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.     

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.     

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.     

Phosphorylation of HPr and Crh by HprK, early steps in the catabolite repression signalling pathway for the Bacillus subtilis levanase operon

Carbon catabolite repression (CCR) of Bacillus subtilis catabolic genes is mediated by CcpA and in part by P-Ser-HPr. For certain operons, Crh, an HPr-like protein, is also implicated in CCR. In this study we demonstrated that in ptsH1 crh1 and hprK mutants, expression of the lev operon was completely relieved from CCR and that both P-Ser-HPr and P-Ser-Crh stimulated the binding of CcpA to the cre sequence of the lev operon.     

In vitro DNA binding of purified CcpA protein from Lactococcus lactis IL1403

During this study His-tagged CcpA protein purified under native conditions to obtain a biologically active protein was used for molecular analysis of CcpA-dependent regulation. Using electrophoretic mobility shift assays it was demonstrated that CcpA of L. lactis can bind DNA in the absence of the HPr-Ser-P corepressor and exhibits DNA-binding affinity for nucleotide sequences lacking cre sites. However, purified HPr-Ser-P protein from Bacillus subtilis was shown to slightly increase the DNA-binding capacity of the CcpA protein. It was also observed that CcpA bound to the cre box forms an apparently more stable complex than that resulting from unspecific binding. Competition gel retardation assay performed on DNA sequences from two PEP:PTS regions demonstrated that the ybhE, bglS, rheB, yebE, ptcB and yecA genes situated in these regions are most probably directly regulated by CcpA.     

trans-acting factors affecting carbon catabolite repression of the hut operon in Bacillus subtilis

In Bacillus subtilis, CcpA-dependent carbon catabolite repression (CCR) mediated at several cis-acting carbon repression elements (cre) requires the seryl-phosphorylated form of both the HPr (ptsH) and Crh (crh) proteins. During growth in minimal medium, the ptsH1 mutation, which prevents seryl phosphorylation of HPr, partially relieves CCR of several genes regulated by CCR. Examination of the CCR of the histidine utilization (hut) enzymes in cells grown in minimal medium showed that neither the ptsH1 nor the crh mutation individually had any affect on hut CCR but that hut CCR was abolished in a ptsH1 crh double mutant. In contrast, the ptsH1 mutation completely relieved hut CCR in cells grown in Luria-Bertani medium. The ptsH1 crh double mutant exhibited several growth defects in glucose minimal medium, including reduced rates of growth and growth inhibition by high levels of glycerol or histidine. CCR is partially relieved in B. subtilis mutants which synthesize low levels of active glutamine synthetase (glnA). In addition, these glnA mutants grow more slowly than wild-type cells in glucose minimal medium. The defects in growth and CCR seen in these mutants are suppressed by mutational inactivation of TnrA, a global nitrogen regulatory protein. The inappropriate expression of TnrA-regulated genes in this class of glnA mutants may deplete intracellular pools of carbon metabolites and thereby result in the reduction of the growth rate and partial relief of CCR.