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

Abstract

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 cesPTABCD 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.

FIG. 1.

Transcriptional analysis of the ces gene cluster. (A) Bars indicate overlapping primer pairs used for transcriptional analysis of the ces operon (listed in Table S1 in the supplemental material). Bent arrows indicate promoters as determined by primer extension. (B) RT-PCR showed the presence of consecutive transcripts between cesP and cesD (lane 3 to 9) but no transcripts between cesH and cesP (lane 2). There were no transcripts from a forward primer in cesD and reverse primer downstream of cesD after the predicted hairpin termination structure (lane 10). Negative controls (RNA; see lane H-) and positive controls (DNA; see lane H+). M, marker ladder mixture (Fermentas).

FIG. 2.

5′ RACE mapping of ces promoter sites. RACE products detected for the ces genes indicate intracistronic promoters present (cesT and cesB). Central promoters upstream of cesP (P₁ and P₂), indicated by an asterisk, and the cesP promoter region are depicted. Transcription (+1) sites and translation start sites are boxed. A putative ribosome binding site (RBS) and putative −10 and −35 σ^A recognition sites (deduced from the consensus sites for B. subtilis) are underlined. Putative transcription starts determined by RACE were confirmed by primer extension. No intracistronic promoter was detected for cesA (by RACE or by PE). M, marker ladder mixture (Fermentas).

FIG. 3.

Real-time monitoring of cereulide synthetase promoter activity in food. (A) The bioluminescent reporter strain B. cereus F4810/72(pMDX[P₁/luxABCDE]) was inoculated into various foods. After incubation for 24 h at 24°C, the luciferase gene expression controlled by the main cereulide synthetase promoter P₁ was visualized with a photon-counting ICCD camera. According to the intensity of luminescence signals, foods were designated as low-risk, risk, and high-risk products depending on their potential to support cereulide synthesis (Table 2). (B) P₁ promoter activity in different emetic strains on cooked rice. Depicted are the emetic reference strain F4810/72, one clinical isolate, and one isolate from a recent food-borne emetic outbreak transformed with the pMDX[P₁/luxABCDE] luciferase reporter plasmid.