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 DeltaripA 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 DeltaripA mutant and the wild type revealed that the acn mRNA level was increased in the DeltaripA 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 DeltaripA 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). From 13 identified binding sites, the RipA consensus binding motif RRGCGN(4)RYGAC was deduced. Expression of ripA itself is repressed under iron excess by DtxR, since purified DtxR binds to a well conserved binding site upstream of ripA. Thus, repression of acn and the other target genes indicated above under iron limitation involves a regulatory cascade of two repressors, DtxR and its target RipA. The modulation of the intracellular iron usage by RipA supplements mechanisms for iron acquisition that are directly regulated by DtxR.
Highlights
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
A candidate gene that might be responsible for iron-dependent regulation of acn was identified in the DNA microarray experiments used to compare the gene expression profile of C. glutamicum under iron excess and iron limitation
Its mRNA level was always found to be increased under iron-limiting conditions, and it behaved like typical iron starvation genes
Summary
Bacterial Strains, Media, and Growth Conditions—All strains and plasmids used in this work are listed in supplemental Table S1. The purified PCR product was cloned in the modified expression vector pET28b-Streptag [14], resulting in plasmid pET28bStreptag-ripA. Overproduction and Purification of RipA—E. coli BL21(DE3) carrying the plasmid pET28b-strep-ripA was grown at 30 °C in 200 ml of LB medium with 50 g/ml kanamycin to an A600 of ϳ1.2 before adding 1 mM isopropyl -D-thiogalactoside. After cultivation for another 4 h, cells were harvested by centrifugation, washed once, and stored at Ϫ20 °C. All PCR products used in the gel shift assays were purified with the PCR purification Kit (Qiagen, Hilden, Germany) and eluted in EB buffer (10 mM Tris/HCl, pH 8.5)
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