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Molecular cloning and characterization of the hblA gene encoding the B component of hemolysin BL from Bacillus cereus.

ABSTRACT: 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 hblA 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 hblA gene are being pursued to resolve whether hemolysin BL is indeed the enterotoxin.

SUBMITTER: Heinrichs JH

PROVIDER: S-EPMC206798 | biostudies-other | 1993 Nov

REPOSITORIES: biostudies-other

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Molecular cloning and characterization of the hblA gene encoding the B component of hemolysin BL from Bacillus cereus.

Heinrichs J H JH Beecher D J DJ MacMillan J D JD Zilinskas B A BA

Journal of bacteriology 19931101 21

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 part ...[more]

PMID: 7693651

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Project description:BackgroundBacillus cereus group isolates that produce diarrheal or emetic toxins are frequently isolated from raw milk and, in spore form, can survive pasteurization. Several species within the B. cereus group are closely related and cannot be reliably differentiated by established taxonomical criteria. While B. cereus is traditionally recognized as the principal causative agent of foodborne disease in this group, there is a need to better understand the distribution and expression of different toxin and virulence genes among B. cereus group food isolates to facilitate reliable characterization that allows for assessment of the likelihood of a given isolate to cause a foodborne disease.ResultsWe performed whole genome sequencing of 22 B. cereus group dairy isolates, which represented considerable genetic diversity not covered by other isolates characterized to date. Maximum likelihood analysis of these genomes along with 47 reference genomes representing eight validly published species revealed nine phylogenetic clades. Three of these clades were represented by a single species (B. toyonensis -clade V, B. weihenstephanensis - clade VI, B. cytotoxicus - VII), one by two dairy-associated isolates (clade II; representing a putative new species), one by two species (B. mycoides, B. pseudomycoides - clade I) and four by three species (B. cereus, B. thuringiensis, B. anthracis - clades III-a, b, c and IV). Homologues of genes encoding a principal diarrheal enterotoxin (hemolysin BL) were distributed across all, except the B. cytotoxicus clade. Using a lateral flow immunoassay, hemolysin BL was detected in 13 out of 18 isolates that carried hblACD genes. Isolates from clade III-c (which included B. cereus and B. thuringiensis) consistently did not carry hblACD and did not produce hemolysin BL. Isolates from clade IV (B. cereus, B. thuringiensis) consistently carried hblACD and produced hemolysin BL. Compared to others, clade IV was significantly (p = 0.0001) more likely to produce this toxin. Isolates from clade VI (B. weihenstephanensis) carried hblACD homologues, but did not produce hemolysin BL, possibly due to amino acid substitutions in different toxin-encoding genes.ConclusionsOur results demonstrate that production of diarrheal enterotoxin hemolysin BL is neither inclusive nor exclusive to B. cereus sensu stricto, and that phylogenetic classification of isolates may be better than taxonomic identification for assessment of B. cereus group isolates risk for causing a diarrheal foodborne disease.

Dataset Information

Molecular cloning and characterization of the hblA gene encoding the B component of hemolysin BL from Bacillus cereus.

Publications

Molecular cloning and characterization of the hblA gene encoding the B component of hemolysin BL from Bacillus cereus.

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