Cereulide synthetase gene cluster from emetic Bacillus cereus: structure and location on a mega virulence plasmid related to Bacillus anthracis toxin plasmid pXO1

Abstract

Background: Cereulide, a depsipeptide structurally related to valinomycin, is responsible for the emetic type of gastrointestinal disease caused by Bacillus cereus. Recently, it has been shown that this toxin is produced by a nonribosomal peptide synthetase (NRPS), but its exact genetic organization and biochemical synthesis is unknown.

Results: The complete sequence of the cereulide synthetase (ces) gene cluster, which encodes the enzymatic machinery required for the biosynthesis of cereulide, was dissected. The 24 kb ces gene cluster comprises 7 CDSs and includes, besides the typical NRPS genes like a phosphopantetheinyl transferase and two CDSs encoding enzyme modules for the activation and incorporation of monomers in the growing peptide chain, a CDS encoding a putative hydrolase in the upstream region and an ABC transporter in the downstream part. The enzyme modules responsible for incorporation of the hydroxyl acids showed an unusual structure while the modules responsible for the activation of the amino acids Ala and Val showed the typical domain organization of NRPS. The ces gene locus is flanked by genetic regions with high homology to virulence plasmids of B. cereus, Bacillus thuringiensis and Bacillus anthracis. PFGE and Southern hybridization showed that the ces genes are restricted to emetic B. cereus and indeed located on a 208 kb megaplasmid, which has high similarities to pXO1-like plasmids.

Conclusion: The ces gene cluster that is located on a pXO1-like virulence plasmid represents, beside the insecticidal and the anthrax toxins, a third type of B. cereus group toxins encoded on megaplasmids. The ces genes are restricted to emetic toxin producers, but pXO1-like plasmids are also present in emetic-like strains. These data might indicate the presence of an ancient plasmid in B. cereus which has acquired different virulence genes over time. Due to the unusual structure of the hydroxyl acid incorporating enzyme modules of Ces, substantial biochemical efforts will be required to dissect the complete biochemical pathway of cereulide synthesis.

Figure 1

Biosynthetic gene cluster for cereulide synthesis. The domain organization of the structural cereulide synthetase genes cesA and cesB is indicated (see Results for details) and the flanking regions showing homologies to toxin plasmids from B. cereus group members are printed as hatched boxes. For details on CDS designation see Table 2. The bars refer to probes used to test the conservation of ces genes in the B. cereus group (see Table 3) Inset: Structure of cereulide according to [13].

Figure 2

Genetic analysis of A domains. (A3 motif to the A6 motif) from nonribosomal peptide synthetases. The tree was constructed with TREECON [48] using the neighbor-joining method. All bootstrap values > 70% (1000 replicates) are shown next to the nodes. A domains from the cereulide synthetase (Ces) are printed in bold type. Abbreviations: Bac: bacitracin synthetase; Bar: barbamide synthetase; Css: cyclosporine synthetase; Esyn: Enniatin; Fen: fengycin synthetase; Grs: gramicidin S synthetase; Hts: HC-toxin; LicD: lichenysin synthetase; Myc: mycosubtilin synthetase; Nda: nodularin synthetase; Saf: Saframycin synthetase; Srf: surfactin synthetase; Tyc: tyrocidine A synthetase.

Figure 3

(A) Alignment of the adenylation. (A) domain core motifs lining the substrate binding pocket of Ces and unusual core motifs from other bacterial NRPS. Consensus sequence of core motifs A4 to A7 (according to [53]) is depicted. Residues identical to amino acids from Ces core motifs are printed in boldface type. (B) Insertions in A domains from CesA2 (D-O-Leu) and CesB1 (L-O-Val) were aligned to short chain dehydrogenases (SDR) and ketoreductases (KR); partial sequences including putative NADPH binding sites (solid bar) and the catalytic residues of SDRs/KR (printed in boldface type). Numbers between hyphens in the first lines refer to residues not shown before residues depicted in the second lines. JamL: KR from Jamaicamides synthetase (GenBank accession no. AY522504) of Lyngbya majuscula; EryA: KR from erythronolide synthetase of Saccharopolyspora erythraea (GenBank accession no. M63676). GlcDh: Glucose 1-dehydrogenase from B. megaterium (Swissprot accession no. P39482).

Figure 4

Hybridization of emetic and emetic-like B. cereus with probes targeting the ces operon (a) and a pXO1 related CDS (b). Total DNA was separated by PFGE, transferred to a membrane and hybridized with a cesB specific probe and a probe derived from pXO1-11 (for details on probes see Supplemental Materials Table S1). Hybridization with both probes revealed a single band for emetic strains which has the same size as the pBc10987 plasmid from B. cereus ATCC 10987 (lane 2).

Figure 5

Comparison of partial sequenced toxin plasmid pBCE4810 to toxin plasmids from B. cereus group members. The genetic region responsible for cereulide production in emetic strains was compared to the B. anthracis toxin-encoding plasmid pXO1, to pBCXO1 from B. cereus G9241 capable of causing an anthrax-like illness, to pBc10987 from B. cereus ATCC 10987 and to pBCEL1519 from the emetic-like strain NVH1519-00. For CDS designation of pBCE and pBCEL see Table 2. I_I: Group I intron; I_II: Group II intron.