CesH Represses Cereulide Synthesis as an Alpha/Beta Fold Hydrolase in Bacillus cereus

Abstract

Cereulide is notorious as a heat-stable emetic toxin produced by Bacillus cereus and glucose is supposed to be an ingredient supporting its formation. This study showed that glucose addition benefited on cell growth and the early transcription of genes involved in substrate accumulation and toxin synthesis, but it played a negative role in the final production of cereulide. Meanwhile, a lasting enhancement of cesH transcription was observed with the addition of glucose. Moreover, the cereulide production in ΔcesH was obviously higher than that in the wild type. This indicates that CesH has a repression effect on cereulide production. Bioinformatics analysis revealed that CesH was an alpha/beta hydrolase that probably associated with the cell membrane, which was verified by subcellular localization. The esterase activity against para-nitrophenyl acetate (PNPC2) of the recombinant CesH was confirmed. Although no sign of ester bond cleavage in cereulide or valinomycin was demonstrated in in vitro assays, CesH could reverse the cereulide analogue sensitivity of Bacillus subtilis in vivo, by which toxin degradation was facilitated. Moreover, site directed mutations identified that the conserved catalytic triad of CesH might consist of Serine 86, Glutamate 199, and Histidine 227. These results help us to understand the regulation of cereulide production and provide clues for developing control measurements.

Keywords: Bacillus cereus; alpha/beta hydrolase; cereulide; cesH.

Figure 1

(a–f) Effects of glucose and cesH on the cereulide productivity of B. cereus AH187. (a) 72 h growth curve; (b) Cereulide productivity in CADM containing 0% (dash line), 1% (grey line), and 2% (black line) (v/v) glucose at the 8th, 16th, and 24th hour, respectively; Relative mRNA levels of (c) ilvB, (d) cesA, and (e) cesH in CADM containing 0% (white column), 1% (grey column), and 2% (black column) (v/v) glucose at the 8th, 16th, and 24th hour, respectively; (f) Cereulide productivity of AH187 wild type (white column), ΔcesH (black column), and the complementary strain Cm.ΔcesH (grey column) at the 8th, 16th, and 24th hour, respectively. Data were displayed as means and standard deviations from three biological replicates, where the significant differences are indicated by asterisks (* p < 0.1, ** p < 0.05 and *** p < 0.01).

Figure 2

(a–d) Prediction and characterization of CesH as a membrane associated esterase. (a) The predicted 3D structure of CesH. 1, homologous modeling result of CesH via Phyre2.0. The alpha helix and the beta strand were separately labeled in cyan and magenta, and the yellow helix represents the amphipathic alpha at the C terminal. 2, the possible catalytic pocket. S86, E199, and H227 were labeled in Red. 3 and 4, the 3D model and helical diagram of region from 236-247, the hydrophobic residues (L236, F237, M240, L241, W244, and L245) were labeled in blue and hydrophilic residues (K235, N238, K239, E242, E243, and D246) were labeled in white; (b) Subcellular localization of CesH in two recombinant strains B. cereus AH187 gfp and B. cereus AH187 cesH-gfp. (c) Relative activity of CesH against PNPC2 under different temperature. (d) Lineweaver–Burk plot of CesH.

Figure 3

(a–f) Expression of CesH in B. subtilis contributed to an acquisition of valinomycin resistance in the host. (a,b) Comparison of valinomycin sensitivities of B. subtilis 168 (dash line) and B. subtilis 168H (line) by OD measuring and viable cell counting in LB medium containing 9 μM valinomycin (black), or not (grey); (c) chromatogram results of the reaction mixture containing valinomycin at the 0 min, 30 min, and 60 min, respectively; and (d) the relative degradation ratio of valinomycin in vivo counting by HPLC results, relative degradation ratio (E) at any time (t) was calculated according to the equation: E = [(Conc_{168H, T = 0}–Conc_{168H, T = t}) − (Conc_{168, T = 0}–Conc_{168, T = t})]/Conc_{168H, T = 0} × 100%; (e,f) Proliferations and viabilities of the four mutated strains of B. subtilis 168H: S86A (square), H189A (down triangle), E199A (circle), H227A (up triangle) as well as the positive strain of B. subtilis 168H (diamond) were accessed under the valinomycin exposure condition.