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

NTP019Identification and metabolic activity of single cellssimultaneously measured with NanoSIMSH.-U. Ehrke* 1 , F. Horréard 2 , F. Hillion 21 CAMECA GmbH, Unterschleissheim, Munich, Germany2 CAMECA, Gennevilliers Cedex, FranceHISH-SIMS (Halogen In Situ Hybridization) allows quantitativemeasurements of microbes without cultivation and pre-selection [1].Similar to the well known FISH (Fluorescent In Situ Hybridization) methodthe microbes of interest are selected with a gene-tag. Instead of a fluorescentdye a halogen-marker (e.g. fluorine) is used. In an isotopic enrichmentexperiment the nanoSIMS is then recording simultaneously element imagesof F and other isotopes of interest (12C, 13C, 14N, 15N ...) thus allowingidentifying the microbes and measuring their metabolic activity with highprecision and a lateral resolution down to 50nm.This contribution shows application examples of the Exchanges of nitrogenand carbon in a dual-species microbial consortium, Ecophysiology ofanaerobic phototrophic bacteria and others.Secondary Ion Mass Spectrometry (SIMS) technique provides direct in situmeasurement of elemental and isotopic composition in selected μm-sizeareas of the sample. Similar to a scanning electron microscope (SEM), aprimary beam of reactive ions is rastered on the surface of the sample. Thematerial sputtered by the primary beam is collected and mass filtered by amagnetic sector mass analyzer. Up to seven mass selected images ofdifferent elements or isotopes can be simultaneously recorded, originatingfrom the exact same sputtered volume, ensuring reliable isotopic ratio andperfect image registration [2].[1] Musat, N. et al (2008): A single-cell view on the ecophysiology of anaerobic phototrophicbacteria, PNAS November 18, 2008 vol. 105 no. 46 17861-17866.[2] Slodzian, G. et al (1987): High Sensitivity and High Spatial Resolution Ion Probe Instrument,Proceedings of the 6th SIMS Conference, Versailles Sept.OTV001The C-terminal domain DUF1521 of the Bradyrhizobiumjaponicum protein and its functional stabilityJ. Schirrmeister*, S. Zehner, M. Wenzel, L. Friedrich, M. Hoppe,M. GöttfertInstitute of Genetics, University of Technology, Dresden, GermanyBradyrhizobium japonicum is known as soybean symbiont. The effectorprotein NopE1 is secreted via its type III-secretion system and exhibitsautocleavage activity in the presence of calcium. NopE1 consists of 484amino acids and contains two domains of unknown function (DUF1521),each comprising about 170 amino acids [1]. To characterise the minimalprotein domain with autocleavage activity, deletion derivatives were created.This revealed that the minimal functional domain covers the 170 aminoacids of the DUF1521. To test if this domain can be used as a self-cleavinglinker, it was fused with a Strep tag at its C-terminal end and GST at the N-terminal end. The recombinant protein still exhibited self-cleavage afteraddition of calcium. Then, cleavage was analysed under different conditions.Cleavage took place at room temperature, on ice and partially at 60 ºC. Afterincubation for 20 min at 75 ºC, followed by incubation at room temperature,the protein still showed partial cleavage. Cleavage was also observed at a pHrange from 4.5 to 9. These properties suggest, that NopE1 is useful for thedevelopment of a self-cleaving linker for biotechnology purposes. Furthertests with alternative fusion partners are ongoing.[1] Wenzel et al (2010): The type III-secreted protein NopE1 affects symbiosis and exhibits acalcium-dependent autocleavage activity. Mol. Plant-Microbe Interact., 23, 124-129.OTV002Culture Collections' Provision of Continuity forAcademic and Industrial Research - Meeting theEmerging ChallengesD. FritzeGerman Collection of Microorganisms and Cell Cultures (DSMZ),Braunschweig, Germanyc/o JKI, GBRCN, Braunschweig, GermanyMicroorganisms provide essential raw material for biotechnology - but todate less than 0.1% of the estimated number of species are described andavailable from culture collections to be harnessed by man. Microbial culturecollections have to reconcile the interests of providers of living biologicalmaterial (scientists, institutions, countries of origin) and the various kinds ofrecipients/users of cultures of microorganisms from academia and industry.Providing access to high-quality material, related data and scientific serviceswhile, at the same time, observing donor countries' rights (CBD-ABS),intellectual property rights, and biosafety/biosecurity aspects, posesdemanding challenges. A similarly challenging task is to keep abreast ofdevelopments in taxonomy and systematics, as well as new methods for theauthentication and identification, cultivation and maintenance of cultures.Recognising that these challenges are best met by collaborative work,collections organised themselves, e.g., in the European Culture CollectionOrganisation (ECCO). Before this background of understanding, a numberof successful scientific-technical projects emerged (e.g. MINE and CABRI:agreed procedures on quality issues related to biological material and data;EBRCN: information documents to help implementing regulations; MTA:harmonising modes of supply of cultures; EMbaRC: training and research inmicrobial collection matters). In the Global BRC Network initiative, thepartners will - in an era of globalisation - work toward common policies andprinciples with a view to existing and emerging legal frameworks and inhouseprocedures when handling living biological material. The recentlylaunched ESFRI initiative MIRRI (Microbial Resources ResearchInfrastructure) will complement this global effort on the European level.Both latter initiatives take up common interests between culture collectionsand researchers to bring issues forward for discussion and initiate betterinteraction of the culture collections' and the scientific and biotechnologycommunities.OTV003Authenticity of Microbiological Material - The Impact inthe Research Environment.B.J. TindallGerman Collection of Microorganisms and Cell Cultures (DSMZ),Braunschweig, GermanyThe dictionary definition of science indicates that it relates to knowledge. Inbiology that knowledge is directly linked to testable explanations andpredictions about the organisms we study. While much emphasis is nowbeing put on adequate record keeping and archiving of data (obtained duringthe study of biological entities) in order to combat elements such as forgeryor falsification of results, little attention is actually given to the fate of theobjects that we actually study. Both the literature and databases are full oferrors based on the incorrect/mistaken identity of the biological materialunder study. While in most cases the erroneous data has not been collectedwith malicious intent, the consequences are significant because they are nothighlighted by scandals and they are often only identified by experts, withthe broad masses often blissfully unaware of the problem. The consequencesmay well be incorrect interpretation of results that make reference to theincorrect data or significant financial effort being put into research either toverify or disprove the published data. Strangely little attention is given tovery basic, simple aspects in research with microorganisms relating to thelongterm storage and routine checking of the identity of strains used inresearch projects. In instances where data is published or deposited indatabases the fate of the strains under study beyond the duration of theproject or the career of the scientist involved is rarely considered by thosewho are providing funding. The problem is accentuated by the deceasingnumbers of experts to cover the myriad of aspects of a hugely diverse rangeof organisms coupled with an exponentially growing dataset.OTV004Capacity Building, Transnational Access andEncouragement for the Deposit of MicrobiologicalMaterial - the EU Project EMbaRCD. SmithCABI, Bioservices, Egham, United KingdomGBRCN Demonstration Project Secretariat, Braunschweig, United KingdomEuropean Consortium of Microbial Resources Centers (EMbaRC) is an EUproject funded under the Seventh Framework Programme ResearchInfrastructures (INFRA-2008-1.1.2.9: Biological Resources Centers(BRCs)) for microorganisms. It aims to improve, coordinate and validatemicrobial resource Center (MRC) delivery to European and Internationalresearchers from both public and private sectors. The EMbaRC project is amixture of networking, access, training and research. To ensurespektrum | Tagungsband 2011