Developing controllable hypermutable Clostridium cells through manipulating its methyl-directed mismatch repair system

Luan, Guodong; Zhang, Cai; Gong, Fuyu; Dong, Hongjun; Zhao, Lin; Zhang, Yanping; Li, Yin

doi:10.1007/s13238-013-3079-9

Cited by 17 publications

(8 citation statements)

References 44 publications

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“…While both the wt and ΔmutS-adapted strains (AE and MAE, respectively) each evolved to grow significantly better than unadapted wt cells at low pH, genome resequencing of the mutants confirmed that mutS inactivation decreased DNA replication fidelity and yielded derivatives with a greater number of genetic changes (Table S2 and S3). Previous studies investigating the effect of inactive MMR on the mutation rate have shown increased mutation rates of 10-to 1,000-fold (12,13,23,24). Our observed 3.3-fold increase in the number of mutations in L. casei 12A-MAE is lower than the rates previously reported.…”

Section: Discussioncontrasting

confidence: 82%

“…Under stressed conditions, hypermutability can be leveraged to increase an organism's fitness and achieve a desired phenotype through adaptive laboratory evolution (1-3). High mutation rates resulting from a deficient MMR process have been previously shown to increase the fitness of an organism in defined environments (13,23). In this study, we explored the potential for coupling transient inactivation of the MMR process with adaptive evolution to obtain L. casei strains with increased lactic acid resistance at low pH.…”

Section: Discussionmentioning

confidence: 99%

“…However, we believe this difference is explained by the differences between the methods used to identify mutations. In previous studies, the mutation rate was calculated for hypermutable cells using reversion assays (12,13,23,24), whereas we determined the number of mutations using genome resequencing after the adaptive evolution process. We expect that many mutations not selected for under our strong selective pressure were incidentally filtered out during the extended adaptive evolution process, reducing the final number of mutations observed in L. casei 12A-MAE.…”

Section: Discussionmentioning

confidence: 99%

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Transient MutS-Based Hypermutation System for Adaptive Evolution of Lactobacillus casei to Low pH

Overbeck

Welker

Hughes

et al. 2017

Appl Environ Microbiol

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This study explored transient inactivation of the gene encoding the DNA mismatch repair enzyme MutS as a tool for adaptive evolution of MutS deletion derivatives of 12A and ATCC 334 were constructed and subjected to a 100-day adaptive evolution process to increase lactic acid resistance at low pH. Wild-type parental strains were also subjected to this treatment. At the end of the process, the Δ lesion was repaired in representative 12A and ATCC 334 Δ mutant isolates. Growth studies in broth at pH 4.0 (titrated with lactic acid) showed that all four adapted strains grew more rapidly, to higher cell densities, and produced significantly more lactic acid than untreated wild-type cells. However, the adapted Δ derivative mutants showed the greatest increases in growth and lactic acid production. Further characterization of the 12A-adapted Δ derivative revealed that it had a significantly smaller cell volume, a rougher cell surface, and significantly better survival at pH 2.5 than parental 12A. Genome sequence analysis confirmed that transient inactivation decreased DNA replication fidelity in both strains, and it identified genetic changes that might contribute to the lactic acid-resistant phenotypes of adapted cells. Targeted inactivation of three genes that had acquired nonsense mutations in the adapted 12A Δ mutant derivative showed that NADH dehydrogenase (), phosphate transport ATP-binding protein PstB (), and two-component signal transduction system (TCS) quorum-sensing histidine protein kinase () genes act in combination to increase lactic acid resistance in 12A. Adaptive evolution has been applied to microorganisms to increase industrially desirable phenotypes, including acid resistance. We developed a method to increase the adaptability of 12A and ATCC 334 through transient inactivation of the DNA mismatch repair enzyme MutS. Here, we show this method was effective in increasing the resistance of to lactic acid at low pH. Additionally, we identified three genes that contribute to increased acid resistance in 12A. These results provide valuable insight on methods to enhance an organism's fitness to complex phenotypes through adaptive evolution and targeted gene inactivation.

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Section: Discussioncontrasting

confidence: 82%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Transient MutS-Based Hypermutation System for Adaptive Evolution of Lactobacillus casei to Low pH

Overbeck

Welker

Hughes

et al. 2017

Appl Environ Microbiol

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“…Several studies have shown that in microbial cells, elevated mutation rates can be exploited to facilitate ALE toward better fitness compared with non-mutator cells (Sniegowski et al, 1997; Giraud et al, 2001a; Notley-McRobb et al, 2002; Barrick et al, 2009; LaCroix et al, 2015). Often, MMR-impaired cells have been used as the mutator strain in numbers of ALE applications (Luan et al, 2013; Overbeck et al, 2017; Wannier et al, 2018) because MMR-inactivation were known to generate moderate mutators in E. coli (Taddei et al, 1997; Rosche and Foster, 1999; Tenaillon et al, 1999; Loh et al, 2010; Sprouffske et al, 2018). In laboratory settings, the desired ALE results can be achieved when strains become best-adapted for survival in an unfavorable condition.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2019

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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.

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“…So by placing mutS and/or mutL under the control of inducible promoters as well it may be possible to extend the range of tunable mutation rates downward; ( Foster 1999 ) found that overexpression of mutS, mutL, or both reduced the number of reversion mutations of the lac or tet alleles. ( Luan et al 2013a ) adjusted the mutation rate of Clostridium acetobutylicum by altering mutS and mutL levels, and did not attain lower than wildtype mutation rates.…”

Section: Discussionmentioning

confidence: 99%

Escherichia coli with a Tunable Point Mutation Rate for Evolution Experiments

Sherer

Kuhlman

2020

G3 Genes|Genomes|Genetics

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The mutation rate and mutations' effects on fitness are crucial to evolution. Mutation rates are under selection due to linkage between mutation rate modifiers and mutations' effects on fitness. The linkage between a higher mutation rate and more beneficial mutations selects for higher mutation rates, while the linkage between a higher mutation rate and more deleterious mutations selects for lower mutation rates. The net direction of selection on mutations rates depends on the fitness landscape, and a great deal of work has elucidated the fitness landscapes of mutations. However, tests of the effect of varying a mutation rate on evolution in a single organism in a single environment have been difficult. This has been studied using strains of antimutators and mutators, but these strains may differ in additional ways and typically do not allow for continuous variation of the mutation rate. To help investigate the effects of the mutation rate on evolution, we have genetically engineered a strain of E. coli with a point mutation rate that can be smoothly varied over two orders of magnitude. We did this by engineering a strain with inducible control of the mismatch repair proteins MutH and MutL. We used this strain in an approximately 350 generation evolution experiment with controlled variation of the mutation rate. We confirmed the construct and the mutation rate were stable over this time. Sequencing evolved strains revealed a higher number of single nucleotide polymorphisms at higher mutations rates, likely due to either the beneficial effects of these mutations or their linkage to beneficial mutations.

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Developing controllable hypermutable Clostridium cells through manipulating its methyl-directed mismatch repair system

Cited by 17 publications

References 44 publications

Transient MutS-Based Hypermutation System for Adaptive Evolution of Lactobacillus casei to Low pH

Transient MutS-Based Hypermutation System for Adaptive Evolution of Lactobacillus casei to Low pH

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

Escherichia coli with a Tunable Point Mutation Rate for Evolution Experiments

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