Vibrio cholerae embraces two major evolutionary traits as revealed by targeted gene sequencing

Vibrio cholerae inhabits aquatic environments worldwide and has over 200 recognized serogroups classified by O-polysaccharide specificity. Here, we report that V. cholerae selects either of two genetic traits during their evolution. Sequencing of the specific gene locus MS6_A0927 revealed that 339 of 341 strains of V. cholerae and closely related Vibrio species originating from 34 countries over a century carried either metY (M) (~1, 269 bp) or luxR-hchA (LH) (~1, 600 bp) genes, and consequently those vibrios were separated into two clusters, M (45.4%) and LH (54.6%). Only two strains contained both M and LH in the same locus. Moreover, extensive polymorphisms in those genes were detected in M and LH with 79 and 46 sequence variations, respectively. V. cholerae O1 strains isolated from cholera outbreaks worldwide, and some non-O1 strains evolving from O1 via exchange of genes encoding cell surface polysaccharides possessed LH alleles. Analysis of polymorphisms in the gene locus implicated a high degree of genetic diversity and identical subpopulations among the V. cholerae species

Hypothetical evolutionary relationship among clades of <i>Vibrio cholerae</i> with reference to strain MS6.

<p>Hypothetical ancestral <i>Vibrio</i> organisms are indicated by open circles. Although <i>V. cholerae</i> O1 possesses the partially overlapping <i>hchA/luxR</i>, they are replaced by <i>metY</i> in strains MS6 and 2740-80.</p

Comparative Genomic Characterization of a Thailand–Myanmar Isolate, MS6, of <i>Vibrio cholerae</i> O1 El Tor, Which Is Phylogenetically Related to a “US Gulf Coast” Clone

<div><p>Background</p><p>The cholera outbreaks in Thailand during 2007–2010 were exclusively caused by the <i>Vibrio cholerae</i> O1 El Tor variant carrying the cholera toxin gene of the classical biotype. We previously isolated a <i>V. cholerae</i> O1 El Tor strain from a patient with diarrhea and designated it MS6. Multilocus sequence-typing analysis revealed that MS6 is most closely related to the U. S. Gulf Coast clone with the exception of two novel housekeeping genes.</p><p>Methodology/Principal Findings</p><p>The nucleotide sequence of the genome of MS6 was determined and compared with those of 26 <i>V. cholerae</i> strains isolated from clinical and environmental sources worldwide. We show here that the MS6 isolate is distantly related to the ongoing seventh pandemic <i>V. cholerae</i> O1 El Tor strains. These strains differ with respect to polymorphisms in housekeeping genes, seventh pandemic group-specific markers, CTX phages, two genes encoding predicted transmembrane proteins, the presence of <i>metY</i> (MS6_A0927) or <i>hchA/luxR</i> in a highly conserved region of the <i>V. cholerae</i> O1 serogroup, and a superintegron (SI). We found that <i>V. cholerae</i> species carry either <i>hchA/luxR</i> or <i>metY</i> and that the <i>V. cholerae</i> O1 clade commonly possesses <i>hchA/luxR,</i> except for MS6 and U. S. Gulf Coast strains. These findings illuminate the evolutionary relationships among <i>V. cholerae</i> O1 strains. Moreover, the MS6 SI carries a quinolone-resistance gene cassette, which was closely related with those present in plasmid-borne integrons of other gram-negative bacteria.</p><p>Conclusions/Significance</p><p>Phylogenetic analysis reveals that MS6 is most closely related to a U. S. Gulf Coast clone, indicating their divergence before that of the El Tor biotype strains from a common <i>V. cholerae</i> O1 ancestor. We propose that MS6 serves as an environmental aquatic reservoir of <i>V. cholerae</i> O1.</p></div

<i>Vibrio cholerae</i> O1 genomes can be divided into two clusters that possess either <i>hchA/luxR</i> or <i>metY</i> in a conserved syntenic region of the small chromosome.

<p>Dendrograms were constructed based on <i>hchA</i>/<i>luxR</i> or <i>metY</i> using the method described in the legend to Fig. 3. Strains highlighted in blue belong to serogroup O1. However, four strains enclosed in the purple square may have undergone lateral gene exchange of O-antigen gene clusters; thus, strains V52 and MO10 were converted into O37 and O139 serogroups, respectively, while strains 12129(1) and TM11079-80 gained the O1-antigen gene cluster <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098120#pone.0098120-Chun1" target="_blank">[25]</a>.</p

MS6, but not U. S. Gulf Coast strain 2740-80, carries the <i>Vibrio</i> seventh pandemic island-1 (VSP-1) between <i>VC0174</i> and <i>VC0186</i>.

<p>Dendrograms were constructed based on the genes flanking VSP-1 (<i>VC0174</i> and <i>VC0186</i>) using the neighbor-joining method using MEGA version 5.2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098120#pone.0098120-Tamura1" target="_blank">[37]</a>. VSP-1 was identified in the eight <i>V. cholerae</i> strains shown in red. MS6 as well as the seventh pandemic strains carry the full VSP-1 sequence between <i>VC0174</i> and <i>VC0186</i>, which is closely related to that of strain 2740-80. Scale bars indicate nucleotide substitutions per site.</p

Comparison of the large and small chromosomes of <i>Vibrio cholerae</i> O1 El Tor MS6 and those of three reference strains.

<p>The first and second outermost circles of each chromosome show the COG functional categories of the MS6 coding regions, in the clockwise and anticlockwise directions, respectively. The next three circles compare the coding regions of <i>V. cholerae</i> O1 M66-2, N16961, and 2010EL-1786 with those of MS6. The sixth and seventh circles show the GC content of the MS6 sequence and the percent G+C deviation by strand, respectively.</p

Maximum-likelihood tree showing the phylogenetic relationships among MS6 and 26 <i>Vibrio cholerae</i> strains representing diverse serogroups.

<p>The tree was rooted by treating the VL426 strain as an outlier. Bootstrap supports (%) are indicated at the branching points. Branch lengths are proportional to the sequence differences.</p