Fig. 4.

Dependent mutations within resistance-associated genes. We measured the identity and prevalence of significant dependent mutations occurring after initial resistance evolution. (A) Mutations that occur after mutations in known antibiotic resistance genes, visualized on the genome using pyCircos (github.com/ponnhide/pyCircos), with colors corresponding to the antibiotics in B. Antibiotics with shared genetic basis of resistance are shown in the same color. Only mutations that happen sequentially at least five times are shown. (B) Fraction of resistant strains that display one or more pairs of sequential dependent mutations. (C) Example of pairs of dependent mutations within the kanamycin resistance pathway, shown on a per-gene basis. Kanamycin's inhibition of the ribosome is blunted by ribosomal RNA mutations, while cellular kanamycin levels are reduced by increased levels of Eis, putatively caused by both mutations in the eis promoter region and mutations in the regulatory proteins WhiB7 and WhiB6. Dependencies between these mutations demonstrate multistep resistance evolution.