Long-term experimental evolution in Escherichia coli. XII. DNA topology as a key target of selection

Abstract

The genetic bases of adaptation are being investigated in 12 populations of Escherichia coli, founded from a common ancestor and serially propagated for 20,000 generations, during which time they achieved substantial fitness gains. Each day, populations alternated between active growth and nutrient exhaustion. DNA supercoiling in bacteria is influenced by nutritional state, and DNA topology helps coordinate the overall pattern of gene expression in response to environmental changes. We therefore examined whether the genetic controls over supercoiling might have changed during the evolution experiment. Parallel changes in topology occurred in most populations, with the level of DNA supercoiling increasing, usually in the first 2000 generations. Two mutations in the topA and fis genes that control supercoiling were discovered in a population that served as the focus for further investigation. Moving the mutations, alone and in combination, into the ancestral background had an additive effect on supercoiling, and together they reproduced the net change in DNA topology observed in this population. Moreover, both mutations were beneficial in competition experiments. Clonal interference involving other beneficial DNA topology mutations was also detected. These findings define a new class of fitness-enhancing mutations and indicate that the control of DNA supercoiling can be a key target of selection in evolving bacterial populations.

Figure 1.—

Parallel changes in DNA topology during experimental evolution. Plasmid DNA was isolated from ancestral and evolved cells carrying reporter plasmid pUC18 and analyzed by electrophoresis on gels that allow visualization of the topoisomer distribution, with more tightly supercoiled topoisomers migrating faster (Higgins et al. 1988). (A) Gel for population Ara−1 with pUC18 extracted from the ancestor clone (lane 1) and evolved clones from 2000, 10,000, and 20,000 generations (lanes 2–4, respectively). Quantitative comparisons between each evolved clone and the ancestor are based on densitometric analyses of the gels (see materials and methods). The indicated σ-value for the ancestor clone (−0.066) was obtained relative to the midpoint topoisomer of plasmid completely relaxed with calf-thymus topoisomerase I (see materials and methods). (B) The change in supercoiling level is shown by bars for each evolved population, labeled as +1–+6 (Ara+1–Ara+6) and −1 to −6 (Ara−1–Ara−6). Open, hatched, and solid bars represent clones isolated at 2000, 10,000, and 20,000 generations, respectively. The σ-values calculated in evolved clones are compared to the ancestral level (−0.066), such that higher absolute values show increased supercoiling and lower absolute values indicate relaxation of DNA. The absence of a bar for +6 at 10,000 generations indicates that no measurement could be obtained (see text). Otherwise, short bars indicate the absence of any change in supercoiling.

Figure 2.—

Changes in DNA supercoiling generated by evolved topA and fis mutations. The changes in σ-values relative to the ancestral level (−0.066) are shown for various strains. The strain 606 is the ancestral strain; 2K and 20K are clones isolated from population Ara−1 at 2000 and 20,000 generations, respectively. All others are isogenic constructs made by replacing ancestral alleles with evolved alleles (denoted by superscript e) or by replacing an evolved allele with its ancestral counterpart (superscript +).

Figure 3.—

Relative levels of Fis protein. Extracts were prepared from the ancestral strain (606), an isogenic strain with the evolved fis allele (606 fis^e), and a fis-deleted derivative of the ancestor (606 fis^Δ) after 1 and 1.5 hr of exponential growth in LB medium. Western blots and immunodetection assays are shown for an anti-Fis antibody (top) and, as a control, an anti-RpoA antibody (middle). (Bottom) The ratio of Fis to RpoA levels in the same strains, based on three replicate experiments with error bars indicating standard deviations. The ratio is arbitrarily set to 1 for the ancestral strain after 1 hr of exponential growth. No Fis protein was detected in the control strain with the fis gene deleted.

Figure 4.—

Fitness effects of the evolved DNA topology-altering mutations in the ancestral genetic background. Competition experiments were performed in the same medium used in the long-term evolution. Error bars are 95% confidence intervals based on 16, 11, 4, and 6 replicate competition assays for each genotype (left to right). The strain 606 is the ancestor; topA^e and fis^e are alleles that evolved in focal population Ara−1 and then were moved, alone or in combination, into the ancestral chromosome. For comparison, we also show the mean fitness for population Ara−1 at three time points: 2000 (2K), 10,000 (10K), and 20,000 (20K) generations. The value shown for 2K is the average of values obtained by Lenski et al. (1991) and by Lenski and Travisano (1994); the value for 10K is the average of values obtained by Lenski and Travisano (1994) and by Cooper and Lenski (2000); and the value for 20K is from Cooper and Lenski (2000).