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. 2023 Feb 10;23(1):37.
doi: 10.1186/s12866-023-02783-3.

SigB modulates expression of novel SigB regulon members via Bc1009 in non-stressed and heat-stressed cells revealing its alternative roles in Bacillus cereus

Kah Yen Claire Yeak #  1   2 , Marcel Tempelaars #  2 , Jia Lun Wu  2 , Wouter Westerveld  2 , Alexander Reder  3 , Stephan Michalik  3 , Vishnu M Dhople  3 , Uwe Völker  3 , Jan Pané-Farré  4 , Marjon H J Wells-Bennik  1 , Tjakko Abee  5
Affiliations

SigB modulates expression of novel SigB regulon members via Bc1009 in non-stressed and heat-stressed cells revealing its alternative roles in Bacillus cereus

Kah Yen Claire Yeak et al. BMC Microbiol. .

Abstract

Background: The Bacillus cereus Sigma B (SigB) dependent general stress response is activated via the two-component RsbKY system, which involves a phosphate transfer from RsbK to RsbY. It has been hypothesized that the Hpr-like phosphocarrier protein (Bc1009) encoded by bc1009 in the SigB gene cluster may play a role in this transfer, thereby acting as a regulator of SigB activation. Alternatively, Bc1009 may be involved in the activation of a subset of SigB regulon members.

Results: We first investigated the potential role of bc1009 to act as a SigB regulator but ruled out this possibility as the deletion of bc1009 did not affect the expression of sigB and other SigB gene cluster members. The SigB-dependent functions of Bc1009 were further examined in B. cereus ATCC14579 via comparative proteome profiling (backed up by transcriptomics) of wt, Δbc1009 and ΔsigB deletion mutants under heat stress at 42 °C. This revealed 284 proteins displaying SigB-dependent alterations in protein expression levels in heat-stressed cells, including a subgroup of 138 proteins for which alterations were also Bc1009-dependent. Next to proteins with roles in stress defense, newly identified SigB and Bc1009-dependent proteins have roles in cell motility, signal transduction, transcription, cell wall biogenesis, and amino acid transport and metabolism. Analysis of lethal stress survival at 50 °C after pre-adaptation at 42 °C showed intermediate survival efficacy of Δbc1009 cells, highest survival of wt, and lowest survival of ΔsigB cells, respectively. Additional comparative proteome analysis of non-stressed wt and mutant cells at 30 °C revealed 96 proteins with SigB and Bc1009-dependent differences in levels: 51 were also identified under heat stress, and 45 showed significant differential expression at 30 °C. This includes proteins with roles in carbohydrate/ion transport and metabolism. Overlapping functions at 30 °C and 42 °C included proteins involved in motility, and ΔsigB and Δbc1009 cells showed reduced motility compared to wt cells in swimming assays at both temperatures.

Conclusion: Our results extend the B. cereus SigB regulon to > 300 members, with a novel role of SigB-dependent Bc1009 in the activation of a subregulon of > 180 members, conceivably via interactions with other transcriptional regulatory networks.

Keywords: Adaptive general stress response; Amino acid metabolisms; Metabolic crosstalk; Motility; Phosphocarrier protein; SigB baseline function; SigB subregulon; Sporulation; Virulence.

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Conflict of interest statement

The authors declare that there are no conflicts of interest.

Figures

Fig. 1
Fig. 1
SigB gene cluster in Bacillus cereus and differential gene expression after heat shock. A- SigB gene cluster arrangement in B. cereus ATCC14579. B- Change in expression of genes belonging to the sigB gene cluster for wt and ΔsigB mutant after 20 min of heat shock at 42 °C compared to the non-heat-stressed condition at 30 °C. N = 4, p < 0.001. C- Change in expression of genes belonging to the SigB gene cluster expression for wt and Δbc1009 mutant after heat shock at 42 °C compared to the non-heat-stressed condition at 30 °C; Black bar - wt; white bar - ΔsigB mutant; grey bar - Δbc1009 mutant. * indicates the absence of gene activity. N = 4, p < 0.001, refers to the difference in expression values of wt vs. either ΔsigB or Δbc1009 mutant
Fig. 2
Fig. 2
Proteomics analyses of B. cereus ATCC14579 wt, ΔsigB, and Δbc1009 mutants upon heat shock. Left: Venn diagram showing 429 significantly induced (upregulated) proteins in B. cereus heat-stressed wt cells (black circle) compared to the non-heat-stressed wt cells at 30 °C (see Supplementary Table S2A). White circle: 175 heat-induced proteins in wt that are SigB-dependent, i.e., proteins that show upregulation of > 0.6 log2 fold change in wt/ΔsigB cells upon heat shock compared to the non-heat-stressed condition at 30 °C. Grey circle: For 98 proteins, the heat-mediated increase in level in the wt is dependent on SigB and Bc1009, i.e., proteins that show upregulation of > 0.6 log2 fold change in wt/ΔsigB and wt/Δbc1009 cells upon heat shock compared to the non-heat-stressed condition at 30 °C (see Table 2 for the complete list of proteins). Right: Venn diagram showing 435 significantly downregulated proteins in B. cereus heat-stressed wt cells (black circle) compared to the non-heat-stressed wt cells at 30 °C (Supplementary Table S2A). White circle: 109 downregulated proteins in heat-stressed wt cells vs. non-heat-stressed wt cells are SigB-dependent, i.e., proteins that show downregulation of > 0.6 log2 fold change in wt/ΔsigB cells upon heat shock compared to the non-heat-stressed condition at 30 °C. Grey circle: For 40 proteins, reduction in level in wt cells upon heat shock was dependent on SigB and Bc1009, i.e., proteins that show downregulation of > 0.6 log2 fold change in wt/ΔsigB and wt/Δbc1009 cells upon heat shock vs. non-heat-stressed condition (see Table 3). The underlying transcriptome data supporting this figure are presented in Supplementary Table S2B
Fig. 3
Fig. 3
SigB and SigB (and Bc1009)-dependent induced and downregulated proteins in wt cells after heat shock (30 °C to 42 °C) versus before heat shock and their cluster of orthologous group (COG). A- log2 fold change in protein expression. Positive and negative fold change values indicate induced and downregulated proteins, respectively. Numbers indicate the top 30 induced (Table 2) or top 20 downregulated (Table 3) proteins, respectively. The p-value threshold for each protein was < 0.05, N = 4. Grey symbols: induced/downregulated proteins that are SigB (and Bc1009-dependent; i.e., proteins that show up(or down) regulation of > 0.6 log2 fold change in wt/ΔsigB and wt/Δbc1009 cells upon heat shock vs. non-heat-stressed condition. White symbols: induced/downregulated proteins that are SigB-dependent; i.e., proteins that show up(or down) regulation of > 0.6 log2 fold change in wt/ΔsigB cells upon heat shock vs. non-heat-stressed condition. B- cluster of orthologous group (COG) function for both SigB-dependent induced (Right) and downregulated proteins (Left) in wt cells upon heat shock. The number on the x-axis indicates the total number of induced/downregulated proteins, and the negative sign indicates downregulation. Grey bar: induced/downregulated proteins after heat shock that are SigB (and Bc1009)-dependent, i.e., proteins that show up(or down) regulation of > 0.6 log2 fold change in wt/ΔsigB and wt/Δbc1009 cells; White bar: induced/downregulated proteins that are SigB-dependent, i.e., proteins that show up(or down) regulation of > 0.6 log2 fold change only in wt/ΔsigB cells respectively, compared to the expression in the non-heat-stressed condition at 30 °C. The underlying transcriptome data supporting this figure are presented in Supplementary Table S3B
Fig. 4
Fig. 4
The relative survival of B. cereus wt, ΔsigB, and Δbc1009 mutants upon lethal heat exposure at 50°C. The relative survival at 50 °C of heat-preadapted cells (30 °C to 42 °C for 45 min) of B. cereus wt (filled circle), ΔsigB (filled triangle), and Δbc1009 cells (filled square) compared to cells that were not preadapted to heat (42 °C) (wt- open circle; ΔsigB- open triangle; Δbc1009- open square) for 120 min. N = 4. p < 0.001 for time point at 100 min and 120 min when comparing wt and the two mutant strains. Error bars show the standard deviation of four biological replicates
Fig. 5
Fig. 5
SigB-dependent induced and downregulated proteins for ΔsigB and Δbc1009 mutants compared to wt cells at 30°C and their cluster of orthologous group (COG). A- log2 fold change in protein expression. Positive and negative fold change values indicate induced and downregulated proteins, respectively. Grey symbols: induced/downregulated proteins that are SigB (and Bc1009)- dependent, i.e., differentially expressed proteins in ΔsigB and Δbc1009 mutants compared to wt cells at 30 °C; the numbers indicate the top 30 induced proteins (positive x-axis) or top 20 downregulated proteins (negative x-axis), with details listed in Table 4. White symbols: induced/downregulated proteins at 30 °C that are either only SigB-dependent or Bc1009-dependent, i.e., differentially expressed proteins in ΔsigB or Δbc1009 mutants compared to wt cells at 30 °C (see details in Supplementary Table S5A). The threshold of the p-value for each protein was < 0.05. N = 4. B- cluster of orthologous group (COG) function for SigB (and Bc1009)-induced and downregulated proteins. The number on the x-axis indicates the number of induced/downregulated proteins in respective COG groups. The underlying transcriptome data supporting this figure are presented in Supplementary Table S5B
Fig. 6
Fig. 6
Motility phenotype of wt, ΔsigB, and Δbc1009 mutants. A- the motility of wt, ΔsigB, and Δbc1009 was compared on Brain Heart Infusion (BHI) agar with 0.25% agar and indicated by the colony diameter (mm) formed on the agar after 24 h incubation. Black bar: wt; Grey bar: ΔsigB mutant; white bar: Δbc1009 mutant; light grey bar: ΔflgG mutant (negative control without flagella). The motility of all cells was tested under three different conditions, 1) at 30 °C for 24 h; 2) upon heat shock from 30 °C to 42 °C for 30 min, and back to 30 °C for 24 h; and 3) at 42 °C for 24 h. The dotted line shows the maximum plate size. B- Colony of wt, ΔsigB, Δbc1009, and ΔflgG cells on 0.25% BHI agar at 30 °C after 24 h. Error bars indicates the standard deviation of colony diameters of four biological replicates
Fig. 7
Fig. 7
Activation model of Bacillus cereus SigB and the SigB-dependent Bc1009 subregulon. The RsbKY two-component system mediates the SigB general stress response in B. cereus. Under heat shock (temperature shift from 30 °C to 42 °C), SigB is induced (bold arrow) via the RsbKY signaling cascade and directly regulates the Hpr-like phosphocarrier protein Bc1009 to mediate amino acid transport and metabolism, cell motility, signal transduction, cell wall/membrane/envelope biogenesis and transcription. At 30 °C, the baseline level of SigB also appears to regulate Bc1009 (non-bold arrow) to mediate similar functions as described for the heat shock condition, except for amino acid transport and metabolism (which switch to carbohydrate/ion transport and metabolism at 30 °C instead, indicated in dark grey and light grey box, respectively). Upon SigB activation, other SigB regulon members not controlled by Bc1009 are also activated (see main text, Tables 2 and 3, and Supplementary Tables S5A and B). White color circles: known SigB signalling pathway as described in the literature [5, 12, 15, 16], Grey and Black circles and boxes: new insight obtained in the current study (see text for details)
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