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bioRxiv · 10.1101/2025.11.06.686970

Effects of ΔmotY mutations on motility behavior of Pseudomonas aeruginosa chimeric periplasmic stator variants

Ferguson, N.·Desai, M.·Silva, J. F.·Nguyen, H.·Rodenborn, B.·Shindell, O.·Healy, F.
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Abstract

Pseudomonas aeruginosa utilizes dual flagellar stator systems for motility; and dual stator bacteria possess auxiliary flagellar rotor ring components in their periplasm. In P. aeruginosa MotAB and MotCD comprise the dual stator system and MotY is the auxiliary periplasmic ring component. We investigated motility of strains and their isogenic {Delta}motY derivatives which expressed chimeric MotB/MotD periplasmic domains and characterized differences in motility behaviors. We found in general motility is severely impaired in strains carrying motY deletions and which express C-terminal periplasmic regions of MotD, compared with strains expressing MotB C-terminal counterparts. Motility in soft agar is slightly increased in strains expressing N-terminal MotB transmembrane domains and MotD C-terminal periplasmic plug, PGB, and extensions, but motility is severely impaired in {Delta}motY strains. Addition of the extended 24-residue C-terminus of MotB to the C-terminus of MotD does not significantly affect either motility or compensate for the deleterious effect of {Delta}motY mutation, but does significantly increase motility in motY+ backgrounds. The soft agar motility results for organisms with wild type MotAB stators was not different from those with MotAB stators carrying 24 residue MotB C-terminal deletions; however, motility of these mutants was significantly lower in {Delta}motY mutants compared to {Delta}motY mutants expressing wild type MotAB stators. We discuss contributions of stator functional domains to motility and the effects of {Delta}motY deletion. Lastly, we speculate on possible mechanistic roles for the two stator plug types based on thermodynamic considerations related to differences in composition of the hydrophobic surfaces of the two amphipathic helices. IMPORTANCEPseudomonas aeruginosa uses two torque-generating stators, MotAB and MotCD, to drive flagellar rotation. Dual stator bacteria have additional rotor components; in P. aeruginosa, this component has been identified as MotY. This study investigates the interaction between the C-terminal plug and periplasmic regions of the MotB and MotD components of the MotAB and MotCD stator complexes with MotY. Motility assays of periplasmic chimeric strains expressing variants with MotB and MotD C-terminal plug and peptidoglycan binding domains reveal an enhanced sensitivity of MotD C-terminus to MotY deletions. These data suggest that critical interactions, either direct or indirect, must take place between the basal body MotY ring complex and MotD periplasmic regions for proper function of the MotCD stator in flagellar motility.

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Ferguson, N., Desai, M., Silva, J. F., Nguyen, H., Rodenborn, B., Shindell, O., Healy, F.. 2025-11-07. Effects of ΔmotY mutations on motility behavior of Pseudomonas aeruginosa chimeric periplasmic stator variants. https://doi.org/10.1101/2025.11.06.686970

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