Cereulide, a
depsipeptide structurally related to
valinomycin, is responsible for the
emetic type of
gastrointestinal disease caused by Bacillus cereus. Due to its chemical structure, (D-O-Leu-D-Ala-L-O-Val-L-Val)(3),
cereulide might be synthesized nonribosomally. Therefore, degenerate PCR primers targeted to conserved sequence motifs of known
nonribosomal peptide synthetase (NRPS) genes were used to amplify gene fragments from a
cereulide-producing B. cereus strain. Sequence analysis of one of the amplicons revealed
a DNA fragment whose putative gene product showed significant homology to
valine activation NRPS modules. The sequences of the flanking regions of this
DNA fragment revealed a complete module that is predicted to activate
valine, as well as a putative carboxyl-terminal thioesterase domain of the NRPS gene. Disruption of the
peptide synthetase gene by insertion of a
kanamycin cassette through homologous recombination produced
cereulide-deficient mutants. The
valine-activating module was highly conserved when sequences from nine
emetic B. cereus strains isolated from diverse geographical locations were compared. Primers were designed based on the NRPS sequence, and the resulting PCR assay, targeting the ces gene, was tested by using a panel of 143 B. cereus group strains and 40 strains of other bacterial species showing PCR bands specific for only the
cereulide-producing B. cereus strains.