Multiple pathways of selected gene amplification during adaptive mutation

Abstract

In a phenomenon referred to as "adaptive mutation," a population of bacterial cells with a mutation in the lac operon (lac-) accumulates Lac+ revertants during prolonged exposure to selective growth conditions (lactose). Evidence was provided that selective conditions do not increase the mutation rate but instead favor the growth of rare cells with a duplication of the leaky lac allele. A further increase in copy number (amplification) improves growth and increases the likelihood of a sequence change by adding more mutational targets to the clone (cells and lac copies per cell). These duplications and amplifications are described here. Before selection, cells with large (134-kb) lac duplications and long junction sequences (>1 kb) were common (0.2%). The same large repeats were found after selection in cells with a low-copy-number lac amplification. Surprisingly, smaller repeats (average, 34 kb) were found in high-copy-number amplifications. The small-repeat duplications form when deletions modify a preexisting large-repeat duplication. The shorter repeat size allowed higher lac amplification and better growth on lactose. Thus, selection favors a succession of gene-amplification types that make sequence changes more probable by adding targets. These findings are relevant to genetic adaptation in any biological systems in which fitness can be increased by adding gene copies (e.g., cancer and bacterial drug resistance).

Fig. 1.

Structures of amplification intermediates and the F′ plasmid. (A) Formation of duplications and amplifications by sister-strand exchanges. Boxes indicate sequence elements that recombine to form a duplication. Exchanges between the extensive repeated segments can lead to further increases and decreases in copy number. (B) Plasmid F'₁₂₈ contains F plasmid sequence (dark portion of circle) with an IS3-flanked 134-kb fragment of the E. coli chromosome including lac, dinB, and REP elements (12). Each REP element (triangle) belongs to one of the three classes indicated (16).

Fig. 2.

Pathways of selected change during growth under selection. At the top above the horizontal line, are cell types with various structures of the lac region (black dots) and their frequencies before selection. Before selection, duplications (dup) arise between IS3 elements (boxes), and deletions (del) convert them to SJ duplications. Numbers at top indicate how many cells of each type are plated on selective medium (lactose). Events below the horizontal line occur under selection. Genotypes farther below the line allow better growth under selection. Copy number is indicated by the “n” after the diagrammed repeat. The final colonies are clonal mixtures of the stable lac⁺ revertant cells and its progenitor amplification cell; dotted lines indicate the minority type.

Fig. 3.

Duplications found before and after selection. The central box represents the bacterial chromosome segment on F'₁₂₈; vertical bars indicate the position of REP element clusters. The top section depicts the repeat found in 24 duplications found in cells grown in rich medium. The second section presents the repeated segments of 34 amplifications found in the rare (<3%) unstable Lac⁺ cells from stable-rich Lac⁺ revertants. The bottom section shows the amplified segments of selected revertant colonies with mostly (>97%) unstable Lac⁺ cells. Junction sequences are described in supporting information. ∗, The total of 48 amplifications includes five inversion duplications, which are not diagrammed.

Fig. 4.

Formation of an SJ duplication by a deletion. Boxes are large sequences (e.g., IS3) able to support recombination events that form the initial duplication. The black dots between b and c indicate the lac operon. The short arrows designate a repeated sequence (3–12 bp) that can mediate deletion formation.

Fig. 5.

Accumulation of stable- and unstable-rich Lac⁺ revertant colonies. The Salmonella version of the Cairns strain (TT18302) was grown and plated as described in Materials and Methods.