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In recent years Bacillus cereus has gained increasing importance as a food poisoning pathogen. It is the eponymous member of the B. cereus sensu lato group that consists of eight closely related species showing impressive diversity of their pathogenicity. The high variability of cytotoxicity and the complex regulatory network of enterotoxin expression have complicated efforts to predict the toxic potential of new B. cereus isolates. In this study, comprehensive analyses of enterotoxin gene sequences, transcription, toxin secretion and cytotoxicity were performed. For the first time, these parameters were compared in a whole set of B. cereus strains representing isolates of different origin (food or food poisoning outbreaks) and of different toxic potential (enteropathogenic and apathogenic) to elucidate potential starting points of strain-specific differential toxicity. While toxin gene sequences were highly conserved and did not allow for differentiation between high and low toxicity strains, comparison of nheB and hblD enterotoxin gene transcription and Nhe and Hbl protein titers revealed not only strain-specific differences but also incongruence between toxin gene transcripts and toxin protein levels. With one exception all strains showed comparable capability of protein secretion and so far, no secretion patterns specific for high and low toxicity strains were identified. These results indicate that enterotoxin expression is more complex than expected, possibly involving the orchestrated interplay of different transcriptional regulator proteins, as well as posttranscriptional and posttranslational regulatory mechanisms plus additional influences of environmental conditions.

Bacillus cereus is a ubiquitous bacterial pathogen increasingly reported to be the causative agent of foodborne infections and intoxications. Since the enterotoxins linked to the diarrheal form of food poising are foremost produced in the human intestine, the toxic potential of enteropathogenic B. cereus strains is difficult to predict from studies carried out under routine cultivation procedures. In this study, toxigenic properties of a panel of strains (n = 19) of diverse origin were compared using cell culture medium pre-incubated with CaCo-2 cells to mimic intestinal growth conditions. Shortly after contact of the bacteria with the simulated host environment, enterotoxin gene expression was activated and total protein secretion of all strains was accelerated. Although the signal stimulating enterotoxin production still needs to be elucidated, it could be shown that it originated from the CaCo-2 cells. Overall, our study demonstrates that the currently used methods in B. cereus diagnostics, based on standard culture medium, are not allowing a conclusive prediction of the potential health risk related to a certain strain. Thus, these methods should be complemented by cultivation procedures that are simulating intestinal host conditions.

The copper-regulated Rhodobacter capsulatus cutO (multicopper oxidase) gene confers copper tolerance and is carried in the tricistronic orf635-cutO-cutR operon. Transcription of cutO strictly depends on the promoter upstream of orf635, as demonstrated by lacZ reporter fusions to nested promoter fragments. Remarkably, orf635 expression was not affected by copper availability, whereas cutO and cutR were expressed only in the presence of copper. Differential regulation was abolished by site-directed mutations within the orf635-cutO intergenic region, suggesting that this region encodes a copperresponsive mRNA element. Bioinformatic predictions and RNA structure probing experiments revealed an intergenic stem-loop structure as the candidate mRNA element. This is the first posttranscriptional copper response mechanism reported in bacteria.

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