Abstract
Expression of the alpha-amylase gene (amyE) of Bacillus subtilis is subject to CcpA (catabolite control protein A)-mediated catabolite repression, a global regulatory mechanism in Bacillus and other Gram-positive bacteria. To determine effectors of CcpA, we tested the ability of glycolytic metabolites, nucleotides, and cofactors to affect CcpA binding to the amyE operator, amyO. Those that stimulated the DNA-binding affinity of CcpA were tested for their effect on transcription. HPr-P (Ser-46), proposed as an effector of CcpA, also was tested. In DNase I footprint assays, the affinity of CcpA for amyO was stimulated 2-fold by fructose-1,6-diphosphate (FDP), 1.5-fold by oxidized or reduced forms of NADP, and 10-fold by HPr-P (Ser-46). However, the triple combinations, CcpA/NADP/HPr-P (Ser-46) and CcpA/FDP/HPr-P (Ser-46) synergistically stimulated DNA-binding affinity by 120- and 300-fold, respectively. NADP added to CcpA specifically stimulated transcription inhibition of the amyE promoter by 120-fold. CcpA combined with HPr (Ser-46) inhibited transcription from the amyE promoter, but it also inhibited several control promoters. FDP did not stimulate transcription inhibition by CcpA nor did the triple combinations. The finding that NADP had little effect on CcpA DNA binding but increased the ability of CcpA to inhibit transcription suggests that catabolite repression is not simply caused by CcpA binding amyO but rather a result of interactions with the transcription machinery enhanced by NADP.