Rifampicin suppresses thymineless death by blocking the transcription-dependent step of chromosome initiation

Thymineless death (TLD), a phenomenon in which thymine auxotrophy becomes lethal when cells are starved of thymine, can be prevented by the presence of rifampicin, an RNA polymerase inhibitor. Several lines of evidence link TLD to chromosome initiation events. This suggests that rifampicin-mediated TLD suppression could be due to the inhibition of RNA synthesis required for DNA chromosomal initiation at oriC, although other mechanisms cannot be discarded. In this work, we show that the addition of different rifampicin concentrations to thymine-starved cells modulates TLD and chromosomal initiation capacity (ChIC). Time-lapse experiments find increasing levels of ChIC during thymine starvation correlated with the accumulation of simple-Y, double-Y and bubble arc replication intermediates at the oriC region as visualized by two-dimensional DNA agarose gel electrophoresis. None of these structures were observed following rifampicin addition or under genetic-physiological conditions that suppress TLD, indicating that abortive chromosome replication initiations under thymine starvation are crucial for this lethality. Significantly, the introduction of mioC and gid mutations which alter transcription levels around oriC, reduces ChIC and alleviates TLD. These results show that the impairment of transcription-dependent initiation caused by rifampicin addition, is responsible for TLD suppression. Our findings here may provide new avenues for the development of improved antibacterial treatments and chemotherapies based on thymine starvation-induced cell death.