Oxidative damage blocks thymineless death and trimethoprim poisoning in Escherichia coli

Cells that cannot synthesize one of the DNA precursors, dTTP, due to thyA mutation or metabolic poisoning, undergo thymineless death (TLD), — a chromosome-based phenomenon of unclear mechanisms. In E. coli , thymineless death is caused either by denying thyA mutants thymidine supplementation or by treating wild type cells with trimethoprim. Two recent reports promised a potential breakthrough in TLD understanding, suggesting significant oxidative damage during thymine starvation. Oxidative damage in vivo comes from Fenton's reaction, when hydrogen peroxide meets ferrous iron to produce hydroxyl radical. Therefore, TLD could kill via irreparable double-strand breaks behind replication forks, when starvation-caused single-strand DNA gaps are attacked by hydroxyl radicals. We tested the proposed Fenton-TLD connection, in both thyA mutants denied thymidine, as well as in trimethoprim-treated WT cells, under three conditions: 1) intracellular iron chelation; 2) mutational inactivation of hydrogen peroxide (HP) scavenging; 3) acute treatment with sublethal HP concentrations. We found that TLD kinetics are affected by neither iron chelation, nor HP stabilization in cultures, indicating no induction of oxidative damage during thymine starvation. Moreover, acute exogenous HP treatments completely block TLD, apparently by blocking cell division — which may be a novel TLD prerequisite. Separately, the acute trimethoprim sensitivity of the rffC and recBCD mutants demonstrates how bactericidal power of this antibiotic could be amplified by inhibiting the corresponding enzymes. Importance Mysterious thymineless death strikes cells that are starved for thymine and therefore replicating their chromosomal DNA without dTTP. After 67 years of experiments testing various obvious and not so obvious explanations, thymineless death is still without a mechanism. Recently, oxidative damage via in vivo Fenton's reaction was proposed as a critical contributor to the irreparable chromosome damage during thymine starvation. We have tested this idea by either blocking in vivo Fenton's reaction (expecting no thymineless death) or by amplifying oxidative damage (expecting hyper thymineless death). Instead, we found that blocking Fenton's reaction has no influence on thymineless death, while amplifying oxidative damage prevents thymineless death altogether. Thus, oxidative damage does not contribute to thymineless death, while the latter remains enigmatic.

Double-Strand Break Generation under Deoxyribonucleotide Starvation in Escherichia coli

ABSTRACT Stalled replication forks produced by three different ways of depleting deoxynucleoside triphosphate showed different capacities to undergo “replication fork reversal.” This reaction occurred at the stalled forks generated by hydroxyurea treatment, was impaired under thermal inactivation of ribonucleoside reductase, and did not take place under thymine starvation.

Role of DNA Replication and Repair in Thymineless Death in Escherichia coli

ABSTRACT Inhibition of DNA replication with hydroxyurea during thymine starvation of Escherichia coli shows that active DNA synthesis is not required for thymineless death (TLD). Hydroxyurea experiments and thymine starvation of lexA3 and uvrA DNA repair mutants rule out unbalanced growth, the SOS response, and nucleotide excision repair as explanations for TLD.

Gyrase Inhibitors and Thymine Starvation Disrupt the Normal Pattern of Plasmid RK2 Localization in Escherichia coli

ABSTRACT Multicopy plasmids in Escherichia coli are not randomly distributed throughout the cell but exist as defined clusters that are localized at the mid-cell, or at the 1/4 and 3/4 cell length positions. To explore the factors that contribute to plasmid clustering and localization, E. coli cells carrying a plasmid RK2 derivative that can be tagged with a green fluorescent protein-LacI fusion protein were subjected to various conditions that interfere with plasmid superhelicity and/or DNA replication. The various treatments included thymine starvation and the addition of the gyrase inhibitors nalidixic acid and novobiocin. In each case, localization of plasmid clusters at the preferred positions was disrupted but the plasmids remained in clusters, suggesting that normal plasmid superhelicity and DNA synthesis in elongating cells are not required for the clustering of individual plasmid molecules. It was also observed that the inhibition of DNA replication by these treatments produced filaments in which the plasmid clusters were confined to one or two nucleoid bodies, which were located near the midline of the filament and were not evenly spaced throughout the filament, as is found in cells treated with cephalexin. Finally, the enhanced yellow fluorescent protein-RarA fusion protein was used to localize the replication complex in individual E. coli cells. Novobiocin and nalidixic acid treatment both resulted in rapid loss of RarA foci. Under these conditions the RK2 plasmid clusters were not disassembled, suggesting that a completely intact replication complex is not required for plasmid clustering.

The Escherichia coli mazEF Suicide Module Mediates Thymineless Death

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 P2 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.