Abstract
The transcription factor RpoS (σS) of Escherichia coli controls many genes important for tolerance of a variety of stress conditions. IraD promotes the post-translation stability of RpoS by inhibition of RssB, an adaptor protein for ClpXP degradation. We have previously documented DNA damage induction of iraD expression, independent of the SOS response. Both iraD and rpoS are required for tolerance to DNA damaging treatments such as H2O2 and the replication inhibitor azidothymidine (AZT) in the log phase of growth. Using luciferase gene fusions to the 672 bp iraD upstream region, we show here that both promoters of iraD are induced by AZT. Genetic analysis suggests that both promoters are repressed by DnaA-ATP, partially dependent on a putative DnaA box at -81 bp and are regulated by RIDA (regulatory inactivation of DnaA), dependent on the DnaN processivity clamp. By electrophoretic mobility shift assays we show that purified DnaA protein binds to the iraD upstream region, so DnaA regulation of IraD is likely to be direct. DNA damage induction of iraD during log phase growth is abolished in the dnaA-T174P mutant, suggesting that DNA damage, in some way, relieves DnaA repression, possibly through the accumulation of replication clamps and enhanced RIDA. We also demonstrate that the RNA-polymerase associated factor, SspA (stringent starvation protein A), induced by the accumulation of ppGpp, also affects iraD expression, with a positive effect on constitutive expression and a negative effect on AZT-induced expression.