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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

A residue in σE involved in the interaction with CsfB.A: alignment of regions 2.1, 2.2 and the beginning of region 2.3 from the σA, σF, σG, σE, and σK proteins of the indicated species (the sequence of σA from T. thermophilus is identical to that of T. aquaticus for the segments represented). The residue in B. subtilis σG important for the interaction with CsfB is shown in a blue box. Note that the homologous position in σF and in the other sigma proteins is invariantly occupied by a glutamic acid (yellow box). The residue herein identified in region 2.3 of B. subtilis σE (N100) that is important for binding by CsfB is shown in a blue box. This residue is conserved among Bacillus orthologues of σE whereas in other sigma factors the homologous position is occupied by and acidic residue (yellow box). Residues in region 2.2. of E. coli σA implicated in core binding are highlighted in grey; residues, at the end of region 2.3, involved in promoter meting are also indicated (reviewed by [55]). The residues boxed in green were shown to affect sporulation in B. subtilis [20] and promoter melting in E. coli [68,69]. The accession numbers of the aligned sequences are given in the Material and Methods section. B: GST pull-down assays to investigate the role of E100 of σE in the interaction with CsfB. GST, GST-σE and GST-σE N100E fusions were bound to glutathione beads and incubated with purified CsfB-Strep II (100 nM). Protein complexes were captured on glutathione sepharose beads, and visualized, following elution, by immunoblotting with anti-GST, or anti-Strep tag antibodies. C: colony lift assays (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 σE wt, or σE N100E, σK wt, or σK E93N, to the GAL4 binding domain (BD), as indicated. Assays in which the BD and AD were produced from empty vectors were used as negative controls (“-“). D: effect of the sigEN100E and sigKE73N alleles on σE- and σK-dependent gene expression. The figure shows the expression of PspoIVCB-lacZ (σE-dependent) and PcotG-lacZ (σK-dependent) transcriptional fusions during sporulation. Samples were withdrawn from cultures of the represented strains, at the indicated times, in hours after the onset of sporulation in re-suspension (denoted as T0), and assayed for β-galactosidase activity (shown in Miller units).
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pgen.1005104.g007: A residue in σE involved in the interaction with CsfB.A: alignment of regions 2.1, 2.2 and the beginning of region 2.3 from the σA, σF, σG, σE, and σK proteins of the indicated species (the sequence of σA from T. thermophilus is identical to that of T. aquaticus for the segments represented). The residue in B. subtilis σG important for the interaction with CsfB is shown in a blue box. Note that the homologous position in σF and in the other sigma proteins is invariantly occupied by a glutamic acid (yellow box). The residue herein identified in region 2.3 of B. subtilis σE (N100) that is important for binding by CsfB is shown in a blue box. This residue is conserved among Bacillus orthologues of σE whereas in other sigma factors the homologous position is occupied by and acidic residue (yellow box). Residues in region 2.2. of E. coli σA implicated in core binding are highlighted in grey; residues, at the end of region 2.3, involved in promoter meting are also indicated (reviewed by [55]). The residues boxed in green were shown to affect sporulation in B. subtilis [20] and promoter melting in E. coli [68,69]. The accession numbers of the aligned sequences are given in the Material and Methods section. B: GST pull-down assays to investigate the role of E100 of σE in the interaction with CsfB. GST, GST-σE and GST-σE N100E fusions were bound to glutathione beads and incubated with purified CsfB-Strep II (100 nM). Protein complexes were captured on glutathione sepharose beads, and visualized, following elution, by immunoblotting with anti-GST, or anti-Strep tag antibodies. C: colony lift assays (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 σE wt, or σE N100E, σK wt, or σK E93N, to the GAL4 binding domain (BD), as indicated. Assays in which the BD and AD were produced from empty vectors were used as negative controls (“-“). D: effect of the sigEN100E and sigKE73N alleles on σE- and σK-dependent gene expression. The figure shows the expression of PspoIVCB-lacZ (σE-dependent) and PcotG-lacZ (σK-dependent) transcriptional fusions during sporulation. Samples were withdrawn from cultures of the represented strains, at the indicated times, in hours after the onset of sporulation in re-suspension (denoted as T0), and assayed for β-galactosidase activity (shown in Miller units).

Mentions: The N45E substitution in σG results in loss of CsfB binding, whereas the E39N substitution in σF is sufficient for conferring CsfB binding ability [20]. However, the residue homologous to N45 in σE corresponds to a glutamate, E64 (Fig. 7A). Together with the mapping experiments described in the preceding section, this observation suggests that the interaction of CsfB with σE differs from that with σG. To more precisely delineate the determinants for CsfB binding to σE, we sought to identify residues that are necessary for the interaction. An inspection of the amino acid sequence of σE in regions 2.2 and 2.3 revealed an asparagine residue located at position 100 (i.e., at the beginning of region 2.3) and conserved among all of Bacillus σE orthologues. Strikingly, the position homologous to N100 in σK is occupied by an aspartate (E93) in B. subtilis and invariably (with the exception of the σE proteins) by an acidic residue in all other Bacillus sigma factors (Fig. 7A). N100 in σE is located in the vicinity of several residues involved in promoter melting (Fig. 7A). By contrast, E64 in σE and N45 in σG are located at the beginning of a helix within which several contacts are established with the β´ subunit of RNA polymerase (S6 Fig). Thus, depending on which sigma factor is present in the complex, CsfB appears to bind to two distinct functional surfaces of the RNA polymerase holoenzyme.


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)

A residue in σE involved in the interaction with CsfB.A: alignment of regions 2.1, 2.2 and the beginning of region 2.3 from the σA, σF, σG, σE, and σK proteins of the indicated species (the sequence of σA from T. thermophilus is identical to that of T. aquaticus for the segments represented). The residue in B. subtilis σG important for the interaction with CsfB is shown in a blue box. Note that the homologous position in σF and in the other sigma proteins is invariantly occupied by a glutamic acid (yellow box). The residue herein identified in region 2.3 of B. subtilis σE (N100) that is important for binding by CsfB is shown in a blue box. This residue is conserved among Bacillus orthologues of σE whereas in other sigma factors the homologous position is occupied by and acidic residue (yellow box). Residues in region 2.2. of E. coli σA implicated in core binding are highlighted in grey; residues, at the end of region 2.3, involved in promoter meting are also indicated (reviewed by [55]). The residues boxed in green were shown to affect sporulation in B. subtilis [20] and promoter melting in E. coli [68,69]. The accession numbers of the aligned sequences are given in the Material and Methods section. B: GST pull-down assays to investigate the role of E100 of σE in the interaction with CsfB. GST, GST-σE and GST-σE N100E fusions were bound to glutathione beads and incubated with purified CsfB-Strep II (100 nM). Protein complexes were captured on glutathione sepharose beads, and visualized, following elution, by immunoblotting with anti-GST, or anti-Strep tag antibodies. C: colony lift assays (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 σE wt, or σE N100E, σK wt, or σK E93N, to the GAL4 binding domain (BD), as indicated. Assays in which the BD and AD were produced from empty vectors were used as negative controls (“-“). D: effect of the sigEN100E and sigKE73N alleles on σE- and σK-dependent gene expression. The figure shows the expression of PspoIVCB-lacZ (σE-dependent) and PcotG-lacZ (σK-dependent) transcriptional fusions during sporulation. Samples were withdrawn from cultures of the represented strains, at the indicated times, in hours after the onset of sporulation in re-suspension (denoted as T0), and assayed for β-galactosidase activity (shown in Miller units).
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Related In: Results  -  Collection

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Show All Figures
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pgen.1005104.g007: A residue in σE involved in the interaction with CsfB.A: alignment of regions 2.1, 2.2 and the beginning of region 2.3 from the σA, σF, σG, σE, and σK proteins of the indicated species (the sequence of σA from T. thermophilus is identical to that of T. aquaticus for the segments represented). The residue in B. subtilis σG important for the interaction with CsfB is shown in a blue box. Note that the homologous position in σF and in the other sigma proteins is invariantly occupied by a glutamic acid (yellow box). The residue herein identified in region 2.3 of B. subtilis σE (N100) that is important for binding by CsfB is shown in a blue box. This residue is conserved among Bacillus orthologues of σE whereas in other sigma factors the homologous position is occupied by and acidic residue (yellow box). Residues in region 2.2. of E. coli σA implicated in core binding are highlighted in grey; residues, at the end of region 2.3, involved in promoter meting are also indicated (reviewed by [55]). The residues boxed in green were shown to affect sporulation in B. subtilis [20] and promoter melting in E. coli [68,69]. The accession numbers of the aligned sequences are given in the Material and Methods section. B: GST pull-down assays to investigate the role of E100 of σE in the interaction with CsfB. GST, GST-σE and GST-σE N100E fusions were bound to glutathione beads and incubated with purified CsfB-Strep II (100 nM). Protein complexes were captured on glutathione sepharose beads, and visualized, following elution, by immunoblotting with anti-GST, or anti-Strep tag antibodies. C: colony lift assays (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 σE wt, or σE N100E, σK wt, or σK E93N, to the GAL4 binding domain (BD), as indicated. Assays in which the BD and AD were produced from empty vectors were used as negative controls (“-“). D: effect of the sigEN100E and sigKE73N alleles on σE- and σK-dependent gene expression. The figure shows the expression of PspoIVCB-lacZ (σE-dependent) and PcotG-lacZ (σK-dependent) transcriptional fusions during sporulation. Samples were withdrawn from cultures of the represented strains, at the indicated times, in hours after the onset of sporulation in re-suspension (denoted as T0), and assayed for β-galactosidase activity (shown in Miller units).
Mentions: The N45E substitution in σG results in loss of CsfB binding, whereas the E39N substitution in σF is sufficient for conferring CsfB binding ability [20]. However, the residue homologous to N45 in σE corresponds to a glutamate, E64 (Fig. 7A). Together with the mapping experiments described in the preceding section, this observation suggests that the interaction of CsfB with σE differs from that with σG. To more precisely delineate the determinants for CsfB binding to σE, we sought to identify residues that are necessary for the interaction. An inspection of the amino acid sequence of σE in regions 2.2 and 2.3 revealed an asparagine residue located at position 100 (i.e., at the beginning of region 2.3) and conserved among all of Bacillus σE orthologues. Strikingly, the position homologous to N100 in σK is occupied by an aspartate (E93) in B. subtilis and invariably (with the exception of the σE proteins) by an acidic residue in all other Bacillus sigma factors (Fig. 7A). N100 in σE is located in the vicinity of several residues involved in promoter melting (Fig. 7A). By contrast, E64 in σE and N45 in σG are located at the beginning of a helix within which several contacts are established with the β´ subunit of RNA polymerase (S6 Fig). Thus, depending on which sigma factor is present in the complex, CsfB appears to bind to two distinct functional surfaces of the RNA polymerase holoenzyme.

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