The Origin of Mutants Under Selection: How Natural Selection Mimics Mutagenesis (Adaptive Mutation)

Abstract

Selection detects mutants but does not cause mutations. Contrary to this dictum, Cairns and Foster plated a leaky lac mutant of Escherichia coli on lactose medium and saw revertant (Lac(+)) colonies accumulate with time above a nongrowing lawn. This result suggested that bacteria might mutagenize their own genome when growth is blocked. However, this conclusion is suspect in the light of recent evidence that revertant colonies are initiated by preexisting cells with multiple copies the conjugative F'lac plasmid, which carries the lac mutation. Some plated cells have multiple copies of the simple F'lac plasmid. This provides sufficient LacZ activity to support plasmid replication but not cell division. In nongrowing cells, repeated plasmid replication increases the likelihood of a reversion event. Reversion to lac(+) triggers exponential cell growth leading to a stable Lac(+) revertant colony. In 10% of these plated cells, the high-copy plasmid includes an internal tandem lac duplication, which provides even more LacZ activity—sufficient to support slow growth and formation of an unstable Lac(+) colony. Cells with multiple copies of the F'lac plasmid have an increased mutation rate, because the plasmid encodes the error-prone (mutagenic) DNA polymerase, DinB. Without DinB, unstable and stable Lac(+) revertant types form in equal numbers and both types arise with no mutagenesis. Amplification and selection are central to behavior of the Cairns-Foster system, whereas mutagenesis is a system-specific side effect or artifact caused by coamplification of dinB with lac. Study of this system has revealed several broadly applicable principles. In all populations, gene duplications are frequent stable genetic polymorphisms, common near-neutral mutant alleles can gain a positive phenotype when amplified under selection, and natural selection can operate without cell division when variability is generated by overreplication of local genome subregions.

Figure 1.

The strains used in the Cairns–Foster system. The Escherichia coli tester strain carries an F′lac plasmid with a mutant lac allele; the chromosomal lac region has been deleted. On the F′lac plasmid, the lacI and lacZ genes have been fused so they encode a single protein with β-galactosidase (LacZ) activity. This hybrid gene is transcribed from a constitutively expressed lacI^Q promoter. The ability of the tester to grow on lactose is blocked by a +1 frameshift mutation within the lacI portion of the hybrid gene. This mutation is leaky and allows production of about 2% of the revertant enzyme activity (Cairns and Foster 1991). The F′lac plasmid also carries the dinB gene, which encodes an SOS-inducible error-prone (mutagenic) DNA repair polymerase. The transfer (tra) functions of plasmid F′lac₁₂₈ that allow conjugation are constitutively expressed. Conjugal replication and transfer processes are initiated at a transfer origin (oriT) by the single-strand endonuclease TraI.

Figure 2.

The Cairns–Foster reversion experiment. Cells of the strain described above are pregrown on glycerol, washed, and plated on lactose medium. The few revertant colonies that appear on lactose within 2 days are initiated by fully lac⁺ revertant cells that arose during prior nonselective pregrowth. More Lac⁺ colonies accumulate on the plate over the next 4 days. On day 5, 90% of new late colonies are made up of stable lac⁺ cells that have acquired a compensating frameshift mutation and, therefore, a functional lac⁺allele. The other 10% of revertant colonies are made up of cells with an amplified array of the original mutant lac allele. Amplification-bearing cells are unstable and frequently lose their Lac⁺ phenotype. When these revertants are streaked on rich, nonselective medium containing the chromogenic LacZ substrate X-gal, they form sectored (blue/white) colonies that reveal their frequent loss of ability to use lactose (see Fig. 3).

Figure 3.

The time course of accumulating stable and unstable revertants in the Cairns–Foster experiment. After the tester strain is plated on lactose medium (on day 0), revertant colonies accumulate over several days. Stable lac⁺ colonies accumulate linearly with time, whereas unstable Lac⁺ colonies accumulate exponentially with time. The genotype of the parent tester strain is diagrammed at the top right. Stable Lac⁺ colonies have acquired a compensating (−1) frameshift mutation and thereby a lac⁺ allele. Cells in unstable Lac⁺ revertant colonies have multiple copies (n) of the original leaky mutant lac allele, arranged either as tandem direct-order repeats (short junction [SJ]) or expanded tandem inversion duplications (TIDs) with copies in an alternating orientation. Cells with sufficient copies of the partially functional mutant lac allele can grow on lactose and form a sectored blue/white colony on rich X-gal medium, owing to loss of lac copies.

Figure 4.

Major contributors to revertant yield. A considerable body of data has been accumulated to test the effect of various chromosome- and plasmid-encoded functions on revertant yield. Top left shows dependence of revertant yield on the error-prone repair polymerase DinB (4- to 5-fold). Top center shows that, in a DinB⁺ strain, stable revertants are about 90% of total and unstable revertants are 10%. Top right shows that, without DinB, the number of stable revertants is reduced about 10-fold and becomes roughly equal to the number of unstable revertants (Foster 2000; McKenzie et al. 2001). That is, even without the mutagenic DinB polymerase, the number of stable revertants increases with time under selection. Two of the most important functions for reversion are TraI (bottom left), which nicks and unwinds plasmid DNA during conjugative transfer, and RecA, a strand-exchange protein that is central to homologous recombination. Mutants in recBC and ruvABC genes have similarly strong effects on revertant yield.

Figure 5.

How lac amplification enhances revertant yield without mutagenesis. Each act of lac replication provides an opportunity for a reversion event (frameshift). A cell with only one lac copy has one opportunity to revert for each cell division or plasmid replication (see left column). As the lac allele amplifies, each cell gains additional chances for a reversion event with no increase in mutation rate (right column). If the amplified array improves growth on lactose, then the lineage on the right expands faster than that on the left (without an amplification) and this growth also adds to the likelihood of a reversion event. In effect, amplification directs mutations to the exact base pairs that limit growth, because once a revertant allele forms, selection holds only that allele, whereas the nonrevertants alleles are no longer selectively retained. The likelihood of an unselected mutation near lac is the same with and without selection, because only the revertant allele is kept in the genome.