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 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 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 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 Contrasting Effects of σE on Compartmentalization of σF Activity during Sporulation of Bacillus subtilis

2004 ◽  
Vol 186 (7) ◽  
pp. 1983-1990 ◽  
Author(s):  
David W. Hilbert ◽  
Vasant K. Chary ◽  
Patrick J. Piggot
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Bacillus Subtilis ◽  
Mother Cell ◽  
Cell Fates ◽  
Gene Encoding ◽  
Primitive Form ◽  
Small Proteins ◽  
Dna Translocase

ABSTRACT Spore formation by Bacillus subtilis is a primitive form of development. In response to nutrient starvation and high cell density, B. subtilis divides asymmetrically, resulting in two cells with different sizes and cell fates. Immediately after division, the transcription factor σF becomes active in the smaller prespore, which is followed by the activation of σE in the larger mother cell. In this report, we examine the role of the mother cell-specific transcription factor σE in maintaining the compartmentalization of gene expression during development. We have studied a strain with a deletion of the spoIIIE gene, encoding a DNA translocase, that exhibits uncompartmentalized σF activity. We have determined that the deletion of spoIIIE alone does not substantially impact compartmentalization, but in the spoIIIE mutant, the expression of putative peptidoglycan hydrolases under the control of σE in the mother cell destroys the integrity of the septum. As a consequence, small proteins can cross the septum, thereby abolishing compartmentalization. In addition, we have found that in a mutant with partially impaired control of σF, the activation of σE in the mother cell is important to prevent the activation of σF in this compartment. Therefore, the activity of σE can either maintain or abolish the compartmentalization of σF, depending upon the genetic makeup of the strain. We conclude that σE activity must be carefully regulated in order to maintain compartmentalization of gene expression during development.

Growth Factor Independence 1 (Gfi-1) Plays a Role in Mediating Specific Granule Deficiency (SGD) in a Patient Lacking an Abnormality in the C/EBPε Gene.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3546-3546
Author(s):  
Arati Khanna-Gupta ◽  
Terry Zibello ◽  
Hong Sun ◽  
Laurence A. Boxer ◽  
Nancy Berliner
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Transcription Factor ◽  
Growth Factor ◽  
Western Blot ◽  
Blot Analysis ◽  
Germline Mutations ◽  
Coding Region ◽  
Blood Neutrophils ◽  
Specific Granule

Abstract Neutrophil specific granule deficiency (SGD) is a rare congenital disorder marked by recurrent bacterial infections of the skin and respiratory system. Neutrophils from SGD patients lack secondary and tertiary granules and their content proteins and exhibit defects in chemotaxis and bactericidal activity. A mouse model deficient for the transcription factor CCAAT/enhancer binding protein epsilon (C/EBPε) manifests a similar phenotype as SGD patients and prompted examination of the human C/EBPε gene for mutation in this disease. Mutations in the C/EBPε gene have been identified by others in two patients with SGD, resulting in loss of gene expression. However, other patients with a similar disease phenotype have a normal C/EBPε coding sequence, suggesting that other genetic abnormalities in myelopoiesis can lead to SGD. Studies in our laboratory on one such patient lacking a C/EBPε mutation demonstrated an elevated level of the C/EBPε protein in the patient’s peripheral blood neutrophils as compared to the level in normal control neutrophils. Microarray analysis of this patient’s bone marrow compared with that of a normal control revealed, among other genes, elevated levels of the transcription factors C/EBPε and PU.1. As a consequence, several PU.1 target genes showed increased expression in the SGD patient bone marrow. This observation was confirmed by both real time PCR and western blot analysis. PU.1 is a hematopoietic-specific transcription factor belonging to the Ets family of DNA binding proteins and plays a critical role in B-cell, macrophage and late stage neutrophil development. Sequence analysis of the PU.1 gene from our SGD patient however, did not reveal any mutation in the coding region of the gene. Western blot analysis of nuclear extracts prepared from peripheral blood neutrophils from this patient did however reveal significantly decreased levels of the transcription factor Gfi-1 (Growth factor independent-1), although no mutation has been found thus far in the coding region of the Gfi-1 gene from the SGD patient. Gfi-1 belongs to a family of zinc finger containing transcriptional repressor oncoproteins. Mice lacking Gfi-1 were found not only to be neutropenic, but also demonstrated defects in neutrophil differentiation, including loss of neutrophil secondary and tertiary granule proteins, reminiscent of SGD. More recently, heterozygous germline mutations of Gfi-1 were shown to cause severe congenital neutropenia (SCN) in humans. It has been suggested that Gfi1 represses neutrophil elastase (Ela2), germline mutations within which are a major contributor to hereditary neutropenias. Our data suggest that decreased levels of Gfi1 in our SGD patient result in increased levels of PU.1 and C/EBPε; an effect consistent with observations first made in the neutrophils of Gfi-1-null mice. The increased PU.1 levels might then act to sequester C/EBPε protein via direct protein-protein interaction. This in turn could explain the loss of secondary granule protein gene expression in the SGD patient by inducing functional C/EBPε deficiency.

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