A DNA damage response in Escherichia coli involving the alternative sigma factor, RpoS

Abstract

We isolated an Escherichia coli mutant in the iraD gene, sensitive to various forms of DNA damage. Our data are consistent with the function of IraD to promote accumulation of the alternative transcription sigma factor, RpoS, by binding to the adaptor RssB protein that targets RpoS for degradation. Our results demonstrate the physiological importance of this mode of regulation for DNA damage tolerance. Although RpoS is best known for its regulation of genes induced in stationary phase, our work underscores the importance of the RpoS regulon in a DNA damage response in actively growing cells. We show that iraD transcription is induced by DNA damage by a mechanism independent of the SOS response. The IraD and SOS regulatory pathways appear to act synergistically to ensure survival of cells faced with oxidative or DNA damaging stress during cellular growth.

Fig. 1.

Complementation of iraD with low-copy plasmid-encoded iraD. Plating efficiency of strains after 15-min exposure to 12.5 mM H₂O₂. Each point represents an independent determination. Strains denoted (+) carry the vector with iraD⁺ and its upstream noncoding region, whereas those denoted (−) carry only the upstream noncoding sequences. wt, wild type.

Fig. 2.

Plating efficiency with chronic AZT exposure. Squares represent WT, triangles represent iraDΔ, circles represent rpoSΔ, diamonds represent rssBΔ, and squares with X through represent rssBΔ iraDΔ. Strains in the left panel carry an additional mutation in hsdR, used as a linked selective marker in certain iraD strain constructions, which does not affect survival in these assays. Strains in the right panel are hsdR⁺.

Fig. 3.

RpoS levels and stability in iraD mutants by RpoS Western blot analysis. (A) Western blot of steady-state RpoS levels during early log phase in the iraD mutants versus WT. (B) RpoS stability following chloramphenicol treatment to block new protein synthesis either with (+) or without (−) 5 mM hydrogen peroxide treatment for 10 min. Levels of RpoS are shown at the times indicated after chloramphenicol treatment.

Fig. 4.

Expression of iraD, recA, and dinB reporter constructs in response to DNA damage. (A) Expression of iraD, recA, and dinB luxCDABE reporters in response to H₂O (Ctrl), AZT, H₂O₂, phleomycin (Phleo), and mitomycin C (MMC) for at least 4 isolates. (B) Expression of iraD::luxCDABE in response to H₂O (Ctrl), AZT, H₂O₂, and Phleo for at least 3 isolates in WT, recAΔ, or lexA3 background. RLU, relative luminescence unit (bioluminescence cpm normalized to OD₆₀₀).

Fig. 5.

Plating efficiency under chronic AZT exposure. WT (open squares), iraDΔ (filled squares), lexA3 (open triangles), iraDΔ lexA3 double mutant (filled triangles), recAΔ (open circles), and iraDΔ recAΔ double mutant (filled circles).