Whole genome sequencing and comparative genomic analyses of two Vibrio cholerae O139 Bengal-specific Podoviruses to other N4-like phages reveal extensive genetic diversity

Abstract

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 (ϕVchO139-I) that has similar host range and virion morphology as phage vB_VchP_JA1 (ϕJA1) 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 ϕJA1 and ϕVchO139-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 ϕJA1 has been attributed to the unique capsular O-antigen produced by O139 strains. Plaque morphologies of the two phages were different; ϕVchO139-I produced a larger halo around the plaques than ϕJA1. Restriction profiles of ϕJA1 and ϕVchO139-I genomes were also different. The genomes of ϕJA1 and ϕVchO139-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 ϕJA1 and ϕVchO139-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 ϕJA1 could be demonstrated experimentally. Our data pave the way for potential use of ϕJA1 and ϕVchO139-I in Vibrio cholerae typing and control.

Figure 1

Transmission electron micrographs of phages ϕJA1 and ϕVchO139-I. Plates A-D show images of phage particles negatively stained with 2% uranyl acetate and plate E shows an image of a phage particle negatively stained with 2% phosphotungstate. A) ϕJA1. B) ϕVchO139-I. C) ϕJA1. D) ϕVchO139-I. E) ϕVchO139-I. Scale bar represents 100 nm. Podoviridae morphology of ϕJA1 and ϕVchO139-I particles is evident.

Figure 2

Plaque morphology of phages ϕJA1 and ϕVchO139-I. Bacterial lawns of V. cholerae O139 Bengal strain AI1834 prepared on 2xYT bottom and top agar show differing plaque morphologies of phages ϕJA1 and ϕVchO139-I. A) ϕVchO139-I; B) ϕJA1; C) ϕVchO139-I close-up view of plaques; D) ϕJA1 close-up view of plaques.

Figure 3

Identification of virion structural proteins of phages ϕJA1 and ϕVchO139-I. Proteins were separated on an 8-18% gradient SDS-PAGE (lane A- ϕJA-1 and lane B- ϕVchO139-I) and extracted from gel slices corresponding to specific bands and submitted for LC-ESI-MS/MS peptide mass fingerprinting. Approximate band molecular weight and sequence annotation of corresponding proteins are indicated. Observed molecular mass of the identified proteins is indicated in parenthesis. Other protein bands were contaminated with capsid protein and could not reliably be identified.

Figure 4

Genome maps and protein identity comparison between phages N4, ϕJA1, and ϕVchO139-I. Genetic maps of the genomes of phages N4, ϕJA-1 and ϕVchO139-I. The linear map is based on nucleotide sequences of the phage genomes and predicted open reading frames. CDSs are labeled by locus identifier and colored by functional role categories as noted in the boxed key. BLASTP matches between CDSs are colored by protein percent identity (se e key). Green arrows and red hairpin structures indicate predicted promoters and transcriptional terminators, respectively. Blue vertical lines indicate restriction enzyme recognition sites with the restriction enzyme name marked on top. The location of SNPs is noted on the ϕJA 1 map as small colored tick marks (see key).

Figure 5

Proteome-based phylogenetic analysis of N4-like phages. A dendrogram was constructed based on the mean of the pairwise BLASTP score ratios (BSRs) of the N4-like group of phages.

Figure 6

Phylogenetic tree based on the large terminase protein of various phages. ClustalW alignment of 109 large terminase subunit amino acid sequences computed into a phylogenetic tree using Neighbor Joining method and 1000 bootstrap replicates (CLC Genomics Workbench 6).