The bc1009 gene in the SigB gene cluster (Fig. 1A) encodes a putative Hpr-like phosphocarrier protein (Bc1009) in B. cereus. The potential role of bc1009 acting as a SigB regulator was first examined via gene expression studies, and its SigB-dependent functions were investigated via proteome profiling (complemented with transcriptomic data). The global protein/gene expressions of the Δbc1009 mutant before and after heat shock were analyzed and compared with the protein/gene expressions of the B. cereus wt cells and a marker-free ΔsigB mutant under the same conditions.

Expression of bc1009 is dependent on sigB, but deletion of bc1009 does not affect the expression of sigB

Changes in expression within the SigB gene cluster: bc1002 (rsbV), bc1003 (rsbW), bc1004 (sigB), bc1005, bc1006 (rsbY), bc1008 (rsbK), and bc1009 of B. cereus wt, ΔsigB, and Δbc1009 cells upon heat shock (30 °C → 42 °C) were determined via RT-qPCR. Results were first compared for wt versus the ΔsigB mutant (Fig. 1B), and then for wt versus the Δbc1009 mutant (Fig. 1C).

Upon heat shock of wt cells, the expression of the three known SigB-dependent genes rsbV (bc1002), bc1005, bc1009, and sigB itself increased > 200-fold (~ log2 8) compared to wt cells without heat shock. The rsbY (bc1006) and rsbK (bc1008) genes that encode the two-component system were also expressed ~ 4- fold (~ log2 2) higher in wt cells after heat shock than before heat shock (Figs. 1B and C). Upon heat shock of the ΔsigB mutant, rsbV and bc1005 were mildly expressed (< 4- fold) (~ log2 2), whereas bc1009 was expressed at a lower level after heat shock in the ΔsigB mutant (Fig. 1B), showing that its heat induction was dependent on SigB. The expression of the histidine kinase encoding gene rsbK is controlled by sigA. The observation that its expression was similar for the ΔsigB mutant and the wt cultures after heat shock is in line with this. Its cognate regulator partner rsbY was expressed at a lower level in the ΔsigB mutant than in wt cultures, indicating partial dependency of its expression on SigB (Fig. 1B). Notably, when the bc1009 gene was deleted, the expression of all other genes in the SigB cluster was unaffected and similar to that in wt cultures (Fig. 1C). This implied that sigB expression did not rely on bc1009, ruling out a possible role of bc1009 as an additional phosphocarrier in the activation of SigB via the RsbKY system.

Comparison of proteomic profiles of ΔsigB and Δbc1009 mutants to wt upon heat shock reveals novel SigB-dependent proteins conceivably mediated via Bc1009 in B. cereus

To further explore the role of Bc1009 and SigB in B. cereus, we compared the proteome profiles of B. cereus cultures of wt, ΔsigB, and Δbc1009 mutants grown at 30 °C with cultures that were heat-shocked at 42ºC after growth at 30 °C. The proteomics results of wt, ΔsigB, and Δbc1009 cells obtained in this study are presented below and complemented by transcriptomics results (Supplementary files).

Heat-induced changes of proteome profiles in wt cells

After heat shock treatment, ~ 1500 proteins were detected for wt cells. Among these, 429 proteins displayed significantly increased (≥ 1.5 folds, log2 FC ≥ 0.585), and 435 significantly decreased (≤ 1.5 folds, log2 FC ≤ 0.585) levels in wt cells after heat shock compared to control conditions, respectively (Fig. 2). The complete list of proteins displaying significantly different levels is presented in Supplementary Table S2A, and the clusters of orthologous group (COG) functions are presented in Supplementary Figure S1.

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

As expected, next to previously described SigB regulon members (see below), well-known protein chaperones (GroEL, DnaJ, DnaK), transcription repressors (HrcA, CtsR) that control Clp proteases (ClpC, ClpP, ClpB, ClpY, ClpQ), DNA repair proteins (RadA, MutS) and other well-known heat shock proteins like GrpE, YflT, and FtsH were all present at ≥ 1.5 folds higher levels in wt cultures after heat shock at 42 °C compared to control conditions at 30 °C. These heat shock proteins were also identified in an earlier study on B. cereus heat stress response [51], confirming the congruency of the results obtained in this study. Extended information on the B. cereus heat shock regulon was acquired (Supplementary Table S2A), and ~ 20% of the proteins with temperature-dependent changes in level found in wt cultures upon heat shock were also influenced at the gene expression level (Supplementary Table S2B).

Impact of SigB and Bc1009 on B. cereus protein profiles after heat shock SigB-dependent and SigB/Bc1009-dependent heat shock induction of proteins

The protein profiles of ΔsigB and Δbc1009 mutants were further compared with wt cells. Out of 429 proteins displaying increased levels after heat shock in wt cells, 175 showed lower levels in the ΔsigB mutant, indicating that the expression of the encoding genes was inducible via SigB in wt cells directly or indirectly (Fig. 2, Table 2). Additionally, for 98 of these 175 proteins, this effect was not only dependent on SigB but also on Bc1009 because they also exhibited lower levels in the Δbc1009 mutant than in the wt (Fig. 2, Table 2), hereby referred to as SigB (and Bc1009)-dependent proteins. The complete list of the SigB-induced and SigB (and Bc1009)-induced proteins with their functional annotation is listed in Table 2. The log2 fold changes of the protein levels are presented in the volcano plot in Fig. 3A, and their COG functions in Fig. 3B. The top 30 most significant SigB-induced proteins are indicated with numbers in Fig. 3A on the right (marked in white), and for half of these, the increase in level was also dependent on Bc1009 (marked in Grey in Fig. 3A) (see details in Table 2).

Table 2 SigB-dependent induced proteins upon heat shock in B. cereus ATCC14579a 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

The 175 proteins displaying SigB-dependent increases in level after heat shock include the general stress defense proteins previously described by van Schaik et al. [10] and De Been et al. [12], i.e., Bc0861, Bc0862 (YfkM), Bc0863 (KatE), Bc0998 (YflT), Bc0999 (CsbD), Bc1002 (RsbV), Bc1003 (RsbW), Bc1004 (SigB), Bc1005 (bacterioferritin), Bc1010 (hypothetical), Bc1012 (hypothetical), Bc1154 (ferrochelatase), and Bc3132 (general stress protein) (Table 2). In line with these findings, significantly induced transcription was seen for genes that encode these general stress proteins (Supplementary Table S3B). Although the Bc1009 protein was not detected, the complementary transcriptional results showed that the bc1009 gene was induced (Supplementary Table S3B).

Other newly identified members of the SigB-regulon mainly belong to the COG groups of cell motility, signal transduction mechanisms, transcription, amino acid transport, and metabolisms, or a group without assigned function (Fig. 3B, Tables 2 and 3). Representatives of the cell motility COG group included many flagella or chemotaxis proteins (FliM, FliN, FliG, YvzB, Bc1637, FlgE, PhnB, McpBH, YoaH, CheV, CheA, Bc0404, MotA, Bc0678, TlpA), and the signal transduction and transcription COG groups contained proteins that are related to sporulation (SigK, YndF, Spo0A, Bc4463). Other strongly induced proteins included Bc0409 (carbamate kinase), Bc0566 (endonuclease/exonuclease phosphatase family protein), and Bc0666 (Immune inhibitor A precursor). Strikingly, a large number of transcriptional regulators were shown to be SigB-dependent, including SigK (sporulation sigma factor), Spo0A (sporulation initiation protein), CggR (central glycolytic regulator), ArsR family transcriptional regulator Bc0613, GntR family transcriptional regulator YvfI, PadR, YdgH, and the MerR family transcriptional regulator Bc3356 (Table 2). The most significantly induced protein—Bc0107 (YacN), a 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, has a putative role in lipid transport and metabolism. Together with the strongly induced protein Bc4345 that encodes a lipase, this suggests an additional role of SigB in the modulation of membrane lipid composition. Remarkably, a cluster of proteins (Bc5117—Bc5125) that is likely involved in the transport of nutrients, and Bc0423 (a non-ribosomal peptide synthetase that is involved in secondary metabolite production) also showed significant induction in wt cells and impaired production in ΔsigB cells after heat shock compared to before heat shock (Fig. 3B, Table 2).

Table 3 SigB-dependent downregulated proteins upon heat shock in B. cereus ATCC14579a

A total of 98 of the newly identified SigB-dependent proteins are also Bc1009-dependent, including flagella/chemotaxis proteins, transcriptional activator/repressors, ABC transporters, proteins involved in amino acid transport and metabolism, and cell wall/membrane/envelope biogenesis (Fig. 3B, and bold-highlighted in Table 2). Similarly, as reported above, the Bc5117- Bc5125 cluster and Bc0423 (non-ribosomal peptide synthetase) also showed reduced levels in Δbc1009 culture compared to wt cells. Bc0423 was also downregulated at the gene level in both mutants (complementing transcriptomic data are presented in Supplementary Table S3B. Several other proteins that showed significantly different levels in ΔsigB and Δbc1009 cultures versus wt do not belong to the COG groups mentioned above. For instance, YocJ (FMN-dependent NADH-azoreductase) was induced > 50-fold (log2 ~ 5.6) stronger in wt cultures compared to ΔsigB mutant, and > tenfold (log2 ~ 3.3) stronger in wt cultures compared to Δbc1009 mutant. Similar observations were made for other proteins with unknown functions (Bc4786, Bc5066, Bc3935 cytosolic protein, Bc5077, Bc0666 immune inhibitor A precursor) (see Table 2).

These results were supported by the transcriptomics data (Supplementary Table S3B), which showed differential transcription of genes encoding the reported motility proteins in ΔsigB and Δbc1009 cultures, pointing to a role of SigB and Bc1009 in the control of cell motility. Notably, despite the SigB-induced transcription of a group of phage genes, the corresponding encoded proteins were either not detected or not differentially produced in heat-stressed cells.

SigB-dependent and SigB/Bc1009- dependent downregulated proteins

On the other hand, 109 of 435 proteins that showed lower levels in wt cells after heat shock than before heat shock showed higher levels in the ΔsigB mutant, indicating that the expression of the encoding genes is likely indirectly regulated by SigB in wt cells (Fig. 2, Table 3). 40 of these 109 proteins also showed higher levels in the Δbc1009 mutant than in wt, suggesting that the encoding genes are directly or indirectly regulated by SigB and Bc1009 (Fig. 2, Table 3). The complete list of the SigB-dependent and SigB (and Bc1009)-dependent proteins that displayed lower levels after heat shock with their annotated functions is presented in Table 3. The log2 fold changes of the levels of these proteins are presented in the volcano plot (Fig. 3A), and their COG functions are shown in Fig. 3B. The top 20 SigB-dependent proteins with lower reductions of levels after heat shock in ΔsigB and Δbc1009 cells compared to wt are indicated by numbers in Fig. 3A on the left (marked in white), with five of these being SigB and Bc1009- dependent (marked in Grey in Fig. 3A) (see details in Table 3).

Many of the 109 proteins (Fig. 2, Table 3) that revealed SigB-dependent reductions in levels in wt cells after heat shock compared to control samples have undefined functions or fall into the COG group of proteins with transcription, cell wall biogenesis, and energy production and conversion functions (Fig. 3B, Table 3). For instance, the most prominently downregulated proteins in wt cells (~ 25 to 75 fold; log2 FC =  ~ 4.6—6.2) after heat shock vs. before heat shock include RpsT (SSU ribosomal protein, involved in translation, ribosomal structure, and biogenesis), Bc2026 (oligopeptide-binding protein OppA involved in amino acid transport and mechanism), Bc1699 (ECF-type sigma factor negative effector with unknown function), YkfJ (protein tyrosine phosphatase in signal transduction), and Bc3442 (hypothetical protein). Remarkably, several other proteins involved in transcription were significantly downregulated in wt cells but less so in ΔsigB and Δbc1009 cells, such as the ArgR arginine repressor, Sigma-54-dependent transcriptional activator GlcR regulator, TetR family regulator Bc3592, and ArsR family transcriptional regulator Bc4256 (Table 3).

For 40 of these proteins (Table 3, bold highlighted), the reduction in level after heat shock was not only dependent on SigB but also on Bc1009, and for many of them the function has not been defined yet. Those showing significant differential expression with known functions are mainly engaged in transcription, including YitH acetyltransferase, ArgR arginine repressor, Bc2298 transcriptional repressor, Bc4652 IcaR transcriptional regulator, Bc4256 ArsR family transcriptional regulator, BkdR sigma 54-dependent transcriptional activator, and Bc2369 acetyltransferase.

Transcriptional analysis generally supported proteomics data, except for a group of nar genes involved in anaerobic respiration; these showed SigB dependency in wt cells upon heat shock (Supplementary Table S3B), while no differential expression of corresponding proteins was observed (Table 3). Moreover, comparative proteomics and transcriptomics data of wt vs. Δbc1009 also showed an additional group of Bc1009-induced/downregulated proteins/genes that are not dependent on SigB. Several prominent Bc1009-induced proteins are transcriptional regulators and Bc1009-downregulated proteins are phage or transport proteins (Table S4A, Figure S3), but their exact roles in B. cereus in relation to heat-stress response are yet to be elucidated. As many of these proteins/genes have hypothetical functions and this study focussed on the SigB-mediated responses, these data are not further discussed here, but details are listed in Supplementary Table S4A (Figure S3) and S4B, respectively.

The results presented show more than 300 newly identified SigB-dependent proteins (Tables 2 and 3), and more than 100 of these require both SigB and Bc1009 for changes in their level in heat-stressed cells, indicating a significant extension of the B. cereus SigB regulon and a subregulon additionally requiring the Hpr-like phosphocarrier protein Bc1009. Most of these SigB and Bc1009-dependent proteins are involved in cell motility, signal transduction mechanisms, transcription, amino acid transport and metabolism, and cell wall biogenesis. Other proteins are responsible for DNA replication and repair, protein quality maintenance, and cell wall remodeling, suggesting a role of Bc1009 in adaptive heat stress response in B. cereus as well.

Bc1009 and SigB contribute to survival of severe heat stress

To determine the impact of bc1009 and sigB on survival of severe heat stress, the wt strain and isogenic mutants lacking Bc1009 or SigB were exposed to 50 °C, with or without pre-adaptation at 42 °C (Fig. 4). Pre-adaption of wt, ΔsigB, and Δbc1009 cultures resulted in a significantly higher survival rate at 50 °C than the non-pre-adapted control cells. The wt showed a 1.5 log10 reduction at 120 min. Although the adapted ΔsigB, Δbc1009, and wt cultures showed similar survival during the first 40 min of exposure, the survival of the ΔsigB mutant then rapidly declined, resulting in approximately 2 log10 reductions after 120 min compared to the wt. The thermotolerance of adapted Δbc1009 cells was higher than that of ΔsigB cells. Survival rates were also similar to wt cells for the first 80 min, but a stronger decrease in survival was observed after 120 min at 50 °C with an approximate 1.3 log10 reduction compared to the wt. This points to a modest role of Bc1009 in activating heat stress defense, in line with its role in controlling the expression of a subset of SigB-dependent genes/proteins.

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

Exposure to 50 °C of cultures of wt, ΔsigB and Δbc1009 that were not pre-adapted at 42 °C showed rapid killing. After 20 min of exposure to this lethal heat stress, the wt showed a more prominent reduction than the two mutants however, a 3 log10 reduction was observed for all three strains after 30 min (Fig. 4).

Impact of SigB and Bc1009 on the B. cereus protein profile under control conditions at 30°C

The impact of SigB and Bc1009 on the level of motility proteins after heat shock, and previous studies on the regulation of B. cereus motility at 30 °C by MogR and RpoN [52, 53], prompted us to perform an additional comparative omics analysis of non-stressed wt, ΔsigB and Δbc1009 cells at 30 °C.

In total, 96 proteins (Table 4) showed significant differences in level in both ΔsigB and Δbc1009 mutants compared to wt cells already at 30 °C, indicating that even their basal levels are dependent on SigB and Bc1009. This included 72 proteins with higher levels in the wt compared to the mutants and 24 with lower levels in the wt compared to the mutants (Fig. 5A, Table 4). The proteins displaying the largest SigB and Bc1009-dependent differences in levels are indicated with numbers in Fig. 5A on the right and left, respectively. The COG functions of these proteins are shown in Fig. 5B, and the complete list of differentially regulated proteins is presented in Table 4 (detailed in Supplementary Table S5A), with the complemented transcriptome data shown in Supplementary Table S5B. Interestingly, ~ 50% (41 of 72 induced and 12 of 24 downregulated) of the differentially expressed proteins that were detected at 30 °C were also differentially regulated in heat-stressed cells (bold highlighted in Table 4) and described above in the section Comparison of proteomic profiles of ΔsigB and Δbc1009 mutants to wt upon heat shock.

Table 4 Differentially regulated SigB and Bc1009-dependent proteins at 30°C in B. cereus ATCC14579aFig. 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

SigB and Bc1009-dependently induced proteins at 30°C

SigB and Bc1009-dependent induced proteins at 30 °C include flagella and chemotaxis proteins, transcriptional regulators like Spo0A and CggR, the Bc5117- Bc5125 cluster (conceivably involved in transporting nutrients), and Bc0423 (Non-ribosomal peptide synthetase) (Table 4). Next to proteins that were also differentially expressed under heat shock (described above in the section  Impact of SigB and Bc1009 on B. cereus protein profiles after heat shock), an additional group of proteins involved in carbohydrate/ion transport and metabolisms was present at lower levels in ΔsigB and Δbc1009 cells compared to wt cells at 30 °C (Fig. 5B, Table 4). These were YtzE, Bc2464, and Bc0896- Peptidoglycan endo-beta-N-acetylglucosaminidase, Bc1157- alpha-amylase, CutC- copper homeostasis protein, YclP-ferric transport ATP binding protein, Bc4984 ABC transporter, YtiB carbonic anhydrase, and YvgY copper chaperon associated protein. The remaining uniquely SigB and Bc1009-induced proteins at 30 °C mainly have undefined functions or are additional proteins found in the same COG groups described above, including motility (Table 4). Proteomics results were supported by transcriptional data (Supplementary Table S5B).

SigB and Bc1009-dependently downregulated proteins at 30°C

An additional 24 proteins (Table 4) were present at lower levels in wt cells compared to ΔsigB and Δbc1009 mutants at 30 °C, of which 12 also displayed lower levels in wt cells upon heat shock, and 12 were uniquely regulated at 30 °C (Table 4). Four SigB and Bc1009-dependent downregulated proteins have putative roles in transcription regulation, including LytR family transcriptional regulator (Bc3587), leucine-responsive regulatory protein LrpC (Bc1363), ArsR family transcriptional regulator (Bc4256) and arginine repressor ArgR (Bc0405), of which the latter two were also upregulated in heat-stressed ΔsigB and Δbc1009 mutants compared to wt (Impact of SigB and Bc1009 on B. cereus protein profiles after heat shock section). Remarkably, the arginine repressor ArgR (Bc0405) and Bc0385 thioredoxin reductase were both present at 100-fold lower levels (log2 fold change ~ 6) in wt cells compared to ΔsigB and Δbc1009 mutants (Fig. 5, Table 4). However, genes that encode these proteins did not show differential expression (Supplementary Table S5B), suggesting regulation at the post-transcriptional level.

Taken together, this section provides evidence that SigB may be active already during control conditions at 30 °C, showing its alternative role in cellular functions other than the SigB GSR, potentially via the putative Hpr-like protein, Bc1009.

Δbc1009 and ΔsigB mutants both show a defective motility phenotype

Based on the observation that many motility/chemotaxis proteins were present at higher levels in both non-stressed and heat-stressed wt cells compared to non-stressed and heat-stressed ΔsigB and Δbc1009 mutants, we compared the motility of ΔsigB and Δbc1009 mutants with that of wt cells on BHI agar plates with a low agar percentage (0.25%) under three conditions: 1) at 30 °C (isothermal); 2) at 30 °C, following a heat-shock at 42 °C for 30 min; and 3) at 42 °C (isothermal). A mutant unable to produce flagella (ΔflgG) was used as a negative control. Results that are presented in Fig. 6 show that at 30 °C, the colony diameter of ΔflgG mutant was lowest, followed by that of Δbc1009, then ΔsigB, and with the wt showing the highest motility (Fig. 6 left and right; observed phenotypes). Following a mild heat shock for 30 min at 42 °C and subsequent incubation at 30 °C, again, the colony diameter of ΔflgG mutant was lowest, with both ΔsigB and Δbc1009 mutants displaying intermediate levels of motility, with the wt displaying the highest motility. Similarly, incubation at isothermal 42 °C showed the lowest and highest motility of ΔflgG mutant and wt cells, respectively, while ΔsigB and Δbc1009 cells showed comparable intermediate motility. These results show that SigB-induced motility in non-heat-stressed and heat-stressed cells depends on Bc1009.

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