Analysis of regulatory genes for production of hemolysin of Vibrio cholerae.

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1. We cloned the inner fragment of hlyU which is reported as positive regulator for hlyA to a suicide vector plasmid. The recombinant plasmid was introduced to Vibrio cholerae N86 and hlyU was destroyed by single crossover by the plasmid. Little decrease of the hemolytic activity was observed in the hlyU-deleted mutant. We constructed the hlyU-deleted mutant using the suicide plasmid in several hemolytic V.cholerae non-O1 strains. Hemolytic activities in a few mutants was reduced, but, no effect was observed in most of the strains by destroying hlyU.This suggest that hlyU is not always essential positive regulator in all strains and that an unknown regulatory system other than hlyU may exit in V.cholerae.2. To screen unknown gene for regulation of the hemolysin production, suicide vector containing 0.6-kb DNA fragment of whole DNA of V.cholerae was introduced to hemolytic N86 strain. Two strains (IM1 and IM2) which reduced in hemolytic activities were obtained. The DNA sequences of the insertion sites of plasmids were analyzed using TIGR genome data base. The insertion sites were inside of emrD in IM2 and a ORF in IM1, respectively. mRNAs of hlyA in IM2 was completely diminished and that of IM1 was observed. These results suggest that the region around emrD, which is homologous to the multi-drug resistant gene of Escherichia coli, is related to regulation of hemolysin production.