Mutational hotspots lead to robust but suboptimal adaptive outcomes in certain environments

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The observed mutational spectrum of adaptive outcomes can be constrained by many factors. For example, mutational biases can narrow the observed spectrum by increasing the rate of mutation at isolated sites in the genome. In contrast, complex environments can shift the observed spectrum by defining fitness consequences of mutational routes. We investigate the impact of different nutrient environments on the evolution of motility in Pseudomonas fluorescens Pf0-2x (an engineered non-motile derivative of Pf0-1) in the presence and absence of a strong mutational hotspot. Previous work has shown that this mutational hotspot can be built and broken via six silent mutations, which provide rapid access to a mutation that rescues swimming motility and confers the strongest swimming phenotype in specific environments. Here, we evolved a hotspot and non-hotspot variant strain of Pf0-2x for motility under nutrient-rich (LB) and nutrient-limiting (M9) environmental conditions. We observed the hotspot strain consistently evolved faster across all environmental conditions and its mutational spectrum was robust to environmental differences. However, the non-hotspot strain had a distinct mutational spectrum that changed depending on the nutrient environment. Interestingly, while alternative adaptive mutations in nutrient-rich environments were equal to, or less effective than, the hotspot mutation, the majority of these mutations in nutrient-limited conditions produced superior swimmers. Our competition experiments mirrored these findings, underscoring the role of environment in defining both the mutational spectrum and the associated phenotype strength. This indicates that while mutational hotspots working in concert with natural selection can speed up access to robust adaptive mutations (which can provide a competitive advantage in evolving populations), they can limit exploration of the mutational landscape, restricting access to potentially stronger phenotypes in specific environments.
Original languageEnglish
Article number001395
Number of pages13
Issue number10
Publication statusPublished - 10 Oct 2023

Bibliographical note

Funding Information:
The authors would like to thank the Royal Society for funding this research. They would also like to thank Matthew Shepherd for invaluable advice and guidance with the genetic engineering aspects of this project. They would also like to sincerely thank Kees Wanders for help with statistical analyses in this work.

Funding Information:
This project was funded by a Royal Society Enhancement Grant (RGF\EA\180265; awarded to T.B.T.) supporting L.M.F., BBSRC NI grant (BB/T012994/1; awarded to T.B.T.) supporting J.S.H. and a Royal Society Dorothy Hodgkin Research Fellowship (DH150169) awarded to and supporting T.B.T. Illumina WGS was performed by SeqCenter (LLC), Pittsburgh, PA, USA. Figures S1 and S2 were created using

Publisher Copyright:
© 2023 The Authors.


  • Pseudomonas fluorescens
  • constrained evolution
  • evolutionary rescue
  • mutational bias
  • nutrient environment
  • predicting evolution

ASJC Scopus subject areas

  • Microbiology


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