Abstract
The emetic toxin cereulide produced by Bacillus cereus is synthesized by the modular enzyme complex Ces that is encoded on a pXO1-like megaplasmid. To decipher the role of the genes adjacent to the structural genes cesA/cesB, coding for the non-ribosomal peptide synthetase (NRPS), gene inactivation- and overexpression mutants of the emetic strain F4810/72 were constructed and their impact on cereulide biosynthesis was assessed. The hydrolase CesH turned out to be a part of the complex regulatory network controlling cereulide synthesis on a transcriptional level, while the ABC transporter CesCD was found to be essential for post-translational control of cereulide synthesis. Using a gene inactivation approach, we show that the NRPS activating function of the phosphopantetheinyl transferase (PPtase) embedded in the ces locus was complemented by a chromosomally encoded Sfp-like PPtase, representing an interesting example for the functional interaction between a plasmid encoded NRPS and a chromosomally encoded activation enzyme. In summary, our results highlight the complexity of cereulide biosynthesis and reveal multiple levels of toxin formation control. ces operon internal genes were shown to play a pivotal role by acting at different levels of toxin production, thus complementing the action of the chromosomal key transcriptional regulators AbrB and CodY.
Highlights
The cyclic dodecadepsipeptide cereulide, a heat, acid, and proteolytically stable toxin, is responsible for the emetic type of food borne illness caused by a specific subgroup of Bacillus cereus
Little is known about the adjacent genes cesH, cesP, cesC, and cesD, which code for a putative hydrolase, a phosphopantetheinyl transferase (PPtase) and a putative transport system of the ABC-type, respectively (EhlingSchulz et al, 2006)
Since it was recently shown that the Ces-nonribosomal peptide synthetase (NRPS) represents a novel mechanism for non-ribosomal depsipeptide assembly (Marxen et al, 2015b), information on the functional architecture of the ces gene locus would improve our general understanding of the complex biochemical and the mutant strains F48 cesP (5), F48 cesP/Ippt (6), F48 cesP/polar (4), F48 cesCD (7), F48IcesH (2), and F48pMM/cesH (3). (A) Relative cesA transcription of B. cereus strains grown in LB medium at 30 or 37◦C and harvested at exponential growth phase (OD600 = 8) for RNA isolation
Summary
The cyclic dodecadepsipeptide cereulide, a heat-, acid-, and proteolytically stable toxin, is responsible for the emetic type of food borne illness caused by a specific subgroup of Bacillus cereus. Intoxication with cereulide, which is preformed during vegetative growth of B. cereus in foods, causes nausea and heavy vomiting around 0.5–6 h after consumption of contaminated food (Ehling-Schulz et al, 2004). These symptoms are presumably induced by the interaction of cereulide with 5-HT3 serotonin receptors leading to the stimulation of the afferent vagus nerve (Agata et al, 1995). These symptoms decline after 24 h, but more severe foodborne. Very recently we could show that the enzymatic activity of the Ces-NRPS does not follow the canonical NRPS biosynthesis logic, but represents a novel mechanism of non-ribosomal peptide assembly, by using dipeptides rather than monomers as basic units (Marxen et al, 2015a,b)
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