The Escherichia coli mazEF suicide module mediates thymineless death - PubMed (original) (raw)

The Escherichia coli mazEF suicide module mediates thymineless death

Boaz Sat et al. J Bacteriol. 2003 Mar.

Abstract

In 1954, Cohen and Barner discovered that a thymine auxotrophic (thyA) mutant of Escherichia coli undergoes cell death in response to thymine starvation. This phenomenon, called thymineless death (TLD), has also been found in many other organisms, including prokaryotes and eukaryotes. Though TLD has been studied intensively, its molecular mechanism has not yet been explained. Previously we reported on the E. coli mazEF system, a regulatable chromosomal suicide 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 module can also be activated by thymine starvation. We found that TLD depends on E. coli mazEF and that under thymine starvation, the activity of the mazEF promoter P(2) is significantly reduced. Our results, which describe thymine starvation as a trigger for a built-in death program, have implications for programmed cell death in both prokaryotes and eukaryotes.

PubMed Disclaimer

Figures

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 MC4100_relA+ (WT), MC4100Δ_mazEFrelA_+ (Δ_mazEF_), and MC4100Δ_mazEFrelA_+/p_mazEF_ (Δ_mazEF_/p_mazEF_). MC4100_relA_+ 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 MC4100_relA_+ and its Δ_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 MC4100_relA_+ (▴) and its Δ_mazEF_ derivative (▪) without the addition of trimethoprim. ▵, trimethoprim-treated MC4100_relA_+; □, Δ_mazEF_ derivative.

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 MC4100_relA+ (WT) and its Δ_mazEF_ derivative (Δ_mazEF_) in M9 liquid medium at 37°C. MC4100_relA_+ 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.

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 KL742_thyA748::Tn_10_ (WT), KL742_thyA748_::Tn_10_Δ_mazEF_ (Δ_mazEF_), and KL742_thyA748_::Tn_10_Δ_clpP_ (clpP −) at 37°C in M9 liquid medium lacking thymine. Cell survival was determined as described in the legend to Fig. 1a.

FIG. 4.

(a) Thymine starvation by trimethoprim in E. coli MC4100_relA_+ reduces transcription from the mazEF promoter P2. E. coli strain MC4100_relA_+ harboring plasmid pSK10Δ6-pef was treated with trimethoprim; RNA was extracted at the indicated times, primer

References

1. Ahmad, S. I. 1980. Thymineless death in recombination deficient mutants of Escherichia coli K12. Z. Naturforsch. Teil C 35:279-283. - PubMed
1. Ahmad, S. I., S. H. Kirk, and A. Eisenstark. 1998. Thymine metabolism and thymineless death in prokaryotes and eukaryotes. Annu. Rev. Microbiol. 52:591-625. - PubMed
1. Aizenman, E., H. Engelberg-Kulka, and G. Glaser. 1996. An Escherichia coli chromosomal “addiction module” regulated by guanosine 3′,5′-bispyrophosphate: a model for programmed bacterial cell death. Proc. Natl. Acad. Sci. USA 93:6059-6063. - PMC - PubMed
1. Angehrn, P., and R. Then. 1973. Nature of trimethoprim-induced death in Escherichia coli. Arzneimittelforschung 23:447-451. - PubMed
1. Bachmann, B. J. 1990. Linkage map of Escherichia coli K-12, edition 8. Microbiol. Rev. 54:130-197. - PMC - PubMed

The Escherichia coli mazEF suicide module mediates thymineless death - PubMed (original) (raw)