Cloning and characterization of a periplasmic nuclease of Vibrio vulnificus and its role in preventing uptake of foreign DNA

We have cloned a nuclease gene, vvn, from Vibrio vulnificus, an estuarine b acterium that causes wound infections and septicemia in humans and eels, Th e gene contained a 696-bp open reading frame encoding 232 amino acids (aa), including a signal sequence of 18 aa. The deduced amino acid sequence of t he mature nuclease predicted a molecular mass of 25 kDa, which was confirme d by vital stain, and a pi of 8.6, Vvn was produced in the periplasm of eit her V. vulnificus or recombinant Escherichia coil strains and was active in the oxidized (but not the reduced) form. This nuclease was able to digest DNA and RNA, with differential thermostability in DNase and RNase activitie s. Expression of Vvn in E. coli DH5 alpha reduced the frequencies of transf ormation with the divalent ion-treated cells and electroporation by about 6 and 2 logs, respectively. In addition, the transformation frequency of a V vn-deficient V. vulnificus mutant (ND) was 10-fold higher than that of the parent strain, These data suggested that VM may be involved in preventing u ptake of foreign DNA by transformation. However, VM expressed in the recipi ents had little effect on the conjugation frequency in either E. coli or V. vulnificus, Some other DNase(s) may be present in the periplasm and respon sible for a residual DNase activity, which was about one-fourth of that of the parent strain, detected in the ND mutant. We also demonstrated that VM was not required for the,irulence of V. vulnificus mice.