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.oxidative stress ͉ posttranslational regulation ͉ replication stress ͉ SOS response ͉ DNA repair T hroughout its life cycle, Escherichia coli is faced with different environmental challenges and regulates gene expression accordingly. One way is by changes in the promoter recognition of RNA polymerase via different situation-specific factors (1). In E. coli, the major alternative sigma factor is S (RpoS), which is required for expression of specific genes on entry to stationary phase or as a response to stress (2-4). Although the RpoS dependence of many of these responses and the regulation of RpoS itself have been well studied, the relevance of this to DNA repair has not been a major focus.To find genes important in DNA damage responses, we performed a random Tn5 transposon insertion mutant screen, assaying sensitivity to, among other agents, phleomycin and azidothymidine (AZT). Phleomycin induces random single-or double-strand breaks in the backbone of DNA (5), whereas AZT blocks DNA synthesis, leading to single-strand gaps in the replication fork (6). One insertion mutant in iraD (previously an unknown gene, yjiD) was hypersensitive to phleomycin and AZT.Recent work from Gottesman and coworkers (7) implicated IraD in posttranslational regulation of RpoS. The RssB adaptor protein targets RpoS to ClpXP for degradation during logarithmic growth, keeping RpoS protein levels low in the absence of stress (8-12). IraD was identified in a high-copy plasmid screen for genes promoting accumulation of an RpoS-LacZ fusion protein. The IraD gene product acts as an antiadaptor protein via direct binding and inhibition of the ability of RssB to target RpoS for proteolysis by ClpXP in vitro (7).In the work presented here, we demonstrate that IraD is required for survival to DNA damage, providing evidence of the physiological importance of IraD in particular and the antiadaptor mechanism in general. The data presented suggest that IraD acts as an antagonist of RssB, regulating RpoS levels and stabilization, not only after DNA damage but constitutively. Our results establish the importance of RpoS stabilization in proliferating bacterial cells in which replication has been direc...

Stress sigma factor RpoS degradation and translation are sensitive to the state of central metabolism

“…IraP and IraD are normally present in the cell at low levels; their synthesis increases enough to stabilize RpoS only under specific stress conditions (2,4,18). The fact that IraP and IraD stabilize RpoS in an aceE::kan mutant suggests that their expression is induced in this background.…”

“…IraM, induced via activation of the PhoQ/PhoP two-component system, is similarly critical for RpoS stabilization during low Mg 2+ growth (2). IraD induction is not fully understood, but it clearly plays a role both in the transition to stationary phase, via induction of a ppGpp-dependent promoter, and after DNA damage (4,5). IraM and IraD are also negatively regulated by H-NS (6).…”

RpoS proteolysis is controlled directly by ATP levels in Escherichia coli

“…Accordingly, we conclude that phosphorylation of SprE is not integral to the carbon starvation signaling pathway that regulates RpoS. SprE was recently found to regulate mRNA stability (Carabetta et al 2009), and it is not known if phosphorylation plays a role in that activity.In response to other stresses, such as phosphate starvation, magnesium starvation, and DNA damage, a major mechanism for RpoS accumulation is antagonism of the adaptor SprE by the anti-adaptor proteins IraP, IraM, and IraD, respectively (Bougdour et al 2006(Bougdour et al , 2008Merrikh et al 2009). However, none of the Ira proteins have been associated with carbon starvation, and despite extensive screens, no other proteins have been identified in the carbon starvation signaling pathway.…”

“…In response to other stresses, such as phosphate starvation, magnesium starvation, and DNA damage, a major mechanism for RpoS accumulation is antagonism of the adaptor SprE by the anti-adaptor proteins IraP, IraM, and IraD, respectively (Bougdour et al 2006(Bougdour et al , 2008Merrikh et al 2009). However, none of the Ira proteins have been associated with carbon starvation, and despite extensive screens, no other proteins have been identified in the carbon starvation signaling pathway.…”

Enhanced Promoter Activity by Replenishment of Sigma Factor rpoE in Klebsiella pneumoniae

“…It was reported that an engineered sigma factor library could reprogram metabolic flux and thus led to diverse phenotypes, and some of mutant strains could be used for industrial production purposes [12]. Additionally, sigma factors were shown to be associated with the cell survival under stress conditions [13][14][15]. Based on above information, we speculated that sigma factors may affect promoter activity.…”