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Sequence variation in the thermostable direct hemolysin-related hemolysin (trh) gene of Vibrio parahaemolyticus.

ABSTRACT: Our previous molecular epidemiologic study with gene probes (H. Shirai, H. Ito, T. Hirayama, Y. Nakamoto, N. Nakabayashi, K. Kumagai, Y. Takeda, and M. Nishibuchi, Infect. Immun. 58:3568-3573, 1990) demonstrated that the gene (trh) encoding a thermostable direct hemolysin-related hemolysin was strongly associated with clinical strains of Vibrio parahaemolyticus. Strain-to-strain variation in the intensities of the hybridization signals observed in the above study also suggested that the trh genes in different strains may have significantly divergent nucleotide sequences. To assess the public health significance of the rare environmental strains which exhibited very weak hybridization signals with the trh gene-specific DNA probe, the trh-like sequence was cloned from one of the environmental strains and the nucleotide sequence was determined in this study. A hemolysin gene (trh2) which was 84% homologous to the trh gene (newly named trh1) and 54.8 to 68.8% homologous to the genes (tdh) encoding thermostable direct hemolysins was detected in the cloned sequence. The trh2 gene product showed a profile of hemolytic activities against various animal erythrocytes different from that of the trh1 gene product. The trh2 gene product was antigenically related (partially identical) to the trh1 and tdh gene products. DNA colony blot and Southern blot hybridization analyses with trh1- and trh2-specific DNA probes showed that the trh1 probe-positive strains exhibiting hybridization signals with varying intensities could be clustered into trh1 and trh2 subgroups. In addition, hybridization analysis with oligonucleotide probes demonstrated significant strain-to-strain variation in the trh1 and trh2 gene sequences.(ABSTRACT TRUNCATED AT 250 WORDS)

SUBMITTER: Kishishita M

PROVIDER: S-EPMC195802 | biostudies-other | 1992 Aug

REPOSITORIES: biostudies-other

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Sequence variation in the thermostable direct hemolysin-related hemolysin (trh) gene of Vibrio parahaemolyticus.

Kishishita M M Matsuoka N N Kumagai K K Yamasaki S S Takeda Y Y Nishibuchi M M

Applied and environmental microbiology 19920801 8

Our previous molecular epidemiologic study with gene probes (H. Shirai, H. Ito, T. Hirayama, Y. Nakamoto, N. Nakabayashi, K. Kumagai, Y. Takeda, and M. Nishibuchi, Infect. Immun. 58:3568-3573, 1990) demonstrated that the gene (trh) encoding a thermostable direct hemolysin-related hemolysin was strongly associated with clinical strains of Vibrio parahaemolyticus. Strain-to-strain variation in the intensities of the hybridization signals observed in the above study also suggested that the trh gene ...[more]

PMID: 1514791

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Project description:In an effort to identify the regulatory gene controlling the expression of the tdh gene, encoding the thermostable direct hemolysin of Vibrio parahaemolyticus, we examined total DNA of AQ3815 (a Kanagawa phenomenon-positive strain) for sequences homologous to that of the toxR gene of Vibrio cholerae. The extracted DNA gave a weak hybridization signal under reduced-stringency conditions with a toxR-specific DNA probe. Cloning and sequence analysis of the probe-positive sequence revealed an operon (Vp-toxRS) which was highly similar to the toxRS operon of V. cholerae (Vc-toxRS) (52 and 62% similarities in the two genes, respectively). The deduced amino acid sequences of the Vp-toxRS gene products (Vp-ToxRS) contained regions similar to the proposed transmembrane and activity domains of the Vc-toxRS gene products (Vc-ToxRS). All clinical and environmental strains of V. parahaemolyticus examined possessed the Vp-toxRS genes. In the presence of Vp-ToxS, Vp-ToxR promoted expression of the tdh2 gene, one of two tdh genes (tdh1 and tdh2) carried by Kanagawa phenomenon-positive strains. The DNA sequence located 144 bp upstream of the tdh2 coding region was shown to be important for the Vp-ToxR-stimulated expression of the tdh2 gene in an Escherichia coli background. Comparative analysis of AQ3815 and its isogenic Vp-toxR null mutant gave the following results: (i) Vp-ToxR promoted, in an AQ3815 background, expression of the tdh gene to different degrees in various culture media, with KP broth (2% peptone, 0.5% NaCl, 0.03 M KH2PO4, pH 6.2) being most effective (12-fold); (ii) the promotion of tdh gene expression in KP broth was at the level of transcription; and (iii) Vp-ToxR was essential for demonstration of enterotoxic activity of AQ3815 in the rabbit ileal loop, a model previously used to demonstrate thermostable direct hemolysin-mediated enterotoxic activity of AQ3815. These results demonstrate that Vp-ToxR and Vc-ToxR share a strikingly similar function, i.e., direct stimulation at the transcriptional level of the gene encoding a major virulence determinant (enterotoxin) of a Vibrio species.

Project description: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 clinical V. 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 and ureR. 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 ure genes, there were five ORFs, which are homologous with the nickel transport operon (nik) of Escherichia coli. We disrupted each of the ureR, ureC, and nikD 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 only trh-positive strains and not in Kanagawa phenomenon-positive and environmental V. parahaemolyticus strains. At the end of the region, an insertion sequence-like element existed. These results suggest that the DNA region was introduced into V. 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.

Dataset Information

Sequence variation in the thermostable direct hemolysin-related hemolysin (trh) gene of Vibrio parahaemolyticus.

Publications

Sequence variation in the thermostable direct hemolysin-related hemolysin (trh) gene of Vibrio parahaemolyticus.

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