Fig. 4: Effect of targeted CpaF mutagenesis on TadPS function in vivo.
From: Bidirectional pilus processing in the Tad pilus system motor CpaF
A Position of truncations or highly conserved residues within the CpaF monomer selected for chromosomal mutation. B Phage infection assays using ΦCbK on the mutant strains where TadPS deactivation impairs phage entry, cell lysis and plaque formation in C. crescentus. Images are representative of three biological repeats. C Demonstration of Tad pili labelling using a fluorescent AF488-conjugated thiol-reactive maleimide dye that is unreactive with wildtype PilA (left) but reacts and decorates pili that have a PilA T36C mutation (right). Inset image shows a magnified C. crescentus cell where both a fluorescent pilus (indicating functional extension) and fluorescent cell body (indicating functional retraction) can be observed. Note that only C. crescentus swarmer-type cells are capable of producing Tad pili. Without this capability, other cell types (stalked and predivisional cells) do not fluoresce. Scale bar = 5 μm for all panels. Images are representative of three biological repeats. D Direct visualisation of Tad pili by fluorescence microscopy in different C. crescentus CpaF mutants as described in (C). Scale bars = 2 μm for all panels. Images are representative of three biological repeats. E (Left panel) Quantification of piliated cells observed in the CpaF truncations or mutant strains, relating to (C) and (D). For the proportion of fluorescent cells in the CpaF Δaa1-78 truncation strain P < 0.0001 (right panel). The mean of three biological replicates (N = 500 cells counted per biological repeat) was plotted with standard error of the mean, **** P < 0.0001, ns = not statistically significant. Statistics were determined using Dunnett’s one-way ANOVA test. (Right panel) Quantification of fluorescent cells observed in the CpaF truncations or mutant strains, relating to (C) and (D). The significant reduction in fluorescent cells in the CpaF Δaa1-78 strain (** P = 0.002) indicates a partial retraction mutant. Source data are provided as a Source Data file.
