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. 2011 Mar;7(3):e1001321.
doi: 10.1371/journal.pgen.1001321. Epub 2011 Mar 3.

Initial mutations direct alternative pathways of protein evolution

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Initial mutations direct alternative pathways of protein evolution

Merijn L M Salverda et al. PLoS Genet. 2011 Mar.

Abstract

Whether evolution is erratic due to random historical details, or is repeatedly directed along similar paths by certain constraints, remains unclear. Epistasis (i.e. non-additive interaction between mutations that affect fitness) is a mechanism that can contribute to both scenarios. Epistasis can constrain the type and order of selected mutations, but it can also make adaptive trajectories contingent upon the first random substitution. This effect is particularly strong under sign epistasis, when the sign of the fitness effects of a mutation depends on its genetic background. In the current study, we examine how epistatic interactions between mutations determine alternative evolutionary pathways, using in vitro evolution of the antibiotic resistance enzyme TEM-1 β-lactamase. First, we describe the diversity of adaptive pathways among replicate lines during evolution for resistance to a novel antibiotic (cefotaxime). Consistent with the prediction of epistatic constraints, most lines increased resistance by acquiring three mutations in a fixed order. However, a few lines deviated from this pattern. Next, to test whether negative interactions between alternative initial substitutions drive this divergence, alleles containing initial substitutions from the deviating lines were evolved under identical conditions. Indeed, these alternative initial substitutions consistently led to lower adaptive peaks, involving more and other substitutions than those observed in the common pathway. We found that a combination of decreased enzymatic activity and lower folding cooperativity underlies negative sign epistasis in the clash between key mutations in the common and deviating lines (Gly238Ser and Arg164Ser, respectively). Our results demonstrate that epistasis contributes to contingency in protein evolution by amplifying the selective consequences of random mutations.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Cefotaxime MIC of selected TEM alleles.
(A) Adaptation of TEM-1 β-lactamase towards improved hydrolysis of cefotaxime. Lines show changes in the average Minimal Inhibitory Concentration (MIC) of cefotaxime for the twelve lines shown in Figure 2 (see Table S3 for replicate MIC values). Aberrant lines 3 and 4 are indicated with solid lines and open and closed triangles, respectively. (B) Increase in cefotaxime MIC for TEM-1 (bold line, the average of Figure 1A) and mutants R164S (open diamonds, dashed line), A237T (diamonds, dashed line), R164S/G238S (open squares) and A237T/G238S (squares).
Figure 2
Figure 2. Amino acid substitutions in evolved TEM-1 alleles.
(A) Substitutions are shown as the TEM-1 amino acid according to the IUPAC single-letter code (left), the position in the protein (middle), and the mutant amino acid (right). Substitutions found after the first round (and hence in all subsequent rounds) have no shade, those found after the second round have a light shade and those found after the third round have a dark shade. The three substitutions indicated with an asterisk appeared in the first round, but disappeared again after round 2 (V33I and M155I in line 10) and round 3 (V10I in line 1). Mutation details (including the official nucleotide numbering as in reference [40]) and silent mutations can be found in Table S1. (B) MIC values of the alleles isolated after the different rounds of evolution, as shown in (A). Values shown are the median value across three replicates (see Table S3 for replicate measurements). Shading as above.
Figure 3
Figure 3. Amino acid substitutions in evolved TEM-mutants R164S and A237T.
(A) See legend of Figure 2A for details. The substitutions present in the starting alleles and still present at the end of the experiment have been highlighted by a thick-lined box. Note that line 1 of the A237T alleles acquired substitution R164C after the first round of evolution, and substitution C164Y after the third round. (B) See legend of Figure 2B for details.
Figure 4
Figure 4. Amino acid substitutions in evolved TEM double-mutant A184V/G238S.
(A) See legend of Figure 2A for details. The substitutions present in the starting alleles and still present at the end of the experiment have been highlighted by a thick-lined box. (B) See legend of Figure 2B for details.
Figure 5
Figure 5. Amino acid substitutions in evolved TEM double-mutants R164S/G238S and A237T/G238S.
(A) See legend of Figure 2A for details. The substitutions present in the starting alleles and still present at the end of the experiment have been highlighted by a thick-lined box. Note that all R164S substitutions initially present in the five R164S/G238S lines eventually revert, either after the first (lines 2,3,4 and 5) or the third round of evolution (line 1). Line 1 of the A237T/G238S alleles shows a reversion of A237T after the second round of evolution, while the other four A237T/G238S lines do not show any reversion. (B) See legend of Figure 2B for details.

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