Molecular analysis of an extracellular protease gene from Vibrio parahaemolyticus

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Abstract
Summary: The structural gene prtVp encoding the extracellular protease of Vibrio parahaemolyticus strain 93 was cloned in Escherichia coli and sequenced. The cloned DNA fragment contained a 1761 bp ORF encoding a 587 amino acid protein. The deduced polypeptide is composed of a 25 amino acid signal peptide and a 562 amino acid extracellular polypeptide with a calculated molecular mass of 63 156 Da. Protease analysis using a gelatin-containing SDS-polyacrylamide gel detected the presence of a 62 kDa protease that was present in the culture supernatant fractions of the wild-type V. parahaemolyticus strain and of E. coli bearing a pUC119 recombinant with the prtVp DNA insert. The protease activity was inhibited by zinc- and metal-specific inhibitors such as EDTA and 1,10-phenanthroline, which suggested that it is a metalloprotease. The deduced amino acid sequence of PrtVp has 32% identity with that of the collagenase of Vibrio alginolyticus, but has no identity with those of the bacterial proteases. A conserved zinc-binding domain was also found in PrtVp from homology comparison with other metalloproteases. This PrtVp can cause weak haemolysis on blood agar.

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