Abstract - Benedens

Biofilm formation protects embedded bacteria from antibiotics and desiccation, facilitates capture of nutrients and ensures survival in hostile environments. The human pathogen Pseudomonas aeruginosa produces several exopolysaccharides, including Pel, to build up the biofilm matrix. The dedicated synthesis/regulation unit of Pel is integrated in the inner membrane, whereby the PelBC complex facilitates transport across the outer membrane. To understand the secretion mechanism, we set out to illuminate the structural and functional aspects of the PelBC assembly. Cryo-EM analysis resolved a 2.4 Å structure of in vivo assembled and nanodisc-reconstituted complex of ~250 kDa including a PelB β-barrel, a dodecameric ring of PelC lipoproteins at the periplasmic interface and multiple lipid/LPS densities. Structural analysis and biochemical studies indicated that the TPR domains emerging from the PelB barrel into the periplasm serve as an anchor point of the PelC ring. On the extracellular side, the barrel is covered with sealing loops.  Specific loop deletions and single-channel conductivity measurements combined with molecular dynamic simulations provided first insights into the conformational dynamics of the PelB protein, switching between different conductivity states. The ongoing structure-guided studies of PelC:lipid, PelC:PelB and PelC:exopolysaccharide interactions are further aiming to explain the role played by the essential subunit PelC in Pel secretion.

Marius Benedens 1, Cristian Rosales Hernandez 2, Sabine Straathof 3, Jennifer Loschwitz 1, Tim Kroll 1, Giovanni Maglia 3, Roland Beckmann 2, Alexej Kedrov 1 ; 1 Synthetic Membrane Systems, Institute for Biochemistry, Heinrich Heine University Düsseldorf, Germany 2 Gene Center Munich, Ludwig Maximilian University Munich, Germany 3 Chemical Biology I, University of Groningen, The Netherlands