Adaptation to mutational inactivation of an essential gene converges to an accessible suboptimal fitness peak.

João V Rodrigues,Eugene I Shakhnovich

doi:10.7554/elife.50509

Abstract

The mechanisms of adaptation to inactivation of essential genes remain unknown. Here we inactivate E. coli dihydrofolate reductase (DHFR) by introducing D27G,N,F chromosomal mutations in a key catalytic residue with subsequent adaptation by an automated serial transfer protocol. The partial reversal G27- > C occurred in three evolutionary trajectories. Conversely, in one trajectory for D27G and in all trajectories for D27F,N strains adapted to grow at very low metabolic supplement (folAmix) concentrations but did not escape entirely from supplement auxotrophy. Major global shifts in metabolome and proteome occurred upon DHFR inactivation, which were partially reversed in adapted strains. Loss-of-function mutations in two genes, thyA and deoB, ensured adaptation to low folAmix by rerouting the 2-Deoxy-D-ribose-phosphate metabolism from glycolysis towards synthesis of dTMP. Multiple evolutionary pathways of adaptation converged to a suboptimal solution due to the high accessibility to loss-of-function mutations that block the path to the highest, yet least accessible, fitness peak.

Highlights

When important cellular functions are inactivated, for example by genetic mutations, long ranging disruptions of cellular networks can occur, which poses a major adaptive challenge to cells
We found that D27N and D27G strains, but not D27F, had additional background mutations that must have been acquired prior to starting the evolution experiments; all D27 strains were constructed from the same wild type E. coli parent strain, we could not prevent the appearance of mutations at any given stage of genetic manipulation prior to the evolution experiments
Since we observed that all clones randomly selected from trajectories that adapted to low folAmix concentrations showed very similar fitness profiles, we focused on individual clones arbitrarily chosen among D27F and D27N trajectories as representatives of adaptation to low folAmix

Summary

Introduction

When important cellular functions are inactivated, for example by genetic mutations, long ranging disruptions of cellular networks can occur, which poses a major adaptive challenge to cells. Recent studies investigated evolution of E. coli upon inactivation of non-essential enzymes of carbon metabolism that lead to re-wiring through less efficient pathways (Krusemann et al, 2018; Long et al, 2018; McCloskey et al, 2018a; McCloskey et al, 2018b; McCloskey et al, 2018c; McCloskey et al, 2018) These studies highlight a crucial interplay between regulatory responses and imbalances in metabolite concentrations resulting from gene knockouts, which can be subsequently corrected by mutations elsewhere. While adaptation upon inactivation of essential genes in microbes has been demonstrated, its mechanisms remain unknown

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