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

Derrick E Fouts1, Jochen Klumpp2*, Kimberly A Bishop-Lilly34, Mathumathi Rajavel5, Kristin M Willner34, Amy Butani34, Matthew Henry34, Biswajit Biswas34, Manrong Li6, M John Albert7, Martin J Loessner2, Richard Calendar8 and Shanmuga Sozhamannan349

Author Affiliations

1 J Craig Venter Institute, Rockville, MD, USA

2 Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 7, Zurich, 8092, Switzerland

3 Henry M. Jackson Foundation, Bethesda, MD, USA

4 Biological Defense Research Directorate, Naval Medical Research Center, Ft. Detrick, MD, USA

5 Department of Biology/Medical Technology, School of Computer, Mathematical and Natural Sciences, Morgan State University, 1700 E. Cold Spring Lane, Baltimore, MD, USA

6 Intralytix, Inc., The Columbus Center, 701 E. Pratt St, Baltimore, MD, USA

7 Department of Microbiology, Faculty of Medicine, Kuwait University, Safat, Kuwait

8 Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, USA

9 Current address: GoldBelt Raven, LLC, Frederick, MD, 21701, USA

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Virology Journal 2013, 10:165  doi:10.1186/1743-422X-10-165

Published: 28 May 2013

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.

Keywords:
Vibrio cholerae O139; N4-like virus; Genome comparison; Terminal repeats; Intein; Phylogenetic relationship