Transcriptional kinetic analyses of cereulide synthetase genes with respect to growth, sporulation and emetic toxin production in Bacillus cereus

In light of the increasing number of serious food borne outbreaks caused by emetic Bacillus cereus, a better understanding of the cereulide synthetase (ces) gene expression and toxin synthesis is required. Here, the relative expression levels of three ces genes (cesP, cesA and cesB) were investigated using quantitative real-time reverse transcription PCR in relation to growth, degree of sporulation and toxin production of the emetic reference strain B. cereus F4810/72 and the weakly emetic strain IH41385. The strict co-transcription of all three genes confirmed the operon structure of the ces gene cluster responsible for cereulide formation. ces transcription turned out to be highly temporal and tightly regulated; ces mRNA was only detectable during mid to late exponential growth in both strains. The low toxigenic potential of the weakly emetic strain IH41385 correlated well with its respective ces transcripts, showing reduced activity at a transcriptional level. Two non-sporulating mutants (F4810/72Δspo0A and F4810/72INsigH) demonstrated that cereulide synthesis is part of the Spo0A regulon but independent of later sporulation processes. Besides strain specific intrinsic factors, ces transcription was found to be significantly influenced by the cellular growth state as well as by extrinsic abiotic factors, like salt. An increase of sodium chloride in the media resulted in lower ces transcription and coincided with lower cereulide toxin levels. Interestingly, at 25 g l−1 NaCl, toxin levels were already reduced without strongly affecting the growth of B. cereus, indicating an inhibitory effect of NaCl on cereulide biosynthesis independent of growth. This illustrates that ces gene expression and toxicity cannot be predicted solely from growth rates or cell numbers, but is influenced by complex interactions of various intrinsic as well as extrinsic factors, which remain to be clarified in detail.