RomX, a novel prokaryotic regulator, links the response receiver domain of RomR with GTP-bound MglA for establishing Myxococcus xanthus polarity

Cell polarity specification and reversals are distinctive features of motility of the soil bacterium Myxococcus xanthus. The bacterial small Ras-like GTPase, MglA, serves as a key player orchestrating these polarity oscillations. RomR, a response regulator, along with its partner RomX, has been identified to function as a guanine nucleotide exchange factor (GEF) for MglA, crucial for its polar recruitment. In this study, we determine the crystal structure of RomX, a protein of a hitherto unknown fold. RomX consists of a three-helix bundle, identified to be the same fold as the stalk domain of atlastin, a member of the dynamin family of GTPases. From our structure-based sequence analysis for proteins of a similar fold, we observe the co-occurrence of the RomX fold with response receiver domains in several bacterial response regulators. Based on mutational analysis and affinity measurements, we conclude that the helix-1 of RomX mediates the interaction with MglA-GTP, while helix-3 of RomX interacts with the RomR N-terminal receiver (REC) domain. We demonstrate that the binding between MglA and RomX is exclusively in the presence of GTP. The absence of additional stimulation of RomX GEF activity in the presence of RomR-REC supports the mutually exclusive interface on RomX for RomR and MglA interaction. Collectively, our findings validate the positioning of RomX between MglA and RomR-REC, providing insights into the concerted action of the RomR/RomX complex at both poles in driving MglA localization within polarized cells.