Closely Related Vibrio alginolyticus Strains Encode an Identical Repertoire of Caudovirales-Like Regions and Filamentous Phages.

Many filamentous vibriophages encode virulence genes that lead to the emergence of pathogenic bacteria. Most genomes of filamentous vibriophages characterized up until today were isolated from human pathogens. Despite genome-based predictions that environmental Vibrios also contain filamentous phages that contribute to bacterial virulence, empirical evidence is scarce. This study aimed to characterize the bacteriophages of a marine pathogen, Vibrio alginolyticus (Kiel-alginolyticus ecotype) and to determine their role in bacterial virulence. To do so, we sequenced the phage-containing supernatant of eight different V. alginolyticus strains, characterized the phages therein and performed infection experiments on juvenile pipefish to assess their contribution to bacterial virulence. We were able to identify two actively replicating filamentous phages. Unique to this study was that all eight bacteria of the Kiel-alginolyticus ecotype have identical bacteriophages, supporting our previously established theory of a clonal expansion of the Kiel-alginolyticus ecotype. We further found that in one of the two filamentous phages, two phage-morphogenesis proteins (Zot and Ace) share high sequence similarity with putative toxins encoded on the Vibrio cholerae phage CTXΦ. The coverage of this filamentous phage correlated positively with virulence (measured in controlled infection experiments on the eukaryotic host), suggesting that this phage contributes to bacterial virulence.

Emergence of a Competence-Reducing Filamentous Phage from the Genome of Acinetobacter baylyi ADP1.

Bacterial genomes commonly contain prophage sequences as a result of past infections with lysogenic phages. Many of these integrated viral sequences are believed to be cryptic, but prophage genes are sometimes coopted by the host, and some prophages may be reactivated to form infectious particles when cells are stressed or mutate. We found that a previously uncharacterized filamentous phage emerged from the genome of Acinetobacter baylyi ADP1 during a laboratory evolution experiment. This phage has a genetic organization similar to that of the Vibrio cholerae CTXϕ phage. The emergence of the ADP1 phage was associated with the evolution of reduced transformability in our experimental populations, so we named it the competence-reducing acinetobacter phage (CRAϕ). Knocking out ADP1 genes required for competence leads to resistance to CRAϕ infection. Although filamentous bacteriophages are known to target type IV pili, this is the first report of a phage that apparently uses a competence pilus as a receptor. A. baylyi may be especially susceptible to this route of infection because every cell is competent during normal growth, whereas competence is induced only under certain environmental conditions or in a subpopulation of cells in other bacterial species. It is possible that CRAϕ-like phages restrict horizontal gene transfer in nature by inhibiting the growth of naturally transformable strains. We also found that prophages with homology to CRAϕ exist in several strains of Acinetobacter baumannii These CRAϕ-like A. baumannii prophages encode toxins similar to CTXϕ that might contribute to the virulence of this opportunistic multidrug-resistant pathogen. IMPORTANCE: We observed the emergence of a novel filamentous phage (CRAϕ) from the genome of Acinetobacter baylyi ADP1 during a long-term laboratory evolution experiment. CRAϕ is the first bacteriophage reported to require the molecular machinery involved in the uptake of environmental DNA for infection. Reactivation and evolution of CRAϕ reduced the potential for horizontal transfer of genes via natural transformation in our experiment. Risk of infection by similar phages may limit the expression and maintenance of bacterial competence in nature. The closest studied relative of CRAϕ is the Vibrio cholerae CTXϕ phage. Variants of CRAϕ are found in the genomes of Acinetobacter baumannii strains, and it is possible that phage-encoded toxins contribute to the virulence of this opportunistic multidrug-resistant pathogen.

Complete genome sequence of a filamentous bacteriophage, RS611, that infects the phytopathogen Ralstonia solanacearum.

Filamentous bacteriophage RS611 (ϕRS611), which infects the phytopathogen Ralstonia solanacearum, had a circular single-stranded DNA genome that was characterized as an Ff-type phage belonging to the family Inoviridae. The ϕRS611 genome was composed of 6386 bases with a G + C content of 62.1 % and contained 11 putative open reading frames. The ϕRS611 genome showed high similarity to those of Ralstonia phages RSS0 and RSS1. However, approximately 900-nucleotide deletions were found in the region corresponding to open reading frames 10 and 11 of ϕRSS0 and ϕRSS1.

Isolation and characterization of a novel filamentous phage from Stenotrophomonas maltophilia.

In this study, a novel filamentous phage, φSHP1, of the environmental Stenotrophomonas maltophilia strain P2 was isolated and characterized. Electron microscopy showed that φSHP1 resembled members of the family Inoviridae and was about 2.1 µm long. The 6,867-nucleotide genome of φSHP1 was a circular single-stranded DNA and had a replication form designated pSH1. Ten putative open reading frames (ORFs) were found in the φSHP1 genome, and six predicted proteins showed similarity to proteins in databases. Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis of φSHP1 displayed one major structural polypeptide of approximately 4.0 kDa. N-terminal sequencing showed that it was the mature product of ORF5 and that its N-terminal 27 amino acid residues had been cleaved off from the predicted nascent protein. Finally, phylogenetic trees were constructed to analyze the phylogenetic relationship of φSHP1 to other known filamentous phages. φSHP1 appears to be the first reported Stenotrophomonas filamentous phage.

DNA binding proteins of the filamentous phages CTXphi and VGJphi of Vibrio cholerae.

The native product of open reading frame 112 (orf112) and a recombinant variant of the RstB protein, encoded by Vibrio cholerae pathogen-specific bacteriophages VGJphi and CTXphi, respectively, were purified to more than 90% homogeneity. Orf112 protein was shown to specifically bind single-stranded genomic DNA of VGJphi; however, RstB protein unexpectedly bound double-stranded DNA in addition to the single-stranded genomic DNA. The DNA binding properties of these proteins may explain their requirement for the rolling circle replication of the respective phages and RstB's requirement for single-stranded-DNA chromosomal integration of CTXphi phage dependent on XerCD recombinases.

New bacteriophages that infect the phytopathogen Ralstonia solanacearum.

Four kinds of bacteriophage (phiRSL, phiRSA, phiRSM and phiRSS) were isolated from Ralstonia solanacearum, a soil-borne Gram-negative bacterium that is the causative agent of bacterial wilt in many important crops. The Myovirus-type phages phiRSL1 and phiRSA1 contained dsDNA genomes of 240 kbp and 39 kbp, respectively. These phages have relatively wide host ranges and gave large clear plaques with various host strains; especially phiRSA1 was able to infect all 15 R. solanacearum strains of different races or different biovars tested in this study. Three host strains contained phiRSA1-related sequences in their genomic DNAs, suggesting a lysogenic cycle of phiRSA1. Two phages, phiRSM1 and phiRSS1, were characterized as Ff-type phages (Inovirus) based on their particle morphology, genomic ssDNA and infection cycle. However, despite their similar fibrous morphology, their genome size (9.0 kb for phiRSM1 and 6.6 kb for phiRSS1) and genome sequence were different. Strains of R. solanacearum that were sensitive to phiRSM1 were resistant to phiRSS1 and vice versa. Several R. solanacearum strains contained phiRSM1-related sequences and at least one strain produced phiRSM1 particles, indicating the lysogenic state of this phage. These phages may be useful as a tool not only for molecular biological studies of R. solanacearum pathogenicity but also for specific and efficient detection (phiRSM1 and phiRSS1) and control of harmful pathogens (phiRSL and phiRSA) in cropping ecosystems as well as growing crops.

Bacteriophages that infect the cellulolytic ruminal bacterium Ruminococcus albus AR67.

AIM: To isolate bacterial viruses that infect the ruminal cellulolytic bacterium Ruminococcus albus. METHODS: Four phages infecting R. albus AR67 were isolated under anaerobic conditions using the soft-agar overlay technique. The phages were characterized on morphology, solvent stability, nucleic acid type and digestion characteristics. Two phages, phiRa02 and phiRa04 comprised icosahedral virions with linear double-stranded DNA and appeared to belong to the family Podoviridae [corrected] The other two phages are most likely filamentous phages with circular single-stranded DNA of the family Inoviridae. SIGNIFICANCE OF THE STUDY: Viruses of the family Inoviridae [corrected] have not previously been isolated from rumen bacteria. The phages isolated in this study are the first phages shown to infect the cellulolytic bacteria of the rumen. This suggests that the cellulolytic populations of the rumen are subject to lytic events that may impact on the ability of these bacteria to degrade plant fibre and on the nutrition of the animal.

VGJ phi, a novel filamentous phage of Vibrio cholerae, integrates into the same chromosomal site as CTX phi.

We describe a novel filamentous phage, designated VGJ phi, isolated from strain SG25-1 of Vibrio cholerae O139, which infects all O1 (classical and El Tor) and O139 strains tested. The sequence of the 7,542 nucleotides of the phage genome reveals that VGJ phi has a distinctive region of 775 nucleotides and a conserved region with an overall genomic organization similar to that of previously characterized filamentous phages, such as CTX phi of V. cholerae and Ff phages of Escherichia coli. The conserved region carries 10 open reading frames (ORFs) coding for products homologous to previously reported peptides of other filamentous phages, and the distinctive region carries one ORF whose product is not homologous to any known peptide. VGJ phi, like other filamentous phages, uses a type IV pilus to infect V. cholerae; in this case, the pilus is the mannose-sensitive hemagglutinin. VGJ phi-infected V. cholerae overexpresses the product of one ORF of the phage (ORF112), which is similar to single-stranded DNA binding proteins of other filamentous phages. Once inside a cell, VGJ phi is able to integrate its genome into the same chromosomal attB site as CTX phi, entering into a lysogenic state. Additionally, we found an attP structure in VGJ phi, which is also conserved in several lysogenic filamentous phages from different bacterial hosts. Finally, since different filamentous phages seem to integrate into the bacterial dif locus by a general mechanism, we propose a model in which repeated integration events with different phages might have contributed to the evolution of the CTX chromosomal region in V. cholerae El Tor.