Exopolysaccharide defects cause hyper-thymineless death in Escherichia coli via massive loss of chromosomal DNA and cell lysis

Abstract

Thymineless death in Escherichia coli thyA mutants growing in the absence of thymidine (dT) is preceded by a substantial resistance phase, during which the culture titer remains static, as if the chromosome has to accumulate damage before ultimately failing. Significant chromosomal replication and fragmentation during the resistance phase could provide appropriate sources of this damage. Alternatively, the initial chromosomal replication in thymine (T)-starved cells could reflect a considerable endogenous dT source, making the resistance phase a delay of acute starvation, rather than an integral part of thymineless death. Here we identify such a low-molecular-weight (LMW)-dT source as mostly dTDP-glucose and its derivatives, used to synthesize enterobacterial common antigen (ECA). The thyA mutant, in which dTDP-glucose production is blocked by the rfbA rffH mutations, lacks a LMW-dT pool, the initial DNA synthesis during T-starvation and the resistance phase. Remarkably, the thyA mutant that makes dTDP-glucose and initiates ECA synthesis normally yet cannot complete it due to the rffC defect, maintains a regular LMW-dT pool, but cannot recover dTTP from it, and thus suffers T-hyperstarvation, dying precipitously, completely losing chromosomal DNA and eventually lysing, even without chromosomal replication. At the same time, its ECA+thyA parent does not lyse during T-starvation, while both the dramatic killing and chromosomal DNA loss in the ECA-deficient thyA mutants precede cell lysis. We conclude that: 1) the significant pool of dTDP-hexoses delays acute T-starvation; 2) T-starvation destabilizes even nonreplicating chromosomes, while T-hyperstarvation destroys them; and 3) beyond the chromosome, T-hyperstarvation also destabilizes the cell envelope.

Coupling between DNA replication, segregation, and the onset of constriction in Escherichia coli

“…The agreement between data and models was poorer at later times, in which all of the models predicted the doubling times to return to their original pre-step-down values, but the experimentally measured doubling times were approximately 10% longer. In these later times, thymine limitation is reported to affect beyond the replication speed, also the cell envelope biosynthesis ( Rao and Kuzminov, 2020 ). Consistent with this idea, we observed membrane blebbing in some cells after long periods of thymine limitation.…”

“…Even though thymine limitation in thyA cells allows controllably to perturb the replication period, this limitation also perturbs other cellular processes such as the envelope biosynthesis ( Rao and Kuzminov, 2020 ). These effects appear to dominate in the long-term response to thymine perturbation (beyond 400 min; Figure 5 ).…”

Thymine‐starvation‐induced chromosomal fragmentation is not required for thymineless death in Escherichia coli

“…This idea predicts that, even in the growing cultures, depletion of thymidine to this critical level should lead to chromosomal fragmentation. We have preliminary evidence that the thyA deo rffC mutant, deficient in extracting dTTP from LMW-dT pools due to the rffC defect (Rao & Kuzminov, 2020), exhibits fragmentation without viability loss when grown in limited thymidine (Figure S17). This result further confirms the disconnect between fragmentation and lethality.…”

“…In spite of several inconsistencies among the results from different groups, there are a few less‐disputed characteristics of T‐starvation, one of them being the accumulation of single‐stranded DNA breaks (SSBs) (Freifelder, 1969; Mennigmann & Szybalsky, 1962; Nakayama & Hanawalt, 1975; Reichenbach et al, 1971; Walker, 1970). Double‐strand DNA breaks (DSBs) that fragment the chromosomes were initially detected under some, but not other conditions (Nakayama et al, 1994; Yoshinaga, 1973), until we and others conclusively showed that TLD is accompanied by significant chromosomal fragmentation (Guarino et al, 2007; Kuong & Kuzminov, 2010, 2012; Rao & Kuzminov, 2019, 2020). Since chromosome fragmentation in E. coli is associated with the loss of viability under a variety of DNA damaging conditions (UV, hydrogen peroxide, phleomycin, ligase inactivation, etc.)…”

“…We expanded this investigation to explore whether TiCF played a role in viability loss during hyper-TLD in thyA recBCD and thyA rffC mutants. To characterize TiCF further, we tested whether replication, which was dispensable for TLD in thyA mutant (Khan & Kuzminov, 2019;Rao & Kuzminov, 2020), was required for fragmentation and, if it was, whether its elimination influenced TLD.…”