The catabolite control protein CcpA controls ammonium assimilation in Bacillus subtilis

Carbon catabolite repression of several catabolic operons in Bacillus subtilis is mediated by the repressor CcpA. An inactivation of the ccpA gene has two distinct phenotypes: (i) catabolite repression of catabolic operons is lost and (ii) the growth of bacteria on minimal medium is severely impaired. We have analyzed the physiological properties of a ccpA mutant strain and show that the ccpA mutation does not affect sugar transport. We have isolated extragenic suppressors of ccpA that suppress the growth defect (sgd mutants). Catabolite repression of beta-xylosidase synthesis was, however, not restored suggesting that the suppressor mutations allow differentiation between the phenotypes of the ccpA mutant. A close inspection of the growth requirements of the ccpA mutant revealed the inability of the mutant to utilize inorganic ammonium as a single source of nitrogen. An intact ccpA gene was found to be required for expression of the gltAB operon encoding glutamate synthase. This enzyme is necessary for the assimilation of ammonium. In a sgd mutant, gltAB operon expression was no longer dependent on ccpA, suggesting that the poor expression of the gltAB operon is involved in the growth defect of the ccpA mutant.     

Catabolite repression in the D-serine deaminase system of Escherichia coli K-12

The induced synthesis of d-serine deaminase in Escherichia coli is subject to three catabolic effects: inhibition on inducer uptake, transient repression, and catabolite repression. Inhibition on d-serine uptake is not significant at the d-serine concentration normally used for induction. Transient repression and catabolite repression of d-serine deaminase synthesis are abolished by mutations in dsdCy, which appears to be an operator locus. The decline in the rate of constitutive synthesis observed in dsdCx mutants growing with glycerol as carbon source at temperatures above 37 C is due to catabolite repression. The low level of constitutivity at 37 C and the partial cis dominance of dsdCx mutants are not artifacts of catabolite repression. It is suggested that a product of one of the genes of the dsd operon may regulate the expression of the operon.     

Catabolite repression of the Bacillus subtilis gnt operon mediated by the CcpA protein.

Inducer exclusion was not important in catabolite repression of the Bacillus subtilis gnt operon. The CcpA protein (also known as AlsA) was found to be necessary for catabolite repression of the gnt operon, and a mutation (crsA47, which is an allele of the sigA gene) partially affected this catabolite repression.     

Significance of HPr in catabolite repression of alpha-amylase.

CcpA and HPr are presently the only two proteins implicated in Bacillus subtilis global carbon source catabolite repression, and the ptsH1 mutation in the gene for the HPr protein was reported to relieve catabolite repression of several genes. However, alpha-amylase synthesis by B. subtilis SA003 containing the ptsH1 mutation was repressed by glucose. Our results suggest HPr(Ser-P) may be involved in but is not required for catabolite repression of alpha-amylase, indicating that HPr(Ser-P) is not the sole signaling molecule for CcpA-mediated catabolite repression in B. subtilis.     

Determination of the cis sequence involved in catabolite repression of the Bacillus subtilis gnt operon; implication of a consensus sequence in catabolite repression in the genus Bacillus.

The mechanism underlying catabolite repression in Bacillus species remains unsolved. The gluconate (gnt) operon of Bacillus subtilis is one of the catabolic operons which is under catabolite repression. To identify the cis sequence involved in catabolite repression of the gnt operon, we performed deletion analysis of a DNA fragment carrying the gnt promoter and the gntR gene, which had been cloned into the promoter probe vector, pWP19. Deletion of the region upstream of the gnt promoter did not affect catabolite repression. Further deletion analysis of the gnt promoter and gntR coding region was carried out after restoration of promoter activity through the insertion of internal constitutive promoters of the gnt operon before the gntR gene (P2 and P3). These deletions revealed that the cis sequence involved in catabolite repression of the gnt operon is located between nucleotide positions +137 and +148. This DNA segment contains a sequence, ATTGAAAG, which may be implicated as a consensus sequence involved in catabolite repression in the genus Bacillus.     

Nature of the effector of catabolite repression of beta-galactosidase in Escherichia coli

Loomis, William F., Jr. (Massachusetts Institute of Technology, Cambridge, Mass.), and Boris Magasanik. Nature of the effector of catabolite repression of beta-galactosidase in Escherichia coli. J. Bacteriol. 92:170-177. 1966.-Many carbon sources were found to give rise to catabolite repression of beta-galactosidase in a mutant strain of Escherichia coli lacking hexose phosphate isomerase activity. Compounds containing glucose or galactose cannot be formed from several of these carbon sources in this mutant strain, and, therefore, appear not to be required for catabolite repression of beta-galactosidase. Glucose was observed to elicit catabolite repression of beta-galactosidase in another mutant strain under conditions in which the formation of compounds of the citric acid cycle is inhibited. If catabolite repression of the lac operon is mediated by a single compound, it appears that the compound is related to the pentoses and trioses of intermediary metabolism. The repression of beta-galactosidase by galactose in galactokinase negative strains was shown to be independent of the gene, CR, which determines catabolite sensitivity of the lac operon, and to be dependent on a functional i gene.     

Catabolite regulation of the cytochrome c550-encoding Bacillus subtilis cccA gene

In Gram-positive bacteria, catabolite control protein A (CcpA)-mediated catabolite repression or activation regulates not only the expression of a great number of catabolic operons, but also the synthesis of enzymes of central metabolic pathways. We found that a constituent of the Bacillus subtilis respiratory chain, the small cytochrome c550 encoded by the cccA gene, was also submitted to catabolite repression. Similar to most catabolite-repressed genes and operons, the Bacillus subtilis cccA gene contains a potential catabolite response element cre, an operator site recognized by CcpA. The presumed cre overlaps the -35 region of the cccA promoter. Strains carrying a cccA'-IacZ fusion formed blue colonies when grown on rich solid medium, whereas white colonies were obtained when glucose was present. beta-Galactosidase assays with cells grown in rich medium confirmed the repressive effect of glucose on cccA'-lacZ expression. Introduction of a ccpA or hprK mutation or of a mutation affecting the presumed cccA cre relieved the repressive effect of glucose during late log phase. An additional glucose repression mechanism was activated during stationary phase, which was not relieved by the ccpA, hprK or cre mutations. An interaction of the repressor/corepressor complex (CcpA/seryl-phosphorylated HPr (P-Ser-HPr)) with the cccA cre could be demonstrated by gel shift experiments. By contrast, a DNA fragment carrying mutations in the presumed cccA cre was barely shifted by the CcpA/P-Ser-HPr complex. In footprinting experiments, the region corresponding to the presumed cccA cre was specifically protected in the presence of the CcpA/P-Ser-HPr complex.     

Mutations in Escherichia coli that relieve catabolite repression of tryptophanase synthesis. Mutations distant from the tryptophanase gene.

Two mutants are described in which the synthesis of tryptophanase is unusually insensitive to catabolite repression. Neither mutation is linked by transduction to the tryptophane structural gene, neither mutation renders the synthesis of beta-galactosidase insensitive to catabolite repression, and the mutations do not permit tryptophanase to be synthesized in strains deficient in adenyl cyclase. During growth in glucose-minimal medium the mutants maintained a similar intracellular concentration of cyclic AMP to their wild-type parent; but since in the wild type the concentration of cyclic AMP was the same in glycerol-minimal medium as in glucose-minimal medium, it is doubtful whether catabolite repression is mediated by measurable changes in the concentration of this nucleotide.     

Absence of involvement of glutamine synthetase and of NAD-linked glutamate dehydrogenase in the nitrogen catabolite repression of arginase and other enzymes in Saccharomyces cerevisiae

The escape of several enzymes from “ammonia catabolite repression” in gdhA^? (NADP-linked glutamate-dehydrogenase-less) mutants, as well as in gdhCR mutants of Saccharomyces cerevisiae, does not involve glutamine synthetase, either as a positive or as a negative control element. A glutamine-synthetase-less mutant (gln^?) was used in this demonstration.In addition to its derepressing effect on the NAD-linked glutamate dehydrogenase, the gdhCR mutation releases “nitrogen catabolite repression” on arginase and allatoinase, as well as glutamine repression on glutamine synthetase. A gdhCS mutation was used to demonstrate that these effects are not mediated through the NAD-linked glutamate dehydrogenase.     

Altered end-product patterns and catabolite repression in Escherichia coli

Dobrogosz, Walter J. (North Carolina State University, Raleigh). Altered end-product patterns and catabolite repression in Escherichia coli. J. Bacteriol. 91:2263-2269. 1966.-End products formed during growth of Escherichia coli ML30 on glucose were examined under various conditions known to promote or prevent catabolite repression of the inducible beta-galactosidase system in this organism. Cultures were grown under these conditions in the presence of C(14)-glucose or C(14)-pyruvate. The products formed were assayed isotopically after separation on columns of silicic acid. Under conditions known to promote catabolite repression, glucose was degraded primarily to acetate and CO(2). When repression was turned off by anaerobic shock, glucose metabolism was characterized by the accumulation of ethyl alcohol in addition to acetate and CO(2). The results presented in this report indicate that oxidative decarboxylation of pyruvate may markedly affect the amount of energy that can be derived from glucose catabolism. In turn, the amount of energy derived from catabolic processes may play a key role in the mechanism of catabolite repression.     

The mechanism of sugar-mediated catabolite repression of the propionate catabolic genes in Escherichia coli

Carbon catabolite repression (CCR) is a well-known phenomenon that involves the preferential utilization of glucose as a carbon source. Cyclic adenosine monophosphate (cAMP) and the cAMP receptor protein (CRP) mediate CCR. Recently, a second CCR hierarchy that leads to the preferential consumption of arabinose over xylose, mediated by arabinose-bound AraC, has been identified. In this study, we report yet another CCR hierarchy that causes the preferential utilization of sugars (arabinose, galactose, glucose, mannose, and xylose) over a short-chain fatty acid (propionate). Expression of the propionate catabolic (prpBCDE) genes is down-regulated in the presence of these sugars. Sugar-mediated repression of the propionate catabolic genes is independent of sugar-specific regulators such as AraC and dependent on global regulators of sugar transport such as the cAMP-CRP complex and the Phosphotransferase System (PTS). Inhibition of the prpBCDE promoter is encountered during rapid sugar uptake and metabolism. This unique regulatory crosstalk between sugar metabolism and fatty acid metabolism may help provide new insights into CRP-dependent catabolite repression acting in conjunction with non-carbohydrate metabolism.     

Catabolite repression resistance of gnt operon expression in Bacillus subtilis conferred by mutation of His-15, the site of phosphoenolpyruvate-dependent phosphorylation of the phosphocarrier protein HPr.

Carbon catabolite repression of the gnt operon of Bacillus subtilis is mediated by the catabolite control protein CcpA and by HPr, a phosphocarrier protein of the phosphotransferase system. ATP-dependent phosphorylation of HPr at Ser-46 is required for carbon catabolite repression as ptsH1 mutants in which Ser-46 of HPr is replaced with an unphosphorylatable alanyl residue are resistant to carbon catabolite repression. We here demonstrate that mutation of His-15 of HPr, the site of phosphoenolpyruvate-dependent phosphorylation, also prevents carbon catabolite repression of the gnt operon. A strain which expressed two mutant HPrs (one in which Ser-46 is replaced by Ala [S46A HPr] and one in which His-15 is replaced by Ala [H15A HPr]) on the chromosome was barely sensitive to carbon catabolite repression, although the H15A mutant HPr can be phosphorylated at Ser-46 by the ATP-dependent HPr kinase in vitro and in vivo. The S46D mutant HPr which structurally resembles seryl-phosphorylated HPr has a repressive effect on gnt expression even in the absence of a repressing sugar. By contrast, the doubly mutated H15E,S46D HPr, which resembles the doubly phosphorylated HPr because of the negative charges introduced by the mutations at both phosphorylation sites, had no such effect. In vitro assays substantiated these findings and demonstrated that in contrast to the wild-type seryl-phosphorylated HPr and the S46D mutant HPr, seryl-phosphorylated H15A mutant HPr and H15E,S46D doubly mutated HPr did not interact with CcpA. These results suggest that His-15 of HPr is important for carbon catabolite repression and that either mutation or phosphorylation at His-15 can prevent carbon catabolite repression.     

The role of the regulator-gene product (repressor) in catabolite repression of β-galactosidase synthesis in Escherichia coli

1. The specific role of the lac repressor (i-gene product) in transient catabolite repression evoked by the introduction of glucose into the medium has been investigated in Escherichia coli by using mutants of the i-gene. 2. A temperature-sensitive mutant (i(TL)) is normally inducible and demonstrates transient repression when grown at 32 degrees . At 42 degrees it is about 20% constitutive and transient catabolite repression is abolished. 3. A strain carrying an amber suppressor-sensitive mutation in the i-gene is phenotypically constitutive and also fails to show transient catabolite repression. 4. Insertion of Flaci(+) into this strain restores both inducibility and transient repression. 5. It is concluded that the i-gene product interacts with the catabolite co-repressor in such a way that its affinity for the operator is increased.