Cloning and expression in Escherichia coli of Vibrio parahaemolyticus thermostable direct hemolysin and thermolabile hemolysin genes

Abstract

Two hemolysin genes of Vibrio parahaemolyticus WP1, a thermostable direct (TSD) hemolysin gene and a thermolabile henlolysin gene, were cloned into the pBR322 vector in Escherichia coli K-12 C600. A large amount of the TSD hemolysin produced in E. coli K-12 accumulated in the periplasmic space. The TSD hemolysin gene was localized on a 0.9-kilobase HindIII-BamHI fragment by identifying qualitatively the production of the TSD hemolysin by a reverse passive hemagglutination assay in the osmotic shock fluid. The thermolabile hemolysin gene was isolated on a 1.3-kilobase HindIII-PstI fragment by selection with the hemolysin on blood agar. Southern blot hybridization and colony hybridization experiments indicated that the TSD hemolysin gene was present in the chromosomal DNA of 15 Kanagawa phenomenon-positive strains but not in 14 negative strains, whereas the thermolabile hemolysin gene was detected in all strains. No homologous DNA sequences to TSD and themolabile hemolysin genes were detected in the chromosomes of Vibrio cholerae, Vibrio vulnificus, non-O1 V. cholerae, and Vibrio anguillarum.

Haemolysins in Vibrio species

“…This proteinaceous toxin, which does not have a lipid or carbohydrate moiety , is a dimer which is composed of two identical subunits each having a molecular mass of c. 21 kDa (Takeda et al 1978). Purified Vp-TDH is heat-stable, even at 100°C for 10 min (Taniguchi et al 1985;Iida and Honda 1997), and has an amino acid sequence of 165 amino acid residues, with one disulfide bond near the carboxyl terminus (Tsunasawa et al 1987). These data are in good agreement with that obtained from the nucleotide sequence of the tdh gene (Nishibuchi and Kaper 1985).…”

“…In addition to Vp-TDH and Vp-TRH, a thermolabile haemolysin (TLH), also coined the lecithin-dependent haemolysin (LDH), was discovered in V. parahaemolyticus (Taniguchi et al 1985(Taniguchi et al , 1986Shinoda et al 1991). TLH was heat labile (60°C for 10 min; Taniguchi et al 1985), and had phospholipase A2/lysophospholipase activity (Shinoda et al 1991). The TLH haemolysin gene (tlh) contained an ORF of 1254 bp (Shinoda et al 1991).…”

“…Using computer analysis, homology was not found between the nucleotide sequences of the tdh gene and the tlh gene of V. parahaemolyticus (Taniguchi et al 1986). Yet, the tlh gene has been found in the genomes of all V. parahaemolyticus isolates examined, regardless of whether they have been derived from clinical or environmental sources (Taniguchi et al 1985;Bej et al 1999;McCarthy et al 1999). However, the role of this haemolysin in the enteropathogenicity of V. parahaemolyticus is unknown (Shinoda et al 1991).…”

“…Overall there are four representative haemolysin families, including the thermostable direct haemolysin (TDH) of V. parahaemolyticus, the El Tor haemolysin of V. cholerae (HlyA), the thermolabile haemolysin (TLH) of V. parahaemolyticus and another thermostable haemolysin, that is, the d-VPH of V. parahaemolyticus (e.g. Taniguchi et al 1985Taniguchi et al , 1986Taniguchi et al , 1990Yamamoto et al 1990a;Zhang et al 2001;Fallarino et al 2002; Table 2).…”

Comparison of different biochemical and molecular methods for the identification of Vibrio parahaemolyticus

“…Other targets proposed for PCR detection were a cloned HindIII DNA fragment of V. parahaemolyticus chromosomal DNA, namely the pR72H fragment (Lee et al 1995;Robert-Pillot et al 2002) and a thermolabile haemolysin gene (tl), which is not associated with illness in humans. This haemolysin was shown to be present in all of the V. parahaemolyticus tested (Taniguchi et al 1985(Taniguchi et al , 1986 and was therefore used to detect this species in shellfish (Bej et al 1999).…”