Genetic diversity in cultured and wild marine cyanomyoviruses reveals phosphorus stress as a strong selective agent

Viruses that infect marine cyanobacteria–cyanophages–often carry genes with orthologs in their cyanobacterial hosts, and the frequency of these genes can vary with habitat. To explore habitat-influenced genomic diversity more deeply, we used the genomes of 28 cultured cyanomyoviruses as references to identify phage genes in three ocean habitats. Only about 6–11% of genes were consistently observed in the wild, revealing high gene-content variability in these populations. Numerous shared phage/host genes differed in relative frequency between environments, including genes related to phosphorous acquisition, photorespiration, photosynthesis and the pentose phosphate pathway, possibly reflecting environmental selection for these genes in cyanomyovirus genomes. The strongest emergent signal was related to phosphorous availability; a higher fraction of genomes from relatively low-phosphorus environments–the Sargasso and Mediterranean Sea–contained host-like phosphorus assimilation genes compared with those from the N. Pacific Gyre. These genes are known to be upregulated when the host is phosphorous starved, a response mediated by pho box motifs in phage genomes that bind a host regulatory protein. Eleven cyanomyoviruses have predicted pho boxes upstream of the phosphate-acquisition genes pstS and phoA; eight of these have a conserved cyanophage-specific gene (PhCOG173) between the pho box and pstS. PhCOG173 is also found upstream of other shared phage/host genes, suggesting a unique regulatory role. Pho boxes are found upstream of high light-inducible (hli) genes in cyanomyoviruses, suggesting that this motif may have a broader role than regulating phosphorous-stress responses in infected hosts or that these hlis are involved in the phosphorous-stress response.     

Multiple density classes of phage P1 due to tetramer formation

Summary Stocks of coliphage P1 contain infective phage particles (P1), a smaller morphological variant (pP1) and single phage tails. In crude stocks and under certain conditions these particles form stable aggregates of four by adhesion of their base plates. It happens that all the aggregates of P1, pP1 and tails having densities above 1.41 g/cm³ are distributed around five values: 1.422, 1.435, 1.450, 1.459 and 1.473 g/cm³. Consequently they form five rather distinct bands when examined by analytical centrifugation. Formation and dissociation of tetramers results in loss and recovery of phage infectivity. Tetramers formed of four P1 particles have a sedimentation constant of 1,185 S as compared to 715 S for single P1 particles. Within the limits of our methods we could not detect any P1 or pP1 particles containing amounts of DNA different from 10^–16 g and 4×10^–17 g, respectively.     

Cluster J Mycobacteriophages: Intron Splicing in Capsid and Tail Genes

Bacteriophages isolated on Mycobacterium smegmatis mc²155 represent many distinct genomes sharing little or no DNA sequence similarity. The genomes are architecturally mosaic and are replete with genes of unknown function. A new group of genomes sharing substantial nucleotide sequences constitute Cluster J. The six mycobacteriophages forming Cluster J are morphologically members of the Siphoviridae, but have unusually long genomes ranging from 106.3 to 117 kbp. Reconstruction of the capsid by cryo-electron microscopy of mycobacteriophage BAKA reveals an icosahedral structure with a triangulation number of 13. All six phages are temperate and homoimmune, and prophage establishment involves integration into a tRNA-Leu gene not previously identified as a mycobacterial attB site for phage integration. The Cluster J genomes provide two examples of intron splicing within the virion structural genes, one in a major capsid subunit gene, and one in a tail gene. These genomes also contain numerous free-standing HNH homing endonuclease, and comparative analysis reveals how these could contribute to genome mosaicism. The unusual Cluster J genomes provide new insights into phage genome architecture, gene function, capsid structure, gene mobility, intron splicing, and evolution.     

A DNA repair system specific for thermophilic Archaea and bacteria predicted by genomic context analysis

During a systematic analysis of conserved gene context in prokaryotic genomes, a previously undetected, complex, partially conserved neighborhood consisting of more than 20 genes was discovered in most Archaea (with the exception of Thermoplasma acidophilum and Halobacterium NRC-1) and some bacteria, including the hyperthermophiles Thermotoga maritima and Aquifex aeolicus. The gene composition and gene order in this neighborhood vary greatly between species, but all versions have a stable, conserved core that consists of five genes. One of the core genes encodes a predicted DNA helicase, often fused to a predicted HD-superfamily hydrolase, and another encodes a RecB family exonuclease; three core genes remain uncharacterized, but one of these might encode a nuclease of a new family. Two more genes that belong to this neighborhood and are present in most of the genomes in which the neighborhood was detected encode, respectively, a predicted HD-superfamily hydrolase (possibly a nuclease) of a distinct family and a predicted, novel DNA polymerase. Another characteristic feature of this neighborhood is the expansion of a superfamily of paralogous, uncharacterized proteins, which are encoded by at least 20–30% of the genes in the neighborhood. The functional features of the proteins encoded in this neighborhood suggest that they comprise a previously undetected DNA repair system, which, to our knowledge, is the first repair system largely specific for thermophiles to be identified. This hypothetical repair system might be functionally analogous to the bacterial–eukaryotic system of translesion, mutagenic repair whose central components are DNA polymerases of the UmuC-DinB-Rad30-Rev1 superfamily, which typically are missing in thermophiles.     

Defective Lysogeny in Erwinia carotovora

The electron microscopic study of several Erwinia carotovora strains showed that the SOS-induced cells of this pectolytic phytopathogenic bacterium produce particular phage parts (tails, heads, and baseplates) but do not assemble them into fully functional phage particles. E. carotovora cells produced several times greater amounts of phage tails in response to induction by mitomycin C than in response to induction by nalidixic acid. The tails were 128–192 nm in length and 13–21 nm in diameter. Phage heads were characterized by four discrete ranges of diameters: 18, 55–59, 66–75, and 92–98 nm. The diameters of phage baseplates varied from 39 to 53 nm, depending on the particular strain. It was shown that cells of the same species may contain several different types of phage tails and heads. The structural organization of phage tails and baseplates in the nalidixic acid–induced lysate of E. carotovora J2 was studied in more detail. The data obtained suggest that pectolytic phytopathogenic erwinia are characterized by defective polylysogeny.     

Comparative Genomic Analysis of 60 Mycobacteriophage Genomes: Genome Clustering, Gene Acquisition, and Gene Size

Mycobacteriophages are viruses that infect mycobacterial hosts. Expansion of a collection of sequenced phage genomes to a total of 60—all infecting a common bacterial host—provides further insight into their diversity and evolution. Of the 60 phage genomes, 55 can be grouped into nine clusters according to their nucleotide sequence similarities, 5 of which can be further divided into subclusters; 5 genomes do not cluster with other phages. The sequence diversity between genomes within a cluster varies greatly; for example, the 6 genomes in Cluster D share more than 97.5% average nucleotide similarity with one another. In contrast, similarity between the 2 genomes in Cluster I is barely detectable by diagonal plot analysis. In total, 6858 predicted open-reading frames have been grouped into 1523 phamilies (phams) of related sequences, 46% of which possess only a single member. Only 18.8% of the phams have sequence similarity to non-mycobacteriophage database entries, and fewer than 10% of all phams can be assigned functions based on database searching or synteny. Genome clustering facilitates the identification of genes that are in greatest genetic flux and are more likely to have been exchanged horizontally in relatively recent evolutionary time. Although mycobacteriophage genes exhibit a smaller average size than genes of their host (205 residues compared with 315), phage genes in higher flux average only 100 amino acids, suggesting that the primary units of genetic exchange correspond to single protein domains.     

The lambda head-tail joining reaction: purification, properties and structure of biologically active heads and tails

Procedures were developed to obtain biologically active lambda heads and tails at high purity with 20 to 40% recovery. Free heads, free tails and phage particles differ markedly in stability. Phage are stable in solutions containing Mg²⁺ but tails are not. The protein subunits which form the shaft of the tail dissociate in the presence of Mg²⁺ and form multisubunit spherical structures. EDTA protects free tails against inactivation but disrupts heads and phage particles. The four carbon diamine, putrescine, stabilizes heads against inactivation; the three and five carbon diamines are less effective. Electron micrographs reveal a new “knob” structure at the distal end of the tail fiber of phage and of free tails. Tails released from EDTA-disrupted phage possess a “head-tail connector”, a structure not present on the tail before its joining with a head.     

Core and accessory genome architecture in a group of Pseudomonas aeruginosa Mu-like phages

Background

Bacteriophages that infect the opportunistic pathogen Pseudomonas aeruginosa have been classified into several groups. One of them, which includes temperate phage particles with icosahedral heads and long flexible tails, bears genomes whose architecture and replication mechanism, but not their nucleotide sequences, are like those of coliphage Mu. By comparing the genomic sequences of this group of P. aeruginosa phages one could draw conclusions about their ontogeny and evolution.

Results

Two newly isolated Mu-like phages of P. aeruginosa are described and their genomes sequenced and compared with those available in the public data banks. The genome sequences of the two phages are similar to each other and to those of a group of P. aeruginosa transposable phages. Comparing twelve of these genomes revealed a common genomic architecture in the group. Each phage genome had numerous genes with homologues in all the other genomes and a set of variable genes specific for each genome. The first group, which comprised most of the genes with assigned functions, was named “core genome”, and the second group, containing mostly short ORFs without assigned functions was called “accessory genome”. Like in other phage groups, variable genes are confined to specific regions in the genome.

Conclusion

Based on the known and inferred functions for some of the variable genes of the phages analyzed here, they appear to confer selective advantages for the phage survival under particular host conditions. We speculate that phages have developed a mechanism for horizontally acquiring genes to incorporate them at specific loci in the genome that help phage adaptation to the selective pressures imposed by the host.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1146) contains supplementary material, which is available to authorized users.     

Morphological and genetic diversity of temperate phages in Clostridium difficile

Eight temperate phages were characterized after mitomycin C induction of six Clostridium difficile isolates corresponding to six distinct PCR ribotypes. The hypervirulent C. difficile strain responsible for a multi-institutional outbreak (NAP1/027 or QCD-32g58) was among these prophage-containing strains. Observation of the crude lysates by transmission electron microscopy (TEM) revealed the presence of three phages with isometric capsids and long contractile tails (Myoviridae family), as well as five phages with long noncontractile tails (Siphoviridae family). TEM analyses also revealed the presence of a significant number of phage tail-like particles in all the lysates. Southern hybridization experiments with restricted prophage DNA showed that C. difficile phages belonging to the family Myoviridae are highly similar and most likely related to previously described prophages phiC2, phiC5, and phiCD119. On the other hand, members of the Siphoviridae phage family are more genetically divergent, suggesting that they originated from distantly related ancestors. Our data thus suggest that there are at least three genetically distinct groups of temperate phages in C. difficile; one group is composed of highly related myophages, and the other two groups are composed of more genetically heterogeneous siphophages. Finally, no gene homologous to genes encoding C. difficile toxins or toxin regulators could be identified in the genomes of these phages using DNA hybridization. Interestingly, each unique phage restriction profile correlated with a specific C. difficile PCR ribotype.     

Analysis of six prophages in Lactococcus lactis IL1403: different genetic structure of temperate and virulent phage populations

We report the genetic organisation of six prophages present in the genome of Lactococcus lactis IL1403. The three larger prophages (36–42 kb), belong to the already described P335 group of temperate phages, whereas the three smaller ones (13–15 kb) are most probably satellites relying on helper phage(s) for multiplication. These data give a new insight into the genetic structure of lactococcal phage populations. P335 temperate phages have variable genomes, sharing homology over only 10–33% of their length. In contrast, virulent phages have highly similar genomes sharing homology over >90% of their length. Further analysis of genetic structure in all known groups of phages active on other bacterial hosts such as Escherichia coli, Bacillus subtilis, Mycobacterium and Streptococcus thermophilus confirmed the existence of two types of genetic structure related to the phage way of life. This might reflect different intensities of horizontal DNA exchange: low among purely virulent phages and high among temperate phages and their lytic homologues. We suggest that the constraints on genetic exchange among purely virulent phages reflect their optimal genetic organisation, adapted to a more specialised and extreme form of parasitism than temperate/lytic phages.     

Comparison of Vero-cytotoxin-encoding phages from Escherichia coli of human and bovine origin

Phages encoding production of Vero cytotoxins VT1 or VT2 were isolated from strains of Escherichia coli of human and bovine origin. Two human strains of serotype O157: H7 produced both VT1 and VT2 and each carried two separate phages encoding either VT1 or VT2. The phages were morphologically similar to each other and to a VT2 phage previously isolated from a strain of serotype O157: H-; all had regular hexagonal heads and short tails. The phages had similar genome sizes and DNA hybridization and restriction enzyme digestion showed that the DNAs were very closely related. This contrasts with another report that one of the strains tested (933) released two clearly distinguishable phages separately encoding VT1 and VT2. The O157 phages differed from a VT1 phage isolated from a bovine E. coli strain belonging to serotype O26: H11 and from the reference VT1 phage isolated previously from a human strain, H19, of serotype O26: H11. The two O26 phages were morphologically similar with elongated heads and long tails. They had similar genome sizes and DNA hybridization indicated a high level of homology between them. Hybridization of an O157 phage DNA probe to DNA of the O26 phages, and vice versa, showed there was some cross-hybridization between the two types of phage. A phage from a bovine strain of serotype O29: H34 had a regular hexagonal head and short tail resembling those of the O157 phages. The DNA was distinguishable from that of all the other phages tested in restriction digest patterns but hybridized significantly to that of an O157 phage. Hybridization of the phage genomes with VT1 and VT2 gene probes showed that sequences encoding these toxins were highly conserved in the different phages from strains belonging to the three serogroups.     

Origins of the Xylella fastidiosa Prophage-Like Regions and Their Impact in Genome Differentiation

Xylella fastidiosa is a Gram negative plant pathogen causing many economically important diseases, and analyses of completely sequenced X. fastidiosa genome strains allowed the identification of many prophage-like elements and possibly phage remnants, accounting for up to 15% of the genome composition. To better evaluate the recent evolution of the X. fastidiosa chromosome backbone among distinct pathovars, the number and location of prophage-like regions on two finished genomes (9a5c and Temecula1), and in two candidate molecules (Ann1 and Dixon) were assessed. Based on comparative best bidirectional hit analyses, the majority (51%) of the predicted genes in the X. fastidiosa prophage-like regions are related to structural phage genes belonging to the Siphoviridae family. Electron micrograph reveals the existence of putative viral particles with similar morphology to lambda phages in the bacterial cell in planta. Moreover, analysis of microarray data indicates that 9a5c strain cultivated under stress conditions presents enhanced expression of phage anti-repressor genes, suggesting switches from lysogenic to lytic cycle of phages under stress-induced situations. Furthermore, virulence-associated proteins and toxins are found within these prophage-like elements, thus suggesting an important role in host adaptation. Finally, clustering analyses of phage integrase genes based on multiple alignment patterns reveal they group in five lineages, all possessing a tyrosine recombinase catalytic domain, and phylogenetically close to other integrases found in phages that are genetic mosaics and able to perform generalized and specialized transduction. Integration sites and tRNA association is also evidenced. In summary, we present comparative and experimental evidence supporting the association and contribution of phage activity on the differentiation of Xylella genomes.     

A computational approach to identify genes for functional RNAs in genomic sequences

Currently there is no successful computational approach for identification of genes encoding novel functional RNAs (fRNAs) in genomic sequences. We have developed a machine learning approach using neural networks and support vector machines to extract common features among known RNAs for prediction of new RNA genes in the unannotated regions of prokaryotic and archaeal genomes. The Escherichia coli genome was used for development, but we have applied this method to several other bacterial and archaeal genomes. Networks based on nucleotide composition were 80–90% accurate in jackknife testing experiments for bacteria and 90–99% for hyperthermophilic archaea. We also achieved a significant improvement in accuracy by combining these predictions with those obtained using a second set of parameters consisting of known RNA sequence motifs and the calculated free energy of folding. Several known fRNAs not included in the training datasets were identified as well as several hundred predicted novel RNAs. These studies indicate that there are many unidentified RNAs in simple genomes that can be predicted computationally as a precursor to experimental study. Public access to our RNA gene predictions and an interface for user predictions is available via the web.     

Heterogeneous Evolution of HIV-1 CRF01_AE in Men Who Have Sex with Men (MSM) and Other Populations in China

Introduction

The HIV epidemic in men who have sex with men (MSM) continues to grow in most countries. However, the phylodynamic and virological differences among HIV-1 strains circulating in MSM and other populations are not well characterized.

Methods

Nearly full-length genomes (NFLGs) of the HIV-1 CRF01_AE were obtained from the Los Alamos HIV database. Phylogenetic analyses were conducted using the NFLG, gag, pol and env genes, using the maximum likelihood method. Selection pressure analyses at the codon level were performed for each gene in the phylogenetic clusters using PAML.

Results

Sequences isolated from MSM in China clustered in Clusters 1 (92.5%) and 2 (85.71%). The major risk factor for Cluster 3 was heterosexual transmission (62.16%). The ratio of non-synonymous to synonymous substitutions in the env gene (0.7–0.75) was higher than the gag (0.26–0.34) or pol (0.21–0.26) genes. In env gene, Cluster 1 (4.56×10^-3subs/site/year) and 2 (6.01×10^-3subs/site/year) had higher evolutionary rates than Cluster 3 (1.14×10^-3subs/site/year). Positive selection affected 4.2–6.58% of the amino acid sites in the env gene. Two sites (HXB2:136 and 316) evolved similarly in Clusters 1 and 2, but not Cluster 3.

Conclusion

The HIV-1 CRF01_AE in MSM is evolving differently than in other populations.     

Exploring the mycobacteriophage metaproteome: phage genomics as an educational platform

Bacteriophages are the most abundant forms of life in the biosphere and carry genomes characterized by high genetic diversity and mosaic architectures. The complete sequences of 30 mycobacteriophage genomes show them collectively to encode 101 tRNAs, three tmRNAs, and 3,357 proteins belonging to 1,536 “phamilies” of related sequences, and a statistical analysis predicts that these represent approximately 50% of the total number of phamilies in the mycobacteriophage population. These phamilies contain 2.19 proteins on average; more than half (774) of them contain just a single protein sequence. Only six phamilies have representatives in more than half of the 30 genomes, and only three—encoding tape-measure proteins, lysins, and minor tail proteins—are present in all 30 phages, although these phamilies are themselves highly modular, such that no single amino acid sequence element is present in all 30 mycobacteriophage genomes. Of the 1,536 phamilies, only 230 (15%) have amino acid sequence similarity to previously reported proteins, reflecting the enormous genetic diversity of the entire phage population. The abundance and diversity of phages, the simplicity of phage isolation, and the relatively small size of phage genomes support bacteriophage isolation and comparative genomic analysis as a highly suitable platform for discovery-based education.     

Evolutionary Relationships among Actinophages and a Putative Adaptation for Growth in Streptomyces spp.

The genome sequences of eight Streptomyces phages are presented, four of which were isolated for this study. Phages R4, TG1, ϕHau3, and SV1 were isolated previously and have been exploited as tools for understanding and genetically manipulating Streptomyces spp. We also extracted five apparently intact prophages from recent Streptomyces spp. genome projects and, together with six phage genomes in the database, we analyzed all 19 Streptomyces phage genomes with a view to understanding their relationships to each other and to other actinophages, particularly the mycobacteriophages. Fifteen of the Streptomyces phages group into four clusters of related genomes. Although the R4-like phages do not share nucleotide sequence similarity with other phages, they clearly have common ancestry with cluster A mycobacteriophages, sharing many protein homologues, common gene syntenies, and similar repressor-stoperator regulatory systems. The R4-like phage ϕHau3 and the prophage StrepC.1 (from Streptomyces sp. strain C) appear to have hijacked a unique adaptation of the streptomycetes, i.e., use of the rare UUA codon, to control translation of the essential phage protein, the terminase. The Streptomyces venezuelae generalized transducing phage SV1 was used to predict the presence of other generalized transducing phages for different Streptomyces species.     

Improving gene annotation of complete viral genomes

Gene annotation in viruses often relies upon similarity search methods. These methods possess high specificity but some genes may be missed, either those unique to a particular genome or those highly divergent from known homologs. To identify potentially missing viral genes we have analyzed all complete viral genomes currently available in GenBank with a specialized and augmented version of the gene finding program GeneMarkS. In particular, by implementing genome-specific self-training protocols we have better adjusted the GeneMarkS statistical models to sequences of viral genomes. Hundreds of new genes were identified, some in well studied viral genomes. For example, a new gene predicted in the genome of the Epstein–Barr virus was shown to encode a protein similar to α-herpesvirus minor tegument protein UL14 with heat shock functions. Convincing evidence of this similarity was obtained after only 12 PSI-BLAST iterations. In another example, several iterations of PSI-BLAST were required to demonstrate that a gene predicted in the genome of Alcelaphine herpesvirus 1 encodes a BALF1-like protein which is thought to be involved in apoptosis regulation and, potentially, carcinogenesis. New predictions were used to refine annotations of viral genomes in the RefSeq collection curated by the National Center for Biotechnology Information. Importantly, even in those cases where no sequence similarities were detected, GeneMarkS significantly reduced the number of primary targets for experimental characterization by identifying the most probable candidate genes. The new genome annotations were stored in VIOLIN, an interactive database which provides access to similarity search tools for up-to-date analysis of predicted viral proteins.