Involvement of the LuxR-Type Transcriptional Regulator RamA in Regulation of Expression of thegapAGene, Encoding Glyceraldehyde-3-Phosphate Dehydrogenase ofCorynebacterium glutamicum

Abstract

SugR, RamA, GlxR, GntR1, and a MarR-type transcriptional regulator bind to the promoter region of the gapA gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), essential for glycolysis in Corynebacterium glutamicum. We previously showed that SugR, a transcriptional repressor of phosphotransferase system genes for the sugar transport system, is involved in the downregulation of gapA expression in the absence of sugar. In this study, the role of RamA in the expression of the gapA gene was examined. Comparing the gapA expression and GAPDH activity of a ramA mutant with those of the wild type revealed that RamA is involved in upregulation of gapA expression in glucose-grown cells. DNase I footprint analyses and electrophoretic mobility shift assays revealed that RamA binds with different affinities to three sites in the gapA promoter. lacZ reporter assays with mutated RamA binding sites in the gapA promoter showed that the middle binding site is the most important for RamA to activate gapA expression and that binding of RamA to the gapA promoter activates the gene expression not only in glucose-grown cells but also in acetate-grown cells. Furthermore, RamA also directly activates sugR expression, indicating that two global regulators, RamA and SugR, are coordinately involved in the complex regulation of gapA expression in C. glutamicum.

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

“…Thus, the changes in the level of sugR expression in the pCRC622-carrying strain seemed to be correlated with those in the level of ramA expression. This is consistent with the finding that RamA directly activates sugR expression (31).…”

“…This is supported by the findings that the activity of the ramA promoter in the ramA mutant background was comparable to that in the sugR-ramA mutant background (Table 2). In this context, it should be noted that RamA acts as a transcription activator of sugR and sugR mRNA is markedly downregulated by ramA deletion (31). We further confirmed that ␤-galactosidase activity of the sugR promoter-lacZ fusion in the ramA mutant background was undetectable (data not shown).…”

“…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 ramA gene is upregulated by an unknown factor in the presence of acetate (43).…”

“…Multiple transcription regulators are involved in the expression of these genes. For example, expression of the gapA-pgk-tpi operon, encoding the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and triose phosphate isomerase, respectively, is coordinately regulated by SugR, RamA, and GlxR (15,31,32). SugR is a global repressor of genes for sugar uptake and metabolism, including phosphotransferase systems, glycolysis, and fermentative lactate dehydrogenase (32)(33)(34)(35)(36)(37).…”

“…GlxR, a cAMP-responsive regulator, and RamA, a LuxR-type regulator, were first identified as the transcriptional repressor and activator, respectively, of the aceB gene, encoding malate synthase of the glyoxylate cycle (14,38). Both regulators not only activate gapA expression (15,31) but are also involved in expression of a number of genes for various physiological functions, including carbon and nitrogen metabolism, respiration, SOS and stress responses, and cell division (15,(39)(40)(41). However, in contrast to the mechanism by which SugR switches gene expression, how GlxR and RamA switch expression of genes involved in glucose and acetate metabolism in response to a carbon source provided is not fully understood.…”