Duplication and variation of the thermostable direct haemolysin (tdh) gene in Vibrio parahaemolyticus

Abstract

The relationship between phenotypic variation and nucleotide sequence variation of the gene encoding Vibrio parahaemolyticus thermostable direct haemolysin (tdh gene) was examined. Strains showeing a typical haemolysin-positive phenotype carried two chromosomal gene copies (designated tdh1 and tdh2) whild tdh-gene-positive strains showing a weakly positive or negative haemolysin phenotype possessed only a single chromosomal gene copy. Both gene copies from a typical haemolysin-positive strain were cloned and sequenced n dpossessed 97.2% homology. Comparison of the amino acid sequence predicted from the nucleotide sequence with the protein sequence determined by Edman degraduation as ell as construction of a tdh1-deficient yet haemolytic strain of V. parahaemolyticus suggest that the tdh2 locus is primarily responsible for the heamoloytic phenotype. Two other tdh gene copies were cloned from a phenotypically negative straifn which was unusual in that it contained one gene copy on a plasmid (designated tdh4) in addition to a single copy on the chromosome (tdh3). Both ted3 and tdh4 were expressed in Escherichia coli and TDHs with haemolytic activity were produced. These gene copies were sequenced and shared 96.7% homology with the tdh1 gene. The V. parahaemolyticus strain carrying tdh3 and tdh4 gene copies did not produce detectable amounts of tdh-specific RNA transcript. It seems, therefore, that differences in the transcriptional control are primarily responsible for the differences seen in haemolytic phenotype.