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Dual-specificity anti-sigma factor reinforces control of cell-type specific gene expression in Bacillus subtilis.

Serrano M, Gao J, Bota J, Bate AR, Meisner J, Eichenberger P, Moran CP, Henriques AO - PLoS Genet. (2015)

Bottom Line: We also show that CsfB prevents activation of σG in the mother cell and the premature σG-dependent activation of σK.The capacity of CsfB to directly block σE activity may also explain how CsfB plays a role as one of the several mechanisms that prevent σE activation in the forespore.Thus the capacity of CsfB to differentiate between the highly similar σF/σG and σE/σK pairs allows it to rinforce the cell-type specificity of these sigma factors and the transition from early to late development in B. subtilis, and possibly in all sporeformers that encode a CsfB orthologue.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Estação Agronómica Nacional, Oeiras, Portugal.

ABSTRACT
Gene expression during spore development in Bacillus subtilis is controlled by cell type-specific RNA polymerase sigma factors. σFand σE control early stages of development in the forespore and the mother cell, respectively. When, at an intermediate stage in development, the mother cell engulfs the forespore, σF is replaced by σG and σE is replaced by σK. The anti-sigma factor CsfB is produced under the control of σF and binds to and inhibits the auto-regulatory σG, but not σF. A position in region 2.1, occupied by an asparagine in σG and by a glutamate in οF, is sufficient for CsfB discrimination of the two sigmas, and allows it to delay the early to late switch in forespore gene expression. We now show that following engulfment completion, csfB is switched on in the mother cell under the control of σK and that CsfB binds to and inhibits σE but not σK, possibly to facilitate the switch from early to late gene expression. We show that a position in region 2.3 occupied by a conserved asparagine in σE and by a conserved glutamate in σK suffices for discrimination by CsfB. We also show that CsfB prevents activation of σG in the mother cell and the premature σG-dependent activation of σK. Thus, CsfB establishes negative feedback loops that curtail the activity of σE and prevent the ectopic activation of σG in the mother cell. The capacity of CsfB to directly block σE activity may also explain how CsfB plays a role as one of the several mechanisms that prevent σE activation in the forespore. Thus the capacity of CsfB to differentiate between the highly similar σF/σG and σE/σK pairs allows it to rinforce the cell-type specificity of these sigma factors and the transition from early to late development in B. subtilis, and possibly in all sporeformers that encode a CsfB orthologue.

No MeSH data available.


Related in: MedlinePlus

Sigma-CsfB interactions.A: pull-down assays using GBP. Whole cell extracts were prepared for cultures of different strains 4 hours after the onset of sporulation in resuspension medium. The extracts were cleared and incubated with GBP bound to sepharose beads. Samples of the whole cell extracts, as well as the proteins bound to the GBP beads were visualized, following elution, by immunoblotting with anti-σG, anti-σE, anti-σK and anti-GFP antibodies. Strains in A: a wild type strain, a strain producing GFP from the xylose-inducible PxylA promoter; strains producing CsfB-GFP from its native promoter region (PcsfB), from the σF-type promoter (PsigF) or the σK-type promoter (PsigK), as indicated. The asterisk indicates a likely degradation product of σG. B: Ni2+-NTA affinity chromatography assay for CsfB-σ interactions. His6-CsfB, and untagged σE and σK, were purified from E. coli cells and analysed by SDS-PAGE (first three lanes). The three proteins were then individually applied to a column and eluted with an imidazole buffer. The eluted proteins were detected following SDS-PAG by Coomassie-staining. His6-CsfB bound to the Ni2+-NTA column whereas σE or σK did not. σE but not σK, bound to the column in the presence of His6-CsfB. The asterisk indicates a likely degradation product of σE. Molecular weight markers (M, in kDa) are shown on the left side of panels B. The asterisks in A and B indicate likely degradation products of σG or σE. C: colony lift assay (left) and assays in liquid medium (right) for the detection of β-galactosidase activity in yeast strains expressing fusions of CsfB to the GAL4 activation domain (AD) and fusions of σF, σE, σG, σK, and σA to the GAL4 binding domain (BD), as indicated. Assays in which the BD and AD were expressed from empty vectors were used as negative controls (“-“).
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pgen.1005104.g004: Sigma-CsfB interactions.A: pull-down assays using GBP. Whole cell extracts were prepared for cultures of different strains 4 hours after the onset of sporulation in resuspension medium. The extracts were cleared and incubated with GBP bound to sepharose beads. Samples of the whole cell extracts, as well as the proteins bound to the GBP beads were visualized, following elution, by immunoblotting with anti-σG, anti-σE, anti-σK and anti-GFP antibodies. Strains in A: a wild type strain, a strain producing GFP from the xylose-inducible PxylA promoter; strains producing CsfB-GFP from its native promoter region (PcsfB), from the σF-type promoter (PsigF) or the σK-type promoter (PsigK), as indicated. The asterisk indicates a likely degradation product of σG. B: Ni2+-NTA affinity chromatography assay for CsfB-σ interactions. His6-CsfB, and untagged σE and σK, were purified from E. coli cells and analysed by SDS-PAGE (first three lanes). The three proteins were then individually applied to a column and eluted with an imidazole buffer. The eluted proteins were detected following SDS-PAG by Coomassie-staining. His6-CsfB bound to the Ni2+-NTA column whereas σE or σK did not. σE but not σK, bound to the column in the presence of His6-CsfB. The asterisk indicates a likely degradation product of σE. Molecular weight markers (M, in kDa) are shown on the left side of panels B. The asterisks in A and B indicate likely degradation products of σG or σE. C: colony lift assay (left) and assays in liquid medium (right) for the detection of β-galactosidase activity in yeast strains expressing fusions of CsfB to the GAL4 activation domain (AD) and fusions of σF, σE, σG, σK, and σA to the GAL4 binding domain (BD), as indicated. Assays in which the BD and AD were expressed from empty vectors were used as negative controls (“-“).

Mentions: The analysis of the global transcriptional profiling data raised the possibility that CsfB could act as an inhibitor of σEand/or σK. This prompted us to determine whether the anti-sigma factor could form complexes with either sigma factor. To isolate CsfB-GFP and interacting proteins from sporulating cultures of a strain expressing csfB-gfp, we used a GFP-binding protein (GBP) coupled to a chromatographic matrix (GFP-Trap beads). As controls, we examined a wild type strain carrying no gfp fusion and a strain producing GFP under the control of the xylose-inducible PxylA promoter. The extracts were prepared 4 hours after the onset of sporulation, when CsfB-GFP is known to accumulate in the mother cell (Fig. 2B; above). Control experiments also confirmed the accumulation of σE, σG, σK or GFP in the whole-cell extracts prepared from the various strains (Fig. 4A). The extracts from all strains were incubated with GFP-Trap beads, the bound proteins eluted and identified by immunoblot analysis with antibodies raised against σE, σG, σK or GFP. We found σE but not σK, to be pulled down efficiently from extracts of the strain producing CsfB-GFP. By contrast, σE was not recovered from extracts of the strain containing no GFP fusion or from the strain that produced unfused GFP (Fig. 4A). Thus, retention of σE by the GFP-trap beads depended on formation of a complex with CsfB-GFP. As expected, CsfB-GFP was able to pull down σG [20], a property used here as a positive control for the experiment (Fig. 4A). Interestingly, a role for CsfB in inhibiting σE activity in the forespore soon after asymmetric division was previously suggested [34]. Under the conditions of our experiments, however, σE is only expected to accumulate in the mother cell and the expression of csfB has switched to the mother cell in most sporangia in the population when the cells were harvested for the pull down assays (see above). Therefore, the result reported in Fig. 4A most likely reflects an interaction occurring between σE and CsfB in the mother cell and not in the forespore.


Dual-specificity anti-sigma factor reinforces control of cell-type specific gene expression in Bacillus subtilis.

Serrano M, Gao J, Bota J, Bate AR, Meisner J, Eichenberger P, Moran CP, Henriques AO - PLoS Genet. (2015)

Sigma-CsfB interactions.A: pull-down assays using GBP. Whole cell extracts were prepared for cultures of different strains 4 hours after the onset of sporulation in resuspension medium. The extracts were cleared and incubated with GBP bound to sepharose beads. Samples of the whole cell extracts, as well as the proteins bound to the GBP beads were visualized, following elution, by immunoblotting with anti-σG, anti-σE, anti-σK and anti-GFP antibodies. Strains in A: a wild type strain, a strain producing GFP from the xylose-inducible PxylA promoter; strains producing CsfB-GFP from its native promoter region (PcsfB), from the σF-type promoter (PsigF) or the σK-type promoter (PsigK), as indicated. The asterisk indicates a likely degradation product of σG. B: Ni2+-NTA affinity chromatography assay for CsfB-σ interactions. His6-CsfB, and untagged σE and σK, were purified from E. coli cells and analysed by SDS-PAGE (first three lanes). The three proteins were then individually applied to a column and eluted with an imidazole buffer. The eluted proteins were detected following SDS-PAG by Coomassie-staining. His6-CsfB bound to the Ni2+-NTA column whereas σE or σK did not. σE but not σK, bound to the column in the presence of His6-CsfB. The asterisk indicates a likely degradation product of σE. Molecular weight markers (M, in kDa) are shown on the left side of panels B. The asterisks in A and B indicate likely degradation products of σG or σE. C: colony lift assay (left) and assays in liquid medium (right) for the detection of β-galactosidase activity in yeast strains expressing fusions of CsfB to the GAL4 activation domain (AD) and fusions of σF, σE, σG, σK, and σA to the GAL4 binding domain (BD), as indicated. Assays in which the BD and AD were expressed from empty vectors were used as negative controls (“-“).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383634&req=5

pgen.1005104.g004: Sigma-CsfB interactions.A: pull-down assays using GBP. Whole cell extracts were prepared for cultures of different strains 4 hours after the onset of sporulation in resuspension medium. The extracts were cleared and incubated with GBP bound to sepharose beads. Samples of the whole cell extracts, as well as the proteins bound to the GBP beads were visualized, following elution, by immunoblotting with anti-σG, anti-σE, anti-σK and anti-GFP antibodies. Strains in A: a wild type strain, a strain producing GFP from the xylose-inducible PxylA promoter; strains producing CsfB-GFP from its native promoter region (PcsfB), from the σF-type promoter (PsigF) or the σK-type promoter (PsigK), as indicated. The asterisk indicates a likely degradation product of σG. B: Ni2+-NTA affinity chromatography assay for CsfB-σ interactions. His6-CsfB, and untagged σE and σK, were purified from E. coli cells and analysed by SDS-PAGE (first three lanes). The three proteins were then individually applied to a column and eluted with an imidazole buffer. The eluted proteins were detected following SDS-PAG by Coomassie-staining. His6-CsfB bound to the Ni2+-NTA column whereas σE or σK did not. σE but not σK, bound to the column in the presence of His6-CsfB. The asterisk indicates a likely degradation product of σE. Molecular weight markers (M, in kDa) are shown on the left side of panels B. The asterisks in A and B indicate likely degradation products of σG or σE. C: colony lift assay (left) and assays in liquid medium (right) for the detection of β-galactosidase activity in yeast strains expressing fusions of CsfB to the GAL4 activation domain (AD) and fusions of σF, σE, σG, σK, and σA to the GAL4 binding domain (BD), as indicated. Assays in which the BD and AD were expressed from empty vectors were used as negative controls (“-“).
Mentions: The analysis of the global transcriptional profiling data raised the possibility that CsfB could act as an inhibitor of σEand/or σK. This prompted us to determine whether the anti-sigma factor could form complexes with either sigma factor. To isolate CsfB-GFP and interacting proteins from sporulating cultures of a strain expressing csfB-gfp, we used a GFP-binding protein (GBP) coupled to a chromatographic matrix (GFP-Trap beads). As controls, we examined a wild type strain carrying no gfp fusion and a strain producing GFP under the control of the xylose-inducible PxylA promoter. The extracts were prepared 4 hours after the onset of sporulation, when CsfB-GFP is known to accumulate in the mother cell (Fig. 2B; above). Control experiments also confirmed the accumulation of σE, σG, σK or GFP in the whole-cell extracts prepared from the various strains (Fig. 4A). The extracts from all strains were incubated with GFP-Trap beads, the bound proteins eluted and identified by immunoblot analysis with antibodies raised against σE, σG, σK or GFP. We found σE but not σK, to be pulled down efficiently from extracts of the strain producing CsfB-GFP. By contrast, σE was not recovered from extracts of the strain containing no GFP fusion or from the strain that produced unfused GFP (Fig. 4A). Thus, retention of σE by the GFP-trap beads depended on formation of a complex with CsfB-GFP. As expected, CsfB-GFP was able to pull down σG [20], a property used here as a positive control for the experiment (Fig. 4A). Interestingly, a role for CsfB in inhibiting σE activity in the forespore soon after asymmetric division was previously suggested [34]. Under the conditions of our experiments, however, σE is only expected to accumulate in the mother cell and the expression of csfB has switched to the mother cell in most sporangia in the population when the cells were harvested for the pull down assays (see above). Therefore, the result reported in Fig. 4A most likely reflects an interaction occurring between σE and CsfB in the mother cell and not in the forespore.

Bottom Line: We also show that CsfB prevents activation of σG in the mother cell and the premature σG-dependent activation of σK.The capacity of CsfB to directly block σE activity may also explain how CsfB plays a role as one of the several mechanisms that prevent σE activation in the forespore.Thus the capacity of CsfB to differentiate between the highly similar σF/σG and σE/σK pairs allows it to rinforce the cell-type specificity of these sigma factors and the transition from early to late development in B. subtilis, and possibly in all sporeformers that encode a CsfB orthologue.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Estação Agronómica Nacional, Oeiras, Portugal.

ABSTRACT
Gene expression during spore development in Bacillus subtilis is controlled by cell type-specific RNA polymerase sigma factors. σFand σE control early stages of development in the forespore and the mother cell, respectively. When, at an intermediate stage in development, the mother cell engulfs the forespore, σF is replaced by σG and σE is replaced by σK. The anti-sigma factor CsfB is produced under the control of σF and binds to and inhibits the auto-regulatory σG, but not σF. A position in region 2.1, occupied by an asparagine in σG and by a glutamate in οF, is sufficient for CsfB discrimination of the two sigmas, and allows it to delay the early to late switch in forespore gene expression. We now show that following engulfment completion, csfB is switched on in the mother cell under the control of σK and that CsfB binds to and inhibits σE but not σK, possibly to facilitate the switch from early to late gene expression. We show that a position in region 2.3 occupied by a conserved asparagine in σE and by a conserved glutamate in σK suffices for discrimination by CsfB. We also show that CsfB prevents activation of σG in the mother cell and the premature σG-dependent activation of σK. Thus, CsfB establishes negative feedback loops that curtail the activity of σE and prevent the ectopic activation of σG in the mother cell. The capacity of CsfB to directly block σE activity may also explain how CsfB plays a role as one of the several mechanisms that prevent σE activation in the forespore. Thus the capacity of CsfB to differentiate between the highly similar σF/σG and σE/σK pairs allows it to rinforce the cell-type specificity of these sigma factors and the transition from early to late development in B. subtilis, and possibly in all sporeformers that encode a CsfB orthologue.

No MeSH data available.


Related in: MedlinePlus