Architecture of the type IVa pilus machine

Abstract

Type IVa pili are filamentous cell surface structures observed in many bacteria. They pull cells forward by extending, adhering to surfaces, and then retracting. We used cryo-electron tomography of intact Myxococcus xanthus cells to visualize type IVa pili and the protein machine that assembles and retracts them (the type IVa pilus machine, or T4PM) in situ, in both the piliated and nonpiliated states, at a resolution of 3 to 4 nanometers. We found that T4PM comprises an outer membrane pore, four interconnected ring structures in the periplasm and cytoplasm, a cytoplasmic disc and dome, and a periplasmic stem. By systematically imaging mutants lacking defined T4PM proteins or with individual proteins fused to tags, we mapped the locations of all 10 T4PM core components and the minor pilins, thereby providing insights into pilus assembly, structure, and function.

Fig. 1. Visualizing the T4PM in intact M. xanthus cells

(A) A frozen-hydrated M. xanthus cell on an EM grid. (B) Slice through tomogram of the cell pole. White arrows, piliated T4PM basal bodies; black arrow, an empty T4PM basal body. Outer and inner membranes (OM and IM) are indicated. (C and D) Examples of slices through subtomograms containing piliated and empty T4PM basal body structures, respectively. Red asterisks denote periplasmic and cytoplasmic density layers. (E and H) Schematic envelopes of the subtomogram averages of wild-type piliated (E) and ΔpilB empty (H) T4PM basal bodies, showing their molecular dimensions. (F and G) Slices through subtomogram averages of wild-type piliated (F) and ΔpilB empty (G) T4PM basal bodies, with annotations of structural features. Scale bars, 500 nm (A), 50 nm (B), 5 nm [(C) and (D)], 10 nm [(F) and (G)].

Fig. 2. Mapping T4PM components

First and third columns: Central slices of subtomogram averages of piliated and empty T4PM basal bodies, respectively, from different M. xanthus strains. Second and fourth columns: Differences in the T4PM mutant structures versus the wild type (red and yellow colors respectively denote addition and omission of densities, with opacities of 10%, 20%, 30%, 40%, and 50% corresponding to density differences of 1, 1.5, 2, 2.5, and 3 standard deviations, respectively, overlaid on the wild-type subtomogram averages). White arrows indicate the component locations identified by the difference maps. Fifth column: Schematic representations of piliated (left) and empty (right) T4PM basal bodies showing each identified component location. Scale bar in A2, 10 nm (applies to columns 1 through 4).

Fig.3. Architectural models of the T4PM

(A) Summary schematics showing the component locations identified in the piliated and empty T4PM basal body structures. (B and C) Central slices of the architectural models of piliated and empty T4PM basal bodies, respectively, in which atomic models of T4PM components are placed in the in vivo envelopes according to the component maps in (A) and previously reported constraints and filtered to 3-nm resolution. (The process of how each component was placed is detailed in Movie 1 and the supplementary materials.) Models of each component are colored as in (A), with the transmembrane segments of PilN and PilO shown as cylinders; “x3” indicates three AMIN domains per PilQ monomer, only one of which is shown. Note that the empty T4PM basal body is shown with five PilA major pilin subunits in the short stem; however, the short stem likely also contains minor pilins. (D) Top view of the PilP HR domains and the PilQ N0 and N1 domains in the architectural model [colored as in (B) and (C)], with PG model (colored green) as background; 36 AMIN (β) domain models from 12 PilQ proteins are randomly placed on PG and connected by long flexible linkers (black) with lengths within 20 nm between β1 and β2 domains (70 residues), 12 nm between β2 and β3 domains (40 residues), and 12 nm between β3 and N0 domains (40 residues). (E) Overall architectural models of piliated (left) and empty (right) T4PM basal bodies. For clarity, the PilQ AMIN domains displayed in (D) are not shown.