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Adaptive benefits from small mutation supplies in an antibiotic resistance enzyme

  1. J. Arjan G. M. de Vissera,3
  1. aLaboratory of Genetics, Department of Plant Sciences, Wageningen University, 6708PB Wageningen, The Netherlands;
  2. bInstitute for Translational Vaccinology, 3721MA Bilthoven, The Netherlands;
  3. cInstitute for Theoretical Physics, University of Cologne, 50937 Cologne, Germany
  1. Edited by Bruce R. Levin, Emory University, Atlanta, GA, and approved October 20, 2017 (received for review July 21, 2017)


Breeding protocols in biotechnology focus on fast short-term responses by selecting extreme phenotypes from maximally diverse pools of heritable variants. However, when interactions among alleles are unpredictable, optimizing long-term responses may require intermediate stages with low or modest improvement. By subjecting mutant libraries of an antibiotic-degrading enzyme of different sizes to selection with a new antibiotic, we find support for this prediction: despite slower initial improvement due to limited genetic variation, the final alleles from two small libraries reached the highest antibiotic-resistance levels overall due to a broader exploration of the adaptive landscape. Our results present a cautionary tale for “greedy” adaptation protocols in biotechnology and call for quantitative data on mutation supplies in the clinical evolution of antibiotic resistance.


Populations with large mutation supplies adapt via the “greedy” substitution of the fittest genotype available, leading to fast and repeatable short-term responses. At longer time scales, smaller mutation supplies may in theory lead to larger improvements when distant high-fitness genotypes more readily evolve from lower-fitness intermediates. Here we test for long-term adaptive benefits from small mutation supplies using in vitro evolution of an antibiotic-degrading enzyme in the presence of a novel antibiotic. Consistent with predictions, large mutant libraries cause rapid initial adaptation via the substitution of cohorts of mutations, but show later deceleration and convergence. Smaller libraries show on average smaller initial, but also more variable, improvements, with two lines yielding alleles with exceptionally high resistance levels. These two alleles share three mutations with the large-library alleles, which are known from previous work, but also have unique mutations. Replay evolution experiments and analyses of the adaptive landscape of the enzyme suggest that the benefit resulted from a combination of avoiding mutational cohorts leading to local peaks and chance. Our results demonstrate adaptive benefits from limited mutation supplies on a rugged fitness landscape, which has implications for artificial selection protocols in biotechnology and argues for a better understanding of mutation supplies in clinical settings.


  • ?1Present address: Laboratory of Food Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, 6708 WG Wageningen, The Netherlands.

  • ?2Present address: Department of Microbial Ecology, Netherlands Institute of Ecology, 6708 PB Wageningen, The Netherlands.

  • ?3To whom correspondence should be addressed. Email: arjan.devisser{at}wur.nl.
  • Author contributions: M.L.M.S. and J.A.G.M.d.V. designed research; M.L.M.S., J.K., B.K., and M.P.Z. performed research; B.K. contributed new reagents/analytic tools; M.L.M.S., J.K., M.P.Z., and J.A.G.M.d.V. analyzed data; and M.L.M.S., M.P.Z., and J.A.G.M.d.V. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online at www.danielhellerman.com/lookup/suppl/doi:10.1073/pnas.1712999114/-/DCSupplemental.

Published under the PNAS license.

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