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.
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 ptaack 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/TG 4-6 T/C or AC 4-5 A/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.Corynebacterium glutamicum is a nonpathogenic, aerobic grampositive soil bacterium that is widely used for the large-scale production of amino acids such as L-glutamate and L-lysine (21,24,28,30,35). In addition, the organism has gained increasing interest as a suitable model organism for the Corynebacterineae, a suborder of the actinomycetes which also includes the medically important genus Mycobacterium (51).C. glutamicum is able to grow on a variety of carbohydrates and organic acids as single or combined sources of carbon and energy, and among these substrates are glucose and acetate (31, 34). Based on biochemical, genetic, and regulatory studies; on quantitative determination of metabolic fluxes during utilization of acetate and/or glucose; and on genome-wide comparative expression analyses, there is considerable knowledge of enzymes and genes involved in acetate metabolism of C. glutamicum (reviewed in references 9 and 17). The utilization of acetate involves its uptake and subsequent activation to acetyl coenzyme A (CoA) and, when acetate is the sole carbon substrate, also requires the operation of the glyoxylate cycle as anaplerotic pathway. The key enzymes of acetate activation, acetate kinase (AK) and phosphotransacetylase (PTA), and those of the glyoxylate cycle, isocitrate lyase (ICL) and malate synthase (MS)...
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.
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.Corynebacterium glutamicum is an aerobic gram-positive soil bacterium well known for its use in large-scale biotechnological production of L-glutamate and lysine (10-14). We are interested in acetate metabolism and its regulation in this organism (7), and we recently identified and characterized two regulatory proteins, designated regulator of acetate metabolism A and B (RamA and RamB, respectively) from C. glutamicum ATCC 13032 (4, 6). RamA binds to single or tandem stretches of A/C/TG 4-6 T/C or AC 4-5 A/G/T and thereby acts as an activator of the pta, ack, aceA, and aceB genes, encoding phosphotransacetylase, acetate kinase, isocitrate lyase, and malate synthase, all involved in acetate metabolism of C. glutamicum. RamA was also shown to activate transcription of the surface layer protein gene (cspB) in C. glutamicum ATCC 14067 (9). Furthermore, RamA was shown to bind to the promoter region of its own gene and to act as a repressor there (3). The RamB protein specifically binds to a highly conserved 13-bp motif (AA/GAACTTTGCAAA) and represents a repressor of the pta, ack, aceA, and aceB genes when C. glutamicum is grown on glucose as a carbon and energy source (6). Inspection of the ramB promoter region revealed the presence of putative RamA and RamB binding sites, and this observation prompted us to study expression of ramB in cells growing in media containing glucose and/or acetate and to test for a regulatory function of RamA and RamB in ramB expression.The bacterial strains and the plasmids, their relevant characteristics and sources, and the oligonucleotides used in this study are given in Table 1. The media used and the methods not outlined explicitly (DNA preparation, promoter binding assays with hexahistidyl-tagged RamA and RamB fusion proteins, enzyme assays, RNA preparation and identification of the transcriptional start site by the RACE [rapid amplification of cDNA ends] method, generation of polyclonal antibodies, and Western blotting) were described previously (3, 4, 6).Western blot experiments with RamB-specific antibodies and crude extracts from C. glutamicum wild-type (WT) cells grown in minimal medium containing glucose, glucose plus acetate, and acetate showed that RamB is present in cells grown on either carbon source (Fig. 1). In agreement with the results of DNA affinity chromatography experiments (4), the largest amounts of RamB were observed in the cells grown on glucose alone.To test for ramB promoter activities in C. glutamicum cells grown in minimal media with glucose and/or acetate, we constructed reporter gene fusions by cloning a 593-bp ramB promoter fragment (covering the region 521 bp upstream to 72 bp downstream of the ramB sta...
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