Effective polyploidy causes phenotypic delay and influences bacterial evolvability

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 wild-type gene copies transiently mask the phenotype of recessive mutant gene copies in the same cell. Using modeling and simulation methods, we explored the consequences of effective polyploidy for mutation rate estimation by fluctuation tests and sequencing-based methods. For recessive mutations, despite the substantial phenotypic delay, the per-copy or per-genome mutation rate is accurately estimated. However, the per-cell rate cannot be estimated by existing methods. Finally, with a mathematical model, we showed that effective polyploidy increases the frequency of costly recessive mutations in the standing genetic variation, and thus their potential contribution to evolutionary adaptation, while drastically reducing the chance that de novo recessive mutations can rescue populations facing a harsh environmental change 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. Author summaryWhat is the time delay between the occurrence of a genetic mutation in a bacterial cell and manifestation of its phenotypic effect? We show that antibiotic resistance mutations in E.coli show a remarkably long phenotypic delay of 3-4 bacterial generations. The primary underlying mechanism of this delay is effective polyploidy. In a polyploid cell with multiple chromosomes, once a mutation arises on one of the chromosomes, the presence of non-mutated, wild-type gene copies on other chromosomes may mask the . CC-BY 4.0 International license not peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/226654 doi: bioRxiv preprint first posted online Nov. 29, 2017; 2 phenotype of the mutation. One implication of this finding is that conventional methods to determine the mutation rates of bacteria do not detect polyploidy and thus underestimate their potential for adaptation. More generally, the effect that a new mutation may become useful only in the "grand-children of the grand-children" suggests that pre-existing mutations are more importan...

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“…Mediating the high-fidelity repair of DSBs in bacteria, HDR is accepted to be the main mechanism by which most prokaryotes circumvent death by DSBs 55,53 , and it requires an intact copy of the genetic region to repair 56 . While several enzymes are involved in this type of repair, the RecBCD complex is generally considered its main actor, processing DSBs and generating DNA ends with a 3' extension that is recognized by the RecA recombinase, in turn, responsible for promoting strand invasion into the intact DNA template, DNA repair taking place and the initial sequence being restored thereafter 57 .…”

“…Additionally, effective polyploidy 56 (that is, E. coli having more than one plasmid at the same time during fast exponential phase) might be another reason by which cells bypass having multiple deletions. While one or multiple copies of a chromosome might be edited within a cell while grown under selection for targeting of a certain locus, once these cells are selected for the next antibiotic in the cycle, cells originating from the polyploid cell that now carry an intact copy of the chromosome will likely have a considerable growth advantage, which will ultimately result in cells only maintaining one deletion in their genome after having undergone our genome editing cycle.…”

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