Hybrid Exb/Mot stators require substitutions distant from the chimeric pore to power flagellar rotation

Abstract

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.

Molecular and structural innovations of the stator motor complex at the dawn of flagellar motility

“…Stator variants were tested in stator-deleted derivative strains of E. coli RP437 (E. coli ΔmotAB) (36). The pDB108 (pBAD33 backbone, Cm+) plasmid encoding motA and motB (37) was used as the vector to clone and express all constructs.…”