Fig. 8.

Effect of H₂O₂ on promoter occupancy by Irr and expression of Irr-regulated genes in heme-deficient strain ΔhemAH grown in iron-limited media. Cultures were grown to mid-log phase in media not supplemented with iron and supplemented with 150 nM hemin to satisfy heme auxotrophy. The actual iron concentration was 0.3 μM. At time 0, 2 mM H₂O₂ was added to the cultures and were continually aerated at 29°C, and harvested at the times shown. (A) Steady-state levels of Irr and GroEL were detected by immunoblotting as described in Fig. 1. (B–G) Promoter occupancy of respective genes by Irr in cells treated with H₂O₂ were carried out as described in the Fig. 1 legend. The data are expressed as the relative starting quantities (SQ) of immunoprecipitated DNA normalized to the input DNA and are presented as average of three replicates ± the standard deviation. (H–M) Steady state transcript levels of the respective genes obtained from cells treated with H₂O₂ 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.