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

Abstract

RpoS, the stationary phase/stress sigma factor of Escherichia coli, regulates a large cohort of genes important for the cell to deal with suboptimal conditions. Its level increases quickly in the cell in response to many stresses and returns to low levels when growth resumes. Increased RpoS results from increased translation and decreased RpoS degradation. Translation is positively regulated by small RNAs (sRNAs). Protein stability is positively regulated by anti-adaptors, which prevent the RssB adaptor-mediated degradation of RpoS by the ClpXP protease. Inactivation of aceE, a subunit of pyruvate dehydrogenase (PDH), was found to increase levels of RpoS by affecting both translation and protein degradation. The stabilization of RpoS in aceE mutants is dependent on increased transcription and translation of IraP and IraD, two known anti-adaptors. The aceE mutation also leads to a significant increase in rpoS translation. The sRNAs known to positively regulate RpoS are not responsible for the increased translation; sequences around the start codon are sufficient for the induction of translation. PDH synthesizes acetyl-CoA; acetate supplementation allows the cell to synthesize acetyl-CoA by an alternative, less favored pathway, in part dependent upon RpoS. Acetate addition suppressed the effects of the aceE mutant on induction of the anti-adaptors, RpoS stabilization, and rpoS translation. Thus, the bacterial cell responds to lowered levels of acetyl-CoA by inducing RpoS, allowing reprogramming of E. coli metabolism.

Keywords: ClpXP; RpoS; RssB; acetyl CoA; pyruvate dehydrogenase.

Fig. 1.

Mutations in aceE lead to increased RpoS levels. (A) WT (MG1655) and ΔaceE (BA334) were grown to an OD₆₀₀ of ∼0.3, protein synthesis was inhibited by chloramphenicol (Cm) addition, and RpoS levels were analyzed by Western blotting using an anti-RpoS antiserum. (B) Strains containing rpoS-lacZ translational fusions (WT, SG30013; ΔaceE, BA455; ΔrssB, SG30018; ΔrssB ΔaceE, BA679) were grown to an OD₆₀₀ of ∼0.3 and assayed for β-galactosidase activity. This fusion has the promoters of rpoS and enough of the RpoS ORF to be subject to RssB-dependent degradation. The mean from three replicates is presented, and the standard error of the mean (SEM) is indicated by the error bars. M.U., Miller units. (C) Cells were grown to an OD₆₀₀ of 0.2 in LB, and samples were removed for RNA analysis. Samples were probed with biotinylated probes for SdsR (Top), ChiX (Middle), or SsrA (Bottom). Strains: WT (NM1100), ΔaceE::kan (BA334), and rpoS::tet ΔaceE::kan (BA530).

Fig. 2.

RpoS stabilization in an aceE mutant is dependent on the anti-adaptors IraP and IraD. (A) Strains were grown and RpoS levels analyzed by Western blotting as in Fig. 1A. The RpoS level was quantified, with intensity measured at time 0 for each strain set at 100%. The mean from three replicates is presented, and the error bars indicate the SEM. The following strains were used: WT (MG1655); ΔaceE (BA334); and the isogenic derivatives ΔaceE ΔiraP ΔiraD (BA449), ΔaceE ΔiraP ΔiraM (BA447), ΔaceE ΔiraD ΔiraM (BA467), and ΔaceE ΔiraP ΔiraD ΔiraM (BA385). The gels and other mutants are in Fig. S2A. (B) Strains containing transcriptional fusions iraP-lacZ (WT, AB060; ΔaceE, BA407), iraD-lacZ (WT, AB050; ΔaceE, BA411), or iraM-lacZ (WT, AB042; ΔaceE, BA409) were grown in LB to an OD₆₀₀ of ∼0.3 and assayed for β-galactosidase activity. The mean from three replicates is presented; the SEM is indicated by the error bars. M.U., Miller units. The level of expression of the iraP fusion is significantly higher than that for the other two fusions. (C) Strains were grown as in Fig. 1A, and RpoS levels were analyzed by Western blotting using anti-RpoS antiserum (Upper) or anti–EF-Tu antiserum (Lower). The following strains were used: −10–2 dis2 iraP ΔiraD::tet (BA946) and the isogenic aceE derivative (BA948).

Fig. 3.

Regions in rpoS needed for translational induction in the aceE mutant. All strains carried an RpoS-LacZ translational fusion driven by a Cp17 promoter; the portion of RpoS present does not include the region necessary for RssB-dependent degradation. Without the promoter there was no expression of lacZ. (A) Cells were grown to an OD₆₀₀ of 0.3 and were assayed for β-galactosidase. Isogenic pairs of aceE⁺ and aceE::kan strains were assayed. Strains used were the following: lac (control fusion, with 38-nt leader of lac and lacZ: BA926, BA928); rpoS FL (full-length 567-nt leader of rpoS and 477 nt of rpoS ORF: BA938, BA940); C125T (derivative of rpoS FL with leader mutation C125T, disrupting hairpin: BA942, BA944); hfq (Δhfq derivatives of rpoS FL: BA960, BA984); C125T hfq (Δhfq derivatives of C125T rpoS FL: BA962, 986). (B) Cells were grown and assayed as for A. Strains used were the following: 24rpoS + 8rpoS (BA964, BA966); 24rpoS + lac (BA992, BA996); and lac + 8rpoS (BA994, BA998).

Fig. 4.

Acetate overcomes effect of aceE mutant. (A) WT (MG1655) and ΔaceE (BA334) strains were grown and RpoS levels were analyzed as for Fig. 1A. (B) Strains contain the rpoS-lacZ translational fusion used in Fig. 1B (under the control of rpoS promoters and subject to RssB-dependent degradation). ΔrssB (SG30018) and ΔaceE ΔrssB (BA679) strains were grown at 37° in LB in the absence or presence of acetate (30 mM) and assayed as for Fig. 1B.