Fig 1. Experimental approach to isolate and map rescue (sup) strains.

Strains harboring one of five deletions (Δzwf, ΔdapF, Δdgk, ΔentC, or Δppk) were suspended in M9 supplemented with 0.4% (w/v) of glucose (M9G) in triplicate. The deletion strains were allowed to grow at 37° C with shaking for 12 hours, at which point they were diluted to OD₆₀₀ = 0.01. Adaptive evolution proceeded in this manner for 21–28 days (216–611 generations). At the end of this AE period, the culture was plated, colonies were recovered and resuspended in liquid media, and log-phase growth rates were measured, identifying sup strains. The genetic lesions associated with rescued growth were identified by whole genome sequencing (WGS).