NUCLEOTIDE-SEQUENCE AND EXPRESSION OF AN EXTRACELLULAR HEMOLYSIN GENE OF AEROMONAS-HYDROPHILA

The extracellular hemolysin (AHH1) gene of Aeromonas hydrophila ATCC7966 was cloned into Charomid928 in Escherichia coli DH1, and its complete nucleotide sequence determined. Escherichia coli carrying this gene expressed an extracellular heat-labile hemolysin for rabbit red blood cells. The minimum size of the coding region of the 2.6 kilobase-pair BamHI-SphI fragment was subcloned into pUC118 and pUC119, selecting for hemolytic activity. The nucleotide sequence of this region contained a single open reading frame of 1734 base pairs, corresponding to a protein of 577 amino acid residues (63658 daltons). A consensus promoter sequence was present upstream of the AHH1 open reading frame. Maxicell analysis of [35S] methionine-labelled proteins in E. coli CSR603 carrying the AHH1 plasmid suggested that AHH1 gene codes for an approximately 60000 dalton polypeptide. By colony DNA-DNA hybridization analysis, the AHH1 gene was detected in 43 of 62 hemolysin-producing strains of A. hydrophila (isolated from various sources and areas) and in all 43 hemolysin-producing strains of A. salmonicida (isolated from fish). Three hemolysin-negative strains of A. hydrophila did not react with the AHH1 probe, whereas three non-hemolytic A. salmonicida strains hybridized with the probe. © 1991.