FliL and its paralog MotF have distinct roles in the stator activity of the Sinorhizobium meliloti flagellar motor

The bacterial flagellum is a complex macromolecular machine that drives bacteria through diverse fluid environments. Although many components of the flagellar motor are conserved across species, the roles of FliL are numerous and species‐specific. Here, we have characterized an additional player required for flagellar motor function in Sinorhizobium meliloti, MotF, which we have identified as a FliL paralog. We performed a comparative analysis of MotF and FliL, identified interaction partners through bacterial two‐hybrid and pull‐down assays, and investigated their roles in motility and motor rotation. Both proteins form homooligomers, and interact with each other, and with the stator proteins MotA and MotB. The ∆motF mutant exhibits normal flagellation but its swimming behavior and flagellar motor activity are severely impaired and erratic. In contrast, the ∆fliL mutant is mostly aflagellate and nonmotile. Amino acid substitutions in cytoplasmic regions of MotA or disruption of the proton channel plug of MotB partially restored motor activity to the ∆motF but not the ∆fliL mutant. Altogether, our findings indicate that both, MotF and FliL, are essential for flagellar motor torque generation in S. meliloti. FliL may serve as a scaffold for stator integration into the motor, and MotF is required for proton channel modulation.