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

Abstract

Thymine or thymidine starvation induces robust chromosomal fragmentation in Escherichia coli thyA deoCABD mutants and is proposed to be the cause of thymineless death (TLD). However, fragmentation kinetics challenges the idea that fragmentation causes TLD, by peaking before the onset of TLD and disappearing by the time TLD accelerates. Quantity and kinetics of fragmentation also stay unchanged in hyper-TLD-exhibiting recBCD mutant, making its faster and deeper TLD independent of fragmentation as well. Elimination of fragmentation without affecting cellular metabolism did not abolish TLD in the thyA mutant, but reduced early TLD in the thyA recBCD mutant, suggesting replication-dependent, but undetectable by pulsed-field gel, double-strand breaks contributed to TLD. Chromosomal fragmentation, but not TLD, was eliminated in both the thyA and thyA recBCD mutants harboring deoCABD operon. The expression of a single gene, deoA, encoding thymidine phosphorylase, was sufficient to abolish fragmentation, suggesting thymidine-to-thymine interconversion during T-starvation being a key factor. Overall, this study reveals that chromosomal fragmentation, a direct consequence of T-starvation, is either dispensable or redundant for the overall TLD pathology, including hyper-TLD in the recBCD mutant. Replication forks, unlike chromosomal fragmentation, may provide a minor contribution to TLD, but only in the repair-deficient thyA deoCABD recBCD mutant.

Keywords: deoCABD; recBCD; double-strand breaks; nucleotide salvage pathway; replication forks.

FIGURE 1.

TLD and TiCF as a function of recBCD deletion and protein synthesis inhibition. (a) TLD in the thyA and thyA recBCD mutants. Data points in all figures, when shown with standard errors, are means of three or more independent experiments. (b) An autoradiogram showing the qualitative comparison of the kinetics of chromosomal fragmentation in the thyA and thyA recBCD strains under the conditions TLD is determined in (a). (c) An ethidium bromide-stained pulsed field gel showing 1H TiCF with lambda ladder MW markers. For this gel, electrophoresis was done for 40 hours at 4.5V/cm with 5-120 second switch time ramp. (d) Quantitation of fragmentation in the thyA and thyA recBCD strains under the T-starvation conditions. Difference in fragmentation at 1H time is statistically significant in t-test (P value = 0.03). (e) Effect of chloramphenicol, added at the time of onset of T-starvation (time 0), on chromosomal fragmentation in the thyA cells. (f) TLD in the thyA cells under the conditions fragmentation is determined in (e). RES, Resistance phase; RED, Rapid exponential death phase. All strains used in this figure are deoCABD mutants.

FIGURE 2.

Dependence of TiCF on replication forks. Comparison of fragmentation between the thyA and thyA dnaA601(Ts) (a), and the thyA and thyA dnaC7(Ts) (b) mutants, when the strains grown at 28°C were starved at 42°C (a) or 38°C (b), respectively. (c) Comparison of viability loss in the thyA and thyA dnaC7(Ts) mutants under the conditions described in (b). (d) TLD and TiCF in the thyA mutant under the ‘awakening protocol’ done at 37 °C. Details of the experiment are described in the text. All strains used in this figure are deoCABD mutants.

FIGURE 3.

Role of pre-existing forks and new initiations in extra TLD observed in the thyA recBCD mutant. Comparison of early fragmentation (a) and TLD (b) between the thyA and thyA recBCD mutants when chloramphenicol was added at the time of starvation initiation (time 0). Comparison of TLD (c) and early fragmentation (d) between the thyA recBCD and thyA recBCD dnaC7(Ts) mutants after pre-growing them at 28°C and shifting them to non-permissive temperature (38°C) at the onset of starvation. All strains used in this figure are deoCABD mutants.

FIGURE 4.

Involvement of pre-existing forks and protein synthesis in TiCF and TLD in the repair-deficient recBCD mutant. (a) TLD in the thyA and thyA recBCD mutants under the ‘awakening protocol’ done at 37 °C. (b) Effect of addition of chloramphenicol at the time of starvation on TLD in the thyA and thyA recBCD mutants under the ‘awakening protocol’. (c) Comparison of TiCF in the thyA mutant when chloramphenicol was added at the time of starvation-initiation (time 0) or after one hour of starvation. Experiment was done using the standard conditions. (d) Kinetics of TLD in the thyA mutant under the conditions described in (c). (e) Kinetics of TLD in the thyA recBCD mutant when the strain was starved in the absence of chloramphenicol or with chloramphenicol added at 0H or 1H post-starvation. All strains used in this figure are deoCABD mutants.

FIGURE 5.

Genomic profiling of the fragmented DNA and the DNA retained in agarose plugs upon 0H and 1H T-starvation of the thyA (a), and thyA recBCD mutants (b). DNA was purified from agarose using β-agarase. Details of the samples used for sequencing are outlined in supplementary figure S7. Strains used in this figure are deoCABD mutants.

FIGURE 6.

Role of DNA degradation on TLD and stability of TiCF. Comparison of TLD (a), and TiCF (b) and (c), among the thyA, thyA recBCD and thyA recBCD sbcBC mutants under standard conditions. Details of experiments are described in the text. All strains used in this figure are deoCABD mutants.

FIGURE 7.

Role of the Deo proteins of the nucleoside salvage pathway in TiCF and TLD. (a) Qualitative (left panel) and quantitative (right panel) comparison of fragmentation between the thyA and thyA deoCABD+ strains during T-starvation. (b) TLD in the thyA and thyA deoCABD+ strains. (c) Schematic representation of part of deoxy-ribonucleoside salvage pathway showing enzymatic reactions catalyzed by the Deo proteins. tdk, thymidine kinase; tmk, thymidylate kinase; ndk, nucleoside diphosphate kinase. (d) Effect of deoCABD expressing plasmid on TiCF in fragmentation proficient thyA strain and its comparison with fragmentation-deficient thyA deoCABD⁺ strain. Right panel shows fragmentation quantification from multiple gels like the one shown on the left.

FIGURE 8.

Confirmation of the critical Deo protein and determination of the role of deo operon on TLD and TiCF under RecBCD⁻ conditions. Qualitative comparison (a) and quantification of TiCF (b) in the thyA, its deoA expressing derivative, thyA deoCABD⁺ and thyA deoCBD⁺(deoA::kan) strains. Gel picture of the fragmentation profiles (0-5 h) is shown in supplementary figure S15. (c) TLD in strains assessed for TiCF in (b). (d) Comparison of TiCF between the thyA recBCD and thyA deoCABD⁺ recBCD strains and their RecBCD+ derivatives. (e) TLD shown by strains used in (d).

FIGURE 9.

A hypothetical scheme explaining how utilization of the residual thymidine at the time of starvation could lead to TiCF in deo mutants (top panel) by supporting limited movement of replication forks. Presence of thymidine phosphorylase (DeoA) (bottom panel) could push thymidine into central metabolism, inhibiting it from channeling into DNA metabolism and starving replication forks of dTMP causing snap stall and TiCF loss. More details are provided in the text.