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

Abstract

The mRNA level of the aconitase gene acn of Corynebacterium glutamicum is reduced under iron limitation. Here we show that an AraC-type regulator, termed RipA for "regulator of iron proteins A," is involved in this type of regulation. A C. glutamicum ⌬ripA mutant has a 2-fold higher aconitase activity than the wild type under iron limitation, but not under iron excess. Comparison of the mRNA profiles of the ⌬ripA mutant and the wild type revealed that the acn mRNA level was increased in the ⌬ripA mutant under iron limitation, but not under iron excess, indicating a repressor function of RipA. Besides acn, some other genes showed increased mRNA levels in the ⌬ripA mutant under iron starvation (i.e. those encoding succinate dehydrogenase (sdhCAB), nitrate/nitrite transporter and nitrate reductase (narKGHJI), isopropylmalate dehydratase (leuCD), catechol 1,2-dioxygenase (catA), and phosphotransacetylase (pta)). Most of these proteins contain iron. Purified RipA binds to the upstream regions of all operons mentioned above and in addition to that of the catalase gene (katA). Corynebacterium glutamicum is a nonpathogenic, aerobic Grampositive soil bacterium that is used for large scale industrial production of amino acids, predominantly L-glutamate (1.5 million tons/year) and L-lysine (0.7 million tons/year). In addition, C. glutamicum has gained interest as a suitable model organism for the Corynebacterineae, a suborder of the actinomycetes that includes the genus Mycobacterium. An overview on C. glutamicum biology, genetics, physiology, and biotechnology can be found in a recent monograph (1).The citric acid cycle is of central importance for metabolism in general and for amino acid production in particular, because it provides the biosynthetic precursors of the aspartate and glutamate family of amino acids. Despite its key role, knowledge about the genetic regulation of this pathway in C. glutamicum is scarce. We recently could show that the 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). 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. In the ⌬acnR mutant, this decrease was even larger (4.8-fold), presumably because the increased expression of aconitase, which contains a 4Fe-4S cluster, leads to an enhanced iron starvation.We now have identified a new transcriptional regulator, designated RipA, which is responsible for iron-dependent re...

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

“…However, expression of genes encoding enzymes of the TCA cycle and glyoxylate bypass in C. glutamicum has been shown previously to be subject to at least the following five transcriptional regulators: AcnR, a TetR-type repressor of the acn gene ; RipA, a repressor of the acn gene under iron limitation (Wennerhold et al, 2005); RamB, a negative regulator of glyoxylate bypass genes (aceA and aceB) involved in acetate metabolism (Cramer et al, 2006;Gerstmeir et al, 2004); DtxR, a transcriptional regulator of the sdhCAB gene in iron metabolism (Brune et al, 2006); and GlxR, the repressor of the glyoxylate bypass genes (Kim et al, 2004). It is possible that multiple regulators control TCA cycle genes to manifest a complex response to the environment.…”

The Bacillus subtilis iron-sparing response is mediated by a Fur-regulated small RNA and three small, basic proteins

“…Aconitase contains a 4Fe-4S cluster and is a known target of iron-sparing responses in C. glutamicum (mediated by the RipA protein, ref. 23) and in S. cerevisiae (mediated by Cth2p; 22). It is interesting to note that B. subtilis aconitase, like its mammalian counterparts that function as iron-regulatory element binding proteins (IRE-BP), has an iron-regulated RNA-binding activity (39).…”

“…Ironsparing responses are also likely to be present in Gram-positive bacteria. In Corynebacterium spp., the iron sensor DtxR represses the RipA protein, which, in turn, is a repressor of iron-using proteins (23). Analysis of the S. aureus proteome as a function of iron availability also suggests the presence of an iron-sparing response, but the mechanism is not yet known (24).…”

Regulation of ldh expression during biotin-limited growth of Corynebacterium glutamicum

“…To our knowledge, only iron (Wennerhold et al, 2005), phosphate (Ishige et al, 2003), ammonium (Silberbach et al, 2005a) and nitrogen (Silberbach et al, 2005b) starvation, and glucose limitation (CocaignBousquet et al, 1996), have been investigated. The effects of biotin limitation on C. glutamicum metabolism have not been studied to date, even though biotin was postulated to be essentially supplied by plant roots and then limiting in soils (Rovira & Harris, 1961).…”