Identification of ligands affecting the activity of the transcriptional repressor CcpN from Bacillus subtilis

Carbon catabolite repression in Bacillus subtilis is mediated primarily by the major regulator CcpA. However, sugar-dependent repression of three genes, sr1 encoding a small nontranslated RNA and two genes coding for gluconeogenic enzymes, gapB and pckA, is carried out by the transcriptional repressor CcpN (control catabolite protein of gluconeogenic genes). It has previously been shown that ccpN is constitutively expressed, which leads to a constant occupation of all operators with CcpN. Since this would not allow for specific regulation, a ligand that modulates CcpN activity is required. In vitro transcription assays demonstrated that CcpN is able to specifically repress transcription to a small extent at the three mentioned promoters in the absence of an activating ligand. Upon testing of several ligands, including nucleotides and glycolysis intermediates, it could be shown that ATP is able to specifically enhance the repressing activity of CcpN, and this effect was more pronounced at a slightly acidic pH. Furthermore, ADP was found to specifically counteract the repressive effect of ATP. Circular dichroism measurements demonstrated a significant alteration of CcpN structure in the presence of ATP at acidic pH and in the presence of ADP. Electrophoretic mobility shift assays revealed that neither ATP nor ADP altered the affinity of CcpN for its operators. Therefore, we hypothesise that the effect of ligand-bound CcpN on the RNA polymerase might be due to a conformational switch that alters the interaction between the two proteins. Based on these results, a working model for CcpN action is discussed.