Cereulide synthesis in emetic Bacillus cereus is controlled by the transition state regulator AbrB, but not by the virulence regulator PlcR

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Cereulide, the emetic Bacillus cereus toxin, is synthesized by cereulide synthetase via a nonribosomal peptide synthetase (NRPS) mechanism. Previous studies focused on the identification, structural organization, and biochemical characterization of the ces gene locus encoding cereulide synthetase; however, detailed information about the transcriptional organization of the ces genes was lacking. The present study shows that the ces-PTABCD genes are transcribed as a 23-kb polycistronic transcript, while cesH, encoding a putative hydrolase, is transcribed from its own promoter. Transcription initiation was mapped by primer extension and rapid amplification of cDNA ends (RACE). Deletion analysis of promoter elements revealed a main promoter located upstream of the cesP coding sequence, encoding a 4-phosphopantetheinyl transferase. This promoter drives transcription of cesPTABCD. In addition, intracistronic promoter regions in proximity to the translational start sites of cesB and cesT were identified but were only weakly active under the chosen assay conditions. The identified main promoter was amplified from the emetic reference strain B. cereus F4810/72 and fused to luciferase genes in order to study promoter activity in complex environments and to establish a biomonitoring system to assess cereulide production in different types of foods. ces promoter activity was strongly influenced by the food matrix and varied by 5 orders of magnitude. The amount of cereulide toxin extracted from spiked foods correlated well with the bioluminescence data, thus illustrating the potential of the established reporter system for monitoring of ces gene expression in complex matrices.Bacillus cereus is the causative agent of two types of food poisoning: diarrhea and emesis. The toxicoinfection referred to as diarrheal disease occurs after consumption of B. cereus spores or vegetative cells, most likely due to the action of heat-labile enterotoxins, which are produced by cells multiplying in the small intestine (4,22,30,39). Contrarily, the emetic type of food-borne illness is caused by intoxication with the heat-stable peptide cereulide, which is preformed in foods and elicits vomiting a few hours after ingestion (18,51,58).Regulation of enterotoxin expression of B. cereus has been studied in some detail (e.g., references 16, 29, and 46; for a review, see reference 58), revealing a major role of the pleiotropic transcription regulator PlcR in activation of enterotoxin genes and other virulence factors (1,29,45). Cereulide synthesis in emetic B. cereus, in contrast to synthesis of B. cereus enterotoxins, is not controlled by PlcR but by the Spo0A phosphorelay. The global transition state factor AbrB was identified as one factor repressing cereulide production in early exponential phase (38). Although B. cereus enterotoxins and the emetic toxin cereulide seem to belong to completely different regulatory networks, production of both types of toxins depends substantially on nutritional and environmental factors (3,46,55), and it is expe...

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confidence: 99%

“…The HEp-2 cell-based bioassay was carried out as described previously (38). Additionally, extracts of control food samples were measured in order to exclude an interfering toxic background effect of the different ethanolic extracts on the HEp-2 cells.…”

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confidence: 99%

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

Dommel

Frenzel

Strasser

et al. 2010

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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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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 ...

“…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,...