Fig. 3.

TfcP is important for stability of PilY1.1 and minor pilins of cluster_1. (A) Accumulation of proteins of the T4aPM and cluster_1. Cells were grown in 1.0% CTT suspension culture. Relative protein amounts were determined using targeted proteomics with one to five heavy-labeled reference peptides for each protein spiked into the trypsin-digested cell lysates (SI Appendix, SI Materials and Methods). To calculate relative protein amounts, each light-to-heavy intensity ratio of the endogenous (light) and reference (heavy) peptide was calculated. Individual data points represent the mean of the log₂ ratios of the relative amount of all peptides of one protein in one biological replicate to the mean relative amount of the same peptide in the WT_Δ2Δ3 strain. Center marker and error bars in black: Mean and SD from four biological replicates. Statistical analyses were performed by comparing WT_Δ2Δ3 to the mutants using Welch’s test, *P < 0.01. (B) Immunoblot analysis of TfcP and PilY1.1 accumulation. Cells were grown in 1.0% CTT suspension culture. Total cell extracts from the same number of cells were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. PilC was used as loading control. (C) Localization of mCherry-PilM. Strains were grown in 1.0% CTT suspension culture, placed on 1.0% agarose supplemented with Tris/phosphate/magnesium (TPM) buffer, and immediately imaged by fluorescence microscopy. (Scale bar, 5 µm.)