Composite conserved promoter-terminator motifs (PeSLs) that mediate modular shuffling in the diverse T4-like myoviruses.

The diverse T4-like phages (Tquatrovirinae) infect a wide array of gram-negative bacterial hosts. The genome architecture of these phages is generally well conserved, most of the phylogenetically variable genes being grouped together in a series hyperplastic regions (HPRs) that are interspersed among large blocks of conserved core genes. Recent evidence from a pair of closely related T4-like phages has suggested that small, composite terminator/promoter sequences (promoterearly stem loop [PeSLs]) were implicated in mediating the high levels of genetic plasticity by indels occurring within the HPRs. Here, we present the genome sequence analysis of two T4-like phages, PST (168 kb, 272 open reading frames [ORFs]) and nt-1 (248 kb, 405 ORFs). These two phages were chosen for comparative sequence analysis because, although they are closely related to phages that have been previously sequenced (T4 and KVP40, respectively), they have different host ranges. In each case, one member of the pair infects a bacterial strain that is a human pathogen, whereas the other phage's host is a nonpathogen. Despite belonging to phylogenetically distant branches of the T4-likes, these pairs of phage have diverged from each other in part by a mechanism apparently involving PeSL-mediated recombination. This analysis confirms a role of PeSL sequences in the generation of genomic diversity by serving as a point of genetic exchange between otherwise unrelated sequences within the HPRs. Finally, the palette of divergent genes swapped by PeSL-mediated homologous recombination is discussed in the context of the PeSLs' potentially important role in facilitating phage adaption to new hosts and environments.

Phage morphology recapitulates phylogeny: the comparative genomics of a new group of myoviruses.

Among dsDNA tailed bacteriophages (Caudovirales), members of the Myoviridae family have the most sophisticated virion design that includes a complex contractile tail structure. The Myoviridae generally have larger genomes than the other phage families. Relatively few "dwarf" myoviruses, those with a genome size of less than 50 kb such as those of the Mu group, have been analyzed in extenso. Here we report on the genome sequencing and morphological characterization of a new group of such phages that infect a diverse range of Proteobacteria, namely Aeromonas salmonicida phage 56, Vibrio cholerae phages 138 and CP-T1, Bdellovibrio phage φ1422, and Pectobacterium carotovorum phage ZF40. This group of dwarf myoviruses shares an identical virion morphology, characterized by usually short contractile tails, and have genome sizes of approximately 45 kb. Although their genome sequences are variable in their lysogeny, replication, and host adaption modules, presumably reflecting differing lifestyles and hosts, their structural and morphogenesis modules have been evolutionarily constrained by their virion morphology. Comparative genomic analysis reveals that these phages, along with related prophage genomes, form a new coherent group within the Myoviridae. The results presented in this communication support the hypothesis that the diversity of phages may be more structured than generally believed and that the innumerable phages in the biosphere all belong to discrete lineages or families.

Morphology and genome sequence of phage ϕ1402: A dwarf myovirus of the predatory bacterium Bdellovibrio bacteriovorus.

Phages are among the simplest biological entities known and simultaneously the most numerous and ubiquitous members of the biosphere. Among the three families of tailed dsDNA phages, the Myoviridae have the most structurally sophisticated tails which are capable of contraction, unlike the simpler tails of the Podoviridae and Siphoviridae. Such "nanomachines" tails are involved in both efficient phage adsorption and genome injection. Their structural complexity probably necessitates multistep morphogenetic pathways, involving non-structural components, to correctly assemble the structural constituents. For reasons probably related, at least in part, to such morphological intricacy, myoviruses tend to have larger genomes than simpler phages. As a consequence, there are no well-characterized myoviruses with a size of less than 40 kb. Here we report on the characterization and sequencing of the 23,931 bp genome of the dwarf myovirus ϕ1402 of Bdellovibrio bacteriovorus. Our genomic analysis shows that ϕ1402 differs substantially from all other known phages and appears to be the smallest known autonomous myovirus.

Isolation and genomic characterization of the first phage infecting Iodobacteria: ϕPLPE, a myovirus having a novel set of features.

The aquatic phage ϕPLPE infects a bacterium of the genus Iodobacter that are common inhabitants of rivers, streams and canals that produce violacein-like pigments. Our characterization of ϕPLPE reveals it to be a small, contractile-tailed phage whose 47.5 kb genome sequence is phylogenetically distant from all previously characterized phages. The genome has a generally modular organization (e.g. replication/recombination, structure/morphogenesis, lysis/lysogeny) and approximately half of its 84 open reading frames have no known homologues. It behaves as a virulent phage under the host growth conditions we have employed and, with the exception of an anti-repressor (ant) homologue, the genome lacks all the genes associated with a lysogenic lifestyle. Thus, either ϕPLPE was once a temperate phage that has lost most of its lysogeny cassette or it is a virulent phage that acquired an ant-like gene presumably for some function other than the control of lysogeny. The ϕPLPE genome has few bacterial gene homologues with the interesting exception of a putative acylhydrolase (acylase). This function has been implicated in bacterial quorum sensing since it degrades homoserine-lactone signalling molecules and can disrupt or modulate quorum signalling from either the emitter or its competitors. ϕPLPE may be an example of a phage co-opting components of the bacterial quorum-sensing apparatus to its own advantage.

Exploring the prokaryotic virosphere.

The world of prokaryotic viruses, including the "traditional" bacteriophages and the viruses of Archaea, is currently in a period of renaissance, brought about largely by our new capabilities in (meta)genomics and by the isolation of diverse novel virus-host systems. In this review, we highlight some of the directions where we believe research on the prokaryotic virosphere will lead us in the near future.

The capsid of the T4 phage superfamily: the evolution, diversity, and structure of some of the most prevalent proteins in the biosphere.

The Escherichia coli bacteriophage T4 has served as a classic system in phage biology for more than 60 years. Only recently have phylogenetic analyses and genomic comparisons demonstrated the existence of a large, diverse, and widespread superfamily of T4-like phages in the environment. We report here on the T4-like major capsid protein (MCP) sequences that were obtained by targeted polymerase chain reaction (PCR) of marine environmental samples. This analysis was then expanded to include 1,000 s of new sequences of T4-like capsid genes from the metagenomic data obtained during the Sorcerer II Global Ocean Sampling (GOS) expedition. This data compilation reveals that the diversity of the major and minor capsid proteins from the GOS metagenome follows the same general patterns as the sequences from cultured phage genomes. Interestingly, the new MCP sequences obtained by PCR targeted to MCP sequences in environmental samples are more divergent (deeper branching) than the vast majority of the MCP sequences coming from the other sources. The marine T4-like phage population appears to be largely dominated by the T4-like cyanophages. Using approximately 1,400 T4-like MCP sequences from various sources, we mapped the degree of sequence conservation on a structural model of the T4-like MCP. The results indicate that within the T4 superfamily there are some clear phylogenetic groups with regard to the more conserved and more variable domains of the MCP. Such differences can be correlated with variations in capsid morphology, the arrangement of the MCP lattice, and the presence of different capsid accessory proteins between the subgroups of the T4 superfamily.

Genetic diversity among five T4-like bacteriophages.

BACKGROUND: Bacteriophages are an important repository of genetic diversity. As one of the major constituents of terrestrial biomass, they exert profound effects on the earth's ecology and microbial evolution by mediating horizontal gene transfer between bacteria and controlling their growth. Only limited genomic sequence data are currently available for phages but even this reveals an overwhelming diversity in their gene sequences and genomes. The contribution of the T4-like phages to this overall phage diversity is difficult to assess, since only a few examples of complete genome sequence exist for these phages. Our analysis of five T4-like genomes represents half of the known T4-like genomes in GenBank. RESULTS: Here, we have examined in detail the genetic diversity of the genomes of five relatives of bacteriophage T4: the Escherichia coli phages RB43, RB49 and RB69, the Aeromonas salmonicida phage 44RR2.8t (or 44RR) and the Aeromonas hydrophila phage Aeh1. Our data define a core set of conserved genes common to these genomes as well as hundreds of additional open reading frames (ORFs) that are nonconserved. Although some of these ORFs resemble known genes from bacterial hosts or other phages, most show no significant similarity to any known sequence in the databases. The five genomes analyzed here all have similarities in gene regulation to T4. Sequence motifs resembling T4 early and late consensus promoters were observed in all five genomes. In contrast, only two of these genomes, RB69 and 44RR, showed similarities to T4 middle-mode promoter sequences and to the T4 motA gene product required for their recognition. In addition, we observed that each phage differed in the number and assortment of putative genes encoding host-like metabolic enzymes, tRNA species, and homing endonucleases. CONCLUSION: Our observations suggest that evolution of the T4-like phages has drawn on a highly diverged pool of genes in the microbial world. The T4-like phages harbour a wealth of genetic material that has not been identified previously. The mechanisms by which these genes may have arisen may differ from those previously proposed for the evolution of other bacteriophage genomes.

Transcription of a 'photosynthetic' T4-type phage during infection of a marine cyanobacterium.

The transcription of S-PM2 phage following infection of Synechococcus sp. WH7803, a marine cyanobacterium, was analysed by quantitative real-time PCR. Unlike the distantly related coliphage T4, there were only two (early and late) instead of three (early, middle and late) classes of transcripts during the developmental cycle of the phage. This difference is consistent with the absence from the S-PM2 genome of T4-like middle mode promoter sequences and the transcription factors associated with their recognition. Phage S-PM2 carries the 'photosynthetic' genes psbA and psbD that encode homologues of the host photosystem II proteins D1 and D2. Transcripts of the phage psbA gene appeared soon after infection and remained at high levels until lysis. Throughout the course of infection, the photosynthetic capacity of the cells remained constant. A considerable transient increase in the abundance of the host psbA transcripts occurred shortly after infection, suggesting that the host responds to the trauma of phage infection in a similar way as it does to a variety of other environmental stresses. The very substantial transcription of the phage psbA gene during the latter phase of phage infection suggests that S-PM2 has acquired this cellular gene to ensure that D1 levels and thus photosynthesis are fully maintained until the infected cell finally lyses. Unexpectedly, transcripts of a phage-encoded S-layer protein gene were among the earliest and most abundant detected, suggesting that this partial homologue of a host protein plays an important role in the S-PM2 infection process.

War is peace--dispatches from the bacterial and phage killing fields.

Large-scale sequence analyses of phage and bacteria have provided new insights into the diverse and multifaceted interactions of these genomes. Such interactions are important because they determine the partitioning of a large fraction of global biomass. Furthermore, the struggle between phage and bacteria has had a significant impact on the evolution of the biosphere. This competition for resources has created an enormous pool of genetic diversity. Eons of horizontal genetic transfer have permitted the entire biosphere to directly benefit from a bargain-basement source of evolutionary innovation.

The diversity and evolution of the T4-type bacteriophages.

Recent studies suggest that viruses are the most numerous entities in the biosphere; bacteriophages, the viruses that infect Eubacteria and Archaea, constitute a substantial fraction of this population. In spite of their ubiquity, the vast majority of phages in the environment have never been studied and nothing is known about them. For the last 10 years our research has focused on an extremely widespread group of phages, the T4-type. It has now become evident that phage T4 has a myriad of relatives in nature that differ significantly in their host range. The genomes of all these phages have homology to the T4 genes that determine virion morphology. Although phylogenetically related, these T4-type phages can be subdivided into four groups that are increasingly distant from T4: the T-evens, the pseudo T-evens, the schizo T-evens and the exo T-evens. Genomic comparisons between the various T4-type phages and T4 indicate that these genomes share homology not only for virion structural components but also for most of the essential genes involved in the T4 life cycle. This suggests that horizontal transmission of the genetic information may have played a less general role in the evolution of these phages than has been supposed. Nevertheless, we have identified several regions of the T4-type genome, such as the segment containing the tail fiber genes that exhibit evidence of extensive modular shuffling during evolution. The T4-type genomes appear to be a mosaic containing a large and fixed group of essential genes as well as highly variable set of non-essential genes. These non-essential genes are probably important for the adaptation of these phages to their particular life-style. Furthermore, swapping autonomous domains within the essential proteins may slightly modify their function(s) and contribute to the adaptive ability of the T4-type phage family. Regulatory sequences also display considerable evolutionary plasticity and this too may facilitate the adaptation of phage gene expression to new environments and stresses.