Fig 1.
CupE1 subunits within the CupE pilus are arranged in a zigzag architecture.
(A) Raw micrograph and 2D class average of CupE pili show zigzag-shaped filaments. (B-C) A 3.5 Å resolution cryo-EM structure of the CupE pilus reveals how CupE1 subunits are arranged in a zigzag pattern, with the donor strand of the (n+1)th subunit enveloped by the incomplete Ig-like fold of each nth subunit. Five CupE1 subunits form a longer repeat of ~155 Å.
Fig 2.
Architecture of the CupE1 subunit within the CupE pilus.
(A) The donor strand from the (n+1)th subunit completes a β-sheet in the nth subunit by providing a 13-residue β-strand. (B) Subunit topology demonstrating β-sheet architecture of the CupE1 subunit. A yellow line denotes a disulfide bridge. (C) Extensive hydrophobic interactions anchor the donor strand of the (n+1)th subunit into the Ig-like fold of the nth subunit. (D) Compared to the extensive interactions of the donor strand with the complemented subunit, the globular subunit:subunit interface between Ig-like folds shows few strong interactions at the interface. A disulfide bridge is observed in each CupE1 subunit (marked).
Fig 3.
Cryo-ET imaging of CupE pili on P. aeruginosa cells.
(A-B) Tomographic slice of cells expressing CupE pili. CupE pili adopt significant curvature on cells. The pilus can be seen going in and out of plane in (B). Inset: Atomic model of CupE fitted in the subtomogram averaging map produced from cellular cryo-ET data. (C-D) Segmentation of cell membranes and CupE pili illustrates how CupE pili adopt variable curvatures. See also S2 Movie.
Fig 4.
Co-occurrence of cupA and cupE gene clusters.
(A) Bioinformatic analysis reveals co-occurrence of the cupA and cupE gene clusters with preserved gene order in most P. aeruginosa strains. Overlapping sequences of different CUP genes indicate possible translational coupling; of the usher gene cupE5 to the adhesive tip subunit gene cupE6, of the two minor pilin subunits cupE2 and cupE3, and of all genes cupA2-cupA6. A phosphodiesterase (CupA6) is encoded after cupA5 in every strain in which cupA occurs. (B) Outside of P. aeruginosa, examples of co-occurrence can be found in pathogenic proteobacteria such as P. fluorescens, Y. pestis, A. baumannii, and B. pseudomallei. Gene order in CUP clusters outside P. aeruginosa is not preserved; e.g. in P. fluorescens ATCC_13525, an additional pilin is encoded, and in B. pseudomallei K96243, the cupA6-like gene is encoded on the opposite strand. Accession data and gene loci are provided in S5 Table and supplementary file S1 Data.