Genetic variation in the Vibrio vulnificus group 1 capsular polysaccharide operon

Abstract

Vibrio vulnificus produces human disease associated with raw-oyster consumption or wound infections, but fatalities are limited to persons with chronic underlying illness. Capsular polysaccharide (CPS) is required for virulence, and CPS expression correlates with opaque (Op) colonies that show "phase variation" to avirulent translucent (Tr) phenotypes with reduced CPS. The results discussed here confirmed homology of a V. vulnificus CPS locus to the group 1 CPS operon in Escherichia coli. However, two distinct V. vulnificus genotypes or alleles were associated with the operon, and they diverged at sequences encoding hypothetical proteins and also at unique, intergenic repetitive DNA elements. Phase variation was examined under conditions that promoted high-frequency transition of Op to Tr forms. Recovery of Tr isolates in these experiments showed multiple genotypes, which were designated TR1, TR2, and TR3: CPS operons of TR1 isolates were identical to the Op parent, and cells remained phase variable but expressed reduced CPS. TR2 and TR3 showed deletion mutations in one (wzb) or multiple genes, respectively, and deletion mutants were acapsular and locked in the Tr phase. Complementation in trans restored the Op phenotype in strains with the wzb deletion mutation. Allelic variation in repetitive elements determined the locations, rates, and extents of deletion mutations. Thus, different mechanisms are responsible for reversible phase variation in CPS expression versus genetic deletions in the CPS operon of V. vulnificus. Repetitive-element-mediated deletion mutations were highly conserved within the species and are likely to promote survival in estuarine environments.

FIG. 1.

Genetic organization of group 1 CPS operons. Group 1 CPS operon structure includes conserved transport and polymorphic biosynthetic regions for (A) V. vulnificus MO6-24/O (allele 1), (B) V. vulnificus YJO16 (allele 2), (C) V. fischeri, and (D) E. coli K30. The directions of transcription are indicated by arrows, and the predicted stem-loop transcriptional terminator in shown for V. vulnificus operons.

FIG. 2.

Alignment of group 1 CPS alleles for V. vulnificus. DNA alignment of the conserved transport regions are shown for (A) allele 1 and (B) allele 2 of the group 1 CPS operon for multiple strains. The strain numbers refer to CDC strain designations listed in Table 1. The brackets indicate multicopy R1 identical repeats (ACAGGACC) in allele 1 or R2 variable repeats (A/CCTAGAG/AGAA/C) in allele 2.

FIG. 3.

PCR analysis of CPS transport region in V. vulnificus phase variants. PCR amplicons derived from primers that spanned the transport region (wza to wzc) of the CPS operon, as described in Materials and Methods, are shown for phase variants of V. vulnificus MO6-24/O, MO6-24/TR1 (TR1), LC4/TR2 (TR2), 345/TR3 (TR3), and YJ016/TR2A (TR2A). Approximate amplicon sizes (arrows) are derived from DNA standards (not shown).

FIG. 4.

Allelic variation and deletion mutations in the group 1 CPS operon. Group 1 CPS operon structure and corresponding TR2 and TR2A deletion mutants are shown for (A) allele 1 and (B) allele 2. The directions of transcription are indicated by arrows, and genes removed by deletion mutations are shaded.