Allosteric adaptation in the stator complex rescues bacterial motility in Exb/Mot chimeras

Powered by ion transport across the cell membrane, conserved ion-powered rotary motors (IRMs) drive bacterial motility by generating torque on the rotor of the bacterial flagellar motor. Homologous heteroheptameric IRMs have been structurally characterized in ion pumps such as Tol/Ton/Exb/Gld, and most recently in phage defense systems such as Zor. Functional stator complexes synthesized from chimeras of PomB/MotB (PotB) have been used to slow flagellar rotation via reduced external sodium concentration. Such chimeras are highly sensitive to the location of the cut-site and thus far have been arbitrarily designed. To date, no chimeras have been constructed using interchange of components from Tol/Ton/Exb/Gld and other ion powered motors with more distant homology.

Here we synthesised chimeras of MotAB, PomAPotB and ExbBD to assess their capacity for cross-compatibility. We obtained motile B-unit chimeric stator complexes whose motility was further optimised by directed evolution. Whole genome sequencing of these revealed epistatic adaptations in the A-subunit and at the peptidoglycan binding domain of the B-unit could improve motility. Overall, our work highlights the role of allostery and the challenges associated with rational design of chimeric IRMs.

Importance

Ion-powered rotary motors (IRMs) underpin the rotation of nature’s oldest wheel, the flagellar motor. Indeed, they themselves appear to rotate to drive flagellar motor and may be the oldest molecular wheel. Recent structures have shown this complex drives ion pumping and even phage defence and thus appears to be a fundamental module in bionanotechnology with diverse biological utility where electrical energy can be coupled to rotational force to execute work.

Here we attempted to rationally design chimeric ‘Frankenstein’ IRMs to explore the cross-compatibility of these ancient motors, to try to better understand what a common ancestor may have looked like, as well as to explore possibilities to engineer this fundamental component of bionanotechnology for future application.