Fig. 1.

Effects of manganese on promoter occupancy and expression of Irr-regulated genes. Cells were grown in media supplemented with 20 μM FeCl₃ (Fe) or 50 μM MnCl₂ (Mn) (+) or not supplemented with metal (−). The actual Fe and Mn concentrations in the unsupplemented media were 0.3 μM and 0.4 μM, respectively. (A) Steady state levels of Irr were detected by immunoblotting using anti-Irr antibodies. GroEL was used as a control for an unregulated protein, and was detected using anti-GroEL antibodies. Fifteen micrograms of protein was loaded per lane. (B–G) In vivo promoter occupancy of the respective genes in cells grown under different metal conditions was measured by crosslinking and immunoprecipitation using anti-Irr antibodies as described in Materials and Methods. The precipitated DNA was analyzed by qPCR using primers delimiting the promoter regions of the respective genes. The data are expressed as the relative starting quantities (SQ) of immunoprecipitated DNA normalized to the input DNA and are presented as the average of three replicates ± the standard deviation. (H–M) Steady-state transcript levels of the respective genes obtained from cells grown under different metal conditions were analyzed by qPCR. The data are expressed as relative starting quantities (SQ) of respective mRNAs normalized to the housekeeping gene gapA, and presented as average of three replicates ± the standard deviation.