Abstract
Whether mutations in bacteria exhibit a noticeable delay before expressing their corresponding mutant phenotype was discussed intensively in the 1940s-50s, but the discussion eventually waned for lack of supportive evidence and perceived incompatibility with observed mutant distributions in fluctuation tests. Phenotypic delay in bacteria is widely assumed to be negligible, despite lack of direct evidence. Here we revisited the question using recombineering to introduce antibiotic resistance mutations into E. coli at defined time points and then tracking expression of the corresponding mutant phenotype over time. Contrary to previous assumptions, we found a substantial median phenotypic delay of 3-4 generations. We provided evidence that the primary source of this delay is multifork replication causing cells to be effectively polyploid, whereby the presence of wild-type gene copies transiently masks the mutant phenotype in the same cell. Using mathematical models, we showed that this multigenerational delay has profound consequences for mutation rate estimation by fluctuation tests, standing genetic variation and evolutionary adaptation to rapidly shifting selection pressure such as antibiotic treatment. Overall, we have identified phenotypic delay and effective polyploidy as previously overlooked but essential components in bacterial evolvability, including antibiotic resistance evolution.