The oceanic cyanobacteria Prochlorococcus are globally important, ecologically diverse primary producers. It is thought that their viruses (phages) mediate population sizes and affect the evolutionary trajectories of their hosts. Here we present an analysis of genomes from three Prochlorococcus phages: a podovirus and two myoviruses. The morphology, overall genome features, and gene content of these phages suggest that they are quite similar to T7-like (P-SSP7) and T4-like (P-SSM2 and P-SSM4) phages. Using the existing phage taxonomic framework as a guideline, we examined genome sequences to establish “core” genes for each phage group. We found the podovirus contained 15 of 26 core T7-like genes and the two myoviruses contained 43 and 42 of 75 core T4-like genes. In addition to these core genes, each genome contains a significant number of “cyanobacterial” genes, i.e., genes with significant best BLAST hits to genes found in cyanobacteria. Some of these, we speculate, represent “signature” cyanophage genes. For example, all three phage genomes contain photosynthetic genes (psbA, hliP) that are thought to help maintain host photosynthetic activity during infection, as well as an aldolase family gene (talC) that could facilitate alternative routes of carbon metabolism during infection. The podovirus genome also contains an integrase gene (int) and other features that suggest it is capable of integrating into its host. If indeed it is, this would be unprecedented among cultured T7-like phages or marine cyanophages and would have significant evolutionary and ecological implications for phage and host. Further, both myoviruses contain phosphate-inducible genes (phoH and pstS) that are likely to be important for phage and host responses to phosphate stress, a commonly limiting nutrient in marine systems. Thus, these marine cyanophages appear to be variations of two well-known phages—T7 and T4—but contain genes that, if functional, reflect adaptations for infection of photosynthetic hosts in low-nutrient oceanic environments.
Citation: Sullivan MB, Coleman ML, Weigele P, Rohwer F, Chisholm SW (2005) Three Prochlorococcus Cyanophage Genomes: Signature Features and Ecological Interpretations. PLoS Biol 3(5): e144. doi:10.1371/journal.pbio.0030144
Academic Editor: Nancy A. Moran, University of Arizona, United States of America
Received: June 19, 2004; Accepted: February 23, 2005; Published: April 19, 2005
Copyright: © 2005 Sullivan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: DNAP, deoxyribonucleic acid polymerase; HL, high-light-adapted; HN, hemagglutinin neuraminidase; LL, low-light-adapted; LPS, lipopolysaccharide; ORF, open reading frame; PSII, photosystem II; RNAP, ribonucleic acid polymerase; RNR, ribonucleotide reductase; ssRNA, single-stranded ribonucleic acid
Prochlorococcus is the numerically dominant primary producer in the temperate and tropical surface oceans . These cyanobacteria are the smallest known photosynthetic organisms (less than a micron in diameter), yet are significant contributors to global photosynthesis [2,3] because they occur in high abundance (as many as 105 cells/ml) throughout much of the world's oceans. They are adapted to living in low-nutrient oceanic regions  and are physiologically and genetically diverse with at least two “ecotypes” that have distinctive light physiology , nitrogen  and phosphorus (L. R. Moore, personal communication) utilization, and copper  and virus (phage)  sensitivity. Cyanobacterial phages are also abundant in these environments [8,9,10,11,12] and have a small, but significant, role in mediating population sizes [9,10]. Further, cyanophages likely play a role in maintaining the extensive microdiversity within marine cyanobacteria [9,10] through keeping “competitive dominants” (sensu ) in check, as well as by carrying photosynthetic “host” genes [14,15,16] and mediating horizontal transfer of genetic material between cyanobacterial hosts .
Although there are more than 430 completed double-stranded DNA phage genomes in GenBank, only nine phages with published genomes infect marine hosts (cyanophage P60; vibriophages VpV262, KVP40, VP16T, VP16C, K139, and VHML; roseophage SIO1; and Pseudoalteromonas phage PM2). Of those nine, only one infects cyanobacteria (cyanophage P60, a member of the Podoviridae). P60 was isolated from estuarine waters using Synechococcus WH7803 as a host and appears most closely related to the T7-like phages . It contains 11 T7-like phage genes and has no genes with homology to non-T7-like phages. However, it lacks the conserved T7-like genome architecture. Thus, P60 is thought to be only distantly related to the T7-like phages, but still part of a T7 supergroup  proposed by Hardies et al. . The T7 supergroup also contains two other marine phages (roseophage SIO1 and vibriophage VpV262) that show similarity to some (three) T7-like genes. However, these phages lack many T7-like genes including the hallmark T7-like RNA polymerase (RNAP) gene . Thus, there is clearly a gradient in relatedness among the T7 supergroup, with these newer marine phage genomes at the distant, less-similar end of the group.
Marine phages are subject to different selection pressures (e.g., dispersal strategies, encounter rates, limiting nutrients, and environmental variability) than their relatively well-studied terrestrial counterparts. Thus, beyond informing phage taxonomy, the analysis of their genomes should unveil “signatures” of these selective agents. For example, genomic analysis of two marine phages, roseophage SIO1  and vibriophage KVP40 , has revealed phosphate-inducible genes. It is thought that these genes play an important regulatory role in the phosphorus-limited waters from which they were isolated. Similarly, some Prochlorococcus and Synechococcus phages (including the three cyanophage genomes presented here) contain core photosynthetic genes that are full-length, conserved, and cyanobacterial in origin [14,15,16]. They are hypothesized to be important for maintaining active photosynthetic reaction centers—and hence the flow of energy—during phage infection [14,15,16].
With a large collection of phages from which to choose , we used host range and phage morphology to select strains for sequencing. The selected podovirus (P-SSP7) is very host-specific, infecting a single high-light-adapted (HL) Prochlorococcus strain of 21 Prochlorococcus and Synechococcus strains tested. In contrast, the two myoviruses that were selected cross-infect between Prochlorococcus (but not Synechococcus) hosts: P-SSM2 can infect three low-light-adapted (LL) host strains, and P-SSM4 can infect two HL and two LL hosts . We had no prior knowledge of the gene content of these phages; thus, with regard to their genomes, these phages were selected randomly.
As mentioned earlier, our first survey of these phage genomes led to the surprising discovery of photosynthetic genes in all three Prochlorococcus phages , similar to the findings in Synechococcus cyanophages [15,16,22]. In this report, we present a more thorough analysis of these three cyanophage genomes, which, we argue, appear to be T7-like (P-SSP7) and T4-like (P-SSM2 and P-SSM4) phages.
General Features of the Podovirus P-SSP7
P-SSP7 is morphologically similar to the Podoviridae (tails are short and noncontractile; Figure 1A). It also includes a rectangular region of electron transparency (Figure 1A) that is similar to the gp14/gp15/gp16 core located at the unique portal vertex found in coliphage T7 . Its genome contains 44,970 bp (54 open reading frames [ORFs]; 38.7% G+C content; Figure 1B), including a T7-like RNAP and a phage-related integrase gene (a more detailed analysis of this feature is discussed later). Thus, the P-SSP7 genome is more T7-like or P22-like than ϕ29-like among the Podoviridae (Table 1). Thirty-five percent of the translated ORFs have best hits to phage proteins; nearly all of these are T7-like, whereas none are P22-like (Figure 1C). Together, these data suggest that P-SSP7 is most closely related to the T7-like phages. Surprisingly, 11% of the translated ORFs have best hits to bacterial proteins, with well over half of these being cyanobacterial (see later discussion). Roughly half (54%) of the translated ORFs could not be assigned a function (Figure 1C).
(A) Electron micrograph of negative-stained podovirus P-SSP7. Note the distinct T7-like capsid and tail structure. Scale bar indicates 100 nm.
(B) Genome arrangement of Prochlorococcus podovirus P-SSP7. The ORFs are sequentially numbered within the boxes, and gene names are designated above the boxes. Gene designations use T7 nomenclature for T7-like genes  or microbial nomenclature for non-phage genes. Class I, II, and III genes refer to those in T7  that belong to gene regions primarily involved in host transcription of phage genes (class I), DNA replication (class II), and the formation of the virion structure (class III). The ORFs are designated by boxes, and in this genome, all ORFs are oriented in the same direction. Although the phage genome is one molecule of DNA, the representation is broken to fit on a single page. Note that the P-SSP7 genome is most similar to genomes of the T7-like phages.
(C) Taxonomy of best BLASTp hits for P-SSP7. Each predicted coding sequence from the phage genomes was used as a query against the nonredundant database to identify the taxon of the best hit (details in Materials and Methods). Blue slices indicate phage hits, while yellow slices indicate cellular hits.
(D) Diagrammatic representation of the genomic regions surrounding a putative phage and host integration site. This site consists of a 42-bp exact match between the podovirus P-SSP7 and its host Prochlorococcus MED4 located directly downstream of the phage integrase gene and the noncoding strand of a host tRNA gene.
An examination of the genomes of coliphage T7 and its closest coliphage relatives (T3, gh-1, ΦYe03–12, ΦA1122) revealed that they share 26 genes, which we define as core genes (Table 2). P-SSP7 has 15 of these 26 core genes and an additional gene (0.7) that is common, but not universal, among T7-like phages (Table 2). Further, only two non-T7-like phage genes were identified in this genome: hypothetical gene 12 from a Burkholderia phage, Bcep1, of the Myoviridae family, and the phage-related integrase gene discussed later. Strikingly, the T7-like genes found in P-SSP7 are arranged in exactly the same order as in other T7-like phages (Figure 1B). The gene content and genome architecture of P-SSP7 contrast with those from the three other sequenced marine podovirus genomes in the T7 supergroup [17,19,20]. SIO1 and VpV262 lack the hallmark T7-like RNAP and contain only three T7-like core genes (Table 2), whereas cyanophage P60 contains 11 core genes (Table 2) but clearly lacks the conserved T7-like genome architecture .
The putative functions of the 16 T7-like genes in P-SSP7 would allow for the majority of host interactions and phage production as follows (T7-like gene designations are shown in parentheses): shutdown of host transcription (0.7), phage gene transcription (1), degradation of host DNA (3, 6), DNA replication (1, 2.5, 4, 5), formation of a channel across the cell envelope via an extensible tail (15, 16) , DNA packaging (19), and virion formation (8, 9, 10, 11, 12, 17). We found two stretches of DNA (frame +1 from nucleotides 9994–10525, then frame +3 from nucleotides 10485–11759) with matches to T7 gp5 (DNA polymerase [DNAP]): one corresponding to the 3′-exonuclease and one to the polymerase (nucleotidyl transferase) segments of the T7 enzyme. This region may encode a split variant of T7 family DNAP (V. Petrov and J. Karam, personal communication), an arrangement that has been shown to be functional in archaea  and some T4-like phages (V. Petrov and J. Karam, personal communication).
As described earlier, we identified only 15 of the 26 core T7-like genes in P-SSP7. What are the functions of the absent gene set? It includes genes that in T7 are involved in ligation of DNA fragments (1.3), inhibition of host RNAP (2), interactions that are specific to the host cell envelope during virion formation (6.7, 13, 14), lysis events (3.5, 17.5), small-subunit terminase activity (18), and unknown functions (5.7, 6.5, 18.5) . These same genes are also absent in the marine podovirus genomes in the T7 supergroup (cyanophage P60, vibriophage VpV262, and roseophage SIO1; Table 3). If we assume a conserved genomic architecture among the T7-like phages, we find hypothetical ORFs in homologous positions to these T7 core genes in P-SSP7 (Figure 1B) that may fulfill these core (e.g., 5.7, 6.5, 6.7, 13, 14, 17.5, 18, 18.5) and common (e.g., antirestriction gene 0.3) T7-like gene functions. Alternatively, their functions may be unnecessary for this phage.
The P-SSP7 genome assembled as a circular chromosome, suggesting that it is circularly permuted, thus lacking the terminal repeats that are common among T7-like phages . Confirmation of this hypothesis would require direct sequencing of the genome ends (I. Molineux, personal communication), which was not possible in this study because of the difficulty of obtaining significant quantities of purified DNA .
Hypothesized Lysogeny in P-SSP7
One of the more interesting discoveries in the podovirus genome is the presence of a tyrosine site-specific recombinase (int) gene (Figure 1B), which in temperate phages encodes a protein that enables the phage to integrate its genome into the host genome . T7 is a classically lytic phage, and there has been only one other report of int genes in a T7-like phage: in an integrated prophage in the Pseudomonas putida KT2440 genome . The P-SSP7 int contains conserved amino acid motifs previously identified for site-specific recombinases (Arg-His-Arg-Tyr, Leu-Leu-Gly-His, and Gly-Thr ) suggesting it is functional. Downstream of int, we find a 42-bp sequence that is identical to part of the noncoding strand of the leucine tRNA gene in the phage's host genome (Prochlorococcus MED4) (Figure 1D). tRNA genes are a common integration site for phages and other mobile elements , adding support to the hypothesis that this int gene is functional.
P-SSP7 was isolated from surface ocean waters at the end of summer stratification , when nutrients are extremely limiting. We have hypothesized  that the integrating phase of the temperate-phage life cycle may be selected for under these conditions; thus, finding the int gene in this particular phage is consistent with this hypothesis. None of the complete genome sequences of cyanobacterial hosts reported to date have intact prophages [4,32,33,34]. Moreover, temperate phages have not been induced from unicellular freshwater or marine cyanobacterial cultures [9,35,36]. Although some field experiments suggest that temperate cyanophages can be induced from Synechococcus [37,38], prophage integration has not been demonstrated. Thus, experimental validation that P-SSP7 is capable of integration would confirm indirect evidence and establish a valuable experimental system.
General Features of the Myoviruses P-SSM2 and P-SSM4
P-SSM2 and P-SSM4 are morphologically similar to the Myoviridae (tails are long and contractile; Figure 2). Both have an isometric head, contractile tail, baseplate, and tail fiber structures (Figure 2) that are most consistent (but see isometric head discussion later) with the morphological characteristics of the T4-like phages . Their genomes also have general characteristics that are fully consistent with T4-like status within the Myoviridae (Table 3). Both genomes are relatively large: P-SSM2 has 252,401 bp (327 ORFs; 35.5% G+C content; Figure 3) and P-SSM4 has 178,249 bp (198 ORFs; 36.7% G+C content; Figure 4). An apparent strand bias is noteworthy because only 12 (of 327) and six (of 198) ORFs are predicted on the minus strand in the P-SSM2 and P-SSM4 genomes, respectively. Similar to the lytic T4-like phages, integrase genes were absent. Both genomes assembled and closed, suggesting the circularly permuted chromosome common among the T4-like phages (Table 3). A large portion of the nonhypothetical ORFs have best hits to phage proteins (14% and 21%, respectively) and bacterial proteins (26% and 21%, respectively; Figure 5). The phage hits were most similar to T4-like phage proteins, and about half of the bacterial ORFs were most similar to those from cyanobacteria. As with P-SSP7, most of the translated ORFs from P-SSM2 and P-SSM4 could not be assigned a function (60% and 58%, respectively). The majority of the differences between these two phages are due to the presence of two large clusters of genes (24 total) in P-SSM2 (see Figure 3) that are absent from P-SSM4. These clusters contain many sugar epimerase, transferase, and synthase genes that we hypothesize to be involved in lipopolysaccharide (LPS) biosynthesis. The large genome size, collective gene complement, and morphology suggest both P-SSM2 and P-SSM4 are most closely related to T4-like phages.
Myovirus P-SSM2 with (A) non-contracted tail and (B) contracted tail, and myovirus P-SSM4 with (C) contracted tail and (D) non-contracted tail. Note the T4-like capsid, baseplate, and tail structure in both myoviruses. Scale bars indicate 100 nm.
Gene names are designated above the box representing the ORF where genes were identified; descriptions of genes are in Table 4. The ORFs located above the centering line are on the forward DNA strand, whereas those below the line are on the reverse strand. Although the genome is one molecule, the representation is broken to fit the page. Colors indicate the putative role for the identified genes as inferred from T4 phage. Gene designations use T4 nomenclature for T4-like genes  or microbial nomenclature for non-phage genes.
Gene nomenclature is as in Figure 3.
Each predicted coding sequence from both phage genomes was used as a query against the nonredundant database to identify the taxon of the best hit (details in Materials and Methods). Blue slices indicate phage hits, while yellow slices indicate cellular hits.
The six sequenced T4-like phage genomes (T4, RB69, RB49, 44RR2.8t, KVP40, and Aeh1; available as of 15 May 2004 at http://phage.bioc.tulane.edu/) share 75 genes (Table 4), which suggests a core gene complement required for T4-like phage infection. This core contains 18 genes involved in DNA replication, recombination, and repair, seven regulatory genes, ten nucleotide metabolism genes, 34 virion structure and assembly genes, and six genes involved in chaperonin, lysis exclusion, and other activities. Again, despite cyanobacterial hosts being quite divergent from the hosts of these other T4-like phages, our myoviruses contained 43 and 42 of the 75 T4-like core genes, as well as other noncore T4-like genes in each phage (uvsX, uvsY, and possibly dam, 42, and hoc in P-SSM2; uvsX, uvsY, and possibly dam, 42, and denV in P-SSM4; Table 4). Furthermore, aside from the low-complexity tail fiber related genes (see “Tail-Fiber-Related Genes in the Myoviruses” below), we found no genes with sequence similarity to any phage type other than T4-like phages.
Slightly fewer than half of the core T4-like genes were absent in both myoviruses P-SSM2 and P-SSM4. P-SSM2 and P-SSM4 lack the genes required for anaerobic nucleotide biosynthesis (nrdD, nrdG, and nrdH), which is perhaps not surprising because these phages were isolated from the well-mixed, oxygenated surface oceans. Both myoviruses also lack homologs to the prohead core-encoding genes (67 and68) of the T4-like phages (Table 4). However, we note that the capsids of both Prochlorococcus myoviruses are isometric (see Figure 2), rather than prolate as is often observed for other T4-like phage capsids . In T4, mutations in the prohead core proteins (gp67 and gp68) are known to cause a capsid structural defect whereby isometric heads are observed [40,41,42]. Thus, functional homologs of prohead core proteins may not be required for the formation of isometric heads in these Prochlorococcus myoviruses.
Other T4-like phage gene functions may be represented by divergent homologs filling the T4-like phage role in these cyanomyophages. P-SSM2 and P-SSM4 lack core T4-like chaperonin genes (rnlA, 31, and 57A; Table 4) and nucleotide metabolism genes (T4-like pyrimidine biosynthesis: cd, frd, 1, and tk; Table 4). However, both P-SSM2 and P-SSM4 contain non-T4-like hsp20-family chaperonins, as well as a non-T4-like gene (mazG) that in bacteria is involved in degradation of DNA (Table 5) [43,44]. Furthermore, P-SSM2 contains ORFs with high sequence similarity to host-encoded homologs of five genes involved in pyrimidine (pyrE) and purine (purH, purL, purM, and purN) biosynthesis (Table 5). These non-T4-like genes might compensate for T4-like nucleotide metabolism and/or chaperone genes that are absent. Despite the structural similarities between our myophages (see Figure 2) and the T4-like phages, some core virion structural genes (e.g., head genes, 2, 24, 67, 68, and inh; tail/tail fiber genes, 10, 11, 12, 34, 35, 37, and wac) have yet to be identified in these myophage genomes (see Table 4). Similarly, genes involved in transcriptional regulation (dsbA, rnlA, and pseT), lysis events (rIIa and rIIb), and replication, recombination, and repair (DNA ligase, 30; topoisomerases, 39 and 52; RNase H, rnh; and an exonuclease, dexA) also have yet to be identified.
Tail-Fiber-Related Genes in the Myoviruses
Sequence analysis of phage tail fiber genes has revealed extensive swapping of gene fragments between loci [45,46]. Such exchanges yield phages with altered host ranges . Although this mosaic gene construction makes computational identification of tail fiber genes by sequence homology difficult, we have attempted to do so in the two Prochlorocococcus T4-like genomes. The analysis is motivated by the belief that understanding mechanisms of attachment and host range is critical for developing assays for studying phage–host interactions in wild populations—one of the underlying motivations of our work with this system.
We identified ORFs as potential tail fiber genes by a three-tiered bioinformatics approach using sequence similarity, repeat analysis, and paralogy (details in Materials and Methods). First, sequence similarity to known tail fiber genes was used to add ORFs to the pool of possible tail fiber genes (Figure 6). Seven ORFs in P-SSM2 and three ORFs in P-SSM4 had similarity to known tail fiber genes. In T4, the long tail fiber of T4 is composed of four protein subunits including a proximal-end subunit (gp34) anchoring the fiber to the phage baseplate and a distal-end subunit (gp37) responsible for host recognition and attachment (reviewed in ). Thus P-SSM2 and P-SSM4 ORFs contained regions similar to T4-like phage distal tail fiber genes (gp37; P-SSM2 orf023, orf033, orf295, and orf298; P-SSM4 orf087) and proximal tail fiber genes (gp34; P-SSM2 orf295 and orf315; P-SSM4 orf026 and orf087). Further, two P-SSM2 ORFs (orf034 and orf315) and a P-SSM4 ORF (orf027) are similar to other known tail fiber genes, albeit with low sequence similarity, and for only a small portion of the ORF.
Red bars indicate P-SSM2 ORFs (labeled as M2); blue bars indicate P-SSM4 ORFs (labeled as M4). Due to space constraints, P-SSM2 orf67 and P-SSM4 orf10 are broken as indicated.
Second, ORFs containing repeat sequences were added to the pool of possible tail fiber genes. Both simple (amino acid triplets) and complex (longer amino acid motifs) repeats are associated with phage tail fiber genes [49,50]. Simple repeats are found in two P-SSM2 ORFs (orf23 and orf28; Figure 6), with nearly 49% of orf028 encoding the simple triplet repeat Gly-X-Y (where X and Y are often proline, serine, or threonine). Proteins with extended runs of these collagen-like amino acid motifs are thought to fold into trimeric coiled coils, consistent with a tail-fiber-like structure . Complex repeat motifs of 15 to 51 amino acids in length are found in P-SSM2 (orf111 and orf298) and P-SSM4 (orf087; Figure 6). Some of these motifs are similar to those found in the long distal tail fiber (gp37) and short tail fiber (gp12) genes in T4, where they encode tandem, beta-strand-rich, supersecondary structural elements that are correlated with the beaded or knobbed shaft structure of these tail fibers [49,51].
Third, possible tail-fiber-encoding ORFs were identified through paralogy to other Prochlorococcus phage tail fiber ORFs already identified (Figure 6). This approach follows the observation of homology between three T4 tail fiber genes (gp12, gp34, and gp37) , which are thought to have arisen via gene duplication events . These analyses added four ORFs to the pool of possible tail fiber genes for P-SSM2 (orf021, orf022, orf293, and orf301) and two for P-SSM4 (orf080 and orf082).
After identification of a pool of putative tail fiber genes, we used sequence similarity to known tail fiber and/or baseplate genes as a guideline to annotate ORFs according to the known T4 phage architecture. Three tail-fiber-like ORFs of P-SSM2 (orf111, orf295, and orf298) have N-terminal domains that are similar to T4 baseplate proteins (Figure 6). In T4, the N-terminus of the proximal long tail fiber (gp34) is bound to the baseplate via the baseplate protein gp9 and possibly gp10 [53,54,55]. The N-terminus of P-SSM2 orf298 is similar to the P-SSM4 orf081 (a gp9 homolog by sequence), suggesting that P-SSM2 orf298 could be analogous to a T4 proximal long tail fiber subunit (gp34), albeit fused to the baseplate socket in P-SSM2. Although such a fused protein does not appear to exist for the other myophage, P-SSM4, the adjacent reading frame to orf081 encodes a possible tail fiber ORF with significant similarity to C-terminal stretches of P-SSM2 orf298. Thus, it appears that P-SSM4 orf081 and orf082 are orthologous with the PSSM2 orf298 N- and C-terminal regions, respectively. P-SSM2 orf295 also appears to be a tail fiber fused to a baseplate protein, gp10, which, in T4, may also play a role in binding tail fiber proteins, although this role is less clear. Similarly, the very large homologous genes (>15,000 nt) P-SSM2 orf113 and P-SSM4 orf080 appear fused to baseplate wedge initiator (gp7) homologs, which are not known to bind tail fiber in T4 . Regardless of their precise assignments relative to T4 tail fiber genes, these putative fusions likely encode tail fiber subunits that bind directly to the baseplate through incorporation of their N-termini into the baseplate complex. Assuming that the long tail fibers of P-SSM2 or P-SSM4 are composed of more than one kind of protein subunit, as in T4 , we hypothesize that these baseplate-domain-containing tail fibers are unlikely to determine host specificity, but rather are analogous to the proximal long tail fiber (gp34) or short tail fiber (gp12) of T4.
Thus we identify a pool of 12 and five putative tail-fiber-related genes (awaiting experimental confirmation) in the P-SSM2 and P-SSM4 genomes, respectively. Some are quite large relative to those in T4, whereas others appear fused to baseplate genes, which has not been observed for the T4-like phages.
Metabolic Genes Uncommon among Phages
All three cyanophages contained genes that are not commonly found in phages. We have selected the following cyanobacterial genes for discussion because we hypothesize that they could play defining functional roles in the marine cyanophage–cyanobacterium phage–host system.
Photosynthesis-related genes in cyanophages.
We previously reported photosynthesis-related genes (psbA and hli) in all three of these Prochlorococcus phages, as well as other photosynthesis genes (petE, petF, and psbD) in one of the two Prochlorococcus myovirus genomes . In addition, genomic analyses have revealed that P-SSM2 contains pebA and ho1, whereas P-SSM4 contains pcyA and speD (see Table 5). In cyanobacteria these genes are involved in phycobilin biosynthesis (ho1, pebA, and pcyA) [56,57] and polyamine biosynthesis (speD). Although the phycobilin biosynthesis genes are found in Prochlorococcus [4,34], their function is unclear because Prochlorococcus does not have the intact phycobilisomes characteristic of most cyanobacteria. These genes are thought to be a remnant of the evolutionary reduction of the phycobilisome-based antenna to a chlorophyll-b-based antenna [4,58,59,60]. Although low levels of phycoerythrin occur in some LL Prochlorococcus strains , they have, as yet, no known function in the host.
The polyamine biosynthesis gene speD found in the phage has a homolog in all of the marine cyanobacteria with complete genome sequences. Although its function has not been confirmed in these organisms, SpeD is known to catalyze the terminal step in polyamine synthesis in other prokaryotes, and polyamines affect the structure and oxygen evolution rate of the photosystem II (PSII) reaction center in higher plants . Therefore, SpeD, if expressed, may play a role in maintaining the host PSII reaction center during phage infection.
Nucleotide metabolism genes.
The podovirus P-SSP7 contains an ORF (orf20) with a putative ribonucleotide reductase (RNR) domain (see Table 5). In prokaryotes and T4-like phages, RNRs provide the building blocks for DNA synthesis through catalyzing a thioredoxin-mediated reduction of diphosphates (e.g., rNDP → dNDP) during nucleotide metabolism . Among T7-like genomes, these domains have been observed only in marine phages (see Table 5) including cyanophage P60 and roseophage SIO1 [17,20]. An examination of the two genes (nrdA and nrdB) in P60 that contain homology to RNRs suggests that they represent a split RNR (as described earlier for DNAP): nrdA is similar to the 5′-end and nrdB is similar to the 3′-end of cyanobacterial class II RNRs (data not shown). When analyzed for the presence of a class II RNR diagnostic motif , all three marine T7-like phage putative RNRs were found to contain homology to this motif (seven of nine residues in SIO1, P-SSP7; eight of nine residues in P60; as compared to eight of nine residues in the marine cyanobacteria) (Figure S1). Furthermore, the putative RNRs are located in the genomes at the distal end of a region homologous to the nucleotide metabolism region in T7 . It is plausible that T7-like phage infection in phosphorus-limited environments requires extra nucleotide-scavenging genes.
Both Prochlorococcus myoviruses contain the alpha and beta RNR subunits that are found in all known T4-like phages (see Table 4). The genes have closer sequence homology to those in T4-like phages than cyanobacterial hosts (Figure S2). Interestingly, our myoviruses also contain a noncyanobacterial cobS gene, which has never been found in phages. This gene encodes a protein that catalyzes the final step in cobalamin (vitamin B12) biosynthesis in bacteria [66,67], and cobalamin is an RNR cofactor during nucleotide metabolism in cyanobacteria . Both physiological assays [69,70] and genomic evidence [4,34] indicate that Prochlorococcus synthesizes its own cobalamin. It is tempting to speculate that the phage cobS gene serves to boost cobalamin production in the host during infection, thus improving the activity of RNRs. However, these phage RNRs clearly contain the α2 and β2 subunits (typical of class I RNRs) and lack the class II motif described earlier. Thus, if the phage cobS does increase cobalamin production and if this production increase is important, then either the phage class I RNRs are cobalamin dependent (which is unprecedented) or cobalamin must be useful for some other process.
Carbon metabolism genes.
In cyanobacteria, the pentose phosphate pathway oxidizes glucose to produce NADPH for biosynthetic reactions (oxidative branch) and ribulose-5-phosphate for nucleotides and amino acids (non-oxidative branch). This pathway (both branches) is particularly important in cyanobacteria for metabolizing the products of photosynthesis during dark metabolism . Long ago, it was hypothesized that cyanophages utilize this pathway as a source of energy and carbon when the host is not photosynthesizing . Interestingly, genomic sequencing has recently revealed that Synechococcus cyanophage S-RSM2  and the Prochlorococcus cyanophages P-SSM2 and P-SSM4  contain a transaldolase gene (talC). In Escherichia coli, transaldolase is a key enzyme in the non-oxidative branch of the pentose phosphate pathway . It has been suggested that the product of the phage talC gene may facilitate phage access to stored carbon pools during the dark period .
Recent work in E. coli has revealed two genes (mipB/fsa and talC) that are divergent from the bona fide transaldolases (talA and talB) , but encode a structurally similar enzyme . Members of this new subfamily (MipB/TalC) of aldolases, which have a striking sequence similarity to each other, can have distinctly different functions, acting either as a transaldolase or fructose-6-phosphate aldolase, but not both . All three of the genes previously reported as “transaldolase” genes in cyanophages [14,16], as well as an ORF in the podovirus P-SSP7, are most similar to these MipB/TalC aldolase genes (see Table 5; Figure S3). The translated cyanophage genes contain 26 (P-SSM2), 28 (P-SSP7 and S-RSM2), and 29 (P-SSM4) of 32 diagnostic (as designated by Thorell et al. ) amino acid residues (Figure S4). In the active site of this enzyme, as inferred from the crystal structure of E. coli fructose-6-phosphate aldolase, eight of 14 residues are not conserved between the MipB/TalC subfamily, varying depending on enzyme specificity (fructose-6-phosphate aldolase versus transaldolase) . When aligned with MipB/TalC members of known substrate specificity, the cyanophage putative active site residues match all eight of those enzyme sequences with transaldolase activity (Figure S4). Thus, it appears that each of the four cyanophage talC genes encodes an enzyme with transaldolase activity. If functional, these genes are likely to be important for metabolizing carbon substrates—which is central to biosynthesis and energy production—during phage infection of cyanobacterial hosts.
Phosphate stress genes in the myoviruses.
Phosphorus is a scarce resource in the oligotrophic oceans [76,77]. It is often growth limiting for cyanobacteria  and is required in significant amounts for phage replication. Thus it is perhaps not surprising that the phosphate-inducible phoH gene, which has been found in two marine phage genomes [20,21], is also found in both Prochlorococcus myoviruses (see Table 5; see Figures 3 and 4). Although the phoH gene is found widely distributed among both eubacteria and archaea , including all cyanobacteria, and is known to be induced under phosphate stress in E. coli , its function has not been experimentally determined. Bioinformatic analyses suggest that these phoH genes are part 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) .
Both P-SSM2 and P-SSM4 also contain a phosphate-inducible pstS gene—which is also widespread among the archaea and eubacteria, including all known cyanobacteria—that has not been reported in phages. In bacteria, the pstS gene encodes a periplasmic phosphate-binding protein involved in phosphate uptake . If expressed by the phage, it might serve to enhance phosphorus acquisition during infection of phosphate-stressed cells.
LPS biosynthesis genes in P-SSM2.
The myovirus P-SSM2 contains 24 LPS genes that form two major clusters in the genome (see Figure 3). Reports of phage-encoded LPS genes have previously been limited to temperate phages . Such temperate phage LPS genes are thought to be used during infection and establishment of the prophage state to alter the cell-surface composition of the host, preventing other phages from attaching to the host cell. Although T4-like phages are commonly thought of as lytic phages, the lytic process can be stalled upon infection (sometimes termed “pseudolysogeny”) during suboptimal host growth . If this phenomenon occurs in marine phages, as has been suggested [22,84,85], then a phage-encoded LPS gene cluster, even in a lytic phage, might maintain a similar functional role.
Signature genes for oceanic cyanophages?
Although data are too limited to be conclusive (Table 6), some of the host genes that appear common in oceanic cyanophages may ultimately represent signature genes for these phages. For example, the genomes of all three cyanophages presented here and five partial genomes (<5 kb) of Synechococcus cyanomyophages presented by Millard et al.  all contain a psbA gene. Further, all three cyanophages presented here contain at least one hli and a talC gene, and both myoviruses presented here are unique among the phages in that they contain pstS and cobS (Table 6). As more phages are sequenced, will we find that these genes are specifically characteristic of oceanic cyanophages? If true, this would provide us with a powerful tool for studying these phages in the wild because quantitative PCR could be used to differentiate between cyanophages and other phages in environmental samples.
Hypothesized Transient Genes
There are genes of interest, found in only one of the myoviruses, that we hypothesize are not functional, but rather were obtained by cyanomyophages through packaging random DNA, probably by illegitimate recombination [86,87] with DNA from a common phage genome pool .
P-SSM2 contains a gene (prnA) that is known to exist in only nine species of bacteria, in which it encodes a tryptophan halogenase that catalyzes the NADH-consuming first step of four that are involved in converting tryptophan to the antibiotic pyrrolnitrin [89,90,91]. Although this gene is full length (Figure S5), prnA is part of a unique metabolic pathway missing in most bacteria, including cyanobacteria.
Archaeal and eukaryotic genes.
The other myovirus, P-SSM4, contains three grouped genes with homology only to eukaryotic prion-like proteins (orf32), an archaeal protease (orf35), and a hypothetical protein from a eukaryotic slime mold (orf36) (see Figure 4). Other eukaryotic and prion-like genes have been predicted in the genomes of mycobacteriophages that infect actinobacterial hosts , although they have no similarity to those found in P-SSM4.
P-SSM4 contains a possible hemagglutinin neuraminidase (HN), which has only been observed in single-stranded RNA (ssRNA) viruses and Prochlorococcus MED4 (orf1400). In ssRNA viruses, HN cleaves sialic acid from glycolipids on the host cell surface, which enables these viruses to attach. Protein alignments show, however, that both the MED4 and P-SSM4 HN genes are only partial genes—they are missing the N- and C-termini (approximately 200 amino acids)—relative to other ssRNA HNs (Figure S6). It is noteworthy that the HN gene occurs nowhere else in the prokaryotic world except for MED4. Could this gene have been obtained by P-SSP7 through the phage genome pool (sensu Hendrix et al. ), then transferred to MED4? This postulate is buttressed by the observation that the HN gene in MED4 is found next to three hli genes (which encode high-light-inducible proteins)—genes which we have argued earlier are susceptible to horizontal gene transfer in this phage–host system .
Ecological and Evolutionary Implications of Phages Carrying Host Genes
Prochlorococcus cells are slow-growing (doubling times range from 1 to 10 d), oxygenic phototrophs that thrive in nutrient-poor, aerobic surface waters —conditions that are fundamentally different from those of most of the host cells of the phages sequenced to date. Thus, oceanic cyanophages are subject to substantially different selective pressures than most other sequenced phages in the database. The presence in these phages of host genes that are likely involved in the maintenance of photosynthesis, response to phosphate stress, and mobilization of carbon stores during infection may be interpreted as evidence of such unique pressures (see Table 5).
If phage genomes interact as “local neighborhoods” (sensu Hendrix et al. ) within a “global phage metagenome” (sensu Rohwer ), one would expect to find biologically cohesive units akin to species, defined by local gene transfers as proposed for “microbial species” . Such cohesive units would be characterized by core genes that determine a general phage infection lifestyle (e.g., T4-like or T7-like), as well as host-specific genes within phages that infect similar hosts. Indeed, 26 and 75 such core genes exist among the T7-like and T4-like phages, respectively (see Tables 3 and 4), and host-specific genes abound among these cyanophages (see Figures 1C, 5A, and 5B). That these core genes represent mostly morphological and DNA replication genes suggests a T7-like or T4-like lifestyle that would involve a specific means of delivering DNA from host to host (in a tailed, capsid structure) as well as converting the host into a phage factory. Based upon the presence of many such core genes in our Prochlorococcus phages, one would predict they would behave as T7-like (P-SSP7; although probably with the ability to integrate into its host) and T4-like phages (P-SSM2 and P-SSM4) during cyanobacterial infection.
Beyond these core genes, our Prochlorococcus phages contain many “nonphage” genes that are of greatest sequence similarity to cyanobacterial genes (see Figures 1C, 5A, and 5B). We speculate that the acquisition and use of some host genes by phages plays an important role in phage ecology, even shaping the evolution of the phage host range. The initial host range alterations are likely to occur by phage tail fiber switching , but beyond that, these co-opted host genes could either shift or expand the phage's host range depending upon whether they affect fitness of the phage in the original hosts. Understanding this dynamic fitness landscape will require modeling efforts directed by a thorough knowledge of the mechanisms and relative rates for this complex genetic shuffling—factors that likely underpin the complexity of phage–host interactions in the environment.
Materials and Methods
Prochlorococcus phages were concentrated using ultracentrifugation. Concentrates were prepared for microscopy by spotting phage lysates onto freshly glow-discharged carbon/formvar–coated copper grids. Grids were negatively stained with 1% uranyl acetate, dried, and viewed in a JEOL (Peabody, Massachusetts, United States) 1200 EXII transmission electron microscope operated at 80 kV.
Preparation of cyanophages for genome sequencing.
Three Prochlorococcus phages were chosen for sequencing based upon their host ranges, which were restricted to Prochlorococcus hosts (see Introduction).
Phages were prepared for genomic sequencing as previously described [14,95]. Briefly, phage particles were concentrated from phage lysates using polyethylene glycol. Concentrated DNA-containing phage particles were purified from other material in phage lysates using a density cesium chloride gradient. Purified phage particles were broken open (SDS/proteinase K), and DNA was extracted (phenol:chloroform) and precipitated (ethanol) yielding small amounts of DNA (<1 μg). A custom 1- to 2-kb insert linker-amplified shotgun library was constructed by Lucigen (Middletown, Wisconsin, United States) as described previously . Additional larger insert (3–8 kb) clone libraries were constructed from genomic DNA by the Department of Energy (Joint Genome Institute, Walnut Creek, California, United States) using a similar protocol to provide larger scaffolds during assembly. Inserts were sequenced by the Department of Energy Joint Genome Institute from all of these clone libraries and used for initial assembly of these phage genomes. The Stanford Human Genome Center Finishing Group (Palo Alto, California, United States) closed the genomes using primer walking.
Gene identification and characterization.
Protein coding genes were predicted using GeneMark  and manual curation. Translated ORFs were compared to known proteins in the nonredundant GenBank database (http://www.ncbi.nlm.nih.gov/BLAST/) and in the KEGG database (http://www.genome.ad.jp/kegg/kegg2.html) using the BLASTp program (ftp://ftp.ncbi.nih.gov/blast). Translated ORFs were also analyzed for signal sequences and transmembrane regions using the Web-based software SignalP and TMHMM, respectively (available at the CBS prediction servers; http://www.cbs.dtu.dk/services/). Where BLASTp e-values were high (>0.001) or no sequence similarity was observed, ORF annotation was aided by the use of PSI-BLAST, gene size, domain conservation, and/or synteny (gene order), the last as suggested for highly divergent genes encountered during phage genome annotation . Identification of tRNA genes was done using tRNAscan-SE .
Taxonomy of best hits.
For global genome comparison, we used BLASTp (e-values < 0.001) or manual annotation to classify to which group of organisms or phages each predicted coding sequence was most similar. In most cases this was obvious. However, approximately 2% of the coding sequences were less obvious, so we established an operational definition of “most similar” as the query sequence having e-values within four orders of magnitude of the top cluster of organismal types. For example, if a query sequence was similar to noncyanobacterial sequences with e-values of 10–29 to 10–25 and to cyanobacterial sequences with e-values of 10–20 or greater, then, despite sequence similarity to cyanobacterial sequences, the query would be considered noncyanobacterial.
Tail fiber gene identification.
Tail fiber genes were identified by generating alignments (stand-alone Basic Local Alignment Search Tool, BLAST , 2.2.8 release) of conceptually translated, computationally identified ORFs from the P-SSM2 and P-SSM4 genomes against a database consisting of 33,270 sequences encompassing all known phage sequences obtained from the NCBI NR database in April 2004. Only ORFs whose alignments to known tail fiber genes were longer than 100 residues and had e-values less than 0.001 were designated as tail-fiber-like. Sequences close to this cutoff were re-aligned using the bl2seq command of BLAST, which computes e-values independently of database size. Tail-fiber-like paralogs were identified by individually aligning the set of tail-fiber-like ORFs with all other ORFs in the genomes. All ORFs with alignments greater than 100 residues and e-values less than 0.001, were designated as tail fiber paralogs. All BLAST searches and alignments were performed with the low-complexity sequence filter and default parameters. Amino acid sequence repeats were identified by self-alignment matrices using the program Dotter .
Sequence manipulation and phylogenetic analyses.
Alignments were generated using Clustal X  and edited manually as necessary. PAUP V4.0b10  was used for the construction of distance and maximum parsimony trees. Amino acid distance trees were inferred using minimum evolution as the objective function, and mean distances. Heuristic searches were performed with 100 random addition sequence replicates and the tree bisection and reconnection branch-swapping algorithm. Starting trees were obtained by stepwise addition of sequences. Bootstrap analyses of 1,000 resamplings were carried out. Maximum likelihood trees were constructed using TREE-PUZZLE 5.0 . Evolutionary distances were calculated using the JTT model of substitution assuming a gamma-distributed model of rate heterogeneities with 16 gamma-rate categories empirically estimated from the data. Quartet puzzling support was estimated from 10,000 replicates.
Figure S1. Class II RNR Motif Compared Against Cyanobacterial and Non-T4-Like Phage RNRs
A question mark indicates this sequence data is not known; a period indicates identical residue to the reference sequence; and a dash indicates a gap in the alignment. Anab, Anabaena; Pro, Prochlorococcus; Syn, Synechococcus; Syncy, Synechocystis.
(10 KB PDF).
Figure S2. Distance Tree of RNR Family Proteins, Including Phage Sequences from P-SSM2, P-SSM4, and P-SSP7
Sequences from P-SSM2, P-SSM4, and P-SSP7 are shown in bold. Trees were generated from 900 amino acids. Bootstrap values for distance and maximum parsimony analyses and quartet puzzling values for maximum likelihood analysis, greater than 50%, are shown at the nodes (distance/maximum likelihood/maximum parsimony). Trees were unrooted; abbreviations as in Figure S1.
(14 KB PDF).
Figure S3. Distance Tree of Tal Proteins, Including Phage Sequences from P-SSM2, P-SSM4, and P-SSP7
Sequences from P-SSM2, P-SSM4, and P-SSP7 are shown in bold. Trees were generated from 566 amino acids. Bootstrap values for distance and maximum parsimony analyses and quartet puzzling values for maximum likelihood analysis, greater than 50%, are shown at the nodes (distance/maximum likelihood/maximum parsimony). Trees were unrooted; abbreviations as in Figure S1.
(14 KB PDF).
Figure S4. Alignment of TalC Subfamily Aldolases, Including Phage Sequences from P-SSM2, P-SSM4, P-SSP7, and S-RSM2
The 32 amino acid residues suggested to be diagnostic by Thorell et al.  are labeled with an asterisk and shaded where identical to bona fide TalC proteins, whereas the active site residues are labeled with an “at” symbol. Note the active site residues in the cyanophage TalC sequences exclusively match those from enzymes known to have transaldolase activity rather than fructose-6 phosphate aldolase activity.
(14 KB PDF).
Figure S5. Alignment of Tryptophan Halogenase Amino Acid Sequences Deduced from Phage and Cellular Encoded prnA Gene Sequences
Note the phage gene appears full-length relative to the other cellular genes. Bdellovibrio, Bdellovibrio bacteriovorus; Bordtella, Bordetella pertussis; Burkpyrro, Burkholderia pyrrocinia; Caulobacter, Caulobacter crescentus; Myxfulvus, Myxococcus fulvus; Pschloro, Pseudomonas chlororaphis; Pseud_fl, Pseudomonas fluorescens; Shewanella, Shewanella oneidensis MR-1; Xanaxon, Xanthomonas axonopodis; Xancamp, Xanthomonas campestris.
(35 KB PDF).
Figure S6. Alignment of HN Amino Acid Sequences Deduced from Phage and ssRNA Viral Gene Sequences
Note the Prochlorococcus phage and host gene appears to contain only the central region of the gene relative to the other ssRNA viral genes.APMV6, avian paramyxovirus 6; BPIV3, bovine parainfluenza virus 3; Gparamyxovirus, goose paramyxovirus; HPIV1,2,3, human parainfluenza virus 1,2,3; ProMED4, Prochlorococcus MED4.
(36 KB PDF).
The GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) accession numbers for the genomes discussed in this paper are MED4 (BX548174), P-SSM2 (AY939844), P-SSM4 (AY940168), and P-SSP7 (AY939843).
We thank David Mead (Lucigen) and Chris Detter (Department of Energy Joint Genome Institute [DOE JGI]) for clone library construction from minimal DNA. The sequencing and assembly of the phage genomes was performed by the production sequencing group at the DOE JGI through the Sequence-for-Others Program under the auspices of the US DOE's Office of Science, Biological, and Environmental Research Program and the University of California, Lawrence Livermore National Laboratory, under contract number W-7405-ENG-48; Lawrence Berkeley National Laboratory under contract number DE-AC03–76SF00098; Los Alamos National Laboratory under contract number W-7405-ENG-36; and Stanford University under contract number DE-FC02–99ER62873. This research was supported by the US DOE under grant numbers DE-FG02–99ER62814 and DE-FG02–02ER63445, and the National Science Foundation under grant number OCE-9820035 (to SWC). We thank Sherwood Casjens, Drew Endy, Hector Hernandez, and Roger Hendrix for discussions about phage biology, evolution, and RNRs, as well as Virginia Rich, Debbie Lindell, and Erik Zinser for valuable comments on the manuscript.
Particular thanks go to Ian Molineux for providing access to his unpublished T7 Group review chapter and extensive suggestions on the manuscript; the teams of Henry Krisch and Jim Karam for providing data at the T4-like Genome Web site (http://phage.bioc.tulane.edu; Jim Karam and Vasiliy Petrov for their analysis of the gp5 DNAP split in P-SSP7; Luke Thompson for analytical assistance with the cyanophage transaldolase family genes; and Anca Segall for finding the 42-bp exact match sequence in P-SSP7 and Prochlorococcus MED4 that supported our hypothesis that the P-SSP7 integrase gene might be functional.
MBS grew, purified, and extracted DNA from the phages. Non-authors (see Acknowledgments) prepared clone libraries, sequenced the inserts, and assembled the genomes. MBS, MLC, and FR did the majority of the genome annotation, while PW evaluated tail-fiber-related genes and provided electron micrographs of the particles. MBS and SWC wrote the majority of the paper with significant contributions from all authors, as well as non-authors (detailed in the Acknowledgments).
- 1. Partensky F, Hess WR, Vaulot D (1999) Prochlorococcus a marine photosynthetic prokaryote of global significance. Microbiol Mol Biol Rev 63: 106–127.
- 2. Liu H, Nolla HA, Campbell L (1997) Prochlorococcus growth rate and contribution to primary production in the equatorial and subtropical North Pacific Ocean. Aquatic Microb Ecol 12: 39–47.
- 3. Liu H, Campbell L, Landry MR, Nolla HA, Brown SL, et al. (1998) Prochlorococcus and Synechococcus growth rates and contributions to production in the Arabian Sea during the 1995 Southwest and Northeast Monsoons. Deep-Sea Res II 45: 2327–2352.
- 4. Rocap G, Larimer FW, Lamerdin J, Malfatti S, Chain P, et al. (2003) Genome divergence in two Prochlorococcus ecotypes reflects oceanic niche differentiation. Nature 424: 1042–1047.
- 5. Moore LR, Rocap G, Chisholm SW (1998) Physiology and molecular phylogeny of coexisting Prochlorococcus ecotypes. Nature 393: 464–467.
- 6. Moore LR, Post AF, Rocap G, Chisholm SW (2002) Utilization of different nitrogen sources by the marine cyanobacteria Prochlorococcus and Synechococcus. Limnol Oceanogr 47: 989–996.
- 7. Mann EL, Ahlgren N, Moffett JW, Chisholm SW (2002) Copper toxicity and cyanobacteria ecology in the Sargasso Sea. Limnol Oceanogr 47: 976–988.
- 8. Sullivan MB, Waterbury JB, Chisholm SW (2003) Cyanophages infecting the oceanic cyanobacterium Prochlorococcus. Nature 424: 1047–1051.
- 9. Waterbury JB, Valois FW (1993) Resistance to co-occurring phages enables marine Synechococcus communities to coexist with cyanophage abundant in seawater. Appl Environ Microbiol 59: 3393–3399.
- 10. Suttle CA, Chan AM (1994) Dynamics and distribution of cyanophages and their effects on marine Synechococcus spp. Appl Environ Microbiol 60: 3167–3174.
- 11. Marston MF, Sallee JL (2003) Genetic diversity and temporal variation in the cyanophage community infecting marine Synechococcus species in Rhode Island's coastal waters. Appl Environ Microbiol 69: 4639–4647.
- 12. Lu J, Chen F, Hodson RE (2001) Distribution, isolation, host specificity, and diversity of cyanophages infecting marine Synechococcus spp. in river estuaries. Appl Environ Microbiol 67: 3285–3290.
- 13. Thingstad TF (2000) Elements of a theory for the mechanisms controlling abundance, diversity, and biogeochemical role of lytic bacterial viruses in aquatic ecosystems. Limnol Oceanogr 45: 1320–1328.
- 14. Lindell D, Sullivan MB, Johnson ZI, Tolonen AC, Rohwer F, et al. (2004) Transfer of photosynthesis genes to and from Prochlorococcus viruses. Proc Natl Acad Sci U S A 101: 11013–11018.
- 15. Mann NH, Cook A, Millard A, Bailey S, Clokie M (2003) Marine ecosystems: Bacterial photosynthesis genes in a virus. Nature 424: 741.
- 16. Millard A, Clokie MR, Shub DA, Mann NH (2004) Genetic organization of the psbAD region in phages infecting marine Synechococcus strains. Proc Natl Acad Sci U S A 101: 11007–11012.
- 17. Chen F, Lu J (2002) Genomic sequence and evolution of marine cyanophage P60: A new insight on lytic and lysogenic phages. Appl Environ Microbiol 68: 2589–2594.
- 18. Scholl D, Kieleczawa J, Kemp P, Rush J, Richardson CC, et al. (2004) Genomic analysis of bacteriophages SP6 and K1–5, an estranged subgroup of the T7 supergroup. J Mol Biol 335: 1151–1171.
- 19. Hardies SC, Comeau AM, Serwer P, Suttle CA (2003) The complete sequence of marine bacteriophage VpV262 infecting Vibrio parahaemolyticus indicates that an ancestral component of a T7 viral supergroup is widespread in the marine environment. Virology 310: 359–371.
- 20. Rohwer F, Segall A, Steward G, Seguritan V, Breitbart M, et al. (2000) The complete genomic sequence of the marine phage Roseophage SIO1 shares homology with nonmarine phages. Limnol Oceanogr 45: 408–418.
- 21. Miller ES, Heidelberg JF, Eisen JA, Nelson WC, Durkin AS, et al. (2003) Complete genome sequence of the broad-host-range vibriophage KVP40: Comparative genomics of a T4-related bacteriophage. J Bacteriol 185: 5220–5233.
- 22. Mann NH (2003) Phages of the marine cyanobacterial picophytoplankton. FEMS Microbiol Rev 27: 17–34.
- 23. Molineux I (2005) The T7 group. In: Calendar R, editor. The bacteriophages. New York: In press.
- 24. Molineux IJ (2001) No syringes please, ejection of phage T7 DNA from the virion is enzyme driven. Mol Microbiol 40: 1–8.
- 25. Kelman Z, Pietrokovski S, Hurwitz J (1999) Isolation and characterization of a split B-type DNA polymerase from the archaeon Methanobacterium thermoautotrophicum deltaH. J Biol Chem 274: 28751–28761.
- 26. Lavigne R, Burkal'tseva MV, Robben J, Sykilinda NN, Kurochkina LP, et al. (2003) The genome of bacteriophage phiKMV, a T7-like virus infecting Pseudomonas aeruginosa. Virology 312: 49–59.
- 27. Paul JH, Sullivan MB, Segall AM, Rohwer F (2002) Marine phage genomics. Comp Biochem Physiol B Biochem Mol Biol 133: 463–476.
- 28. Groth AC, Calos MP (2004) Phage integrases: Biology and applications. J Mol Biol 335: 667–678.
- 29. Nelson KE, Weinel C, Paulsen IT, Dodson RJ, Hilbert H, et al. (2002) Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environ Microbiol 4: 799–808.
- 30. Nunes-Duby SE, Kwon HJ, Tirumalai RS, Ellenberger T, Landy A (1998) Similarities and differences among 105 members of the Int family of site-specific recombinases. Nucleic Acids Res 26: 391–406.
- 31. Williams KP (2002) Integration sites for genetic elements in prokaryotic tRNA and tmRNA genes: Sublocation preference of integrase subfamilies. Nucleic Acids Res 30: 866–875.
- 32. Canchaya C, Proux C, Fournous G, Bruttin A, Brussow H (2003) Prophage genomics. Microbiol Mol Biol Rev 67: 238–276.
- 33. Casjens S (2003) Prophages and bacterial genomics: What have we learned so far? Mol Microbiol 49: 277–300.
- 34. Dufresne A, Salanoubat M, Partensky F, Artiguenave F, Axmann IM, et al. (2003) Genome sequence of the cyanobacterium Prochlorococcus marinus SS120, a nearly minimal oxyphototrophic genome. Proc Natl Acad Sci U S A 100: 10020–10025.
- 35. Adolph KW, Haselkorn RH (1973) Isolation and characterization of a virus infecting a blue-green alga of the genus Synechococcus. Virology 54: 230–236.
- 36. Sherman LA, Connelley M (1976) Isolation and characterization of a cyanophage infecting the unicellular blue-green algae A. nidulans and S. cedorum. Virology 72: 540–544.
- 37. Ortmann AC, Lawrence JE, Suttle CA (2002) Lysogeny and lytic viral production during a bloom of the cyanobacterium Synechococcus spp. Microb Ecol 43: 225–231.
- 38. McDaniel L, Houchin LA, Williamson SJ, Paul JH (2002) Lysogeny in marine Synechococcus. Nature 415: 496.
- 39. Ackermann HW, Krisch HM (1997) A catalogue of T4-type bacteriophages. Arch Virol 142: 2329–2345.
- 40. Keller B, Dubochet J, Adrian M, Maeder M, Wurtz M, et al. (1988) Length and shape variants of the bacteriophage T4 head: Mutations in the scaffolding core genes 68 and 22. J Virol 62: 2960–2969.
- 41. Volker TA, Gafner J, Bickle TA, Showe MK (1982) Gene 67, a new, essential bacteriophage T4 head gene codes for a prehead core component, PIP. I. Genetic mapping and DNA sequence. J Mol Biol 161: 479–489.
- 42. Volker TA, Kuhn A, Showe MK, Bickle TA (1982) Gene 67, a new, essential bacteriophage T4 head gene codes for a prehead core component, PIP. II. The construction in vitro of unconditionally lethal mutants and their maintenance. J Mol Biol 161: 491–504.
- 43. Zhang J, Inouye M (2002) MazG, a nucleoside triphosphate pyrophosphohydrolase, interacts with Era, an essential GTPase in Escherichia coli. J Bacteriol 184: 5323–5329.
- 44. Zhang J, Zhang Y, Inouye M (2003) Thermotoga maritima MazG protein has both nucleoside triphosphate pyrophosphohydrolase and pyrophosphatase activities. J Biol Chem 278: 21408–21414.
- 45. Haggard-Ljungquist E, Halling C, Calendar R (1992) DNA sequences of the tail fiber genes of bacteriophage P2: Evidence for horizontal transfer of tail fiber genes among unrelated bacteriophages. J Bacteriol 174: 1462–1477.
- 46. Xue Q, Egan JB (1995) Tail sheath and tail tube genes of the temperate coliphage 186. Virology 212: 218–221.
- 47. Tetart F, Desplats C, Krisch HM (1998) Genome plasticity in the distal tail fiber locus of the T-even bacteriophage: Recombination between conserved motifs swaps adhesin specificity. J Mol Biol 282: 543–556.
- 48. Leiman PG, Kostyuchenko VA, Shneider MM, Kurochkina LP, Mesyanzhinov VV, et al. (2000) Structure of bacteriophage T4 gene product 11, the interface between the baseplate and short tail fibers. J Mol Biol 301: 975–985.
- 49. Cerritelli ME, Wall JS, Simon MN, Conway JF, Steven AC (1996) Stoichiometry and domainal organization of the long tail-fiber of bacteriophage T4: A hinged viral adhesin. J Mol Biol 260: 767–780.
- 50. Smith MC, Burns N, Sayers JR, Sorrell JA, Casjens SR, et al. (1998) Bacteriophage collagen. Science 279: 1834.
- 51. van Raaij MJ, Schoehn G, Jaquinod M, Ashman K, Burda MR, et al. (2001) Identification and crystallisation of a heat- and protease-stable fragment of the bacteriophage T4 short tail fibre. Biol Chem 382: 1049–1055.
- 52. Kutter E, Gachechiladze K, Poglazov A, Marusich E, Shneider M, et al. (1995) Evolution of T4-related phages. Virus Genes 11: 285–297.
- 53. Kostyuchenko VA, Leiman PG, Chipman PR, Kanamaru S, van Raaij MJ, et al. (2003) Three-dimensional structure of bacteriophage T4 baseplate. Nat Struct Biol 10: 688–693.
- 54. Kostyuchenko VA, Navruzbekov GA, Kurochkina LP, Strelkov SV, Mesyanzhinov VV, et al. (1999) The structure of bacteriophage T4 gene product 9: The trigger for tail contraction. Structure Fold Des 7: 1213–1222.
- 55. King J (1968) Assembly of the tail of bacteriophage T4. J Mol Biol 32: 231–262.
- 56. Frankenberg N, Lagarias JC (2003) Phycocyanobilin:ferredoxin oxidoreductase of Anabaena sp. PCC 7120. Biochemical and spectroscopic. J Biol Chem 278: 9219–9226.
- 57. Frankenberg N, Mukougawa K, Kohchi T, Lagarias JC (2001) Functional genomic analysis of the HY2 family of ferredoxin-dependent bilin reductases from oxygenic photosynthetic organisms. Plant Cell 13: 965–978.
- 58. Ting CS, Rocap G, King J, Chisholm SW (2002) Cyanobacterial photosynthesis in the oceans: The origins and significance of divergent light-harvesting strategies. Trends Microbiol 10: 134–142.
- 59. Ting CS, Rocap G, King J, Chisholm SW (2001) Phycobiliprotein genes of the marine photosynthetic prokaryote Prochlorococcus: Evidence for rapid evolution of genetic heterogeneity. Microbiology 147: 3171–3182.
- 60. Hess WR, Rocap G, Ting CS, Larimer FW, Stilwagen S, et al. (2001) The photosynthetic apparatus of Prochlorococcus Insights through comparative genomics. Photosynth Res 70: 53–71.
- 61. Penno S, Campbell L, Hess WR (2000) Presence of phycoerythrin in two strains of Prochlorococcus (cyanobacteria) isolated from the subtropical North Pacific Ocean. J Phycol 36: 723–729.
- 62. Bograh A, Gingras Y, Tajmir-Riahi HA, Carpentier R (1997) The effects of spermine and spermidine on the structure of photosystem II proteins in relation to inhibition of electron transport. FEBS Lett 402: 41–44.
- 63. Madigan MT, Martinko JM, Parker J (2003) Brock biology of microorganisms. Upper Saddle River: Prentice Hall. 1019 p.
- 64. Borovok I, Kreisberg-Zakarin R, Yanko M, Schreiber R, Myslovati M, et al. (2002) Streptomyces spp. contain class Ia and class II ribonucleotide reductases: Expression analysis of the genes in vegetative growth. Microbiology 148: 391–404.
- 65. Dunn JJ, Studier FW (1983) Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J Mol Biol 166: 477–535.
- 66. Maggio-Hall LA, Escalante-Semerena JC (1999) In vitro synthesis of the nucleotide loop of cobalamin by Salmonella typhimurium enzymes. Proc Natl Acad Sci U S A 96: 11798–11803.
- 67. Lawrence JG, Roth JR (1995) The cobalamin (coenzyme B12) biosynthetic genes of Escherichia coli. J Bacteriol 177: 6371–6380.
- 68. Gleason FK, Olszewski NE (2002) Isolation of the gene for the B12-dependent ribonucleotide reductase from Anabaena sp. strain PCC 7120 and expression in Escherichia coli. J Bacteriol 184: 6544–6550.
- 69. Moore LR, Chisholm SW (1999) Photophysiology of the marine cyanobacterium Prochlorococcus Ecotypic differences among cultured isolates. Limnol Oceanog 44: 628–638.
- 70. Waterbury JB, Watson SW, Valois FW, Franks DG (1986) Biological and ecological characterization of the marine unicellular cyanobacterium Synechococcus. Can Bull Fish Aquat Sci 214: 71–120.
- 71. Stanier RY (1973) Autotrophy and heterotrophy in unicellular blue-green algae. In: Carr NG, Whitton BA, editors. The biology of blue-green algae. Berkeley: University of California Press. pp. 501–518.
- 72. Sherman LA (1976) Infection of Synechococcus cedrorum by the cyanophage AS-1M. III. Cellular metabolism and phage development. Virology 71: 199–206.
- 73. Sprenger GA (1995) Genetics of pentose-phosphate pathway enzymes of Escherichia coli K-12. Arch Microbiol 164: 324–330.
- 74. Schurmann M, Sprenger GA (2001) Fructose-6-phosphate aldolase is a novel class I aldolase from Escherichia coli and is related to a novel group of bacterial transaldolases. J Biol Chem 276: 11055–11061.
- 75. Thorell S, Schurmann M, Sprenger GA, Schneider G (2002) Crystal structure of decameric fructose-6-phosphate aldolase from Escherichia coli reveals inter-subunit helix swapping as a structural basis for assembly differences in the transaldolase family. J Mol Biol 319: 161–171.
- 76. Karl DM (1999) A sea of change: Biogeochemical variability in the North Pacific Subtropical Gyre. Ecosystems 2: 181–214.
- 77. Wu J, Sunda W, Boyle EA, Karl DM (2000) Phosphate depletion in the western North Atlantic Ocean. Science 289: 759–762.
- 78. Scanlan DJ, Silman NJ, Donald KM, Wilson WH, Carr NG, et al. (1997) An immunological approach to detect phosphate stress in populations and single cells of photosynthetic picoplankton. Appl Environ Microbiol 63: 2411–2420.
- 79. Kazakov AE, Vassieva O, Gelfand MS, Osterman A, Overbeek R (2003) Bioinformatics classification and functional analysis of PhoH homologs. In Silico Biol 3: 3–15.
- 80. Kim SK, Makino K, Amemura M, Shinagawa H, Nakata A (1993) Molecular analysis of the phoH gene, belonging to the phosphate regulon in Escherichia coli. J Bacteriol 175: 1316–1324.
- 81. Wanner BL (1996) Phosphorus assimilation and control of the phosphate regulon. In: Neidhardt FC, editor. Escherichia coli and Salmonella Cellular and molecular biology, 2nd ed. Washington (DC): ASM Press. pp. 1357–1381.
- 82. Calendar R (1988) The bacteriophages. New York: Plenum Press. editor.
- 83. Los M, Wegrzyn G, Neubauer P (2003) A role for bacteriophage T4 rI gene function in the control of phage development during pseudolysogeny and in slowly growing host cells. Res Microbiol 154: 547–552.
- 84. Moebus K (1996) Marine bacteriophage reproduction under nutrient-limited growth of host bacteria. II. Investigations with phage-host system [H3:H3/1]. Mar Ecol Prog Ser 144: 13–22.
- 85. Williamson SJ, McLaughlin MR, Paul JH (2001) Interaction of the PhiHSIC virus with its host: Lysogeny or pseudolysogeny? Appl Environ Microbiol 67: 1682–1688.
- 86. Mosig G (1998) Recombination and recombination-dependent DNA replication in bacteriophage T4. Annu Rev Genet 32: 379–413.
- 87. Mosig G, Gewin J, Luder A, Colowick N, Vo D (2001) Two recombination-dependent DNA replication pathways of bacteriophage T4, and their roles in mutagenesis and horizontal gene transfer. Proc Natl Acad Sci U S A 98: 8306–8311.
- 88. Hendrix RW, Smith MC, Burns RN, Ford ME, Hatfull GF (1999) Evolutionary relationships among diverse bacteriophages and prophages: All the world's a phage. Proc Natl Acad Sci U S A 96: 2192–2197.
- 89. Hammer PE, Burd W, Hill DS, Ligon JM, van Pee K (1999) Conservation of the pyrrolnitrin biosynthetic gene cluster among six pyrrolnitrin-producing strains. FEMS Microbiol Lett 180: 39–44.
- 90. Kirner S, Hammer PE, Hill DS, Altmann A, Fischer I, et al. (1998) Functions encoded by pyrrolnitrin biosynthetic genes from Pseudomonas fluorescens. J Bacteriol 180: 1939–1943.
- 91. Hammer PE, Hill DS, Lam ST, Van Pee KH, Ligon JM (1997) Four genes from Pseudomonas fluorescens that encode the biosynthesis of pyrrolnitrin. Appl Environ Microbiol 63: 2147–2154.
- 92. Pedulla ML, Ford ME, Houtz JM, Karthikeyan T, Wadsworth C, et al. (2003) Origins of highly mosaic mycobacteriophage genomes. Cell 113: 171–182.
- 93. Rohwer F (2003) Global phage diversity. Cell 113: 141.
- 94. Lawrence JG, Hendrickson H (2003) Lateral gene transfer: When will adolescence end? Mol Microbiol 50: 739–749.
- 95. Breitbart M, Salamon P, Andresen B, Mahaffy JM, Segall AM, et al. (2002) Genomic analysis of uncultured marine viral communities. Proc Natl Acad Sci U S A 99: 14250–14255.
- 96. Besemer J, Lomsadze A, Borodovsky M (2001) GeneMarkS: A self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions. Nucleic Acids Res 29: 2607–2618.
- 97. Brussow H, Hendrix RW (2002) Phage genomics: Small is beautiful. Cell 108: 13–16.
- 98. Lowe TM, Eddy SR (1997) tRNAscan-SE: A program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 25: 955–964.
- 99. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215: 403–410.
- 100. Sonnhammer EL, Durbin R (1995) A dot-matrix program with dynamic threshold control suited for genomic DNA and protein sequence analysis. Gene 167: GC1–GC10.
- 101. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25: 4876–4882.
- 102. Swofford DL (2002) PAUP: Phylogenetic analysis using parsimony (and other methods), version 4 [computer program]. Sunderland (Massachusetts): Sinauer.
- 103. Schmidt HA, Strimmer K, Vingron M, von Haeseler A (2002) TREE-PUZZLE: Maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics 18: 502–504.
- 104. Miller ES, Kutter E, Mosig G, Arisaka F, Kunisawa T, et al. (2003) Bacteriophage T4 genome. Microb Mol Biol Rev 67: 86–156.
- 105. van Regenmortel MHV, Fauquet CM, Bishop DHL, Carstens EB, Estes MK, et al. (2000) Virus taxonomy: The classification and nomenclature of viruses. San Deigo: Academic Press. 1167 p.
- 106. Desplats C, Krisch HM (2003) The diversity and evolution of the T4-type bacteriophages. Res Microbiol 154: 259–267.
- 107. Tetart F, Desplats C, Kutateladze M, Monod C, Ackermann HW, et al. (2001) Phylogeny of the major head and tail genes of the wide-ranging T4-type bacteriophages. J Bacteriol 183: 358–366.
- 108. Mann NH, Clokie MR, Millard A, Cook A, Wilson WH, et al. The genome of S-PM2, a “photosynthetic” T4-type bacteriophage that infects marine Synechococcus. J Bacteriol. In press.