Genetic Characterization of DNA Region Containing the trh and ure Genes of Vibrio parahaemolyticus

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Abstract
We have demonstrated that possession of the gene for thermostable direct hemolysin-related hemolysin (trh) coincides with the presence of the urease gene among clinical Vibrio parahaemolyticus strains and that the location of the two genes are in close proximity on the chromosome. Here, we cloned and sequenced the 15,754-bp DNA region containing the trh gene and the gene cluster for urease production from the chromosome of clinicalV. parahaemolyticus (TH3996). We found 16 open reading frames (ORFs) and a lower G+C content (41%) compared with the total genome of this bacterium (46 to 47%). The ure cluster consisted of eight genes, namely, ureDABCEFG andureR. ureR was located 5.2 kb upstream of the other seven genes in the opposite direction. The genetic organization and sequences of the ure genes resembled those found in Proteus mirabilis. Between ureR and the other uregenes, there were five ORFs, which are homologous with the nickel transport operon (nik) of Escherichia coli. We disrupted each of the ureR, ureC, andnikD genes in TH3996 by homologous recombination and analyzed the phenotype of the mutants. In the presence of urea these mutant strains had dramatically less urease activity than the strain they were derived from. Disruption of ureR,nikD, or ureC, however, had no effect on TRH production. The DNA region containing the trh,nik, and ure genes was found in onlytrh-positive strains and not in Kanagawa phenomenon-positive and environmental V. parahaemolyticusstrains. At the end of the region, an insertion sequence-like element existed. These results suggest that the DNA region was introduced intoV. parahaemolyticus in the past through a mechanism mediated by insertion sequences. This is the first reported case that the genes for an ATP-binding cassette-type nickel transport system, which may play a role in nickel transport through bacterial cytoplasmic membrane, are located adjacent to the ure cluster on the genome of an organism.