Exploring Thymineless Death Using Systems Biology and Laboratory Evolution

Cells die when they are starved of thymidine, one of the four DNA nucleotides. Since the discovery of this killing phenomenon, termed thymineless death (TLD), researchers have been trying to understand why. The goal of the work presented here is to use systems level approaches to shed light on this process. Because DNA synthesis is the only cellular process that requires thymidine, it is logical that the focus has been mainly on DNA stability and damage. My work expands the focus to new frontiers: acetate metabolism, the cytoplasm and the inner membrane. I generated thymidine auxotrophs in two genetic backgrounds by inactivating the thymidylate synthase enzyme, thyA. These mutants need supplementation with exogenous thymidine in order to survive. I used these strains in three experimental approaches to explore the mechanisms of TLD. Fitness profiling of a transposon insertion library in a thyA- strain, long-term laboratory evolution during thymidine-limitation, and RNA sequencing of TLD-sensitive and TLD-resistant strains identified genes in previously known processes as well as genes in novel processes. These approaches allowed me to gather rich data sets that identified many contributing genes. 52 genes showed consistent effects across approaches. My work confirms that ROS is a key contributor to killing during thymidine starvation and reveals that putrescine biosynthesis enzymes, an acetate overflow kinase, and the proton-transporting ATP synthase are novel players in TLD. I suggest that these three novel players contribute through their shared role in modulating cytoplasmic pH and propose a model in which DNA damage, ROS accumulation, and cytoplasmic acidification converge on the killing process during thymidine starvation. My findings expand the sites of critical action during TLD from the DNA to the cell’s inner and outer membranes and the cytoplasm. Theories on active vs. passive and specific vs. general bacterial death pathways will be discussed at the end