Complex expression control of the Corynebacterium glutamicum aconitase gene: Identification of RamA as a third transcriptional regulator besides AcnR and RipA

Abstract

Expression of the aconitase gene acn of Corynebacterium glutamicum was previously shown to be repressed by the TetR-type regulator AcnR in response to a yet unknown stimulus and by the AraC-type regulator RipA in response to iron limitation. Here we have identified a third transcriptional regulator of aconitase, RamA. The RamA protein was enriched by DNA affinity chromatography with the acn promoter region from protein extracts of acetate-grown cells but not or only weakly from extracts of glucose-grown cells. In the wild type, aconitase activity is about 3-fold higher in acetate-grown cells compared to glucose-grown cells. In extracts of a ramA deletion mutant, acetate-grown cells possess the same aconitase activity as glucose-grown cells. Inspection of the acn promoter region led to the identification of a RamA binding motif (TGGGGGTGAGTAAGGGGGT), which was shown by electrophoretic mobility shift assays to be essential for binding of purified RamA. Furthermore, the functional relevance of this motif, which is located −180 to −162 bp upstream of the transcriptional start site, for RamA-dependent activation of acn expression was confirmed by promoter fusion assays. Thus, RamA was shown to be responsible for activation of acn expression in the presence of acetate. Furthermore, evidence was obtained in this work that RamB negatively regulates acn expression, but in an indirect manner.