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 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 The Timing of cotE Expression Affects Bacillus subtilis Spore Coat Morphology but Not Lysozyme Resistance

2006 ◽  
Vol 189 (6) ◽  
pp. 2401-2410 ◽  
Author(s):  
Teresa Costa ◽  
Mónica Serrano ◽  
Leif Steil ◽  
Uwe Völker ◽  
Charles P. Moran ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Mother Cell ◽  
Spore Coat ◽  
Outer Coat ◽  
Bacillus Subtilis Spore ◽  
Spore Resistance ◽  
Early Expression ◽  
Cell Gene Expression ◽  
Coat Layer

ABSTRACT The synthesis of structural components and morphogenetic factors required for the assembly of the Bacillus subtilis spore coat is governed by a mother cell-specific transcriptional cascade. The first two temporal classes of gene expression, which involve RNA polymerase sigma σE factor and the ancillary regulators GerR and SpoIIID, are deployed prior to engulfment of the prespore by the mother cell. The two last classes rely on σK, whose activation follows engulfment completion, and GerE. The cotE gene codes for a morphogenetic protein essential for the assembly of the outer coat layer and spore resistance to lysozyme. cotE is expressed first from a σE-dependent promoter and, in a second stage, from a promoter that additionally requires SpoIIID and that remains active under σK control. CotE localizes prior to engulfment completion close to the surface of the developing spore, but formation of the outer coat is a late, σK-controlled event. We have transplanted cotE to progressively later classes of mother cell gene expression. This created an early class of mutants in which cotE is expressed prior to engulfment completion and a late class in which expression of cotE follows the complete engulfment of the prespore. Mutants of the early class assemble a nearly normal outer coat structure, whereas mutants of the late class do not. Hence, the early expression of CotE is essential for outer coat assembly. Surprisingly, however, all mutants were fully resistant to lysozyme. The results suggest that CotE has genetically separable functions in spore resistance to lysozyme and spore outer coat assembly.

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 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 Capsid Protein of Eastern Equine Encephalitis Virus Inhibits Host Cell Gene Expression

2007 ◽  
Vol 81 (8) ◽  
pp. 3866-3876 ◽  
Author(s):  
Patricia V. Aguilar ◽  
Scott C. Weaver ◽  
Christopher F. Basler
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Host Cell ◽  
Encephalitis Virus ◽  
Eastern Equine Encephalitis Virus ◽  
Eastern Equine Encephalitis ◽  
Equine Encephalitis Virus ◽  
Antiviral Effects ◽  
Cell Gene Expression ◽  
Cell Gene

ABSTRACT Eastern equine encephalitis virus (EEEV) causes sporadic but often severe cases of human and equine neurological disease in North America. To determine how EEEV may evade innate immune responses, we screened individual EEEV proteins for the ability to rescue the growth of a Newcastle disease virus expressing green fluorescent protein (NDV-GFP) from the antiviral effects of interferon (IFN). Only expression of the EEEV capsid facilitated NDV-GFP replication. Inhibition of the antiviral effects of IFN by the capsid appears to occur through a general inhibition of cellular gene expression. For example, the capsid inhibited the expression of several reporter genes under the control of RNA polymerase II promoters. In contrast, capsid did not inhibit expression from a T7 RNA polymerase promoter construct, suggesting that the inhibition of gene expression is specific and is not a simple manifestation of toxicity. The inhibition correlated both with capsid-induced phosphorylation of eukaryotic initiation factor 2 alpha and with capsid-mediated inhibition of cellular mRNA accumulation. Mapping analysis identified the N terminus as the region important for the inhibition of host gene expression, suggesting that this inhibition is independent of capsid protease activity. Finally, when cell lines containing EEEV replicons encoding capsid were selected, replicons consistently acquired mutations that deleted all or part of the capsid, for example, amino acids 18 to 135. Given that the amino terminus of the capsid is required to inhibit host cell gene expression, these data suggest that capsid expression from the replicons is ultimately toxic to host cells, presumably because of its ability to inhibit gene expression.

A forespore checkpoint for mother cell gene expression during development in B. subtilis

Cell ◽  
1990 ◽  
Vol 62 (2) ◽  
pp. 239-250 ◽  
Author(s):  
Simon Cutting ◽  
Valerie Oke ◽  
Adam Driks ◽  
Richard Losick ◽  
Sijie Lu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mother Cell ◽  
Cell Gene Expression ◽  
Cell Gene

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.

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