Using CRISPR/cas9 Editing to Explore the Spontaneous Adaptation of Ion Selectivity in a Bacterial Flagellar Motor

Motility provides a selective advantage to many bacterial species and is often achieved by rotation of flagella that propel the cell towards more favourable conditions. In most species, the rotation of the flagellum, driven by the Bacterial Flagellar Motor (BFM), is powered by H⁺ or Na⁺ ion transit through the torque-generating stator subunit of the motor complex. The ionic requirements for motility appear to have adapted to environmental changes throughout history but the molecular basis of this adaptation remains unknown. Here we used CRISPR engineering to replace the native E. coli H⁺-powered stator with Na⁺-powered stator genes and report the rapid and spontaneous reversion to H⁺-power in a low sodium environment. We followed the evolution of the stators during their reversion to H⁺-powered motility and used whole genome sequencing to identify both flagellar- and non-flagellar-associated genes involved in the cell's adaptation to new power sources. This work warns of the rapid reversion of CRISPR edits in rapidly evolving settings and highlights the utility of the flagellar stator system for studying the molecular mechanisms underlying adaptation, specifically demonstrating how environmental change can rapidly alter the function of an ion transporter.