Cloning and characterization of the catabolite control protein A gene from Bacillus stearothermophilus No. 236.

Abstract

The gene encoding the catabolite control protein A (CcpA) of Bacillus stearothermophilus No. 236, a strong xylanolytic bacterium, was cloned, sequenced, and expressed in Escherichia coli. The nucleotide sequence of the ccpA gene corresponded to an open reading frame of 1,005 bp that encodes a polypeptide of 334 amino acid residues with a calculated molecular mass of 36,902 kDa. The CcpA protein belonging to the LacI/GalR family of transcriptional regulators was produced by a recombinant E. coli strain expressing the B. stearothermophilus No. 236 ccpA gene and purified to apparent homogeneity. The transcription start site was mapped at a position 63 nucleotides upstream of the translation initiation codon, and a presumed promoter sequence was also identified. The deduced amino acid sequence of the ccpA gene product contained the helix-turn-helix motif found in many DNA-binding proteins, and showed the highest identity (62%) with CcpA from B. subtilis. The B. stearothermophilus No. 236 ccpA gene was demonstrated to be able to complement a B. subtilis ccpA mutant that exhibited two distinct mutant phenotypes: a growth defect and a release of carbon catabolite repression (CCR). These results indicate that the ccpA gene product of B. stearothermophilus No. 236 is functionally active also in B. subtilis. Electrophoretic mobility shift assay with the purified CcpA revealed that the CcpA of B. stearothermophilus No. 236 bound specifically to the xynA creB (catabolite responsive element B) sequence. Taken together, these results strongly suggest that the CcpA protein participates in CCR of B. stearothermophilus No. 236 xynA gene.