Impact of a Novel PagR-like Transcriptional Regulator on Cereulide Toxin Synthesis in Emetic Bacillus cereus.

The emetic type of foodborne disease caused by Bacillus cereus is produced by the small peptide toxin cereulide. The genetic locus encoding the Ces nonribosomal peptide synthetase (CesNRPS) multienzyme machinery is located on a 270 kb megaplasmid, designated pCER270, which shares its backbone with the Bacillus anthracis toxin plasmid pXO1. Although the ces genes are plasmid-borne, the chromosomally encoded pleiotropic transcriptional factors CodY and AbrB are key players in the control of ces transcription. Since these proteins only repress cereulide synthesis during earlier growth phases, other factors must be involved in the strict control of ces expression and its embedment in the bacterial life cycle. In silico genome analysis revealed that pCER270 carries a putative ArsR/SmtB family transcription factor showing high homology to PagR from B. anthracis. As PagR plays a crucial role in the regulation of the protective antigen gene pagA, which forms part of anthrax toxin, we used a gene-inactivation approach, combined with electrophoretic mobility shift assays and a bacterial two-hybrid system for dissecting the role of the PagR homologue PagRBc in the regulation of cereulide synthesis. Our results highlight that the plasmid-encoded transcriptional regulator PagRBc plays an important role in the complex and multilayered process of cereulide synthesis.

Detection and Isolation of Emetic Bacillus cereus Toxin Cereulide by Reversed Phase Chromatography.

The emetic toxin cereulide is a 1.2 kDa dodecadepsipeptide produced by the food pathogen Bacillus cereus. As cereulide poses a serious health risk to humans, sensitive and specific detection, as well as toxin purification and quantification, methods are of utmost importance. Recently, a stable isotope dilution assay tandem mass spectrometry (SIDA-MS/MS)-based method has been described, and an method for the quantitation of cereulide in foods was established by the International Organization for Standardization (ISO). However, although this SIDA-MS/MS method is highly accurate, the sophisticated high-end MS equipment required for such measurements limits the method's suitability for microbiological and molecular research. Thus, we aimed to develop a method for cereulide toxin detection and isolation using equipment commonly available in microbiological and biochemical research laboratories. Reproducible detection and relative quantification of cereulide was achieved, employing reversed phase chromatography (RPC). Chromatographic signals were cross validated by ultraperformance liquid chromatography-mass spectrometry (UPLC-MS/MS). The specificity of the RPC method was tested using a test panel of strains that included non-emetic representatives of the B. cereus group, emetic B. cereus strains, and cereulide-deficient isogenic mutants. In summary, the new method represents a robust, economical, and easily accessible research tool that complements existing diagnostics for the detection and quantification of cereulide.

Revealing details of stays abroad by sequential stable isotope analyses along human hair strands.

Multi-element stable isotope analyses of δ13C, δ15N, δ34S and δ2H values were performed along scalp hair strands to detect isotopic changes resulting from different stays abroad. One hair strand with a hair length of more than 50 cm originated from a German woman, who frequently made long-distance travels of 1 to 4 weeks. The second hair strand with a length of 15 cm was taken from a Japanese woman who went to Germany for a period of some months. Stable isotopic influences due to the stays abroad were clearly reflected in the 5-mm segments along the proximal part of the hair strand; whereas in the more distal parts, the isotopic influences were blurred. This can be regarded as the result of the highly variable intra-individual hair growth rate of single hairs of at least ± 30% compared to the mean growth rate. Consequently, the initial isotope signal obtained by short stays abroad became rapidly attenuated in the more distal parts of the hair strand. Furthermore, decreasing sulphur content associated with higher sulphur isotope values was observed with increasing hair length. The isotope shifts along the scalp hair strand, provoked by dietary changes at new locations, appeared at such points of hair length, which correspond well with the maximum growth rate of single hairs. Consequently, the exact date for any changes coming along with isotopic shifts may be calculated by best approach considering a hair growth value of 1.4 cm per 30 days, instead of the commonly used mean monthly hair growth rate of 1.1 cm. This may be important in forensics, if detailed information about a person's recent lifetime should be figured out by segmental scalp hair analyses.