| Iron is an essential nutrient, but can be toxic at high concentrations as it can induce oxidative stress. Therefore, intracellular iron levels must be tightly regulated. The Irr protein in the bacterium Bradyrhizobium japonicum is a conditionally stable transcriptional regulator that accumulates under iron-deplete, manganese-replete conditions, but degrades in a heme-dependent manner under iron-replete, manganese-deplete conditions, or upon exposure to H2O2. Irr controls the expression of a large cohort of genes, protein products of which are involved in a variety of cellular functions. It seemed plausible that Irr-dependent gene regulation may require additional levels of control than those possible by heme-dependent degradation alone. Here, we identified Irr regulon genes that were unresponsive to factors that promote Irr degradation. The promoters of those genes bound Irr with 200-fold greater affinity than promoters of responsive genes, resulting in maintenance of promoter occupancy over a wide range of cellular Irr concentrations.;For Irr-repressible genes, the promoter occupancy correlated with transcriptional repression, resulting in differential levels of expression based on Irr affinity for the target promoters. The genes that were less responsive to changes in Irr encoded protein involved in iron export and iron storage, which were required specifically under iron stress conditions. The genes that were responsive to changes in Irr encoded housekeeping proteins. Based on these findings, we suggest that the wide range of affinities Irr has for different promoters allows for differential derepression of housekeeping genes and stress-response genes, based on the prevailing metabolic state of the cell.;Inactivation of positively-controlled genes under manganese-limitation, in the presence of iron, or following exposure to H2O2 required neither promoter vacancy nor loss of Irr DNA-binding activity. Under these conditions, Irr still maintained repressor activity. Thus, activation and repression of Irr can be uncoupled from each other under certain conditions. Abrogation of Irr activation function was heme-dependent, thus heme modulates Irr activity by degradation and inactivation. Most of the Irr-activated genes encode iron- and heme-uptake proteins. We suggest that under conditions of oxidative stress, by uncoupling activation and repression functions, Irr prevents iron uptake while still repressing the transcription of iron-utilizing proteins. |
