Control of enterotoxin gene expression in Bacillus cereus F4430/73 involves the redox-sensitive ResDE signal transduction system

Abstract

In contrast to Bacillus subtilis, the role of the two-component regulatory system ResDE has not yet been investigated in the facultative anaerobe Bacillus cereus. We examined the role of ResDE in the food-borne pathogen B. cereus F4430/73 by constructing resDE and resE mutants. Growth performances, glucose metabolism, and expression of hemolysin BL (Hbl) and nonhemolytic enterotoxin (Nhe) were analyzed in the three strains under distinct oxygenation and extracellular oxidoreduction potential (ORP) conditions. We show that growth and glucose metabolism were only moderately perturbed in both resDE and resE mutants under aerobiosis, microaerobiosis, and anaerobiosis generated under N(2) atmosphere (initial ORP = +45 mV). The major effects of resDE and resE mutations were observed under low-ORP anaerobic conditions generated under hydrogen atmosphere (iORP = -148 mV). These conditions normally favor enterotoxin production in the wild type. The resE mutation was more deleterious to the cells than the resDE mutation, causing growth limitation and strong deregulation of key catabolic genes. More importantly, the resE mutation abolished the production of enterotoxins under all of the conditions examined. The resDE mutation only decreased enterotoxin expression under anaerobiosis, with a more pronounced effect under low-ORP conditions. Thus, the ResDE system was found to exert major control on both fermentative growth and enterotoxin expression, and it is concluded that the ResDE system of B. cereus should be considered an anaerobic redox regulator. The data presented also provide evidence that the ResDE-dependent regulation of enterotoxins might function at least partially independently of the pleiotropic virulence gene regulator PlcR.

FIG. 1.

Major routes of the anaerobic and aerobic catabolic pathways in Bacillus cereus. For clarity, catabolic enzymes are indicated by their gene names. Gene names and functions are described in Table 1. Q, (mena)quinone.

FIG. 2.

(A) Gene organization of the B. cereus chromosome region containing resDE. Arrowhead and stem-loop-type structures indicate putative promoters for σ_A showing the directions of transcription and of the ρ-independent transcriptional terminators. The arrows show the positions and directions of the gene-specific primers used in RT-PCR to amplify internal regions within resA (1), resC (2), resD (3), and resE (4) and the intergenic region between resC and resD (IG). (B) RT-PCR analysis for detecting resA (1), resC (2), resDE (3), and resE (4) transcripts and the intergenic region (IG) between resC and resDE in strain F4430/73 grown under N₂ anaerobiosis (pO₂ = 0%) or full aerobiosis (pO₂ = 100%). Labels on the left indicate the size of the amplified fragments in base pairs. (C) Predicted membrane topology of the kinase sensor ResE. Putative transmembrane domains (amphipathic helices) are depicted as black rectangles. Putative PAS and histidine kinase cytoplasmic domains are also indicated. Coordinates of the amino acid residues that are expected to face the cell wall and constitute the two cytoplasmic domains are indicated in parentheses. Phosphoryl transfer from ResE to the response regulator ResD is also shown.

FIG. 3.

Comparison of specific enterotoxin production between the resDE and resE mutant strains and their parent strain B. cereus F4430/73. Hbl and Nhe levels were measured in culture supernatants at the end of pH-controlled batch cultures conducted under anaerobiosis at low initial ORP (−148 mV) and high initial ORP (+45 mV), under microaerobiosis (pO₂ = 1%, iORP = +174 mV), 21% aerobiosis (pO₂ = 21%; iORP = +222 mV), and under full aerobiosis (pO₂ = 100%; iORP = +280 mV). Bars indicate the standard error of the mean.

FIG. 4.

Comparison of catabolic gene transcript levels between resDE and resE mutant strains and the wild-type strain grown under anaerobic, microaerobic, and aerobic conditions at different initial oxidoreduction potentials (iORP). The data for each gene are normalized relative to the ssu transcript and are shown as the transcript ratios from the resDE mutant (▪) or the resE mutant (░⃞) strain relative to the wild type. The order of genes from ptsG to resA is the same as that described in Table 3. Two independent experiments were performed for each strain and growth condition. For each culture, two measurements from two independent RNA samples taken from the mid-exponential growth phase (μ = μ_max) were analyzed in parallel. Each datum point is an average of the results for the combined experiments. Only ratios of ≤0.5 and ≥2 were considered to be significant (i.e., P ≤ 0.05) according to the precision of the method. Note that no resD transcript was detected in resDE mutant.

FIG. 5.

Changes in specific transcript levels of the PlcR-regulated genes hblC (A), nheA (B), and plcR (C) induced by resDE and resE mutations under anaerobic, microaerobic, and aerobic growth conditions at different initial oxidoreduction potentials (iORP). The change in mRNA level of each gene in resDE or resE was calculated relative to that of the wild type. Each datum point is an average of three independent measurements. RNA sampling and statistical methods were identical to those described in Fig. 4 and in Materials and Methods.