An epimerase gene essential for capsule synthesis in Vibrio vulnificus

Abstract

The extracellular capsule polysaccharide (CPS) of Vibrio vulnificus is a primary virulence factor which allows survival of the bacteria in the human host. To study the genes involved in expression of the capsule, we generated mutants that lost the ability to produce CPS following the insertion of a minitransposon into the genome of an encapsulated, clinical strain of V. vulnificus. A genomic region, from one nonencapsulated mutant, containing the transposon and flanking V. vulnificus DNA was cloned, and a probe complementary to the chromosomal DNA immediately adjacent to the transposon was used to locate this fragment in the genome of the encapsulated parent strain. The fragment, which contained a putative capsule gene, was cloned and, when supplied in trans, complemented the mutation in the nonencapsulated mutant to restore capsule production. In addition, virulence studies, using the 50% lethal dose assay, showed that the restoration of capsule production also restored the virulence of the organism. Sequence analysis of the gene disrupted by the transposon revealed that it matched a nucleotide-sugar epimerase of Vibrio cholerae O139, with 75 and 85% identities at the nucleotide and amino acid levels, respectively. In addition, computer analysis recognized epimerases of various organisms as highly similar to the putative epimerase of V. vulnificus. Finally, a combination of PCR amplification and Southern blotting showed that this epimerase is common to at least 10 strains of V. vulnificus that each express a serologically distinct CPS. Our results indicate that the epimerase gene is essential for capsule expression in V. vulnificus.

FIG. 1

Southern blot of epimerase-probe A hybridized to a 6.5-kb fragment of V. vulnificus 1003 (lane 1) and a 21-kb fragment of V. cholerae O139 (lane 2), both digested with EcoRI. Lane MW contains molecular weight markers, sizes of which are shown in kilobases at the left.

FIG. 2

Map of the pEpiBS vector, which consists of a 6.5-kb insert of V. vulnificus 1003(O) chromosomal DNA cloned into pBluescript at EcoRI restriction enzyme sites. Genes originating from the pBluescript vector itself are designated by open arrows. The positions and orientations of the epimerase gene and rfbQRS are shown by the striped and solid arrows, respectively.

FIG. 3

(a) PCR amplification of the 139-bp region of the putative epimerase gene in V. vulnificus C7184 (lane 1), 1010 (lane 2), 1014 (lane 3), 1007 (lane 4), 1657 (lane 5), 1866 (lane 6), 1002 (lane 7), 549 (lane 8), 938 (lane 9), 1456 (lane 10), and 1003 (lane 11). (b) Southern blot of the PCR products (see above) hybridized by an 83-bp probe (epimerase-probe B) specific for a region of the epimerase gene of V. vulnificus.