RamA, the Transcriptional Regulator of Acetate Metabolism in Corynebacterium glutamicum, Is Subject to Negative Autoregulation

Abstract

The RamA protein represents a LuxR-type transcriptional activator of genes involved in acetate metabolism of Corynebacterium glutamicum. Here we analyze the expression of the respective ramA gene and its regulation. Transcription was found to start 71 nucleotides upstream of the translational start codon and to be two- to threefold up-regulated in the presence of acetate in the growth medium. Accordingly, about twofold higher amounts of RamA were observed in C. glutamicum cells grown on acetate instead of glucose. Using cell extracts of C. glutamicum and employing DNA affinity chromatography, we found RamA itself as the main protein which binds to the ramA promoter region. By electrophoretic mobility shift analysis with the ramA promoter region and His-tagged RamA protein, multiple RamA-binding sites were identified in front of the ramA transcriptional start site. Transcriptional cat fusion experiments revealed that ramA promoter activity was about threefold higher in a RamA-deficient mutant of C. glutamicum than in the wild-type, however, acetate-dependent up-regulation of ramA expression was not affected in the RamA-negative mutant. These results indicate that RamA negatively controls the expression of its own gene, but is not involved in acetate-dependent up-regulation of ramA expression.