Inactivation of a Mismatch-Repair System Diversifies Genotypic Landscape of Escherichia coli During Adaptive Laboratory Evolution

Abstract

Adaptive laboratory evolution (ALE) is used to find causal mutations that underlie improved strain performance under the applied selection pressure. ALE studies have revealed that mutator populations tend to outcompete their non-mutator counterparts following the evolutionary trajectory. Among them, mutS- inactivated mutator cells, characterize d by a dysfunctional methyl-mismatch repair system, are frequently found in ALE experiments. Here, we examined mutS inactivation as an approach to facilitate ALE of Escherichia coli. The wild-type E. coli MG1655 and mutS knock-out derivative (Δ mutS ) were evolved in parallel for 800 generations on lactate or glycerol minimal media in a serial-transfer experiment. Whole-genome re-sequencing of each lineage at 100-generation intervals revealed that (1) mutations emerge rapidly in the Δ mutS compared to in the wild-type strain; (2) mutations were more than fourfold higher in the Δ mutS strain at the end-point populations compared to the wild-type strain; and (3) a significant number of random mutations accumulated in the Δ mutS strains. We then measured the fitness of the end-point populations on an array of non-adaptive carbon sources. Interestingly, collateral fitness increases on non-adaptive carbon sources were more pronounced in the Δ mutS strains than the parental strain. Fitness measurement of single mutants revealed that the collateral fitness increase seen in the mutator lineages can be attributed to a pool of random mutations. Together, this study demonstrates that short-term mutator ALE extensively expands possible genotype space, resulting in versatile bacteria with elevated fitness levels across various carbon sources.

Spatiotemporal Manipulation of the Mismatch Repair System of Pseudomonas putida Accelerates Phenotype Emergence

“…12,18 This rationale has been applied to certain ALE experiments, where the genetic diversity of a microbial population was increased before and/or during growth under restrictive culture conditions. 5,15,19,20 Chemical and/or physical mutagenesis techniques have been traditionally used due to their simplicity and wide applicability, 21,22 but other genome-wide random mutagenesis techniques can also be applied for this purpose. 5 Mutator strains, i.e., bacteria displaying higher mutation rates, frequently have mutations in one or several genes encoding DNA repair or error-avoidance systems.…”

Spatiotemporal manipulation of the mismatch repair system ofPseudomonas putidaaccelerates phenotype emergence

“…Since intrinsic DNA mutation rates are typically very low (ranging in the order of 10 −9 –10 −10 per base pair per generation) 17-18 , small and transient increases in mutation frequency can significantly improve the accumulation of beneficial mutations in microbial populations 13,19 . This rationale has been applied to certain ALE experiments in which the genetic diversity of a microbial population was increased before and/or during growth under restrictive culture conditions 6,16,20-21 . Chemical and/or physical mutagenesis techniques have been traditionally used due to their simplicity and wide applicability 22-23 , but other genome-wide random mutagenesis techniques can be also applied for this purpose 6 .…”

Developing rapid growing Bacillus subtilis for improved biochemical and recombinant protein production

“…Adaptive laboratory evolution (ALE) is an important strategy to improve the fitness of microorganisms through selected environmental conditions and was widely used in metabolic engineering ( Papapetridis et al., 2018 ; Lee et al., 2016 ; Choe et al., 2019 ; Phaneuf et al., 2019 ; Gibson et al., 2020 ). Five major application areas of ALE are growth rate optimization ( Sandberg et al., 2014 ; Pfeifer et al., 2017 ), increasing tolerance of the strain ( Qi et al., 2019 ; Wang et al., 2018 ; Pereira et al., 2019 ), increase of substrate utilization ( Kawai et al., 2019 ; Gonzalez-Villanueva et al., 2019 ), increase of product yield/titer ( Gibson et al., 2020 ; Lee et al., 2019 ; Vasconcellos et al., 2019 ) and mechanism discovery ( Tenaillon et al., 2012 ; Kang et al., 2019 ). Fast growth phenotypes of Escherichia coli have been obtained through ALE, and the underlying mechanism has been studied ( LaCroix et al., 2015 ; Long et al., 2017 ; McCloskey et al., 2018 ; Wannier et al., 2018 ; Sandberg et al., 2016 ).…”