INTRODUCTION

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Vibrio parahaemolyticus is a gram-negative, halophilic marine bacterium which causes gastroenteritis in humans who are infected through consumption of raw or inadequately cooked seafood (15). V. parahaemolyticus strains that are isolated from diarrheal patients produce either the thermostable direct hemolysin (TDH) or the TDH-related hemolysin (TRH), or both, while hardly any isolates from the environment have these properties (15, 41). TDH and TRH, encoded by tdh and trh, respectively, are each composed of 165-amino-acid residues and show approximately 67% identity in their amino acid sequences. TDH and TRH show several common biological activities, such as hemolytic activity, enterotoxicity, cytotoxicity, and cardiotoxicity, and are considered to be the major virulence factors in the pathogenesis of V. parahaemolyticus (15, 35, 41, 47).

Urease is an enzyme that catalyzes the hydrolysis of urea to ammonia and carbon dioxide. It is found in a number of bacteria, plants, fungi, and algae (29). Several bacterial urease genes have been isolated and characterized. The types of organization of gene clusters for urease in most bacteria are basically similar, comprising structural genes, accessory genes involved in the incorporation of the nickel ion into the apoenzyme and, in some cases, genes involved in a nickel uptake or regulation of urease expression (30). In common with other Vibrio species, generally only a small population (several percent) of clinical V. parahaemolyticus strains produce urease (7, 13, 16, 46). The relatively rare urease-positive phenotype of V. parahaemolyticus is always associated with the possession of the trh gene (26, 36, 37, 46), making urease production a reasonably good clinical diagnostic marker for virulent (trh-positive) V. parahaemolyticus (46). Our previous research has shown that the association is due to a genetic linkage between the structural gene for urease (ureC) and trh on the chromosome of virulent V. parahaemolyticus strains (17). In one such strain, using long and accurate PCR (LA-PCR), the distance between trh and ureC was shown to be less than 8.5 kb (18). We also recently reported that V. parahaemolyticus and other Vibrio species possess two chromosomes and that, when present, the trh and ureC genes are localized on the smaller replicon (52).

In this study, to precisely understand the nature of the genetic linkage between the trh and ure genes in V. parahaemolyticus, we cloned and sequenced a DNA region (ca. 16 kb) containing the genes from a clinical V. parahaemolyticus strain. Furthermore, we constructed mutant strains with disruption in the genes on this DNA region to examine the role of the genes to urease production and hemolytic activity of V. parahaemolyticus.

	MATERIALS AND METHODS

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Bacterial strains and growth conditions. Table 1 shows the bacterial strains and plasmids used in this study. Clinical V. parahaemolyticus strains, including TH3996 (51), were isolated at the Osaka International Airport quarantine station from patients with traveller's diarrhea. The bacterium was cultured at 37°C with shaking in Luria-Bertani (LB) broth (tryptone, 1%; yeast extract, 0.5%) with 3% NaCl. The Escherichia coli JM109 (53) and SM10pir (28) strains that we used for general manipulation of plasmids and mobilization of plasmid into V. parahaemolyticus were grown in LB broth or on LB agar. TCBS agar (Nissui, Tokyo, Japan) was used for the screening of mutant strains. Antibiotics were used at the following concentrations: ampicillin (100 µg/ml), kanamycin (50 µg/ml), and chloramphenicol (5 µg/ml).

DNA manipulation. Chromosomal DNA from V. parahaemolyticus TH3996 was extracted from overnight culture of the organism in LB broth with 3% NaCl using a previously described method (50). DNA used for subcloning or nucleotide sequence analysis was extracted from E. coli by using the QIAPrep Spin Plasmid Kit (Qiagen) according to the manufacturer's recommendations. Cloning, restriction endonuclease procedures, DNA ligation, and transformation of E. coli by plasmids were carried out using previously described standard protocols (40). All of the restriction enzymes and DNA ligation kits were purchased from Takara Shuzo (Otsu, Japan).

Southern blot analysis. Digested chromosomal DNA was resolved in 0.8% agarose gel in 1× TBE buffer (0.09 M Tris-borate, 2 mM EDTA [pH 8.0]) and transferred onto a nylon membrane (GeneScreen Plus; Dupont). DNA was fixed to the membrane by UV cross-linking using a GS Gene Linker UV chamber (Bio-Rad). We prepared probes using the PCR DIG Probe Synthesis Kit (Boehringer Mannheim) with specific primers for each gene. Hybridization was carried out at 40°C, and hybridized DNA was detected with an alkaline phosphatase-labeled anti-digoxigenin monoclonal antibody (Boehringer Mannheim).

DNA sequence analysis. The nucleotide sequences were determined with a Licor model 4000L automated DNA sequencer (Lincoln, Nebr.) using universal M13 IRD41 infrared-dye-labeled primers. Sequence data were analyzed using the DNASIS program (Hitachi Software, Tokyo, Japan) and the GENETYX sequence analysis program (Software Development, Tokyo, Japan). The homology of the open reading frames (ORFs) was ascertained via the National Center for Biotechnology Information using the BLAST network service to search GenBank.

Oligonucleotide primers and PCR conditions. The following oligonucleotide primers were used: for the cloning of pKS3, 5'-TCGCATAATTGGTGTTGAA-3' and 5'-TTTTGGCAGCATACCTTTG-3'; for the Cm probe, 5'-TTGCCCGCCTGATGAATGCTC-3' and 5'-CCCTGCCACTCATCGCAGTA-3'; for the nikD mutant, 5'-GTCAAATTGTCAACTCGT-3' and 5'-CAATGGTTTCGCCATTAG-3'; for the ureR mutant, 5'-CACAAAGAAAATGGCATAT-3' and 5'-CGAAATTGCAGTGGTGTT-3'; for the ureC mutant, 5'-ACTAATGCTACAACAGTCAC-3' and 5'-ATGCTGGAATGATGTTAGGT-3'; and for LA-PCR, 5'-CATTTCCGCTCTCATATGC-3' and 5'-ATGCTGGAATGATGTTAGGT-3'.

Construction of mutant strains. Mutant strains, derived from V. parahaemolyticus TH3996, were constructed as previously described (51), except that the homologous recombination was done by a single-crossover event. Briefly, to construct the mutants, using the chromosomal DNA of V. parahaemolyticus TH3996 as a template, we amplified partial sequences of ureR, nikD, and ureC by PCR. Each amplified fragment was cloned into a pT7-Blue vector and then digested with BamHI and PstI. These fragments were cloned into a R6K-ori suicide vector, pKY719 (33), digested with BamHI and PstI. These plasmids were introduced into E. coli SM10pir and then mated with V. parahaemolyticus TH3996. Conjugation was performed on nitrocellulose membrane laid over LB agar plates with incubation for 4 h at 37°C. The membrane was suspended in LB broth and inoculated onto TCBS agar plates containing 5 µg of chloramphenicol per ml and incubated overnight at 37°C. Chloramphenicol-resistant colonies were isolated and screened by Southern blot analysis for single-crossover mutation of ureR, nikD, or ureC.

Urease assay. Bacteria were grown at 37°C with shaking in 100 ml of LB broth (3% NaCl) supplemented with 0.1% sterile urea after autoclaving of the medium. The organisms were harvested by centrifugation at 10,000 × g for 10 min, washed three times with 20 mM sodium phosphate (pH 7.0)-5 mM dithiothreitol-1 mM EDTA, and disrupted by sonication. Subsequently, supernatant was obtained after centrifugation at 4°C (12,000 × g, 20 min). The supernatant was used both for the urease assay and for the determination of protein quantity using the Bio-Rad DC protein assay kit (Bio-Rad). Urease activity was determined using an ammonia test kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan) to measure the release of ammonia. The amount of ammonia produced was calculated by referring to a standard curve made for known concentrations of ammonium chloride. Urease activity was expressed as micromoles of urea hydrolyzed per minute per milligram of protein.

Hemolytic activity assay. Wild-type and mutant strains were cultured at 37°C for 16 h with shaking in SPP medium (14) (5 g of NaCl, 10 g of peptone, 2 g of glucose, and 5 g of dibasic sodium phosphate per liter in distilled water, pH 7.6). The supernatant was obtained by centrifugation (12,000 × g, 10 min) at 4°C. The hemolytic activity was determined by a modification of the previously described method (14). Briefly, sheep erythrocytes were washed in phosphate-buffered saline (PBS) three times and adjusted to a hematocrit of 4% with PBS. Equal volumes (100 µl each) of supernatant serially diluted with PBS and erythrocyte suspension were mixed and incubated at 37°C in a water bath for 1 h and then centrifuged at 2,000 × g for 2 min. Supernatant samples (160 µl) of the reaction mixtures were taken for spectrophotometric measurement of released hemoglobin at 540 nm on a 96-well plate using a Multiskan Mcc/340 (Labosystems, Tokyo, Japan).

Rabbit ileal loop test. We assessed the enterotoxic activity of mutant strains and the wild-type strain by using a previously described method (51).

Nucleotide sequence accession number. The nucleotide sequence data reported here will appear in the DDBJ, EMBL and the GenBank sequence databases under accession no. AB038238.

	RESULTS

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Cloning and sequencing of the region containing the trh and ure cluster. For the cloning of the region containing the trh and ure cluster, total chromosomal DNA was isolated from V. parahaemolyticus TH3996 and completely digested with several restriction enzymes. The DNA fragments were Southern blotted with a digoxigenin-labeled ure probe (17). The 11.8-kb XhoI fragment and the 7.3-kb BglII fragment which hybridized with the ure probe were cloned to the pUC119 vector at the SalI and BamHI sites (designated pKS1 and pKS2, respectively). The adjacent region of the 11.8-kb XhoI fragment, which as a 4.0-kb SpeI fragment hybridized with probe A (Fig. 1), was cloned into the XbaI site of the pUC19 vector (designated pKS3). In the end, the total region of approximately 16 kb that contains the trh and ure cluster was cloned (Fig. 1). After analysis of the restriction maps, each fragment was digested with appropriate restriction enzymes and subcloned into the corresponding sites of the pUC119 vector. The complete nucleotide sequence was determined in both directions.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Schematic representation of the region containing the trh and ure gene cluster and ORF assignment.

Nucleotide sequence analysis. Nucleotide sequence analysis of the DNA region revealed that the region is 15,754 bp in size and possesses a total of 16 ORFs (Fig. 1 and Table 2). ORF1 as homologous with a transposase found in Vibrio anguillarum (88% identity; L40498) and was flanked by 18-bp inverted repeats (5'-GGCTTTGTTGCGTAATTC-3') in both sides. ORF2 was the trh gene. ORF3 had some homology with two positive regulators for urease expression, P. mirabilis UreR (51% identity; A40644) and E. coli UreR (50% identity; P32326). In addition, the ORF contained a typical bacterial regulatory AraC family signature located at amino acid positions 215 to 257. Therefore, ORF3 was designated UreR.

Construction of ureR, nikD, and ureC mutant strains. To determine the possible contribution of a putative nickel transport gene operon and urease gene cluster to urease production and hemolytic activity, each of the genes (ureR, nikD, and ureC) was disrupted by homologous recombination in V. parahaemolyticus TH3996.

Urease activity of wild and mutant strains. In P. mirabilis, which possesses the ureR gene, urease production is not regulated by the nitrogen regulatory system (ntr) or catabolite repression but is induced by urea and the induction is mediated by ureR (8). Because V. parahaemolyticus TH3996 has the ureR gene in common with P. mirabilis, we investigated whether or not the urease production of the TH3996 strain is affected by the presence of urea. In the LB broth with 3% NaCl without urea, there was little urease production. With 0.1% urea supplementing the medium, however, urease production was 100 times greater. These data suggest that urea is involved in the induction of V. parahaemolyticus urease. Therefore, the following experiments for examining urease production were conducted with media containing 0.1% urea. The urease activities of ureR, nikD, and ureC mutant strains were very low, even in the presence of 0.1% urea (Table 3). In contrast, under the same conditions the urease activity of the trh mutant strain (51) did not change compared with the wild-type strain. These results suggest that ureR, nikD, and ureC genes are essential for active urease production. From these results, we concluded that the ureR, nikD, and ureC genes are important for urease expression, whereas trh is not.

Hemolytic activity of mutant strains. Next, we investigated the possible contribution of the ureR, nikD, and ureC genes to hemolysin (TRH) production by the TH3996 strain by comparing the hemolytic activity of wild-type and mutant strains. As shown in Table 3, the nikD, ureR, and ureC mutant strains retained the same hemolytic activity.

Enterotoxicity of mutants as shown by the rabbit ileal loop test. To investigate the enterotoxicity of the mutant strains, we examined the enterotoxic activity of the wild-type and mutant strains in rabbit ileum. The wild-type or mutant strains (10⁸ CFU) were injected into the ligated ileal loops of rabbits which were sacrificed after 16 h. After removal of the small intestine the fluid accumulation in the ligated ileal loops was measured (Table 3). Except for the trh mutant strain, no significant differences in fluid accumulation were observed in samples from wild-type and mutant strains. These results indicate that the presence or absence of urease production by the V. parahaemolyticus strain does not affect the enterotoxicity of the organism under these test conditions.

Distribution of ureR and nik operon in clinical V. parahaemolyticus strains. In previous studies, we demonstrated that the possession of trh completely coincided with the presence of the ureC (a structural gene of urease) by clinical V. parahaemolyticus strains and that, in strain AQ4673, which possesses the two genes, the distance between trh and ureC is within several kilobases (17, 18). Consistent with these previous results, nucleotide sequence analysis of TH3996 showed that the distance between the two genes is 7,945 bp. To investigate the distance between the two genes of the other trh-positive clinical strains of V. parahaemolyticus, we performed LA-PCR using oligonucleotide primer pairs targeting trh and ureC (18). All of the strains for testing were amplified accurately to the same amplicon size as the AQ4673 strain (data not shown). Next, we used colony hybridization with specific probes for ureR and nikD to test for the presence or absence of the ureR and nik operon in clinical and environmental strains of V. parahaemolyticus. The ureR and nikD genes were present in all of the trh-positive strains (n = 8) but not in Kanagawa phenomenon (KP)-positive strains (n = 12) and the environmental strains (n = 6) (data not shown).

	DISCUSSION

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Urease, which requires the nickel ion in the active site, catalyzes the hydrolysis of urea to ammonia and carbon dioxide, resulting in net increase in environmental pH (30). So far, urease gene clusters have been isolated in several bacteria, such as P. mirabilis (19, 20, 44), K. aerogenes (23, 31, 32), Helicobacter pylori (2, 4, 22), Bacillus sp. strain TB-90 (25), Streptococcus salivarius (42), and Yersinia enterocolitica (5, 43). The expression of bacterial urease genes is regulated by different mechanisms (3). For example, urease expression in K. aerogenes is activated only under nitrogen-limiting conditions (24). P. mirabilis urease is induced by urea and is mediated by the positive transcriptional regulator ureR (8). S. salivarius urease is regulated by pH (42).

We previously reported that the urease production by clinical V. parahaemolyticus strains correlates completely with the possession of the trh gene, and research by others has shown similar results (26, 36, 37). We have also found that the distance between the two genes is within several kilobases (17, 18), suggesting the genetic linkage between the two genes. In the present study, we demonstrated this genetic linkage. To investigate the coexistence and proximity of the trh and ure genes, we cloned and sequenced the region containing the trh and ure gene cluster on the chromosome of TH3996, a clinical V. parahaemolyticus strain that has the trh gene and produces urease.

Nucleotide sequencing showed that the ure gene cluster of V. parahaemolyticus is comprised of eight genes. The structural genes, ureA, ureB, and ureC, which encode subunits of the enzyme, are flanked immediately upstream by ureD, encoding a chaperone-like protein, and downstream by the ureE, ureF, and ureG accessory genes that encode the proteins required for incorporating the nickel ion into the metallocenter within the active site (30). In addition, ureR, the positive regulatory gene, lies 5.2 kb upstream of ureD and is oriented opposite the other seven genes. The predicted polypeptide sequence from this gene contains a putative helix-turn-helix DNA-binding motif, suggesting that this protein belongs to the AraC family of positive regulators. Members of the AraC family are primarily involved in the positive regulation of a number of genes (10).

As our data show, in the absence of urea, expression of V. parahaemolyticus urease is very low, but when 0.1% urea is present in the medium, urease expression is about 100 times greater. This increase was not seen in samples of the mutant strain which had undergone disruption of ureR. From this we conclude that V. parahaemolyticus urease is induced by urea and that this induction is mediated by ureR. The organization and sequences of the ure gene cluster of V. parahaemolyticus were similar to that of P. mirabilis. Furthermore, the regulation of the expression of urease is similar in the two species (Table 3). The major difference between the V. parahaemolyticus and P. mirabilis urease gene clusters was the presence or absence of a nickel transporter operon between ureR and ureD.

Between ureR and ureD of V. parahaemolyticus there were five overlapping genes that had significant homology to the nik operon of E. coli (34). These genes appear to encode a typical ABC transporter system, including ATP-binding polypeptides and integral cytoplasmic membrane proteins (9). NikB and NikC were highly hydrophobic proteins with six potential transmembrane domains, which may form the channel for the substrate transport. NikD and NikE contain Walker A and B motifs responsible for coupling energy to the nickel transport system (49). To investigate the role of the ABC transport system, we constructed a nikD disrupted mutant by homologous recombination. The mutant strain showed almost complete absence of urease activity compared with the wild-type strain. Consequently, it is plausible to suggest that the ABC transport system that we found contributes to nickel transport through the bacterial cytoplasmic membrane. ABC-type transporters of nickel ions from the extracellular milieu into bacterial cells, such as an ABC transporter in H. pylori (11), have also been found; however, similar genes have not been reported in other bacteria that possess the urease gene cluster. In H. pylori, the ABC transporter genes are not located close to the urease gene cluster on the genome (48). Interestingly, the nik genes of V. parahaemolyticus are located between the ureR and the ure genes. Thus, this is the first report of finding genes for the ABC-type nickel transport system adjacent to the urease gene cluster on the genome of an organism.

LA-PCR analysis showed the distance between the trh and ureC of all the trh-positive V. parahaemolyticus samples we tested to be precisely 7.9 kb. ureR and nik operon were consistently present in trh-positive strains (regardless of tdh possession) but not in KP-positive strains, which possess two copies of tdh, or in environmental strains. The sequencing results showed ORF1 to be a transposase. These findings suggest the possibility that the trh, nik operon, and ure gene cluster were transmitted into V. parahaemolyticus strains through a mechanism mediated by insertion sequences in the past.

Although the rabbit ileal loop test showed that the enterotoxicity of the ureR, ureC, and nikD mutant strains did not change compared with the TH3996 strain that they were derived from, we cannot completely rule out the possibility that the V. parahaemolyticus urease plays a certain role in the pathogenesis of this bacterium. In the case of Y. enterocolitica, urease appears to act as a virulence factor by enhancing the survival of bacteria during their passage through the stomach, presumably by neutralizing hydrogen ions which penetrate the bacterial cell wall (6). The contribution of urease to the pathogenesis of V. parahaemolyticus is currently being analyzed in our laboratory.