Cereulide-producing Bacillus cereus isolates can cause serious emetic (vomiting) syndrome and even acute lethality. As mobile genetic elements, the exploration of prophages derived from emetic B. cereus isolates will help in our understanding of the genetic diversity and evolution of these pathogens. In this study, five temperate phages derived from cereulide-producing B. cereus strains were induced, with four of them undergoing genomic sequencing. Sequencing revealed that they all belong to the Siphoviridae family, but presented in different forms in their hosts. PfNC7401 and PfIS075 have typical icosahedral heads, probably existing alone as phagemids in the host with self-replicating capability in the lysogenic state. PfEFR-4, PfEFR-5, and PfATCC7953 have elongated heads, with the genomes of the former two identified as linear dsDNA, which could be integrated into the host genome during the lysogenic state. Genomic comparison of the four phages with others also derived from emetic B. cereus isolates showed similar genome structures and core genes, thus displaying host spectrum specificity. In addition, phylogenic analysis based on the complete genome and conserved tail fiber proteins of 36 Bacillus species-derived phages confirmed that the phages derived from emetic B. cereus strains were highly similar. Furthermore, one endolysin LysPfEFR-4 was cloned and showed lytic activity against all tested emetic B. cereus strains and cross-lytic activity against some other pathogenic bacteria, implying a potential to control bacterial contamination in the food supply.

Cereulide is notorious as a heat-stable emetic toxin produced by Bacillus cereus and glucose is supposed to be an ingredient supporting its formation. This study showed that glucose addition benefited on cell growth and the early transcription of genes involved in substrate accumulation and toxin synthesis, but it played a negative role in the final production of cereulide. Meanwhile, a lasting enhancement of cesH transcription was observed with the addition of glucose. Moreover, the cereulide production in ΔcesH was obviously higher than that in the wild type. This indicates that CesH has a repression effect on cereulide production. Bioinformatics analysis revealed that CesH was an alpha/beta hydrolase that probably associated with the cell membrane, which was verified by subcellular localization. The esterase activity against para-nitrophenyl acetate (PNPC2) of the recombinant CesH was confirmed. Although no sign of ester bond cleavage in cereulide or valinomycin was demonstrated in in vitro assays, CesH could reverse the cereulide analogue sensitivity of Bacillus subtilis in vivo, by which toxin degradation was facilitated. Moreover, site directed mutations identified that the conserved catalytic triad of CesH might consist of Serine 86, Glutamate 199, and Histidine 227. These results help us to understand the regulation of cereulide production and provide clues for developing control measurements.