VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

dependent polar flagellum. The torque generating unit in the flagellumconsists of the proteins PomA, PomB, MotX and MotY in which thePomA/B complex plays a role in the transport of Na + . It has been shown thatthis complex consists of two PomA homodimers and one PomB homodimer[1]. Biochemical studies of the native complex have not yet been reported.In this work I will focus on the characterisation of PomA and PomB of theflagellar stator complex. In a recent publication [2] the expression andpurification of PomA with a N-terminal His-tag and PomB with a C-terminal Strep-tag was described. Here, the purification of the PomA/Bcomplex using a new construct with a Strep-tag to the N-terminal end ofPomA and a His-tag to the C-terminal end of PomB is reported. In addition,a vector introducing a GFP fusion to the N-terminus of PomB wasconstructed.The vector functionally complemented a pomAB deletion strain, indicatingthat PomA-GFP-PomB complex was inserted into the stator complex. Polarlocalization of the complex was confirmed by fluorescence microscopy.Introducing the D23N mutation in PomB did result in a non-motilephenotype of V. cholerae, demonstrating a functional role of D23 for statorfunction.[1] Sato, K. et al (2000): Multimeric structure of PomA, a compinent of the Na + -driven polar flagellarmotor of Vibrio alginolyticus, J. Biol. Chem. 275 (2000) 20223-20228.[2] Vorburger, T. et al (2009): Functional role of a conserved aspartatic acid residue in the motor ofthe Na + -driven flagellum from Vibrio cholerae, BBA 1787 1198-1204.MPP007Localization and function of ubiquinone-8 in the Na + -translocating NADH: quinone oxidoreductase (Na + -NQR)from Vibrio choleraeS. Vossler* 1 , J. Steuber 1Department of Microbiology, University of Hohenheim Stuttgart, GermanyThe sodium ion-translocating NADH:quinone oxidoreductase (Na + -NQR)from Vibrio cholerae is a respiratory membrane protein complex thatcouples the oxidation of NADH and the reduction of membrane-boundquinone to the transport of Na + across the bacterial membrane [1]. The Na + -NQR is composed of the six subunits NqrA-F and contains at least fiveredox active cofactors: FAD and a 2Fe-2S cluster on NqrF which alsoharbours the binding site for NADH, covalently attached FMN on NqrC, andcovalently attached FMN and riboflavin on NqrB.A specific binding site for quinones was identified on the peripheral NqrAsubunit by modification with photoactivatable, biotinylated quinone whichwas prevented by ubiquinone-8 (Q 8). From a titration which monitored thefluorescence of 1-anilino-naphthalene-8-sulfonate (ANS), a dissociationconstant of 76 mM for ubiquinone-1 (Q 1) was determined. This indicatedthat the affinity of NqrA towards short-chain ubiquinone was high. CDspectroscopy revealed pronounced structural changes of NqrA upon bindingof Q 1. In our new model describing electron transfer in the Na + -NQR,ubiquinone-8 on subunit NqrA is proposed to act as the ultimate acceptor.[1] Casutt, M.S. et al (2010): Localization and function of the membraneboundriboflavin in the Na + -translocating NADH:quinone oxidoreductase(Na + -NQR) from Vibrio cholerae, J. Biol. Chem., 285:27088-27089.MPP008Biosynthesis of the membrane lipid phosphatidylcholinein bacteria interacting with eukaryotesR. Moser*, M. Aktas, F. NarberhausDepartment of Microbial Biology, Ruhr-Universiy, Bochum, GermanyPhosphatidylcholine (PC, lecithin) is the most abundant membranephospholipidin eukaryotes, whereas many prokaryotes lack PC. Based on insilico-studies, about 10 % of all bacteria synthesize PC [3]. Most of the PCsynthesizingbacteria are known to interact with eukaryotic hosts in acommensalic, symbiotic or pathogenic manner. Amongst others, PC and thePC-synthesizing enzymes were identified in the plant-pathogenAgrobacterium tumefaciens and the soybean-symbiont Bradyrhizobiumjaponicum [1]. Here, PC plays a crucial role in the bacterium-plantinteractionand is important for the virulence and symbiosis [2, 4]. Inbacteria, PC-biosynthesis is carried out via two pathways, the N-methylationpathway and the phosphatidylcholine synthase (Pcs) pathway. The stepwisemethylation of phosphatidylethanolamine (PE) to PC via the intermediatesmonomethyl-PE (MMPE) and dimethyl-PE (DMPE) in the N-methylationpathway requires phospholipid N-methyltransferases. While theN-methylation route in A. tumefaciens is accomplished by a single PmtA, inB. japonicum this pathway involves five phospholipid N-methyltransferaseswith distinct substrate specifities [1].Our recent data provide evidence for PC biosynthesis in severalplant-commensalic and plant-growth promoting bacteria (Methylobacteriumextorquens, Pseudomonas fluorescens) as well as in differentplant-pathogenic bacteria (Xanthomonas campestris, Pseudomonas syringaepv. tomato) that cause diseases of different economically important plants.The investigated Pseudomonads seem to prefer the Pcs pathway.Heterologous expression of the M. extorquens and X. campestris pv.campestris pmt-genes in Escherichia coli clearly suggest a bipartiteN-methylation pathway similar to the one in B. japonicum.[1] Aktas, M. et al (2010): Eur. J. Cell Biol. 89: 888-894.[2] Minder, A. C. et al (2001): Mol. Microbiol. 39: 1186-1198.[3] Sohlenkamp, C. et al (2003) Prog. Lipid Res. 42: 115-162.[4] Wessel, M. et al (2006) Mol. Microbiol. 62: 906-915.MPP009Trimeric autotransporter adhesin-dependent adherenceof Bartonella henselae, Bartonella quintana and Yersiniaenterocolitica to matrix components and endothelial cellsunder static and dynamic flow conditionsN. Müller 1 , P. Kaiser 2 , D. Linke 3 , H. Schwarz 3 , T. Riess 2 , A. Schäfer 1 ,H. Eble 4 , V. Kempf* 21 Institute for Medical Microbiology and Hygiene, University HospitalTübingen, Tübingen, Germany2 Institute for Medical Microbiology and Infection Control, UniversityHospital Frankfuirt am Main, Frankfurt am Main, Germany3 Max Planck Institut for Developmental Biology, Tübingen, Germany4 Center for Molecular Medicine, Cluster of Excellence CardiopulmonarySystem, Frankfurt am Main, GermanyTrimeric autotransporter adhesins (TAAs) are important virulence factors ofGram-negative bacteria responsible for adherence to extracellular matrix(ECM) and host cells. Here, we analyzed three different TAAs [Bartonellaadhesin A (BadA) of Bartonella henselae, variably expressed outermembrane proteins (Vomps) of Bartonella quintana, Yersinia adhesin A(YadA) of Yersinia enterocolitica] for mediating bacterial adherence toECM and endothelial cells. Using static (cell culture vials) and dynamic(capillary flow chambers) experimental settings, adherence of wildtypebacteria and the respective TAA-negative strains were compared. Understatic conditions, ECM adherence of B. henselae, B. quintana and Y.enterocolitica was strongly dependent on the expression of their particularTAAs. YadA of Y. enterocolitica did neither mediate bacterial binding toplasma nor cellular fibronectin both under static and dynamic conditions.TAA-dependent host cell adherence appeared more significant underdynamic conditions although the total number of bound bacteria wasdiminished compared to static conditions. The herein described results allowto dissect the biological role of particular TAAs in ECM and host celladherence and to identify differences in bacterial binding under static anddynamic conditions. Dynamic models expand the methodology to performbacterial adherence experiments under more realistic blood-stream likeconditions.MPP010In vitro production of neutrophil extracellular trapsagainst Aspergillus fumigatusS. Bruns* 1,2 , M. Hasenberg 3,4 , O. Kniemeyer 1,2 , A. Thywißen 1,2 ,M. Gunzer 3,4 , A. Brakhage 1,21 Department of Molecular and Applied Microbiology, Hans-Knöll-Institute(HKI), Jena, Germany2 Institute for Microbiology and Molecular Biology, Friedrich-Schiller-University Jena, Jena, Germany3 Institute for Molecular and Clinical Immunology, Magdeburg, Germany4 Medical Faculty, Otto-von-Guericke-University, Mageburg, GermanyThe opportunistic pathogenic mold Aspergillus fumigatus is an increasingcause of morbidity and mortality in immunocompromised and in partimmunocompetent patients. The very small conidia, acting as infectiousagent, infiltrate the lungs and get in contact with alveolar macrophages andneutrophil granulocytes, which repesent the first line of defense. Both arephagocytic cells and kill the conidia via phagocytosis. Besides, neutrophilsare able to form neutrophil extracellular traps (NETs) against A. fumigatusspektrum | Tagungsband 2011