Dataset Information

Ancestral reconstruction of the MotA stator subunit reveals that conserved residues far from the pore are required to drive flagellar motility.

ABSTRACT: The bacterial flagellar motor (BFM) is a rotary nanomachine powered by the translocation of ions across the inner membrane through the stator complex. The stator complex consists of two membrane proteins: MotA and MotB (in H⁺-powered motors), or PomA and PomB (in Na⁺-powered motors). In this study, we used ancestral sequence reconstruction (ASR) to probe which residues of MotA correlate with function and may have been conserved to preserve motor function. We reconstructed 10 ancestral sequences of MotA and found four of them were motile in combination with contemporary Escherichia coli MotB and in combination with our previously published functional ancestral MotBs. Sequence comparison between wild-type (WT) E. coli MotA and MotA-ASRs revealed 30 critical residues across multiple domains of MotA that were conserved among all motile stator units. These conserved residues included pore-facing, cytoplasm-facing, and MotA-MotA intermolecular facing sites. Overall, this work demonstrates the role of ASR in assessing conserved variable residues in a subunit of a molecular complex.

SUBMITTER: Islam MI

PROVIDER: S-EPMC10117855 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Ancestral reconstruction of the MotA stator subunit reveals that conserved residues far from the pore are required to drive flagellar motility.

Islam Md Imtiazul MI Ridone Pietro P Lin Angela A Michie Katharine A KA Matzke Nicholas J NJ Hochberg Georg G Baker Matthew A B MAB

microLife 20230403

The bacterial flagellar motor (BFM) is a rotary nanomachine powered by the translocation of ions across the inner membrane through the stator complex. The stator complex consists of two membrane proteins: MotA and MotB (in H<sup>+</sup>-powered motors), or PomA and PomB (in Na<sup>+</sup>-powered motors). In this study, we used ancestral sequence reconstruction (ASR) to probe which residues of MotA correlate with function and may have been conserved to preserve motor function. We reconstructed 1 ...[more]

PMID: 37223728

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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

Ancestral reconstruction of the MotA stator subunit reveals that conserved residues far from the pore are required to drive flagellar motility.

Publications

Ancestral reconstruction of the MotA stator subunit reveals that conserved residues far from the pore are required to drive flagellar motility.

Similar Datasets

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