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Insights on Bacterial Filament Regrowth: Findings from Osaka University Research

This study investigates the regrowth potential of bacterial filaments through cutting experiments conducted with advanced laser technology. Despite the presence of overexpressed cap proteins like FliD, no regrowth of cut filaments was observed after two hours under controlled conditions. The research sheds light on the complex assembly and functionality of bacterial motility, emphasizing the crucial role of the filament cap for growth and the implications for understanding bacterial virulence and antibiotic resistance.

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Insights on Bacterial Filament Regrowth: Findings from Osaka University Research

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  1. Can bacterial filaments regrow? Guillaume Paradis Ismaël Duchesne Simon Rainville Namba Protonic NanoMachine Project Osaka Univsersity, Japan.

  2. Whystudybacterialmotility? • Toxicity and virulence increaseswithmotility • Antibioticscrisis • Nanotechnology

  3. E.colinear a surface

  4. 3D biasedrandomwalk JM Berg, JL Tymoczko, and L Stryer. Biochemistry

  5. How do bacteria move? • Complexassemby of over 20 types of proteins • 45nm-diameter rotative motor • Rotation in both direction up 18000 rpm • Proton powered

  6. Filament structure David E. Tanner and al. Theoretical and computational investigation of flagellin translocation and bacterial flagellum growth. Biophysical Journal

  7. Methodology • Cells are grown to the exponential phase of growth • A first Fluorescent labelling isdone • Filaments are cutusing a femtosecond laser • Coordinates of the bacteria are noted • A second labelling isdoneduring a second growthperiod of 2h (37°C) • Cells are revisited

  8. Experimental setup • Pulsed laser (1 khz)80 fs, Near infrared • Fluorescent microscopy at 546nm and 488nm • Custom-made flow-cell • Bacteriawith 1 or 2 filaments

  9. Cutting the filament • Laser isopened • Filament cutitself on the laser beam • Acceleration of the rotation shows cutting

  10. Resultsafter 2h growthperiod • 44 filaments werecut and revisited • No regrowthwasobserved

  11. Whatwe know • When the filament iscut, the cap islost • The cap isneeded for filament growth • FliDproteinisconstantlysecreted • HAP3 (FlgL) isneeded for the cap to form • If therewasregrowth, shouldtake minutes to beseen

  12. Overexpression of FliD (HAP2) • A geneticallymodifiedstraincontrolling the expression of the FliDproteinwasused • The proteinwasoverexpressed (doubled) during the 2h growthperiod • 18 filaments werecut and revisited • No regrowthwasobserved

  13. Controls • Bacteria in the same flow-cellleftuntouchedwereused as controls • 90-95% of filaments stillturningwerebicolor

  14. Controls (2) • In one instance, a second filament hadgrown on a cellwith a cut filament

  15. Previousstudy • Turner and Berg (2012) • Cellswereshearedmechanically • Let overnight at 7°C • Tends to show regrowth Turner L et al. J. Bacteriol. 2012;194:2437-2442

  16. Conclusions • No regrowthwasobserved • Bicoloruncut filaments in the sameflowcellswereobserved • Evenwithhigherlevels of cap protein, cut filaments don’tregrow • In these conditions, bacterial filaments don’tregrow

  17. Future and ongoingexperiments • Rate of growth vs length of the filament • Study of a proposedchainmechanism model A chainmechanism for flagellum growth, Nature, 2013

  18. Thankyou • Dr Simon Rainville • phDstudents • Guillaume Paradis • Ismael Duchesne • Dr Kelly HuguesUniversity of Utah • Dr Marc ErhardtHelmholtz Centre for infection research, Germany

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