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.     

Efficient utilization and operation of the gluconate-inducible system of the promoter of the Bacillus subtilis gnt operon in Escherichia coli.

A DNA fragment containing the promoter of the Bacillus subtilis gluconate (gnt) operon and its first gene (gntR) was cloned into Escherichia coli. E. coli recognized this promoter efficiently and precisely. Moreover, the gluconate-inducible system of this operon operated even in E. coli.     

Role of the DNA sequence downstream of the Bacillus subtilis hut promoter in regulation of the hut operon

To identify the role of the downstream region of a hut promoter in regulation of the Bacillus subtilis hut operon, three single-base substitutions (+9G-->A, +14C-->T, and +23T-->G) were introduced into the hut operon. Analysis of expression of the hut operon containing each of these three single-base substitutions and the hut-lacZ fusions with the single-base substitutions at position +14 showed that the position at +14 and probably the position at +23 were required for amino acid repression at the hut promoter, while the position at +14 was not required for catabolite repression at the hut promoter. The position at +9 was required for a histidine-dependent increase of activity of the hut promoter. Analysis of expression of the hut-lacZ fusions and the hut operon in the codY mutant indicated that the position at +14 and probably the position at +23 were involved in CodY-mediated amino acid repression at the hut promoter and that CodY was not required for catabolite repression at the hut promoter.     

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 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.