No abstract

Bacterial pili are involved in a host of activities, including motility, adhesion, transformation, and immune escape. Structural studies of these pili have shown that several distinctly different classes exist, with no common origin. Remarkably, it is now known that the archaeal flagellar filament appears to have a common origin with the bacterial type IV pilus, and assembly in both systems involves hydrophobic N-terminal alpha-helices that form three-stranded coils in the center of these filaments. Recent work has identified further genes in archaea as being similar to bacterial type IV pilins, but the function or structures formed by such gene products was unknown. Using electron cryo-microscopy, we show that an archaeal pilus from Methanococcus maripaludis has a structure entirely different from that of any of the known bacterial pili. Two subunit packing arrangements were identified: one has rings of four subunits spaced by approximately 44 A and the other has a one-start helical symmetry with approximately 2.6 subunits per turn of a approximately 30 A pitch helix. Remarkably, these schemes appear to coexist within the same filaments. For the segments composed of rings, the twist between adjacent rings is quite variable, while for the segments having a one-start helix there is a large variability in both the axial rise and the twist per subunit. Since this pilus appears to be assembled from a type IV pilin-like protein with a hydrophobic N-terminal helix, it provides yet another example of how different quaternary structures can be formed from similar building blocks. This result has many implications for understanding the evolutionary divergence of bacteria and archaea.

Exchange of DNA between bacteria involve conjugative pili. While the prevailing view has been that F-pili are completely retracted before single-stranded DNA is passed from one cell to another, it has recently been reported that the F-pilus, in addition to establishing the contact between mating cells, serves as a channel for passing DNA between spatially separated cells during conjugation. The structure and function of F-pili are poorly understood. They are built from a single subunit having only 70 residues, and the small size of the subunit has made these filaments difficult to study. Here, we have applied electron cryo-microscopy and single particle methods to solve the long-existing ambiguity in the packing geometry of F-pilin subunits. We show that the F-pilus has an entirely different symmetry from any of the known bacterial pili as well as any of the filamentous bacteriophages, which have been suggested to be structural homologs. Two subunit packing schemes were identified: one has stacked rings of four subunits axially spaced by ∼ 12.8 Å; while the other has a one-start helical symmetry with an axial rise of ∼ 3.5 Å per subunit and a pitch of ∼ 12.2 Å. Both structures have a central lumen of ∼ 30 Å diameter that is more than large enough to allow for the passage of single-stranded DNA. Remarkably, both schemes appear to coexist within the same filaments, in contrast to filamentous phages that have been described as belonging to one of two possible symmetry classes. For the segments composed of rings, the twist between adjacent rings is quite variable, while the segments having a one-start helix are in multiple states of both twist and extension. This coexistence of two very different symmetries is similar to what has recently been reported for an archaeal Methanococcus maripaludis pili filament and an archaeal Sulfolobus shibatae flagellar filament.

The traditional Fourier-Bessel approach to three-dimensional reconstruction from electron microscopic (EM) images of helical polymers involves averaging over filaments, assuming a homogeneous structure and symmetry. We have used a real-space reconstruction approach to study the EspA filaments formed by enteropathogenic E. coli. In negative stain, the symmetry of these filaments is ambiguous, and we suggest that such ambiguities may be more prevalent than realized. Using cryo-EM of frozen-hydrated filaments, we find that these filaments have a fixed twist with 5.6 subunits per turn but an axial rise per subunit that varies from about 3.6 A to 5.6 A. Reconstructions at approximately 15 A resolution show a switching between the more compressed and extended filaments in the packing of putative alpha helices around the hollow lumen. Outside of a crystal, where there is nothing to maintain long-range order, the structural polymorphism in helical polymers may be much greater than has been assumed.

The E. coli RecBCD enzyme facilitates the loading of RecA onto single-stranded DNA produced by the combined helicase/nuclease activity of RecBCD. The nuclease domain of RecB protein, RecBnuc, has been previously shown to bind RecA. Surprisingly, RecBnuc also binds to phage and eukaryotic homologs of RecA, leading to the suggestion that RecBnuc interacts with the polymerization motif that is present in all three proteins. This mode of interaction could only be with monomeric RecA, as this motif would be buried in filaments. We show that RecBnuc binds extensively to the outside of RecA-DNA filaments. Three-dimensional reconstructions suggest that RecBnuc binds to the ATP-binding core of RecA, with a displacement of the C-terminal domain of RecA. Solution experiments confirm that the interaction of RecBnuc is only with the RecA core. Since the RecA C-terminal domain has been shown to be regulatory, the interaction observed may be part of the loading mechanism where RecB displaces the RecA C-terminal domain and activates a RecA monomer for polymerization.