MOLECULAR-CLONING AND CHARACTERIZATION OF THE HBLA GENE ENCODING THE B-COMPONENT OF HEMOLYSIN BL FROM BACILLUS-CEREUS

Previous evidence suggests that hemolysin BL, which consists of a binding component, B, and two lytic components, L1 and L2, is the enterotoxin responsible for the diarrheal form of gastroenteritis caused by food-borne strains of Bacillus cereus. To prove that hemolysin BL and the enterotoxin are the same requires large amounts of these components free of other B. cereus proteins. For this purpose, we sought to clone the gene encoding the B component and to express it in Escherichia coli. A partial genomic library was constructed and a 29-base, 1,152-fold-degenerate oligonucleotide probe, designed from the N-terminal amino acid sequence of the B component, was used to identify recombinant clones containing the gene. Detection of gene products reactive with a monoclonal antibody specific for the B component and analysis of the nucleotide sequence confirmed that isolated clones, reactive with the oligonucleotide probe, did contain the gene encoding the B component. The protein, expressed in E. coli, apparently from the B. cereus promoter, produces a ring-shaped zone of hemolysis when combined with purified L components from B. cereus, a reaction typical of hemolysin BL. Northern (RNA) blot analysis of B. cereus RNA showed a large (5.1-kb) transcript which hybridized with a 500-bp probe internal to the B-component-coding sequence, suggesting that the hbl4 gene encoding the B component may be transcribed as part of a polycistronic message, possibly including the structural genes for the two lytic components. Higher levels of expression and disruption of the kblA gene are being pursued to resolve whether hemolysin BL is indeed the enterotoxin.