Genome-Wide Identification of In Vivo Binding Sites of GlxR, a Cyclic AMP Receptor Protein-Type Regulator in Corynebacterium glutamicum

Abstract

Corynebacterium glutamicum GlxR is a cyclic AMP (cAMP) receptor protein-type regulator. Although over 200 GlxR-binding sites in the C. glutamicum genome are predicted in silico, studies on the physiological function of GlxR have been hindered by the severe growth defects of a glxR mutant. This study identified the GlxR regulon by chromatin immunoprecipitation in conjunction with microarray (ChIP-chip) analyses. In total, 209 regions were detected as in vivo GlxR-binding sites. In vitro binding assays and promoter-reporter assays demonstrated that GlxR directly activates expression of genes for aerobic respiration, ATP synthesis, and glycolysis and that it is required for expression of genes for cell separation and mechanosensitive channels. GlxR also directly represses a citrate uptake gene in the presence of citrate. Moreover, ChIP-chip analyses showed that GlxR was still able to interact with its target sites in a mutant with a deletion of cyaB, the sole adenylate cyclase gene in the genome, even though binding affinity was markedly decreased. Thus, GlxR is physiologically functional at the relatively low cAMP levels in the cyaB mutant, allowing the cyaB mutant to grow much better than the glxR mutant.

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

“…A major control system for utilization of carbon sources, namely, carbon catabolite repression, is mediated by the cyclic AMP (cAMP) receptor protein (CRP) via intracellular cAMP levels in E. coli, while in B. subtilis, it is mediated by carbon control protein A (CcpA) via phosphorylation states of HPr, a component of a phosphotransferase system. In contrast to the situation in E. coli, where the phosphotransferase system for glucose uptake modifies adenylate cyclase activity to decrease the intracellular cAMP levels in the presence of glucose (12,13), the C. glutamicum intracellular cAMP levels are increased in the presence of glucose by an unknown mechanism(s) (14)(15)(16). Besides, no HPr kinase/phosphatase system is found in the C. glutamicum genome.…”

“…GlxR was expressed with an N-terminal His tag and purified by affinity chromatography as described previously (15). DNA fragments containing the ramA promoter region with a native or mutated GlxR binding site were amplified by PCR using primers PramAFW and PramARV and promoter-lacZ fusion plasmids, which were constructed as described below, as the templates and cloned into the pGEM-T Easy vector (Promega).…”

“…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.…”

“…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.…”

“…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).…”