CcpN Controls Central Carbon Fluxes in Bacillus subtilis

Abstract

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...

13C-flux Analysis Reveals NADPH-balancing Transhydrogenation Cycles in Stationary Phase of Nitrogen-starving Bacillus subtilis

“…2A and supplemental Table S2). Because these genes are normally fully repressed by CcpN in the presence of glucose during exponential growth (49,50), nitrogen starvation appears to alleviate glucose repression, despite the presence of high glucose concentrations.…”

The Bacillus subtilis ywjI ( glpX ) Gene Encodes a Class II Fructose-1,6-Bisphosphatase, Functionally Equivalent to the Class III Fbp Enzyme

“…We have shown that ywjI constitutes with the upstream gene murAB an operon whose transcription does not depend on the glycolytic or neoglucogenic condition. Thus, like fbp, and contrary to the two other genes essential for gluconeogenesis, gapB and pckA, ywjI (glpX) is not regulated by CcpN (20,23).…”

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

“…Indeed, the burst size scales with the lifetime of the active state of the operator, which for a self-repressed promoter, scales inversely with the concentration of repressor and is smaller under permissive conditions when the feedback is weak. In contrast to the strong transcriptional bursting observed for the glycolytic promoter, in the case of the gluconeogenic promoters, because derepression under glycolytic conditions greatly impairs growth (16,19), strict control by CcpN and limited noise are essential. Hence, the repressive mechanism of CcpN has evolved to limit bursting in the repressed state by impairing promoter escape by the RNAP, leading to very strong catabolite repression.…”

“…The operator site for CggR is located downstream of the transcription start site and upstream of the translation initiation region (RBS), and hence CggR is thought to function as a roadblock to the transcribing RNA Polymerase (RNAP) (17,18). The GapB and PckA enzymes are both required for the utilization of gluconeogenic carbon sources, such as malate, but their expression is deleterious under glycolytic regimes (15,16,19). CcpN is the repressor responsible for the very strong catabolite repression of the gapB and pckA promoters in the presence of glucose or other glycolytic substrates (16).…”

“…CcpN is the repressor responsible for the very strong catabolite repression of the gapB and pckA promoters in the presence of glucose or other glycolytic substrates (16). It plays a dominant role in the control of carbon fluxes through central metabolic pathways in B. subtilis and is obligate for opti-mal growth under glycolytic conditions (16,19). CcpN activity is linked to the energy charge of the cell (20) and is also negatively controlled by the coexpressed regulatory protein, YqfL (16).…”