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Can bacterial filaments regrow ?

Can bacterial filaments regrow ?. Guillaume Paradis Ismaël Duchesne Simon Rainville. Namba Protonic NanoMachine Project Osaka Univsersity , Japan . Why study bacterial motility ? . Toxicity and virulence increases with motility Antibiotics crisis Nanotechnology.

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Can bacterial filaments regrow ?

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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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