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英文摘要
Vibrio vulnificus is a halophilic gram-negative marine
bacterium that when infected could cause fulminant septicemia
with a high mortality rate and serious wound infection in
persons who have underlying conditions, especially those with
liver cirrhosis or hemochromatosis. This organism exhibits
DNase activity on the DNase test agar. In order to study the
role of the DNase activity in the pathogenesis and physiology
of V. vulnificus, we cloned and determined the nucleotide
sequence of the gene encoding this enzyme. From the gene
library of V. vulnificus YJ016, 8 nuclease-producing clones
were screened and were proved to contain the same nuclease gene.
From these clones, SL006 was selected for extensive studies.
The nuclease gene was mapped to a 1.7-kb MluI-BamHI fragment by
deletion analysis. This fragment was then subcloned to pUC19
to facilitate nucleotide sequence determination. Two open
reading frames were identified in this sequence. One was 696 bp
long encoding a protein of 232 amino acids（aa）, and the
deduced amino acid sequence of it shared 78 % homology with
that of the mature Vibrio cholerae DNase. Its gene product
（designated Vvn） was able to digest both DNA, either circular
or linear form, and RNA, and remained active for digesting DNA
after a heat treatment at 100℃for 30 min. The molecular weight
of Vvn estimated either by protein gel electrophoresis or from
the deduced aa sequence was approximately 24 kDa, and the pI
value estimated from the deduced aa sequence was 8.61. Vvn
contains a signal sequence of 18 aa with a signal peptidase
cleavage site between two alanine residues. The nuclease
acativity was detected in the periplasmic fraction of either
V. vulnificus or the recombinant E. coli strain, and was lost
when Vvn was reduced by treatment with 2-mercaptoethanol.
E. coli K12 strain DH5a expressing Vvn from a high copy number
plasmid showed a lower frequency, when served as the recipient,
in conjugation, electroporation, and especially, transformation
, compared with that was not expressing the nuclease. This
suggests that Vvn might hamper the gene transfer processes,
perhaps would not allow the recipient to retain the transferred
gene. Therefore, disruption of the nuclease gene in
V. vulnificus should render gene transfer to this organism
easier and facilitate further genetic studies of this organism.
Such mutants should also be useful for determining the role of
V. vulnificus nuclease in the pathogenesis.
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