Functional and metabolic effects of adaptive glycerol kinase (GLPK) mutants in Escherichia coli.

Herein we measure the effect of four adaptive non-synonymous mutations to the glycerol kinase (glpK) gene on catalytic function and regulation, to identify changes that correlate to increased fitness in glycerol media. The mutations significantly reduce affinity for the allosteric inhibitor fructose-1,6-bisphosphate (FBP) and formation of the tetramer, which are structurally related, in a manner that correlates inversely with imparted fitness during growth on glycerol, which strongly suggests that these enzymatic parameters drive growth improvement. Counterintuitively, the glpK mutations also increase glycerol-induced auto-catabolite repression that reduces glpK transcription in a manner that correlates to fitness. This suggests that increased specific GlpK activity is attenuated by negative feedback on glpK expression via catabolite repression, possibly to prevent methylglyoxal toxicity. We additionally report that glpK mutations were fixed in 47 of 50 independent glycerol-adapted lineages. By far the most frequently mutated locus (nucleotide 218) was mutated in 20 lineages, strongly suggesting this position has an elevated mutation rate. This study demonstrates that fitness correlations can be used to interrogate adaptive processes at the protein level and to identify the regulatory constraints underlying selection and improved growth.

Whole-genome resequencing of Escherichia coli K-12 MG1655 undergoing short-term laboratory evolution in lactate minimal media reveals flexible selection of adaptive mutations.

BACKGROUND: Short-term laboratory evolution of bacteria followed by genomic sequencing provides insight into the mechanism of adaptive evolution, such as the number of mutations needed for adaptation, genotype-phenotype relationships, and the reproducibility of adaptive outcomes. RESULTS: In the present study, we describe the genome sequencing of 11 endpoints of Escherichia coli that underwent 60-day laboratory adaptive evolution under growth rate selection pressure in lactate minimal media. Two to eight mutations were identified per endpoint. Generally, each endpoint acquired mutations to different genes. The most notable exception was an 82 base-pair deletion in the rph-pyrE operon that appeared in 7 of the 11 adapted strains. This mutation conferred an approximately 15% increase to the growth rate when experimentally introduced to the wild-type background and resulted in an approximately 30% increase to growth rate when introduced to a background already harboring two adaptive mutations. Additionally, most endpoints had a mutation in a regulatory gene (crp or relA, for example) or the RNA polymerase. CONCLUSIONS: The 82 base-pair deletion found in the rph-pyrE operon of many endpoints may function to relieve a pyrimidine biosynthesis defect present in MG1655. In contrast, a variety of regulators acquire mutations in the different endpoints, suggesting flexibility in overcoming regulatory challenges in the adaptation.

Impact of individual mutations on increased fitness in adaptively evolved strains of Escherichia coli.

We measured the relative fitness among a set of experimentally evolved Escherichia coli strains differing by a small number of adaptive mutations by directly measuring allelic frequencies in head-to-head competitions using a mass spectrometry-based method. We compared the relative effects of mutations to the same or similar genes acquired in multiple strains when expressed in allele replacement strains. We found that the strongest determinant of fitness among the evolved strains was the impact of beneficial mutations to the RNA polymerase beta and beta' subunit genes. We also identified several examples of epistatic interactions between rpoB/C and glpK mutations and identified two other mutations that are beneficial only in the presence of previously acquired mutations but that have little or no adaptive benefit to the wild-type strain. Allele frequency estimation is shown to be a highly sensitive method for measuring selection rates during competitions between strains differing by as little as a single-nucleotide polymorphism and may be of great use for investigating epistatic interactions.

Comparative genome sequencing of Escherichia coli allows observation of bacterial evolution on a laboratory timescale.

We applied whole-genome resequencing of Escherichia coli to monitor the acquisition and fixation of mutations that conveyed a selective growth advantage during adaptation to a glycerol-based growth medium. We identified 13 different de novo mutations in five different E. coli strains and monitored their fixation over a 44-d period of adaptation. We obtained proof that the observed spontaneous mutations were responsible for improved fitness by creating single, double and triple site-directed mutants that had growth rates matching those of the evolved strains. The success of this new genome-scale approach indicates that real-time evolution studies will now be practical in a wide variety of contexts.