Single channel analysis of bacterial outer membrane transport: Porins and secretion systems

The translocation of hydrophilic molecules across the outer membrane of Gram-negative bacteria is handled by specific membrane proteins which are organized as beta-barrels. Despite a common architecture, these transporter proteins have very different and specific functions.;We then present the first electrophysiological study of a two partner secretion transporter (TpsB) from Haemophilus influenzae, HMW1B. This protein belongs to the growing family of the Omp85/TpsB proteins which are essential for protein secretion and membrane insertion. Our experiments show that HMW1B forms a large pore with a low open probability, and that this pore contains titrable acidic residues. We also demonstrate that the C-terminal domain of the protein is sufficient for pore formation, and that the N-terminal domain has no impact on the closed-open transitions of this pore. These results provide a foundation for further experiments aimed at deciphering the molecular mechanisms for protein secretion through this translocon.;The first part of this dissertation addresses the modulation and biophysical properties of the two major general diffusion porins of Vibrio cholerae OmpU and OmpT. Porins are transmembrane proteins of the outer membrane involved in the transport of hydrophilic solutes between the external medium and the periplasm. Here, we show that these porins are modulated differently at the channel level by conditions that can be encountered by V. cholerae in the host, i.e. acidic pH and the presence of bile salts. These environmental factors are known to affect the physiology of the bacterium, and here we uncover the molecular basis of these effects at the level of porins. Using patch clamp and planar lipid bilayer approaches, we show that the major bile component deoxycholic acid has a visible impact on the channel kinetic of OmpT but not OmpU, indicating that it permeates OmpT more favorably than OmpU. Moreover, while studying the effect of acidic pH, we uncover a very unique proton-induced modulation of OmpU, where the porin shows closures at the level of the full trimer in opposition to the usual modulation at the individual pore level. Finally, we estimate the effective radius of OmpU and OmpT and find differences that may be physiologically relevant.