Crowding depression of UV-mutagenesis in E. coli.

Strains of E. coli B/r were exposed to UV radiation and assayed for reversion mutation, using a standard selection medium. If more irradiated bacteria were assayed per petri dish, a proportional increase in the number of indicated reversion mutants was found only up to a limiting plating density. Beyond a density of about 10(8) viable bacteria per petri dish, the number of indicated revertants per viable bacteria assayed (the mutation frequency) decreased as the plating density was increased. The crowding depression of mutagenesis was more severe for de novo and converted suppressor mutations, the mutation frequency being reduced 100-fold at a plating density of about 6 x 10(9) viable bacteria per plate. The effect on backmutation was 10 times less. Crowding depression of mutagenesis occurred in excision-proficient and -deficient strains, with identical effects in the 2 strains on de novo and converted suppressor mutation, but different effects on backmutations. There were no accompanying effects on viability. Irreversible loss of potential mutants during crowded growth was indicated in wash-off experiments. The kinetics suggested a half-life of approximately 1 h. Kinetics for accumulation by the bacteria of the limiting metabolite (tyrosine) on the assay plate indicated a short period of time for protein synthesis, but direct examination of the proteins synthesized during early growth on a crowded plate demonstrated successful induction of recA protein. The results suggested a possible disruption in the rec/lex respondency system somewhere between induction of recA protein and the various end points, including mutational repair, in cells plated close to one another.