The redox regulator Fnr is required for fermentative growth and enterotoxin synthesis in Bacillus cereus F4430/73

Abstract

Glucose-grown cells of Bacillus cereus respond to anaerobiosis and low extracellular oxidoreduction potentials (ORP), notably by enhancing enterotoxin production. This response involves the ResDE two-component system. We searched the B. cereus genome for other redox response regulators potentially involved in this adaptive process, and we identified one gene encoding a protein predicted to have an amino acid sequence 58% identical (80% similar) to that of the Bacillus subtilis Fnr redox regulator. The fnr gene of the food-borne pathogen B. cereus F4430/73 has been cloned and partially characterized. We showed that fnr was up-regulated during anaerobic fermentation, especially when fermentation occurred at low ORP (under highly reducing conditions). The expression of fnr was down-regulated in the presence of O(2) and nitrate which, unlike fumarate, stimulated the respiratory pathways. The inactivation of B. cereus fnr abolished fermentative growth but only moderately affected aerobic and anaerobic nitrate respiratory growth. Analyses of glucose by-products and the transcription profiles of key catabolic genes confirmed the strong regulatory impact of Fnr on B. cereus fermentative pathways. More importantly, the fnr mutation strongly decreased the expression of PlcR-dependent hbl and nhe genes, leading to the absence of hemolysin BL (Hbl) and nonhemolytic enterotoxin (Nhe) secretion by the mutant. These data indicate that fnr is essential for both fermentation and toxinogenesis. The results also suggest that both Fnr and the ResDE two-component system belong to a redox regulatory pathway that functions at least partially independently of the pleiotropic virulence gene regulator PlcR to regulate enterotoxin gene expression.

FIG. 1.

Architecture of fermentative and respiratory pathways in B. cereus. Genomic data (24) and information from references (12, 26, 45, 60) were used for this scheme. Most of the genes and functions are described in Table 4. TCA, tricarboxylic acid; Q, (mena)quinone; DHAP, dihydroxyacetone phosphate; e⁻, electron; c, cytochrome c; acetyl-CoA, acetyl coenzyme A; FAD, flavin adenine dinucleotide; FADH₂, reduced flavin adenine dinucleotide. The glpD gene encoding sn-glycerol-3-phosphate dehydrogenase was repressed by glucose and induced during aerobic and anaerobic growth on glycerol (reference and data not shown). Respiratory complex III encoded by the qcrABC operon was required for growth on glycerol, while it made only a minor contribution to growth on glucose (45).

FIG. 2.

Genetic organization of the fnr chromosomal region of B. cereus F4430/73 and ATCC 14579. (A) Large arrows represent the open reading frames identified in strain ATCC 14579 (ERGO; Integrated Genomics, Inc.), and their orientation shows the transcriptional direction. Only the names of open reading frames encoding proteins with predicted metabolic functions are indicated. (B) The nucleotide sequences of the promoter region and the putative terminator of F4430/73 fnr are shown. Primers F and R were used for the amplification of the overall DNA of the fnr locus, and primers SP1, SP2, and SP3 were used to map the transcriptional start site. The transcriptional start site (+1) and the putative −35 and −10 motifs are boxed. Putative CRP boxes are underlined. RBS, ribosome-binding site.

FIG. 3.

Effect of growth conditions on specific levels of fnr transcripts. Wild-type F4430/73 cells were grown in regulated batch cultures (pH 7.2) under aerobic (pO₂ = 100% and 21%), microaerobic (pO₂ = 1%), and anaerobic (pO₂ = 0%) conditions at different initial ORPs (iORP) as described in Materials and Methods. Relative specific levels can be compared to the 100% value that was arbitrarily fixed for the fully aerobic condition (pO₂ = 100%). For each experiment, two measures from two independent RNA samples from the same culture taken during the mid-exponential growth phase (μ = μ_max) were analyzed in parallel. Each data point represents an average of the results for the combined experiments. RNA sampling and statistical methods were identical to those described in Table 4 and in Materials and Methods.

FIG. 4.

Changes in specific levels of transcripts of the PlcR-regulated genes hbl, nhe, and plcR induced by fnr mutation under aerobic (respiratory) and anaerobic nitrate (respirofermentative) growth conditions. The change in the mRNA level for each gene in the fnr mutant was calculated in relation to the mRNA level for the wild-type. Data presentation is as described in the legend to Fig. 3.