Abstract
Four integral membrane proteins, PomA, PomB, MotX, and MotY, are thought to be directly involved in torque generation of the Na(+)-driven polar flagellar motor of Vibrio alginolyticus. Our previous study showed that PomA and PomB form a complex, which catalyzes sodium influx in response to a potassium diffusion potential. PomA forms a stable dimer when expressed in a PomB null mutant. To explore the possible functional dependence of PomA domains in adjacent subunits, we prepared a series of PomA dimer fusions containing different combinations of wild-type or mutant subunits. Introduction of the mutation P199L, which completely inactivates flagellar rotation, into either the first or the second half of the dimer abolished motility. The P199L mutation in monomeric PomA also altered the PomA-PomB interaction. PomA dimer with the P199L mutation even in one subunit also had no ability to interact with PomB, indicating that the both subunits in the dimer are required for the functional interaction between PomA and PomB. Flagellar rotation by wild-type PomA dimer was completely inactivated by phenamil, a sodium channel blocker. However, activity was retained in the presence of phenamil when either half of the dimer was replaced with a phenamil-resistant subunit, indicating that both subunits must bind phenamil for motility to be fully inhibited. These observations demonstrate that both halves of the PomA dimer function together to generate the torque for flagellar rotation.
Highlights
Bacterial flagella are the organelles responsible for motility
We describe a fusion protein that contains different combinations of wild-type PomA or mutant (P199L or D148Y) subunits that we have used to evaluate whether adjacent PomA domains may be required for torque generation
All PomA dimers containing the P199L mutation in either half of the dimer reduced the swarming ability of VIO5 (PomAϩ) cells, whereas PomA homodimer resulted in no significant reduction in swarm size compared with wild-type cells. These results suggest that PomA dimers with the P199L mutation compete with the wild-type PomA for occupying the functional site of the torque-generating unit
Summary
Bacterial flagella are the organelles responsible for motility. Flagellar rotation is driven by a reversible rotary motor embedded in the cytoplasmic membrane at the base of each flagellar filament [1,2,3]. Together with FliM and FliN [19, 20], FliG forms the switch complex, which is essential for torque generation, flagellar assembly, and controlling the direction of motor rotation [21,22,23,24,25] Bacteria such as alkalophilic Bacillus and Vibrio species use the electrochemical gradient of sodium to drive flagellar rotation [4]. PomA and PomB are homologous to MotA and MotB and contain four and one transmembrane segments, respectively Both MotX and MotY, which were first identified in Vibrio parahaemolyticus [30, 31], have a putative single transmembrane segment; they are unique to the sodium-type motor, and their function is unknown so far. Our results demonstrate that PomA functions as an even number of subunits in a single functional complex and that both subunits contribute to PomA-PomB assembly
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