Amplification-mutagenesis: evidence that "directed" adaptive mutation and general hypermutability result from growth with a selected gene amplification

Abstract

When a particular lac mutant of Escherichia coli starves in the presence of lactose, nongrowing cells appear to direct mutations preferentially to sites that allow growth (adaptive mutation). This observation suggested that growth limitation stimulates mutability. Evidence is provided here that this behavior is actually caused by a standard Darwinian process in which natural selection acts in three sequential steps. First, growth limitation favors growth of a subpopulation with an amplification of the mutant lac gene; next, it favors cells with a lac(+) revertant allele within the amplified array. Finally, it favors loss of mutant copies until a stable haploid lac(+) revertant arises and overgrows the colony. By increasing the lac copy number, selection enhances the likelihood of reversion within each developing clone. This sequence of events appears to direct mutations to useful sites. General mutagenesis is a side-effect of growth with an amplification (SOS induction). The F' plasmid, which carries lac, contributes by stimulating gene duplication and amplification. Selective stress has no direct effect on mutation rate or target specificity, but acts to favor a succession of cell types with progressively improved growth on lactose. The sequence of events--amplification, mutation, segregation--may help to explain both the origins of some cancers and the evolution of new genes under selection.

Figure 1

Amplification–mutagenesis model for reversion during growth under selection and associated hypermutability. (Left) The basic amplification model without general mutagenesis. (Right) Induction of SOS mutagenesis is a side effect of growth with an unstable amplification.

Figure 2

Frequency of unstable Lac⁺ cells in revertant colonies. For each histogram, about 20 revertant colonies were classified based on the percentage of Lac⁺ cells (3,000 from each colony) with an unstable phenotype. (A) Early colonies formed by full revertants that arose before selection. (B and D) A set of microscopic new colonies were identified on day 4; half were picked and scored immediately (B) and the other half remained under selection for 2 more days before scoring (D). (C and E) A set of new colonies identified on day 6 (not visible on day 5). The colonies in C were tiny at the moment of identification and scoring; those in E were large. The tester strain used was FC40 = TR7178 [ara thiA Rif^R Del13(lac proB)/F′₁₂₈ lacI33(fs) lacIZfusion(Ω) pro⁺] and the scavenger strain was FC29 = TR7177 [ara thiA Rif^S Del13(lac proB)/F′₁₂₈ Del(lacI, lacZ) pro⁺].

Figure 3

Variable maturation of microclones during reversion under nonlethal selection. For the reconstruction, 400 Lac⁺ revertants of FC40 were plated with scavengers on selective medium. Each of the other lines describes new revertant colonies (>100) that arose on 20 plates on each of a series of days (absent the previous day); each set was scored for color over several days.

Figure 4

Reversion is reduced by inhibiting growth of cells with a lac amplification. (A) Tester strains were FC40 (no Tn10), TT23242 (Tn10 in the chromosome), TT22951 (Tn10 in the plasmid mhpC gene 4.6 kb from lac). Strain TT23241 served as scavenger. “+Tc” and “−Tc” indicate presence or absence of Tc (10 μg/ml) in selection plates. (B) Tester strains were FC40 (no Tn10), TT23242 (Tn10 in chromosome), TT22951 (Tn10 4.6 kb from lac). Strains TT23243, TT23244, and TT23245 carried Tn10 >30 kb, >30 kb, and 20 kb from lac, respectively. Without chlortetracycline (CTc), all strains showed reversion like FC40.