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 bacterium that causes wound infections and septicemia in humans and eels. The 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 the mature nuclease predicted a molecular mass of 25 kDa, which was confirmed by vital stain, and a pI of 8.6. Vvn was produced in the periplasm of either V. vulnificus or recombinant Escherichia coli 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 activities. Expression of Vvn in E. coli DH5alpha reduced the frequencies of transformation with the divalent ion-treated cells and electroporation by about 6 and 2 logs, respectively. In addition, the transformation frequency of a Vvn-deficient V. vulnificus mutant (ND) was 10-fold higher than that of the parent strain. These data suggested that Vvn may be involved in preventing uptake of foreign DNA by transformation. However, Vvn expressed in the recipients 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 responsible 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 Vvn was not required for the virulence of V. vulnificus mice.
Wu
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SI
Department of Microbiology and Immunology, College of Medicine, National Cheng-Kung University, Tainan 701, Taiwan, Republic of China.
Lo
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SK
Shao
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CP
Tsai
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HW
Hor
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LI
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D003227:Conjugation, Genetic; D004247:DNA / Q000378:metabolism; D003851:Deoxyribonucleases / Q000235:genetics* / Q000378:metabolism*; D018274:Electroporation / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D004950:Esterases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D019897:Periplasm / Q000201:enzymology*; D012335:RNA, Ribosomal / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013347:Subcellular Fractions; D014169:Transformation, Bacterial; D014733:Vibrio / Q000201:enzymology* / Q000235:genetics; D014735:Vibrio Infections / Q000382:microbiology
D001426:Bacterial Proteins; D012335:RNA, Ribosomal; D004247:DNA; D003851:Deoxyribonucleases; D004950:Esterases; C425771:Vvn protein, Vibrio vulnificus