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--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.
A 2-deoxy-~-glucose-resistant mutant of a pLZl5-cured derivative of Lactobadhs casei ATCC 393 was isolated on agar medium containing 10 mM 2-deoxy-~-glucose and 5 g lactose I-'. The mutant was impaired in the main glucose transport mechanism, a PTSman-type system. Additionally a protonmotive-force-dependent glucose permease was detected. The growth response and the sugar consumption rates of the wild-type and the PTSmn-deficient mutant suggested that the mutated element of the complex IIABCman was, in the wild-type, responsible for a strong repression by glucose and mannose of the lactose and ribose assimilation genes, while assimilation of galactose was only weakly repressed. It is postulated that they are regulated by a different mechanism of catabolite repression.
In Lactobacillus casei ATCC 393, the chromosomally encoded lactose operon, lacTEGF, encodes an antiterminator protein (LacT), lactose-specific phosphoenolpyruvate-dependent phosphotransferase system (PTS) elements (LacE and LacF), and a phospho-β-galactosidase. lacT, lacE, andlacF mutant strains were constructed by double crossover. The lacT strain displayed constitutive termination at a ribonucleic antiterminator (RAT) site, whereas lacE andlacF mutants showed an inducer-independent antiterminator activity, as shown analysis of enzyme activity obtained from transcriptional fusions of lac promoter (lacp) and lacpΔRAT with the Escherichia coli gusAgene in the different lac mutants. These results strongly suggest that in vivo under noninducing conditions, the lactose-specific PTS elements negatively modulate LacT activity. Northern blot analysis detected a 100-nucleotide transcript starting at the transcription start site and ending a consensus RAT sequence and terminator region. In a ccpA mutant, transcription initiation was derepressed but no elongation through the terminator was observed in the presence of glucose and the inducing sugar, lactose. Full expression oflacTEGF was found only in a man ccpA double mutant, indicating that PTS elements are involved in the CcpA-independent catabolite repression mechanism probably via LacT.
The chromosomally encoded lactose-specific phosphoenol pyruvate-dependent phosphotransferase system (PTS) has been investigated in Lactobacillus casei ATCC 393 [pLZ15-] and it was considered an excellent system to study the regulation of the lactose operon. This chromosomal operon has been cloned and sequenced, being 99% homologous to that encoded on the plasmid pLZ64. Expression of the lactose operon in different mutants of L. casei ATCC 393 [pLZ15-] and primer extension analysis revealed that it is subject to a dual regulation: (i) glucose repression possibly mediated by CcpA and PTS elements, and (ii) induction by lactose through transcriptional antitermination.
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