Identification of the Main Promoter Directing Cereulide Biosynthesis in Emetic
            <i>Bacillus cereus</i>
            and Its Application for Real-Time Monitoring of
            <i>ces</i>
            Gene Expression in Foods

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Severe intoxications caused by the Bacillus cereus emetic toxin cereulide can hardly be prevented due to the ubiquitous distribution and heat resistance of spores and the extreme thermal and chemical stability of cereulide. It would therefore be desirable to inhibit cereulide synthesis during food manufacturing processes or in prepared foods, which are stored under time-temperature abuse conditions. Toward this end, the impacts of three long-chain polyphosphate (polyP) formulations on growth and cereulide production were examined. The inhibition was dependent on the concentration and the type of the polyP blend, indicating that polyPs and not the orthophosphates were effective. Quantitative PCR (qPCR) monitoring at sublethal concentrations revealed that polyPs reduced the transcription of ces nonribosomal peptide synthetase (NRPS) genes by 3-to 4-fold along with a significantly reduced toxin production level. At lower concentrations, toxin synthesis was decreased, although the growth rate was not affected. These data indicate a differential effect on toxin synthesis independent of growth inhibition. The inhibition of toxin synthesis in food was also observed. Despite the growth of B. cereus, toxin synthesis was reduced by 70 to 100% in two model food systems (reconstituted infant food and oat milk), which were analyzed with HEp-2 cell culture assays and high-performance liquid chromatography (HPLC)/electrospray ionization-time of flight mass spectrometry (ESI-TOF-MS). Accordingly, ces promoter activity was strongly downregulated, as visualized by using a lux-based reporter strain. These data illustrate the potential of polyphosphate formulations to reduce the risk of cereulide synthesis in food and may contribute to targeted hurdle concepts.

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confidence: 99%

Inhibition of Cereulide Toxin Synthesis by Emetic Bacillus cereus via Long-Chain Polyphosphates

Frenzel

Letzel

Scherer

et al. 2011

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confidence: 99%

“…These elements contain a 23-kb gene cluster (ces) involved in the cereulide synthesis. The two largest genes, named cesA (ϳ10 kb) and cesB (ϳ8 kb), lead to the incorporation and modification of D-O-Leu, D-Ala (cesA), L-O-Val, and D-Val (cesB), which compose the basic tetradepsipeptide motif of cereulide (7,14).Cereulide can cause stomach pain and vomiting, respiratory distress, and occasional loss of consciousness, possibly leading to coma and ultimately death of the individual (6). This toxin is also an ionophoric molecule with a high degree of affinity for K ϩ ions (26).…”

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confidence: 99%

Antifungal Activity Displayed by Cereulide, the Emetic Toxin Produced by Bacillus cereus

Ladeuze

Lentz

Delbrassinne

et al. 2011

In this study, the fungistatic activity of Bacillus cereus cereulide-producing strains was demonstrated against nine fungal species. The role of cereulide was confirmed using plasmid-cured derivatives and ces knockout mutants. The fungistatic spectra of cereulide and valinomycin, a chemically related cyclododecadepsipeptide, were also compared and found to be similar but distinct.Food poisoning by emetic Bacillus cereus strains has been shown to cause fatal liver failure (6,15,25). Cereulide, the emetic toxin of B. cereus, is a cyclic dodecadepsipeptide composed of three repetitions of two amino acids and two hydroxyIts chemical structure is closely related to that of valinomycin, which is produced by various Streptomyces spp. (16). Cereulide is a heat-stable toxin (4,21,24,27) and is synthesized enzymatically by a nonribosomal peptide synthesis (NRPS) whose genetic determinants are located on the pCERE01 plasmid of the B. cereus emetic strain Kinrooi 5975c (9), the pBC270 element from B. cereus strain AH187 (23), and the pBCE4810 plasmid from B. cereus reference strain F4810/72 (8). These elements contain a 23-kb gene cluster (ces) involved in the cereulide synthesis. The two largest genes, named cesA (ϳ10 kb) and cesB (ϳ8 kb), lead to the incorporation and modification of D-O-Leu, D-Ala (cesA), L-O-Val, and D-Val (cesB), which compose the basic tetradepsipeptide motif of cereulide (7,14).Cereulide can cause stomach pain and vomiting, respiratory distress, and occasional loss of consciousness, possibly leading to coma and ultimately death of the individual (6). This toxin is also an ionophoric molecule with a high degree of affinity for K ϩ ions (26). It is lipophilic and specifically targets mitochondrial membranes (4), where it leads to cellular dysfunctions, such as swelling mitochondria and blocking oxidative phosphorylation (2, 17).It has been shown that valinomycin and cereulide display similar activities, since they block the motility of boar spermatozoa through the dissipation of mitochondrial internal membrane potential, a property often used for the detection of cereulide (3, 4, 22, 23a). Given these similarities, it was interesting to consider the known activities of valinomycin to hypothesize on the actual "raison d'être" of cereulide. Valinomycin is toxic for some insects, nematodes, and Gram-positive bacteria at very low concentrations (18,19,20). Although the antifungal activity of valinomycin has been reported (18), Altayar and Sutherland (1) showed that cereulide does not have any antifungal activity on rich medium. Since fungi are ubiquitous organisms living in the same environment as B. cereus (11, 12), a possible competition between these organisms could, however, be expected. The purpose of this study was to assess the potential antifungal activity of three emetic B. cereus strains and to highlight the specific contribution of cereulide using isogenic cereulide-producing and cereulide-minus (cured or mutated) strains. Comparison with the antifungal activity spectrum of valinomycin was also ...

“…Cereulide is a so-called cyclic dodecadepsipeptide containing a three-repeat sequence [cyclo(L-O-Val-L-Val-D-O-Leu-D-Ala-)3], and its K ϩ ionophoric properties have been established in black-lipid membranes and by assessing its impact on mitochondrial swelling and function (1,32,45,68). Recent research focused mainly on the genetic factors involved in the production of cereulide, the identification of transcriptional regulators, detection methods, and the impact of environmental conditions on cereulide levels reached, including a variety of (model) foods (13,15,17,38,69,71,78). Yet, besides its role in food poisoning and its effect on mammalian cells, the biological relevance of cereulide production in the environment, including its impact on other bacteria, has not been addressed.…”

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confidence: 99%

“…Recent research focused mainly on the genetic factors involved in the production of cereulide, the identification of transcriptional regulators, detection methods, and the impact of environmental conditions on cereulide levels reached, including a variety of (model) foods (13,15,17,38,69,71,78). Yet, besides its role in food poisoning and its effect on mammalian cells, the biological relevance of cereulide production in the environment, including its impact on other bacteria, has not been addressed.…”

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confidence: 99%

Comparative Analysis of Antimicrobial Activities of Valinomycin and Cereulide, the Bacillus cereus Emetic Toxin

Tempelaars

Rodrigues

Abee

2011

Cereulide and valinomycin are highly similar cyclic dodecadepsipeptides with potassium ionophoric properties. Cereulide, produced by members of the Bacillus cereus group, is known mostly as emetic toxin, and no ecological function has been assigned. A comparative analysis of the antimicrobial activity of valinomycin produced by Streptomyces spp. and cereulide was performed at a pH range of pH 5.5 to pH 9.5, under anaerobic and aerobic conditions. Both compounds display pH-dependent activity against selected Gram-positive bacteria, including Staphylococcus aureus, Listeria innocua, Listeria monocytogenes, Bacillus subtilis, and Bacillus cereus ATCC 10987. Notably, B. cereus strain ATCC 14579 and the emetic B. cereus strains F4810/72 and A529 showed reduced sensitivity to both compounds, with the latter two strains displaying full resistance to cereulide. Both compounds showed no activity against the selected Gram-negative bacteria. Antimicrobial activity against Gram-positive bacteria was highest at alkaline pH values, where the membrane potential (⌬⌿) is the main component of the proton motive force (PMF). Furthermore, inhibition of growth was observed in both aerobic and anaerobic conditions. Determination of the ⌬⌿, using the membrane potential probe DiOC 2 (3) (in the presence of 50 mM KCl) in combination with flow cytometry, demonstrated for the first time the ability of cereulide to dissipate the ⌬⌿ in sensitive Gram-positive bacteria. The putative role of cereulide production in the ecology of emetic B. cereus is discussed.Members of the Bacillus cereus group, also known as Bacillus cereus sensu lato, are Gram-positive, rod-shaped, facultative anaerobic, spore-forming bacteria (30,31). This group consists of the species B. cereus, B. weihenstephanensis, B. mycoides, B. pseudomycoides, B. anthracis, and B. thuringiensis (16, 30, 31, 70). B. cereus is found predominantly in the soil (3,30,70,75) and has been isolated from the rhizosphere (3, 5), the air (70), the gut of invertebrates (30,39), and a wide range of foods (16,70). It is a notorious food spoilage organism (4, 70) and the causative agent of two types of food-borne disease (16,22,70), the emetic and diarrheal syndromes. The emetic toxin cereulide is produced by emetic B. cereus (1, 2, 22, 70) and some psychrotolerant B. weihenstephanensis strains (72,73). This food intoxication, expressed by vomiting, is usually mild but occasionally results in fatalities (12,42,70). The toxin was found to be proteolytic resistant and highly heat and pH stable (57,67,68,74). Cereulide is a so-called cyclic dodecadepsipep-, and its K ϩ ionophoric properties have been established in black-lipid membranes and by assessing its impact on mitochondrial swelling and function (1,32,45,68). Recent research focused mainly on the genetic factors involved in the production of cereulide, the identification of transcriptional regulators, detection methods, and the impact of environmental conditions on cereulide levels reached, including a variety of (model) foods (13,15,17,38,...