Regulator CcpA Research Articles

Assessment of CcpA-mediated catabolite control of gene expression in Bacillus cereus ATCC 14579

BackgroundThe catabolite control protein CcpA is a transcriptional regulator conserved in many Gram-positives, controlling the efficiency of glucose metabolism. Here we studied the role of Bacillus cereus ATCC 14579 CcpA in regulation of metabolic pathways and expression of enterotoxin genes by comparative transcriptome analysis of the wild-type and a ccpA-deletion strain.ResultsComparative analysis revealed the growth performance and glucose consumption rates to be lower in the B. cereus ATCC 14579 ccpA deletion strain than in the wild-type. In exponentially grown cells, the expression of glycolytic genes, including a non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase that mediates conversion of D-glyceraldehyde 3-phosphate to 3-phospho-D-glycerate in one single step, was down-regulated and expression of gluconeogenic genes and genes encoding the citric acid cycle was up-regulated in the B. cereus ccpA deletion strain. Furthermore, putative CRE-sites, that act as binding sites for CcpA, were identified to be present for these genes. These results indicate CcpA to be involved in the regulation of glucose metabolism, thereby optimizing the efficiency of glucose catabolism. Other genes of which the expression was affected by ccpA deletion and for which putative CRE-sites could be identified, included genes with an annotated function in the catabolism of ribose, histidine and possibly fucose/arabinose and aspartate. Notably, expression of the operons encoding non-hemolytic enterotoxin (Nhe) and hemolytic enterotoxin (Hbl) was affected by ccpA deletion, and putative CRE-sites were identified, which suggests catabolite repression of the enterotoxin operons to be CcpA-dependent.ConclusionThe catabolite control protein CcpA in B. cereus ATCC 14579 is involved in optimizing the catabolism of glucose with concomitant repression of gluconeogenesis and alternative metabolic pathways. Furthermore, the results point to metabolic control of enterotoxin gene expression and suggest that CcpA-mediated glucose sensing provides an additional mode of control in moderating the expression of the nhe and hbl operons in B. cereus ATCC 14579.

Structural Analysis of the Bacterial HPr Kinase/Phosphorylase V267F Mutant Gives Insights into the Allosteric Regulation Mechanism of This Bifunctional Enzyme

The HPr kinase/phosphorylase (HPrK/P) is a bifunctional enzyme that controls the phosphorylation state of the phospho-carrier protein HPr, which regulates the utilization of carbon sources in Gram-positive bacteria. It uses ATP or pyrophosphate for the phosphorylation of serine 46 of HPr and inorganic phosphate for the dephosphorylation of Ser(P)-46-HPr via a phosphorolysis reaction. HPrK/P is a hexameric protein kinase of a new type with a catalytic core belonging to the family of nucleotide-binding protein with Walker A motif. It exhibits no structural similarity to eukaryotic protein kinases. So far, HPrK/P structures have shown the enzyme in its phosphorylase conformation. They permitted a detailed characterization of the phosphorolysis mechanism. In the absence of a structure with bound nucleotide, we used the V267F mutant enzyme to assess the kinase conformation. Indeed, the V267F replacement was found to cause an almost entire loss of the phosphorylase activity of Lactobacillus casei HPrK/P. In contrast, the kinase activity remained conserved. To elucidate the structural alterations leading to this drastic change of activity, the x-ray structure of the catalytic domain of L. casei HPrK/P-V267F was determined at 2.6A resolution. A comparison with the structure of the wild type enzyme showed that the mutation induces conformation changes compatible with the switch from phosphorylase to kinase function. Together with nucleotide binding fluorescence measurements, these results allowed us to decipher the cooperative behavior of the protein and to gain new insights into the allosteric regulation mechanism of HPrK/P.

Effects of Histidine Protonation and Phosphorylation on Histidine-Containing Phosphocarrier Protein Structure, Dynamics, and Physicochemical Properties

Previous structural studies of the histidine-containing phosphocarrier protein (HPr) have shown that active site residue His15 can adopt two distinct conformations which were termed OPEN and CLOSED. Using molecular dynamics simulations and protonation probability calculations, we were able to show that these two conformations correspond to different protonation forms of the histidine ring. The CLOSED-to-OPEN transition requires His15 to adopt a conformation with higher energy, which is compensated by the favorable energetic consequences of protonation. Calculations of the conformational energy of His15 show that HPr exists mainly in the CLOSED form at pH 7. The very low apparent pKa value (3.2-4.5) of the CLOSED conformation and the fact that the imidazole ring of residue 15 is primarily unprotonated at Ndelta1 at neutral pH ensure that His15 is ideally primed to be specifically phosphorylated at Ndelta1. In contrast to unphosphorylated HPr, the phosphorylated form exhibits no conformational transitions, and the CLOSED state is stable even for the protonated imidazole ring due to favorable interactions between the phosphate group and the backbone of Ala16 and Arg17. These observations from MD simulations are confirmed by a simple four-microstate model which can explain both the pH-dependent conformational change of unphosphorylated HPr and the conformational rigidity of phosphorylated HPr. Our study suggests that the predominant CLOSED conformation is relevant for HPr function in the phosphotransfer reaction, while the OPEN form of unphosphorylated HPr might be important for its additional regulatory function, in which an OPEN conformation of His15 is recognized by the transcriptional regulator CcpA.

Structure of full-length transcription regulator CcpA in the apo form

The catabolite control protein A (CcpA) from Bacillus megaterium is a member of the bacterial repressor protein family GalR–LacI. CcpA functions as master transcriptional regulator of carbon catabolite repression/regulation in firmicutes. Here we present the crystal structure of full-length apo CcpA at 2.5 Å resolution from B. megaterium. The structure reveals the location of the helix–turn–helix domain as well as the hinge region, which were not visible due to their high flexibility in earlier crystallographic studies on CcpA molecules. The structure of the apo CcpA homodimer in the present form is in contrast to other reported structures for CcpA.

Loss of penicillin tolerance by inactivating the carbon catabolite repression determinant CcpA in Streptococcus gordonii

Antibiotic tolerance is a phenomenon allowing bacteria to withstand drug-induced killing. Here, we studied a penicillin-tolerant mutant of Streptococcus gordonii (Tol1), which was shown to be deregulated in the expression of the arginine deiminase operon (arc). arc was not directly responsible for tolerance, but is controlled by the global regulator CcpA. Therefore, we sought whether CcpA might be implicated in tolerance. The ccpA gene was characterized and subsequently inactivated by PCR ligation mutagenesis in both the susceptible wild-type (WT) and Tol1. The minimal inhibitory concentration and time-kill curves for the strains were determined and the outcome of penicillin treatment in experimental endocarditis assessed. ccpA sequence and expression were similar between the WT and Tol1 strains. In killing assays, the WT lost 3.5 +/- 0.6 and 5.3 +/- 0.6 log(10) cfu/mL and Tol1 lost 0.4 +/- 0.2 and 1.4 +/- 0.9 log(10) cfu/mL after 24 and 48 h of penicillin exposure, respectively. Deletion of ccpA almost totally restored Tol1 kill susceptibility (loss of 2.5 +/- 0.7 and 4.9 +/- 0.7 log(10) cfu/mL at the same endpoints). In experimental endocarditis, penicillin treatment induced a significant reduction in vegetation bacterial densities between Tol1 (4.1 log(10) cfu/g) and Tol1DeltaccpA (2.4 log(10) cfu/g). Restitution of ccpA re-established the tolerant phenotype both in vitro and in vivo. CcpA, a global regulator of the carbon catabolite repression system, is implicated in penicillin tolerance both in vitro and in vivo. This links antibiotic survival to bacterial sugar metabolism. However, since ccpA sequence and expression were similar between the WT and Tol1 strains, other factors are probably involved in tolerance.

Control of the Phosphorylation State of the HPr Protein of the Phosphotransferase System in Bacillus subtilis: Implication of the Protein Phosphatase PrpC

In the Gram-positive bacterium Bacillus subtilis as well as in other firmicutes, the HPr protein of the phosphotransferase system (PTS) has two distinct phosphorylation sites, His-15 and Ser-46. These sites are phosphorylated by the Enzyme I of the PTS and by the ATP-dependent HPr kinase/phosphorylase, respectively. As a result, the phosphorylation state of HPr reflects the nutrient supply of the cell and is in turn involved in several responses at the levels of transport activity and expression of catabolic genes. Most important, HPr(Ser-P) serves as a cofactor for the pleiotropic transcription regulator CcpA. In addition to the proteins that phosphorylate HPr, those that are involved in the dephosphorylation are important in controlling the overall HPr phosphorylation state and the resulting regulatory and physiological outputs. In this study, we found that in addition to the phosphorylase activity of the HPr kinase/phosphorylase, the serine/threonine protein phosphatase PrpC uses HPr(Ser-P) as a target.

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

Carbon catabolite repression in Bacillus subtilis is carried out mainly by the major regulator CcpA. In contrast, sugar-dependent repression of three genes, sr1 encoding a small untranslated RNA, and two genes, gapB and pckA, coding for gluconeogenic enzymes is mediated by the recently identified transcriptional repressor CcpN. Since previous DNase I footprinting yielded only basic information on the operator sequences of CcpN, chemical interference footprinting studies were performed for a precise contact mapping. Methylation interference, potassium permanganate and hydroxylamine footprinting were used to identify all contacted residues in both strands in the three operator sequences. Furthermore, ethylation interference experiments were performed to identify phosphate residues essential for CcpN binding. Here, we show that each operator has two binding sites for CcpN, one of which was always contacted more strongly than the other. The three sites that exhibited close contacts were very similar in sequence, with only a few slight variations, whereas the other three corresponding sites showed several deviations. Gel retardation assays with purified CcpN demonstrated that the differences in contact number and strength correlated well with significantly different K D values for the corresponding single binding sites. However, quantitative DNase I footprinting of whole operator sequences revealed cooperative binding of CcpN that, apparently, compensated the asymmetric contact distribution. Based on these data, possible consequences for the repression mechanism of CcpN are discussed.

Structural Basis for Allosteric Control of the Transcription Regulator CcpA by the Phosphoprotein HPr-Ser46-P

Carbon catabolite repression (CCR) is one of the most fundamental environmental-sensing mechanisms in bacteria and imparts competitive advantage by establishing priorities in carbon metabolism. In gram-positive bacteria, the master transcription regulator of CCR is CcpA. CcpA is a LacI-GalR family member that employs, as an allosteric corepressor, the phosphoprotein HPr-Ser46-P, which is formed in glucose-replete conditions. Here we report structures of the Bacillus megaterium apoCcpA and a CcpA-(HPr-Ser46-P)-DNA complex. These structures reveal that HPr-Ser46-P mediates a novel two-component allosteric DNA binding activation mechanism that involves both rotation of the CcpA subdomains and relocation of pivot-point residue Thr61, which leads to juxtaposition of the DNA binding regions permitting “hinge” helix formation in the presence of cognate DNA. The structure of the CcpA-(HPr-Ser46-P)- cre complex also reveals the elegant mechanism by which CcpA family-specific interactions with HPr-Ser46-P residues Ser46-P and His15 partition the high-energy CCR and low-energy PTS pathways, the latter requiring HPr-His15-P.

Transcriptional profiling of gene expression in response to glucose in Bacillus subtilis: regulation of the central metabolic pathways

Chemoheterotrophic bacteria use a few central metabolic pathways for carbon catabolism and energy production as well as for the generation of the main precursors for anabolic reactions. All sources of carbon and energy are converted to intermediates of these central pathways and then further metabolized. While the regulation of genes encoding enzymes used to introduce specific substrates into the central metabolism has already been studied to some detail, much less is known about the regulation of the central metabolic pathways. In this study, we investigated the responses of the Bacillus subtilis transcriptome to the presence of glucose and analyzed the role of the pleiotropic transcriptional regulator CcpA in these responses. We found that CcpA directly represses genes involved in the utilization of secondary carbon sources. In contrast, induction by glucose seems to be mediated by a variety of different mechanisms. In the presence of glucose, the genes encoding glycolytic enzymes are induced. Moreover, the genes responsible for the production of acetate from pyruvate with a concomitant substrate-level phosphorylation are induced by glucose. In contrast, the genes required for the complete oxidation of the sugar (Krebs cycle, respiration) are repressed if excess glucose is available for the bacteria. In the absence of glucose, the genes of the Krebs cycle as well as gluconeogenic genes are derepressed. The genes encoding enzymes of the pentose phosphate pathway are expressed both in the presence and the absence of glucose, as suggested by the central role of this pathway in generating anabolic precursors.

Direct and indirect roles of CcpA in regulation of Bacillus subtilis Krebs cycle genes.

Carbon catabolite repression of the Bacillus subtilis citrate synthase (citZ) and aconitase (citB) genes, previously known to be regulated by CcpC, was shown to depend on CcpA as well. Transcription of the citZ gene was partially derepressed in ccpA and ccpC single mutants and fully derepressed in a ccpA ccpC double mutant. DNase I footprinting studies showed that CcpA binds to a catabolite-responsive element (cre) site located at positions +80 to +97 with respect to the transcription start site, whereas CcpC binds at positions -14 to +6 and +16 to +36. Mutations in the citZ cre site greatly altered CcpA binding and repression. A ccpA null mutation also caused partial derepression of citB. Disruption of citrate synthase activity, however, suppressed the effect of the ccpA mutation, suggesting that increased citrate accumulation in a ccpA mutant partially inactivates CcpC and causes partial derepression of citB. Therefore, CcpA controls expression of Krebs cycle genes directly by regulating transcription of citZ and in-directly by regulating availability of citrate, the inducer for CcpC.

Relationship between carbon catabolite repression and the biosynthesis regulation of the prolidase PepQ from Lactobacillus delbrueckii ssp. bulgaricus CNRZ 397

Lactobacillus delbrueckiissp. bulgaricus CNRZ 397 (L. bulgaricus) displays several enzymes specific of proline-containing peptides. We focused on the prolidase PepQ which specifi- cally cleaves X-Pro dipeptides. PepQ biosynthesis was previously shown to be independent of the pep- tide concentration of the culture medium in contrast to the cell surface proteinase PrtB and several aminopeptidases. Regulation of PepQ biosynthesis can be explained by the genetic organization of the region pepR1-cre-pepQ. The pepR1 gene encodes a CcpA-like regulator and its promoter harbors a cre site located immediately upstream of pepQ. Expression of fusions cre-pepQ-lacZand pepQ-lacZ in Bacillus subtilis showed that, under glucose conditions, the regulator CcpA acts as a transcriptional activator of pepQ expression. Analysis of PepQ biosynthesis in L. bulgaricus cells grown in differ- ent media is in agreement with a regulation dependent on carbohydrates. Lactobacillus delbrueckii ssp. bulgaricus / catabolite repression / PepQ / PepR1

Bacillus subtilis ccpA gene mutants specifically defective in activation of acetoin biosynthesis.

A large number of carbon source utilization pathways are repressed in Bacillus subtilis by the global regulator CcpA, which also acts as an activator of carbon excretion pathways during growth in media containing glucose. In this study, CcpA mutants defective in transcriptional activation of the alsSD operon, which is involved in acetoin biosynthesis, were identified. These mutants retained normal glucose repression of amyE, encoding alpha-amylase, and acsA, encoding acetyl-coenzyme A synthetase, and normal activation of ackA, which is involved in acetate excretion; in these ccpA mutants the CcpA functions of activation of the acetate and acetoin excretion pathways appear to be separated.

Role of CcpA in regulation of the central pathways of carbon catabolism in Bacillus subtilis.

The Bacillus subtilis two-dimensional (2D) protein index contains almost all glycolytic and tricarboxylic acid (TCA) cycle enzymes, among them the most abundant housekeeping proteins of growing cells. Therefore, a comprehensive study on the regulation of glycolysis and the TCA cycle was initiated. Whereas expression of genes encoding the upper and lower parts of glycolysis (pgi, pfk, fbaA, and pykA) is not affected by the glucose supply, there is an activation of the glycolytic gap gene and the pgk operon by glucose. This activation seems to be dependent on the global regulator CcpA, as shown by 2D polyacrylamide gel electrophoresis analysis as well as by transcriptional analysis. Furthermore, a high glucose concentration stimulates production and excretion of organic acids (overflow metabolism) in the wild type but not in the ccpA mutant. Finally, CcpA is involved in strong glucose repression of almost all TCA cycle genes. In addition to TCA cycle and glycolytic enzymes, the levels of many other proteins are affected by the ccpA mutation. Our data suggest (i) that ccpA mutants are unable to activate glycolysis or carbon overflow metabolism and (ii) that CcpA might be a key regulator molecule, controlling a superregulon of glucose catabolism.