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 bind ing component, B, and two lytic components, L1 and L2, is the enteroto xin responsible for the diarrheal form of gastroenteritis caused by fo od-borne strains of Bacillus cereus. To prove that hemolysin BL and th e 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 co li. A partial genomic library was constructed and a 29-base, 1,152-fol d-degenerate oligonucleotide probe, designed from the N-terminal amino acid sequence of the B component, was used to identify recombinant cl ones containing the gene. Detection of gene products reactive with a m onoclonal antibody specific for the B component and analysis of the nu cleotide sequence confirmed that isolated clones, reactive with the ol igonucleotide probe, did contain the gene encoding the B component. Th e protein, expressed in E. coli, apparently from the B. cereus promote r, produces a ring-shaped zone of hemolysis when combined with purifie d L components from B. cereus, a reaction typical of hemolysin BL. Nor thern (RNA) blot analysis of B. cereus RNA showed a large (5.1-kb) tra nscript which hybridized with a 500-bp probe internal to the B-compone nt-coding sequence, suggesting that the hbl4 gene encoding the B compo nent may be transcribed as part of a polycistronic message, possibly i ncluding the structural genes for the two lytic components. Higher lev els of expression and disruption of the kblA gene are being pursued to resolve whether hemolysin BL is indeed the enterotoxin.