Complex regulation of the Bacillus subtilis aconitase gene

The roles of the CcpC, CodY, and AbrB proteins in regulation of the Bacillus subtilis aconitase (citB) gene were found to be distinct and to vary with the conditions and phase of growth. CcpC, a citrate-inhibited repressor that is the primary factor regulating citB expression in minimal-glucose-glutamine medium, also contributed to repression of citB during exponential-phase growth in broth medium. A null mutation in codY had no effect on citB expression during growth in minimal medium even when combined with ccpC and abrB mutations. However, a codY mutation slightly relieved repression during exponential growth in broth medium and completely derepressed citB expression when combined with a ccpC mutation. An abrB mutation led to decreased expression of citB during stationary phase in both broth and minimal medium. All three proteins bound in vitro to specific and partially overlapping sites within the citB regulatory region. Interaction of CcpC and CodY with the citB promoter region was partially competitive.     

Catabolite repression in Lactobacillus casei ATCC 393 is mediated by CcpA.

The chromosomal ccpA gene from Lactobacillus casei ATCC 393 has been cloned and sequenced. It encodes the CcpA protein, a central catabolite regulator belonging to the LacI-GalR family of bacterial repressors, and shows 54% identity with CcpA proteins from Bacillus subtilis and Bacillus megaterium. The L. casei ccpA gene was able to complement a B. subtilis ccpA mutant. An L. casei ccpA mutant showed increased doubling times and a relief of the catabolite repression of some enzymatic activities, such as N-acetylglucosaminidase and phospho-beta-galactosidase. Detailed analysis of CcpA activity was performed by using the promoter region of the L. casei chromosomal lacTEGF operon which is subject to catabolite repression and contains a catabolite responsive element (cre) consensus sequence. Deletion of this cre site or the presence of the ccpA mutation abolished the catabolite repression of a lacp::gusA fusion. These data support the role of CcpA as a common regulatory element mediating catabolite repression in low-GC-content gram-positive bacteria.     

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.     

Regulators of the Bacillus subtilis cydABCD operon: identification of a negative regulator, CcpA, and a positive regulator, ResD

The cydABCD operon of Bacillus subtilis encodes products required for the production of cytochrome bd oxidase. Previous work has shown that one regulatory protein, YdiH (Rex), is involved in the repression of this operon. The work reported here confirms the role of Rex in the negative regulation of the cydABCD operon. Two additional regulatory proteins for the cydABCD operon were identified, namely, ResD, a response regulator involved in the regulation of respiration genes, and CcpA, the carbon catabolite regulator protein. ResD, but not ResE, was required for full expression of the cydA promoter in vivo. ResD binding to the cydA promoter between positions -58 and -107, a region which includes ResD consensus binding sequences, was not enhanced by phosphorylation. A ccpA mutant had increased expression from the full-length cydA promoter during stationary growth compared to the wild-type strain. Maximal expression in a ccpA mutant was observed from a 3'-deleted cydA promoter fusion that lacked the Rex binding region, suggesting that the effect of the two repressors, Rex and CcpA, was cumulative. CcpA binds directly to the cydA promoter, protecting the region from positions -4 to -33, which contains sequences similar to the CcpA consensus binding sequence, the cre box. CcpA binding was enhanced upon addition of glucose-6-phosphate, a putative cofactor for CcpA. Mutation of a conserved residue in the cre box reduced CcpA binding 10-fold in vitro and increased cydA expression in vivo. Thus, CcpA and ResD, along with the previously identified cydA regulator Rex (YdiH), affect the expression of the cydABCD operon. Low-level induction of the cydA promoter was observed in vivo in the absence of its regulatory proteins, Rex, CcpA, and ResD. This complex regulation suggests that the cydA promoter is tightly regulated to allow its expression only at the appropriate time and under the appropriate conditions.     

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.     

CcpA-mediated repression of Clostridium difficile toxin gene expression

The presence of glucose or other rapidly metabolizable carbon sources in the bacterial growth medium strongly represses Clostridium difficile toxin synthesis independently of strain origin. In Gram-positive bacteria, carbon catabolite repression (CCR) is generally regarded as a regulatory mechanism that responds to carbohydrate availability. In the C. difficile genome all elements involved in CCR are present. To elucidate in vivo the role of CCR in C. difficile toxin synthesis, we used the ClosTron gene knockout system to construct mutants of strain JIR8094 that were unable to produce the major components of the CCR signal transduction pathway: the phosphotransferase system (PTS) proteins (Enzyme I and HPr), the HPr kinase/phosphorylase (HprK/P) and the catabolite control protein A, CcpA. Inactivation of the ptsI, ptsH and ccpA genes resulted in derepression of toxin gene expression in the presence of glucose, whereas repression of toxin production was still observed in the hprK mutant, indicating that uptake of glucose is required for repression but that phosphorylation of HPr by HprK is not. C. difficile CcpA was found to bind to the regulatory regions of the tcdA and tcdB genes but not through a consensus cre site motif. Moreover in vivo and in vitro results confirmed that HPr-Ser45-P does not stimulate CcpA-dependent binding to DNA targets. However, fructose-1,6-biphosphate (FBP) alone did increase CcpA binding affinity in the absence of HPr-Ser45-P. These results showed that CcpA represses toxin expression in response to PTS sugar availability, thus linking carbon source utilization to virulence gene expression in C. difficile.