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Contribution of reactive oxygen species to thymineless death in Escherichia coli

Abstract

Nutrient starvation usually halts cell growth rather than causing death. Thymine starvation is exceptional, because it kills cells rapidly. This phenomenon, called thymineless death (TLD), underlies the action of several antibacterial, antimalarial, anticancer, and immunomodulatory agents. Many explanations for TLD have been advanced, with recent efforts focused on recombination proteins and replication origin (oriC) degradation. Because current proposals account for only part of TLD and because reactive oxygen species (ROS) are implicated in bacterial death due to other forms of harsh stress, we investigated the possible involvement of ROS in TLD. Here, we show that thymine starvation leads to accumulation of both single-stranded DNA regions and intracellular ROS, and interference with either event protects bacteria from double-stranded DNA breakage and TLD. Elevated levels of single-stranded DNA were necessary but insufficient for TLD, whereas reduction of ROS to background levels largely abolished TLD. We conclude that ROS contribute to TLD by converting single-stranded DNA lesions into double-stranded DNA breaks. Participation of ROS in the terminal phases of TLD provides a specific example of how ROS contribute to stress-mediated bacterial self-destruction.

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Acknowledgements

The authors thank G.C. Walker, S.M. Rosenberg, K. Gerdes, D. Jin, J. Imlay and H. Aiba for strains and D. Dubnau, J. Freundlich, M. Gennaro, M. Neiditch and B. Shopsin for critical comments on the manuscript. The authors also acknowledge support from grants from the National Institutes of Health (DP2OD007423 and R01 AI07341) and the National Natural Science Foundation of China (81473251).

Author information

Y.H. conducted most of the experiments. Y.H., L.L. and G.L. conducted flow cytometry and microscopy assays. Y.H., L.L., G.L., K.D. and X.Z. analysed the data. Y.H., K.D. and X.Z. designed the study and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Correspondence to Xilin Zhao.

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Further reading

Fig. 1: Association of ROS with TLD.
Fig. 2: Suppression of ROS accumulation during T-starvation by rifampicin, chloramphenicol or deficiencies in respiratory-chain genes.
Fig. 3: ssDNA regions are necessary but insufficient for TLD.
Fig. 4: dsDNA breaks associated with TLD.
Fig. 5: Conversion of ssDNA regions into lethal breaks by exogenous ROS.
Fig. 6: Scheme describing ROS-mediated conversion of ssDNA regions into lethal DSBs during TLD.