Previous studies with Corynebacterium diphtheriae and Mycobacterium species revealed that the transcriptional regulator DtxR and its ortholog IdeR play a central role in the control of iron metabolism. In the present work, we used genome-based approaches to determine the DtxR regulon of Corynebacterium glutamicum, a nonpathogenic relative of C. diphtheriae. First, global gene expression of a dtxR deletion mutant was compared with that of the wild type using DNA microarrays. Second, we used a computer-based approach to identify 117 putative DtxR binding sites in the C. glutamicum genome. In the third step, 74 of the corresponding genome regions were amplified by PCR, 51 of which were shifted by the DtxR protein. Finally, we analyzed which of the genes preceded by a functional DtxR binding site showed altered mRNA levels in the transcriptome comparison. Fifty-one genes organized in 27 putative operons displayed an increased mRNA level in the ⌬dtxR mutant and thus are presumably repressed by DtxR. The majority of these genes are obviously involved in iron acquisition, three encode transcriptional regulators, e.g., the recently identified repressor of iron proteins RipA, and the others encode proteins of diverse or unknown functions. Thirteen genes showed a decreased mRNA level in the ⌬dtxR mutant and thus might be activated by DtxR. This group included the suf operon, whose products are involved in the formation and repair of iron-sulfur clusters, and several genes for transcriptional regulators. Our results clearly establish DtxR as the master regulator of iron-dependent gene expression in C. glutamicum.Corynebacterium glutamicum is a nonpathogenic, aerobic, gram-positive soil bacterium used for the large-scale biotechnological production of amino acids, mainly L-glutamate (1.5 million tons/year) and L-lysine (0.7 million tons/year). In addition, this species has gained interest as a model organism for the Corynebacterineae, a suborder of the Actinomycetales, which also includes the genus Mycobacterium (34). An overview on the current knowledge on C. glutamicum can be found in a recent monograph (9).Our group has initiated studies on the regulation of C. glutamicum genes and enzymes involved in the citric acid cycle, which is of central importance for metabolism in general and for amino acid production in particular because it provides the precursors of the aspartate and glutamate family of amino acids. We identified and characterized AcnR, a member of the TetR family of transcriptional regulators, which functions as a repressor of the aconitase gene acn (24). In the course of these studies, it became evident that acn expression is controlled by iron in an AcnR-independent manner, being reduced under iron limitation. Subsequently, we were able to show that the iron-dependent transcriptional regulation of aconitase is exerted by the AraCtype regulator RipA, which represses aconitase under iron limitation but not under iron excess (40). RipA stands for "regulator of iron proteins A," and this name was given because ...
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...
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