A GENETIC STRATEGY TO DEMONSTRATE THE OCCURRENCE OF SPONTANEOUS MUTATIONS IN NONDIVIDING CELLS WITHIN COLONIES OF ESCHERICHIA-COLI

A genetic strategy was designed to examine the occurrence of mutations in stationary-phase populations. In this strategy, a parental populat ion of cells is able to survive under both permissive and restrictive conditions whereas mutants at a particular target locus exhibit a cond itional-lethal phenotype. Thus, by growing the population to stationar y phase under restrictive conditions and then shifting it to permissiv e conditions, mutations that had arisen in stationary phase can be stu died without confounding effects caused by the occurrence of similar m utations during growth of the population. In two different application s of this strategy, we have studied the reversion to Lac(+) in station ary phase of several Lac-mutations in Escherichia coli. Our results in dicate that a variety of spontaneous point mutations and deletions, pa rticularly those that are sensitive to the mechanisms of replication s lippage (for their generation) and methyl-directed mismatch repair (fo r their correction), can arise in nondividing populations of cells wit hin a colony. The frequency of their occurrence was also elevated in m utS Strains, which are defective in such mismatch repair. These data h ave relevance to the ongoing debate on adaptive or directed mutations in bacteria.