Structural bases for F plasmid conjugation and F pilus biogenesis in Escherichia coli

Abstract

Bacterial conjugation systems are members of the large type IV secretion system (T4SS) superfamily. Conjugative transfer of F plasmids residing in the Enterobacteriaceae was first reported in the 1940s, yet the architecture of F plasmid-encoded transfer channel and its physical relationship with the F pilus remain unknown. We visualized F-encoded structures in the native bacterial cell envelope by in situ cryoelectron tomography (CryoET). Remarkably, F plasmids encode four distinct structures, not just the translocation channel or channel-pilus complex predicted by prevailing models. The F1 structure is composed of distinct outer and inner membrane complexes and a connecting cylinder that together house the envelope-spanning translocation channel. The F2 structure is essentially the F1 complex with the F pilus attached at the outer membrane (OM). Remarkably, the F3 structure consists of the F pilus attached to a thin, cell envelope-spanning stalk, whereas the F4 structure consists of the pilus docked to the OM without an associated periplasmic density. The traffic ATPase TraC is configured as a hexamer of dimers at the cytoplasmic faces of the F1 and F2 structures, where it respectively regulates substrate transfer and F pilus biogenesis. Together, our findings present architectural renderings of the DNA conjugation or "mating" channel, the channel-pilus connection, and unprecedented pilus basal structures. These structural snapshots support a model for biogenesis of the F transfer system and allow for detailed comparisons with other structurally characterized T4SSs.

Keywords: DNA conjugation; cryoelectron tomography; pilus; protein transport; type IV secretion.

Fig. 1.

In situ structure of the E. coli F-plasmid type IV secretion machine revealed by CryoET and subtomogram averaging. (A) A tomographic slice from a representative E. coli minicell showing multiple T4SS machines embedded in the cell envelope; the boxed region was magnified to show an F1-CH complex. (B) A 3D surface view of the E. coli minicell in A showing T4SS machines and F pili. (C) A central slice of the averaged structure of the F1-CH complex in the cell envelope. Diameters of the flare and cylinder are shown. (D) After refinement, details of the OMC are visible, including a 2-nm gap in the OM. (E) A cross-section view of the region in D marked by a yellow arrow shows 13-fold symmetry of the OMC. (F) After refinement, details of the IMC are visible, including a periplasmic collar, cylinder and central channel (yellow arrowheads), and cytoplasmic inverted “V” structures. (G) A cross-section view of the region in F marked by a yellow arrow shows sixfold symmetry of the IMC. (H–J) Three-dimensional surface renderings of the F1-CH complex shown in different views.

Fig. 2.

Architectures of mutant F1-CH machines. The top row shows the 3D reconstructions of F1-CH complexes visualized in WT, ΔtraC, traC-GFP, traC.K487Q, and ΔtraD mutant cells. The second row shows refinements of the IMCs, and the third and fourth rows show surface renderings from side and bottom views, respectively. (A) Native F1-CH complex (WT) for comparisons. (B) The ΔtraC mutant machine lacks the periplasmic collar and cytoplasmic inverted “V” structures. (C) The mutant machine with TraC-GFP in place of native TraC shows the presence of GFP densities (green dots shown in surface renderings) associated with the inner and outer arms of the inverted “V” structures. (D and E) Mutant machines with TraC.K487Q in place of native TraC or deleted of TraD align well with the WT F1-CH structure.

Fig. 3.

In situ structure of the F-encoded F2-CH/Pilus complex. (A) A tomographic slice from a representative E. coli minicell and a high magnification view showing the F2-CH/Pilus complex with associated F pilus. (B) Cross-section views show differences in widths of the pilus, cylinder, and inner membrane complex at positions in C indicated by yellow arrows. (C) A central slice of the averaged structure of the F2-CH/Pilus complex across the cell envelope. (D) The OMC shows a 13-fold symmetry in a cross view, and the IMC shows sixfold symmetry in a cross view. (E) Three-dimensional surface rendering of the F2-CH/Pilus and cutaway view with architectural features indicated.

Fig. 4.

In situ structures of F-encoded F3-ST/Pilus and F4-OM/Pilus complexes. (A) Tomographic slices from representative E. coli minicells showing the F pilus associated with an envelope-spanning stalk (F3-ST/Pilus) or the OM in the absence of an associated periplasmic density (F4-OM/Pilus). (B) A central slice of the averaged structure of the F3-ST/Pilus complex and cross views of the pilus, “mushroom-cap,” and stalk showing the absence of a central channel; cross-sections views and dimensions are presented for the various structures at positions indicated by yellow arrows. (C) Three-dimensional surface rendering of the F3-ST/Pilus complex and cutaway view with architectural features indicated. (D) MS2 bacteriophage binds to the sides of pOX38-encoded F pili docked onto F2 and F3 basal structures elaborated by intact E. coli cells.

Fig. 5.

A structure-driven assembly pathway for the F transfer system. Step I: Tra subunits assemble initially as a quiescent F1-Channel complex. Step II: The F1 complex transitions to the F2-Channel/Pilus for dynamic extension and retraction of the F pilus in a “mate-seeking” mode. Dynamic F pilus extension and retraction by the F2 structure favors formation of distant cell-cell contacts during low cell density, e.g., planktonic, growth. Step III: Assembled F pili are alternatively deposited onto distinct platforms, yielding the F3-Stalk/Pilus or F4-OM/Pilus structures. The presumptively static F3 and F4 structures promote nonspecific aggregation and biofilm development; in polymicrobial settings, these dense growth conditions favor formation of mating junctions. Step IV: Recipient cell contact (lightning bolts) triggers F pilus retraction and recruitment of the TraD receptor and F plasmid substrate to activate the DNA transfer or “mating” mode. Step V: Following plasmid transfer, donor and recipient cells disengage and the “mating” channel reverts to the F1 quiescent complex. The relative percentages of each complex visualized on E. coli minicells by in situ CryoET are shown.