The biogenesis of diverse adhesive structures in a variety of Gram-negative bacterial species is dependent on the chaperone/usher pathway. Very little is known about how the usher protein translocates protein subunits across the outer membrane or how assembly of these adhesive structures occurs. We have discovered several mechanisms by which the usher protein acts to regulate the ordered assembly of type 1 pili, specifically through critical interactions of the chaperone-adhesin complex with the usher. A study of association and dissociation events of chaperone-subunit complexes with the usher in real time using surface plasmon resonance revealed that the chaperone-adhesin complex has the tightest and fastest association with the usher. This suggests that kinetic partitioning of chaperone-adhesin complexes to the usher is a defining factor in tip localization of the adhesin in the pilus. Furthermore, we identified and purified a chaperoneadhesin-usher assembly intermediate that was formed in vivo. Trypsin digestion assays showed that the usher in this complex was in an altered conformation, which was maintained during pilus assembly. The data support a model in which binding of the chaperoneadhesin complex to the usher stabilizes the usher in an assembly-competent conformation and allows initiation of pilus assembly.

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Bacterial virulence factors are typically surface-associated or secreted molecules that in Gram-negative bacteria must cross the outer membrane (OM). Protein translocation across the bacterial OM is not well understood. To elucidate this process we studied P pilus biogenesis in Escherichia coli. We present high-resolution electron micrographs of the OM usher PapC and show that it forms an oligomeric complex containing a channel approximately 2 nm in diameter. This is large enough to accommodate pilus subunits or the linear tip fibrillum of the pilus but not large enough to accommodate the final 6.8-nm-wide helical pilus rod. We show that P pilus rods can be unraveled into linear fibers by incubation in 50% glycerol. Thus, they are likely to pass through the usher in this unwound form. Packaging of these fibers into their final helical structure would only occur outside the cell, a process that may drive outward growth of the pilus organelles. The usher complex appears to be similar to complexes formed by members of the PulD͞pIV family of OM proteins, and thus these two protein families, previously thought to be unrelated, may share structural and functional homologies.

Type 1 pilus biogenesis was used as a paradigm to investigate ordered macromolecular assembly at the outer cell membrane. The ability of Gram-negative bacteria to secrete proteins across their outer membrane and to assemble adhesive macromolecular structures on their surface is a defining event in pathogenesis. We elucidated genetic, biochemical, and biophysical requirements for assembly of functional type 1 pili. We discovered that the minor pilus protein FimG plays a critical role in nucleating the formation of the adhesive tip fibrillum. Genetic methods were used to trap pilus subunits during their translocation through the outer membrane usher protein, providing data on the structural interactions that occur between subunit components during type 1 pilus formation. Electron microscopic and biochemical analyses of these stepwise assembly intermediates demonstrated that translocation of pilus subunits occurs linearly through the usher's central channel, with formation of the pilus helix occurring extracellularly. Specialized pilin subunits play unique roles both in this multimerization and in the final ultrastructure of the adhesive pilus.

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