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

Abstract

The central carbon metabolism genes in Corynebacterium glutamicum are under the control of a transcriptional regulatory network composed of several global regulators. It is known that the promoter region of ramA, encoding one of these regulators, interacts with its gene product, RamA, as well as with the two other regulators, GlxR and SugR, in vitro and/or in vivo. Although RamA has been confirmed to repress its own expression, the roles of GlxR and SugR in ramA expression have remained unclear. In this study, we examined the effects of GlxR binding site inactivation on expression of the ramA promoter-lacZ fusion in the genetic background of single and double deletion mutants of sugR and ramA. In the wild-type background, the ramA promoter activity was reduced to undetectable levels by the introduction of mutations into the GlxR binding site but increased by sugR deletion, indicating that GlxR and SugR function as the transcriptional activator and repressor, respectively. The marked repression of ramA promoter activity by the GlxR binding site mutations was largely compensated for by deletions of sugR and/or ramA. Furthermore, ramA promoter activity in the ramA-sugR double mutant was comparable to that in the ramA mutant but was significantly higher than that in the sugR mutant. Taken together, it is likely that the level of ramA expression is dynamically balanced by GlxR-dependent activation and repression by RamA along with SugR in response to perturbation of extracellular and/or intracellular conditions. These findings add multiple regulatory loops to the transcriptional regulatory network model in C. glutamicum.

Identification of two mutations increasing the methanol tolerance of Corynebacterium glutamicum

“…Indeed, induction of these genes was not observed in Tol1 (Additional file 1 : Table S2). The genes ack , pta , aceA are directly repressed by RamB [ 54 , 55 ] and activated by RamA [ 56 , 57 ]. AceA and aceB are directly repressed by GlxR, but regulation of the pta-ack operon by GlxR has not yet been demonstrated in vivo.…”

“…AceA and aceB are directly repressed by GlxR, but regulation of the pta-ack operon by GlxR has not yet been demonstrated in vivo. GlxR and SugR indirectly control these genes by regulation of ramA expression [ 56 ]. C. glutamicum mutants lacking RamA cannot grow with acetate or ethanol as sole carbon sources [ 55 ].…”

The extracytoplasmic function σ factor σC regulates expression of a branched quinol oxidation pathway in Corynebacterium glutamicum

“…TSPs of the σ C regulon were determined by 5′‐RACE analysis as described previously (Toyoda et al ., ). Briefly, total RNA extracted from the sigC ‐overexpressing strain was poly(A)‐tailed.…”

“…To construct a sigC overexpression strain, the coding region of the sigC gene was amplified by PCR using the primers shown in Supporting Information Table S2. The PCR product was cloned into the KpnI site of the IPTG‐inducible vector pCRB12iP, which carries the LacI repressor gene lacI q and the tac promoter (Toyoda et al ., ), yielding pCRC634. sigC overexpression in strains carrying pCRC634 was induced by supplementation with 0.5 mM IPTG.…”

Expanding the Regulatory Network Governed by the Extracytoplasmic Function Sigma Factor σHin Corynebacterium glutamicum

“…5= RACE. For the identification of the transcriptional start points (TSPs), 5= rapid amplification of cDNA ends (RACE) was carried out as described previously (33). Briefly, the total RNA extracted from the wild type after heat treatment was poly(A) tailed.…”