Neutral drift upon threshold-like selection promotes variation in antibiotic resistance phenotype

Heritable phenotypic variation plays a central role in evolution by conferring rapid adaptive capacity to populations. Mechanisms that can explain genetic diversity by describing connections between genotype and organismal fitness have been described. However, the difficulty of acquiring comprehensive data on genotype-phenotype-environment relationships has hindered the efforts to explain how the ubiquitously observed phenotypic variation in populations emerges and is maintained. To address this challenge, we establish an experimental system where we can examine the genotype-phenotype relationships in a controlled environment. We perform long-term experimental evolution on VIM-2 β-lactamase, an antibiotic-resistance enzyme, to explore the conditions that promote the emergence and maintenance of phenotypic variation. We found that evolution in a static environment with low antibiotic concentrations can promote and maintain significant phenotypic variation within populations. Notably, evolution of VIM-2 under selection with a low antibiotic concentration led to variants that conferred resistance to over 100-fold higher antibiotic concentrations than used in selection. A model based on the previously described threshold-like relationship between enzyme phenotype and fitness generated using VIM-2’s all single amino acid variants, sufficiently explains the emergence of standing phenotypic variation under static environmental conditions. Overall, our approach provides a tractable model for studying phenotypic variation and evolvability at the population level.