Fig. 1. Schematic overview of the mutation accumulation experiment.

P. aeruginosa mutS::Tn (the starting cell line) was streaked on LB agar. Six colonies were picked and used to inoculate the overnight cultures (the starting cultures). Each of the starting cultures was used to inoculate a weak bottleneck (WBN) cell line with 100 cells and a strong bottleneck (SBN) cell line with one cell. WBN and SBN cell lines were propagated for 24 days. Six single-colony-derived clones for each WBN cell line were saved in liquid nitrogen. Subsequently, whole-genome sequencing of cryo-stocked starting cultures, last passage of the SBN cell lines and the six WBN clones per each WBN cell line was performed. We also performed an analogous mutation accumulation experiment with S. enterica LT2 ∆mutS using 250 cells for passaging WBN cell lines.