Figure 6. Effect of CsgE on CsgA Polymerization and Pellicle Formation.

(A) Immuno-gold labeling of overexpressed CsgE. MC4100 csgE mutant cells with empty vector (left) or overexpressing CsgE (middle and right) were incubated with CsgE antibody (left and middle) or buffer (right) followed by incubation with gold particle conjugated secondary antibody prior to uranyl acetate staining and TEM. Scale bar represents 200 nm.

(B) CsgE interacts with CsgA fibers. Surface plasmon resonance sensograms of 1 μM purified CsgE (solid line) or a mock purification (dotted line) were injected over immobilized sonicated CsgA fibers on a CM5 sensor chip. Flow of phosphate buffer was resumed after 120 s.

(C) CsgE added to freshly purified CsgA or CsgA incubated for 1 hr inhibits CsgA polymerization. CsgE added to CsgA incubated for 7 hr or 8 hr efficiently inhibits further polymerization.

(D) SDS-PAGE after CsgA overnight incubation with or without equimolar concentration of the chaperone CsgE. No CsgA is left in solution when incubated alone (lane 2) whereas CsgA incubated with CsgE (lane 3) was still in a soluble form.

(E) Extracellular addition of CsgE prevents formation of curli-dependent pellicle biofilm in a concentration-dependent manner.