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

OTP037Identification of an acidic lipase activity fromPhialemonium curvatum and comparison with two otherfungal lipases in a newly developed two-layer assayS. Barig* 1 , R. Alisch 1,2 , J. Schubert 1 , S. Nieland 1 , A. Wuttke 1,3 , K.-P. Stahmann 11 Department of Biology, Chemistry and Process Technology, University ofApplied Sciences Lausitz, Senftenberg, Germany2 Department of Infectious Diseases, University Hospital, Heidelberg,Germany3 Department of Medical Cell Biology, Uppsala University, Uppsala,GermanyLipases catalyse the hydrolysis of long-chain triglycerides at interfacesbetween oil and water. Additionally they have interesting properties makingthem useable in different fields of industrial production. Most lipases used inindustry are produced by microorganisms. Still the screening and productionof lipases with specific properties such as activity at acidic or alkaline pH aswell as a wide temperature range is of high interest. Acidic lipases areinvolved for example in food and flavour industries [1] or as a substitute forgastric lipase in enzyme therapy [2].A fast and reliable test to analyse the pH range of newly identified ormutated lipases is valuable in lipid research. A two-layer lipase activityassay was established in microtiter plates for rapid activity test as well as onPetri dishes to compare the activity at a specific pH. Two layers wereestablished, bottom was 2 % agar melted in appropriate buffer system, forpH3-5 0.05 M acetic acid-sodium acetate; pH6-7 0.05 M phosphoric acidsodiumphosphate; pH8-9 0.05 M Tris-HCl, top layer contained additionallyto the components of the bottom layer 1 % tributyrin as substrate. Two wellcharacterizedlipases were applied to the system, Rhizomucor miehei lipasestable between pH7-10 with an optimum at pH8 [3] and Thermomyceslanuginosus lipase acting between pH5-9, optimum at pH7 [4], respectively.Cell-free lipase as well as homogenized mycelium of Phialemoniumcurvatum, reported to grow under acidic conditions in minimal medium withplant triglycerides as sole carbon source [5], were used to determine pHdependence. In microtiter plates R. miehei lipase, as well as T. lanuginosuslipase showed activity between pH4-9. Only lipase of P. curvatum showedactivity starting at pH3 to pH9. Comparing these results with the same twolayeractivity test in Petri dishes determination of the clearance zonediameter led to the exact pH optima. For the lipases of T. lanuginosus and R.miehei the literature values were confirmed. The newly characterised lipaseactivity of P. curvatum had its optimum at pH7.[1] Hassan, F. et al (2006): Industrial application of microbial lipases, Enzyme Microb Technol,39:235-251.[2] Aloulou, A. (2007): Purification and biochemical characterization of the LIP2 lipase fromYarrowia lipolytic, Biochimica et Biophysica Acta 1771:228-237.[3] Wu, X. et al (1996): Purification and Partial Characterization of Rhizomucor miehei Lipase forEster Synthesis, Applied Biochemistry and Biotechnology, 59:145-158.[4] Omar, I.C. et al (1986): Purifications and some Properties of a Thermostable Lipase fromHumicola lanuginosa No. 3, Agri. Biol. Chem. 51(1):37-45.[5] Stahmann, K.-P. et al (2008): Mikrobielles Verfahren zur Herstellung von Enzymen, Patent DE 102006 057 724 A1.OTP038Site-Specific Cross-Linking Between the A and T Units ofan ECF-Type Biotin TransporterO. Neubauer*, T. EitingerInstitute for Biology/Microbiology, Humboldt-University, Berlin, GermanyEnergy-coupling factor (ECF) transporters are a class of micronutrientimporters in prokaryotes composed of a substrate-specific transmembraneprotein (S unit) and an energy-coupling module. The latter consists of aconserved transmembrane protein (T unit) and pairs of ABC-ATPases (Aunits) [1, 2]. Although highly diverse on the sequence level, recentelucidation of the 3D structures of the riboflavin-specific S unit RibU [3]and the thiamine-specific equivalent ThiT [D.J. Slotboom, personalcommunication] uncovered a conserved fold with six transmembranedomains (TMD). Oligomeric-state analyses of BioY, a biotin-specific S unit,in living bacteria suggested that S units are organized as oligomers [4].Choosing the biotin transporter BioMNY (BioM=A; BioN=T) we haveinvestigated the role of T units. BioN forms stable BioMNY holotransportercomplexes, and is contained - in vitro and in vivo - in stable BioMNcomplexes in the absence of BioY [4]. Two well-conserved three amino-acidmotifs with Ala-Arg-Gly as the consensus, found in a cytoplasmic helicalloop of T units, are essential for complex stability and intersubunit signaling[5]. In analogy to canonical ABC transporters, we hypothesized that theARG-containing stretches may function as coupling domains for interactionwith the A units. A cysteine-less BioMNY variant was constructed and usedto generate sets of double Cys variants with individual Cys residues in theARS-ARG region of BioN and the Q loop of BioM. Cu-phenanthrolineinducedCys cross-linking in isolated membranes confirmed the predictedBioN-BioM interaction. Specifically, our present data show that (i) bothARG signatures (163-ARS-165; 194-ARG-196) interact with the Q loop(s),(ii) they interact in particular with the N-terminus of the Q loop and (iii) Cysresidues adjacent to ARS-ARG do not crosslink with Q loop.[1] Eitinger, T. et al (2010): FEMS Microbiol. Rev. 35:3-67.[2] Rodionov, D.A. et al (2009): J. Bacteriol. 191:42-51.[3] Zhang, P. et al (2010): Nature 468:717-20.[4] Finkenwirth, F. et al (2010): Biochem. J. 431:373-380.[5] Neubauer, O. et al (2009): J. Bacteriol. 191:6482-6488.OTP039Identification and structure resolution (2.3 Å) of a novelmetagenome-derived short chain oxidoreductase (SDR)involved in quorum quenching phenotypes in P.aeruginosaP. Bijtenhoorn* 1 , H. Meyerhofer 2 , J. Müller-Dieckmann 2 , C. Utpatel 1 ,C. Hornung 1 , M. Szesny 3 , S. Grond 3 , W.R. Streit 11 Microbiology and Biotechnology,University of Hamburg, Hamburg,Germany2 EMBL Hamburg Outstation, Hamburg, Germany3 Institute for Organic Chemistry, Eberhard-Karls-University, Tübingen,GermanyHere we report on the identification and structural characterization of anovel short chain oxidoreductase from a soil metagenome. Thecorresponding gene bpiB09 was identified through a screening formetagenome clones interfering with bacterial quorum sensing. The bpiB09gene encoded for a 239 aa protein which was weakly similar (Identity 58 %,blastx E-value 6e-60) to a predicted short-chain dehydrogenase fromAcidobacteria. Heterologous expression as a 10x his-fusion protein in E. coliresulted in the production of a 30 kDa protein. Additional crystallographicstudies established BpiB09 as an NADPH-dependent reductase. Structuraland phylogenetic analyses revealed that it belongs to the classical SDRfamily of proteins. There it falls within the subgroup cP3. Interestingly,expression of bpiB09 in P. aeruginosa PAO1 resulted in significantlyreduced pyocyanin production, decreased motility and poor biofilmformation. Furthermore HPLC-MS analyses suggested that autoinducersynthesis of N-3-oxo-acyl-L-homoserine lactone was strongly affected incells expressing the bpiB09 gene suggesting a possible role of the proteinduring the early steps of autoinducer biosynthesis.OTP040Novel lactonases from Rhizobium sp. NGR234M. Rodriguez Orbegoso* 1 , D. Krysciak 1 , S. Preuss 1 , M. Quitschau 2 ,S. Grond 2 , W. Streit 11 University of Hamburg, Microbiology and Biotechnology, Hamburg,Germany2 Institute of Organic Chemistry, University of Tübingen, Tübingen,GermanyQuorum sensing (QS) is an important area of application, when it comes tofighting microbial infection. Quenching of QS-signal molecules, such as theautoinducer I- family of N-acyl-L-homoserine lactones, is a useful strategyto inhibit QS-mediated processes e.g. biofilm formation.Here we report on the analysis of the genes and enzymes involved inautoinducer I hydrolysis in Rhizobium sp. NGR234. Using a previouslypublished function-based screening with the biosensor strain Agrobacteriumtumefaciens NTL4, which carries a traI-lacZ gene fusion for the detection ofautoinducer I hydrolase genes, we identified a total of five cosmid clonesthat repeatedly gave positive results in our assay. Two of these loci werelocated on the megaplasmid pNGR234b and three were encoded by thechromosome cNGR234. Subcloning and transposon mutagenesis incombination with blast analyses identified the corresponding ORFs,designated dlhR, qsdR1, qsdR2, aldR and hitR. Employing recombinant andpurified DlhR and QsdR1 protein, we showed that both enzymes inhibitedbiofilm formation and other QS-dependent processes in Pseudomonasaeruginosa PAO1, Chromobacterium violaceum ChV26 and Agrobacteriumtumefaciens NTL4. Using high performance liquid chromatography-massspectrometry (HPLC-MS) analysis, we demonstrate the cleavingspektrum | Tagungsband 2011