Evolution and structural diversity of the MotAB stator: insights into the origins of bacterial flagellar motility.

The rotation of the bacterial flagellum is powered by the MotAB stator complex, which converts ion flux into torque. Despite its central role in flagellar function, the evolutionary origin and structural diversity of this system remain poorly understood. Here, we present the first comprehensive phylogenetic and structural characterization of MotAB and its closest non-flagellar homologs. We gathered homologs from 205 genomes across 27 bacterial phyla, estimated phylogenies, inferred ancestral sequences, and predicted structures for both extant and inferred ancestral proteins using AlphaFold. Our analyses characterized two structurally distinct groups: flagellar ion transporters (FIT) and generic ion transporters (GIT). FIT proteins are structurally conserved, including a characteristic square fold domain and a torque-generating interface (TGI). We further delineate FIT proteins into two subgroups, TGI4 and TGI5s, based on the presence of 4 or 5 short helices within the TGI region. TGI5 motors, such as those found in the Escherichia coli K12 system, are primarily restricted to Pseudomonadota, whereas TGI4 motors, such as the Na⁺-powered polar motors of Vibrio (PomAB), are distributed across a broader range of bacterial lineages. In contrast, GIT proteins exhibit substantial structural and functional heterogeneity and lack features associated with flagellar motility. Nevertheless, a conserved interaction between the A and B subunits is retained across FIT and GIT proteins, with their corresponding genes typically adjacent to operons. Functional assays in E. coli show that FIT-specific structural elements are indispensable for flagellar motility. Our results suggest that the flagellar stator motor complex evolved once from a common ancestral ion transporter, acquiring unique structural traits to support motility. This work provides a robust framework for understanding the evolutionary diversification of stator complexes and their mechanistic specialization.IMPORTANCEFlagellar motility allows bacteria to propel themselves and direct movement according to environmental conditions. It plays a key role in bacterial pathogenicity and survival. We investigated the molecular and structural diversity of the stator motor proteins that provide the ion motive force to power flagellar rotation. This study uses a comparative approach that integrates phylogenetics, 3D protein structure, motility assays, and ancestral state reconstruction (ASR) to provide insights into the structural mechanisms that first powered the flagellar motor. We provide the first phylogenetic and structural characterization and classification of MotAB and relatives.