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In response to low iron availability, Vibrio parahaemolyticus synthesizes and secretes a polyhydroxycarboxylate-type siderophore vibrioferrin which is composed of 1 mol each of 2-ketoglutaric acid, L-alanine, ethanolamine, and citric acid. We have previously reported the cloning and characterization of the pvuA gene, which encodes the 78-kDa outer membrane receptor protein for ferric vibrioferrin. In this study, nine genes involved in the biosynthesis and transport of vibrioferrin have been identified in the genomic regions surrounding the pvuA gene. The genes were sequenced, and gene disruptants were constructed by insertion mutation for phenotype analysis. Five of the genes, named pvsABCDE, constitute an operon that is expressed under iron-limiting conditions. Homology searches of their predicted protein products suggested that the four genes pvsABDE are implicated in the biosynthesis of the siderophore. Another gene in the same operon, pvsC, encodes a putative exporter that is homologous to members of the major facilitator superfamily of multidrug efflux pumps. The remaining four genes, named pvuBCDE, encode proteins strongly homologous to Escherichia coli FecBCDE, respectively, which are components of the ATP-binding cassette transporter system for ferric dicitrate. Reverse transcriptase PCR analysis revealed that these transport genes are transcribed as a single mRNA with the upstream genes, psuA and pvuA. Phenotypic comparison between the wild-type strain and its targeted gene disruptants supported the biological functions for the respective operons that were expected on the basis of the homology search.

A protease was purified from a strain of Vibrio vulnificus isolated from the blood of a septicemic human. The vibrio was cultured in bacto peptone-yeast extract medium, and the protease was purified by a purification procedure including ultrafiltration of the culture supernatant with an Amicon YM 5 membrane, diethylaminoethyl-Sephacel column chromatography, Sephacryl S-200 column chromatography and fast protein liquid chromatography on Mono Q column. The protease preparation revealed homogeneity on polyacrylamide gel electrophoresis and about 30,000-fold purification was achieved, with a yield of about 30% . The isoelectric point of the purified V. vulnificus protease was about 5.80 and its molecular weight was ca. 45,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH of the protease activity was 8.0. The V. vulnificus protease was inhibited by a metalloprotease inhibitor and zinc ion and/or ferrous ion were essential for its enzyme activity. No cysteine residue was detected in the V. vulnificus protease. The protease had caseinolytic, elastolytic and collagenolytic activities.The illnesses caused by Vibrio vulnificus, a marine bacterium, are divided into two groups, wound-infection group and primary septicemia group (2, 3). In the former, edema and erythema are formed around a new wound in contact with sea water. In the latter group, cutaneous lesions, such as bullae, erythema or necrotic ulcer, often appear on one or more extremities.V. vulnificus is known to produce many toxic or pathogenic factors, such as cytolysin (8, 13, 23), protease (11, 13, 25), phospholipase A2 (27), and siderophores (24). However, in the pathogenesis of V. vulnificus infection, the actual role of any product has not been clarified yet. Kreger and Lockwood (13) reported that V. vulnificus cytolysin activity in the culture filtrate decreased in the stationary phase of the growth and the decrease correlated with the appearance of protease in the culture filtrate. We also observed significant excretion of protease from many strains of V. vulnificus isolated from patients. The pathogenic significance of proteases by some bacteria, such as pseudomonal protease (7,10,12) and serratial protease (9,16,19), has been reported. These proteases have vascular permeability-enhancing and hemorrhagic activities which are closely related to the above mentioned cutaneous lesions. We also found that the crude V. vulnificus protease preparation

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Twenty‐three strains of Salmonella spp. isolated from healthy humans in Guangdong, China, were examined for their susceptibility to ten common antibiotics and the presence of antibiotic resistance integrons. All the strains were resistant to at least one antibiotic, and 4 strains were positive for the intI1 gene. Polymerase chain reaction using in‐F and in‐B primers showed the existence of amplicons of 1,009 bp in two, 1,664 bp in one, and 1,009 bp and 1,664 bp in one of the intI1‐positive isolates, respectively. Sequence analysis revealed that the 1,009‐bp amplicon harbored gene cassette aadA2, conferring resistance to spectinomycin, and the 1,664‐bp amplicon harbored genes aadA5 and dfr17, conferring resistance to spectinomycin, streptomycin and trimethoprim. Meanwhile the experiments of plasmid conjugation and Southern hybridization with intI1 as the DNA probe indicated that all the integrons found in these strains were chromosomal. Because the strains carrying class 1 integrons were isolated from healthy humans, it suggests the need for all‐round surveillance of the antibiotic resistance of pathogens.