ppGpp regulation of RpoS degradation via anti-adaptor protein IraP

Abstract

IraP is a small protein that interferes with the delivery of sigma(S) (RpoS) to the ClpXP protease by blocking the action of RssB, an adaptor protein for sigma(S) degradation. IraP was previously shown to mediate stabilization of sigma(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 sigma(S) levels.

Fig. 1.

Analysis of iraP expression. Cells were grown at 37°C, and total RNA was isolated as described in Materials and Methods. (A) Northern blot analysis of RNA extracted from exponentially growing cells in Mops glucose in the presence of phosphate (time point 0) and after removal of phosphate at the time points indicated. Strains were as follows: wt (MG1655), AB030 (P_{iraP(−10⁻¹)}), and AB031 (P_{iraP(−10⁻²)}). Quantification of the major iraP mRNA signal (arrowhead) is shown. No signal was detected in the mock experiment (filtration without phosphate starvation; data not shown). (B) Primer extension analysis of iraP mRNA. Primer extension assays were carried out as described in supporting information (SI) Materials and Methods, with total RNA extracted from MG1655 cells grown in exponential phase (lane 1) and stationary phase (lane 2) in LB or before (lane 5) and after 15 min (lane 4) and 30 min (lane 3) of phosphate starvation in Mops glucose. Signals specific for stationary-phase and phosphate-starvation conditions are indicated by arrowheads. (C) Sequence of iraP promoter region in E. coli K12. The 5′ end of iraP mRNA mapped in B is indicated with an arrow. Putative −35 and −10 sequences are underlined. Base substitutions introduced within the promoter region of iraP in strains AB030 (P_{iraP(−10⁻¹)}), AB031 (P_{iraP(−10⁻²)}), AB033 (P_iraP(dis2)), and AB034 (P_{iraP(−10⁻² dis2)}) are indicated in bold. The holoenzyme-σ⁷⁰ consensus sequences are shown. (D) Comparison of σ^S half-lives in AB031 (P_{iraP(−10⁻²)}) and wt (MG1655) strains. The half-life of σ^S was measured in cells grown at 37°C in Mops glucose. Protein synthesis was inhibited with chloramphenicol at OD₆₀₀ of ≈0.3 for σ^S half-life in exponential phase. Samples were removed at specific time points and analyzed by immunoblot with an anti-σ^S antibody. Half-lives (t_1/2) were calculated by regression analysis of the exponential decay of σ^S. (E) Effects of phoB and glucose starvation on iraP expression. Northern blot analysis of RNA extracted from exponentially growing cells in Mops minimal medium (time point 0) and after removal of glucose or phosphate at the time points indicated. Strains used in these experiments are wt (MG1655) and phoB:cm (AB026). The same RNA extracts were analyzed by using biotinylated probes to detect phoA mRNA and iraP mRNA. Representative data are shown.

Fig. 2.

Effect of ppGpp on iraP expression. Cells were grown at 37°C in Mops complete medium (A, B, and D), Mops minimal medium (C), or LB (E), and starved for phosphate (A–C), and total RNA was isolated as described in Materials and Methods. Northern blot analyses were conducted as described in Fig. 1. (A) Effect of ppGpp on iraP promoter activity. The expression of iraP was compared in wt (MG1655) and ppGpp° strains (AB029, ΔrelA:kn ΔspoT:cm; AB036, ΔrelA:kn spoT_E319Q). (B) Effect of ppGpp on the activity of the promoter P_{iraP(−10⁻²)}. Strains used were as follows: P_{iraP(−10⁻²)} ppGpp⁺ (AB031); P_{iraP(−10⁻²)} ppGpp° (AB035). iraP transcripts (arrowhead) were normalized to SsrA levels (data not shown); relative amounts are given below the gel. (C) SpoT activity is required for iraP induction in response to phosphate starvation. mRNA levels in wt (MG1655), ΔrelA:kn (AB028), and spoT_E319Q (AB037) were analyzed. (D) Effect of the stringent response on iraP expression. Cells were cultured in Mops glucose medium supplemented with all amino acids (40 μg/ml each) except serine. Serine hydroxamate (Ser-Hx) (final concentration of 500 μg/ml) was added at T = 0 to exponentially growing cells (OD₆₀₀, ≈0.3) to induce starvation, and samples were removed at the indicated time points. iraP mRNA levels were compared in wt (MG1655) and ΔrelA:kn (AB028) strains. (E) Effect of ppGpp overproduction on iraP expression. Induction of ppGpp was achieved by adding 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) to exponentially growing cells (AB028) bearing plasmids pALS13 or pALS10 to overexpress a fragment of the RelA protein, RelA′, which is constitutive for ppGpp synthetic activity or wt RelA protein, respectively. A plasmid pALS14, encoding a truncated and inactive RelA protein, RelA*, was used as a control. (F) Immunoblot analysis of IraP expression. Cells (AB040, iraP-HA, or AB040 carrying pALS13) were grown as described above for C (phosphate starvation), D (serine hydroxamate), and E (RelA′), and an immunoblot of IraP-HA was conducted as described in SI Materials and Methods.

Fig. 3.

Effects of promoter mutations on iraP expression and activity. Cells were grown at 37°C in Mops glucose medium and starved for phosphate. (A) Northern blots for iraP mRNA were performed as described in Fig. 1. The expression of iraP was compared in wt (MG1655), AB031 (P_{iraP(−10⁻²)}), AB033 (P_iraP(dis2)), and AB034 (P_{iraP(−10⁻²dis2)}). (B) The half-life of σ^S was determined in exponentially growing cells and after 1 h of phosphate starvation as indicated. Protein synthesis was inhibited with chloramphenicol at an OD₆₀₀ of ≈0.3. Samples were removed at specific times and analyzed by immunoblot with an anti-σ^S antiserum. To obtain similar amounts of σ^S at time 0 of each of the chase experiments, the amount of cell extract used for exponential phase cultures was twice the volume used for starved cells. σ^S half-lives (t_1/2) were determined from plots of intensity vs. time. σ^S degradation was monitored in the wt (MG1655) and AB033 (P_iraP(dis2)) strains.