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

OTP022c-type cytochromes from Geobacter sulfurreducens asmodel system for spectroscopic studiesA. Seidel* 1 , J. Seidel 1 , M. Hoffmann 2 , P. Lukat 1 , D. Heitmann 1 , O. Einsle 11 Institute for Biochemistry and Molecular Biology, Albert-Ludwigs-University, Freiburg, Germany2 Institute for Biochemistry and Biotechnology, University of Technology,Braunschweig, GermanyThe genome of the δ-proteobacterium Geobacter sulfurreducens includes111 genes encoding for c-type cytochromes that contain between one and 40heme groups [1]. From these, three small-size cytochromes are chosen asmodel systems to determine the orientation of heme groups by single-crystalEPR spectroscopy. The selected c-type cytochromes from Geobactersulfurreducens are OmcF [2], DHC2 [3] and MacA [4]. A single hemegroup yields a distinct axial signal in the EPR spectrum. Here we present anew approach for detecting multiple heme group arrangements bymonocrystal EPR spectroscopy.OmcF is an 11 kDa c-type cytochrome which contains one single hemegroup and serves as a reference system. The 11 kDa diheme c-typecytochrome DHC2 contains the conserved parallel heme-packing motif. Theiron atoms have a short distance of 9.4 Å and thus are expected to beengaged in magnetic coupling. The third model system is the 35 kDa dihemecytochrome MacA containing two domains with one heme group each. Theheme iron atoms have a distance of 21 Å and the orientation of the porphyrinplains in MacA is virtually perpendicular.These model systems can be used as a general basis for further studies oncytochromes containing multiple heme groups in more complex structures.[1] Methé, B. A. et al. (2003): Genome of Geobacter sulfurreducens: metal reduction in subsurfaceenvironments. Science 302, 1967-1969.[2] Lukat, P. et al (2008): Crystal packing of the c6-type cytochrome OmcF from Geobactersulfurreducens is mediated by an N-terminal Strep-tag II. Acta Cryst. D64, 919-926.[3] Heitmann, D. and O. Einsle (2005): Structural and biochemical characterization of DHC2, a noveldi-heme cytochrome c from Geobacter sulfurreducens. Biochemistry 44, 12411-12419.[4] Seidel, J. et al (unpublished data).OTP023Inhibition of quorum sensing in Pseudomonas aeruginosaand Serratia marcescens by a staphylococcal compoundY.-Y. chu* 11 Institute of Microbiology and Infection Medicine (IMIT), MicrobialGenetics, Eberhard-Karls-University, Tübingen, GermanyQuorum sensing is a process of bacterial communication, which is usingsecreted membrane-diffusible signaling moleculars called autoinducers toregulate intercellular interaction. This process plays critical roles inregulating various physiological activities, including production ofantibiotics, secretion of virulence factors, formation of biofilms, swarmingmotility, bioluminescence, sporulation as well as symbiosis. Similarly, it isfound that various bacteria are able to secrete compounds for inhibiting,inactivating or stimulating quorum sensing signals in other bacteria. In ourprevious study on coinfection of Staphylococcus and Pseudomonasaeruginosa, we observed that P. aeruginosa could repress the growth ofpathogenic staphylococcal species but not of nonpathogenic staphylococcalspecies by respiratory inhibitors. Meanwhile, to our surprise, two strains ofthe nonpathogenic staphylococcal species exhibit unknown compound X tointerrupt the function of quorum sensing-controlled factors in gram-negativebacteria, such as the red prodigiosin pigment in Serratia marcescens, theblue-green pyocyanin pigment and biofilm formation in P. aeruginosa.Physical analysis using XAD-16 resin demonstrated that the molecularweight of compound X is below 2 kDa. Moreover, compound X resistsalkaline and acid pH, high temperature and proteinase K treatment, whichmight exclude compound X as a peptide. However, the mechanism ofcompound X expression is still unknown since it is independent of thegrowth temperature, oxygen, NaCl and glucose concentration in themedium. In further study, not only purification and identification of thecompound X using high-performance liquid chromatography (HPLC) andmass spectrometry (MS) are essential. It also needs to indentify thecorresponding genes by transposon mutagenesis or cloning randomchromosomal DNA of compound producing staphylococcal stain into anonproducing strain. In the end, investigation of how compound X disruptsthe quorum sensing signaling system in gram-negative bacteria would be animportant and interesting issue for new generation of antibiotics.OTP024Protein-Protein Interactions in PHB-Metabolism ofRalstonia eutropha as Revealed by Two-Hybrid AnalysisD. Pfeiffer* 1 , D. Jendrossek 11 Institute of Microbiology, University of Stuttgart, Stuttgart, GermanyQuestion: R. eutropha is the model organism for studying metabolism ofpoly(3-hydroxyalkanoates) (PHA). Recently, it was found that morepolypeptides are present on the surface of PHA-granules in R. eutropha andin Pseudomonas putida than would be essential for PHA-synthesis [1, 2].However, little is known whether and how PHA granule associated proteinsinteract with others proteins. A bacterial two-hybrid system was used tostudy protein-protein interaction in R. eutropha.Methods: The bacterial adenylate cyclase-based two-hybrid system(BACTH) was used that is based on genetic fusions of two putativeinteracting proteins with two complementary fragments (T25 and T18) ofBordetella pertussis adenylate cyclase. An association of the two-hybridproteins in vivo results in functional complementation between T25 and T18fragments and leads to cAMP synthesis in an adenylate cyclase deficientreporter strain [3].To study protein-protein interactions in R. eutropha the genes for severalproteins involved in PHB-metabolism including PHB-synthase (PhaC1), β-ketothiolase (PhaA), acetoacetyl-CoA-reductase (PhaB), phasins (PhaP1-P4), regulator of phasin expression (PhaR) and PHB-depolymerase (PhaZa1)were cloned into the two-hybrid vectors pUT18C and pKT25 and thecorresponding plasmids were co-transformed in the reporter strain E. coliBTH101. The efficiencies of interactions between different hybrid proteinswere analyzed by colour formation on LB X-Gal and MacConkeymaltose/lactose medium and quantified by determination of β-galactosidaseactivity in liquid cultures.Results: In this study two-hybrid experiments using nine different proteinsof R. eutropha, with particular functions in PHB metabolism wereperformed. Nearly all tested proteins showed a more or less intense abilityfor homo-oligomerisation. A strong interaction was found between phasinPhaP2 and phasin PhaP4 and other phasins as well as moderate interactionsbetween PHB-depolymerase PhaZa1 and phasins. PHB synthase PhaC1apparently did not significantly interact with any of the phasins.Conclusions: Our data indicate that PhaP2 and other phasins play animportant role in the spatial organisation of PHB granule associated proteinsand that PHB depolymerase PhaZa1 and phasins may influence each other.[1] Jendrossek, D. (2009): Polyhydroxyalkanoate granules are complex subcellular organelles(carbonosomes). J Bacteriol, 191:3195-3202.[2] Ruth, K. et al (2008): Identification of two acyl-CoA synthetases from Pseudomonas putida GPo1:one is located at the surface of polyhydroxyalkanoates granules Biomacromolecules, 9:1652-1659.[3] Karimova, G. et al (1998): A bacterial two-hybrid system based on a reconstituted signaltransduction pathway. Proc Natl Acad Sci USA, 95:5752-5756.OTP025Escherichia coli growth and biofilm development areinfluenced by secreted metabolite products of otherEnterobacteriaceae speciesA. Vacheva*, R. Ivanova, S. StoitsovaStephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences,Morphology of Microorganisms and Electron Microscopy, Sofia, BulgariaIn nature, microorganisms are predominantly associated with surfaces andorganized in communities known as biofilms. Most of them are complexconsortia of different microbial species, where complicated interstrain andinterspecies interactions exist. They are often mediated by metaboliteproducts secreted in the environment, including exopolysaccharides (EPS),proteins, etc. Autoinducers (AI), used for cell-to-cell communication, areinvolved also. All these metabolites are components of biofilm matrix whichis a defining feature of microbial biofilms and contributes to their resistance.This study aims at the examination of the effects of sterile late stationaryphasesupernatants, containing secretory metabolite products byEnterobacteriaceae species (SMP-E), on growth and biofilm developmentof E. coli.Methods: Eight strains E. coli K-12 were tested. Sterile supernatants wereisolated from E. coli K-12, E. coli O157, and Yersinia enterocolitica. 96-well microtiter plate assay was applied for estimation of the effect of SMP-Eon bacterial growth and biofilm development. The presence of AI-2 in thesupernatants was detected using Vibrio harveyi bioluminescence assay. Theproduction of the EPS colanic acid (CA) and poly N-acetyl-D-glucosamine(PNAG) was registered by Enzyme-linked lectinosorbent assay.Comparision of the effect of the supernatants and the crude EPS, extractedspektrum | Tagungsband 2011