In situ imaging of the bacterial flagellar motor disassembly and assembly processes

Mohammed Kaplan; Poorna Subramanian; Debnath Ghosal; Catherine M. Oikonomou; Sahand Pirbadian; Ruth Starwalt-Lee; Shrawan Kumar Mageswaran; Davi R. Ortega; Jeffrey A. Gralnick; Mohamed Y. El-Naggar; Grant J. Jensen

doi:10.15252/embj.2018100957

In situ imaging of the bacterial flagellar motor disassembly and assembly processes

Mohammed Kaplan, Poorna Subramanian, Debnath Ghosal, Catherine M. Oikonomou, Sahand Pirbadian, Ruth Starwalt-Lee, Shrawan Kumar Mageswaran, Davi R. Ortega, Jeffrey A. Gralnick, Mohamed Y. El-Naggar, Grant J. Jensen

Biotechnology Institute

Research output: Contribution to journal › Article › peer-review

32 Scopus citations

Abstract

The self-assembly of cellular macromolecular machines such as the bacterial flagellar motor requires the spatio-temporal synchronization of gene expression with proper protein localization and association of dozens of protein components. In Salmonella and Escherichia coli, a sequential, outward assembly mechanism has been proposed for the flagellar motor starting from the inner membrane, with the addition of each new component stabilizing the previous one. However, very little is known about flagellar disassembly. Here, using electron cryo-tomography and sub-tomogram averaging of intact Legionella pneumophila, Pseudomonas aeruginosa, and Shewanella oneidensis cells, we study flagellar motor disassembly and assembly in situ. We first show that motor disassembly results in stable outer membrane-embedded sub-complexes. These sub-complexes consist of the periplasmic embellished P- and L-rings, and bend the membrane inward while it remains apparently sealed. Additionally, we also observe various intermediates of the assembly process including an inner-membrane sub-complex consisting of the C-ring, MS-ring, and export apparatus. Finally, we show that the L-ring is responsible for reshaping the outer membrane, a crucial step in the flagellar assembly process.

Original language	English (US)
Article number	e100957
Journal	EMBO Journal
Volume	38
Issue number	14
DOIs	https://doi.org/10.15252/embj.2018100957
State	Published - Jul 15 2019

Bibliographical note

Funding Information:
This work was supported by the National Institutes of Health (NIH, grant R01 AI127401 to G.J.J.). M.K. is supported by a postdoctoral Rubicon fellowship from De Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO). S.P. and M.Y.E.-N. are supported by the Air Force Office of Scientific Research Presidential Early Career Award for Scientists and Engineers (FA955014-1-0294, to M.Y.E.-N.). R.S.L. and J.A.G acknowledge support from the Office of Naval Research (N00014-18-1-2632 to J.A.G.). We would like to thank Dr. Spiros Garbis, Dr. Annie Moradian, Dr. Michael Sweredoski, and Dr. Brett Lomenick from the Caltech Proteome Exploration Laboratory for their help in mass spectrometry experiments and data analysis. All P. aeruginosa mutants were kindly supplied by Dianne Newman’s laboratory at Caltech. ECT was performed in the Beckman Institute Resource Center for Cryo-EM.

Funding Information:
This work was supported by the National Institutes of Health (NIH, grant R01 AI127401 to G.J.J.). M.K. is supported by a postdoctoral Rubicon fellowship from De Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO). S.P. and M.Y.E.-N. are supported by the Air Force Office of Scientific Research Presidential Early Career Award for Scientists and Engineers (FA955014-1-0294, to M.Y.E.-N.). R.S.L. and J.A.G acknowledge support from the Office of Naval Research (N00014-18-1-2632 to J.A.G.). We would like to thank Dr. Spiros Garbis, Dr. Annie Moradian, Dr. Michael Sweredoski, and Dr. Brett Lomenick from the Caltech Proteome Exploration Laboratory for their help in mass spectrometry experiments and data analysis. All P. aeruginosa mutants were kindly supplied by Dianne Newman's laboratory at Caltech. ECT was performed in the Beckman Institute Resource Center for Cryo-EM.

Publisher Copyright:
© 2019 The Authors

Keywords

assembly
bacterial flagellar motor
disassembly
electron cryo-tomography
in situ imaging

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10.15252/embj.2018100957

https://authors.library.caltech.edu/95599/10/embj.2018100957.reviewer-comments.pdf

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@article{9430fe8253a04432b61eda0bed227622,

title = "In situ imaging of the bacterial flagellar motor disassembly and assembly processes",

abstract = "The self-assembly of cellular macromolecular machines such as the bacterial flagellar motor requires the spatio-temporal synchronization of gene expression with proper protein localization and association of dozens of protein components. In Salmonella and Escherichia coli, a sequential, outward assembly mechanism has been proposed for the flagellar motor starting from the inner membrane, with the addition of each new component stabilizing the previous one. However, very little is known about flagellar disassembly. Here, using electron cryo-tomography and sub-tomogram averaging of intact Legionella pneumophila, Pseudomonas aeruginosa, and Shewanella oneidensis cells, we study flagellar motor disassembly and assembly in situ. We first show that motor disassembly results in stable outer membrane-embedded sub-complexes. These sub-complexes consist of the periplasmic embellished P- and L-rings, and bend the membrane inward while it remains apparently sealed. Additionally, we also observe various intermediates of the assembly process including an inner-membrane sub-complex consisting of the C-ring, MS-ring, and export apparatus. Finally, we show that the L-ring is responsible for reshaping the outer membrane, a crucial step in the flagellar assembly process.",

keywords = "assembly, bacterial flagellar motor, disassembly, electron cryo-tomography, in situ imaging",

author = "Mohammed Kaplan and Poorna Subramanian and Debnath Ghosal and Oikonomou, {Catherine M.} and Sahand Pirbadian and Ruth Starwalt-Lee and Mageswaran, {Shrawan Kumar} and Ortega, {Davi R.} and Gralnick, {Jeffrey A.} and El-Naggar, {Mohamed Y.} and Jensen, {Grant J.}",

note = "Funding Information: This work was supported by the National Institutes of Health (NIH, grant R01 AI127401 to G.J.J.). M.K. is supported by a postdoctoral Rubicon fellowship from De Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO). S.P. and M.Y.E.-N. are supported by the Air Force Office of Scientific Research Presidential Early Career Award for Scientists and Engineers (FA955014-1-0294, to M.Y.E.-N.). R.S.L. and J.A.G acknowledge support from the Office of Naval Research (N00014-18-1-2632 to J.A.G.). We would like to thank Dr. Spiros Garbis, Dr. Annie Moradian, Dr. Michael Sweredoski, and Dr. Brett Lomenick from the Caltech Proteome Exploration Laboratory for their help in mass spectrometry experiments and data analysis. All P. aeruginosa mutants were kindly supplied by Dianne Newman{\textquoteright}s laboratory at Caltech. ECT was performed in the Beckman Institute Resource Center for Cryo-EM. Funding Information: This work was supported by the National Institutes of Health (NIH, grant R01 AI127401 to G.J.J.). M.K. is supported by a postdoctoral Rubicon fellowship from De Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO). S.P. and M.Y.E.-N. are supported by the Air Force Office of Scientific Research Presidential Early Career Award for Scientists and Engineers (FA955014-1-0294, to M.Y.E.-N.). R.S.L. and J.A.G acknowledge support from the Office of Naval Research (N00014-18-1-2632 to J.A.G.). We would like to thank Dr. Spiros Garbis, Dr. Annie Moradian, Dr. Michael Sweredoski, and Dr. Brett Lomenick from the Caltech Proteome Exploration Laboratory for their help in mass spectrometry experiments and data analysis. All P. aeruginosa mutants were kindly supplied by Dianne Newman's laboratory at Caltech. ECT was performed in the Beckman Institute Resource Center for Cryo-EM. Publisher Copyright: {\textcopyright} 2019 The Authors",

year = "2019",

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doi = "10.15252/embj.2018100957",

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AU - Kaplan, Mohammed

AU - Subramanian, Poorna

AU - Ghosal, Debnath

AU - Oikonomou, Catherine M.

AU - Pirbadian, Sahand

AU - Starwalt-Lee, Ruth

AU - Mageswaran, Shrawan Kumar

AU - Ortega, Davi R.

AU - Gralnick, Jeffrey A.

AU - El-Naggar, Mohamed Y.

AU - Jensen, Grant J.

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KW - in situ imaging

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In situ imaging of the bacterial flagellar motor disassembly and assembly processes

Abstract

Bibliographical note

Keywords

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