VAAM-Jahrestagung 2012 18.–21. März in Tübingen

151bodies. Hence, we started to develop a novel bacterial expression systemfor the synthesis of membrane proteins that is based on the phototsyntheticbacterium Rhodobacter capsulatus. Due to its unique physiologicalproperties the photosynthetic bacterium R. capsulatus is particularly suitedfor the high-level expression of membrane bound enzymes in an activeform: Phototrophic growth conditions induce an intracellulardifferentiation of the inner membrane, leading to the formation ofmembrane vesicles in R. capsulatus. The membrane vesicles in turnprovide an intrinsically high protein folding and incorporation capacity.In order to evaluate the optimal growth conditions for heterologousmembrane protein expression we started to express two differentmembrane proteins, the bacteriorhodopsin from Halobacterium salinariumas well as the squalene epoxidase from Stigmatella aurantiaca, underphototrophic, non-phototrophic as well as shifted conditions. Furthermore,integration of the heterologous membrane proteins into the photosyntheticmembrane vesicles was confirmed by protein localization studies. Thenovel R. capsulatus expression system will now be used to identify novelmembrane bound monooxgenases from metagenomic libraries.OTP061Dehalococcoides sp. strain CBDB1 reductively dehalogenatesbromobenzenes to benzene in a respiratory processM. Cooper* 1 , A. Wagner 2 , S. Ferdi 2 , J. Seifert 1 , L. Adrian 11 Helmholtz Centre for Environmental Research, Isotope Biogeochemistry,Leipzig, Germany2 Technische Universität Berlin, Angewandte Biochemie, Berlin, GermanyBrominated aromatics have broad applications in industry as flameretardants and fumigants or as intermediates during the synthesis of dyes,agrochemicals, pharmaceuticals and herbicides. By now, many brominatedcompounds are widespread contaminants in the environment and areregarded as potentially harmful to humans and the environment. However,brominated aromatics are also released naturally, particularly in marineecosystems by algae, polychaets, sponges and molluscs. The completeremoval of all halogen substituents is a crucial step in the degradationprocess and for further mineralization of halogenated compounds. Abacterial group which is known for its ability to reductively dehalogenate abroad range of toxic chlorinated compounds such as chloroethenes,chlorobenzenes, chlorobiphenyls and dioxins is the genus of theDehalococcoides.In this study we investigated whether the pure Dehalococcoides sp. strainCBDB1 is able to dehalogenate brominated benzenes, which were chosenas ‘model’ molecules for other more complex brominated compounds fromnatural or anthropogenic sources. Cultivation of strain CBDB1 with 1,2,4-tribromobenzene, three different dibromobenzene congeners ormonobromobenzene revealed that all tested bromobenzenes werereductively dehalogenated to benzene in a respiratory process. Growthyields of 1.8 x 10 14 to 2.8 x 10 14 cells per mol of bromide released wereobtained. Additionally a newly designed methylviologen based enzymeactivity test was established to assess enzyme activity towardsbromobenzenes. Furthermore mass spectrometric analyses of reductivedehalogenases were carried out to gain deeper insight into expressionpatterns of reductive dehalogenases after cultivation with differentbromobenzenes. Our findings indicate that the same enzymes are involvedduring bromobenzene reduction as during chlorobenzene reduction, andsuggest that Dehalococcoides sp. strain CBDB1 can be used forremediation of brominated aromatic contaminants.OTP062BlueTox: A novel genetically encoded photosensitizerS. Endres* 1 , J. Walter 2 , J. Potzkei 1 , M. Wingen 1 , A. Heck 1 , K.-E. Jaeger 3 ,T. Drepper 11 Heinrich-Heine-University, Institute of Molecular Enzyme Technology, WGDrepper, Düsseldorf, Germany2 Heinrich-Heine-University, Department of Neurology, Düsseldorf, Germany3 Heinrich-Heine-University, Institute of Molecular Enzyme Technology,Düsseldorf, GermanyFluorescent active dyes and proteins like the green fluorescent protein(GFP), isolated from the jellyfish Aequorea victoria and members of theGFP-like protein family generates reactive oxygen species (ROS) as abyproduct of its fluorescence activity (1) . Thereby, the amount of generatedROS is strongly dependent on the proteins structure (2,3) . One example for ahigh-level ROS-producing fluorescent protein is KillerRed, a derivate ofthe non-fluorescent chromoprotein anm2CP isolated from Anthemedusaesp. (4) . This photosensitizer enables the light-mediated directed inactivationof targeted cell-structures and/or whole cells by application of thechromophore-assisted-light-inactivation (CALI-) technique(5) . As analternative to this red fluorescent photosensitizer we developed, on basis ofa FMN-based-fluorescent-protein (FbFP) (6) , the novel photosensitizerBlueTox. BlueTox harbors a LOV-domain (light, oxygen, voltage) thatbinds flavinmononucleotide (FMN) as fluorophore and shows thecharacteristic excitation and emission maxima at 450nm ex /495nm em , respectively.We demonstrated the blue-light induced, ROS-mediated photosensitizingeffect of BlueTox by heterologous expression of the photosensitizer inEscherichia coli and subsequent time-resolved irradiation studies. Theresults of our in vivo analyses revealed a significant correlation betweendecrease of the amount of living cells and irradiation time. Therefore,BlueTox is a powerful tool for light-mediated inactivation of bacteria withhigh spatio-temporal resolution.1Jiménez-Banzo, A., S. Nonell, et al. (2008). "Singlet Oxygen Photosensitization by EGFP and itsChromophore HBDI." Biophysical journal 94 (1): 168-172.2Pletnev, S., N. G. Gurskaya, et al. (2009). "Structural basis for phototoxicity of the genetically encodedphotosensitizer KillerRed." The Journal of biological chemistry 284 (46): 32028-32039.3Carpentier, P., S. Violot, et al. (2009). "Structural basis for the phototoxicity of the fluorescent proteinKillerRed. "FEBS letters 583 (17): 2839-2842.4Bulina, M. E., D. M. Chudakov, et al. (2006). "A genetically encoded photosensitizer." Naturebiotechnology 24 (1): 95-99.5Bulina, M. E., K. A. Lukyanov, et al. (2006). "Chromophore-assisted light inactivation (CALI) using thephototoxic fluorescent protein KillerRed. "Nature protocols 1 (2): 947-953.6Drepper, T., T. Eggert, et al. (2007). "Reporter proteins for in vivo fluorescence without oxygen." Naturebiotechnology 25 (4): 443-445.OTP063Production of the liposomase in Clostridium sporogenes for thetherapeutic use in tumor therapyK. Riegel* 1 , D. Meisohle 2 , P. Dürre 11 Universität Ulm, Institut für Mikrobiologie und Biotechnologie, Ulm, Germany2 Universität Ulm, Institut für Medizinische Mikrobiologie and Hygiene, Ulm,GermanySolid tumors and their environment possess certain features that are uniquein the human body. The most striking one is oxygen deprivation. Theseregions offer obligate anaerobic bacteria, such as clostridia, optimalconditions for growth. However, the colonization of the tumors alone isnot sufficient for a complete tumor regression (Ryan et al., 2006). Bygenetic modifications, these bacteria can function as vectors deliveringtherapeutic proteins or prodrug-converting enzymes to their targetsresulting in a direct effect on the remaining tumor tissue.In this project, the liposomase is used for this purpose. The liposomase is aprotein originally isolated from Clostridium novyi that can destroyliposomes (Cheong et al., 2006). Liposomes are membranous vesicleswhich can function as carrier for anticancer drugs such as doxorubicin, asthese vesicles specifically accumulate in tumor tissues. However, the drugrelease from the liposomes is very slow due to their chemical and physicalstability (Gabizon et al., 2006). Therefore, a genetically engineered strainof Clostridium sporogenes producing this enzyme should greatly enhancedrug delivery from liposomes. C. sporogenes is a proteolytic and sporeformingorganism that proved to be an excellent colonizer of hypoxictumor tissue (Brown and Liu, 2004). For the expression of the liposomasegene in this organism a protein expression system based on the T7 systemwas constructed. The resulting expression mutant of C. sporogenes shouldproduce and secrete the liposomase in the surrounding medium in asufficient concentration providing a more effective strategy in the fightagainst cancer.Brown, J.M., & Liu, S.C., 2004. Use of anaerobic bacteria for cancer therapy. In: Nakano, M.M., & Zuber P.Strict and facultative anaerobes - medical and environmental aspects. Horizon Bioscience, Wymondham,England, 211-220.Cheong I., Huang X., Bettegowda C., Diaz L.A. Jr., Kinzler K.W., Zhou S. and Vogelstein B., 2006. Abacterial protein enhances the release and efficacy of liposomal cancer drugs. Science, 314, 1308-1311.Gabizon A.A., Shmeeda H. and Zakipsky S., 2006. Pros and Cons of the liposome platform in cancer drugtargeting. Journal of Liposome Research, 16, 175-183.Ryan, R.M., Green, J., & Lewis, C.E., 2006. Use of bacteria in anti-cancer therapies. BioEssays, 28, 84-94.OTP064ClubSub-P: cluster-based subcellular localization predictionfor Gram-negative bacteria and archaeaN. Paramasivam*, D. LinkeMPI Developmental Biology, Protein Evolution, Tuebigen, GermanyThe subcellular localization (SCL) of proteins provides important clues totheir function in a cell. In our efforts to predict useful vaccine targetsagainst Gram-negative bacteria, we noticed that misannotated start codonsfrequently lead to wrongly assigned SCLs. This and other problems inSCL prediction, such as the relatively high false-positive and falsenegativerates of some tools, can be avoided by applying multipleprediction tools to groups of homologous proteins.Here we present ClubSub-P, an online database that combines existingSCL prediction tools into a consensus pipeline from more than 600proteomes of fully sequenced microorganisms. On top of the consensusprediction at the level of single sequences, the tool uses clusters ofhomologous proteins from Gram-negative bacteria and from Archaea toeliminate false-positive and false-negative predictions. ClubSub-P canassign the SCL of proteins from Gram-negative bacteria and Archaea withhigh precision. The database is searchable, and can easily be expandedusing either new bacterial genomes or new prediction tools as they becomeavailable. This will further improve the performance of the SCL prediction, aswell as the detection of misannotated start codons and other annotation errors.ClubSub-P is available online athttp://toolkit.tuebingen.mpg.de/clubsubp/Paramasivam N and Linke D (2011) ClubSub-P: cluster-based subcellular localization predictionfor Gram-negative bacteria and archaea. Front. Microbio. 2:218. doi: 10.3389/fmicb.2011.00218BIOspektrum | Tagungsband 2012