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Birgit E. Scharf, Email: bscharf@vt.edu
E.H.E., B.E.S., M.M.K., M.A.B.K., and R.C.S. designed the study and experiments. Achromobacter sp. MFA1 R4 flagellar filaments were provided by V.P.C. EHEC O157:H7 flagellar filaments were prepared by J.A.G. Initial EPEC O127:H6 flagellar filaments were prepared by A.P., T.R.D.C., and G.F. Additional EPEC O127:H6 flagellar filaments were prepared by S.C. and G.F. S. meliloti flagellar filaments were prepared by B.E.S. M.A.B.K. screened and prepared samples for cryoEM as well as performed the image processing and helical and asymmetric reconstructions. Atomic models were generated by M.A.B.K. F.W. assisted with cryoEM image processing and model building. A.B.S. and M.M.K. constructed the FF and RR mutants, performed soft agar motility assays, and prepared EHEC samples for analysis by negative stain TEM and cryoEM. R.C.S. performed the western blot analysis, soft agar motility assays, and the phase-contrast microscopy experiments to measure the velocity and tumbling of the various E. coli strains. M.A.B.K. and R.C.S. analyzed EHEC and E. coli K-12 cells tumbling. Flagellar filaments were labeled by M.A.B.K., and fluorescence microscopy experiments were done by M.A.B.K. and V.K. Figures were prepared by M.A.B.K. M.A.B.K., R.C.S., B.E.S., and E.H.E. wrote the manuscript.
CryoEM images were collected at the UVA Molecular Electron Microscopy Core (MEMC). Mass spectrometry was done at the UVA Biomolecular analysis facility.We thank Howard C. Berg and Karen Fahrner for the anti-flagellin antiserum. We thank Chris Calladine and Ben Luisi at Cambridge University for helpful discussions. We also thank Clay Fuqua and Melene Thompson at the University of Indiana for help in the identification and analysis of the Achromobacter flagellar filament. We thank Prof. Ilan Rosenshine of the Hebrew University for the flhDC plasmid. We thank Weronika Tomaka for assistance with fluorescence microscopy. We thank Dr. Joyce Loeper of Oregon State University and Dr. Virginia Stockwell at the USDA-ARS in Corvalis Oregon for providing us with pure Achromobacter sp MFA1 R4 cell stocks.
The authors declare no competing interests.
This work was supported by NIH GM122510 (to E.H.E.), NIH grants NIH/ NIAID grants: AI154355 and AI163565 (to M.M.K.), by the Welcome Trust Award 215164/Z/18/Z (to T.R.D.C.), the Robert R. Wagner Fellowship (to M.A.B.K.), NIH Grant T32 GM080186, and NIH P01 GM072694. Work done in the GF lab was supported by a grant from the Wellcome Trust. We thank Howard C. Berg and Karen Fahrner for the anti-flagellin antiserum.
© The Author(s) 2022