A manually curated compendium of expression profiles for the microbial cell factory Corynebacterium glutamicum.

Corynebacterium glutamicum is the major host for the industrial production of amino acids and has become one of the best studied model organisms in microbial biotechnology. Rational strain construction has led to an improvement of producer strains and to a variety of novel producer strains with a broad substrate and product spectrum. A key factor for the success of these approaches is detailed knowledge of transcriptional regulation in C. glutamicum. Here, we present a large compendium of 927 manually curated microarray-based transcriptional profiles for wild-type and engineered strains detecting genome-wide expression changes of the 3,047 annotated genes in response to various environmental conditions or in response to genetic modifications. The replicates within the 927 experiments were combined to 304 microarray sets ordered into six categories that were used for differential gene expression analysis. Hierarchical clustering confirmed that no outliers were present in the sets. The compendium provides a valuable resource for future fundamental and applied research with C. glutamicum and contributes to a systemic understanding of this microbial cell factory. Measurement(s) Gene Expression Analysis Technology Type(s) Two Color Microarray Factor Type(s) WT condition A vs. WT condition B • Plasmid-based gene overexpression in parental strain vs. parental strain with empty vector control • Deletion mutant vs. parental strain Sample Characteristic - Organism Corynebacterium glutamicum Sample Characteristic - Environment laboratory environment Sample Characteristic - Location Germany.

RamA and RamB are global transcriptional regulators in Corynebacterium glutamicum and control genes for enzymes of the central metabolism.

In Corynebacterium glutamicum, the transcriptional regulators of acetate metabolism RamA (encoded by cg2831) and RamB (encoded by cg0444) play an important role in expression control of genes involved in acetate and ethanol metabolism. Both regulators were speculated to have broader significance in expression control of further genes in the central metabolism of C. glutamicum. Here we investigated the RamA and RamB regulons by genome-wide transcriptome analysis with special emphasis on genes encoding enzymes of the central carbon metabolism. When compared to the parental wild-type, 253 genes and 81 genes showed different mRNA levels in defined RamA- and RamB-deficient C. glutamicum strains, respectively. Among these were genes involved in sugar uptake, glycolysis, gluconeogenesis, acetate, l-lactate or ethanol metabolism. The direct interaction of RamA and RamB proteins with the respective promoter/operator fragments was demonstrated in vitro by electrophoretic mobility shift assays. Taken together, we present evidence for an important role of RamA and RamB in global gene expression control in C. glutamicum.

RamB is an activator of the pyruvate dehydrogenase complex subunit E1p gene in Corynebacterium glutamicum.

In Corynebacterium glutamicum, the transcriptional regulator RamB negatively controls the expression of genes involved in acetate metabolism. Here we show that during growth in media containing glucose and in complex medium without glucose RamB activates expression of the aceE gene, encoding the E1p subunit of the pyruvate dehydrogenase complex. Thus, RamB functions both as repressor and as activator in C. glutamicum.

RamA, the transcriptional regulator of acetate metabolism in Corynebacterium glutamicum, is subject to negative autoregulation.

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.

RamB, the transcriptional regulator of acetate metabolism in Corynebacterium glutamicum, is subject to regulation by RamA and RamB.

In Corynebacterium glutamicum, the transcriptional regulator RamB negatively controls the expression of genes involved in acetate metabolism. Here we show that RamB represses its own expression by direct interaction with a 13-bp motif in the ramB promoter region. Additionally, ramB expression is subject to carbon source-dependent positive control by RamA.

Identification of RamA, a novel LuxR-type transcriptional regulator of genes involved in acetate metabolism of Corynebacterium glutamicum.

In Corynebacterium glutamicum, the acetate-activating enzymes phosphotransacetylase and acetate kinase and the glyoxylate cycle enzymes isocitrate lyase and malate synthase are coordinately up-regulated in the presence of acetate in the growth medium. This regulation is due to transcriptional control of the respective pta-ack operon and the aceA and aceB genes, brought about at least partly by the action of the negative transcriptional regulator RamB. Using cell extracts of C. glutamicum and employing DNA affinity chromatography, mass spectrometry, and peptide mass fingerprinting, we identified a LuxR-type transcriptional regulator, designated RamA, which binds to the pta-ack and aceA/aceB promoter regions. Inactivation of the ramA gene in the genome of C. glutamicum resulted in mutant RG2. This mutant was unable to grow on acetate as the sole carbon and energy source and, in comparison to the wild type of C. glutamicum, showed very low specific activities of phosphotransacetylase, acetate kinase, isocitrate lyase, and malate synthase, irrespective of the presence of acetate in the medium. Comparative transcriptional cat fusion experiments revealed that this deregulation takes place at the level of transcription. By electrophoretic mobility shift analysis, purified His-tagged RamA protein was shown to bind specifically to the pta-ack and the aceA/aceB promoter regions, and deletion and mutation studies revealed in both regions two binding motifs each consisting of tandem A/C/TG4-6T/C or AC4-5A/G/T stretches separated by four or five arbitrary nucleotides. Our data indicate that RamA represents a novel LuxR-type transcriptional activator of genes involved in acetate metabolism of C. glutamicum.

RamB, a novel transcriptional regulator of genes involved in acetate metabolism of Corynebacterium glutamicum.

The adaptation of Corynebacterium glutamicum to acetate as a carbon and energy source involves transcriptional regulation of the pta-ack operon coding for the acetate-activating enzymes phosphotransacetylase and acetate kinase and of the aceA and aceB genes coding for the glyoxylate cycle enzymes isocitrate lyase and malate synthase, respectively. Deletion and mutation analysis of the respective promoter regions led to the identification of highly conserved 13-bp motifs (AA/GAACTTTGCAAA) as cis-regulatory elements for expression of the pta-ack operon and the aceA and aceB genes. By use of DNA affinity chromatography, a 53-kDa protein specifically binding to the promoter/operator region of the pta-ack operon was purified. Mass spectrometry and peptide mass fingerprinting identified the protein as a putative transcriptional regulator (which was designated RamB). Purified His-tagged RamB protein was shown to bind specifically to both the pta-ack and the aceA/aceB promoter/operator regions. Directed deletion of the ramB gene in the genome of C. glutamicum resulted in mutant strain RG1. Whereas the wild type of C. glutamicum showed high-level specific activities of acetate kinase, phosphotransacetylase, isocitrate lyase, and malate synthase when grown on acetate and low-level specific activities when grown on glucose as sole carbon and energy sources, mutant RG1 showed high-level specific activities with all four enzymes irrespective of the substrate. Comparative transcriptional cat fusion experiments revealed that this deregulation takes place at the level of transcription. The results indicate that RamB is a negative transcriptional regulator of genes involved in acetate metabolism of C. glutamicum.