Efficiency of Vibrio parahaemolyticus tdh gene expression depends upon two point mutations in its promoter region

A molecular study of Vibrio parahaemolyticus clinical isolates containing the thermostable direct hemolysin (Tdh) gene and the Tdh-related hemolysin (Trh) gene have been conducted. Southern blot hybridization revealed that in the genomes of strains carrying the determinants of both hemolysins (tdh + trh +) the tdh gene is presented by a single copy while tdh + trh − strains have two copies (tdh1 and tdh2). All investigated tdh + trh + and some tdh + trh − strains did not express the tdh gene (Kanagawa-negative, KP−) or expressed it weakly and inconstantly (Kanagawa-intermediate, KP±) in contrast to several Kanagawa-positive (KP+) strains. To establish the reasons for the KP−/± phenotypes we sequenced tdh, tdh1 and tdh2 genes of 13 strains isolated in Russia and neighbouring foreign countries followed by bioinformatics analysis of the obtained sequences in comparison with those of a number of strains presented in GenBank. The results revealed that poor expression of the tdh gene depends not only on a single point mutation in the promoter region (substitution of A to G in the −35 sequence) as it was believed earlier, but to the same extent on a second substitution (G to A at −3 nucleotide position from the t-10 sequence) which appeared to be sufficient in the absence of the first one. Therefore, a reversion of KP-/± strains to KP+ actually may occur as a result of a single point back mutation, and such strains are to be considered as potentially dangerous. Those bearing both substitutions may reverse with less probability as this process requires two simultaneous mutations.