Electron Microscopy Reveals Unexpected Cytoplasm and Envelope Changes during Thymineless Death in Escherichia coli

Bacterial rod-shaped cells experiencing irreparable chromosome damage should filament without other morphological changes. Thymineless death (TLD) strikes thymidine auxotrophs denied external thymine/thymidine (T) supplementation. Such T-starved cells cannot produce the DNA precursor dTTP and therefore stop DNA replication. Stalled replication forks in T-starved cells were always assumed to experience mysterious chromosome lesions, but TLD was recently found to happen even without origin-dependent DNA replication, with the chromosome still remaining the main TLD target. T starvation also induces morphological changes, as if thymidine prevents cell envelope or cytoplasm problems that otherwise translate into chromosome damage. Here, we used transmission electron microscopy (TEM) to examine cytoplasm and envelope changes in T-starved Escherichia coli cells, using treatment with a DNA gyrase inhibitor as a control for "pure" chromosome death. Besides the expected cell filamentation in response to both treatments, we see the following morphological changes specific for T starvation and which might lead to chromosome damage: (i) significant cell widening, (ii) nucleoid diffusion, (iii) cell pole damage, and (iv) formation of numerous cytoplasmic bubbles. We conclude that T starvation does impact both the cytoplasm and the cell envelope in ways that could potentially affect the chromosome. IMPORTANCE Thymineless death is a dramatic and medically important phenomenon, the mechanisms of which remain a mystery. Unlike most other auxotrophs in the absence of the required supplement, thymidine-requiring E. coli mutants not only go static in the absence of thymidine, but rapidly die of chromosomal damage of unclear nature. Since this chromosomal damage is independent of replication, we examined fine morphological changes in cells undergoing thymineless death in order to identify what could potentially affect the chromosome. Here, we report several cytoplasm and cell envelope changes that develop in thymidine-starved cells but not in gyrase inhibitor-treated cells (negative control) that could be linked to subsequent irreparable chromosome damage. This is the first electron microscopy study of cells undergoing "genetic death" due to irreparable chromosome lesions.