The ClgR Protein Regulates Transcription of the clpP Operon in Bifidobacterium breve UCC 2003

Fiveclp genes (clpC ,clpB ,clpP1 ,clpP2 , andclpX ), representing chaperone- and protease-encoding genes, were previously identified inBifidobacterium breve UCC 2003. In the present study, we characterize theB. breve UCC 2003clpP locus, which consists of two paralogous genes, designatedclpP1 andclpP2 , whose deduced protein products display significant similarity to characterized ClpP peptidases. Transcriptional analyses showed that theclpP1 andclpP2 genes are transcribed in response to moderate heat shock as a bicistronic unit with a single promoter. The role of aclgR homologue, known to control the regulation ofclpP gene expression inStreptomyces lividans andCorynebacterium glutamicum , was investigated by gel mobility shift assays and DNase I footprint experiments. We show that ClgR, which in its purified form appears to exist as a dimer, requires a proteinaceous cofactor to assist in specific binding to a 30-bp region of theclpP promoter region. In pull-down experiments, a 56-kDa protein copurified with ClgR, providing evidence that the two proteins also interact in vivo and that the copurified protein represents the cofactor required for ClgR activity. The prediction of the ClgR three-dimensional structure provides further insights into the binding mode of this protein to theclpP1 promoter region and highlights the key amino acid residues believed to be involved in the protein-DNA interaction.