Substrate-translocase interactions during two-partner secretion in Gram-negative bacteria

• Applicant: Professor Dr. Matthias Müller
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2008 to 2015
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 43311986

The two-partner secretion (TPS-) pathway found in pathogenic Gram-negative bacteria is dedicated to the secretion of virulence factors (termed TpsA-proteins). TpsA-proteins are translocated via the Sec translocon to the periplasm, from where they are exported through a β-barrel pore that is formed by the cognate TpsB protein in the outer membrane. A related transport system is that of autotransporters, in which the secreted protein is C-terminally fused to a β-barrel domain that mediates translocation across the outer membrane. Recently we have developed a novel in vitro system for the study of translocation of a TpsA protein across its TpsB-pore. We now want to use this system to identify periplasmic proteins involved in chaperoning and targeting a TpsA protein to the TpsB-pore. We will use site-specific photo-crosslinking in combination with the generation of translocation intermediates of a TpsA-protein stalled in the TpsB-pore, to determine the interacting sites between TpsA and TpsB, the actual translocation path across TpsB, and the orientation by which TpsA is threaded into TpsB. By use of inactivating mutations of the TpsA-protein we want to examine if its post-translocational folding provides the driving force and unidirectionality of transport. We will further exploit the cell-free system to study the related autotransporter pathway.

• DFG Programme: Research Units
• Subproject of: FOR 929: Dynamics of Bacterial Membrane Proteins