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发表于 2009-4-23 08:47 |只看该作者 |倒序浏览 |打印

1 Joint Program in Biological Oceanography, Woods Hole Oceanographic Institution and Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America,2 Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America,3 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America,4 Department of Biology, San Diego State University, San Diego, California, United States of America
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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.& J/ x# ]' A4 [
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Introduction* x5 d( R/ Y) b0 _6 X

Prochlorococcus is the numerically dominant primary producer in the temperate and tropical surface oceans [1]. 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 [4] and are physiologically and genetically diverse with at least two ※ecotypes§ that have distinctive light physiology [5], nitrogen [6] and phosphorus (L. R. Moore, personal communication) utilization, and copper [7] and virus (phage) [8] 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 [13]) in check, as well as by carrying photosynthetic ※host§ genes [14,15,16] and mediating horizontal transfer of genetic material between cyanobacterial hosts [14].
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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 [17]. 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 [18] proposed by Hardies et al. [19]. 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 [18]. 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 [20] and vibriophage KVP40 [21], 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].
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With a large collection of phages from which to choose [8], 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 [8]. 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 [14], 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.# j+ ]! d) [+ z; ]5 S

Results/Discussion0 F4 q  f0 l, b' s( e% W+ h! i
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General Features of the Podovirus P-SSP7+ T  d9 p" S& B4 d! N  B
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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 [23]. 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 29-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.
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(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 [24] or microbial nomenclature for non-phage genes. Class I, II, and III genes refer to those in T7 [66] 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.- ?2 D' u3 o4 L" u! f

(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.9 Y9 r( M: C$ ^& j
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An examination of the genomes of coliphage T7 and its closest coliphage relatives (T3, gh-1, 朴Ye03–12, 朴A1122) 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 [17].

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) [24], 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 [25] and some T4-like phages (V. Petrov and J. Karam, personal communication).
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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) [23]. 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 [26]. 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 [27].' _% g7 {7 e4 S& i4 V4 _
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Hypothesized Lysogeny in P-SSP7
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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 [28]. 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 [29]. 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 [30]) 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 [31], adding support to the hypothesis that this int gene is functional.) a  O" N# E: ?5 h0 w2 I

P-SSP7 was isolated from surface ocean waters at the end of summer stratification [8], when nutrients are extremely limiting. We have hypothesized [8] 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.
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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 [39]. 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.
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Myovirus P-SSM2 with (A) non-contracted tail and (B) contracted tail, and myovirus P-SSM4 with (C) contracted tail(Matthew B. Sullivan, Maur)

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