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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We have shown previously that ribosome stalling during translation caused by various reasons leads to mRNA cleavage, resulting in non-stop mRNAs that are eliminated in a tmRNA-dependent manner. Amino acid starvation is a physiological condition in which ribosome stalling is expected to occur more frequently. Here we demonstrate that mRNA cleavage is induced by amino acid starvation, resulting in accumulation of truncated mRNAs in cells lacking tmRNA. The truncated mRNAs are eliminated in wild-type cells, indicating that the tmRNA system rapidly degrade the truncated mRNAs. The cleavage pattern of model mRNAs in which serine codons were replaced with threonine codons indicated that mRNA cleavage occurs near serine codons in response to serine starvation. Cells lacking all of the five known toxin loci were proficient in mRNA cleavage, showing that toxin–antitoxin systems are not responsible for the cleavage. A mild serine starvation caused a significant growth inhibition in cells lacking tmRNA but not in wild-type cells. The ribosome-mediated mRNA cleavage along with the tmRNA system is an important mechanism that enables cells to adapt to amino acid starvation conditions.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The bacterial tmRNA system acts to deal with the problems associated with translation of mRNAs lacking a stop codon (non-stop mRNAs) that are frequently produced in cells (Keiler, 2007). When a translating ribosome reaches the 3′ end of a non-stop mRNA, tmRNA charged with alanine along with SmpB and EF-Tu enters the A-site of the stalled ribosome. Then it acts first as a tRNA and then as an mRNA to direct the addition of a short peptide tag to the C-terminus of growing polypeptide (Keiler et al., 1996). As the result of this process called trans-translation (Atkins and Gesteland, 1996; Jentsch, 1996) both the tagged polypeptide and the stalled ribosome are released. The tagged polypeptide is rapidly degraded by ATP-dependent proteases (Keiler et al., 1996; Gottesman et al., 1998). The rescue of the stalled ribosome and the degradation of aberrant polypeptides are major biological roles of the tmRNA-mediated quality-control system (Karzai et al., 2000; Withey and Friedman, 2002). In addition, the tmRNA system facilitates the release of the truncated mRNA from the stalled ribosome and allows its rapid degradation by 3′ to 5′ exonucleases to prevent further production of aberrant polypeptides (Yamamoto et al., 2003; Richards et al., 2006).

Non-stop mRNAs can be generated in cells under various situations such as premature termination of transcription (Abo et al., 2000), degradation by ribonucleases (Yamamoto et al., 2003) and chemical or physical damages to the message. A ribosome also reaches the 3′ end of an mRNA and becomes a target for the tmRNA system when a normal stop codon is erroneously translated either by suppressor tRNAs (Ueda et al., 2002) or by miscoding drugs (Abo et al., 2002). In addition, it has been demonstrated that the tmRNA tagging occurs in response to ribosome stalling caused by a cluster of rare codons (Roche and Sauer, 1999; Li et al., 2006) or by particular sequences of nascent peptides (Hayes et al., 2002; Sunohara et al., 2002; 2004a,b), or by reduced action of release factors (Li et al., 2007). Importantly, the ribosome stalling within an mRNA has been shown to induce mRNA cleavage to generate non-stop mRNAs prior to tmRNA action in all of these cases. As the result, the tmRNA system acts on the empty A-site of a ribosome stalled at the 3′ end of non-stop mRNA.

Amino acid starvation is a natural physiological condition in which ribosome stalling is expected to occur frequently due to depletion of aminoacyl-tRNAs (Cashel et al., 1996). Recent studies demonstrated that amino acid starvation leads to activation of bacterial toxins such as RelE and MazF, and that these toxins inhibit translation by cleaving mRNAs at several specific sequences/codons (Christensen et al., 2001; 2003; Christensen and Gerdes, 2003). On the other hand, the mRNA cleavage mediated by ribosome stalling under various conditions mentioned above has been shown to occur independently of bacterial toxins (Hayes and Sauer, 2003; Sunohara et al., 2004a,b). This leads a question: to what extent do toxin-dependent and toxin-independent mRNA cleavages occur during amino acid starvation conditions? In this article, we investigated the mechanism of mRNA cleavage induced by amino acid starvation focusing on this question. We found that significant mRNA cleavages are induced in response to serine starvation conditions in which RelE is not activated yet, resulting in non-stop mRNAs that are recognized by tmRNA system. The mRNA cleavage occurs efficiently in cells lacking all of the five toxin–antitoxin (TA) loci. These results have led us to conclude that mRNA cleavage under amino acid starvation occurs primarily in a toxin-independent manner. We also show that the quality control mediated by ribosome and tmRNA system plays a significant role in cellular physiology during amino acid starvation.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Serine starvation induces mRNA cleavage

The exposure of serine hydroxamate (SHX), a competitive inhibitor of seryl-tRNA synthetase, to growing cells leads to depletion of seryl-tRNA (serine starvation) because charging of serine tRNA is prevented (Tosa and Pizer, 1971). We first examined the effect of serine starvation on mRNA metabolism focusing on ompA mRNA. Isogenic ssrA+ and ssrA strains were grown to mid-log phase in Luria–Berani (LB) medium. Increasing concentrations of SHX were added and the incubation was continued for 20 min. Total RNAs were prepared and subjected to Northern blot analysis using a 5′ompA DNA probe. Remarkably, smaller ompA mRNA fragments were generated with increasing concentrations of SHX (Fig. 1A). The mRNA fragments were markedly reduced in ssrA+ cells when SHX concentrations were less than 0.1 mg ml−1 (Fig. 1A, lanes 2, 4 and 6), implying that they were rapidly degraded by the tmRNA system. This means that the major fragments generated by SHX treatment are truncated non-stop ompA mRNAs. Interestingly, the truncated mRNAs became resistant to the tmRNA system when SHX concentration was more than 0.2 mg ml−1, suggesting that higher concentrations of SHX somehow prevent the tmRNA action. A possible reason for the decreased action of tmRNA system in the presence of higher concentrations of SHX would be toxin-dependent cleavage of tmRNA because RelE and MazF are known to be able to cleave tmRNA and they are induced at a high concentration of SHX (Christensen and Gerdes, 2003; Christensen et al., 2003).

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Figure 1. Cleavage of ompA mRNA in response to serine starvation. A. Generation of 5′ompA mRNA fragments by SHX treatment. W3110 (wild-type) and TA331 (ΔssrA) cells were grown in LB medium at 37°C to A600 = 0.5. Indicated concentrations of SHX were added and incubation was continued for 20 min. Total RNAs were prepared and 5 μg of each RNA was resolved by electrophoresis on a 1.5% agarose-formaldehyde gel. Northern blot analysis was performed using the DIG-labelled 5′ompA probe. B. Generation of 3′ mRNA fragments by SHX treatment. Northern blot analysis of total RNAs was performed using the DIG-labelled 3′ompA probe. C. Time-course of mRNA cleavage after SHX treatment. Cells were grown in LB medium at 37°C. At A600 = 0.5, 0.1 mg ml−1 SHX was added and incubation was continued. Total RNAs were prepared at indicated times and 5 μg of each RNA was resolved by electrophoresis on a 1.5% agarose-formaldehyde gel. Northern blot analysis was performed using the DIG-labelled 5′ompA probe.

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We also carried out Northern blot analysis using a 3′ompA DNA probe. A series of mRNA fragments that are distinct from those detected by the 5′ompA probe were generated with SHX treatment in both ssrA+ and ssrA- strains (Fig. 1B). The insensitivity of these mRNA fragments to tmRNA is consistent with that these fragments are derived from the 3′ portion of ompA. Concomitant production of 5′ and 3′ mRNA fragments suggests that these mRNA fragments were generated by endonucleolytic cleavages of ompA mRNAs. Taken together, we propose that serine starvation leads to ribosome stalling resulting in endonucleolytic cleavages of mRNAs although we do not exclude completely the possibility that serine starvation generates truncated mRNAs by other mechanisms such as exonucleolytic digestion or premature termination of transcription.

Next, we carried out a time-course experiment of SHX effect. Cells were grown to mid-log phase in LB medium. Then, SHX was added at a final concentration of 0.1 mg ml−1 and the incubation was continued. Total RNAs were prepared at various times after the addition of SHX and subjected to Northern blot analysis using the 5′ompA DNA probe (Fig. 1C). Truncated ompA mRNAs began to be observed shortly after SHX addition with a peak of accumulation at 20 min in cells lacking tmRNA (Fig. 1C, lanes 1–6). The truncated mRNAs were barely detected in ssrA+ cells (Fig. 1C, lanes 7–12), indicating again that they were eliminated in a tmRNA-dependent fashion.

We also carried out Northern analysis of ompF and ompC mRNAs by using RNA samples prepared 20 min after SHX addition (Fig. 2A and B). Truncated mRNA fragments were accumulated in cells lacking tmRNA (Fig. 2A and B, lane 3) but not in ssrA+ cells (Fig. 2A and B, lane 4). Few truncated mRNAs were produced without SHX addition (Fig. 2A and B, lanes 1 and 2). Taken together, we conclude that serine starvation caused by SHX treatment induces significantly mRNA cleavage in general and the resulting truncated mRNAs are rapidly degraded in a tmRNA-dependent manner.

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Figure 2. Cleavage of ompC and ompF mRNA in response to serine starvation. W3110 (wild-type) and TA331 (ΔssrA) cells were grown in LB medium at 37°C. At A600 = 0.5, 0.1 mg ml−1 SHX was added and cells were further incubated for 20 min. Total RNAs were prepared at indicated times and 5 μg of each RNA was resolved by electrophoresis on a 1.5% agarose-formaldehyde gel. Northern blot analysis was performed using DIG-labelled 5′ompF (A) and ompC (B) probes.

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The mRNA cleavage occurs independently of bacterial toxins and ppGpp

It is known that the activity and expression of bacterial toxins such as RelE and MazF increase upon amino acid starvation and that these toxins act to cleave translated mRNAs in codon-specific manners (Christensen and Gerdes, 2003; Christensen et al., 2003). Therefore, the mRNA cleavage induced by SHX addition could be a result of RelE and/or MazF action. In addition or alternatively, the mRNA cleavage under the serine starvation condition could occur independently of toxins. To test these possibilities, we first examined the effect of overexpression of RelE or MazF on ompA mRNA by Northern analysis. The PCR-amplified relE or mazF gene was cloned into a plasmid in which the transcription of toxin genes is under the control of an arabinose-inducible promoter. Each of these plasmids was introduced into cells lacking tmRNA. Cells were grown in LB medium to mid-log phase and RelE or MazF was induced for 5 min by arabinose. Total RNAs were prepared and subjected to Northern blotting. The ompA mRNA was significantly degraded by the induction of RelE or MazF as expected (Fig. 3A, lanes 2 and 4). However, the cleavage patterns of ompA mRNA by RelE or MazF are apparently different from that caused by serine starvation (Fig. 3A, lane 5), suggesting that the mRNA cleavage caused by serine starvation is not the result of RelE and/or MazF action.

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Figure 3. Cleavage of mRNA is independent on bacterial toxins and ppGpp. A. Effect of overexpression of RelE and MazF on ompA mRNA. TA331 (ΔssrA) cells harbouring pRelE (lanes 1 and 2) or pMazF (lanes 3 and 4) were grown in LB medium at 37°C. At A600 = 0.5, 0. 01% arabinose was added and cells were incubated for 5 min. Total RNAs were prepared before (lanes 1 and 3) and after (lanes 2 and 4) arabinose addition. Five micrograms of each RNA was resolved by electrophoresis on a 1.5% agarose-formaldehyde gel. Northern blot analysis was performed using the DIG-labelled 5′ompA probe. Lane 5 represent the RNA sample prepared from TA331 (ΔssrA) cells treated with 0.1 mg ml−1 SHX for 20 min. B. Effect of SHX on relE expression. TA331 (ΔssrA) cells were grown in LB medium at 37°C. At A600 = 0.5, 0.1 mg ml−1 (lanes 1–4) or 1 mg ml−1 (lanes 5–8) SHX was added and incubation was continued. Total RNAs were prepared at indicated times and 5 μg of each RNA was resolved by electrophoresis on a 1.5% agarose-formaldehyde gel. Northern blot analysis was performed using the DIG-labelled relE probe. C. Generation of mRNA fragments by SHX treatment in cells lacking bacterial toxins. SC301467 (Δ5) and XL001 (Δ5 (ΔssrA) cells were grown in LB medium at 37°C. At A600 = 0.5, 0.1 mg ml−1 SHX was added and incubation was continued. Total RNAs were prepared at indicated times and 5 μg of each RNA was resolved by electrophoresis on a 1.5% agarose-formaldehyde gel. Northern blot analysis was performed using the DIG-labelled 5′ompA probe. D. Generation of mRNA fragments by SHX treatment in cells lacking ppGpp. MY001 (relA spoT) and MY002 (relA spoT ssrA) cells were grown in LB medium at 37°C. At A600 = 0.5, indicated concentrations of SHX were added and incubation was continued for 20 min. Total RNAs were prepared and 5 μg of each RNA was resolved by electrophoresis on a 1.5% agarose-formaldehyde gel. Northern blot analysis was performed using the DIG-labelled 5′ompA probe.

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Next, we examined the expression of relE mRNA during serine starvation by Northern blotting. The transcription of relE mRNA increased in response to serine starvation when the concentration of SHX was 1 mg ml−1 (Fig. 3B, lanes 5–8) as reported previously (Christensen et al., 2001). Importantly, however, the level of relE mRNA remained unchanged when the concentration of SHX was 0.1 mg ml−1 (Fig. 3B, lanes 1–4) where the mRNA cleavages already occurred efficiently, suggesting that RelE is not activated yet therefore not involved in the mRNA cleavage in response to mild serine starvation conditions.

Finally, we examined the effect of serine starvation on ompA mRNA in cells lacking five TA loci (Christensen et al., 2004). Total RNAs prepared at various times after SHX addition were subjected to Northern blotting. Truncated mRNAs were efficiently produced by SHX treatment in cells lacking all five TA genes in the absence of tmRNA (Fig. 3C, lanes 1–5) but not in the presence of tmRNA (Fig. 3C, lanes 6–10). Thus, the deletion of all five TA genes essentially did not affect the extent and pattern of ompA mRNA cleavage by SHX treatment (see Fig. 2A and 3C). Taken together, we conclude that the mRNA cleavage induced by serine starvation is independent of bacterial toxins including RelE and MazF.

We also tested whether ppGpp is involved in the mRNA cleavage in response to serine starvation. For this, relA spoT (ppGpp°) strains both in ssrA+ and in ssrA- background were grown to mid-log phase in LB medium and treated with low concentrations of SHX for 20 min. Total RNAs were prepared and subjected to Northern blot analysis using a 5′ompA DNA probe. Truncated mRNAs were again efficiently produced by SHX treatment in ssrA- background (Fig. 3D, lanes 3 and 5), implying that the mRNA cleavage under amino acid starvation occurs in the absence of ppGpp. Interestingly, significant amounts of truncated mRNAs were still observed even in ssrA+ background (Fig. 3D, lanes 4 and 6), suggesting that non-stop mRNAs become more resistant to the tmRNA system without ppGpp.

The mRNA cleavage occurs around serine codons

Our data strongly suggest that the serine starvation leads to ribosome stalling at serine codons which in turn causes the mRNA cleavage in a toxin-independent manner. If this is the case, the mRNA cleavage should be serine codon dependent and is expected to occur at or near serine codons. To test this, we analysed the cleavage pattern of mRNAs derived from a series of plasmid-born mutant crp genes. The wild-type crp gene contains 11 serine codons that are located at various positions (Aiba et al., 1982). We classified these serine codons into five groups: group 1 (codons 16, 25 and 27); group 2 (codons 46 and 62); group 3 (codons 83 and 98); group 4 (codons 117 and 128); group 5 (codons 179 and 197) (Fig. 4). The crp gene on pHA7M (Abo et al., 2000) was manipulated to construct a series of mutant crp genes in which serine codons of each group were progressively replaced to threonine codons. The constructed plasmids are pXL11, pXL12, pXL13, pXL14 and pXL15, carrying a variant crp lacking serine codons of group 1, groups 1–2, groups 1–3, groups 1–4 and groups 1–5 respectively (Fig. 4).

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Figure 4. Schematic drawing of the location of serine codons in crp variants. Plasmid pHA7M carries the wild-type crp gene which contains 11 serine codons. These serine codons, represented by S with the position number, are classified into five groups. Plasmids pXL11, pXL12, pXL13, pXL14 and pXL15 carry a variant crp gene in which all serine codons of clusters 1, 2, 3, 4 and 5 are replaced to threonine codons (T) respectively. Plasmids pXL34 carries a variant crp gene in which a nucleotide sequence corresponding to four serine codons is inserted just after the serine codon 179 of pXL14.

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Each of these plasmids was introduced into cells with or without tmRNA and the effect of serine starvation on mRNAs was analysed by Northern blotting. Truncated crp mRNAs of various sizes were produced upon the addition of SHX along with the full-length crp mRNA of about 700 bases from pHA7M carrying the wild-type crp gene. Some of the truncated fragments were sensitive to tmRNA while others were not affected by the presence of tmRNA (Fig. 5, lanes 1 and 2). The shortest tmRNA-sensitive mRNA fragments derived from pHA7M (cluster 1) were no longer produced when the crp variant lacking group 1 serine codons (pXL11) was expressed (Fig. 5, lane 3 and 4), suggesting that these mRNA fragments correspond to group 1 serine codons. Similarly, mRNA fragments represented by clusters 2, 3, 4 and 5, derived from pXL11, pXL12, pXL13 and pXL14, seem to correspond to groups 2, 3, 4 and 5 serine codons because each of these clusters disappeared progressively when pXL12, pXL13, pXL14 and pXL15 were expressed respectively (Fig. 5, lanes 5–12). Essentially, no tmRNA-sensitive mRNA fragments were produced when the mutated crp gene carrying no serine codon was expressed (Fig. 5, lanes 11 and 12). These data strongly suggest that mRNA cleavage induced by serine starvation occurs at or near serine codons where ribosome stalling is expected to occur.

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Figure 5. Cleavage of mRNA occurs around serine codons. A. Cleavage of crp mRNA by SHX treatment. TA341 (Δcrp) and TA501 (ΔcrpΔssrA) cells harbouring indicated plasmids were grown in LB medium at 37°C. At A600 = 0.5, 0.1 mg ml−1 SHX was added and cells were further incubated for 20 min. Total RNAs were prepared and 5 μg of each RNA was resolved by electrophoresis on a 1.8% agarose-formaldehyde gel. Northern blot analysis was performed using the DIG-labelled 5′crp probe. Clusters 1, 2, 3, 4 and 5 correspond to the shortest tmRNA-sensitive mRNA fragments generated from pHA7M, pXL11, pXL12, pXL13 and pXL14 respectively. B. Introduction of consecutive serine codons enhances mRNA cleavage by SHX treatment. TA341 (Δcrp) and TA501 (ΔcrpΔssrA) cells harbouring pXL34 were grown in LB medium at 37°C. At A600 = 0.5, 0.1 mg ml−1 SHX was added and cells were further incubated for 20 min. Total RNAs were prepared and 5 μg of each RNA was resolved by electrophoresis on a 1.8% agarose-formaldehyde gel. Northern blot analysis was performed using the DIG-labelled 5′crp probe.

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We also constructed plasmid pXL34 in which four consecutive serine codons were introduced right after the serine codon 179 of pXL14 (Fig. 4). A clear tmRNA-sensitive fragment was produced when pXL34 was expressed in ssrA- cells, indicating that increasing number of serine codons induced a more efficient ribosome stalling therefore mRNA cleavage (Fig. 5B). These results are consistent with our previous finding that consecutive rare codons lead to ribosome stalling resulting in mRNA cleavage and protein tagging (Li et al., 2006).

Serine starvation enhances tmRNA-mediated protein tagging

Major truncated mRNAs generated by serine starvation are sensitive to tmRNA. Therefore, the C-terminus of proteins translated from these truncated mRNAs would be tagged by the tmRNA system. To detect the tmRNA-mediated protein tagging, pXL14 carrying the crp gene possessing serine codons at positions 179 and 197 was expressed in three isogenic strains with different ssrA allele (ssrA+, ΔssrA and ssrADD). Cell lysates were prepared and analysed by Western blotting with anti-CRP antibody. A very weak signal corresponding to a DD-tagged truncated CRP was detected in ssrADD cells (data not shown). Then, plasmid pXL34 was expressed in ssrA+, ΔssrA and ssrADD cells, and the lysates were analysed by Western blotting using anti-CRP and ant-DD-tag antibodies (Fig. 6A). A significant tmRNA taggingof a truncated CRP was observed when pXL34 was expressed in ssrADD cells (Fig. 6A). We confirmed that the DD-tagging occurs within the serine clusters by mass spectrometry analysis of the purified DD-tagged CRP (data not shown).

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Figure 6. Serine starvation enhances tmRNA-mediated protein tagging. A. Western blot analysis of CRP expressed from pXL34. TA501 (ΔcrpΔssrA), TA341 (Δcrp ssrA+) and TA481 (Δcrp ssrADD) cells harbouring pXL34 were grown in LB medium at 37°C. At A600 = 0.5, 0.1 mg ml−1 SHX was added and cells were further incubated for 30 min. The crude extracts were prepared and analysed by Western blotting using anti-CRP (lanes 1–3) and anti-DD-tag (lane 4) antibodies. Lysates equivalent to 0.02 OD600 units were loaded onto the gel. B and C. Effect of SHX treatment on tagging of cellular proteins. TA481 (Δcrp ssrADD) cells were grown in LB medium at 37°C. At A600 = 0.5, 0.1 mg ml−1 SHX was added and incubation was continued. The crude extracts were prepared at indicated times after the addition of SHX and analysed by Western blotting using anti-DD antibodies (B) and SDS-polyacrylamide gel electrophoresis followed by Coomassie Blue staining (C). Lysates equivalent to 0.002 and 0.02 OD600 units were loaded onto the gel for Western blotting and Coomassie staining respectively.

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Ribosome stalling therefore mRNA cleavage should occur more or less at every serine codon in all mRNAs being translated under serine starvation. Therefore, serine starvation would generate a large number of truncated mRNAs that are recognized by the tmRNA system. Thus, serine starvation is expected to enhance overall protein tagging mediated by tmRNA. To test this, TA371 strain (ssrADD) was treated with SHX and cell extracts were analysed by Western blotting using anti-DD-tag antibodies. The SHX treatment indeed significantly enhanced tmRNA-mediated tagging of many proteins (Fig. 6B). Under the same conditions, the profile of total proteins detected by Coomassie staining remained unchanged (Fig. 6C).

The tmRNA system rescues growth inhibition caused by serine starvation

To examine how serine starvation affects growth of both ssrA+ and ssrA- strains, cells were grown to mid-log phase in LB medium. The cultures were divided and growth was continued in the absence or presence of increasing concentrations of SHX. The addition of a high concentration of SHX (0.4 mg ml−1) completely inhibited cell growth of irrespective of ssrA allele (Fig. 7). On the other hand, two strains exhibited different sensitivities to SHX when its concentration was less than 0.2 mg. For example, growth of ssrA- cells was significantly inhibited when 0.1 mg ml−1 SHX was added (Fig. 7B) while no growth inhibition was observed in ssrA+ cells under the same condition (Fig. 7A). This implies that the tmRNA system is able to rescue the growth inhibition caused by a moderate serine starvation. It should be noted that the toxin-independent mRNA cleavage efficiently occurs without activation of toxins under this condition (Figs 1 and 3B). These results strongly suggest that generation of non-stop mRNAs by the toxin-independent mRNA cleavage is primarily responsible for the growth inhibition in ssrA+ cells under a moderate serine starvation condition and that the tmRNA-mediated quality-control system helps cells cope with problems caused by non-stop mRNAs. On the other hand, when 0.4 mg ml−1 SHX was added, cell growth was almost completely inhibited irrespective of ssrA allele. Both strong translational inhibition by a serious depletion of seryl-tRNA and activation of toxins would be responsible for the severe growth defect under a strong serine starvation condition. Interestingly, the growth defect under this condition is not rescued by tmRNA system. This is consistent with the observation that the tmRNA itself is destructed under a strong serine starvation condition (Christensen and Gerdes, 2003; Christensen et al., 2003).

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Figure 7. Effect of SHX on cell growth. W3110 (wild-type) and TA331 (ΔssrA) cells were grown in LB medium at 37°C. At A600 = 0.4 (arrow), indicated concentrations of SHX were added and incubation was continued. Cell growth was monitored by measuring optical density at 600 nm.

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Depletion of casamino acid induces mRNA cleavages

So far, we focused on the effect of serine starvation on mRNAs. We also examined whether general amino acid starvation could induce mRNA cleavage. For this, both ssrA+ and ssrA- strains were grown to mid-log phase in M9-glucose medium supplemented with casamino acid. Cells were collected by centrifugation, re-suspended in fresh M9-glucose medium with or without casamino acid, and the incubation was continued. Total RNAs were prepared 20 min after the medium change and subjected to Northern blotting using appropriate DNA probes. The results of ompA and ompC mRNAs were shown in Fig. 8A and B respectively. When casamino acid was depleted, truncated fragments of ompA and ompC mRNAs with various lengths were observed in ssrA- cells lacking tmRNA (Fig. 8A and B, lane 3). These truncated mRNAs were no longer observed in ssrA+ cells possessing tmRNA (Fig. 8A and B, lane 4), suggesting that the truncated mRNAs were rapidly degraded depending on tmRNA, as demonstrated previously. Essentially no truncated mRNAs were observed in cells grown in the presence of casamino acid (Fig. 8A and B, lanes 1 and 2). Thus, generation of truncated mRNAs is associated with the depletion of casamino acid. We conclude that general amino acid starvation also induces mRNA cleavages resulting in truncated mRNAs.

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Figure 8. Effect of depletion casamino acid on mRNAs. W3110 (wild-type) and TA331 (ΔssrA) cells were grown in M9-glucose medium containing casamino acid at 37°C to A600 = 0.5. The cells were collected by a centrifugation, re-suspended in fresh M9-glucose medium with or without casamino acid and incubated for 20 min. Total RNAs were prepared and 5 μg of each RNA was resolved by electrophoresis on a 1.5% agarose-formaldehyde gel. Northern blot analysis was performed using DIG-labelled 5′ompA (A) and ompC (B) probes.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

In bacteria, amino acid starvation causes pleiotropic physiological changes, called stringent response (Cashel et al., 1996). The well-known physiological outcomes of stringent response, inhibition of transcription of stable RNAs and stimulation of transcription of amino acid biosynthetic operons, are mediated by a rapid increase in the synthesis of a signalling molecule guanosine tetraphosphate (ppGpp). The rapid synthesis of ppGpp under amino acid starvation depends on the relA encoding ppGpp synthetase, and requires the presence of stalled ribosomes and uncharged tRNAs. Another profound ppGpp-dependent event under amino acid starvation is degradation of ribosomal proteins that is an important source of free amino acids required for the synthesis of enzymes needed (Kuroda et al., 2001). The degradation of ribosomal proteins under amino acid starvation is caused through activation of Lon protease that is mediated by ppGpp-dependent accumulation of inorganic polyphosphate. Some physiological consequences of stringent response such as the activation of toxins seem to occur independently of ppGpp. For example, the relEB encodes the RelE toxin and RelB antitoxin that neutralizes the RelE toxicity under normal growth conditions. RelB also acts as an autorepressor of the relEB operon. Amino acid starvation causes Lon-mediated degradation of RelB, which in turn leads to activation of RelE as well as increase of the relEB transcription (Christensen et al., 2001). RelE was shown to inhibit translation by cleaving mRNAs being translated (Christensen and Gerdes, 2003). Other toxins, ChpAK and ChpBK, were also shown to inhibit translation by inducing cleavage of mRNAs (Christensen et al., 2003). Interestingly, the activation of toxins is independent of ppGpp (Christensen et al., 2001; 2003).

The present study has shed a light on an additional aspect of stringent response. The major finding is that ribosome stalling caused by amino acid starvation is likely to induce endonucleolytic cleavages of mRNAs in a toxin-independent manner resulting in non-stop mRNAs that are recognized by the tmRNA system. This conclusion is based on the following observations: (i) serine starvation caused by SHX treatment leads to concomitant accumulation of both 5′ and 3′ mRNA fragments (Fig. 1), (ii) the 5′ but not 3′ mRNA fragments are degraded in tmRNA-dependent fashion (Figs 1 and 2), (iii) mRNA cleavage patterns induced by overproduction of RelE or MazF are distinguished from that produced under serine starvation (Fig. 3A), (iv) RelE is not activated in the presence of a moderate concentration of SHX in which the mRNA cleavages occur efficiently (Fig. 3B), (v) cells lacking all of five TA loci are proficient in mRNA cleavage in response to serine starvation (Fig. 3C), and (vi) the substitution of serine codons to threonine codons within a model gene eliminates the cleavage at the corresponding positions (Fig. 5).

How does our conclusion reconcile with the previous reports that bacterial toxins such as RelE and MazF are activated and/or induced in response to amino acid starvation and act to cleave translated mRNAs (Christensen et al., 2001; 2003; Christensen and Gerdes, 2003)? It is certainly true that artificial overproduction of toxins causes mRNA cleavages. An important finding, however, is that RelE is not induced yet under a modest starvation condition where the mRNA cleavages occur already efficiently. The induction of RelE and probably other toxins occurs only in the presence of high concentrations of SHX. In other words, the toxin-mediated mRNA cleavage becomes significant only when cells meet a strong starvation condition. Thus, mRNA cleavage under a moderate amino acid starvation occurs independently of toxins in response to ribosome stalling as observed in other situations associated with ribosome stalling. It is likely that both toxin-independent and toxin-dependent mRNA cleavages occur under a strong amino acid starvation condition in which toxins are activated. However, the toxin-independent mRNA cleavage appears to be predominant even in that case because increasing concentrations of SHX did not alter significantly the cleavage patterns of ompA mRNA (Fig. 1A).

We also found the mRNA cleavage in response to serine starvation is totally independent of ppGpp (Fig. 3D). Although this does not tell us how the ribosome stalling leads to mRNA cleavage, it raises again the possibility that components of ribosome and/or its associated molecules may mediate the cleavage reaction. The identification of factor(s) involved in the mRNA cleavage in response to ribosome stalling is still an important unsolved question to understand the mechanism of the quality-control system mediated by ribosome and tmRNA.

The observation that a mild serine depletion causes a significant growth inhibition in ssrA deletion cells but not in wild-type cells clearly illuminates the biological role of the tmRNA system under a stress condition (Fig. 7). Namely, the tmRNA system is acting to deal with the problems associated with mRNA cleavages due to ribosome stalling under amino acid starvation. This situation is quite similar to what we observed when cells were treated with sublethal concentrations of miscoding antibiotics such as streptomycin (Abo et al., 2002). We showed previously that low concentrations of miscoding drugs generate target mRNAs for tmRNA by translational readthrough of stop codons and that the tmRNA system confers cells a partial resistance to miscoding drugs by dealing with problems associated with translational readthrough (Abo et al., 2002). In addition, it is known that tmRNA-deleted cells recover slowly after carbon starvation back (Oh and Apirion, 1991). It remains to be studied whether carbon starvation also leads to mRNA cleavages. The tmRNA system was also shown to alleviate partially growth defect caused by overexpression of bacterial toxins (Christensen and Gerdes, 2003; Christensen et al., 2003). However, we would like to emphasize again that the expression of chromosomal relE is activated only under a strong serine starvation condition in which the tmRNA system is apparently not functional (Fig. 1A and 3B). This explains well why the tmRNA system no longer alleviates the growth defect under a strong serine starvation condition. Thus, our data strongly suggest that there is no situation in which the tmRNA system acts efficiently to deal with the toxin-mediated mRNA cleavages under physiological stress condition in wild-type cells. Finally, we would like to point out an additional physiological role of tmRNA system under amino acid starvation. Namely, the tmRNA-mediated degradation of aberrant proteins would be certainly useful to provide free amino acids along with PolyP/Lon-mediated degradation of ribosome proteins under amino acid starvation conditions as proposed by Kenn Gardes and colleagues (Christensen et al., 2003).

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Media and growth condition

Cells were grown aerobically in LB or M9 minimum medium supplemented with indicated sugars at indicated temperatures. Antibiotics were used at the following concentrations when needed: ampicillin (50 μg ml−1), kanamycin (30 μg ml−1), tetracycline (15 μg ml−1) and chloramphenicol (15 μg ml−1). Bacterial growth was monitored by determining the optical density at 600 nm.

Strains and plasmids

The Escherichia coli K-12 strains used in this study are listed in Table 1. The ΔssrA::KmR allele was transferred from W3110 ssrA (Komine et al., 1994) to SC301467 (Δ5) (Christensen et al., 2004) by P1 transduction to construct XL001. The spoT204::CmR allele (Xiao et al., 1991) was transferred from W3110 relA251::KmRspoT204::CmR to MC4100 (Casadaban, 1976) and MC4100 ssrA (Abo et al., 2000) by P1 transduction to construct MY001 and MY002 respectively. Plasmids pXL11, pXL12, pXL13, pXL14 were constructed from pHA7M (Abo et al., 2000), pXL11, pXL12 and pXL13 by PCR mutagenesis using appropriate primers respectively. Plasmids pXL15 and pXL34 were constructed from pXL14 by PCR mutagenesis using appropriate primers. To construct plasmids pRelE and pMazF, DNA fragments containing the relE and mazF coding region including ribosome binding site were amplified by PCR using appropriate primers. The amplified fragments were digested with SacI and HindIII and the resulting each fragment containing the relE or mazF region was inserted into the SacI and HindIII region of pBAD33 (Guzman et al., 1995).

Table 1.  Bacterial strains and plasmids used in this study.
Strain/plasmidRelevant genotype and propertySource
Strain
 W3110Wild typeLaboratory stock
 TA331W3110 ΔssrA::FRTAbo et al. (2002)
 TA371W3110 ssrADD-FRTAbo et al. (2002)
 TA341W3110 ΔcrpSunohara et al. (2004b)
 TA501W3110 ΔcrpΔssrA::FRTSunohara et al. (2004b)
 TA481W3110 Δcrp ssrADD-FRTSunohara et al. (2004b)
 SC301467 (Δ5)MG1655 ΔmazFΔchpBΔrelBEΔ(dinJ-yafQ) Δ(yefM-yoeB)Christensen et al. (2004)
 XL001MG1655 ΔmazFΔchpBΔrelBEΔ(dinJ-yafQ) Δ(yefM-yoeB) ΔssrA::KmRThis work
 W3110 ssrAW3110 ΔssrA::KmRKomine et al. (1994)
 MC4100relA1Casadaban (1976)
 MC4100 ssrArelA1ΔssrAAbo et al. (2000)
 W3110relA spoTrelA251::KmRspoT204::CmR 
 MY001relA1 spoT204::CmRThis work
 MY002relA1 spoT204::CmRΔssrAThis work
Plasmid
 pHA7MpBR322 derivative carrying Pbla-crpAbo et al. (2000)
 pXL11pHA7M derivative (first 3 serine codons of crp are changed to threonine codons)This work
 pXL12pHA7M derivative (first 5 serine codons of crp are changed to threonine codons)This work
 pXL13pHA7M derivative (first 7 serine codons of crp are changed to threonine codons)This work
 pXL14pHA7M derivative (first 9 serine codons of crp are changed to threonine codons)This work
 pXL15pHA7M derivative (all of serine codons of crp are changed to threonine codons)This work
 pXL34pHA7M derivative (4 consecutive serine codons are inserted after S179 codon of crpThis work
 pBAD33pACYC184 derivative carrying PBADGuzman et al. (1995)
 pRelEpBAD33 derivative carrying PBAD-relEThis work
 pMazFpBAD33 derivative carrying PBAD-mazFThis work

RNA analyses

Total RNA was isolated from cells grown in LB medium or M9 minimum medium to mid-log phase as described (Aiba et al., 1981). For Northern blot analysis, indicated amount of total RNA was resolved by 1.5% agarose-gel electrophoresis in the presence of formaldehyde and blotted onto a Hybond-N+ membrane (Amersham). The mRNAs were visualized using digoxigenin (DIG) reagents and kits for non-radioactive nucleic acid labelling and detection system (Roche) according to the procedure specified by the manufacturer. The DIG-labelled DNA probes used were 200 bp fragment corresponding to the 5′ompA mRNA, 280 bp fragment corresponding to the 3′ompA mRNA, 200 bp fragment corresponding to the 5′ompF mRNA, 200 bp fragment corresponding to the 5′ompC mRNA, 150 bp fragment corresponding to the 5′relE mRNA and 120 bp fragment corresponding to the 5′crp mRNA. The DIG-labelled RNA marker III (Roche) was used to estimate the size of RNA bands.

Protein analyses

Cells were grown under the conditions described. The culture samples (1 ml) were harvested and suspended in 50 μl of H2O. The cell suspensions were mixed with 50 μl of 2× loading buffer (125 mM Tris-HCl at pH 6.8, 4% SDS, 10% glycerol, 10% β-mercaptoethanol, 0.2% bromophenol blue) and heated at 100°C for 5 min. Indicated amount of protein samples were subjected to a 12% or 15% polyacrylamide-0.1% SDS gel electrophoresis and transferred to Immobilon membrane (Milipore). The membranes were probed with anti-CRP and anti-DD-tag antibodies (Ishizuka et al., 1993; Abo et al., 2000) using ECL system (Amersham Life Science). The total cellular proteins were also analysed by a 0.1% SDS-12% polyacrylamide gel electrophoresis followed by Coomassie brilliant blue staining.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We thank Dr K. Gerdes (Newcastle University) for strain SC301467 (Δ5) and Dr T. Oshima (Nara Institute of Science and Technology) for strain W3110relA spoT. This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by Ajinomoto Co., Inc.

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  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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