Identification and dynamics of a beneficial mutation in a long-term evolution experiment with Escherichia coli

Abstract

Background: Twelve populations of E. coli were serially propagated for 20,000 generations in a glucose-supplemented minimal medium in order to study the dynamics of evolution. We sought to find and characterize one of the beneficial mutations responsible for the adaptation and other phenotypic changes, including increased cell size, in one of these populations.

Results: We used transposon-tagging followed by P1-transduction into the ancestor, screening for increased cell size and fitness, co-transduction analysis, and DNA sequencing. We identified a 1-bp insertion in the BoxG1 region located upstream of glmUS, an operon involved in cell-wall biosynthesis. When transduced into the ancestor, this mutation increased competitive fitness by about 5%. This mutation spread through its population of origin between 500 and 1500 generations. Mutations in this region were not found in the other 11 evolving populations, even after 20,000 generations.

Conclusion: The 1-bp insertion in the BoxG1 region near glmUS was demonstrably beneficial in the environment in which it arose. The absence of similar mutations in the other evolved populations suggests that they substituted other mutations that rendered this particular mutation unimportant. These results show the unpredictability of adaptive evolution, whereas parallel substitutions at other loci in these same populations reveal the predictability.

Figure 1

Region upstream of glmUS, including site of beneficial mutation. NagC binds to two 23-bp BoxG elements shown in bold. The beneficial adenine insertion was in a tract of seven existing adenines in BoxG1; it is shown above the sequence as +A. The Shine-Dalgarno sequence (S-D) and the -10 and -35 regions of two promoters, P1 and P2, are underlined. Figure modified from Plumbridge [31].

Figure 2

Biochemical pathways involving GlmS and GlmU. Biochemical pathways involving GlmS and GlmU, along with certain transport and biosynthetic reactions. Not all participating molecules are shown for every reaction. Genes encoding relevant enzymes are indicated next to the reactions. Genes printed in bold, and next to reactions shown as heavier arrows, are regulated by NagC. The dashed horizontal line represents the cell envelope. Dashed arrows indicate multiple steps. This figure is a composite of information from several sources [31-33,41,59].

Figure 3

Effect of BoxG1^8A mutation on NagC binding sequence. The consensus sequence, including alternative bases at some sites, for a 23-bp NagC-binding Box element is shown at the top [31]. The four underlined bases are the only ones that are invariant across all such elements [31], and mutagenesis has shown that these four are also the most important for binding NagC [38]. The next row shows the ancestral BoxG1 sequence. The bottom two rows show the effect of inserting an extra adenine, as in the evolved BoxG1^8A allele, when viewed as shifting the sequence either to the left or to the right. The former eliminates the invariant T at position -5. The latter removes agreement with the consensus at position +11.