Dataset Information

MotI (DgrA) acts as a molecular clutch on the flagellar stator protein MotA in Bacillus subtilis.

ABSTRACT: Stator elements consisting of MotA4MotB2 complexes are anchored to the cell wall, extend through the cell membrane, and interact with FliG in the cytoplasmic C ring rotor of the flagellum. The cytoplasmic loop of MotA undergoes proton-driven conformational changes that drive flagellar rotation. Functional regulators inhibit motility by either disengaging or jamming the stator-rotor interaction. Here we show that the YcgR homolog MotI (formerly DgrA) of Bacillus subtilis inhibits motility like a molecular clutch that disengages MotA. MotI-inhibited flagella rotated freely by Brownian motion, and suppressor mutations in MotA that were immune to MotI inhibition were located two residues downstream of the critical force generation site. The 3D structure of MotI bound to c-di-GMP was solved, and MotI-fluorescent fusions localized as transient MotA-dependent puncta at the membrane when induced at subinhibitory levels. Finally, subinhibitory levels of MotI expression resulted in incomplete inhibition and proportional decreases in swimming speed. We propose a model in which flagellar stators are disengaged and sequestered from the flagellar rotor when bound by MotI.

SUBMITTER: Subramanian S

PROVIDER: S-EPMC5754805 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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MotI (DgrA) acts as a molecular clutch on the flagellar stator protein MotA in Bacillus subtilis.

Subramanian Sundharraman S Gao Xiaohui X Dann Charles E CE Kearns Daniel B DB

Proceedings of the National Academy of Sciences of the United States of America 20171201 51

Stator elements consisting of MotA4MotB2 complexes are anchored to the cell wall, extend through the cell membrane, and interact with FliG in the cytoplasmic C ring rotor of the flagellum. The cytoplasmic loop of MotA undergoes proton-driven conformational changes that drive flagellar rotation. Functional regulators inhibit motility by either disengaging or jamming the stator-rotor interaction. Here we show that the YcgR homolog MotI (formerly DgrA) of Bacillus subtilis< ...[more]

PMID: 29196522

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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.

Project description:While the protein complex responsible for controlling the direction (clockwise [CW] or counterclockwise [CCW]) of flagellar rotation has been fairly well studied in Escherichia coli and Salmonella, less is known about the switch complex in Bacillus subtilis or other Gram-positive species. Two component proteins (FliG and FliM) are shared between E. coli and B. subtilis, but in place of the protein FliN found in E. coli, the B. subtilis complex contains the larger protein FliY. Notably, in B. subtilis the signaling protein CheY-phosphate induces a switch from CW to CCW rotation, opposite to its action in E. coli Here, we have examined the architecture and function of the switch complex in B. subtilis using targeted cross-linking, bacterial two-hybrid protein interaction experiments, and characterization of mutant phenotypes. In major respects, the B. subtilis switch complex appears to be organized similarly to that in E. coli The complex is organized around a ring built from the large middle domain of FliM; this ring supports an array of FliG subunits organized in a similar way to that of E. coli, with the FliG C-terminal domain functioning in the generation of torque via conserved charged residues. Key differences from E. coli involve the middle domain of FliY, which forms an additional, more outboard array, and the C-terminal domains of FliM and FliY, which are organized into both FliY homodimers and FliM heterodimers. Together, the results suggest that the CW and CCW conformational states are similar in the Gram-negative and Gram-positive switches but that CheY-phosphate drives oppositely directed movements in the two cases.IMPORTANCE Flagellar motility plays key roles in the survival of many bacteria and in the harmful action of many pathogens. Bacterial flagella rotate; the direction of flagellar rotation is controlled by a multisubunit protein complex termed the switch complex. This complex has been extensively studied in Gram-negative model species, but little is known about the complex in Bacillus subtilis or other Gram-positive species. Notably, the switch complex in Gram-positive species responds to its effector CheY-phosphate (CheY-P) by switching to CCW rotation, whereas in E. coli or Salmonella CheY-P acts in the opposite way, promoting CW rotation. In the work here, the architecture of the B. subtilis switch complex has been probed using cross-linking, protein interaction measurements, and mutational approaches. The results cast light on the organization of the complex and provide a framework for understanding the mechanism of flagellar direction control in B. subtilis and other Gram-positive species.

Dataset Information

MotI (DgrA) acts as a molecular clutch on the flagellar stator protein MotA in Bacillus subtilis.

Publications

MotI (DgrA) acts as a molecular clutch on the flagellar stator protein MotA in <i>Bacillus subtilis</i>.

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