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

hemagglutinates sheep erythrocytes. Some of its surface proteins have beencharacterised in the early past. The serine-aspartate repeat protein I (SdrI),contains the longest SD repeat region described so far (854 aa) and aLPXTG-motif for cell wall anchoring. It is a member of the MSCRAMMprotein family and shows a typical ABB domain structure. Within the Adomain a specific amino acid sequence (TYTFTNYVD) is found. This motifor it variants is also found in many other fibronectin or collagen bindingproteins from staphylococci.Previous experiments have shown that the purified A domain is able to bindto fibronectin coated on microtiter plates. To localise the area of binding theA domain was divided into three parts: N1, N2 and N3. N2, N3 and N2+3were cloned into the pQE30Xa vector for overexpression and purificationusing a n-terminal His-Tag. The parts of interest were N2 and N3 because inN2 the TYTFTNYVD is located. Bindingassays were carried out with N2,N3, N2+3 and the whole A domain as a positive control were done using acoated microtiter plate. The bound protein was detected by an ELISA. Asexpected the results showed a high binding activity for N2, a slightlyreduced binding for N2+3 compared to N2 and althougth N3 alone showedno binding at all. N2 was further subjected to x-ray structure analysis butdata analysis is still ongoing. We expect to gain new insights into the Adomain conformation of SdrI and its implications for the uro-adherence ofStaphylococcus saprophyticus.MPP057Investigations into the metabolism of LegionellapneumophilaV. Herrmann* 1 , E. Eylert 2 , W. Eisenreich 2 , M. Lautner 1 , K. Heuner 11 Robert-Koch-Institute, P26, Berlin, Germany2 Department of Chemistry, Technical University Munich Garching,GermanyLegionella pneumophila is an aquatic bacterium that replicates withinprotozoa (amoeba), but can also infect human alveolar macrophages causingLegionnaires’ disease, an atypical pneumonia. Many virulence factors of L.pneumophila have been reported, but less is known about nutrition of thebacteria, especially inside host cells. When nutrients become limiting, aregulatory casdade triggers the differentiation from the replicative form,with high metabolic activity, to the transmissive and virulent form. L.pneumophila uses amino acids as primary energy and carbon sources;glucose although assimilated, is thought not to be important for bacterialgrowth. We used 13 C-isotopologue profiling in combination with nuclearmagnetic resonance (NMR) spectroscopy and mass spectrometry (MS) wedemonstrated the use of glucose for de novo-biosynthesis of several aminoacids. We found high 13 C-incorporation rates for the amino acids alanine,aspartate, glutamate, glycine, and proline as well as for 3-hydroxybutyrate inL. pneumophila. The tricarboxylic acid cycle is complete and active.Furthermore, experiments with [1,2- 13 C 2]glucose state the importance of thepentose phosphate pathway for glucose catabolism. In addition, weidentified an active glucoamylase, which is secreted by L. pneumophila anddegrades starch and glycogen. Moreover, we present an experimentalbackground for in vivo-studies of the bacterial metabolism insideAcanthamoeba castellanii host cells.MPP058Antibiotic translocation through bacterial porins -insights from electrophysiologyH. Weingart* 1 , K.R. Mahendran 1 , Q.-T. Tran 1 , W. Suginta 2 ,M. Winterhalter 11 School of Engineering and Science, Jacobs University, Bremen, Germany2 Institute of Science, Suranaree University of Technology, NakhonRatchasima, ThailandThe outer membrane of Gram-negative bacteria contains a number ofchannel-forming hydrophilic proteins called porins. Such channels allowdiffusion of low-molecular weight solutes across the outer membrane. Ourmethod of choice to study the molecular aspects of antibiotic translocationacross such membrane channels is electrophysiology using planar lipidbilayers. In this technique, purified porins are individually inserted into thebilayer. Permeation of molecules inside the channel causes fluctuations inthe ion current, reflecting the molecular interactions with the channel wall.We have been able to characterize facilitated translocation of beta-lactamsand fluoroquinolones through various outer membrane channels includingBpsOmp38 of Burkholderia pseudomallei, OmpPst1 and OmpPst2 ofProvidencia stuartii, and OmpF and OmpC of Escherichia coli. Titrationwith effective antibiotics revealed concentration-dependent blockages of theion flow suggesting interaction with the channel. Noise analysis of ioncurrents through porin in the presence of antibiotics enabled us to determinebinding kinetics and transport parameters at single-molecule level. We alsocharacterized the impact of temperature on the antibiotic passage throughporins reconstituted into bilayer. In vitro activity of antibiotics wasdetermined by MIC assays which correlated with the results obtained frombilayer measurements. Experimental results were compared with moleculardynamics simulations which provide the energy barriers along the diffusionpathway and an atomic description of the antibiotic translocation throughporins. Our study of antibiotic translocation at single-molecule level givesnew insights to design novel drugs with optimal penetration into bacterialcells.MPP059Adhesion of Ustilago maydis filaments at the onset ofpathogenic development.K. Hofmann*, J. KaemperInstitute for Applied Biosciences - Genetics, Karlsruhe Institute ofTechnology (KIT), Karlsruhe, GermanyIn pathogenic fungi, adhesion to surfaces is considered an important event indisease establishment. Adhesion of the phytopathogenic basidiomyceteUstilago maydis is tightly linked to the formation of filaments on the plantsurface that follows the establishment of the infectious dikaryon.Filamentation of the fungus can also be induced solely by contact with agiven hydrophobic surface like Polytetrafluoroethylene. The factorspropagating hyphal adhesion in U. maydis are still largely unknown, andidentification of distinct factors by comparative sequence analyses ishindered by the heterogeneity of involved gene products and their functionalredundancies. In an attempt to circumvent these difficulties we adapted amethod developed for identification of Candida albicans adhesionpromotinggenes. We use a parallel plate shear flow assay to screen a U.maydis cDNA library expressed in adhesion-deficient Saccharomycescerevisiae cells to identify genes that enhance adhesive capabilities of yeastcells to hydrophobic surfaces. The use of a parallel plate flow chamberprovides quantitative reproducible measurements of cell detachment fromthese surfaces by applying a known shear stress under conditions of laminarflow. Identified genes and respective gene products will subsequently beanalyzed and characterized in U. maydis. We expect that this approach willlead to the identification of novel adhesins and regulatory elementscontrolling surface adhesion.MPP060Metabolic flux analysis of enteropathogenic YersiniapseudotuberculosisR. Bücker* 1 , P. Dersch 2 , C. Wittmann 11 Institute of Biochemical Engineering, Technical University, Braunschweig,Germany2 Department of Molecular Infection Biology, Helmholtz Center for InfectionResearch, Braunschweig, GermanyThe pathogenic bacterium Yersinia pseudotuberculosis is the causative agentof self-limiting enteritis, diarrhoea, mesenteric lymphadenitis andautoimmune disorders [1]. It is able to infect animals and humans and isclosely related to Yersinia pestis. Moreover Yersinia pseudotuberculosis isknown to have a complex regulatory network and it is therefore apredestinated model organism for pathogens.The invasion of mammalian cells is controlled by a cascade composed ofregulatory RNAs and proteins some of which are checked by nutritional andenvironmental conditions [2]. Due to this link between virulence andmetabolism a coincident change in central carbon fluxes with formation ofinfection relevant factors is assumed.Metabolic flux analysis has proven as major technology in industrialbiotechnology to perform system-wide pathway analysis and subsequentdesign-based strain optimization [3]. Despite its beneficial application in thisfield, 13 C metabolic flux studies are rarely found in the medical field. Thismethodology is, however, promising to gain a quantitative insight into the invivo activity of enzymes and pathways for analysing the mentioned linkbetween pathogenicity and metabolism.The quantitative analysis of metabolic fluxes was based on a comprehensiveapproach combining an experimental and computational part. This involvedthe development of a reproducible cultivation strategy for Yersiniaspektrum | Tagungsband 2011