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.
Keywords: starvation|activation of transcription|relE|relB
In prokaryotes, the stringent response is defined as the pleiotropic physiological changes triggered by amino acid (aa) starvation (1). In eubacteria, aa limitation elicits a rapid increase in the concentration of ppGpp (guanosine tetraphosphate). This increase has a number of profound consequences for cellular metabolism. A primary effect is an almost instantaneous shutdown of stable RNA (rRNA and tRNA) synthesis, called stringent control (1). ppGpp inhibits transcription of stable RNA operons, probably by means of direct interaction with RNA polymerase (2–5). The coordinate shutdown of stable RNA synthesis reduces energy consumption considerably and is believed to play a major role in adjusting cellular metabolism to nutritional stress (2). However, the stringent response also provokes stimulation of gene expression. Thus, transcription of aa biosynthetic operons increases during the stringent response (6–9), a logical cellular response to aa limitation. Similarly, rpoS expression is positively regulated by ppGpp, leading to an increase in the amount of the starvation sigma factor (σS) and of expression of σS-dependent genes (10). Recently it has been shown that σS-dependent promoters require ppGpp for induction even in the presence of high levels of σS (11). Thus, the stringent response simultaneously elicits positive and negative effects on gene expression.
RelA (ppGpp synthetase I) is encoded by relA located at 59.2 min just upstream of the mazEF toxin–antitoxin locus (12). Escherichia coli contains a second rel locus at 34.4 min called relBE (13). Previously, the relB gene was defined by mutations that conferred the so-called delayed relaxed response (14, 15). In certain relB mutant strains stable RNA synthesis continues after a lag of about 10 min after onset of aa starvation. The underlying molecular mechanism of the delayed relaxed response remained enigmatic; however, activation or synthesis of a translational inhibitor was implicated (15). Interestingly enough, chromosomal relBE loci are present in many Gram-negative and Gram-positive Bacteria, and in Archaea, often in multiple copies (16).
We showed previously that relBE of E. coli encodes a toxin–antitoxin locus (17, 18). Thus, RelE is a toxin that, by an unknown mechanism, severely inhibits cell growth and colony formation. RelB is an antitoxin that neutralizes RelE, probably by means of direct protein–protein interaction. RelB is as an autorepressor of relBE transcription. RelE is a co-repressor of relBE transcription and the relBE promoter is severely repressed during steady-state cell growth. Here we show that relE encodes a global inhibitor of translation that is activated during aa starvation. We also reveal the underlying mechanism of activation. The relBE promoter was strongly activated during aa starvation. Activation of relBE transcription was independent of ppGpp but depended on Lon protease. Consistently, in vivo experiments showed that Lon was required for degradation of RelB. The Lon-mediated degradation of RelB simultaneously explains activation of RelE and increased transcription of relBE during starvation. We observed no cell killing during starvation. Thus, it appears that relBE modulates the global level of translation during starvation without interfering with cell viability.
Materials and Methods
Culture Conditions and Media.
The cultures was grown in either LB or AB minimal medium at 37°C supplemented with 0.2% glucose, 1 μg/μl thiamine, and aa in defined concentrations. [35S]Methionine (1175 Ci/mmol; 1 Ci = 37 GBq), [2-14C]uracil (40–60 mCi/mmol), and [methyl-3H]thymidine (20 Ci/mmol) were obtained from NEN. Serine hydroxamate (SHT) was from SIGMA.
Bacterial Strains and Plasmids Used.
Strains and plasmids are listed in Table 1. E. coli K-12 MC1000 (relBE+) was used as standard strain. MG1 is MC1000 carrying aphA in replacement of relBE (construction to be described elsewhere). The replacement was confirmed by PCR. MG1Δlon was constructed by P1 transduction using SG22095 as donor strain. MG1ΔclpP was constructed according to ref. 19, with pKD3 as template for the PCR reaction and the following primers: ΔclpP1, 5′-GGT GGG CTT TTT TTT GTC ATG AAT TTT GCA TGG AAC CGT GCG AAA AGC CGT GTA GGC TGG AGC TGC TTC; and ΔclpP2, 5′-GCG TTG TGC CGC CCT GGA TAA GTA TAG CGG CAC AGT TGC GCC TCT GGC ACA TAT GAA TAT CCT CCT TAG. The PCR product was electroporated into BW25113/pKD46 and cells spread on LA plates containing 25 μg/ml chloramphenicol (Cml). The deletion of the clpP locus was verified by PCR. This strain was named BW25113ΔclpP. The ΔclpP∷cat alleles were transduced into MC1000. The chloramphenicol resistance allele was deleted as described in ref. 19, resulting in MG1ΔclpP. ER2566 was used for purification of a RelB-intein fusion protein and carries a chromosomal copy of the T7 RNA polymerase gene inserted into lacZ. Expression-vector pTyb3 carries the T7 RNA polymerase promoter upstream of a multiple cloning region and an intein gene. Plasmid pNDM71 is a low-copy-number miniR1 vector stabilized by par of plasmid R1.
Table 1.
Bacterial strains and plasmids used and constructed
Strains/plasmids | Genotypes/plasmid properties* | References |
---|---|---|
MC1000 | Δ(ara-leu) Δlac rpsL150 | 22 |
MG1 | ΔrelBEF | This work |
MG1Δlon | ΔrelBEF, lon146∷tet | This work |
MG1ΔclpP | ΔrelBEF, Δclp | This work |
ER2566 | lon ompT plac∷T7pol gene | New England Biolabs |
SG22025 | Δlac rcsA166∷aphA | Susan Gottesman |
SG22093 | Δlac rcsA166∷aphA clpP1∷cat | Susan Gottesman |
SG22095 | Δlac rcsA166∷aphA lon146∷tet | Susan Gottesman |
CF1648 (MG1655) | wild type E. coli | 23 |
CF1651 | relA251∷aphA | 24 |
CF1693 | relA251∷aphA spoT207∷cat | 23 |
BW25113 | lacIqrrnBT14ΔlacZWJ16hsdR514 ΔaraBADAH33ΔrhaBADLD78 | 19 |
pNDM71 | mini-R1 bla | N. D. Mikkelsen, unpublished data |
pTyb3 | pUC bla, intein expression vector | New England Biolabs |
pMG1904 | pUC bla relBE | 17 |
pMG223 | mini-R1 bla pA1/O4/O3∷relE | 17 |
pMG224 | mini-R1 bla pA1/O4/O3∷relBE | 17 |
pSC302 | pUC bla pT7∷relB∷intein | This work |
pSC7104 | mini-R1 bla relBE | This work |
pKD46 | pSC101reptsbla araC γ β exo | 19 |
pKD3 | R6Koriγ bla cat | 19 |
pCP20 | pSC101reptsbla cat | 19 |
tet denotes the tetracycline-resistance gene, cat the chloramphenicol transacetylase gene, aphA the kanamycin resistance gene, and bla the ampicillin resistance gene.
Construction of Plasmids.
pSC7104: pMG1904 was digested with EcoRI and BamHI and the resulting relBE fragment was inserted into BamHI-EcoRI-digested pNDM71. pSC302: wild-type (wt) relB was PCR amplified with primers relB1NcoI (5′-CCC CCC CCA TGG GTA GCA TTA ACC TGC GTA TTG-3′) and relB2SapI (5′-CCC CCC GCT CTT CCG CAG AGT TCA TCC AGC GTC AC-3′). The PCR product was digested with NcoI and SapI and inserted into pTyb3 digested with the same enzymes. This plasmid, called pSC302, produces a fusion between RelB and Intein.
Rates of Protein, RNA, and DNA Syntheses.
Cells were grown at 37°C in AB minimal medium + 0.2% glucose and aa to an optical density (OD450) of 0.5. The cultures were diluted 10 times and SHT added at an OD of 0.4. Samples of 0.5 ml were added to 5 μCi of [35S]methionine (protein synthesis), 2 μCi [methyl-3H]thymidine (DNA synthesis), or 0.1 μCi [2-14C]uracil (RNA synthesis). After 1 min of incorporation, samples were chased for 10 min with 0.5 mg of cold methionine, 0.5 mg cold thymidine, or 0.5 mg cold uracil, respectively. The samples were harvested and resuspended in 200 μl cold 20% trichloroacetic acid (TCA) and were centrifuged at 20,000 × g for 30 min at 4°C. The samples were washed twice with 200 μl cold 96% ethanol. Precipitates were transferred to vials and the amount of incorporated radioactivity was counted in a liquid scintillation counter.
Purification of RelB.
An overnight culture (ON) of ER2566/pSC302 (pT7∷relB∷intein) was diluted in LB + 500 μg/ml ampicillin and grown at 20°C. At an optical density of 0.5–0.6, isopropyl β-D-thiogalactoside (IPTG) was added to a final concentration of 0.5 mM. The culture was harvested after 5–6 h of induction. Cells were resuspended in column and lysis buffer (20 mM Tris⋅HCl, pH 8.8/500 mM NaCl/0.1% Triton X-100/0.1 mM EDTA) containing protease inhibitors and lysed by sonication. The lysate was centrifuged at 14,000 × g for 30 min. The protein extract was loaded on a column of chitin beads and washed with 10 vol of column and lysis buffer. Then 3 column vol of cleavage buffer (20 mM Tris⋅HCl, pH 8.8/500 mM NaCl/0.1 mM EDTA/30 mM DTT) were added. The cleavage of the fusion protein was induced by the presence of DTT. The column was left at 4°C for 24 h. RelB protein was eluted with 2–3 vol of cleavage buffer without DTT or protease inhibitors in a fraction of 1/10 column volume. The fractions were run on a 16% Tricine-SDS gel essentially as described in ref. 20 and stained with Coomassie blue. The RelB protein was estimated to be 99% pure. The purified protein was dialyzed against 25 mM Tris⋅HCl (pH 8.0), 100 mM KCl, 1 mM DTT, 1 mM EDTA, and 20% glycerol and kept at −80°C. Anti-RelB antibodies were raised in rabbits according to standard procedure and purified by affinity chromatography.
Western Analysis.
Cell samples were resuspended in SDS loading buffer (10% glycerol/100 mM Tris⋅HCl, pH 7.0/0.05% bromphenol blue/1% SDS/10 mM DTT). After SDS/PAGE, proteins were transferred to a hybond-P membrane (Amersham Pharmacia) by use of a semidry blotting apparatus. Membranes were probed with anti-RelB antiserum. The antibodies were used at an OD280 of 0.5 and diluted 500 times before use.
Northern and primer-extension analyses have been described (21). For Northern analysis, total RNA was fractionated by PAGE (4.5% low-bis), blotted to a Zetaprobe nylon membrane, and hybridized with a relBE-specific single-stranded 32P-labeled riboprobe. For primer-extension analysis, the relB15 primer (5′-GGTCATCGTCAATACGCAGG) was labeled with 32P by using PNK and [γ-32P]ATP.
Results
relE Encodes a Global Translational Inhibitor.
Plasmid pMG223 is a low-copy-number miniR1 derivative carrying lacIq and the relE gene downstream of a strong synthetic LacI-regulated promoter [pA1/O4/O3 (25)]. Rates of protein, DNA, and RNA syntheses were measured after the addition of IPTG to growing cells of MC1000/pMG223. As seen from Fig. 1, protein synthesis was rapidly and severely inhibited, whereas DNA and RNA syntheses were not. MC1000 carries a chromosomal copy of relBE that expresses low amounts of RelB. Thus, RelE expressed from pMG223 in the above-described experiment overrode the chromosomal expression of RelB. We were not able to accomplish similar labeling experiments in relBE deletion strains, most probably because of leaky (i.e., toxic) expression of relE from pMG223. One-dimensional gel analyses of pulse-labeled proteins demonstrated that inhibition was almost as rapid and efficient as that obtained with a low-molecular-weight inhibitor of protein synthesis (i.e., tetracycline; data not shown). Two-dimensional gel analyses showed that inhibition appeared uniform, meaning that RelE inhibited synthesis of all proteins to the same extent. Thus, our results show that relE encodes a direct or indirect inhibitor of translation in E. coli.
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%.
relBE Reduces the Global Rate-of-Translation During Amino Acid Starvation.
To investigate the effect, if any, of the chromosomal copy of relBE, we compared rates of translation in strains MC1000 (relBE+) and MG1 (MC1000ΔrelBE) before and after addition of serine hydroxamate (Fig. 2). SHT is a competitive inhibitor of Seryl tRNA synthetase and thus prevents charging of serine tRNA (26). This inhibition, in turn, induces the stringent response. As expected, the rate of translation declined rapidly in both strains after onset of aa starvation. In both strains, new steady-state rates of translation were reached ≈20 min after onset of starvation. However, the poststarvation steady-state levels of translation differed in the two strains in that the rate of MG1 was significantly higher than that of MC1000 (≈100%). That this difference was due solely to the deletion of relBE was shown by complementation analysis. Plasmid pSC7104, a miniR1 replicon carrying the wt relBE locus, was introduced into MG1 (ΔrelBE). As seen in Fig. 2, the poststarvation translation rate of MG1/pSC7104 was similar to that of the wt strain, showing that relBE present on the plasmid complemented the relBE deletion in MG1.
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%.
We also measured cell viability during aa starvation. We did not observe any reduction in viable counts of neither MC1000 nor MG1 during the entire sampling period (data not shown). This observation is in accordance with results presented later (see Fig. 5B). Thus, although relBE appears to inhibit translation during aa starvation, it does not interfere with cell viability.
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.
Amino Acid and Glucose Starvation Induces Transcription of relBE.
The above result indicated that RelE was activated during aa starvation. In turn, this would be consistent with decay of RelB and an increased rate of relBE transcription (because RelB is an autorepressor of relBE transcription). To measure relBE transcription we used Northern analysis. As seen from Fig. 3A (SHT) the level of relBE mRNA increased rapidly and dramatically after addition of SHT. Sixty minutes after onset of starvation, the increase in relBE mRNA was 35-fold as compared with the prestarvation level. A similar, although less pronounced, increase in transcription was also obtained by valine-induced isoleucine starvation (data not shown). That the dramatic activation of relBE transcription was due to an increased transcription rate from the relBE promoter was substantiated by primer extension analysis (Fig. 3D, wt). As seen, the amount of relBE mRNA 5′ ends also increased dramatically after addition of SHT. The transcription start-point was mapped to position +465, one nucleotide upstream of the start-point suggested previously for relBE mRNA (13). The increased level of relBE mRNA was not due to an increase in mRNA stability, because its half-life was ≈5 min both before and after onset of starvation (data not shown). Thus, aa starvation activates the relBE promoter dramatically.
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.
We also investigated the effect of other stress conditions on the relBE transcription pattern. Cells grown in glucose minimal medium exhibited a higher steady-state level of relBE mRNA than cells grown in LB and run-out of glucose conferred a further 4- to 5-fold increase (Fig. 3A, Glucose starvation). Heat shock treatment did not stimulate relBE transcription significantly (Fig. 3A, Heat shock). Addition of chloramphenicol, a global inhibitor of translation, also conferred a dramatic activation of relBE transcription (Fig. 3A, Chloramphenicol).
Transcriptional Activation of relBE Is Independent of ppGpp.
Next, we investigated the relBE transcription pattern in relA and spoT strains. As seen from Fig. 3B (relA), transcriptional activation of relBE also occurred in a relA deletion strain. In the relA spoT double deletion strain, which does not synthesize ppGpp, transcriptional activation was even more pronounced than in the wt strain (Fig. 3B, relA spoT). These results show that activation of relBE transcription occurs independently of ppGpp. The steady-state level of relBE mRNA was higher in the relA spoT strain that in the wt strain, perhaps indicating that ppGpp reduces transcription of relBE in wt cells growing in steady-state (i.e., without starvation).
Transcriptional Activation of relBE Depends on Lon.
The above-described observations are consistent with the proposal that RelB protein decayed during starvation and after inhibition of translation by chloramphenicol. Therefore, we investigated the relBE transcription pattern in lon and clp strains during aa starvation. As seen from Fig. 3C, the transcription pattern in the clp strain was similar to that in the wild type (compare wt and clp). However, in the lon strain, the increase in relBE mRNA was much less severe, indicating that Lon was required for transcriptional activation of relBE (lon). Primer-extension analysis of the relBE mRNA supported this conclusion (Fig. 3D).
Lon Degrades RelB in Vivo.
The metabolic stability of RelB was investigated by Western blot analysis. Using wt E. coli strains that carry a chromosomal copy of relBE, we obtained faint RelB-specific bands only, indicating that, during steady state, wt cells express low amounts of RelB. These bands could not be quantitated reproducibly. Consequently, a low-copy-number plasmid carrying relBE (pSC7104) was introduced into the strains used. We investigated the metabolic stability of RelB in MG1, MG1Δlon, and MG1ΔclpP strains by using chloramphenicol to block translation and thus further production of RelB. In MG1 (lon+ clp+) and MG1ΔclpP, RelB decayed rapidly (Figs. 4 A Left and B Left), with half-lives of ≈15 min in both cases. In MG1Δlon, RelB was much more stable, indicating that Lon degrades RelB in vivo (Fig. 4B Right).
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.
The amount of RelB was also followed during aa starvation (Fig. 4A Right). Initially, the amount of RelB decreased but then increased to a new poststarvation steady-state level that was ≈60% that of the prestarvation level. Thus, the increased relBE promoter activity seen during starvation was accompanied by a lower amount of detectable RelB.
Halting of relBE Transcription During Amino Acid Starvation Leads to Severe Inhibition of Translation and Cell Growth.
We were puzzled by the rapid and severe activation of relBE transcription during aa starvation. To investigate the biological role of the relBE promoter we replaced the promoter with a tight, LacI-regulated promoter (pA1/O4/O3). Thus, low-copy-number plasmid pMG224 carries lacIq and a pA1/O4/O3∷relBE promoter fusion (Fig. 5A). This plasmid was introduced into MG1 (ΔrelBE). Strain MG1/pMG224 was grown in AB minimal medium and treated in three different ways. In the first culture, serine hydroxamate was added at time 0 without any preinduction of relBE transcription (i.e., without IPTG). In this case, the cells stayed fully viable during starvation—i.e., no cell killing was observed (Fig. 5B, diamonds)—and translation was reduced ≈8% of the prestarvation value (Fig. 5C, diamonds), a level similar to that of MG1 without pMG224 (Fig. 2). In the second culture, IPTG was added to growing cells and then removed after 2 h of induction of relBE transcription. Immediately after removal of IPTG, SHT was added to induce aa starvation. In this case, a severe drop of viable counts was observed (Fig. 5B, squares). The reduction in viable counts was accompanied by a severe decrease in the poststarvation steady-state rate-of-translation (Fig. 5C, squares). In the third culture, IPTG was also added 2 h before onset of starvation, but IPTG was not removed before the addition of SHT. Thus, aa starvation was induced and transcription of relBE was allowed to continue during the starvation period. The reduction of viable counts was now much less severe (Fig. 5B, triangles), and the rate of poststarvation translation (Fig. 5C) was close to that of the wt strain carrying native relBE on the chromosome (Fig. 2). The latter experiment mimics the effect of strong ongoing transcription of native relBE during starvation and supports the proposal that continued production of RelB during starvation is required to counteract the very strong inhibitory and toxic effects of RelE (see Discussion).
Discussion
We show here that relE encodes a global inhibitor of translation (Fig. 1). The mechanism by which RelE inhibits translation is not yet known and may be direct or indirect (i.e., by means of activation of a second factor). However, RelE coprecipitates with ribosomes, perhaps indicating a direct interaction (27).
The relBE locus reduced the rate of poststarvation translation in E. coli wt cells starved for amino acids (Fig. 2). The rate of translation remained significantly lower in the wt strain during the entire period of sampling (2 h). Concomitantly, RelB content was reduced (Fig. 4A). Thus, a decreased level of RelB antagonist accompanied the RelE-mediated reduction in translation, thereby yielding a mechanistic explanation of the effect on translation. Glucose starvation also induced relBE transcription, suggesting that relBE might have a general effect during nutrient limitation.
The relBE promoter, which is very strong, is autoregulated by RelB (17). RelE acts in concert with RelB as a co-repressor of transcription and the relBE promoter is highly repressed during steady-state cell growth. Amino acid starvation conferred a dramatic induction in the level of relBE mRNA (Fig. 3A). The increased transcription rate of relBE is consistent with a lower level of RelB in starved cells (Fig. 4A). Moreover, induction of transcription was independent of ppGpp, because a relA spoT (ppGpp0) double-deletion strain also exhibited severe induction of transcription during aa starvation (Fig. 3B). In contrast, transcriptional activation was much less severe in a lon strain (Fig. 3C). Consistently, RelB was much more stable in a lon strain (Fig. 4B). Thus, activation of RelE and the concomitant activation of relBE transcription can both be explained by Lon-mediated degradation of RelB during starvation. Addition of chloramphenicol to growing E. coli cells also induced strong transcription of relBE (Fig. 3A, Chloramphenicol), consistent with the observed decay of RelB after addition of chloramphenicol (Fig. 4A).
We were puzzled by the strong effect of aa starvation on relBE transcription. In an attempt to investigate its function, we replaced the relBE promoter with the LacI-regulated pA1/O4/O3 promoter. In this artificial system, simultaneous block of relBE transcription and induction of aa starvation caused a rapid drop in colony forming units (Fig. 5B) and severe inhibition of translation (Fig. 5C). These two effects both depended on Lon and a functional relE gene (data not shown) and can be explained as follows: in the prestarvation period, a pool of RelB and RelE, and thus RelBE complex, accumulates, and the cells stay viable. The block of relBE transcription prevents further production of RelB, which decays. This, in turn, activates RelE. In contrast, continued transcription of relBE during starvation almost completely abolished RelE activation in that the drop in colony forming units was now much less severe (Fig. 5B) and the translation rate was close to that seen in starved wt cells (Fig. 5C). Based on these results we suggest that the increase of relBE transcription during starvation of wt cells (which does not interfere with colony formation) fine-tunes the RelB:RelE ratio and that continued synthesis of RelB from the increased level of relBE mRNA sustains the appropriate ratio. Thus, too much RelB would prevent RelE activation and too little RelB would confer a complete shutdown of translation, which would have detrimental effects on cellular metabolism.
This line of reasoning raises the question of whether Lon is specifically activated toward RelB during starvation. Regulated activation of proteases has been described—for example, the RssB response regulator mediates ClpXP-dependent turnover of σS during exponential growth (28, 29). The data obtained here do not support regulated degradation of RelB, because chloramphenicol, as well as SHT, mediated rapid degradation of RelB. Chloramphenicol blocks translation instantaneously and consequently abolishes the stringent response. Thus, RelB decay after addition of chloramphenicol probably reflects passive degradation by Lon. However, we do not at the moment exclude that regulatory factors may be involved in RelB proteolysis. Curiously, even moderate overexpression of RelB increased its half-life significantly.
The effect of ppGpp on stable RNA synthesis confers a coordinate reduction in the cellular protein synthesizing apparatus (1) and hence a reduced overall level of translation. However, the effect of ppGpp on translation is indirect. We describe here a mechanism that directly reduces translation and thus energy consumption during starvation. It is thus tempting to speculate that relBE has evolved as a stress-response element assisting the ppGpp-dependent response in keeping energy consumption low during nutrient limitations.
Originally, toxin–antitoxin loci were described as plasmid-encoded genetic elements that enhanced the stability of bacterial plasmids (30–33). Toxin–antitoxin loci express a phenotype called postsegregational killing. In all cases investigated, the antitoxins are labile because of degradation by cellular proteases (Lon or Clp; ref. 31). Because the toxins are metabolically more stable, plasmid-free cells experience decay of the antitoxin, and hence activation of the toxin. In turn, killing of the plasmid-free cells ensues. In growing bacterial cultures, this selective killing yields an advantage for the plasmid-containing cells. However, in many cases, plasmid stabilization by toxin–antitoxin loci is much less efficient than stabilization conferred by true partitioning loci (31). Moreover, we have shown recently that the relBE locus of the E. coli K-12 chromosome mediates plasmid stabilization as efficient as relBE from plasmid P307 (17, 18). Clearly, the function of relBE of E. coli is unlikely to be plasmid stabilization. Rather, the results presented here argue that the primary function of relBE may be related to cellular stress. This argument is further supported by the fact that many bacterial and archaeal chromosomes encode relBE loci, often in multiple copies (16). For example, E. coli K-12 contains three relBE-homologous loci and the archaeons Archaeoglobus fulgidus and Methanococcus jannaschii each contain four such loci.
Remarkably, E. coli encodes two toxin–antitoxin loci that are homologous to pem of plasmids R1/R100 (12, 16, 34, 35). The function of these loci is not yet known. However, in two reports the mazEF toxin–antitoxin locus was described as causing programmed cell death during the stringent response (36, 37).
Acknowledgments
We thank Susan Gottesman, Mike Cashel, and Barry Wanner for the donation of strains. This work was supported by The Danish Biotechnology Instrument Center (DABIC) and European Union Contract BIO4-98-0283.
Abbreviations
- aa
amino acid
- wt
wild type
- ppGpp
guanosine tetraphosphate
- IPTG
isopropyl β-d-thiogalactoside
- SHT
serine hydroxamate
- Cml
chloramphenicol
Footnotes
This paper was submitted directly (Track II) to the PNAS office.
References
- 1.Cashel M, Gentry D, Hernandez V J, Vinella D. In: Escherichia coli and Salmonella. Neidthardt F C, editor. Washington, DC: Am. Soc. Microbiol.; 1996. pp. 1458–1496. [Google Scholar]
- 2.Chatterji D, Kumar O A. Curr Opin Microbiol. 2001;4:160–165. doi: 10.1016/s1369-5274(00)00182-x. [DOI] [PubMed] [Google Scholar]
- 3.Barker M M, Gaal T, Josaitis C A, Gourse R L. J Mol Biol. 2001;305:673–688. doi: 10.1006/jmbi.2000.4327. [DOI] [PubMed] [Google Scholar]
- 4.Kingston R E, Chamberlin M J. Cell. 1981;27:523–531. doi: 10.1016/0092-8674(81)90394-9. [DOI] [PubMed] [Google Scholar]
- 5.Chatterji D, Fujita N, Ishihama A. Genes Cells. 1998;3:279–287. doi: 10.1046/j.1365-2443.1998.00190.x. [DOI] [PubMed] [Google Scholar]
- 6.Aldea M, Garrido T, Pla J, Vicente M. EMBO J. 1990;9:3787–3794. doi: 10.1002/j.1460-2075.1990.tb07592.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Shand R F, Blum P H, Mueller R D, Riggs D L, Artz S W. J Bacteriol. 1989;171:737–742. doi: 10.1128/jb.171.2.737-743.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Stephens J C, Artz S W, Ames B N. Proc Natl Acad Sci USA. 1975;72:4389–4393. doi: 10.1073/pnas.72.11.4389. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Barker M M, Gaal T, Gourse R L. J Mol Biol. 2001;305:689–702. doi: 10.1006/jmbi.2000.4328. [DOI] [PubMed] [Google Scholar]
- 10.Gentry D R, Hernandez V J, Nguyen L H, Jensen D B, Cashel M. J Bacteriol. 1993;175:7982–7989. doi: 10.1128/jb.175.24.7982-7989.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Kvint K, Farewell A, Nystrom T. J Biol Chem. 2000;275:14795–14798. doi: 10.1074/jbc.C000128200. [DOI] [PubMed] [Google Scholar]
- 12.Metzger S, Dror I B, Aizenman E, Schreiber G, Toone M, Friesen J D, Cashel M, Glaser G. J Biol Chem. 1988;263:15699–15704. [PubMed] [Google Scholar]
- 13.Bech F W, Jorgensen S T, Diderichsen B, Karlstrom O H. EMBO J. 1985;4:1059–1066. doi: 10.1002/j.1460-2075.1985.tb03739.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Diderichsen B, Fiil N P, Lavalle R. J Bacteriol. 1977;131:30–33. doi: 10.1128/jb.131.1.30-33.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Lavalle R, Desmarez L, De Hauwer G. In: Control of Ribosome Synthesis. Kjelgaard N O, Maaløe O, editors. Copenhagen: Munksgaard; 1976. pp. 408–418. [Google Scholar]
- 16.Gerdes K. J Bacteriol. 2000;182:561–572. doi: 10.1128/jb.182.3.561-572.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Gotfredsen M, Gerdes K. Mol Microbiol. 1998;29:1065–1076. doi: 10.1046/j.1365-2958.1998.00993.x. [DOI] [PubMed] [Google Scholar]
- 18.Gronlund H, Gerdes K. J Mol Biol. 1999;285:1401–1415. doi: 10.1006/jmbi.1998.2416. [DOI] [PubMed] [Google Scholar]
- 19.Datsenko K A, Wanner B L. Proc Natl Acad Sci USA. 2000;97:6640–6645. doi: 10.1073/pnas.120163297. . (First Published May 30, 2000; 10.1073/pnas.120163297) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Schagger H, von Jagow G. Anal Biochem. 1987;166:368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
- 21.Thisted T, Nielsen A K, Gerdes K. EMBO J. 1994;13:1950–1959. doi: 10.1002/j.1460-2075.1994.tb06464.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Casadaban M J, Cohen S N. J Mol Biol. 1980;138:179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
- 23.Xiao H, Kalman M, Ikehara K, Zemel S, Glaser G, Cashel M. J Biol Chem. 1991;266:5980–5990. [PubMed] [Google Scholar]
- 24.Metzger S, Sarubbi E, Glaser G, Cashel M. J Biol Chem. 1989;264:9122–9125. [PubMed] [Google Scholar]
- 25.Lanzer M, Bujard H. Proc Natl Acad Sci USA. 1988;85:8973–8977. doi: 10.1073/pnas.85.23.8973. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Tosa T, Pizer L I. J Bacteriol. 1971;106:972–982. doi: 10.1128/jb.106.3.972-982.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Galvani C, Terry J, Ishiguro E E. J Bacteriol. 2001;183:2700–2703. doi: 10.1128/JB.183.8.2700-2703.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Muffler A, Fischer D, Altuvia S, Storz G, Hengge-Aronis R. EMBO J. 1996;15:1333–1339. [PMC free article] [PubMed] [Google Scholar]
- 29.Zhou Y, Gottesman S, Hoskins J R, Maurizi M R, Wickner S. Genes Dev. 2001;15:627–637. doi: 10.1101/gad.864401. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Bravo A, de Torrontegui G, Diaz R. Mol Gen Genet. 1987;210:101–110. doi: 10.1007/BF00337764. [DOI] [PubMed] [Google Scholar]
- 31.Jensen R B, Gerdes K. Mol Microbiol. 1995;17:205–210. doi: 10.1111/j.1365-2958.1995.mmi_17020205.x. [DOI] [PubMed] [Google Scholar]
- 32.Ogura T, Hiraga S. Proc Natl Acad Sci USA. 1983;80:4784–4788. doi: 10.1073/pnas.80.15.4784. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Roberts R C, Strom A R, Helinski D R. J Mol Biol. 1994;237:35–51. doi: 10.1006/jmbi.1994.1207. [DOI] [PubMed] [Google Scholar]
- 34.Masuda Y, Miyakawa K, Nishimura Y, Ohtsubo E. J Bacteriol. 1993;175:6850–6856. doi: 10.1128/jb.175.21.6850-6856.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Santos S S, Giraldo R, Diaz O R. FEMS Microbiol Lett. 1998;168:51–58. doi: 10.1111/j.1574-6968.1998.tb13254.x. [DOI] [PubMed] [Google Scholar]
- 36.Aizenman E, Engelberg-Kulka H, Glaser G. Proc Natl Acad Sci USA. 1996;93:6059–6063. doi: 10.1073/pnas.93.12.6059. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Engelberg-Kulka H, Reches M, Narasimhan S, Schoulaker-Schwarz R, Klemes Y, Aizenman E, Glaser G. Proc Natl Acad Sci USA. 1998;95:15481–15486. doi: 10.1073/pnas.95.26.15481. [DOI] [PMC free article] [PubMed] [Google Scholar]