Mutations that improve efficiency of a weak-link enzyme are rare compared to adaptive mutations elsewhere in the genome
- University of Colorado, Boulder, United States
- University of Pittsburgh, United States
Abstract
New enzymes often evolve by gene amplification and divergence. Previous experimental studies have followed the evolutionary trajectory of an amplified gene, but have not considered mutations elsewhere in the genome when fitness is limited by an evolving gene. We have evolved a strain of Escherichia coli in which a secondary promiscuous activity has been recruited to serve an essential function. The gene encoding the 'weak-link' enzyme amplified in all eight populations, but mutations improving the newly needed activity occurred in only one. Most adaptive mutations occurred elsewhere in the genome. Some mutations increase expression of the enzyme upstream of the weak-link enzyme, pushing material through the dysfunctional metabolic pathway. Others enhance production of a co-substrate for a downstream enzyme, thereby pulling material through the pathway. Most of these latter mutations are detrimental in wild-type E. coli, and thus would require reversion or compensation once a sufficient new activity has evolved.
Data availability
The genome sequence of E. coli strain AM187 used in this study has been deposited to NCBI GenBank under accession number CP037857.All other data generated or analyzed during this study are included in the manuscript and supporting files. Source code files have been provided for Figures 3 and 4 and Tables 2 and 3.
Article and author information
Author details
Andrew B Morgenthaler
Christopher C Ebmeier
Funding
National Aeronautics and Space Administration (NNA15BB04A)
- Shelley D Copley
National Aeronautics and Space Administration (NNA15BB04A)
- Vaughn S Cooper
Department of Defense /Defense Advanced Research Projects Agency (13-34-RTA-FP-007)
- William M Old
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2019, Morgenthaler et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Mutations that improve efficiency of a weak-link enzyme are rare compared to adaptive mutations elsewhere in the genome
eLife 8:e53535.
https://doi.org/10.7554/eLife.53535
Further reading
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- Evolutionary Biology
Gene duplication drives evolution by providing raw material for proteins with novel functions. An influential hypothesis by Ohno (1970) posits that gene duplication helps genes tolerate new mutations and thus facilitates the evolution of new phenotypes. Competing hypotheses argue that deleterious mutations will usually inactivate gene duplicates too rapidly for Ohno’s hypothesis to work. We experimentally tested Ohno’s hypothesis by evolving one or exactly two copies of a gene encoding a fluorescent protein in Escherichia coli through several rounds of mutation and selection. We analyzed the genotypic and phenotypic evolutionary dynamics of the evolving populations through high-throughput DNA sequencing, biochemical assays, and engineering of selected variants. In support of Ohno’s hypothesis, populations carrying two gene copies displayed higher mutational robustness than those carrying a single gene copy. Consequently, the double-copy populations experienced relaxed purifying selection, evolved higher phenotypic and genetic diversity, carried more mutations and accumulated combinations of key beneficial mutations earlier. However, their phenotypic evolution was not accelerated, possibly because one gene copy rapidly became inactivated by deleterious mutations. Our work provides an experimental platform to test models of evolution by gene duplication, and it supports alternatives to Ohno’s hypothesis that point to the importance of gene dosage.
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- Cell Biology
- Evolutionary Biology
Maintenance of rod-shape in bacterial cells depends on the actin-like protein MreB. Deletion of mreB from Pseudomonas fluorescens SBW25 results in viable spherical cells of variable volume and reduced fitness. Using a combination of time-resolved microscopy and biochemical assay of peptidoglycan synthesis, we show that reduced fitness is a consequence of perturbed cell size homeostasis that arises primarily from differential growth of daughter cells. A 1000-generation selection experiment resulted in rapid restoration of fitness with derived cells retaining spherical shape. Mutations in the peptidoglycan synthesis protein Pbp1A were identified as the main route for evolutionary rescue with genetic reconstructions demonstrating causality. Compensatory pbp1A mutations that targeted transpeptidase activity enhanced homogeneity of cell wall synthesis on lateral surfaces and restored cell size homeostasis. Mechanistic explanations require enhanced understanding of why deletion of mreB causes heterogeneity in cell wall synthesis. We conclude by presenting two testable hypotheses, one of which posits that heterogeneity stems from non-functional cell wall synthesis machinery, while the second posits that the machinery is functional, albeit stalled. Overall, our data provide support for the second hypothesis and draw attention to the importance of balance between transpeptidase and glycosyltransferase functions of peptidoglycan building enzymes for cell shape determination.
