Date of Award

1997

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Ronald J. Siebeling

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 the production of the polysaccharide capsule, 23 mutants were generated that lost their ability to produce CPS due to the insertion of a mini-transposon into the genome of the encapsulated clinical strain V. vulnificus 1003 (O). The mutants were examined by Southern blot analysis to ensure the transposon had inserted only once in the chromosome. Exoenzymes, which are believed to contribute to the virulence of the organism, produced by all 23 transposon mutants, the encapsulated strain 1003 (O) and a spontaneously-derived translucent strain 1003 (T) were compared. The transposon mutants produced the same exoenzymes as did 1003 (O), but the translucent 1003 (T) varied in its ability to produce certain exoenzymes, suggesting that the mechanism responsible for the loss of capsule production in the environment may effect exoenzyme production. Using a probe complimentary to the genomic region disrupted by the transposon in one mutant, the putative capsule gene was located in the parent strain 1003 (O) and then cloned and sequenced. Sequence analysis determined that the gene disrupted by the transposon matched a nucleotide-sugar epimerase of Vibrio cholerae O139 with 75% and 85% identity at the nucleotide and amino acid level, respectively. Numerous epimerases of various organisms were also recognized by computer analysis to be highly similar to the putative epimerase of V. vulnificus. PCR amplification and Southern blotting have shown that this epimerase is common to at least ten strains of V. vulnificus that each express a serologically distinct CPS, suggesting this gene product provides a precursor necessary for the production of CPS independent of capsule type. Finally, the nonencapsulated mutant was capable of reestablishing capsule formation upon introduction of the epimerase gene in trans, indicating that the loss of CPS was due to the disruption of the epimerase gene and not a polar effect on downstream genes. It may therefore be concluded that the nucleotide-sugar epimerase is essential for capsule production in Vibrio vulnificus.

ISBN

9780591724325

Pages

103

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