Showing papers by "Sumio Shinoda published in 2002"

Environmental investigation of potentially pathogenic Vibrio parahaemolyticus in the Seto‐Inland Sea, Japan

Abstract: Seawater and organic material (live and/or dead matter deposited on any substratum submersed in seawater) were collected during the cool weather season from a coast of the Seto-Inland Sea, Japan, and analyzed to determine Vibrio parahaemolyticus densities and the occurrence of pathogenic strains, defined as those possessing tdh and/or trh genes by the polymerase chain reaction (PCR), using isolated DNA from enrichment culture of the samples. About 95% of the samples were positive for V. parahaemolyticus (with densities of 3 to >1400 cells per 100 ml water or 10 g organic samples) by the most-probable-number (MPN)-PCR technique with species-specific toxR primers, but only 40% were positive by the conventional MPN-culture technique (with densities ranging from 3 to 240 cells per 100 ml water or 10 g organics). Furthermore, the tdh and trh genes were positive in 55% and 20% of samples, respectively, by the MPN-PCR technique. No tdh and trh gene-positive strains were isolated by the conventional MPN-culture procedure. The difference in detection between the MPN-culture and the MPN-PCR techniques appeared to be significant and may be attributed to different detection sensitivities and other factors.

Identification and Characterization of pvuA, a Gene Encoding the Ferric Vibrioferrin Receptor Protein in Vibrio parahaemolyticus

Abstract: We previously reported that Vibrio parahaemolyticus expresses two outer membrane proteins of 78 and 83 kDa concomitant with production of siderophore vibrioferrin in response to iron starvation stress and that these proteins are the ferric vibrioferrin receptor and heme receptor, respectively (S. Yamamoto, T. Akiyama, N. Okujo, S. Matsuura, and S. Shinoda, Microbiol. Immunol. 39:759-766, 1995; S. Yamamoto, Y. Hara, K. Tomochika, and S. Shinoda, FEMS Microbiol. Lett. 128:195-200, 1995). In this study, the Fur titration assay (FURTA) system was applied to isolate DNA fragments containing a potential Fur box from a genomic DNA library of V. parahaemolyticus WP1. Sequencing a 3.2-kb DNA insert in one FURTA-positive clone revealed that an amino acid sequence deduced from a partial gene, which was preceded by a full-length gene (psuA) encoding a receptor for a siderophore of unknown origin, was consistent with the N-terminal amino acid sequence of the 78-kDa ferric vibrioferrin receptor. Then, the full-length gene (pvuA) encoding the ferric vibrioferrin receptor was cloned and characterized. The deduced protein encoded by pvuA displayed the highest similarity (31% identity; 48% similarity) to RumA, a ferric rhizoferrin receptor of Morganella morganii. Primer extension and Northern blot analyses indicated that psuA and pvuA constitute an operon which is transcribed from a Fur-repressed promoter upstream of psuA. The product of the pvuA gene and its function were confirmed by generating a pvuA-disrupted mutant, coupled with genetic complementation studies. A mutant with disruption in the upstream psuA gene also displayed a phenotype impaired in the utilization of ferric vibrioferrin.

Purification of a Serine Protease of Vibrio parahaemolyticus and Its Characterization

Abstract: A 50 kDa protease designated as VPP1 was purified from the culture supernatant of a clinical strain of Vibrio parahaemolyticus by ammonium sulfate fractionation, Sephacryl S-200 HR gel filtration and Fractogel EMD TMAE 650 ion-exchange chromatography. VPP1 was inhibited by EDTA, EGTA and serine protease inhibitors, suggesting that it is a calcium-dependent serine protease. N-terminal amino acid sequence of VPP1 was quite similar to that of V. metschnikovii protease and antibody against VPP1 inhibited the activity of V. metschnikovii protease, suggesting the similarity of the two proteases. It was demonstrated that VPP1 or its related protease widely distribute in not only V. parahaemolyticus but also V. alginolyticus.

Specificity of a heme‐assimilating system of Vibrio vulnificus to synthetic heme compounds

Abstract: Vibrio vulnificus strain L-180, a clinical isolate, can obtain iron from a synthetic heme, iron–tetra(4-sulfonatophenyl)porphyrin (Fe–TPPS), as well as from a natural heme, protoheme. This assimilation of iron bound to TPPS was demonstrated to be a common property of V. vulnificus by testing a total of 27 strains isolated from both clinical and environmental sources. Strain L-180 could also utilize Fe–TCPP, but not Fe–TMPyP, as a sole iron source. TPPS or its complex with a metal ion reduced bacterial multiplication in the broth containing a minimum dose of Fe–TPPS. When inoculated into human serum supplemented with Fe–TCPP, L-180 could grow only in the presence of a protease from the same bacterium. In both TPPS and TCPP, each side chain of a porphyrin ring has a negative charge. Therefore, this negative charge may be important for interaction with an outer membrane receptor involving in a heme-assimilating system of V. vulnificus.