Characterization of the catabolite control protein (CcpA) gene from Leuconostoc mesenteroides SY1

The ccpA gene encoding catabolite control protein A (CcpA) of Leuconostoc mesenteroides SY1, a strain isolated from kimchi, was cloned, sequenced, analyzed for transcript, and overexpressed in Escherichia coli. The ccpA ORF (open reading frame) is 1,011 by in size, which can encode a protein of 336 amino acid residues with a molecular mass of 36,739 Da. The transcription start site was mapped at a position 49 nucleotides upstream of the start codon, and promoter sequences were also identified. The putative ere site overlapped with the -35 promoter sequence. The deduced amino acid sequence of the CcpA contained the helix-turn-helix motif found in many DNA-binding regulatory proteins. CcpA from L. mesenteroides SY1 had 54.6% identity with CcpA from Lactobacillus casei. The Northern blot experiment showed that ccpA was transcribed as a single 1.1 kb transcript, and transcription was repressed when grown on media containing glucose. CcpA was overproduced in E. coli BL21(DE3) cells using the pET expression vector, and purified to an apparent homogeneity. Gel Mobility Shift Assay with purified CcpA and a DNA fragment containing the ere sequence of the alpha-galactosidase gene (aga) from L. mesenteroides SY1 revealed that CcpA bound specifically to the ere site of aga.