Cell Death in Escherichia coli dnaE (Ts) Mutants Incubated at a Nonpermissive Temperature Is Prevented by Mutation in the cydA Gene

Abstract

Cells of the Escherichia coli dnaE(Ts) dnaE74 and dnaE486 mutants die after 4 h of incubation at 40°C in Luria-Bertani medium. Cell death is preceded by elongation, is inhibited by chloramphenicol, tetracycline, or rifampin, and is dependent on cell density. Cells survive at 40°C when they are incubated at a high population density or at a low density in conditioned medium, but they die when the medium is supplemented with glucose and amino acids. Deletion of recA or sulA has no effect. We isolated suppressors which survived for long periods at 40°C but did not form colonies. The suppressors protected against hydroxyurea-induced killing. Sequence and complementation analysis indicated that suppression was due to mutation in the cydA gene. The DNA content of dnaE mutants increased about eightfold in 4 h at 40°C, as did the DNA content of the suppressed strains. The amount of plasmid pBR322 in a dnaE74 strain increased about fourfold, as measured on gels, and the electrophoretic pattern appeared to be normal even though the viability of the parent cells decreased 2 logs. Transformation activity also increased. 4,6-Diamidino-2-phenylindole staining demonstrated that there were nucleoids distributed throughout the dnaE filaments formed at 40°C, indicating that there was segregation of the newly formed DNA. We concluded that the DNA synthesized was physiologically competent, particularly since the number of viable cells of the suppressed strain increased during the first few hours of incubation. These observations support the view that E. coli senses the rate of DNA synthesis and inhibits septation when the rate of DNA synthesis falls below a critical level relative to the level of RNA and protein synthesis.Cell division and DNA synthesis are coupled in all organisms, but the mechanism of the coupling is not clear, notwithstanding the sophisticated description of an operon with multiple promoters determining the production of the FtsZ and other proteins involved in cell division (12,15). In Escherichia coli, the completion of a round of DNA synthesis is usually followed by activation of the cell septation machinery, but synthesis does not trigger septation (4). DNA damage or other blocks to DNA synthesis often lead to inhibition of cell division. For example, induction of the SOS repair pathway activates the sulA gene product, inhibiting cell division (5). Some signal recognizes that the DNA has been damaged (or that synthesis has been inhibited), and as a result division is suspended. Deprivation of thymine necessarily inhibits DNA synthesis and also leads to filamentation (1). Filamentation often accompanies cell death, and there have been numerous studies attempting to elucidate the mechanism of death both as a result of thymine starvation and as a result of other pathways to filamentation.As part of an investigation of the roles of the different DNA polymerases in mutation (35), we studied the behavior of strains carrying a temperature-sensitive (Ts) mutation in the alpha subunit of the replicative DNA polymerase...

Cytochrome Oxidase Deficiency Protects Escherichia coli from Cell Death but Not from Filamentation Due to Thymine Deficiency or DNA Polymerase Inactivation

“…Mutation in the cydA85 gene coding for a subunit of cytochrome bd oxidase of E. coli protects cells carrying a temperature-sensitive mutation in the replicative DNA polymerase from death when incubated at nonpermissive temperature (29). However, by 24 h it is clear that cells of the dnaE74 cydA85 double mutant are protected from death only on LB medium; death of the suppressed strain occurs as rapidly as that of the dnaE74 mutant on minimal medium (Fig.…”

“…These findings imply that events at a stalled replication fork set off a chain of events which lead to cell death. We have started to investigate the steps between inhibition of replication fork movement and the death of the cell in bacteria by identifying a gene, cydA, coding a subunit of the cytochrome bd oxidase which, when in mutant form, protects temperature-sensitive dnaE mutants from death (29). In this paper, we show that this mutation also protects a thymine-requiring mutant from thymineless death.…”

“…We used an allele of the cydA gene with a C3T mutation at nucleotide 770934, resulting in a Ser3Phe change at amino acid 85 (29). The E. coli Genetic Stock Center has assigned this mutation the designation cydA85.…”

“…Unbalanced growth has been invoked as a cause of death after thymine starvation (6) and as a result of restricted DNA synthesis in temperature-sensitive mutants of the Escherichia coli replicative polymerase (29). However, an exact definition in molecular terms of what constitutes unbalanced growth has not been achieved.…”

A simplified method for reconstituting active E. coli DNA polymerase III

“…The subunit can then be replaced by its counterpart from other bacteria during the gap filling experiment to test the interchangeability of the subunits from two DNA pol III systems. This system provides an alternative strategy when in vivo complementation experiments for the DNA pol III subunit do not work (Wechsler and Gross, 1971;Horiuchi et al, 1978;Strauss et al, 2004).…”