Glucose kinase has a regulatory role in carbon catabolite repression in Streptomyces coelicolor.

A glucose kinase (glkA) mutant of Streptomyces coelicolor A3(2) M145 was selected by the ability to grow in the presence of the nonmetabolizable glucose analog 2-deoxyglucose. In this glkA mutant, carbon catabolite repression of glycerol kinase and agarase was relieved on several carbon sources tested, even though most of these carbon sources are not metabolized via glucose kinase. This suggests that catabolite repression is not regulated by the flux through glucose kinase and that the protein itself has a regulatory role in carbon catabolite repression. A 10-fold overproduction of glucose kinase also results in relief of catabolite repression, suggesting that excess glucose kinase can titrate the repressing signal away. This could be achieved directly by competition of excess glucose kinase with its repressing form for binding sites on DNA promoter regions or indirectly by competition for binding of another regulatory protein.     

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.     

Insights into the functioning of Bacillus subtilis HPr kinase/phosphatase: affinity for its protein substrates and role of cations and phosphate

In Bacillus subtilis, carbon catabolite repression is mediated by the HPr kinase/phosphatase (HprK/P) which catalyzes both an ATP-dependent phosphorylation and a dephosphorylation on Ser-46 of either HPr (histidine-containing protein) or Crh (catabolite repression HPr). By using a surface plasmon resonance approach, it was shown here that the presence of magnesium is a prerequisite for the interaction of HprK/P with either HPr or Crh. HprK/P binds both protein substrates with a similar affinity (K(D) of about 40 nM), and addition of nucleotides increases by about 10-fold its affinity for each substrate. In addition, the specificity and the concentration of the cation required for the binding of protein substrates are different from that exhibited by the cation-binding site involved in the nucleotide binding, suggesting the presence of two cation-binding sites on HprK/P. The effects of phosphate on enzymatic activities of HprK/P were also investigated. Phosphate was able to unmask the phosphatase activity, especially in the presence of ATP or both ATP and fructose 1,6-bisphosphate whereas it was shown to inhibit the kinase activity of HprK/P. An apparent competition between phosphate and a fluorescent analogue of nucleotide led to the suggestion that phosphate mediates its effect by binding directly to the ATP-binding site of the enzyme.     

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.     

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.     

Isolation and characterization of a cis-acting mutation conferring catabolite repression resistance to alpha-amylase synthesis in Bacillus subtilis.

Bacillus subtilis 168GR10 was shown to contain a mutation, gra-10, which allowed normal temporal activation of alpha-amylase synthesis in the presence of a concentration of glucose that is inhibitory to activation of amylase synthesis in the parent strain, 168. The gra-10 mutation was mapped by phage PBS-1-mediated transduction and by transformation to a site between lin-2 and aroI906, very tightly linked to amyE, the alpha-amylase structural gene. The gra-10 mutation did not pleiotropically affect catabolite repression of sporulation or of the synthesis of extracellular proteases or RNase and was unable to confer glucose-resistance to the synthesis of chloramphenicol acetyltransferase encoded by the cat-86 gene driven by the amyE promoter region (amyR1) inserted into the promoter-probe plasmid pPL603B. It therefore appears that gra-10 defines a cis-regulatory site for catabolite repression, but not for temporal activation, of amyE expression. The evidence shows that temporal activation and glucose-mediated repression of alpha-amylase synthesis in B. subtilis 168 are distinct phenomena that can be separated by mutation.     

cis sequences involved in modulating expression of Bacillus licheniformis amyL in Bacillus subtilis: effect of sporulation mutations and catabolite repression resistance mutations on expression.

Nutrient conditions which trigger sporulation also activate expression of the Bacillus licheniformis alpha-amylase gene, amyL. Glucose represses both spore formation and expression of amyL. A fusion was constructed between the B. licheniformis alpha-amylase regulatory and 5' upstream sequences (amyRi) and the Escherichia coli lacZ structural gene to identify sequences involved in mediating temporal activation and catabolite repression of the amyL gene in Bacillus subtilis. amyRi-directed expression in a variety of genetic backgrounds and under different growth conditions was investigated. A 108-base-pair sequence containing an inverted repeat sequence, ribosome-binding site, and 26 codons of the structural gene was sufficient to mediate catabolite repression of amyL. spo0 mutations (spo0A, spo0B, spo0E, and spo0H) had no significant effect on temporal activation of the gene fusion when the recipient strains were grown in nonrepressing medium. However, in glucose-grown cultures the presence of a spo0A mutation resulted in more severe repression of amyRi-lacZ. In contrast, a spo0H mutation reduced the repressive effect of glucose on amyRi-lacZ expression. The spo0A effect was relieved by an abrB mutation. Initiation of sporulation is not a prerequisite for either temporal activation or derepression of alpha-amylase synthesis. Mutations causing resistance to catabolite repression in B. subtilis GLU-47, SF33, WLN30, and WLN104 also relieved catabolite repression of amyRi-lacZ.     

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.