(A) Functionality of the MshA[T70C] cysteine variant and its derivatives was assessed by a motility assay alongside the relevant parental controls, as indicated. Surface motility was determined on soft LB agar plates. The swarming diameter (cm) is the mean of three repeats (±S.D.). A flagellin-deficient (ΔflaA) non-motile strain was used as a negative control. (B) Snapshot imaging of MSHA pili in WT parent (A1552-MshA[T70C]), ΔpilT (A1552-MshA[T70C], ΔpilT) and ΔpilU (A1552-MshA[T70C], ΔpilU) backgrounds, as indicated. Cells were stained with AF-488-Mal (Dye). Scale bar = 5 μm. (C-D) Quantification of MSHA piliation in snapshot imaging of WT parent, ΔpilT and ΔpilU backgrounds, as indicated. Bars represent the mean of three repeats (±S.D.). (C) Percentage of piliated cells. (D) Histogram of the number of pili per cell in piliated cells. n = c.a. 200–600 cells per strain per repeat.