ideR , an Essential Gene in Mycobacterium tuberculosis : Role of IdeR in Iron-Dependent Gene Expression, Iron Metabolism, and Oxidative Stress Response

Abstract

The mycobacterial IdeR protein is a metal-dependent regulator of the DtxR (diphtheria toxin repressor) family. In the presence of iron, it binds to a specific DNA sequence in the promoter regions of the genes that it regulates, thus controlling their transcription. In this study, we provide evidence that ideR is an essential gene in Mycobacterium tuberculosis. ideR cannot normally be disrupted in this mycobacterium in the absence of a second functional copy of the gene. However, a rare ideR mutant was obtained in which the lethal effects of ideR inactivation were alleviated by a second-site suppressor mutation and which exhibited restricted iron assimilation capacity. Studies of this strain and a derivative in which IdeR expression was restored allowed us to identify phenotypic effects resulting from ideR inactivation. Using DNA microarrays, the iron-dependent transcriptional profiles of the wild-type, ideR mutant, and ideR-complemented mutant strains were analyzed, and the genes regulated by iron and IdeR were identified. These genes encode a variety of proteins, including putative transporters, proteins involved in siderophore synthesis and iron storage, members of the PE/PPE family, a membrane protein involved in virulence, transcriptional regulators, and enzymes involved in lipid metabolism.

Alternative activation deprives macrophages of a coordinated defense program to Mycobacterium tuberculosis

“…To monitor the status of iron starvation of M. tuberculosis within alternatively activated macrophages, expression levels of marker transcripts encoding proteins involved in mycobactin synthesis (mbtB, Rv2383c; mbtJ, Rv2385; mbtI, Rv2386c) as well as the iron storage protein bacterioferritin (bfrB, Rv3841) were determined by qPCR. During in vitro growth of the bacteria, mycobactin synthesis genes are slightly induced by iron limitation, whereas bacterioferritin expression is repressed [ [51][52][53]. RNA was isolated from M. tuberculosis inside macrophages activated with IL-4 and IFN-c, respectively, as well as resting reference macrophages, 48 h p.i.…”

Lipidomics reveals control of Mycobacterium tuberculosis virulence lipids via metabolic coupling

“…RNA was harvested from log phase grown cells as described (32) and reverse transcribed to generate cDNA. Dye-conjugated cDNA samples were competitively hybridized on microarrays containing oligonucleotide spots representing every gene in M. tuberculosis (Qiagen, Valencia, CA).…”

Neutrophil-mediated innate immune resistance to mycobacteria

“…These media were subsequently adjusted with ferric ammonium citrate to attain specified iron concentrations. Iron-depleted stocks of MTB-lux were grown to log phase in broth containing 2 μM iron (a concentration of iron low enough to induce bacillary siderophore production) (35) and frozen in 15% glycerol. For assays of HNP1-3 and LL-37 activity, inocula of 6 × 10 5 CFU/ml were cultured for 168 hours in medium containing HNP1-3 isolated from human blood (PANATecs) or synthetic LL-37 (PANATecs) at a 10-nM final iron concentration (the concentration of free iron estimated to exist in the phagolysosome) (10).…”