Fig. 3: Cluster_3 minor pilins and PilY1.3 interact.

a T4aP-dependent motility assays for strains with tagged PilY1.3 or PilW3. Strains of the indicated genotypes were incubated 24 h. Scale bar, 1 mm. b Accumulation levels of PilY1.3-FLAG and GFP-FLAG. Protein from 3 × 10⁸ cells were loaded per lane and blots probed with α-FLAG antibodies and α-LonD antibodies (loading control). c PilY1.3 and cluster_3 minor pilins interact. Pull-down experiments with α-FLAG matrix on cell extracts from strain of the indicated genotype expressing PilY1.3-FLAG or GFP-FLAG (negative control). Samples from two biological replicates and two negative controls were analyzed by LFQ-MS; mean iBAQ values and log2-fold enrichment in PilY1.3-FLAG samples compared to GFP-FLAG samples were calculated; enrichment for PilY1.3-FLAG was imputed. Columns represent mean of log2-fold enrichment (n = 2); dots represent the corresponding data points; n.d., not detected in PilY1.3-FLAG samples. d, e Accumulation levels of PilY1.3-sfGFP and PilW3-sfGFP. Protein from 3 × 10⁸ cells was loaded per lane and blots were probed with α-GFP antibodies and α-LonD antibodies (loading control). Gap between lanes indicate lanes that were deleted for presentation purposes. f PilY1.3-sfGFP and PilW3-sfGFP localize bipolarly in the presence of assembled T4aPM. Insets are enlargements of the boxed areas. Note that the PilW3-sfGFP clusters formed in the ∆pilQ strain were smaller and of lower intensity than in WT. N > 100 cells per strain; localization patterns in percentage indicated in schematics (bipolar, unipolar, diffuse (top to bottom)). Scale bars, 5 µm. g PilY1.3-sfGFP and cluster_3 minor pilins interact. Pull-down experiments with α-GFP matrix and cell extracts of cells of the indicated genotype containing PilY1.3-sfGFP or GFP-FLAG (negative control). Samples were analyzed as in c; enrichment was imputed for PilY1.3-sfGFP. h PilW3-sfGFP and cluster_3 proteins interact. Pull-down experiments with α-GFP matrix on cell extracts of the indicated genotype containing PilW3-sfGFP or GFP-FLAG (negative control). Samples were analyzed as in c; enrichment was imputed for PilW3-sfGFP.