Supplementary MaterialsS1 Fig: The reduces the amount of flagella per cell, but does not impact flagellar length. measuring by hand in ImageJ (FIJI) and plotted in Graphpad Prism, with error bars representing the SEM. For B, 45 WT filaments and 30 filaments were measured. For D, 31 WT filaments and 30 filaments were measured.(TIFF) ppat.1008620.s002.tiff (12M) GUID:?2CEE5F33-245E-4E37-915A-75627449A9F9 S3 Fig: Singly-flagellated cells are slower than doubly-flagellated cells. was deleted in the straight-cell background in order to determine how much a helical cell body shape contributes to propulsion in high viscosity media. Similar to the alleles generated for this study. With the exception of strain WPK440 (S3 Movie), all cysteine alleles generated for this study were chromosomally encoded at the native locus. The WT strain for this study, EJC28 (expressed from its native 54 promoter (A). Our initial cysteine allele, locus. In each case, the flagellin is usually expressed from the 28 promoter (D and E).(TIFF) ppat.1008620.s005.tiff (1.3M) GUID:?E5E4F516-B8FE-42C0-B312-6BA4A84E3A8D S6 Fig: Cells in the middle of the sample chamber swim slower than those at the edges. When cells were tracked using 20x magnification phase-contrast microscopy (no fluorescent labeling), cells that were in the middle of the sample chamber swam at approximately half the velocity of cells near the taped edges of the sample chamber. This is presumed to be due to lower oxygen concentration in the middle of the FLT3-IN-2 sample chamber compared to near the porous, double-sided tape used to construct sample CD22 chambers, leading to a reduced proton motive pressure (PMF) to drive flagellar motor rotation.(TIFF) ppat.1008620.s006.tiff (398K) GUID:?63012120-4968-4C70-926B-CC557FCDC3EA S7 Fig: Deletion of impacts swimming velocity and penetrance of high viscosity motility agar. In regular motility agar (MH + 0.4% agar) the mutant was found to swim nearly as well as WT, as judged by the diameter of the swim halo (2.88 cm vs. 3.70 cm, respectively. Values are mean of 5 replicates for each with error bars representing the SEM). In high-viscosity motility agar (MH + 0.4% agar + 0.3% methylcellulose (MC)), however, the mutant was found to be incapable of penetrating and swimming through the agar. Rather, the straight cell mutant spread across the surface of the media (A and B). Using low magnification (20x) phase contrast microscopy, cells in MH + 0.5% MC were found to swim at ~50% the velocity of WT cells, as has been previously reported.(TIFF) ppat.1008620.s007.tiff (5.9M) GUID:?5AAF7825-FC41-4587-9933-69C0D5B46EDF S8 Fig: All-FlaA and all-FlaB are impaired for swimming through complex environments relative to WT. In both regular and high-viscosity motility agar, the all-FlaA and all-FlaB mutants were found to swim with comparable efficiency, but both are inferior to WT with its composite filament assembled from both flagellin types (A FLT3-IN-2 and B). Values in B are the average of 5 replicates for each strain and condition, with error bars representing the SEM.(TIFF) ppat.1008620.s008.tiff (5.8M) GUID:?C6C52182-6AAD-4DCC-9918-FC77223E14DF S1 Movie: The motor rotates at ~100 Hz. Video captured at 1600 frames/second revealed that wraps its leading flagellar filament around the cell body. When fluorescently-labeled cells of EJC28 were observed swimming in MH broth, approximately 50% were found to wrap their leading filament around the cell body during swimming. When the swimming medium was changed to MH + 0.3% MC, almost all cells were wrapped. Area, 31.2 m 26.0 m for 2.75 s.(AVI) ppat.1008620.s010.avi (15M) GUID:?CE915161-8087-4547-A2DE-1E53AE8FE2AD S3 Movie: The leading, wrapped flagellum is actively rotating. Labeled WPK440 (pRY108::cells with wrapped filaments are capable of swimming, albeit more slowly than either singly-flagellated unwrapped cells and doubly-flagellated WT cells. Area, 23.4 m 19.6 m for 0.55 s.(AVI) ppat.1008620.s012.avi (4.6M) GUID:?98754584-FFDB-4A5E-A9FC-7145AA404249 S5 FLT3-IN-2 Movie: Changing swimming direction involves a change in wrapped-filament polarity. By fluorescently labeling EJC28, we were able to observe filament behavior during directional switching events. During a switch in swimming direction, the.