Dataset Information

Intragenic suppressor of a plug deletion nonmotility mutation in PotB, a chimeric stator protein of sodium-driven flagella.

ABSTRACT: The torque of bacterial flagellar motors is generated by interactions between the rotor and the stator and is coupled to the influx of H(+) or Na(+) through the stator. A chimeric protein, PotB, in which the N-terminal region of Vibrio alginolyticus PomB was fused to the C-terminal region of Escherichia coli MotB, can function with PomA as a Na(+)-driven stator in E. coli. Here, we constructed a deletion variant of PotB (with a deletion of residues 41 to 91 [?41-91], called PotB?L), which lacks the periplasmic linker region including the segment that works as a "plug" to inhibit premature ion influx. This variant did not confer motile ability, but we isolated a Na(+)-driven, spontaneous suppressor mutant, which has a point mutation (R109P) in the MotB/PomB-specific ?-helix that connects the transmembrane and peptidoglycan binding domains of PotB?L in the region of MotB. Overproduction of the PomA/PotB?L(R109P) stator inhibited the growth of E. coli cells, suggesting that this stator has high Na(+)-conducting activity. Mutational analyses of Arg109 and nearby residues suggest that the structural alteration in this ?-helix optimizes PotB?L conformation and restores the proper arrangement of transmembrane helices to form a functional channel pore. We speculate that this ?-helix plays a key role in assembly-coupled stator activation.

SUBMITTER: Zhu S

PROVIDER: S-EPMC3510634 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Intragenic suppressor of a plug deletion nonmotility mutation in PotB, a chimeric stator protein of sodium-driven flagella.

Zhu Shiwei S Homma Michio M Kojima Seiji S

Journal of bacteriology 20120928 24

The torque of bacterial flagellar motors is generated by interactions between the rotor and the stator and is coupled to the influx of H(+) or Na(+) through the stator. A chimeric protein, PotB, in which the N-terminal region of Vibrio alginolyticus PomB was fused to the C-terminal region of Escherichia coli MotB, can function with PomA as a Na(+)-driven stator in E. coli. Here, we constructed a deletion variant of PotB (with a deletion of residues 41 to 91 [Δ41-91], called PotBΔL), which lacks ...[more]

PMID: 23024347

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Project description:The bacterial flagellar motor is composed of a rotor and a dozen stators and converts the ion flux through the stator into torque. Each stator unit alternates in its attachment to and detachment from the rotor even during rotation. In some species, stator assembly depends on the input energy, but it remains unclear how an electrochemical potential across the membrane (e.g., proton motive force [PMF]) or ion flux is involved in stator assembly dynamics. Here, we focused on pH dependence of a slow motile MotA(M206I) mutant of Salmonella The MotA(M206I) motor produces torque comparable to that of the wild-type motor near stall, but its rotation rate is considerably decreased as the external load is reduced. Rotation assays of flagella labeled with 1-μm beads showed that the rotation rate of the MotA(M206I) motor is increased by lowering the external pH whereas that of the wild-type motor is not. Measurements of the speed produced by a single stator unit using 1-μm beads showed that the unit speed of the MotA(M206I) is about 60% of that of the wild-type and that a decrease in external pH did not affect the MotA(M206I) unit speed. Analysis of the subcellular stator localization revealed that the number of functional stators is restored by lowering the external pH. The pH-dependent improvement of stator assembly was observed even when the PMF was collapsed and proton transfer was inhibited. These results suggest that MotA-Met206 is responsible for not only load-dependent energy coupling between the proton influx and rotation but also pH-dependent stator assembly.IMPORTANCE The bacterial flagellar motor is a rotary nanomachine driven by the electrochemical transmembrane potential (ion motive force). About 10 stators (MotA/MotB complexes) are docked around a rotor, and the stator recruitment depends on the load, ion motive force, and coupling ion flux. The MotA(M206I) mutation slows motor rotation and decreases the number of docked stators in Salmonella We show that lowering the external pH improves the assembly of the mutant stators. Neither the collapse of the ion motive force nor a mutation mimicking the proton-binding state inhibited stator localization to the motor. These results suggest that MotA-Met206 is involved in torque generation and proton translocation and that stator assembly is stabilized by protonation of the stator.

Dataset Information

Intragenic suppressor of a plug deletion nonmotility mutation in PotB, a chimeric stator protein of sodium-driven flagella.

Publications

Intragenic suppressor of a plug deletion nonmotility mutation in PotB, a chimeric stator protein of sodium-driven flagella.

Similar Datasets

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