Degradation of the Escherichia coli Essential Proteins DapB and Dxr Results in Oxidative Stress, which Contributes to Lethality through Incomplete Base Excision Repair.

Abstract

ABSTRACTVarious lethal stresses, including bactericidal antibiotics, can trigger the production of reactive oxygen species (ROS) that contribute to killing. Incomplete base excision repair (BER) of oxidized nucleotides, especially 8-oxo-dG, has been identified as a major component of ROS-induced lethality. However, the relative contributions of this pathway to death vary widely between stresses, due in part to poorly understood complex differences in the physiological changes caused by these stresses. To identify new lethal stresses that kill cells through this pathway, we screened an essential protein degradation library and found that depletion of either DapB or Dxr leads to cell death through incomplete BER; the contribution of this pathway to overall cell death is greater for DapB than for Dxr. Depletion of either protein generates oxidative stress, which increases incorporation of 8-oxo-dG into the genome. This oxidative stress is causally related to cell death, as plating on an antioxidant provided a protective effect. Moreover, incomplete BER was central to this cell death, as mutants lacking the key BER DNA glycosylases MutM and MutY were less susceptible, while overexpression of the nucleotide sanitizer MutT, which degrades 8-oxo-dGTP to prevent its incorporation, was protective. RNA sequencing of cells depleted of these proteins revealed widely different transcriptional responses to these stresses. Our discovery that oxidative stress-induced incomplete BER is highly dependent on the exact physiological changes that the cell experiences helps explain the past confusion that arose concerning the role of ROS in antibiotic lethality.