Fouts DE, Klumpp J, Bishop-Lilly KA, Rajavel M, Willner KM, Butani A, Henry M, Biswas B, Li M, Albert MJ, Loessner MJ, Calendar R, Sozhamannan S
Whole Genome Sequencing and Comparative Genomic Analyses of Two Vibrio Cholerae O139 Bengal-specific Podoviruses to Other N4-like Phages Reveal Extensive Genetic Diversity.
Virology Journal. 2013 May 28; 10: 165.
BACKGROUND: Vibrio cholerae O139 Bengal is the only serogroup other than O1 implicated in cholera epidemics. We describe the isolation and characterization of an O139 serogroup-specific phage, vB_VchP_VchO139-I ([greek small letter phi]VchO139-I) that has similar host range and virion morphology as phage vB_VchP_JA1 ([greek small letter phi]JA1) described previously. We aimed at a complete molecular characterization of both phages and elucidation of their genetic and structural differences and assessment of their genetic relatedness to the N4-like phage group. METHODS: Host-range analysis and plaque morphology screening were done for both [greek small letter phi]JA1 and [greek small letter phi]VchO139-I. Both phage genomes were sequenced by a 454 and Sanger hybrid approach. Genomes were annotated and protein homologies were determined by Blast and HHPred. Restriction profiles, PFGE patterns and data on the physical genome structure were acquired and phylogenetic analyses were performed. RESULTS: The host specificity of [greek small letter phi]JA1 has been attributed to the unique capsular O-antigen produced by O139 strains. Plaque morphologies of the two phages were different; [greek small letter phi]VchO139-I produced a larger halo around the plaques than [greek small letter phi]JA1. Restriction profiles of [greek small letter phi]JA1 and [greek small letter phi]VchO139-I genomes were also different. The genomes of [greek small letter phi]JA1 and [greek small letter phi]VchO139-I consisted of linear double-stranded DNA of 71,252 and 70,938 base pairs. The presence of direct terminal repeats of around 1974 base pairs was demonstrated. Whole genome comparison revealed single nucleotide polymorphisms, small insertions/deletions and differences in gene content. Both genomes had 79 predicted protein encoding sequences, of which only 59 were identical between the two closely related phages. They also encoded one tRNA-Arg gene, an intein within the large terminase gene, and four homing endonuclease genes. Whole genome phylogenetic analyses of [greek small letter phi]JA1 and [greek small letter phi]VchO139-I against other sequenced N4-like phages delineate three novel subgroups or clades within this phage family. CONCLUSIONS: The closely related phages feature significant genetic differences, in spite of being morphologically identical. The phage morphology, genetic organization, genomic content and large terminase protein based phylogeny support the placement of these two phages in the Podoviridae family, more specifically within the N4-like phage group. The physical genome structure of [greek small letter phi]JA1 could be demonstrated experimentally. Our data pave the way for potential use of [greek small letter phi]JA1 and [greek small letter phi]VchO139-I in Vibrio cholerae typing and control.