The genetic basis of parallel and divergent phenotypic responses in evolving populations of Escherichia coli

Abstract

Pleiotropy plays a central role in theories of adaptation, but little is known about the distribution of pleiotropic effects associated with different adaptive mutations. Previously, we described the phenotypic effects of a collection of independently arising beneficial mutations in Escherichia coli. We quantified their fitness effects in the glucose environment in which they evolved and their pleiotropic effects in five novel resource environments. Here we use a candidate gene approach to associate the phenotypic effects of the mutations with the underlying genetic changes. Among our collection of 27 adaptive mutants, we identified a total of 21 mutations (18 of which were unique) encompassing five different loci or gene regions. There was limited resolution to distinguish among loci based on their fitness effects in the glucose environment, demonstrating widespread parallelism in the direct response to selection. However, substantial heterogeneity in mutant effects was revealed when we examined their pleiotropic effects on fitness in the five novel environments. Substitutions in the same locus clustered together phenotypically, indicating concordance between molecular and phenotypic measures of divergence.

Figure 1

Hierarchical cluster analysis based on estimates of relative fitness of 27 mutant clones on six different resources. The phenotypic distance metric is a normalized Euclidean distance. The identities of the loci where mutations were found in candidate genes are shown on the left. Numbers in parentheses indicate the location of the mutation within the corresponding gene, from the first position of the start codon.

Figure 2

(a) Location of mutations found in the spoT locus after 20 000 generations (top; Cooper et al. 2003) and associated with the first selective sweep within 400 generations (bottom; this study). Clone identification numbers listed in table S1 in the electronic supplementary material are abbreviated here to include only their last two digits, which are unique to each clone (e.g. 9990 is listed as 90). The two shaded regions of the gene correspond to the ppGpp hydrolase and synthetase domains (Gentry & Cashel 1996). Arrows indicate locations of mutations, from the N-terminus (left) to the C-terminus (right); mutations that affect the same codon are indicated by a single arrow with multiple clone labels. (b) Location of mutations found in the nadR locus after 20 000 generations (top; Woods et al. 2006) and associated with the first selective sweep within 400 generations (bottom; this study). The approximate domain boundaries are indicated, as described in Kurnasov et al. (2003): HTH, helix-turn-helix required for the repressor function; NMNAT, NMN adenylyltransferase; RNK, ribosylnicotinamide kinase. Asterisks indicate mutations caused by the insertion of IS150 elements.

Figure 3

Factor loadings plot for the PCA. Variables included in the analysis were relative fitness in six resources: GLU, glucose; NAG, N-acetylglucosamine; MAN, mannitol; MAL, maltose; GAL, galactose; and MEL, melibiose. PTS resources share a common mechanism for transport across the inner membrane of the cell, whereas non-PTS resources use a variety of different mechanisms.

Figure 4

Plot of the genotypes according to their factor scores for the first two principal components. The identity of the genes where mutations were found has been overlaid onto the plot: black triangles, spoT; black squares, nadR; grey circles, pbprod A; grey squares, hokB/sokB; grey triangles, spoT and pykF; open circles, unknown.