The chaperone/usher system is one of the best characterized pathways for protein secretion and assembly of cell surface appendages in Gram-negative bacteria. In particular, this pathway is used for biogenesis of the P pilus, a key virulence factor used by uropathogenic Escherichia coli to adhere to the host urinary tract. The P pilus individual subunits bound to the periplasmic chaperone PapD are delivered to the outer membrane PapC usher, which serves as an assembly platform for subunit incorporation into the pilus and secretion of the pilus fiber to the cell surface. PapC forms a dimeric, twin pore complex, with each monomer composed of a 24-stranded transmembrane -barrel channel, an internal plug domain that occludes the channel, and globular N-and C-terminal domains that are located in the periplasm. Here we have used planar lipid bilayer electrophysiology to characterize the pore properties of wild type PapC and domain deletion mutants for the first time. The wild type pore is closed most of the time but displays frequent short-lived transitions to various open states. In comparison, PapC mutants containing deletions of the plug domain, an ␣-helix that caps the plug domain, or the N-and C-terminal domains form channels with higher open probability but still exhibiting dynamic behavior. Removal of the plug domain results in a channel with extremely large conductance. These observations suggest that the plug gates the usher channel closed and that the periplasmic domains and ␣-helix function to modulate the gating activity of the PapC twin pore.The cell envelope of Gram-negative bacteria contains a vast array of protein machineries dedicated to the translocation of polypeptides across the cytoplasmic membrane, periplasm, and outer membrane (OM) 3 (1, 2). Some of these complexes also participate in the assembly of surface-exposed appendages, such as flagella and pili (fimbriae). One of the most thoroughly studied secretion systems is the chaperone/usher pathway, responsible for the biogenesis of a superfamily of virulenceassociated surface structures, including P and type 1 pili (3). These pili play essential roles in the pathogenesis of uropathogenic Escherichia coli by providing a tool for attachment of the bacteria to host urothelial cells (4 -6). P pili, encoded by the chromosomal pap gene cluster, are critical virulence factors for infection of the kidney by uropathogenic E. coli and the development of pyelonephritis. The P pilus is composed of multiple subunits of PapA, which form a rigid helical rod. A thin linear tip fibrillum is located at the distal end of the pilus and is made of four different subunits (PapK, PapF, PapE, and the adhesin PapG) that assemble in a precise order and stoichiometry (3). The minor pilin, PapH, anchors the pilus rod to the cell surface (7).The pilus subunits are first translocated through the cytoplasmic membrane via the Sec general secretory pathway (8). Once in the periplasm, the subunits form binary complexes with the PapD chaperone. The details of the binding interact...
Together with patch-clamp, the planar lipid bilayer technique is one of the electrophysiological approaches used to study the biophysical properties of bacterial pore-forming proteins. Electrophysiological studies have provided important insight into the mechanistic details underlying the function of this class of proteins. Although there are different apparatus designs and variations to the process of obtaining channel recordings, the general architecture of a planar lipid bilayer setup involves two compartments filled with an ionic solution and separated by a septum with a micro-aperture, where a phospholipid bilayer is formed, and an amplifier used to clamp the membrane potential and record currents. Bacterial outer membrane porins and translocons, among others, can be reconstituted in this bilayer and their electrophysiology probed in different physicochemical conditions or through functional assays with substrates or potential modulators. This chapter describes specifically the reconstitution of detergent purified outer membrane pore-forming proteins into artificial lipid membranes using a laboratory customized planar lipid bilayer apparatus and the subsequent recording of channel activity under voltage clamp.
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