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

139sediments in the Black Sea by Thamdrup et al. 2000. Thus, we identifiedacetate utilizing Arcobacter spp. as potential heterotrophic manganesereducers in pelagic Baltic Sea redoxclines.OTP005Artificial fusion of a two-component styrene monooxygenaseT. Heine*, C. Conrad, J.A.D. Gröning, S.R. Kaschabek, D. Tischler,M. SchlömannTU Bergakademie Freiberg, Environmental Microbiology, Freiberg, GermanyStyrene monooxygenases (SMOs) are external flavoproteinmonooxygenases performing enantioselective oxygenations of styrene andstructurally related compounds (Montersino et al., 2011). In most cases asingle NADH-dependent oxidoreductase (StyB) provides reduced FAD fora single styrene-epoxidizing monooxygenase (StyA). Recently, aremarkable reductase type StyA2B was found and biochemicallycharacterized, in which the oxygenase subunit is naturally fused to theFAD reductase (Tischler et al., 2009). This wild-type single-componentSMO was shown to be self-sufficient but of rather low epoxidationactivity. Cooperation with another single styrene oxygenase subunit StyA1is likely to be necessary to generate high specific epoxidation activity inhost strain R. opacus 1CP (Tischler et al., 2010). Despite a current lack ofknowledge on the biochemical reasons for the evolution of StyA2B, onecomponentSMOs may have several advantages over multicomponentsystems as shown for other monooxygenase classes.Herein, we describe the construction of self-sufficient chimeric styrenemonooxygenases by a molecular genetic approach as well as thepreliminary characterization of the recombinant proteins. The artificialfusion of the genes styA and styB from Pseudomonas fluorescens ST byelimination of the stop codon of styA and by introducing various linkersizes (Lx) provided four styALxB-gene variants. The artificially fused onecomponentSMOs were successfully expressed in E. coli BL21. Activitywas demonstrated from these clones by converting indole into indigo andlater on determined by means of homogeneous protein preparations.The artificial fusion of two-component SMOs was for the first timesuccesfully demonstrated and should provide access to valuablebiocatalysts in the field of fine chemical syntheses.Tischler, D., D. Eulberg, S. Lakner, S. R. Kaschabek, W. J. H. van Berkel, M. Schlömann (2009)Identification of a novel self-sufficient styrene monooxygenase from Rhodococcus opacus 1CP. J. Bacteriol.191:4996-5009.Tischler, D., R. Kermer, J. A. D. Gröning, S. R. Kaschabek, W. J. H. van Berkel, M. Schlömann (2010)StyA1 and StyA2B from Rhodococcus opacus 1CP: A multifunctional styrene monooxygenase system. J.Bacteriol. 192:5220-5227.Montersino, S., D. Tischler, G. T. Gassner, W. J. H. van Berkel (2011) Catalytic and structural features offlavoprotein hydroxylases and epoxidases. Adv. Synth. Catal. 353:2301-2319.OTP006Molecular basis of symbiosis investigated in ChlorochromatiumaggregatumP. Henke*, J. OvermannDSMZ, MÖD, Braunschweig, GermanyThe phototrophic consortium "Chlorochromatium aggregatum" is amulitcellular association between the green sulfur bacterial epibiontsChlorobium chlorochromatii and a central motile chemotrophicBetaproteobacterium. The flagellated central rod moves the entireconsortium towards the light enabling the epibiont to conduct anoxygenicphotosynthesis. The cells are connected through specific cell-cell adhesionstructures and division results in two intact daughter consortia. Theseobservations suggest the exchange of multiple signals between the epibiontand the central bacterium making this culturable association a suitablesystem for understanding the molecular basis of symbiosis betweennonrelated bacteria. The comparison of the Chl. chlorochromatii genomewith eleven available genomes of free-living relatives revealed uniqueopen reading frames. The major fraction of the ORFs code for hypotheticalproteins, but putative large exoproteins and a protein with a RTX toxintypeß-roll were identified. In particular Cag1919 which bears several RTXrepeats which are typically found in Gram-negative pathogenic bacteria isof interest. These putative symbiosis genes (Cag1919, Cag1920, Cag0614and Cag0616) are constitutively transcribed and have been analysedfurther. The whole gene of Cag1919 was cloned into a vector of the pQEseries and expressed heterologously in the E.coli strain XL1Blue. Theprotein can be used in Ca 2+ - binding experiments due to its predicted Ca 2+ -binding region. Cag0614 and Cag0616 represent the largest open readingframes in the prokaryotic world known to date with length of 110418 and61938 bp, respectively. Due to their large size only fragments can becloned and expressed. Interestingly, expression of Cag1919 and 1920 weredeleterious to E. coli strains causing the formation of extremely long,filamented or branched cells. To facilitate the localization of the proteinsin Chl. chlorochromatii, in the free-living and symbiotic state, the resultingrecombinant proteins are used to produce antibodies for immunogoldlabelling and tyramide signal amplification. With these results proteinsrelevant in bacterial symbiosis can be localized and the question how amotif known from pathogenic bacteria operates in symbiosis approached.OTP007Effect of phosphate on a community of iron oxidizing bacteriaJ. Kipry*, C. Wiacek, M. SchlömannTU Bergakademie Freiberg, Environmental Microbiology, Freiberg, GermanyAt the open pit Nochten (Lusatia, East Germany) mine waters arebiotechnologically treated in a pilot plant by microbial iron oxidation withthe subsequent precipitation of schwertmannite. These waters arecharacterized by a low pH value, high concentrations of iron and onlytraces of phosphate. The low phosphate availability appears to be a factorthat influences the microbial activity. To increase the capacity of the pilotplant, the effect of phosphate on the oxidation rate and the microbialcommunity was investigated.The microbial community of the treatment plant was cultivated with andwithout phosphate in a pilot plant-adapted laboratory set up. Besides thedetermination of chemical parameters like pH, iron and phosphateconcentration the microbial community was quantitatively characterizedby cell counting and qualitatively by T-RFLP analysis. The results showthat the iron oxidation rate, the bacterial community and cell numbersdiffered significantly based on the phosphate availability. The cell numberwas doubled in the system with phosphate. The microbial community inthe reactor without phosphate consisted of various iron oxidizing (60%)and non-iron oxidizing bacteria (40%), whereas exclusively the ironoxidizing bacteria ‘Ferrovum myxofaciens’ was present in the reactor withadded phosphate.Together with the higher cell number and higher percentage of ironoxidizer in presence of phosphate also the oxidation rate increasedcompared to the system without phosphate.OTP008L-Sorbitol-Dehydrogenase from Bradyrhizobium japonicumUSDA 110 can be applied in D-Sorbose Production usingElectrochemical Cofactor RegenerationS. Gauer 1 , Z. Wang 2 , M. Etienne 2 , A. Walcarius 2 , F. Giffhorn 1 , G.-W. Kohring* 11 Saarland University, Microbiology, Saarbrücken, Germany2 CNRS, Physical Chemistry and Microbiology for the Environment, Nancy,France, FranceIn the FP7 EU project ERUDESP an enzyme reactor with electrochemicalcofactor regeneration was developed for the production of enantiopurebuilding blocks used in pharmaceutical synthesis. Applying the suitableenzymes, the reactor can also be used for the production of rare sugars likeD-sorbose, which is an interesting synthon for pharmaceutical applicationsand can be used as a low calorie sweetener. An annotated ribitoldehydrogenasegene of Bradyrhizobium japonicum USDA 110 wasidentified in a BLAST search with the N-terminal amino acid sequence ofan earlier described L-sorbitol-dehydrogenase of Stenotrophomonasmaltophilia [1], which oxidizes L-sorbitol to D-sorbose. The gene wasamplified, tagged with histidines and heterologously expressed in E. coliBL21Gold(D3). The biochemical examination of this protein exhibitedcomparable L-sorbitol-dehydrogenase activity to the S.maltophiliaenzyme. The L-sorbitol-dehydrogenase from B. japonicum wasencapsulated together with diaphorase in sol-gel layers on gold electrodesas has been described earlier for a D-sorbitol-dehydrogenase fromRhodobacter sphaeroides [2]. With NAD + and the mediatorferrocenedimethanol in solution the oxidation of D-sorbitol could bedemonstrated by cyclic voltammetry. The results let the enzyme appear asa promising candidate for the production of the rare sugar D-sorbose inenzyme reactors with electrochemical cofactor regeneration.[1]Brechtel E., Huwig A., Giffhorn F., Appl Environ Microbiol.68, 582-587 (2002)[2]Wang, Z., Etienne, M., Kohring, G.W., Bon Saint Côme, Y., Kuhn, A., Walcarius, A.,Electrochimica Acta56, 9032-9040 (2011)OTP009Biofouling of ultrafiltration membranes for drinking watertreatment characterized by Confocal Laser Scanning MicroscopyB. Braun*, U. SzewzykTU Berlin, Environmental Microbiology, Berlin, GermanyBiofouling is known as a major reason for flux decline in the performanceof membrane based water and wastewater treatment plants. The relevanceof biofilm extracellular polymeric substances (EPS) in terms of fouling onmembranes has been indicated in several studies. Therefore, a profoundknowledge of the composition of biofouling is important for thedevelopment of new countermeasures in enhancing membranepermeability.The objective of this investigation was the characterization of microbialaggregates and EPS components in biofilms that contribute to biofoulingof ultrafiltration membranes using confocal laser microscopy (CLMS).Biofouling tests were conducted using an experimental setup, where ahollow-fiber ultrafiltration (UF) membrane module made of polyethylenewas fed with natural water. Dead end filtration was carried outcontinuously by using a constant pressure of 20mbar and an initialBIOspektrum | Tagungsband 2012