Genomic Changes and Genetic Divergence of Vibrio alginolyticus Under Phage Infection Stress Revealed by Whole-Genome Sequencing and Resequencing

Abstract

Bacteriophages (phages) and their bacterial hosts were the most abundant and genetically highly diverse organisms on the earth. In this study, a series of phage-resistant mutant (PRM) strains derived from Vibrio alginolyticus were isolated and Infrequent-restriction-site PCR (IRS-PCR) was used to investigate the genetic diversity of the PRM strains. Phenotypic variations of eight PRM strains were analyzed using profiles of utilizing carbon sources and chemical sensitivity. Genetic variations of eight PRM strains and coevolved V. alginolyticus populations with phages were analyzed by whole-genome sequencing and resequencing, respectively. The results indicated that eight genetically discrepant PRM stains exhibited abundant and abundant phenotypic variations. Eight PRM strains and coevolved V. alginolyticus populations (VE1, VE2, and VE3) contained numerous single nucleotide variations (SNVs) and insertions/indels (InDels) and exhibited obvious genetic divergence. Most of the SNVs and InDels in coding genes were related to the synthesis of flagellar, extracellular polysaccharide (EPS), which often served as the receptors of phage invasion. The PRM strains and the coevolved cell populations also contained frequent mutations in tRNA and rRNA genes. Two out of three coevolved populations (VE1 and VE2) contained a large mutation segment severely deconstructing gene nrdA, which was predictably responsible for the booming of mutation rate in the genome. In summary, numerous mutations and genetic divergence were detected in the genomes of V. alginolyticus PRM strains and in coevolved cell populations of V. alginolyticus under phage infection stress. The phage infection stress may provide an important force driving genomic evolution of V. alginolyticus.

Keywords: Vibrio alginolyticus; genetic divergence; genomic changes; phage-resistant mutations; phages.

FIGURE 1

The phage resistant colonies derived from V. alginolyticus E06333 and normal colonies V. alginolyticus E06333. (A) The phage resistant colonies occurred on a double-layer plate with enriched lytic phages. (B) The normal colonies of V. alginolyticus E06333 on a LB plate.

FIGURE 2

Genotyping of typical PRM strains derived from V. alginolyticus E06333 by IRS-PCR. These PRM strains exhibit obviously different fingerprints from the primary V. alginolyticus E06333. The numbers on the top represent the strain numbers. The black triangles on the bottom present eight PRM strains that are further selected for subsequent analysis. Eight PRM strains were renamed (according to the order from left to right) as VAM01, VAM02, VAM03, VAM04, VAM05, VAM06, VAM07, and VAM08, respectively.

FIGURE 3

Biochemical profiles of eight PRM strains and the primary strain E06333 from V. alginolyticus revealed by Biolog assays. The purple circles in boxes represent a positive reaction (growth). The purple circles with blue background represent six PRM strains obtained the ability of utilizing α-Hydroxy-Butyric Acid. The boxes with light green background represent five tests of chemical sensitivity.

FIGURE 4

The numbers of different mutations in eight PRM strains from V. alginolyticus. The mutations were classed into insertion (INS), deletions (DEL), and single nucleotide variations (SNVs) according to the mutation types and sizes. An 11-bp insertion in the PRM strain VAM03 was not shown in this figure.

FIGURE 5

The distribution and the mutation frequencies of SNVs and InDels in evolved and coevolved V. alginolyticus cell populations revealed by resequencing. T0: the collected sample before grouping at the early exponential stage (OD_{600 nm} = 0.6–1); VC1: collected samples from evolved cell populations at 2 h; VC2: collected samples from evolved cell populations at 13 h; VC3: collected samples from evolved cell populations at 22 h; VE1: collected samples from coevolved cell populations at 2 h; VE2: collected samples from coevolved cell populations at 13 h; VE3: collected samples from coevolved cell populations at 22 h. Mutation sites with yellow background represent 2 unique SNVs in the evolved cell populations. Positions of mutation sites with purple background represent 8 unique mutations in the coevolved cell populations. Mutation frequencies of these sites are shown with different colors.