Subcellular localization of FimX, PilZ, PilB and PilQ.

A) Representative images of fluorescent light microscopy of X. citri cells expressing chromosomal FimX, PilZ, PilB and PilQ fused with msfGFP (N-terminal fusion in FimX, PilZ and PilB and C-terminal fusion in PilQ) in wild type and mutants strains with deletion in indicated T4P regulatory components. The X. citri strains expressing the fluorescent fusion proteins were imaged by phase contrast and epifluorescence microscopy and analyzed at least five times independently with similar results. In these experiments, cells were grown on KB-agarose pads (1.5% w, using 0.2% casamino acids as nitrogen source) supplemented with 2 mM CaCl2. Scale bar, 3 μm. B) Representative time lapse images showing that msfGFP-FimX, msfGFP-PilZ, msfGFP-PilB and PilQ-msfGFP localize to the leading pole in wild type X. citri cells undergoing twitching motility. The time lapse interval (h) is indicated for each frame. Images were taken by using an epi-fluorescence light microscope. Scale bar, 5 μm. See also S5–S8 Movies for time lapse images at shorter intervals. C) Graphical representation of the fluorescence intensity profile over the length of X. citri cells expressing msfGFP-FimX, msfGFP-PilZ, msfGFP-PilB and PilQ-msfGFP. The fluorescence intensity profile was obtained from 100 individual X. citri cells for each case (cell length was normalized). The profiles of all individual cells are shown in S10A Fig. Insets show the foci localizations. A and B show that msfGFP-FimX, msfGFP-PilZ and msfGFP-PilB foci exhibited unipolar localization. PilQ-msfGFP exhibits a mixture of both unipolar and bipolar localization; in the latter case one of the foci was almost always much more intense than the other (see S10A Fig). D) Comparison of the frequency of polar localization of msfGFP-FimX, msfGFP-PilZ, msfGFP-PilB and PilQ-msfGFP foci in wild-type, ΔfimX, ΔpilZ and ΔpilB backgrounds.