DNA Synthesis and Viability of a mutT Derivative of Escherichia coli WP2 under Conditions of Amino Acid Starvation and Relation to Stationary-Phase (Adaptive) Mutation

Abstract

ABSTRACT Escherichia coli WP2 bacteria with an ochre amino acid auxotrophy show no evidence of growth during the first few days after plating at densities above 10 8 on plates lacking the required amino acid. They lose viability for some days, and then a subpopulation recovers and there is cell turnover. At very low plating densities (around 10 2 per plate), almost every cell will eventually form a small but visible colony. At intermediate plating densities (10 6 to 10 7 per plate), there is an immediate increase in the number of viable bacteria. The results are consistent with a model that assumes that growth is dependent on trace amounts of tryptophan or a tryptophan-complementing substance and that death is due to extracellular toxic species in the medium, including active oxygen species. Mutations in mutT bacteria under these conditions result from incorporation of 7,8-dihydro-8-oxo-dGTP into DNA and thus largely reflect DNA synthesis associated with the increase in the number of viable cells at the initial density used (10 7 per plate). We show that the increase in cell number and much of this DNA synthesis can be eliminated by the presence of 10 8 scavenger bacteria and by removal of early-arising mutant colonies that release the required amino acid. The synthesis that remains is equivalent to less than a quarter of a genome per day and is marginally reduced, if at all, in a polA derivative. We cannot exclude the possibility that this residual DNA synthesis is peculiar to mutT bacteria due to transcriptional leakiness, although there is no evidence that this is a major problem in this strain. If such DNA synthesis also occurs in wild-type bacteria, it may well be important for adaptive mutation since use of a more refined agar in selective plates both eliminated the initial increase in cell number seen at low density (10 7 per plate) and reduced the rate of appearance of mutants at plating densities above 10 8 per plate.