Identification of two unique DNA loci from a clinical isolate of vibrio parahaemolyticus by the Differential Subtraction Chain (DSC) technique

Abstract

Vibrio parahaemolyticus occurs in natural marine and estuarine waters worldwide and is the major cause of gastroenteritis where consumption of seafood is high. Existing methods for detecting pathogenic V.parahaemolyticus are primarily based on the thermostable haemolysin (tdh) and/or TDH-related (trh) genes or gene products. Although these genes are thought to determine the virulence of V.parahaemolyticus, the exact pathogenic mechanisms main poorly understood In this project, Differential Subtraction Chain (DSC) technique was used to identify additional Virulence-associated genes in pathogenic V.parahaemolyticus. Two DNA fragments, clone 32 and clone 126, have been identified, and computer analysis indicated that these two clones are derived from different chromosomes of the V.parahaemolyticus genome. 5’- and 3’-genome walking was carried out to obtain additional sequences at both ends for clones 32 and 126, and two contiguous stretches of DNA consisting of 5195 bp and 3599 bp were obtained, respectively. Computer-aided analyses revealed that the 5195-bp sequence (designated as CLONE 32) contained eight open reading frames (ORFs) - ORFl to ORF6, and the VpaIM and HsdR genes. The 3599-bp sequence (designated as CLONE 126) contained ORFs for three genes: a partial mannonate oxidoreductase (UxuB) gene, the complete uronate isomerase (UxaC) and 2-dehydro-3-deoxygluconokinase (KdgK) genes. The complete DNA sequences of VpaIM, HsdR, UxaC and KdgK have been determined and characterized, and expression patterns and prevalence of these genes in clinicaVenvirOnrnenta1 isolates examined using PCR-based approaches. Computer analysis indicated that VpaIM is a methyltransferase (MTase) gene and shares high sequence identity with homologues of V.parahaemolyticus RIMD2210633 and V. cholerae. Detail analysis showed that VpaIM is a member of the N6_N4_MTase family and contains all the sequence motif typical of N-MTam, including Motif I to III and X (which constitute the AdoMet-binding region) and motifs IV to VIII (which constitute the catalytic region). The linear order of the functional domains in VpaIM indcated it is a member of the β group of N6_N4_MTases. Northern blot and RT-PCR analyses showed that VpaIM gene expression was higher at 25°C than at 37°C, and that it was cotranscribed with the upstream ORF6 sequence. Sequence analysis revealed two putative promoters - one is located upstream of the ORF6 start codon while the second is located upstream of the VpaIM start codon. Inverted-repeat sequences without obvious T-stretches were located at the end of the ORF6 and VpuIM genes, which may function as rho-dependent terminator signals. The HsdR (type I) gene has a coding sequence of 2442 bp and results of GenBank search indicated that it is very similar to homologues of other bacterial species. Comparison of the HsdR protein of V.parahaemolyticus to members of the four families of type I restriction and modification (R-M) system of different bacterial species showed that HsdR is a member of the type I B family. Computer analysis indicated that HsdR contains a HSDR_N, DEXDc and Helicase_C domain as well as a segment of low compositional complexity (SLCC). By multiple alignment of HsdR with different type I restriction enzymes, several conserved motifs were identified and included a motif X within the HSDR_ N domain and seven DEAD-box motif clustered in the central part of the polypeptide. The amino acid residues D…P...EXK of motif X are highly conserved in all four families of the type I R-M system, and is reminiscent of the PD…(D/E)XK catalytic motif of type II restriction enzymes. RT-PCR analysis showed that HsdR has an expression pattern similar to the VpaIM gene, which was higher at 25℃ than at 37℃. Promoter analysis identified a typical T-TG- motif extending from the -10 region (TATAGT) together with a putative -35 region (TTCACT) which was located 39 bp upstream of the -10 region. A putative ARBS (CATCCTTTGGTATAGT) sequence was also identified which may be essential for translation initiation. The uxaC and kdgK genes are 1410 bp and 975 bp in length respectively. Homology search showed that they share high sequence identity with the homologues from Vibrios, and are widely distributed among various bacterial species. Computer analysis of the genes flanking the 5’- and 3’-ends of the uxaC and kdgK genes in the genomes of V.parahaemolvticus RlMD2210633 and E. coli K12 indicated that CLONE 126 may be the genomic region harboring the hexuronate utilization gene cluster. RT-PCR analysis showed no differential expression of the uxaaC and kdgK genes at 25°C and 37°C. The genes in CLONE 126 are closely linked and no presumptive promoters were identified upstream of the uxaC and kdgK genes, which indicated that uxaC and kdgK as well as the surrounding genes may be organized and regulated in an operon fashion. Interestingly, a putative SD sequence was found 6 and 8 bp upstream of the start codons of the uxaC and kdgK genes, respectively. The prevalence of the above four genes were examined using gene-specific PCR VpaIM and HsdR genes were detected in 62.5% (20/32) and 0% (0/34) of clinical and environmental V Parahaemolyticus isolates respectively. In contrast, the uxaC/kdgK genes were observed in 81.3% (26/32) and 11.8% (4/32) of clincial and environmental isolates of V. parahaemolyticus, respectively. A strong relationship of these four genes with the tdh virulence gene was observed whereby 89.7% and 65.5% of tdh-positive clinical isolates were also uxaC/kdgK-positive and VpaIM/HsdR-positive, respectively. These observations suggest that CLONE 32 and CLONE 126 genes are likely to be associated with virulence and/or pathogenicity of V.parahaemolyticus.

Date of Award	15 Jul 2004
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Shiu Sun Rudolf WU (Supervisor)