MOLECULAR ANALYSIS OF AN EXTRACELLULAR PROTEASE GENE FROM VIBRIO-PARAHAEMOLYTICUS

The structural gene prtVp encoding the extracellular protease of Vibri o parahaemolyticus strain 93 was cloned in Escherichia coli and sequen ced. The cloned DNA fragment contained a 1761 bp ORF encoding a 587 am ino acid protein. The deduced polypeptide is composed of a 25 amino ac id signal peptide and a 562 amino acid extracellular polypeptide with a calculated molecular mass of 63156 Da. Protease analysis using a gel atin-containing SDS-polyacrylamide gel detected the presence of a 62 k Da protease that was present in the culture supernatant fractions of t he wild-type V. parahaemolyticus strain and of E. coil bearing a pUC11 9 recombinant with the prtVp DNA insert. The protease activity was inh ibited by zinc- and metal-specific inhibitors such as EDTA and 1,10-ph enanthroline, which suggested that it is a metalloprotease. The deduce d amino acid sequence of PrtVp has 32% identity with that of the colla genase 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 PrtV p can cause weak haemolysis on blood agar.