Cloning of a metalloprotease gene involved in the virulence mechanism of Vibrio anguillarum.
Genetic evidence has previously suggested that a zinc metalloprotease is involved in the invasive mechanism of the fish pathogen Vibrio anguillarum NB10. In this study, the metalloprotease gene was cloned and sequenced. The sequence encodes a polypeptide (611 amino acids) that contains a putative signal sequence followed by a large leader sequence and the mature protein (44.6 kDa). Since the purified protein has a molecular mass of 36 kDa instead of the predicted 44.6 kDa, the mature protein is most likely processed a third time. Comparative analyses of the protein sequence showed high homologies to other bacterial metalloproteases within the zinc-binding and active-site regions. The Vibrio cholerae hemagglutinin/protease and the Pseudomonas aeruginosa elastase were exceptions in that the homology extended throughout the entire putative preproprotein. A chromosomal metalloprotease mutant was made via the integration of foreign DNA into the protease gene. This mutant did not secrete the metalloprotease, as determined by sodium dodecyl sulfate (SDS)-polyacrylamide protein analysis and by growth on gelatin agar. Transcomplementation of the chromosomal mutation revived the secretion of the metalloprotease and its activity on gelatin agar. Interestingly, when supernatant proteins were analyzed by gelatin-SDS-polyacrylamide electrophoresis, two different proteases (75 and 30 kDa) were detected in the mutant strain but not in the transcomplemented strain or the wild-type strain. Moreover, fish infection studies were done, and implications for the role of the metalloprotease in the virulence mechanism of V. anguillarum are discussed.
Milton
D L
DL
Department of Cell and Molecular Biology, University of Umeå, Sweden.
Norqvist
A
A
Wolf-Watz
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D005393:Fish Diseases / Q000382:microbiology*; D005798:Genes, Bacterial; D015698:Genomic Library; D007700:Kinetics; D008666:Metalloendopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015345:Oligonucleotide Probes; D010196:Pancreatic Elastase / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014337:Trout / Q000382:microbiology; D014733:Vibrio / Q000201:enzymology / Q000235:genetics* / Q000472:pathogenicity*; D014735:Vibrio Infections / Q000378:metabolism / Q000662:veterinary*; D014774:Virulence / Q000235:genetics*
D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D010196:Pancreatic Elastase; D008666:Metalloendopeptidases