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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

CsfB inhibits in vitro transcription by RNA polymerase associated with σG or σE.A: schematic representation of the promoter-containing PCR fragments used as templates for the in vitro transcription reactions. The expected size (in nucleotides) for each of the run-off products is indicated. B: effect of CsfB on in vitro transcription reactions with the indicated RNA polymerase holoenzymes. CsfB (130 nM) was either added to the reaction after mixing core (E; 13 nM) and the sigma subunit (“a”; 130 nM) or together with the sigma subunit to a mixture already containing core (“b”). The symbol “-”refers to a control reaction lacking CsfB. C and D: effect of the CsfB concentration, shown in molar ratio relative to RNA polymerase (13 nM), on in vitro transcription reactions with the indicated holoenzymes. CsfB was added at the same molar concentration as sigma (130 nM, 1x) or 2, 3, 4 and 8-fold excess and the mixture added to core RNA polymerase (13 nM). In B-D, arrows indicate the position of the expected run-off products, identified with arrows, which maintain the color code for the templates as represented in A. The position of molecular weight markers (in nucleotides) is shown on the left side of the panels.
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pgen.1005104.g005: CsfB inhibits in vitro transcription by RNA polymerase associated with σG or σE.A: schematic representation of the promoter-containing PCR fragments used as templates for the in vitro transcription reactions. The expected size (in nucleotides) for each of the run-off products is indicated. B: effect of CsfB on in vitro transcription reactions with the indicated RNA polymerase holoenzymes. CsfB (130 nM) was either added to the reaction after mixing core (E; 13 nM) and the sigma subunit (“a”; 130 nM) or together with the sigma subunit to a mixture already containing core (“b”). The symbol “-”refers to a control reaction lacking CsfB. C and D: effect of the CsfB concentration, shown in molar ratio relative to RNA polymerase (13 nM), on in vitro transcription reactions with the indicated holoenzymes. CsfB was added at the same molar concentration as sigma (130 nM, 1x) or 2, 3, 4 and 8-fold excess and the mixture added to core RNA polymerase (13 nM). In B-D, arrows indicate the position of the expected run-off products, identified with arrows, which maintain the color code for the templates as represented in A. The position of molecular weight markers (in nucleotides) is shown on the left side of the panels.

Mentions: While CsfB binds to both σG [20] and σE ([34]; see above), no study has shown direct inhibition of transcriptional activity by the anti-sigma factor. To test the ability of purified CsfB to inhibit σG- or σE-directed transcription, core RNA polymerase (E) was purified from B. subtilis, and reconstituted with σF, σE, σG, σK, or σA overproduced and purified from E. coli cells (S2A Fig). As templates for in vitro transcription reactions, we used PCR-generated DNA fragments corresponding to promoters of genes known to be under the control of the sigma factors tested, and whose transcriptional start site has been mapped. As such, the gcaD gene was used as the template for σA-directed transcription [35,36], spoIIQ as a template for EσF [27,37], spoIID, spoIIM, spoIIIA p1 and spoIIIA p2 as templates for EσE [38,39,40], sspB for EσG [30], and gerE for EσK [33] (Fig. 5A and S2B Fig). All forms of RNA polymerase tested directed the production of run-off transcripts of expected sizes (Fig. 5B-D and S2C Fig). No specific transcription products were seen when templates were mixed with core RNA polymerase in the absence of a σ subunit (S2C Fig). CsfB did not inhibit transcription by EσA, EσF (Fig. 5B) or EσK (Fig. 5C), but inhibited the σE-directed utilization of the spoIIM (Fig. 5B), spoIID (Fig. 5C and D), spoIIIA p1 and spoIIIA p2 promoters (S2C Fig). CsfB also inhibited the σG-directed transcription of sspB (Fig. 5B and D). Inhibition of EσA, EσFor EσK by CsfB was not observed even at molar ratios higher than those that inhibited EσE (Fig. 5C and D). Interestingly, inhibition of EσE, and to some extent of EσG, required molar ratios higher than 1 (Fig. 5B and D; S2C Fig). One possibility is that active CsfB is a dimer (or a higher order multimeric form), in agreement with the results of a previous study [21].


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)

CsfB inhibits in vitro transcription by RNA polymerase associated with σG or σE.A: schematic representation of the promoter-containing PCR fragments used as templates for the in vitro transcription reactions. The expected size (in nucleotides) for each of the run-off products is indicated. B: effect of CsfB on in vitro transcription reactions with the indicated RNA polymerase holoenzymes. CsfB (130 nM) was either added to the reaction after mixing core (E; 13 nM) and the sigma subunit (“a”; 130 nM) or together with the sigma subunit to a mixture already containing core (“b”). The symbol “-”refers to a control reaction lacking CsfB. C and D: effect of the CsfB concentration, shown in molar ratio relative to RNA polymerase (13 nM), on in vitro transcription reactions with the indicated holoenzymes. CsfB was added at the same molar concentration as sigma (130 nM, 1x) or 2, 3, 4 and 8-fold excess and the mixture added to core RNA polymerase (13 nM). In B-D, arrows indicate the position of the expected run-off products, identified with arrows, which maintain the color code for the templates as represented in A. The position of molecular weight markers (in nucleotides) is shown on the left side of the panels.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005104.g005: CsfB inhibits in vitro transcription by RNA polymerase associated with σG or σE.A: schematic representation of the promoter-containing PCR fragments used as templates for the in vitro transcription reactions. The expected size (in nucleotides) for each of the run-off products is indicated. B: effect of CsfB on in vitro transcription reactions with the indicated RNA polymerase holoenzymes. CsfB (130 nM) was either added to the reaction after mixing core (E; 13 nM) and the sigma subunit (“a”; 130 nM) or together with the sigma subunit to a mixture already containing core (“b”). The symbol “-”refers to a control reaction lacking CsfB. C and D: effect of the CsfB concentration, shown in molar ratio relative to RNA polymerase (13 nM), on in vitro transcription reactions with the indicated holoenzymes. CsfB was added at the same molar concentration as sigma (130 nM, 1x) or 2, 3, 4 and 8-fold excess and the mixture added to core RNA polymerase (13 nM). In B-D, arrows indicate the position of the expected run-off products, identified with arrows, which maintain the color code for the templates as represented in A. The position of molecular weight markers (in nucleotides) is shown on the left side of the panels.
Mentions: While CsfB binds to both σG [20] and σE ([34]; see above), no study has shown direct inhibition of transcriptional activity by the anti-sigma factor. To test the ability of purified CsfB to inhibit σG- or σE-directed transcription, core RNA polymerase (E) was purified from B. subtilis, and reconstituted with σF, σE, σG, σK, or σA overproduced and purified from E. coli cells (S2A Fig). As templates for in vitro transcription reactions, we used PCR-generated DNA fragments corresponding to promoters of genes known to be under the control of the sigma factors tested, and whose transcriptional start site has been mapped. As such, the gcaD gene was used as the template for σA-directed transcription [35,36], spoIIQ as a template for EσF [27,37], spoIID, spoIIM, spoIIIA p1 and spoIIIA p2 as templates for EσE [38,39,40], sspB for EσG [30], and gerE for EσK [33] (Fig. 5A and S2B Fig). All forms of RNA polymerase tested directed the production of run-off transcripts of expected sizes (Fig. 5B-D and S2C Fig). No specific transcription products were seen when templates were mixed with core RNA polymerase in the absence of a σ subunit (S2C Fig). CsfB did not inhibit transcription by EσA, EσF (Fig. 5B) or EσK (Fig. 5C), but inhibited the σE-directed utilization of the spoIIM (Fig. 5B), spoIID (Fig. 5C and D), spoIIIA p1 and spoIIIA p2 promoters (S2C Fig). CsfB also inhibited the σG-directed transcription of sspB (Fig. 5B and D). Inhibition of EσA, EσFor EσK by CsfB was not observed even at molar ratios higher than those that inhibited EσE (Fig. 5C and D). Interestingly, inhibition of EσE, and to some extent of EσG, required molar ratios higher than 1 (Fig. 5B and D; S2C Fig). One possibility is that active CsfB is a dimer (or a higher order multimeric form), in agreement with the results of a previous study [21].

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