MOLECULAR-CLONING AND CHARACTERIZATION OF THE GENES ENCODING THE L(1)AND L(2) COMPONENTS OF HEMOLYSIN BL FROM BACILLUS-CEREUS

Hemolysin BL, which is composed of a binding component, B, and two lyt ic components, L(1) and L(2), is the enterotoxin responsible for the d iarrheal food poisoning syndrome caused by strains of Bacillus cereus. To further characterize the toxin, we sought to clone and sequence th e genes encoding the L(1) and L(2) proteins, A genomic library was scr eened with polyclonal antibody to the L, and L, proteins to identify r ecombinant clones containing the genes, Five clones reacted with the a ntibody to L(2), but none reacted with the antibody to L(1). Southern hybridization analysis with oligonucleotide probes designed from the N -terminal amino acid sequences of the L(1) and L(2) proteins, in conju nction with immunoblot and nucleotide sequence analysis, revealed that the recombinant plasmid from one of the clones contained two genes, h blC and hblD, which encode L(2) and L(1), respectively, The two genes are arranged in tandem and are separated by only 37 bases, The gene wh ich encodes the B component of hemolysin BL (hblA) is located immediat ely downstream from the gene encoding the L, protein, Northern blot an alysis of B. cereus RNA showed a 5,5-kb transcript which hybridized wi th DNA fragments internal to, or including a portion of, the coding se quences of the B, L(1), and L(2) genes, suggesting that the clustered genes which encode the components of hemolysin BL are cotranscribed an d constitute an operon.