Pilus structure (PDB: 5KUA) showing a cluster of charged amino acids surrounding the protruding K140 involved in aggregation. N138 is in light blue; Q122 and K140 in orange; and E99, K103, K144, and H149 in dark blue.
Piliation per bacterium as measured by flow cytometry using the 20D9 monoclonal antibody and the F10 nanobody.
Visualization of type IV pili by immunofluorescence either using the 20D9 antibody or the F10 nanobody as indicated (magenta). In the case of the K140Q mutant labeled with the 20D9 antibody, the background was made visible to convincingly show the absence of pilus labeling. Scale bar: 2 μm.
Aggregation expressed as a function of piliation per bacterium measured with 20D9 antibody except for pilE
Q122E and pilE
K140Q for which the F10 nanobody value is reported. Each dot represents values for a PilE point mutant relative to the SB strain. Hypo‐aggregative mutants are highlighted in orange, while hyper‐aggregative mutants are in light blue. Solid lines indicate the 95% confidence interval.
Quantification of PilX present in purified pili preparations sheared from the bacterial surface using Western blot. PilX levels were normalized using PilE as a reference and normalized to pilE
SB values. Mean ratio ± SEM is indicated. N = 3 independent experiments. Paired t‐test.
