title: Novel ATP-driven pathway of glycolipid export involving TolC protein.
abstract: Upon depletion of combined nitrogen, N(2)-fixing heterocysts are formed from vegetative cells in the case of the filamentous cyanobacterium Anabaena sp. strain PCC 7120. A heterocyst-specific layer composed of glycolipids (heterocyst envelope glycolipids (HGLs)) that functions as an O(2) diffusion barrier is deposited over the heterocyst outer membrane and is surrounded by an outermost heterocyst polysaccharide envelope. Mutations in any gene of the devBCA operon or tolC result in the absence of the HGL layer, preventing growth on N(2) used as the sole nitrogen source. However, those mutants do not have impaired HGL synthesis. In this study, we show that DevBCA and TolC form an ATP-driven efflux pump required for the export of HGLs across the Gram-negative cell wall. By performing protein-protein interaction studies (in vivo formaldehyde cross-linking, surface plasmon resonance, and isothermal titration calorimetry), we determined the kinetics and stoichiometric relations for the transport process. For sufficient glycolipid export, the membrane fusion protein DevB had to be in a hexameric form to connect the inner membrane factor DevC and the outer membrane factor TolC. A mutation that impaired the ability of DevB to form a hexameric arrangement abolished the ability of DevC to recognize its substrate. The physiological relevance of a hexameric DevB is shown in complementation studies. We provide insights into a novel pathway of glycolipid export across the Gram-negative cell wall.
authors.affiliation: Department of Microbiology/Organismic Interactions, Interfaculty Institute of Microbiology and Infection Medicine Tübingen, University of Tübingen, 72076 Tübingen, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Staron
authors.affiliation: Department of Microbiology/Organismic Interactions, Interfaculty Institute of Microbiology and Infection Medicine Tübingen, University of Tübingen, 72076 Tübingen, Germany.
authors.forename: Karl
authors.initials: K
authors.lastname: Forchhammer
authors.affiliation: Department of Microbiology/Organismic Interactions, Interfaculty Institute of Microbiology and Infection Medicine Tübingen, University of Tübingen, 72076 Tübingen, Germany. Electronic address: iris.maldener@uni-tuebingen.de.
authors.forename: Iris
authors.initials: I
authors.lastname: Maldener
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry*; D017033:Anabaena / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry*; D001692:Biological Transport; D002473:Cell Wall / Q000378:metabolism; D000458:Cyanobacteria / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006017:Glycolipids / Q000737:chemistry*; D006868:Hydrolysis; D008055:Lipids / Q000737:chemistry; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008954:Models, Biological; D020349:Surface Plasmon Resonance
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D006017:Glycolipids; D008055:Lipids; D026901:Membrane Transport Proteins; C073126:tolC protein, E coli; D000255:Adenosine Triphosphate