Transcriptional activation of the Bacillus subtilis ackA gene requires sequences upstream of the promoter

Transcriptional activation of the Bacillus subtilis ackA gene, encoding acetate kinase, was previously shown to require catabolite control protein A (CcpA) and sequences upstream of the ackA promoter. CcpA, which is responsible for catabolite repression of a number of secondary carbon source utilization genes in B. subtilis and other gram-positive bacteria, recognizes a cis-acting consensus sequence, designated cre (catabolite response element), generally located within or downstream of the promoter of the repressed gene. Two sites resembling this sequence are centered at positions -116.5 and -56.5 of the ackA promoter and have been termed cre1 and cre2, respectively. Synthesis of acetate kinase, which is involved in the conversion of acetyl coenzyme A to acetate, is induced when cells are grown in the presence of an easily metabolized carbon source such as glucose. In this study, cre2, the site closer to the promoter, and the region upstream of cre2 were shown to be indispensable for CcpA-dependent transcriptional activation of ackA, whereas cre1 was not required. In addition, insertion of 5 bp between cre2 and the promoter disrupted activation, while 10 bp was tolerated, suggesting face-of-the-helix dependence of the position of cre2 and/or upstream sequences. DNase footprinting experiments demonstrated binding of CcpA in vitro to cre2 but not cre1, consistent with the genetic data. Activation of ackA transcription was blocked in a ptsH1/crh double mutant, suggesting involvement of this pathway in CcpA-mediated transcriptional activation.