RamA, the Transcriptional Regulator of Acetate Metabolism in <i>Corynebacterium glutamicum</i>, 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.

Involvement of Regulatory Interactions among Global Regulators GlxR, SugR, and RamA in Expression of ramA in Corynebacterium glutamicum

“…The promoter activities in the cells cultured for 8 h (in the stationary phase) are summarized in Table 2. The activity of the wild-type promoter was higher in the cells grown on acetate than those grown on glucose (Table 2), which is consistent with previous results (43). The promoter carrying the above-described mutations in the GlxR binding site showed no detectable activity during growth under these conditions (Table 2), suggesting that GlxR plays an essential role in ramA expression.…”

“…It has been reported that four transcription regulators, RamA, SugR, GlxR, and RamB, bind to the ramA promoter in vitro and/or in vivo (15,35,43). RamA is subject to negative autoregulation, as described above.…”

“…SugR, RamA, and GlxR act as transcription repressor of their own genes, sugR, ramA, and glxR, respectively (42)(43)(44). Expression of the sugR gene is directly activated by RamA (31).…”

“…Expression of the sugR gene is directly activated by RamA (31). Expression of the ramA gene is upregulated by an unknown factor in the presence of acetate (43). Thus, the carbon metabolism genes in C. glutamicum are under the control of a complex hierarchical regulatory network consisting of transcription factors responding to various environmental and/or physiological signals.…”

“…RamA is subject to negative autoregulation, as described above. RamB is involved in repression of the acetate metabolism genes in the presence of glucose (45), but disruption of its gene, ramB, has no effect on expression of ramA (43). However, the involvement of SugR and GlxR in ramA expression has not been investigated so far.…”