Fig. 3: Holdfast synthesis timing upon arrest of flagellar rotation.

(A) Construction of FljK-cys mutants. PHYRE predicted structure of the FljK flagellin. The N-terminus of the predicted FljK structure is blue and the C terminus is red. The positions of the mutated amino acids T103 and T176 are depicted as red stars. (B) Motility assay in low percentage PYE (0.3%) agar. (C) Staining of a swarmer cell of NA1000 hfsA+ fljKT103C using AF488-mal under a PYE 0.8% agarose pad. (D-E) Timing of holdfast synthesis by newly divided swarmer cells grown in PYE and constrained by a 0.8% agarose pad containing AF594-WGA. (D) Representative time lapse of swarmer cell with an arrested flagellum and subsequent holdfast synthesis. Cells were imaged using phase contrast microscopy. Flagella and holdfasts were stained using AF488-mal and AF594-WGA respectively. The white arrow indicates the location of expected holdfast synthesis. (E) Timing of holdfast synthesis by newly divided swarmer cells. Cells labeled as “initially rotating” are newly divided cells that exhibited a rotating flagellum that became quickly arrested under the pad; and cells labeled as “never rotating” are newly divided cells in which the flagellum did not rotate at any time. Data are represented by box plots with 5–95% percentile whiskers. n = 72 and 66 cells for rotating and non-rotating flagella respectively. No statistically significant differences between the 2 conditions can be calculated using Mann-Whitney unpaired t-tests (ns).