FIGURE 2.

Effects of manganese and iron on mntH and irr expression in the wild type and fur mutant, and on Fur occupancy of the mntH and irr promoters in those cells. A and B, steady state mRNA levels of the mntH or irr genes were analyzed by quantitative real-time PCR from cells grown in media supplemented with (+) or without (−) 50 μm MnCl₂ and with (+) or without (−) 20 μm FeCl₃. The data are expressed as the SQ of the respective mRNAs normalized (norm) to the housekeeping gene gapA and presented as the average triplicate samples plus S.D. C and D, Fur occupancy of the mntH or irr promoter was carried out by cross-linking of cells grown under the iron and manganese conditions described for the qPCR experiments, followed by immunoprecipitation using anti-Fur antibodies or a mock control lacking the primary antibody. Co-immunoprecipitated DNA was quantified utilizing qPCR with primers used to amplify the promoter regions of irr and mntH. The data are expressed as the SQ of immunoprecipitated DNA normalized to the mock pull down. E, Western blot analysis of Fur protein levels in cells of the wild type (Wt) or fur mutant grown under the iron and manganese conditions described above. 50 μg of protein was loaded per lane.