RelE, a global inhibitor of translation, is activated during nutritional stress

Abstract

The stringent response is defined as the physiological changes elicited by amino acid starvation. Many of these changes depend on the regulatory nucleotide ppGpp (guanosine tetraphosphate) synthesized by RelA (ppGpp synthetase I), the relA-encoded protein. The second rel locus of Escherichia coli is called relBE and encodes RelE cytotoxin and RelB antitoxin. RelB counteracts the toxic effect of RelE. In addition, RelB is an autorepressor of relBE transcription. Here we reveal a ppGpp-independent mechanism that reduces the level of translation during amino acid starvation. Artificial overexpression of RelE severely inhibited translation. During amino acid starvation, the presence of relBE caused a significant reduction in the poststarvation level of translation. Concomitantly, relBE transcription was rapidly and strongly induced. Induction of transcription occurred independently of relA and spoT (encoding ppGpp synthetase II), but instead depended on Lon protease. Consistently, Lon was required for degradation of RelB. Replacement of the relBE promoter with a LacI-regulated promoter indicated that strong and ongoing transcription of relBE is required to maintain a proper RelB:RelE ratio during starvation. Thus relBE may be regarded as a previously uncharacterized type of stress-response element that reduces the global level of translation during nutritional stress.

Figure 1

Protein, RNA, and DNA syntheses after relE induction. Strain MC1000/pMG223 (pA1/O4/O3∷relE) was grown in AB minimal medium at 37°C and IPTG (2 mM) was added at time 0 to induce RelE synthesis. Samples were taken at the time points indicated and the rates of macromolecular syntheses measured. Symbols: ▴, DNA synthesis; ■, RNA synthesis; ♦, protein synthesis. The prestarvation level was set to 100%.

Figure 2

Translation during aa starvation. Strains MC1000 (relBE⁺, ■), MG1 (MC1000ΔrelBE, ♦), and MG1/pSC7104 (relBE⁺, ▴) were grown in AB minimal medium at 37°C, and rates of protein synthesis were determined as described in Materials and Methods. At time 0, SHT (0.4 mg/ml) was added. The prestarvation rate-of-translation was set to 100%.

Figure 3

Activation of relBE transcription during aa starvation and other stress conditions. (A) Northern analysis of total RNA from MC1000 (relBE⁺). SHT: cells were grown in LB medium and SHT (1 mg/ml) added at time 0. Glucose starvation: cells were grown at 37°C in AB minimal medium plus a limiting amount of glucose (0.03%) and onset of starvation was determined from a parallel culture grown in identical medium. Samples were taken before (−10 min) and after onset of starvation. Heat shock: cells were grown in LB medium at 30°C and at time 0 transferred to 42°C. Chloramphenicol: cells were grown in LB medium at 37°C and chloramphenicol (50 μg/ml) was added at an OD₄₅₀ of 0.5 (at time 0). (B) Northern analysis of total RNA from strains CF1648 (relA⁺ spoT⁺), CF1651 (relA⁻ spoT⁺), and CF1693 (relA⁻ spoT⁻) grown in LB medium at 37°C. SHT (1 mg/ml) was added at time 0. (C) Northern analysis of total RNA from strains SG22025 (wt), SG22093 (Δclp), and SG22095 (Δlon) grown in LB medium at 37°C. In A–C, RNA samples were fractionated by 4.5% PAGE, blotted, and hybridized with an anti-relBE riboprobe. (D) Primer-extension analysis of RNA samples from C, using a ³²P-labeled relBE-specific primer as described in Materials and Methods. Numbers above each lane are time points of sampling in minutes.

Figure 4

RelB decay in wt, clp, and lon strains. All strains contained low-copy-number plasmid pSC7104 (relBE⁺). MG1 (A Left), MG1ΔclpP (B Left) and MG1Δlon (B Right) were grown at 37°C in LB medium containing 30 μg/ml ampicillin, and chloramphenicol (50 μg/ml) was added at time 0. Cell samples were removed at the time points indicated (min) and RelB was visualized by Western analysis. “C” denotes control lanes with samples from MG1/pNDM71. In A Right, cells of MG1/pSC7104 (relBE⁺) were grown in LB medium and SHT (1 mg/ml) added at time 0 to induce aa starvation.

Figure 5

Replacement of wt relB promoter with a LacI regulated promoter. (A) Schematic structure of low-copy-number plasmid pMG224 showing relevant genes. pA1/O4/O3 indicates a strong synthetic LacI-regulated promoter. (B) Viable counts of strain MG1/pMG224 grown in AB minimal medium (37°C) and treated with SHT (0.4 mg/ml) at time 0. (C) Translation rates of MG1/pMG224 grown as in B. In B and C, −IPTG indicates that IPTG was not added at all, +IPTG indicates that IPTG (2 mM) was added throughout the entire experiment (including 2 h before sampling), and + IPTG/−IPTG indicates that IPTG was added 2 h before sampling and then removed by centrifugation just before addition of SHT.