<i>Vibrio vulnificus</i> Phage PV94 Is Closely Related to Temperate Phages of <i>V. cholerae</i> and Other <i>Vibrio</i> Species

<div><p>Background</p><p><i>Vibrio vulnificus</i> is an important pathogen which can cause serious infections in humans. Yet, there is limited knowledge on its virulence factors and the question whether temperate phages might be involved in pathogenicity, as is the case with <i>V. cholerae</i>. Thus far, only two phage<i>s</i> (SSP002 and VvAW1) infecting <i>V. vulnificus</i> have been genetically characterized. These phages were isolated from the environment and are not related to <i>Vibrio cholerae</i> phages. The lack of information on temperate <i>V. vulnificus</i> phages prompted us to isolate those phages from lysogenic strains and to compare them with phages of other <i>Vibrio</i> species.</p><p>Results</p><p>In this study the temperate phage PV94 was isolated from a <i>V. vulnificus</i> biotype 1 strain by mitomycin C induction. PV94 is a myovirus whose genome is a linear double-stranded DNA of 33,828 bp with 5′-protruding ends. Sequence analysis of PV94 revealed a modular organization of the genome. The left half of the genome comprising the immunity region and genes for the integrase, terminase and replication proteins shows similarites to <i>V. cholerae</i> kappa phages whereas the right half containing genes for structural proteins is closely related to a prophage residing in <i>V. furnissii</i> NCTC 11218.</p><p>Conclusion</p><p>We present the first genomic sequence of a temperate phage isolated from a human <i>V. vulnificus</i> isolate. The sequence analysis of the PV94 genome demonstrates the wide distribution of closely related prophages in various <i>Vibrio</i> species. Moreover, the mosaicism of the PV94 genome indicates a high degree of horizontal genetic exchange within the genus <i>Vibrio</i>, by which <i>V. vulnificus</i> might acquire virulence-associated genes from other species.</p></div

Analysis of the PV94 cohesive ends.

<p>(A) AdeI, Bgll and SspI restriction patterns of PV94 phage DNA. Lane M, DNA size marker (λ DNA Eco130I), lanes (−), unheated phage DNA, lanes (+), phage DNA that had been heated before digestion. (B) Determination of protruding nucleotides. Chromatograms of run-off sequencing reactions using phage DNA as template and primers (PPNF and PPNR) binding close to the genomic ends. The 16 bp overhanging sequences are shaded and marked by arrows.</p

Comparison of the PV94 and P2 replication proteins and origins of replication.

<p>(A) Conserved motifs (A, B and C) of the replication proteins. The two tyrosine residues within motif C which are part of the active site of P2 protein A are marked by an asterisk. (B) Alignment of the P2 origin of replication located within gene <i>A</i> with the corresponding region of PV94 ORF14. Arrows indicate the cleavage site at the replication origin.</p

Relationship of PV94 to other <i>Vibrio</i> phages.

<p>(A) Dot plots of the genome of PV94 with genomes of <i>V. cholerae</i> phage K139, <i>V. diazotrophicus</i> phage VD1 and a cryptic prophage of the <i>V. furnissii</i> strain NCTC 11218. The axes of abscissas and ordinates show the coordinates of the respective phage genomes. (B) Genome organization of the phages PV94, K139 and the prophage of <i>V. furnissii</i> strain NCTC 11218. Colours indicate the predicted functions of gene products. Related genes of the phages are connected by coloured shading.</p