The Escherichia coli mazEF Suicide Module Mediates Thymineless Death

In 1954, Cohen and Barner discovered that a thymine auxotrophic(thyA) mutant of Escherichia coli undergoes cell death in responseto thymine starvation. This phenomenon, called thymineless death(TLD), has also been found in many other organisms, includingprokaryotes and eukaryotes. Though TLD has been studied intensively,its molecular mechanism has not yet been explained. Previouslywe reported on the E. coli mazEF system, a regulatable chromosomalsuicide module that can be triggered by various stress conditions.MazF is a stable toxin, and MazE is an unstable antitoxin. Here,we show that cell death that is mediated by the mazEF modulecan also be activated by thymine starvation. We found that TLDdepends on E. coli mazEF and that under thymine starvation,the activity of the mazEF promoter P₂ is significantly reduced.Our results, which describe thymine starvation as a triggerfor a built-in death program, have implications for programmedcell death in both prokaryotes and eukaryotes.

INTRODUCTION

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Introduction

As early as 1954, Cohen and Barner discovered that a thymineauxotrophic (thyA) mutant of Escherichia coli undergoes celldeath in response to thymine starvation (10). This phenomenon,called thymineless death (TLD) (7), has also been found in manyother organisms, including prokaryotes and eukaryotes (for arecent review, see reference 2). TLD is a unique effect, sincestarvation of bacteria for the other bases (adenine, guanine,or cytosine) or other nutrients, like amino acids or vitamins,causes growth to stop (i.e., is bacteriostatic) but does notcause cell death (i.e., is not bactericidal) (6). However, despiteintensive research on TLD, its molecular mechanism has not yetbeen explained. Though many molecular events have been implicatedin TLD, no compelling direct relationship has been found amongthem, and thus, no definite hypothesis has been formulated (2).

Most of the studies of TLD have been made in E. coli, wherethe first step in dTTP synthesis is the conversion of dUMP todTMP. This conversion is catalyzed by thymidylate synthetase,which is encoded by the thyA gene (5), and involves the transferof a C-1 unit from N⁵,N¹⁰-methylene tetrahydrofolate to the5 position of dUMP (2); tetrahydrofolate is consumed in thisreaction (15). Thus, thymine starvation in E. coli can be inducedin several ways that inhibit the synthesis of thymidylate eitherdirectly or indirectly by interfering with folate metabolism(2).

We asked whether thymine starvation is a trigger for a built-indeath program. In E. coli, programmed cell death (PCD) is mediatedthrough special genetic elements called addiction modules (forreviews, see references 11, 13, 16, and 31). These modules consistof two genes, where the second gene encodes a stable toxin andthe first gene encodes a labile antitoxin. Addiction moduleswere first discovered in a number of extrachromosomal elements,where they were found to be responsible for the postsegregationalkilling effect, that is, the death of cells from which theseextrachromosomal elements have been lost. Thus, the cells are"addicted" to the short-lived antitoxin product, since its denovo synthesis is essential for cell survival (11, 13, 16, 31).

Pairs of genes homologous to some of these extrachromosomaladdiction modules have been found on the E. coli chromosome(3, 16, 17, 21-23). The E. coli mazEF system, the first knownregulatable prokaryotic chromosomal addiction module, has beenreported (3). The mazEF module consists of two adjacent genes,mazE and mazF, located in the rel operon downstream from therelA gene (23). In the study (3), it was found that mazEF hasthe properties required for an addiction module: (i) MazF istoxic, and MazE is antitoxic; (ii) MazF is long-lived, whileMazE is a labile protein degraded in vivo by the ATP-dependentClpPA serine protease; (iii) MazE and MazF interact; (iv) MazEand MazF are coexpressed; and (v) The two mazEF promoters (P₂and P₃) are strongly negatively autoregulated by the combinedaction of MazE and MazF (20). Another significant characteristicof the mazEF system is that its transcription from the P₂ promoteris inhibited by high concentrations of guanosine 3',5'-bispyrophosphate(ppGpp) (3), which is synthesized by the RelA protein underconditions of extreme amino acid starvation (9). Based on theseproperties of mazEF, and on the requirement for the continuousexpression of MazE to prevent cell death, a model for PCD underconditions of nutritional stress (3) or under conditions inwhich the synthesis of RNA and/or protein are generally inhibited(19, 27) was presented. The model was supported by the resultsof previous experiments showing that mazEF-mediated cell deathcan be triggered by (i) the artificial overproduction of ppGpp(3, 14), (ii) several antibiotics (rifampin, chloramphenicol,and spectinomycin) that are general inhibitors of transcriptionand/or translation (12, 27), or (iii) the Doc protein, the toxicproduct of the addiction module phd-doc of the plasmid prophageP1, which is a general inhibitor of translation (19).

Here, we asked whether mazEF-mediated cell death can be triggeredin E. coli under conditions of thymine starvation. We generatedthe condition of thymine starvation in three ways: (i) we causedthe inhibition of folate metabolism by adding trimethoprim toinhibit dihydrofolate reductase (4), (ii) we caused the inhibitionof folate metabolism by adding sulfonamides to block folatesynthesis (28), and (iii) we grew a thymine auxotrophic (thyA)mutant in a medium lacking thymine (1, 10). We found that inE. coli thymine starvation does trigger mazEF-mediated celldeath. Furthermore, under thymine starvation, the activity ofthe mazEF promoter P₂ is significantly reduced. Thus, the resultsof our work showing that thymine starvation is a trigger fora built-in death program provide a new insight into an old enigma.

MATERIALS AND METHODS

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Materials and Methods

Materials and media. We obtained [6-³H]thymidine (23.0 Ci/mmol) and [5,6-³H]uracil(45 Ci/mmol) from Amersham Pharmacia Biotech (Little Chalfont,United Kingdom) and [³⁵S]methionine (1,175 Ci/mmol) from NEN(Boston, Mass.). Trimethoprim, sulfamethoxazole (SMZ) and inosinewere obtained from Sigma (St. Louis, Mo.). We obtained avianmyeloblastosis virus reverse transcriptase from Promega (Madison,Wis.), we obtained polynucleotide kinase from U.S. BiochemicalCorp. (Cleveland, Ohio), and we obtained [ ${gamma}$ -³²P]ATP (3,000 Ci/mmol)from Amersham Pharmacia Biotech. The bacteria were grown eitherin M9 liquid medium with 1% glucose and a mixture of amino acids(20 µg/ml each) or in Luria-Bertani (LB) liquid mediumand/or agar plates (24).

Bacterial strains and plasmids. We used the following E. coli strains: MC4100relA⁺ [araD139 ${Delta}$ (argF-lac)205 flb5301 pstF25 rpsL150 deoC1) and its derivativeMC4100 ${Delta}$ mazEFrelA⁺ (mazEF::kan) (14). In addition, we used KL742( ${lambda}$ ^- thyA748::Tn10 rph-1 deo-77) (from the E. coli Genetic StockCenter) (K. B. Low, unpublished strains). Its ${Delta}$ mazEF derivativeKL742mazEF::kan and its ${Delta}$ clpP derivative KL742clpP::cat wereconstructed by P1 transduction from strains MC4100mazEF::kanand MC4100clpP::cat (3), respectively.

Plasmid pKK223mazEF (pmazEF) is a pKK223 derivative carryingmazEF (19). Plasmids pSK10 ${Delta}$ 6-p_ef and pIB13 are derivatives ofplasmid pSK10 ${Delta}$ 6, which bears the lac'Z gene lacking its firsteight codons. pSK10 ${Delta}$ 6-p_ef carries the end of the relA gene, themazEF promoter, and the first 17 codons of mazE fused to lac'Z(20). pIB13 carries the trpR promoter and the first 78 codonsof trpR fused to lac'Z (8).

Thymine starvation induced by the addition of trimethoprim or SMZ. Bacteria were grown in M9 minimal medium (with glucose and amixture of amino acids) with shaking (160 rpm) at 37°C.After overnight growth, the cultures were (i) diluted to 10⁶cells/ml in M9 medium supplemented with trimethoprim (0.1 µg/ml)and inosine (50 µg/ml) or (ii) diluted in M9 medium supplementedwith SMZ (10 µg/ml) and inosine (50 µg/ml). Inosinewas added as a purine source (28). While these diluted cultureswere growing at 37°C, samples were withdrawn at 30-min intervals,washed once with saline, and plated on LB plates that were incubatedat 37°C for 18 h for viable-cell counts.

Thymine starvation of a thymine auxotrophic (thyA) mutant. The E. coli thyA mutant strain KL742 was grown in M9 minimalmedium supplemented with thymine (50 µg/ml) plus glucoseand a mixture of amino acids with shaking (160 rpm) at 37°C.After overnight growth, the cultures were diluted 20-fold inthe same medium and allowed to continue growing with shakingat 37°C. When the culture reached the exponential phase(10⁸ cells/ml), 1.0-ml samples were withdrawn, centrifuged,washed two times with saline at 4°C, and resuspended in2.0 ml of fresh M9 medium without thymine. These prepared sampleswere incubated at 37°C with aeration (shaking at 160 rpm).At 30-min intervals, 0.1-ml samples were withdrawn, dilutedappropriately, and plated on LB nutrient agar plates supplementedwith thymine (50 µg/ml).

Assays for the effects of thymine starvation on the synthesis of DNA, RNA, and protein. Synthesis of DNA, RNA, and protein was determined by measuringthe incorporation of [6-³H]thymidine (0.2 µCi/ml), [5,6-³H]uracil(0.1 µCi/ml), or [³⁵S]methionine (24 µCi/ml), respectively,into a trichloroacetic acid (TCA)-insoluble fraction. We addedthis radioactive material to the cultures at the beginning ofthe period of thymine starvation, induced by growing the cellsin M9 medium in the presence of trimethoprim and inosine asdescribed above. At various times during thymine starvation,the reactions were stopped by adding TCA to 1-ml samples toa final concentration of 10%, and the mixtures were incubatedon ice for 30 to 60 min. The precipitates were filtered on Millipore(Bedford, Mass.) filters and washed with TCA (5%). The TCA solutionscontained 100 µg of nonradioactive thymidine or nonradioactiveuracil/ml. The filters were allowed to dry, and the TCA-insolublecounts were determined by using a scintillation counter.

Assay for the effect of thymine starvation on the activity of the mazEF promoter P₂ and the trpR promoter. E. coli strain MC4100relA⁺ harboring either plasmid pSK10 ${Delta}$ 6-p_efor plasmid pIB13 was grown in LB medium to 10⁸ cells/ml, andtrimethoprim (5 µg/ml) was added. At various times, RNAwas extracted, and primer extension was carried out with avianmyeloblastosis virus reverse transcriptase as described previously(20). The oligonucleotide primer used for the pSK10 ${Delta}$ 6-p_ef constructwas from positions -40 to -23 of the lacZ gene toward the transcriptionstart site of mazEF (20). The primer used for pIB13 was frompositions 82 to 59 of the trpR gene toward the promoter regionof trpR (18). The primers were end labeled by polynucleotidekinase with [ ${gamma}$ -³²P]ATP. The reaction products were resolved ona 6% sequencing gel, and DNA-sequencing reactions were performedwith the same primers and were run in parallel with the primerextension reaction as described previously (20). To quantifythe RNA levels, the gels were analyzed and the bands were quantifiedusing the Fujix BAS100 phosphorimager.

RESULTS

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Results

Thymine starvation by trimethoprim induces cell death through the mazEF system. First, we studied the effect on mazEF-mediated cell death ofthymine starvation induced by the addition of trimethoprim.Specifically, we compared the viability of the wild-type E.coli strain MC4100relA⁺ to that of its ${Delta}$ mazEF derivative afterexposure to trimethoprim. Growing the wild-type cells in thepresence of trimethoprim caused 96% of the cells to die by 1.0h of exposure and caused 99% of the cells to die by 2.5 h ofexposure (Fig. 1a). Since the addition of thymine completelyreversed this phenomenon, these data represent a TLD curve.In contrast to the wild-type strain, the ${Delta}$ mazEF derivative didnot die in the presence of trimethoprim, and close to 100% ofthese cells were viable even after 3.0 h (Fig. 1a). We alsotested the effect of trimethoprim in ${Delta}$ mazEF cells in which themazEF gene module was present, on plasmid pKK223mazEF ratherthan on the chromosome. Here again, we observed TLD: 97% ofthe cells died after 1.0 h. Thus, we found that TLD inducedby trimethoprim requires the mazEF system.

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FIG. 1. (a) Thymine starvation by trimethoprim in E. coli induces mazEF-mediated cell death. Cells were grown at 37°C in M9 liquid minimal medium plus 0.1 µg of trimethoprim/ml as described in Materials and Methods. Viability (% survivors) is plotted against the time of exposure to trimethoprim of E. coli MC4100relA⁺ (WT), MC4100 ${Delta}$ mazEFrelA⁺ ( ${Delta}$ mazEF), and MC4100 ${Delta}$ mazEFrelA⁺/pmazEF ( ${Delta}$ mazEF/pmazEF). MC4100relA⁺ was also grown in thepresence of 0.1 µg of trimethoprim/ml plus 50 µg of thymine/ml (WT+thymine). We calculated cell survival by comparing the colony-forming ability of trimethoprim-treated cells to that of untreated cells at zero time. The numbers represent the results of one out of five similar experiments. (b to d) Effect of thymine starvation on the synthesis of DNA (b), RNA (c), and protein (d) in MC4100relA⁺ and its ${Delta}$ mazEF derivative. Cells were grown at 37°C in M9 liquid medium as described in Materials and Methods. After overnight growth, the cultures were diluted to 10⁷ cells/ml and allowed to grow for 1 h in M9 medium plus inosine (50 µg/ml). Then, trimethroprim (2 µg/ml) was added; the cells were immediately labeled with either [6-³H]thymidine (b), [5,6-³H]uracil (c), or [³⁵S]methionine (d); and their incorporation into the acid-insoluble fraction was determined as described in Materials and Methods. As controls, we used MC4100relA⁺ ( ${blacktriangleup}$ ) and its ${Delta}$ mazEF derivative ( ${blacksquare}$ ) without the addition of trimethoprim. ${triangleup}$ , trimethoprim-treated MC4100relA⁺; ${square}$ , ${Delta}$ mazEF derivative.

In addition, we showed that the dependence of TLD on the functionof the mazEF system has no connection with changes in the synthesisof macromolecules, since we found that DNA synthesis was inhibitedin both the wild type and the ${Delta}$ mazEF derivative mutant (Fig.1b), while RNA and protein synthesis were unaffected (Fig. 1cand d).

Thymine starvation by a sulfonamide induces cell death through the mazEF system. We then studied the effect of the induction of thymine starvationby the sulfonamide SMZ on mazEF-mediated cell death. We comparedthe viability of the wild-type E. coli MC4100relA⁺ to the viabilityof its ${Delta}$ mazEF derivative after exposing each of the strains toSMZ (Fig. 2). Death of the wild-type strain occurred later inthe presence of SMZ (Fig. 2) than in the presence of trimethoprim(Fig. 1a). In particular, 90% of the wild-type strain died after6 h of exposure to SMZ (Fig. 2). In contrast, under identicalconditions, we observed no killing of the ${Delta}$ mazEF derivative atall (Fig. 2); in fact, this ${Delta}$ mazEF strain continued growing atthe same rate as the wild-type strain when thymine was addedto reverse the effect of SMZ (Fig. 2). Thus, as when we inducedthymine starvation by the addition of trimethoprim (Fig. 1a),TLD was also clearly mazEF dependent in the presence of SMZ(Fig. 2).

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FIG. 2. Thymine starvation by SMZ in E. coli induces mazEF-mediated cell death. Viability (% survivors) is plotted against time of exposure to SMZ (10 µg/ml) of E. coli MC4100relA⁺ (WT) and its ${Delta}$ mazEF derivative ( ${Delta}$ mazEF) in M9 liquid medium at 37°C. MC4100relA⁺ was also grown in the presence of 10 µg of SMZ/ml plus 50 µg of thymine/ml (WT+Thymine). Cell survival was determined as described in the legend to Fig. 1a.

Thymine starvation of a thyA mutant induces cell death through the mazEF system. Finally, we studied the involvement of the mazEF module in TLDinduced by the lack of thymine in a thymine auxotrophic (thyA)mutant. For this pur pose, we used E. coli strain KL742, whichcarries the mutation thyA748::Tn10. Here, we observed 95% celldeath after 1.5 h and 99% cell death after 2.0 h of growth inthe absence of thymine. However, we observed almost no killingduring this period in either ${Delta}$ mazEF or ${Delta}$ clpP derivatives of E.coli KL742 thyA (Fig. 3). Thus, TLD induced by thymine starvationin a thymine auxotrophic mutant is both mazEF and clpP dependent.

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FIG. 3. Thymine starvation in an E. coli thyA mutant induces mazEF-mediated cell death. Viability (% survivors) is plotted against times of growth of E. coli strains KL742thyA748::Tn10 (WT), KL742thyA748::Tn10 ${Delta}$ mazEF ( ${Delta}$ mazEF), and KL742thyA748::Tn10 ${Delta}$ clpP (clpP -) at 37°C in M9 liquid medium lacking thymine. Cell survival was determined as described in the legend to Fig. 1a.

Thymine starvation in E. coli reduces transcription from the mazEF promoter P_2. We asked whether thymine starvation affects transcription fromthe mazEF promoter. To test the mazEF promoter activity, weused plasmid pSK10 ${Delta}$ 6-p_ef, which was previously constructed forthis purpose (20). It carries a gene fusion in which the beginningof mazE is fused to the eighth codon of lacZ (see Materialsand Methods). We transformed this plasmid into E. coli MC4100relA⁺. The cells harboring the plasmid were starved for thyminewith trimethoprim, and during starvation, the activity of themazEF promoter was determined by primer extension. As shown,the level of transcription from P₂ was reduced to $~$ 25% after2 h of thymine starvation compared to unstarved cells (Fig.4a). We also found that similar conditions of thymine starvationby trimethoprim do not affect the activity of the E. coli trpRpromoter (Fig. 4b).

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FIG. 4. (a) Thymine starvation by trimethoprim in E. coli MC4100relA⁺ reduces transcription from the mazEF promoter P_2. E. coli strain MC4100relA⁺ harboring plasmid pSK10 ${Delta}$ 6-p_ef was treated with trimethoprim; RNA was extracted at the indicated times, primer

DISCUSSION

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Discussion

In summary, we have shown that TLD induced in E. coli K-12 strainsis mediated through the mazEF clpP-dependent system (3, 12-14,19, 20, 27). We used three different procedures to induce thyminestarvation: treatment with trimethoprim or with a sulfonamideantibiotic or thymine starvation of a thyA auxotroph. Moreover,we found that thymine starvation by trimethoprim drasticallyreduces transcription from the mazEF promoter P₂ (Fig. 4a),which was previously described as mainly responsible for transcriptionand regulation of E. coli mazEF (3, 20). mazEF has an additionalpromoter, P₃, located 13 nucleotides downstream from P₂, thathas only 1/10 of the P₂ activity (20). Here, we could hardlydetect the activity of P₃.

Based on these results, we suggest the following model for theinduction of mazEF-mediated cell death by thymine starvation.Previous research has shown that thymine starvation provokesDNA damage that involves a unique breaking or twisting of thechromosome into a configuration that defies all the repair/protectivemechanisms of the cell (references 2 and 26 and references therein).We assume that such substantial damage to the DNA is responsiblefor the reduction of transcription from the mazEF promoter P₂(Fig. 4a). The effect of thymine starvation on the activityof this promoter may result from the direct starvation effecton P₂. Note that the promoter region of P₂ is particularly A:Trich: 20 nucleotides out of 26 (-20 to +6) are A or T (Fig.4a). However, the interference with transcription from P₂ bythymine starvation may also be caused indirectly by the inductionof ppGpp synthesis, known to inhibit the mazEF P₂ promoter (3),and/or by some specific protein(s) that could sense the damageto the DNA. Our experiments indicate that ppGpp might be involved.In these experiments, we compared the mazEF-dependent killingcaused by trimethoprim in MC4100, which carries a defectiverelA gene (relA1) (3) and therefore does not synthesize ppGpp(9), to that of the relA⁺ strain. We found that mazEF-dependentdeath appears in the relA⁺ strain at 0.1 µg of trimethoprim/10⁶cells, while in the relA1 strain it appears only at 0.3 µgof trimethoprim/10⁶ cells (data not shown). Altogether, as aconsequence of thymine starvation, prevention of the continuousexpression of the labile antitoxin MazE results in cell death.Although we have shown that thymine starvation does not affectthe activity of the E. coli trpR promoter (Fig. 4b), other promotersmay be affected; these may include other genes involved in mazEF-mediateddeath.

Our results showing that thymine starvation can trigger a built-indeath program provide new insight into an old enigma and mayhave implications for PCD in both prokaryotes and eukaryotes.(i) Until now, mazEF-directed PCD has been studied only in E.coli. However, this system is not unique to E. coli and is foundon the chromosomes of other bacteria (12, 25). Nevertheless,TLD has been described in a wide range of microorganisms (2),suggesting the existence of analogous suicide modules and PCDnot only in E. coli but in other bacteria as well. (ii) Ourpresent results also increase the repertoire of antibioticsthat operate in various bacteria by triggering a chromosomalsuicide module. In previous work, it was shown that the mazEFsuicide system in E. coli can be triggered by antibiotics likerifampin, chloramphenicol, and spectinomycin that inhibit transcriptionand/or translation (12, 27). Here, we extend this list to includetrimethoprim and sulfonamides that inhibit folic acid metabolismand thereby cause TLD. (iii) Furthermore, our results may haveimplications for both mammalian TLD and cancer research. Fordecades, clinicians have used thymine starvation, leading tomammalian cell death, as a major method of cancer therapy. TLDis induced by treatment with the analogue 5-fluorouracil andwith the folic acid antagonist methotrexate (29, 30). Thus,the knowledge gained from TLD as a trigger of bacterial PCDmay be relevant to apoptosis in mammals and to cancer therapy.

ACKNOWLEDGMENTS

We thank F. R. Warshaw-Dadon (Jerusalem, Israel) for her criticalreading of the manuscript.

The research described here was supported by grant no. 215/99-2from the Israel Science Foundation administered by the IsraelAcademy of Science and Humanities.

FOOTNOTES

* Corresponding author. Mailing address: Department of Molecular Biology, The Hebrew University-Hadassah Medical School, P.O. Box 12272, Jerusalem 91120, Israel. Phone: 972-2-675-8250. Fax: 972-2-678-4010. E-Mail: hanita{at}cc.huji.ac.il.

REFERENCES

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