Identification of the sigmaB regulon of Bacillus cereus and conservation of sigmaB-regulated genes in low-GC-content gram-positive bacteria

Abstract

The alternative sigma factor sigma(B) has an important role in the acquisition of stress resistance in many gram-positive bacteria, including the food-borne pathogen Bacillus cereus. Here, we describe the identification of the set of sigma(B)-regulated genes in B. cereus by DNA microarray analysis of the transcriptome upon a mild heat shock. Twenty-four genes could be identified as being sigma(B) dependent as witnessed by (i) significantly lower expression levels of these genes in mutants with a deletion of sigB and rsbY (which encode the alternative sigma factor sigma(B) and a crucial positive regulator of sigma(B) activity, respectively) than in the parental strain B. cereus ATCC 14579 and (ii) increased expression of these genes upon a heat shock. Newly identified sigma(B)-dependent genes in B. cereus include a histidine kinase and two genes that have predicted functions in spore germination. This study shows that the sigma(B) regulon of B. cereus is considerably smaller than that of other gram-positive bacteria. This appears to be in line with phylogenetic analyses where sigma(B) of the B. cereus group was placed close to the ancestral form of sigma(B) in gram-positive bacteria. The data described in this study and previous studies in which the complete sigma(B) regulon of the gram-positive bacteria Bacillus subtilis, Listeria monocytogenes, and Staphylococcus aureus were determined enabled a comparison of the sets of sigma(B)-regulated genes in the different gram-positive bacteria. This showed that only three genes (rsbV, rsbW, and sigB) are conserved in their sigma(B) dependency in all four bacteria, suggesting that the sigma(B) regulon of the different gram-positive bacteria has evolved to perform niche-specific functions.

FIG. 1.

Validation of microarray results by qPCR. Three independent RNA samples were isolated from cultures of B. cereus strain ATCC 14579 (WT) and the sigB deletion mutant during mid-exponential growth at 30°C (ME) and after a heat shock to 42°C for 10 min (HS). The expression ratios between both strains of the indicated genes were measured using microarrays (closed squares) and qPCR (open circles). Small symbols indicate that no significant difference was measured for the expression levels of the target gene between the two strains.

FIG. 2.

Comparison of differentially expressed genes between B. cereus ATCC 14579 (WT) and the sigB and rsbY deletion mutants. The numbers of genes that are differentially expressed between the parental strain (WT) and the rsbY and sigB mutants upon a heat shock to 42°C for 10 min are indicated in the circles. The black overlapping area indicates that the same genes were identified as being differentially expressed in both the sigB and the rsbY deletion mutants under the indicated conditions. Twenty-four genes were also upregulated upon a heat shock, and these genes form the σ^B regulon of B. cereus.

FIG. 3.

Consensus sequence of the σ^B-dependent promoter sites in B. cereus. The promoter sequences indicated in Table 2 were visualized with WebLogo (2). The height of the nucleotide is indicative of its frequency at that position in the promoter sites. Variable spacing between the −35 and −10 binding sites for σ^B is signified by the absence of a nucleotide at position 22.

FIG. 4.

Conservation of genes from the σ^B regulon in B. cereus, B. subtilis, L. monocytogenes, and S. aureus. The number of genes that are directly regulated by σ^B in a given organism is shown in the large circle. The conservation of σ^B-dependent genes from this organism in three other gram-positive bacteria (B. c., B. cereus; B. s., B. subtilis; L. m., L. monocytogenes; S. a., S. aureus) was determined with TIGR's Multi-Genome Homology Comparison tool (http://cmr.tigr.org/tigr-scripts/CMR/shared/MakeFrontPages.cgi?page=circular_display) with a cutoff for significance of a P value of ≤1.0 × 10⁻⁵. The numbers of conserved genes of the σ^B regulon are indicated in the arrows toward the small circles. The numbers in parentheses are the numbers of genes from the σ^B regulon of the query organism that have a homolog in the target organism. The second number in the arrow indicates the number of genes from this group, which are dependent on σ^B for their expression in both organisms. For comparative purposes, the conservation of all protein-coding genes between the genomes is indicated at the bottom of the figure.