Identification of AcnR, a TetR-type Repressor of the Aconitase Gene acn in Corynebacterium glutamicum

Abstract

In Corynebacterium glutamicum, the activity of aconitase is 2.5-4-fold higher on propionate, citrate, or acetate than on glucose. Here we show that this variation is caused by transcriptional regulation. In search for putative regulators, a gene (acnR) encoding a TetR-type transcriptional regulator was found to be encoded immediately downstream of the aconitase gene (acn) in C. glutamicum. Deletion of the acnR gene led to a 5-fold increased acn-mRNA level and a 5-fold increased aconitase activity, suggesting that AcnR functions as repressor of acn expression. DNA microarray analyses indicated that acn is the primary target gene of AcnR in the C. glutamicum genome. Purified AcnR was shown to be a homodimer, which binds to the acn promoter in the region from ؊11 to ؊28 relative to the transcription start. It thus presumably acts by interfering with the binding of RNA polymerase. The acn-acnR organization is conserved in all corynebacteria and mycobacteria with known genome sequence and a putative AcnR consensus binding motif (CAGNACnnncGTACTG) was identified in the corresponding acn upstream regions. Mutations within this motif inhibited AcnR binding. Because the activities of citrate synthase and isocitrate dehydrogenase were previously reported not to be increased during growth on acetate, our data indicate that aconitase is a major control point of tricarboxylic acid cycle activity in C. glutamicum, and they identify AcnR as the first transcriptional regulator of a tricarboxylic acid cycle gene in the Corynebacterianeae.Corynebacterium glutamicum is a non-pathogenic, aerobic Gram-positive soil bacterium that was described in 1957 (1) as an L-glutamate-excreting bacterium. It has gained considerable interest because of its use in the large scale biotechnological production of L-glutamate (Ͼ1 million tons per year) and Llysine (ϳ0.6 million tons per year) (2-5) and because of its emerging role as a model organism for the Corynebacterineae, a suborder of the actinomycetes, which also includes the genus Mycobacterium (6).The tricarboxylic acid cycle is of central importance for the metabolism of C. glutamicum because it provides energy and biosynthetic precursors and therefore the flux through this cycle is an important aspect for the production of amino acids of the aspartate and glutamate family. Thus, it is not surprising that several tricarboxylic acid cycle enzymes of C. glutamicum have been studied biochemically and/or genetically in the past, No studies have been performed yet in C. glutamicum on aconitase (EC 4.2.1.3), which catalyzes the stereospecific and reversible isomerization of citrate to isocitrate via cis-aconitate in the tricarboxylic acid cycle and in the glyoxylate cycle. It is an unusual enzyme in that it contains a [4Fe-4S] cluster, which is not involved in electron transfer, but in binding of the substrate (13). Besides their catalytic function, a certain class of aconitases can also have a regulatory function by binding to certain mRNAs and inhibiting or increasing their translation into pro...

Effect of carbon source availability and growth phase on expression of Corynebacterium glutamicum genes involved in the tricarboxylic acid cycle and glyoxylate bypass

“…The glyoxylate bypass genes are transcriptionally regulated by a dual mechanism with GlxR and RamB (Gerstmeir et al, 2004;Kim et al, 2004). Krug et al (2005) reported that the increased aconitase activity of cells grown on acetate is partially because of decreased repression by AcnR and that an additional transcriptional regulator (besides AcnR) or another regulatory mechanism for aconitase might exist. The only regulatory system that has ubiquitous effects on many different genes is catabolite derepression by cAMP receptor protein (Eraso & Gancedo, 1984;Kolb et al, 1993;.…”

“…Recently we have reported the transcription of C. glutamicum genes involved in the TCA cycle and glyoxylate bypass (Han et al, 2008). In fact, aspects of the regulation of only three TCA cycle enzymes in C. glutamicum, citrate synthase (encoded by gltA), aconitase (encoded by acn), and isocitrate dehydrogenase (encoded by icd), have been described (Eikmanns et al, 1994(Eikmanns et al, , 1995Krug et al, 2005).…”

The AraC-type Regulator RipA Represses Aconitase and Other Iron Proteins from Corynebacterium under Iron Limitation and Is Itself Repressed by DtxR

“…The factors that control binding of AcnR to its operator are not yet known. DNA microarray experiments revealed that acn expression is not only influenced by the carbon source but also by the iron concentration of the medium (2). Under iron limitation, the acn mRNA level in the wild type was 3-fold lower than under iron excess.…”

“…A repressor of the TetR family, called AcnR, was identified, which represses aconitase by binding to an imperfect inverted repeat within the acn promoter region and interfering with the binding of RNA polymerase (2). The factors that control binding of AcnR to its operator are not yet known.…”

“…activity of aconitase (EC 4.2.1.3), which catalyzes the stereospecific and reversible isomerization of citrate to isocitrate via cis-aconitate, varies depending on the carbon source and that this is caused by transcriptional regulation (2). A repressor of the TetR family, called AcnR, was identified, which represses aconitase by binding to an imperfect inverted repeat within the acn promoter region and interfering with the binding of RNA polymerase (2).…”