from 41 proteins in the purified S-SM1 virion (Table S8, which includes the Synechococcus-enriched gene T4-GC969 described above). These 41 proteins in S-SM1 and their orthologues in the other 15 cyanophage genomes are designated on Fig. 1 as ORF ‘underlining’, along with the data from two other T4-like phage proteomics projects [S-PM2 (Clokie et al., 2008) and Syn9 (Weigele et al., 2007)]. Notably, these include nine proteins known to be encoded in the S-PM2 genome, but not detected in the virion (Clokie et al., 2008). These nine newly detected proteins encode homologues of seven coliphage T4 structural proteins (gp 4, 5, 14, 21, 25, 48, 53), as well as two cyanophage core proteins, including a putative citidylyltransferase (T4-GC190) and a hypothetical protein (T4-GC15). We also identified 18 hypothetical proteins which expand the existing data set of T4-like structural proteins; all of them need structural/functional assignments. We note that 10 virion structural proteins have similar distributions among nine of the cyanophage genomes (Table S8); perhaps these proteins are functionally linked, T4 phage structural components.

Genome evolution in the cyanophages

As discussed above, the ‘cyanophage core’ genes are remarkably syntenic across the 16 cyanophage genomes (Fig. S2), suggesting that most of these cyanophage specialization genes are vertically transmitted and part of general T4 phage strategies for infection of ocean cyanobacteria. Twenty-four ‘core’ genes among non-cyanophages were previously inferred to be vertically transmitted and resistant to horizontal gene transfer (Filee et al., 2006; Comeau et al., 2007). It is thought that such genes might be resistant to horizontal gene transfer due to complexity of the T4 protein–protein interactions required for the complex structure (Leiman et al., 2003) and metabolic function (Miller et al., 2003a) of phage T4 and by analogy, the T4-like phages. In contrast, phylogenies of non-core genes in the T4-like non-cyanophages have conflicting topologies which are interpreted to be due to horizontal gene transfer (Filee et al., 2006). Similarly, our cyanophage core genes are remarkably syntenic, presumably also due to vertical transmission from phage to progeny phage, and the few exceptions to this synteny may be due to the activity of mobile genetic elements (Fig. S2). Such mobile element activity in T4 phages has been previously observed in coliphage T4 (Miller et al., 2003a), as well as ocean cyanophages ranging from T4-like phages (Zeng et al., 2009) to siphoviruses (Sullivan et al., 2009). Specifically, tRNA genes co-occur with many of these altered non-syntenic regions of the genome (Fig. 1), and may serve as substrates for site-specific recombination by mobile genetic elements (Williams, 2002; Campbell, 2003).

The carbon metabolism genes carried by cyanophages may be particularly influenced by the movement of mobile gene cassettes. For example, zwf and gnd co-occur in the genomes of eight phages isolated on Synechococcus as part of a possible mobile gene cassette (Fig. 7): five contain paired, full-length, apparently functional gene cassettes in varied genome locations, while three contain variously degraded gene cassettes including remnants of zwf genes (Fig. S6). The other genes in the cassette include two photosynthetic electron transport genes (petE and PTOX, see above), a hypothetical protein (T4-GC119) and an endonuclease, which may at some point have mobilized the cassette as described below. Notably, a ninth genome (Prochlorococcus phage P-RSM4) lacks zwf and gnd entirely, but appears to have remnants of the rest of this cassette (Fig. 7).

The endonucleases in this region are notable as, in phage T4, such genes are known to be part of selfish DNA elements known as intronless homing endonucleases in both coliphages (Belle et al., 2002; Liu et al., 2003) and T4 cyanophages (Zeng et al., 2009). It is plausible that such selfish genes might lead to highly recombinogenic regions in the T4 genome as the nuclease errs and yields double-strand breaks. Here we observe two forms of endonucleases (Fig. S7) – one of which contains sequences with distant homology to this confirmed homing endonuclease (T4-GC228) where only one member (from P-SSM2) contains the catalytic residues identified by Zeng and colleagues (2009); the second contains sequences that lack any homology to the experimentally determined cyanophage T4 homing endonuclease (T4-GC282). Notably, this endonuclease-flanked cassette is located in variable locations in the genomes (Fig. 7). In four of the genomes the cassette appears in the same gp17–gp18 region that Millard and colleagues (2009) recently described as a hypervariable region. In a fifth genome, S-SM2, the cassette appears near psbA, where it is interrupted by a second gene cassette (the hypothetical T4-GCs cluster described below). The four additional genomes contain degraded forms of this cassette in varied genome locations. Beyond this carbon metabolism cassette, we note that additional carbon metabolism genes, talC and cp12, occupy variable genome positions ranging from locations in the 5′- or 3′-end of the psbA region or near gp5, but are often proximal to tRNAs (Fig. S8).

Two other classes of gene cassettes carry signatures of mobility in these genomes. First, a cluster of five hypothetical proteins (T4-GCs 218, 219, 234, 235, 237), often associated with a plasmid stability protein, was found in all but one (S-PM2) of the cyanophages (Fig. S9). This cluster was similarly positioned and structured across nine genomes, but varies across the other six genomes. We hypothesize that these proteins are clustered for functional reasons, and that the plasmid stability protein may offer mobility of the gene cassette. Second, large clusters of lipopolysaccharide (LPS) genes are present in the larger cyanophage genomes (Fig. 1) located either near hli03 (S-SSM7, S-SM2, P-SSM2) and/or near phoH (P-SSM2), again proximal to tRNAs. It is not known whether these LPS biosynthesis genes are functional or are simply ‘stuffer DNA’ for headful packaging in these larger genome phages. However, seven LPS genes co-occur in three phages that were isolated 2 years apart using source waters hundreds of miles distant from each other (T4-GCs 260, 265, 266, 304, 305, 307, 308 all occur in P-SSM2, S-SM2, and S-SSM7). Either a recent transfer event occurred across these three disparate phages, or, perhaps more likely, these LPS genes are functionally linked and represent convergent evolution.

Conclusion

With this expanded data set we have been able to better define the T4-like phage core genome. The challenge now is to examine more closely the non-core genes required for infection of different hosts and environments. Our analysis reinforces the importance, for cyanophage, of carrying genes involved in the light reactions of photosynthesis, the pentose phosphate pathway, and phosphorus acquisition. In addition, we reveal a possible link to host nitrogen metabolism. Finally, the genome-wide comparison of two phages isolated on the same host from the same sample, offers a first look at intra-population genomic variability that is a critical first step to understanding the biogeography of phage diversity.

Experimental procedures

Phage isolation, purification, DNA extraction and sequencing

Twelve cyanophages were isolated (Waterbury and Valois, 1993; Sullivan et al., 2003; Sullivan et al., 2008), then concentrated and purified for genomic DNA extraction either by CsCl purification (details in Lindell et al., 2004) or using a Lambda Wizard DNA kit (Promega Corp., Madison, WI) directly on phage lysates. This kit precipitates phage particles using a polyethylene glycol solution, followed by DNA extraction using a diatomaceous earth-based resin (Promega Corp., Madison, WI). Total DNA yields were consistently higher using the Wizard DNA kit than using CsCl-purified particles (1–2 µg from 250 ml of lysate versus nanograms from 2 l of lysate). Although host DNA contamination was significant (ranged 11.4–77.5% of total reads) in the Wizard DNA kit preps due to the less rigorous purification, host reads could be filtered out during phage genome assembly. These methods are described in detail elsewhere (Henn et al., 2010).

Construction and pyrosequencing libraries

Pyrosequencing libraries preparations are described in Henn and colleagues (2010). Briefly, 100 µl of cyanophage genomic DNA (1 ng to 2.2 µg) was sheared using Covaris AFA technology and the following conditions: time = 240 s, duty cycle = 5, intensity = 5; cycles per burst = 200 and temperature = 3°C. Post-shearing, the DNA was concentrated and fragments less than 200 bp were removed using AMPure PCR purification beads (Agencourt Bioscience Corporation, Beverly, MA). The DNA shearing profile was determined by running 1 µl of the samples on the Agilent Bioanalyser 2100 using a DNA 1000 chip (Agilent Technologies, Santa Clara, CA) with the optimal size for library construction being 1.2–1.5 kb fragments. The sheared DNA was then used for pyrosequencing library construction with reagents provided in the GS 20 Library Preparation Kit (454 Life Sciences, Branford, CT) according to manufacturer's instructions for fragment end-polishing, adaptor ligation and library immobilization reactions but slightly modified for the clean-up steps, which were performed with the addition of 1.8× AMPure beads.

Genome assembly and annotation

Phage genomes were assembled using the Newbler assembly software package (454 Life Sciences, Branford, CT) with all settings set to default and the ‘-finish’ mode invoked. The ‘-finish’ mode assembles through repetitive regions that form unambiguous paths between contigs, thus some regions that would typically generate an assembly gap were assembled into a contig. Consensus genome sequences reported here represent from 11.9- to 23.8-fold coverage, depending upon the phage, with quality scores better than Q40 for > 99.3% of the bases (Henn et al., 2010).

The assembled genomes were annotated in a pseudo-automated pipeline as follows. ORF predictions were made using GeneMarkS (Besemer et al., 2001), then manually refined based upon synteny and maximizing ORF size where alternate start sites were present. We next used all predicted ORFs from the 26 T4 phages as blastn queries against the genome sequences to pull out all possible ORFs (e-value cut-off < 1e-5). In this way, we identified a small number of cases (< 1%) where the ORF existed in a genome, but had not been predicted by GeneMarkS or manual annotation. Functional annotation to predicted ORFs were assigned using blastp (e-value cut-off < 1e-3) against the NCBI non-redundant database (as of April 2009) in combination with gene size and synteny information and HMM profiles for T4-GCs (described below) were HHsearched against the PFAM database. Identification of tRNA genes were done using tRNA-Scan-SE (Lowe and Eddy, 1997). Bacterial sigma-70 promoters and terminators were predicted using BPROM (LDF > 2.75, Softberry, Mount Kisco, NY) and TransTermHP (confidence score > 80% with an energy score of < −11 and a tail score of < −6; Kingsford et al., 2007), respectively, using default parameters. As well, we specifically searched for known T4 promoters and cyanobacterial nutrient-related promoters as follows. Early T4 phage promoters are sigma-70 promoters that are predicted from the BPROM analysis described above, while to determine T4 late promoters, the known T4 late promoter sequence 5′-TATAAAT-3′ (Miller, 2003a,b;) was used as a query on an initial blastn search (e-value cut-off < 10), over the entire genomes. The resulting sequences were use in a second blastn search (e-value cut-off < 10) to allow for mismatches and obtain further possible promoters. Then only those present in intergenic regions or 10 bp of overlap in the immediate upstream gene were used. Subsequently, known cyanobacterial Pho and ntcA promoters were identified using consensus sequences for known Pho boxes (5′-CTTAN7CTTA-3′; Su et al., 2007) and using the probabilistic model of ntcA binding sites (Su et al., 2005) that was more specifically adapted for use with marine cyanobacteria (5′-GTA-N8-TAC-3′; Su et al., 2006). In addition to probability scoring cut-offs, all promoters or terminators also were required to be intergenic or within 10 bp of the start/stop of an ORF.

The 12 new cyanophage genome annotations (GU071094-GU071099, GU071101, GU071103, GU071105-GU071106, GU071108, GU075905), and the four previously published cyanophage genome annotations (DQ149023, AJ630128, AY940168, AY939844, FM207411) are available at GenBank, while the 10 non-cyanophage genome annotations are available at http://phage.bioc.tulane.edu. Additionally, all 26 T4-like phage genome GenBank accession numbers are available in Table 1, and all 16 new or updated cyanophage genomes are also available as a single project at the CAMERA database (http://web.camera.calit2.net/cameraweb/gwt/org.jcvi.camera.web.gwt.download.ProjectSamplesPage/ProjectSamplesPage.oa?projectSymbol=CAM_PROJ_BroadPhageGenomes).

Whole-genome sequencing of these phages revealed that three previously published gene sequences derived from PCR products from these phages (Sullivan et al., 2006; 2008;) were incorrect: g20 from Syn33 (gene GI:189397306, protein GI:189397307), g20 from S-SSM7 (gene GI:189397276, protein GI:189397277) and psbA from S-SSM5 (gene GI:95115381, protein GI:95115382). These previous GenBank accessions for these sequences have been corrected with the sequences from the genomes.

Protein clustering and divergent sequence annotation

The method for clustering orthologous genes across the 26 T4-like phage genomes was similar to that described previously (Kettler et al., 2007). Briefly, pairwise orthologous relationships were mapped in all T4-like genomes using reciprocal best blastp hit (e-value ≤ 1e-5) to each other where the sequence alignment length was at least 75% of the protein length of the shorter gene of the two compared. T4 Gene Clusters (T4-GCs) were then built by transitively clustering these orthologues together, where if gene A and B are orthologues and gene B and C are orthologues, then genes A, B and C are clustered into an orthologous group. To find divergent orthologues missed by the initial blast-based approach, we built HMM profiles (Durbin et al., 1998) for the T4-GCs, and then searched singleton T4 genes that were not grouped into any T4-GC against the T4-GC HMM profiles. T4-GC HMM profiles were built by aligning each gene in a T4-GC using muscle version 3.7 (Edgar, 2004) with default parameters and then using hmmbuild from hmmer version 2.3.2 (http://hmmer.janelia.org/) to build the HMM profiles from the resulting alignments. The program hmmsearch also from the hmmer version 2.3.2 was used to search a protein sequence against these in-house T4-GC HMM profiles. Those singletons with significant homology (e-value ≤ 1e-5) to T4-GC HMMs were considered for membership in that T4-GC and manually curated to certify membership. A total of 15 single genes were brought into T4-GCs this way.

A multifasta of all ORFs used in this study is provided as a supplementary file which includes in the fasta header the ORF identifier and genome location, T4-GC assignment and functional annotation (File S2).

Gene presence/absence network analysis

A presence/absence table of all T4-GCs in the 26 phage genomes was constructed and displayed as a network using the spring-embedded layout option Cytoscape 2.5 (Fig. 2) (Cline et al., 2007). This layout option treats the connections (edges) between nodes as springs that repel or attract nodes to each other according to a force function; nodes are positioned to minimize the sum of forces in the network. Nodes in the graph represent the T4-GCs (circles) and the genomes (diamonds), and edges represent the presence of a particular T4-GC in a given genome. Each genome node will therefore have a set of T4-GC nodes connected to it. The resulting network highlights the similarities between genomes based on the presence and absence of gene clusters in each genome.

Virion structural proteomics

Structural proteomic experiments were conducted as described previously (Sullivan et al., 2009). Briefly, the samples were incubated in a denaturing solution of 8 M Urea/1% SDS/100 mM ammonium bicarbonate/10 mM DTT pH 8.5 at 37°C for 1 h. Next, the samples were alkylated for 1 h by the addition of iodoacetamide to a final concentration of 40 mM and then quenched with 2 M DTT. Following the addition of 4× LDS loading buffer (Invitrogen), each sample was centrifuged at 14 000 r.p.m. for 5 min at room temperature, and each sample was fractionated on a NuPAGE 10% Bis-Tris 10 lane gel (Invitrogen) for 2.5 h at 125 volts, 50 mA and 8 W. Gels were shrunk overnight by the addition of 50% ethanol and 7% acetic acid, and then allowed to swell for 1 h by the addition of deionized water. Gels were stained with SimplyBlue Safe Stain (Invitrogen) for 2–4 h, imaged, and sliced horizontally into fragments of equal size based on the molecular weight markers.

In-gel digestion was performed after destaining and rinsing the gel sections with two washes of 50% ethanol and 7% acetic acid, followed by two alternating washes with 50 mM ammonium bicarbonate and acetonitrile. After removal of the last acetonitrile wash, 100 µl of sequencing grade trypsin (Promega) was added to each gel slice at a concentration of 6.6 ng µl−1 in 50 mM ammonium bicarbonate/10% acetonitrile. The gel slices were allowed to swell for 30 min on ice, after which the tubes were incubated at 37°C for 24 h. Peptides were extracted with one wash of 100 µl of 50 mM ammonium bicarbonate/10% acetonitrile and one wash of 100 µl of 50% acetonitrile/0.1% formic acid. The extracts were pooled and frozen at −80°C, lyophilized to dryness and redissolved in 40 µl of 5% acetonitrile, 0.1% formic acid.

Samples were then loaded into a 96-well plate (AbGene) for mass spectrometry analysis on a Thermo Fisher Scientific LTQ-FT. For each run, 10 µl of each reconstituted sample was injected with a Famos Autosampler, and the separation was performed on a 75 mM × 20 cm column packed with C18 Magic media (Michrom Biosciences) running at 250 nl min−1 provided from a Surveyor MS pump with a flow splitter with a gradient of 5–60% water, 0.1% formic acid, acetonitrile 0.1% formic acid over the course of 120 min (150 min total run). Between each set of samples, standards from a mixture of five angiotensin peptides (Michrom Biosciences) were run for 2.5 h to ascertain column performance and observe any potential carryover that might have occurred. The LTQ-FT was run in a top five configuration with one MS 200 K resolution full scan and five MS/MS scans. Dynamic exclusion was set to one with a limit of 180 s with early expiration set to two full scans.

Peptide identifications were made using sequest (ThermoFisher Scientific) through the Bioworks Browser 3.3. The data were searched with a 10 ppm window on the MS precursor with 0.5 Da on the fragment ions with no enzyme specificity. A reverse database strategy (Elias and Gygi, 2007) was employed with a six-frame translation of the genomic sequence reversed and concatenated with the forward sequences supplemented with common contaminates and filtered to obtain a false discovery rate of less than or equal to 1%. Peptides passing the filters were mapped back onto the genome and compared with predicted ORFs.

Acknowledgments

Sequencing of the new phage genomes presented here was supported by the Gordon and Betty Moore Foundation MMI Marine Phage, Virus, and Virome Sequencing Initiative through a grant to M.R.H. M.B.S., K.H.H., M.C., A.S.D., S.E.K., L.R.T., R.F., M.S.O., S.W.C. were supported in part by grants to S.W.C. from the Gordon and Betty Moore Foundation, NSF, DOE-GTL, MIT UROP; J.C.I.-E. was supported by a Fulbright Scholarship, as well as University of Arizona BIO5 and Biosphere 2 funds and NSF (DBI-0850105) to M.B.S.; P.R.W. was supported by NIEHS (1-P50-ES012742) and NSF (OCE-0430724). John Waterbury and Freddy Valois kindly provided three cyanophage isolates (Syn1, Syn19, Syn33), and Andy Tolonen and Anton Post kindly collected Red Sea water used for cyanophage isolations.

We thank Brian Binder and the crew of the R/V Endeavor, Dave Karl and the HOT team, Mike Lomas and the BATS team for the sampling opportunities. We also thank Mandy Joye and Matthew Erickson for nitrogen measurements from EN360 cruise, the HOT team and the HOT-DOGS site for nutrient measurements from the HOT179 samples, Jarl Haggerty for assistance automating aspects of genome annotation. As well, the team at Microbes Online (particularly Keith Keller) allowed pre-publication viewing of our cyanophage genomes to leverage the power of their online comparative genomics tools, while Virginia Rich, Melissa Duhaime, Li Deng, Qinglu Zeng and Simon Labrie provided valuable discussion and comments. We thank the Broad Institute Genome Sequencing Platform for their efforts on genome sequencing. Finally, we thank two reviewers and our editor for excellent comments and suggestions on the manuscript, and the editor and Melissa Duhaime for insight into MazG biology.

Note added in proof

After we completed the analyses of the cyanophage genomes described in this article, another Synechococcus phage genome (S-RSM4) became available (Millard et al., 2009). The S-RSM4 genome appears to be a ‘standard Synechococcus T4 phage’ as inferred from its genome-wide %G+C (41%) and gene content (contains all 38 T4 core genes, all 25 cyano T4 core genes, all 12 nearly cyano T4 core genes, 21 Synechococcus-enriched genes and none of the Prochlorococcus-enriched genes).

Both S-RSM4 (Millard et al., 2009) and P-RSM5 (this study) were isolated from the oligotrophic Red Sea, and both contain a notable phosphate-related feature. Specifically, P-RSM5, which contains pstS, was isolated in September, after months of summer stratification (Lindell and Post, 1995; Fuller et al., 2005), which would dramatically reduce nutrient concentrations in surface waters. In contrast, S-RSM4, which lacks pstS, was isolated in April before summer stratification (Lindell and Post, 1995; Fuller et al., 2005), likely resulting in less stressful nutrient limitation. In fact, cyanobacterial pstS expression from these same waters was minimal (Fuller et al., 2005), consistent with a lack of phosphate stress in these waters. We hypothesize therefore that the presence/absence of pstS in these two phages also reflects the nutrient status of the waters from which they were collected.

Supporting information

Additional Supporting Information may be found in the online version of this article:

Fig. S1. Cyanophage genome size plotted as a function of the number of predicted ORFs where original host genera are designated by colour.

Fig. S2. The genome location of four hierarchical ‘core’ gene sets plotted for 26 T4 phage genomes. Lines connect function-based orthologues across genomes, and are coloured as per legend.

Fig. S3. Multiple sequence alignment of the T4 phage gp51 baseplate hub catalyst protein from 26 T4 phage genomes. The cyanophage and marine vibriophage copies of gp51 are significantly reduced, missing the first ~200 amino acids relative to the non-cyano non-marine T4 phage copies (the first 140 amino acids of the alignment are not shown). In spite of this size difference, there is marked similarity in the C-terminal region of the protein shown in the alignment.

Fig. S4. Maximum likelihood tree of the pyrophosphatase MazG protein. The tree was constructed from 271 aligned amino acids, using PhyML and the JTT model of substitution with gamma-distributed rates empirically estimated from the data. The accession numbers for the sequences used in this analysis are available upon request. Numbers at the nodes represent bootstrap values for 1000 replicates.

Fig. S5. Weblogo (http://weblogo.berkeley.edu/) diagrams of the various bioinformatically predicted promoters in the cyanophage genomes.

Fig. S6. Multiple sequence alignment of the cyanophageencoded Zwf proteins identified in varying degrees of preservation across eight cyanophages. While the sequence conservation is minimal for the three highly degraded copies, their position in the genomes is conserved and remnants of sequence similarity remain along the protein.

Fig. S7. Alignment of the endonucleases in T4-GCs 228 and 282.

A. Putative homing endonucleases (T4-GC282) where only the P-SSM2 copy has conserved catalytic residues as compared with the experimentally characterized homing endonuclease present in S-PM2 (S-PM2p177, Zeng et al., 2009). The remaining copies appear to have lost these residues and are likely non-functional, yet are all located at a conserved region suggesting a single evolutionary event of insertion at the 3′-end of gp17 (see upper panel for genome sequence details).

B. Possible endonucleases (T4-GC228) which lack the conserved residues in (A) but nonetheless are highly conserved and proximal to the carbon metabolism genes, suggesting that they may be responsible for genetic shuffling in this region.

Fig. S8. Close-up genome representation of the mobile carbon metabolic gene cluster from cyanophage genomes. Genomic features are as described in Fig. 1, and genome location orientation is as described for Fig. 5.

Fig. S9. Close-up genome representation of the mobile hypothetical genes cluster from cyanophage genomes. Genomic features are as described in Fig. 1, and genome location orientation is as described for Fig. 5.

Table S1. Detailed features of the T4-like ocean cyanophage isolates.

Table S2. T4-like phage core genes determined from 16 cyanophages and 10 non-cyanophages. Numbers listed for each phage represent the size of the genes (bp), with multiple copies separated by a ‘|’. Some T4-GCs were pooled to create a single functional category based upon annotation and genome synteny.

Table S3. Non-cyano T4-like ‘core’ beyond the T4-core. Numbers listed for each phage are as in Table S2.

Table S4. Cyano T4-like core genes. Numbers listed for each phage are as in Table S2.

Table S5. Proteins that are unique to either P-HM1 or P-HM2 phage genome in pairwise comparison of these two co-isolated phages.

Table S6. Synechococcus phage-enriched proteins. Numbers listed for each phage are as in Table S2.

Table S7. Prochlorococcus phage-enriched proteins. Numbers listed for each phage are as in Table S2.

Table S8. Summary of cyano T4 proteomics experiments. Comparative proteomics = experimentally determined protein content in purified virus particles to determine the structural proteins in three sequenced T4-like virus genomes. A ‘Y’ means the protein was detected, ‘–’ means the protein is annotated in the genome but no peptides were detected, ‘NP’ means the protein is not present in the genome, ‘counts’ are the number of peptide fragments detected per protein, ‘copy # in T4’ refers to the biochemically and ultrastructurally determined copy number of proteins in the coliphage T4 particle. Ten of these proteins, in italics, have similar distributions among nine cyanophages and may be functionally linked.

File S1. The spreadsheet used to generate the overview of the cyanophage genome annotations that are presented in Fig. 1.

File S2. Multifasta of all ORFs examined in this study including gene identifiers and genome location, T4-GC assignment and functional annotation.

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

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