PilT and MshE promote distinct activities during MSHA extension.

(A) The speed of MSHA T4P extension was determined in strains containing native motor ATPases (parent), or mutants that slow the ATPase activity of PilT (pilT slow) and/or MshE (mshE slow) as indicated. Because PilU is not required for MSHA piliation, all strains contained a ΔpilU mutation to prevent any confounding effects this motor ATPase might have on extension speed. For all strains, n = 75 from three independent experiments. Each data point indicates the extension speed for a dynamic T4P event. Box plots represent the median and the upper and lower quartile, while the whiskers demarcate the range. Statistical comparisons were made by One way ANOVA with Tukey’s post-test. NS = not significant; *** = p B) Schematic of the experimental setup to test whether PilT and MshE activity can be temporally separated in cells. In the absence of arabinose (Ara) induction (top), PilT-pd2 is left intact during mshE induction via anhydrotetracycline (ATc) (“PilT intact”). When cells are incubated with arabinose (bottom), PilT-pdt2 is degraded by Mf-Lon prior to the expression of mshE (“PilT depleted”). (C) Representative montage of cells subjected to the experiment described in B. Cells were imaged at 0, 20, and 40 minutes after induction of mshE expression. Scale bar = 3 μm. (D) Quantification of extension in the samples shown in C. Graph displays the percentage of cells in each replicate that exhibited pilus extension during a 40-minute window. n ≥ 100 cells analyzed for each of the three biological replicates and data are shown as the mean ± SD. (E) Quantification of imaging-induced retraction in samples shown in C. Graph displays the percentage of cells in each replicate that exhibited any pilus retraction during a three-minute timelapse. n ≥ 25 piliated cells analyzed for each of the three biological replicates and data are shown as the mean ± SD.