Transcriptional Repressor CcpN from Bacillus subtilis Compensates Asymmetric Contact Distribution by Cooperative Binding

Licht, Andreas; Brantl, Sabine

doi:10.1016/j.jmb.2006.09.021

Cited by 26 publications

(32 citation statements)

References 30 publications

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“…At P gapB , a decrease in the concentration of active repressor is consistent with the proposed sequestration of CcpN by YqfL under malate, although the total CcpN concentration is not thought to change (16). The change of k 1 off is minimal upon induction, and hence K D values are constant and in the range of 0.8 nM, in reasonable agreement with the 8 nM affinity measured in vitro (33,34). For CggR, operator affinity is high under repression, K D ¼ 0.1 nM, and decreases about 10-fold upon induction (k 1 off increases), in reasonable agreement with the in vitro operator affinity (<0.5 and 10 nM) of CggR in absence and presence of inducer (35,36).…”

Section: Absolute Quantification Of Expression Levels Under Permissivsupporting

confidence: 82%

Reconciling molecular regulatory mechanisms with noise patterns of bacterial metabolic promoters in induced and repressed states

Ferguson

Coq

Jules

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Assessing gene expression noise in order to obtain mechanistic insights requires accurate quantification of gene expression on many individual cells over a large dynamic range. We used a unique method based on 2-photon fluorescence fluctuation microscopy to measure directly, at the single cell level and with single-molecule sensitivity, the absolute concentration of fluorescent proteins produced from the two Bacillus subtilis promoters that control the switch between glycolysis and gluconeogenesis. We quantified cellto-cell variations in GFP concentrations in reporter strains grown on glucose or malate, including very weakly transcribed genes under strong catabolite repression. Results revealed strong transcriptional bursting, particularly for the glycolytic promoter. Noise pattern parameters of the two antagonistic promoters controlling the nutrient switch were differentially affected on glycolytic and gluconeogenic carbon sources, discriminating between the different mechanisms that control their activity. Our stochastic model for the transcription events reproduced the observed noise patterns and identified the critical parameters responsible for the differences in expression profiles of the promoters. The model also resolved apparent contradictions between in vitro operator affinity and in vivo repressor activity at these promoters. Finally, our results demonstrate that negative feedback is not noise-reducing in the case of strong transcriptional bursting.B. subtilis | central carbon metabolism | promoter activity | stochastic gene expression | gene expression control

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Section: Absolute Quantification Of Expression Levels Under Permissivsupporting

confidence: 82%

Reconciling molecular regulatory mechanisms with noise patterns of bacterial metabolic promoters in induced and repressed states

Ferguson

Coq

Jules

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…Three genetic targets of CcpN, namely, gapB, pckA, and sr1, had been previously identified (28,37). To identify further possible CcpN targets that could contribute to the major flux redistribution observed in a ccpN knockout strain (12), we performed a comparative transcriptome analysis of a ccpN mutant and the wild type in minimal glucose medium.…”

Section: Resultsmentioning

confidence: 99%

CcpN Controls Central Carbon Fluxes in Bacillus subtilis

et al. 2008

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The transcriptional regulator CcpN of Bacillus subtilis has been recently characterized as a repressor of two gluconeogenic genes, gapB and pckA, and of a small noncoding regulatory RNA, sr1, involved in arginine catabolism. Deletion of ccpN impairs growth on glucose and strongly alters the distribution of intracellular fluxes, rerouting the main glucose catabolism from glycolysis to the pentose phosphate (PP) pathway. Using transcriptome analysis, we show that during growth on glucose, gapB and pckA are the only protein-coding genes directly repressed by CcpN. By quantifying intracellular fluxes in deletion mutants, we demonstrate that derepression of pckA under glycolytic condition causes the growth defect observed in the ccpN mutant due to extensive futile cycling through the pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and pyruvate kinase. Beyond ATP dissipation via this cycle, PckA activity causes a drain on tricarboxylic acid cycle intermediates, which we show to be the main reason for the reduced growth of a ccpN mutant. The high flux through the PP pathway in the ccpN mutant is modulated by the flux through the alternative glyceraldehyde-3-phosphate dehydrogenases, GapA and GapB. Strongly increased concentrations of intermediates in upper glycolysis indicate that GapB overexpression causes a metabolic jamming of this pathway and, consequently, increases the relative flux through the PP pathway. In contrast, derepression of sr1, the third known target of CcpN, plays only a marginal role in ccpN mutant phenotypes.Regulation of metabolism enables microbes to grow efficiently on a large variety of carbon sources. Partly, these regulation processes ensure efficient resource allocation by expressing substrate-specific transporters and catabolic enzymes only when the substrate is present (38). However, regulation must also avoid simultaneous activity of incompatible or counteracting reactions. A well-known example is the simultaneous presence of ATP-consuming and ATP-generating enzymes that would lead to ATP-dissipating futile cycles (6, 10, 36). Another relevant case is the expression of isoenzymes with different cofactor specificities (9,21,27,33,38) that may catalyze opposite fluxes through key reactions (7,9,11,16). A particularly challenging situation is therefore the switch from glycolytic to gluconeogenic substrates, where large fluxes through the metabolism backbone have to be reversed because the two groups of substrates enter metabolism at two opposite ends.While allosteric regulation of enzyme activities plays an important role in fine-tuning and rapid adaptation to nutritional changes, transcriptional regulation is probably the main mechanism that allows the organism to respond metabolically to a nutrient shift by promoting the operation of a new optimal subset of reactions and adjusting the fluxes through the different central pathways for the establishment of a new steady state (26). One of the most thoroughly studied control mechanisms is carbon catabolite repression, by which the pre...

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“…C-terminally six-histidinetagged CodY was purified from E. coli overexpression strain KS272(pKT1) as described previously (22). DNase I footprinting was performed as described previously (25). GTP and each of the BCAA isoleucine, leucine, and valine were added prior to DNase I at final concentrations of 2 mM and 10 mM, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…SR1 inhibits translation initiation of ahrC mRNA by a novel mechanism: induction of structural changes downstream from the ribosome binding site (18). Interestingly, SR1 is expressed under gluconeogenic conditions and repressed under glycolytic conditions, and the repression is mediated mainly by CcpN and, to a minor extent, by CcpA (24,25).…”

mentioning

confidence: 99%

CodY Activates Transcription of a Small RNA inBacillus subtilis

et al. 2009

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Regulatory small RNAs (sRNAs) in bacterial genomes have become a focus of research over the past 8 years. Whereas more than 100 such sRNAs have been found in Escherichia coli, relatively little is known about sRNAs in gram-positive bacteria. Using a computational approach, we identified two sRNAs in intergenic regions of the Bacillus subtilis genome, SR1 and SR2 (renamed BsrF). Recently, we demonstrated that SR1 inhibits the translation initiation of the transcriptional activator AhrC. Here, we describe detection of BsrF, its expression profile, and its regulation by CodY. Furthermore, we mapped the secondary structure of BsrF. BsrF is expressed in complex and minimal media in all growth phases in B. subtilis and, with a similar expression profile, also in Bacillus amyloliquefaciens. Neither overexpression nor deletion of bsrF affected the growth of B. subtilis. BsrF was found to be long-lived in complex and minimal media. Analysis of 13 putative transcription factor binding sites upstream of bsrF revealed only an effect for CodY. Here, we showed by using Northern blotting, lacZ reporter gene fusions, in vitro transcription, and DNase I footprinting that the transcription of bsrF is activated by CodY in the presence of branched-chain amino acids and GTP. Furthermore, BsrF transcription was increased 1.5-to 2-fold by glucose in the presence of branched-chain amino acids, and this increase was independent of the known glucose-dependent regulators. BsrF is the second target for which transcriptional activation by CodY has been discovered.

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Transcriptional Repressor CcpN from Bacillus subtilis Compensates Asymmetric Contact Distribution by Cooperative Binding

Cited by 26 publications

References 30 publications

Reconciling molecular regulatory mechanisms with noise patterns of bacterial metabolic promoters in induced and repressed states

Reconciling molecular regulatory mechanisms with noise patterns of bacterial metabolic promoters in induced and repressed states

CcpN Controls Central Carbon Fluxes in Bacillus subtilis

CodY Activates Transcription of a Small RNA inBacillus subtilis

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