scholarly journals Expression of spoIIIJ in the Prespore Is Sufficient for Activation of σG and for Sporulation in Bacillus subtilis

2003 ◽  
Vol 185 (13) ◽  
pp. 3905-3917 ◽  
Author(s):  
Mónica Serrano ◽  
Luísa Côrte ◽  
Jason Opdyke ◽  
Charles P. Moran, ◽  
Adriano O. Henriques
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Sigma Factor ◽  
Vegetative Growth ◽  
Mother Cell ◽  
Asymmetric Division ◽  
Developmental Program ◽  
Inactive Form

ABSTRACT During sporulation in Bacillus subtilis, the prespore-specific developmental program is initiated soon after asymmetric division of the sporangium by the compartment-specific activation of RNA polymerase sigma factor σF. σF directs transcription of spoIIIG, encoding the late forespore-specific regulator σG. Following synthesis, σG is initially kept in an inactive form, presumably because it is bound to the SpoIIAB anti-sigma factor. Activation of σG occurs only after the complete engulfment of the prespore by the mother cell. Mutations in spoIIIJ arrest sporulation soon after conclusion of the engulfment process and prevent activation of σG. Here we show that σG accumulates but is mostly inactive in a spoIIIJ mutant. We also show that expression of the spoIIIGE155K allele, encoding a form of σG that is not efficiently bound by SpoIIAB in vitro, restores σG-directed gene expression to a spoIIIJ mutant. Expression of spoIIIJ occurs during vegetative growth. However, we show that expression of spoIIIJ in the prespore is sufficient for σG activation and for sporulation. Mutations in the mother cell-specific spoIIIA locus are known to arrest sporulation just after completion of the engulfment process. Previous work has also shown that σG accumulates in an inactive form in spoIIIA mutants and that the need for spoIIIA expression for σG activation can be circumvented by the spoIIIGE155K allele. However, in contrast to the case for spoIIIJ, we show that expression of spoIIIA in the prespore does not support efficient sporulation. The results suggest that the activation of σG at the end of the engulfment process involves the action of spoIIIA from the mother cell and of spoIIIJ from the prespore.

scholarly journals Role of the Anti-Sigma Factor SpoIIAB in Regulation of σG during Bacillus subtilis Sporulation

2004 ◽  
Vol 186 (12) ◽  
pp. 4000-4013 ◽  
Author(s):  
Mónica Serrano ◽  
Alexandre Neves ◽  
Cláudio M. Soares ◽  
Charles P. Moran ◽  
Adriano O. Henriques
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Sigma Factor ◽  
Mother Cell ◽  
In Vivo Studies ◽  
Specific Gene ◽  
Rapid Induction ◽  
Inactive Form ◽  
Entire Cell

ABSTRACT RNA polymerase sigma factor σF initiates the prespore-specific program of gene expression during Bacillus subtilis sporulation. σF governs transcription of spoIIIG, encoding the late prespore-specific regulator σG. However, transcription of spoIIIG is delayed relative to other genes under the control of σF, and after synthesis, σG is initially kept in an inactive form. Activation of σG requires the complete engulfment of the prespore by the mother cell and expression of the spoIIIA and spoIIIJ loci. We screened for random mutations in spoIIIG that bypassed the requirement for spoIIIA for the activation of σG. We found a mutation (spoIIIGE156K) that resulted in an amino acid substitution at position 156, which is adjacent to the position of a mutation (E155K) previously shown to prevent interaction of SpoIIAB with σG. Comparative modelling techniques and in vivo studies suggested that the spoIIIGE156K mutation interferes with the interaction of SpoIIAB with σG. The σGE156K isoform restored σG-directed gene expression to spoIIIA mutant cells. However, expression of sspE-lacZ in the spoIIIA spoIIIGE156K double mutant was delayed relative to completion of the engulfment process and was not confined to the prespore. Rather, β-galactosidase accumulated throughout the entire cell at late times in development. This suggests that the activity of σGE156K is still regulated in the prespore of a spoIIIA mutant, but not by SpoIIAB. In agreement with this suggestion, we also found that expression of spoIIIGE156K from the promoter for the early prespore-specific gene spoIIQ still resulted in sspE-lacZ induction at the normal time during sporulation, coincidently with completion of the engulfment process. In contrast, transcription of spoIIIGE156K, but not of the wild-type spoIIIG gene, from the mother cell-specific spoIID promoter permitted the rapid induction of sspE-lacZ expression. Together, the results suggest that SpoIIAB is either redundant or has no role in the regulation of σG in the prespore.

scholarly journals One Perturbation of the Mother Cell Gene Regulatory Network Suppresses the Effects of Another during Sporulation of Bacillus subtilis

2007 ◽  
Vol 189 (23) ◽  
pp. 8467-8473 ◽  
Author(s):  
Lijuan Wang ◽  
John Perpich ◽  
Adam Driks ◽  
Lee Kroos
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Regulatory Network ◽  
Mother Cell ◽  
Feedback Loop ◽  
Network Functions ◽  
Regulatory Loop ◽  
Cell Gene ◽  
Spore Coat Protein

ABSTRACT In the mother cell of sporulating Bacillus subtilis, a regulatory network functions to control gene expression. Four transcription factors act sequentially in the order σE, SpoIIID, σK, GerE. σE and σK direct RNA polymerase to transcribe different regulons. SpoIIID and GerE are DNA-binding proteins that activate or repress transcription of many genes. Several negative regulatory loops add complexity to the network. First, transcriptionally active σK RNA polymerase inhibits early sporulation gene expression, resulting in reduced accumulation of σE and SpoIIID late during sporulation. Second, GerE represses sigK transcription, reducing σK accumulation about twofold. Third, SpoIIID represses cotC, which encodes a spore coat protein, delaying its transcription by σK RNA polymerase. Partially circumventing the first feedback loop, by engineering cells to maintain the SpoIIID level late during sporulation, causes spore defects. Here, the effects of circumventing the second feedback loop, by mutating the GerE binding sites in the sigK promoter region, are reported. Accumulation of pro-σK and σK was increased, but no spore defects were detected. Expression of σK-dependent reporter fusions was altered, increasing the expression of gerE-lacZ and cotC-lacZ and decreasing the expression of cotD-lacZ. Because these effects on gene expression were opposite those observed when the SpoIIID level was maintained late during sporulation, cells were engineered to both maintain the SpoIIID level and have elevated sigK expression late during sporulation. This restored the expression of σK-dependent reporters to wild-type levels, and no spore defects were observed. Hence, circumventing the second feedback loop suppressed the effects of perturbing the first feedback loop. By feeding information back into the network, these two loops appear to optimize target gene expression and increase network robustness. Circumventing the third regulatory loop, by engineering cells to express cotC about 2 h earlier than normal, did not cause a detectable spore defect.

scholarly journals ςK Can Negatively RegulatesigE Expression by Two Different Mechanisms during Sporulation of Bacillus subtilis

1999 ◽  
Vol 181 (13) ◽  
pp. 4081-4088 ◽  
Author(s):  
Bin Zhang ◽  
Paolo Struffi ◽  
Lee Kroos
Keyword(s):  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Sigma Factor ◽  
Mother Cell ◽  
Transcriptional Activity ◽  
The Other ◽  
Mutant Form ◽  
Gene Encoding ◽  
Negative Effect ◽  
Cell Gene Expression

ABSTRACT Temporal and spatial gene regulation during Bacillus subtilis sporulation involves the activation and inactivation of multiple sigma subunits of RNA polymerase in a cascade. In the mother cell compartment of sporulating cells, expression of thesigE gene, encoding the earlier-acting sigma factor, ςE, is negatively regulated by the later-acting sigma factor, ςK. Here, it is shown that the negative feedback loop does not require SinR, an inhibitor of sigEtranscription. Production of ςK about 1 h earlier than normal does affect Spo0A, which when phosphorylated is an activator of sigE transcription. A mutation in thespo0A gene, which bypasses the phosphorelay leading to the phosphorylation of Spo0A, diminished the negative effect of early ςK production on sigE expression early in sporulation. Also, early production of ςK reduced expression of other Spo0A-dependent genes but not expression of the Spo0A-independent ald gene. In contrast, bothsigE and ald were overexpressed late in development of cells that fail to make ςK. Theald promoter, like the sigE promoter, is believed to be recognized by ςA RNA polymerase, suggesting that ςK may inhibit ςA activity late in sporulation. To exert this negative effect, ςKmust be transcriptionally active. A mutant form of ςKthat associates with core RNA polymerase, but does not direct transcription of a ςK-dependent gene, failed to negatively regulate expression of sigE or aldlate in development. On the other hand, the negative effect of early ςK production on sigE expression early in sporulation did not require transcriptional activity of ςK RNA polymerase. These results demonstrate that ςK can negatively regulate sigE expression by two different mechanisms, one observed when ςK is produced earlier than normal, which does not require ςKto be transcriptionally active and affects Spo0A, and the other observed when ςK is produced at the normal time, which requires ςK RNA polymerase transcriptional activity. The latter mechanism facilitates the switch from ςE to ςK in the cascade controlling mother cell gene expression.

scholarly journals Sigma Factor Displacement from RNA Polymerase during Bacillus subtilis Sporulation

1999 ◽  
Vol 181 (16) ◽  
pp. 4969-4977 ◽  
Author(s):  
Jingliang Ju ◽  
Theresa Mitchell ◽  
Howard Peters ◽  
W. G. Haldenwang
Keyword(s):  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Western Blot ◽  
Relative Abundance ◽  
Sigma Factor ◽  
Mother Cell ◽  
Vegetative Cell ◽  
Wild Type

ABSTRACT As Bacillus subtilis proceeds through sporulation, the principal vegetative cell ς subunit (ςA) persists in the cell but is replaced in the extractable RNA polymerase (RNAP) by sporulation-specific ς factors. To explore how this holoenzyme changeover might occur, velocity centrifugation techniques were used in conjunction with Western blot analyses to monitor the associations of RNAP with ςA and two mother cell ς factors, ςE and ςK, which successively replace ςA on RNAP. Although the relative abundance of ςA with respect to RNAP remained virtually unchanged during sporulation, the percentage of the detectable ςAwhich cosedimented with RNAP fell from approximately 50% at the onset of sporulation (T 0) to 2 to 8% by 3 h into the process (T 3). In a strain that failed to synthesize ςE, the first of the mother cell-specific ς factors, approximately 40% of the ςA remained associated with RNAP at T 3. The level of ςA-RNAP cosedimentation dropped to less than 10% in a strain which synthesized a ςE variant (ςECR119) that could bind to RNAP but was unable to direct ςE-dependent transcription. The E-ςE-to-E-ςK changeover was characterized by both the displacement of ςE from RNAP and the disappearance of ςE from the cell. Analyses of extracts from wild-type and mutant B. subtilis showed that the ςK protein is required for the displacement of ςE from RNAP and also confirmed that ςK is needed for the loss of the ςE protein. The results indicate that the successive appearance of mother cell ς factors, but not necessarily their activities, is an important element in the displacement of preexisting ς factors from RNAP. It suggests that competition for RNAP by consecutive sporulation ς factors may be an important feature of the holoenzyme changeovers that occur during sporulation.

scholarly journals Maintaining the Transcription Factor SpoIIID Level Late during Sporulation Causes Spore Defects in Bacillus subtilis

2007 ◽  
Vol 189 (20) ◽  
pp. 7302-7309 ◽  
Author(s):  
Lijuan Wang ◽  
John Perpich ◽  
Adam Driks ◽  
Lee Kroos
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Mother Cell ◽  
Structural Defects ◽  
Spore Coat ◽  
Multicopy Plasmid ◽  
Altered Expression ◽  
Cell Gene Expression ◽  
Cell Gene

ABSTRACT During sporulation of Bacillus subtilis, four regulatory proteins act in the order σE, SpoIIID, σK, and GerE to temporally control gene expression in the mother cell. σE and σK work sequentially with core RNA polymerase to transcribe different sets of genes. SpoIIID and GerE are small, sequence-specific DNA-binding proteins that activate or repress transcription of many genes. Previous studies showed that transcriptionally active σK RNA polymerase inhibits early mother cell gene expression, reducing accumulation of SpoIIID late in sporulation. Here, the effects of perturbing the mother cell gene regulatory network by maintaining the SpoIIID level late during sporulation are reported. Persistent expression was obtained by fusing spoIIID to the σK-controlled gerE promoter on a multicopy plasmid. Fewer heat- and lysozyme-resistant spores were produced by the strain with persistent spoIIID expression, but the number of spores resistant to organic solvents was unchanged, as was their germination ability. Transmission electron microscopy showed structural defects in the spore coat. Reporter fusions to σK-dependent promoters showed lower expression of gerE and cotC and higher expression of cotD. Altered expression of cot genes, which encode spore coat proteins, may account for the spore structural defects. These results suggest that one role of negative feedback by σK RNA polymerase on early mother cell gene expression is to lower the level of SpoIIID late during sporulation in order to allow normal expression of genes in the σK regulon.

scholarly journals Establishment of Prespore-Specific Gene Expression inBacillus subtilis: Localization of SpoIIE Phosphatase and Initiation of Compartment-Specific Proteolysis

1998 ◽  
Vol 180 (13) ◽  
pp. 3276-3284 ◽  
Author(s):  
Peter J. Lewis ◽  
Ling Juan Wu ◽  
Jeffery Errington
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Sigma Factor ◽  
Mother Cell ◽  
Immunofluorescence Microscopy ◽  
The Other ◽  
Specific Gene ◽  
Specific Gene Expression

ABSTRACT Immunofluorescence microscopy was used to study the establishment of compartment-specific transcription during sporulation inBacillus subtilis. Analysis of the distribution of the anti-anti-sigma factor, SpoIIAA, in a variety of mutant backgrounds supports a model in which the SpoIIE phosphatase, which activates SpoIIAA by dephosphorylation, is sequestered onto the prespore face of the asymmetric septum. Thus, prespore-specific gene expression apparently arises as a result of the compartmentalization of SpoIIE protein. The results also suggest the existence of at least two compartment-specific programs of proteolysis, one dependent on the mother cell-specific sigma factor ςE and the other dependent on the prespore-specific sigma factor ςF.

scholarly journals Forespore Signaling Is Necessary for Pro-σK Processing during Bacillus subtilis Sporulation Despite the Loss of SpoIVFA upon Translational Arrest

2002 ◽  
Vol 184 (19) ◽  
pp. 5393-5401 ◽  
Author(s):  
Lee Kroos ◽  
Yuen-Tsu Nicco Yu ◽  
Denise Mills ◽  
Shelagh Ferguson-Miller
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Oxidative Phosphorylation ◽  
Rna Polymerase ◽  
Western Blot ◽  
Blot Analysis ◽  
Mother Cell ◽  
Proteolytic Processing ◽  
Electrochemical Gradient ◽  
Translational Arrest

ABSTRACT The σK checkpoint coordinates gene expression in the mother cell with signaling from the forespore during Bacillus subtilis sporulation. The signaling pathway involves SpoIVB, a serine peptidase produced in the forespore, which is believed to cross the innermost membrane surrounding the forespore and activate a complex of proteins, including BofA, SpoIVFA, and SpoIVFB, located in the outermost membrane surrounding the forespore. Activation of the complex allows proteolytic processing of pro-σK, and the resulting σK RNA polymerase transcribes genes in the mother cell. To investigate activation of the pro-σK processing complex, the level of SpoIVFA in extracts of sporulating cells was examined by Western blot analysis. The SpoIVFA level decreased when pro-σK processing began during sporulation. In extracts of a spoIVB mutant defective in forespore signaling, the SpoIVFA level failed to decrease normally and no processing of pro-σK was observed. Although these results are consistent with a model in which SpoIVFA inhibits processing until the SpoIVB-mediated signal is received from the forespore, we discovered that loss of SpoIVFA was insufficient to allow processing under certain conditions, including static incubation of the culture and continued shaking after the addition of inhibitors of oxidative phosphorylation or translation. Under these conditions, loss of SpoIVFA was independent of spoIVB. The inability to process pro-σK under these conditions was not due to loss of SpoIVFB, the putative processing enzyme, or to a requirement for ongoing synthesis of pro-σK. Rather, it was found that the requirements for shaking of the culture, for oxidative phosphorylation, and for translation could be bypassed by mutations that uncouple processing from dependence on forespore signaling. This suggests that ongoing translation is normally required for efficient pro-σK processing because synthesis of the SpoIVB signal protein is needed to activate the processing complex. When translation is blocked, synthesis of SpoIVB ceases, and the processing complex remains inactive despite the loss of SpoIVFA. Taken together, the results suggest that SpoIVB signaling activates the processing complex by performing another function in addition to causing loss of SpoIVFA or by causing loss of SpoIVFA in a different way than when translation is blocked. The results also demonstrate that the processing machinery can function in the absence of translation or an electrochemical gradient across membranes.

scholarly journals Control of the Expression and Compartmentalization of σG Activity during Sporulation of Bacillus subtilis by Regulators of σF and σE

2005 ◽  
Vol 187 (19) ◽  
pp. 6832-6840 ◽  
Author(s):  
Vasant K. Chary ◽  
Mauro Meloni ◽  
David W. Hilbert ◽  
Patrick J. Piggot
Keyword(s):  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Structural Gene ◽  
Sigma Factor ◽  
Mother Cell ◽  
Cell Formation ◽  
Spore Formation ◽  
Deletion Mutants ◽  
Septum Formation

ABSTRACT During formation of spores by Bacillus subtilis the RNA polymerase factor σG ordinarily becomes active during spore formation exclusively in the prespore upon completion of engulfment of the prespore by the mother cell. Formation and activation of σG ordinarily requires prior activity of σF in the prespore and σE in the mother cell. Here we report that in spoIIA mutants lacking both σF and the anti-sigma factor SpoIIAB and in which σE is not active, σG nevertheless becomes active. Further, its activity is largely confined to the mother cell. Thus, there is a switch in the location of σG activity from prespore to mother cell. Factors contributing to the mother cell location are inferred to be read-through of spoIIIG, the structural gene for σG, from the upstream spoIIG locus and the absence of SpoIIAB, which can act in the mother cell as an anti-sigma factor to σG. When the spoIIIG locus was moved away from spoIIG to the distal amyE locus, σG became active earlier in sporulation in spoIIA deletion mutants, and the sporulation septum was not formed, suggesting that premature σG activation can block septum formation. We report a previously unrecognized control in which SpoIIGA can prevent the appearance of σG activity, and pro-σE (but not σE) can counteract this effect of SpoIIGA. We find that in strains lacking σF and SpoIIAB and engineered to produce active σE in the mother cell without the need for SpoIIGA, σG also becomes active in the mother cell.

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