ppGpp regulation of RpoS degradation via anti-adaptor protein IraP

Abstract

IraP is a small protein that interferes with the delivery of S (RpoS) to the ClpXP protease by blocking the action of RssB, an adaptor protein for S degradation. IraP was previously shown to mediate stabilization of S during phosphate starvation. Here, we show that iraP is transcribed in response to phosphate starvation; this response is mediated by ppGpp. The iraP promoter is positively regulated by ppGpp, dependent on the discriminator region of the iraP promoter. Sensing of phosphate starvation requires SpoT but not RelA. The results demonstrate a target for positive regulation by ppGpp and suggest that the cell use of ppGpp to mediate a variety of starvation responses operates in part by modulating S levels.phosphate starvation ͉ 38 ͉ SpoT B acteria have evolved sophisticated mechanisms to recognize and respond to a variety of environmental stresses. In Escherichia coli, S , the gene product of rpoS, is the factor of the general stress response and adaptation to stationary phase (1, 2). S controls the expression of Ϸ100 genes involved in functions that help cells cope with various kinds of stress. S amounts and activity are tightly regulated (3-5).In exponentially growing cells, the active proteolysis of S is crucial to maintain a low intracellular concentration of this factor (6, 7). S becomes stable as cells are starved or are exposed to certain stresses (reviewed in ref. 4). S protein turnover requires the energy-dependent protease, ClpXP, and the adaptor protein, RssB (SprE), which binds directly to S and targets it to ClpXP (8). RssB has an N-terminal domain of the response regulator family, with a conserved site for phosphorylation. Recently, the identification of an anti-adaptor protein, IraP, provided an explanation for how proteolysis of S can be regulated. In vivo, S is stabilized after phosphate starvation in a manner that depends on IraP and that is independent of RssB phosphorylation. In vitro, IraP inhibits S proteolysis through a direct interaction with RssB (9). Although the inhibitory effect of IraP on S degradation has been observed in response to phosphate starvation, how phosphate starvation is sensed and how this signal affects IraP expression and/or activity has not been described previously. We show in this work that iraP mRNA accumulates in phosphate-starved cells, dependent on the alarmone ppGpp, the global regulator of the stringent response. This induction requires the discriminator sequence of the iraP promoter. Two proteins in E. coli, RelA and SpoT, are capable of synthesizing ppGpp (10, 11); we find that SpoT, but not RelA, is essential for the induction of iraP expression in response to phosphate starvation. The positive regulation of iraP transcription by ppGpp leads to regulation of S proteolysis.Results iraP mRNA Accumulates in Response to Phosphate Starvation. Previous work showed that IraP stabilizes the transcription factor S under phosphate starvation (9). We analyzed by Northern blot the levels of iraP mRNA before and after removal of phosphate. Fig. 1A shows that iraP...

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

“…RpoS was stabilized via two anti-adaptor proteins, IraP and IraD. Induction of these anti-adaptors is in part likely due to increased levels of ppGpp, a positive regulator of both the iraP and iraD promoters (3,5). However, even in the absence of ppGpp (Fig.…”

“…2B). Both iraP and iraD are positively regulated by ppGpp (3,5), suggesting that an increase in this alarmone might explain the induction of these anti-adaptors. Direct measurement of ppGpp in the aceE host was not possible because the strain grew very poorly in the 3-(N-morpholino)propansulfonic acid medium needed for the experiment.…”

“…These proteins, called anti-adaptor proteins, are each induced under a different stress condition (2). IraP, induced via an increase in ppGpp (3), is necessary for stabilization of RpoS during low phosphate growth. IraM, induced via activation of the PhoQ/PhoP two-component system, is similarly critical for RpoS stabilization during low Mg 2+ growth (2).…”

Translation quality control is critical for bacterial responses to amino acid stress

“…These stressresponse genes are direct targets of ppGpp-regulated transcription and are responsible for the expression of rRNAs and tRNAs and RpoS accumulation, respectively (26,27). Expression of iraP has been shown to increase in response to increased ppGpp levels, whereas expression from the rrsB P1 promoter decreases during the stringent response (27)(28)(29). When normalized to Fig.…”

Unfolded protein responses in bacteria and mitochondria: A central role for the ClpXP machine

“…In this case, a specific adaptor protein, regulator of Sigma S protein B (RssB), is required for its ClpXP-mediated degradation (31,32). Under stress conditions, the RssB-mediated delivery of SigmaS is prevented by specific inhibitors of RssB (known as antiadaptors), thereby blocking ClpXP-mediated degradation of SigmaS (33)(34)(35), which results in the transcriptional upregulation of the SigmaS regulon. Here, ClpXP acts as negative regulator of the response, whereas in the envelope stress response (see below) ClpXP acts as a positive regulator, activating transcription of the SigmaE regulon.…”