PMC

Effect of a dinB mutation on reversion in the Pur-Lac system. Pur-Lac tester strain (TT25154) and an isogenic variant that carries a dinB mutation (TT26172) were plated on lactose (adenine) medium and the appearance of Lac⁺ colonies was scored for a period of 6 days.

Time course of a reversion experiment. Tester cells (10⁸) were grown on minimal glycerol medium, washed, and plated on minimal lactose medium. Colony (Lac⁺) number increases over 6 days. The lawn of tester cells increases for 1 day and then remains constant.

Effect of reducing plate growth on revertant number. The tester strain (10⁸ cells) was plated on lactose adenine medium with and without 10⁹ cells of a Lac⁻ scavenger strain. Lawn growth was followed by testing agar plugs for their content of viable tester cells. Revertant number is recorded as in a standard reversion experiment.

Model for development of a single colony. A purR mutant has a 4.5 hr/generation under plate conditions and the second mutation (mDEL1) arises at a rate of 10⁻⁴/cell per generation to produce a doubly mutant cell with a 1.3 hr/generation. Colonies become visible at a population size above 10⁶.

Genetic map of tester strain (TT25154). Growth on lactose is limited by low expression of the chromosomal lacZYA operon. The promoter of the purHD operon, which expresses the lac genes, is repressed by the PurR protein. Mutations that restore a Lac⁺ phenotype affect either the purR gene or several sites in the purHD::lac region (see arrows). These mutations are described in the text.

Effect of reducing plate growth on revertant distribution. Tester cells (10⁸) were plated on selection plates that had first been seeded with 10⁹ Lac⁻ scavenger cells that could consume any nutrients other than lactose contaminating the agar. After 1 day of scavenging, 10⁸ testers cells from each of 10 independent cultures were added and plates were incubated. Scavenging reduced tester growth on the selection plate 10-fold and revertant number ∼2-fold. Note that the distribution of revertant number showed a slope (−1.0)

Fluctuation tests for revertant number in the Pur-Lac system. Cells (10⁹) from 50 independent cultures were plated separately and selected colonies were counted on day 2. To detect double mutants (purR, mDEL1), 10-fold more tester cells (10⁹) were plated. This revealed double mutants that are too rare to appear in the standard experiment. The slope (−1.2) suggests that rare double mutants arise prior to plating. The data for revertants that appeared on days 3 and 6 were obtained by plating 10⁸ cells from each of 25 tubes on selective medium.

Deletion mutations that increase lacZ expression and allow tester growth on lactose. Many of the characterized revertants in the Pur-Lac system have acquired a deletion of the region between the purD coding sequence and the upstream end of the inserted MudJ element (mDEL). Of 22 sequenced mDEL deletions, 21 removed 106 bp (mDEL1) and 1 removed 260 bp (mDEL2). Both deletion types remove material downstream of the purD UGA mutation including an interrupted palindromic sequence element. The mDEL3 and 4 deletions arose secondarily in strains carrying mDEL1 and improved growth further by removing the polar UGA mutation.

β-Galactosidase activity of the Pur-Lac tester strain derivatives. The low LacZ level in the tester depends on the combined effects of operon repression and the polar effect of the purD UGA mutation. The terminator stem has a small effect in the absence of the purD UGA mutation (compare the first two strains) and a large effect in strains with the UGA mutation (compare the third and forth strains). Single mutants and purR, purO double mutants have a partially Lac⁺ phenotype under selective conditions on the reversion plate. Double mutants at the far left are fully Lac⁺. Strains assayed, in order of vertical bars, were: T12306 (No UGA, mDEL⁺), TT26208 (No UGA, mDEL1), TT25154 (tester strain UGA, mDEL1⁺), TT26174 (mDEL1 single), TT26176 (purO single), TT26169 (purR single), TT26209 (purR, purO), TT26175 (purO, mDEL1), and TT25177 (purR, mDEL1).

Reconstruction experiments using purR, purO, and stem deletion mutants. (A) Roughly 100 singly mutant cells (mDEL1 or purR) were plated alone without testers on lactose medium. For comparison, 10⁸ tester cells were plated alone to detect revertants in a standard reversion assay. (B) A mixture of 100 singly mutant revertant cells were genetically marked and plated together with 10⁸ tester cells on selection medium. (C) A control experiment in which 100 doubly mutant cells were plated alone on selective medium. Behavior of tester cells alone is replotted from A. (D) A mixture of 100 doubly mutant cells and 10⁸ tester cells was plated on lactose. Accumulation of colonies from tester cells alone is replotted for comparison. For all parts of the figure, cells were pregrown on minimal glycerol adenine, plated on minimal medium with lactose plus adenine, and incubated at 30° as described in Materials and Methods. Singly mutant cells plated alone or with the tester lawn were genetically marked with a Tn10 insertion near purR⁺. Strains: TT25154 (tester strain), TT26169 (purR), TT26174 (mDEL1), TT26176 (purO), TT26175 (purO mDEL1), and TT25177 (purR mDEL1).