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

166have shown direct evidences, for the first time, of the activity of anaerobicferro-oxidizers within the water column. We have demonstrated that thoseautotrophs thrived by photoferrotrophy (anoxygenic photosynthesis) or bychemo-ferrotrophy (nitrate-dependent respiration). More over, all themetabolisms thought to have existed in the Late Archean Ocean arepresent in Lake La Cruz. Therefore, the results from this study combinedwith those of previous studies allowed us to establish a biogeochemicalmodel that complement the ones describe above. Accordingly, the watercolumn of Lake La Cruz may represent an ecotone between the two mainNeoarchean Ocean compartments and, consequently, be a good modelsystem, or samples source, for studying metabolic activity interactions inexperimental conditions that reflect theoretical models of the ArcheanOcean.OTP132Elimination of indicator bacteria and viruses in open andcovered simulation channels of streaming waterH.-C. Selinka* 1 , H. Dizer 1 , A. Frohnert 1 , R. Schmidt 2 , R. Szewzyk 11 Umweltbundesamt, FG II 1.4, Berlin, Germany2 Umweltbundesamt, FG IV 2.5, Berlin, GermanyIndicator bacteria have been shown to have limited value as indicators forthe presence of viruses in water systems. For this reason, bacteriophages(coliphages) or human adenoviruses have been proposed as additionalindicators for human pathogenic viruses. To test their suitability, survivalof indicator bacteria and viruses after release from a waste water dischargeinto a river was studied in a river water simulation plant. The plantconsisted of elliptical channels each of 100 m length, 100 cm diameter and40 cm water depth, allowing online monitoring of selected physical andchemical parameters. Concentrations of E. coli and intestinal enterococci,as well as somatic coliphages, F+phages, human adenoviruses andnoroviruses were monitored after a peak contamination of channel waterwith 1% or 5% of primary waste water effluent. Special attention wasgiven to the effect of sunlight and its UV components on the survival ofthese bacteria and viruses by using an open channel and a channelprotected from sunlight at different times of the year. Radiation wasmonitored as mJ/cm 2 from meteorological data. The disinfection effect ofradiation was characterized through time and dose dependend kinetics ofelimination of test organisms as log unit per time ( t) or per radiation doseof sunlight ( d). As expected, the effect of solar radiation was dependenton the season with maximum effects during summer. The effect ofradiation differed with regard to the test bacteria and viruses. Exposure tosunlight had a more significant effect on the indicator bacteria than on theviruses. Most prolonged survival was found for somatic coliphages as wellas for human adeno- and noroviruses. These results support previousfindings that indicator bacteria are no good indicators for viruses andsuggest coliphages as suitable viral indicators, especially under adverseenvironmental conditions like high irradiation intensity.OTP133Microvirin - a novel cyanobacterial lectin with broad andpotent anti-HIV activityJ.-C. Kehr* 1 , D. Huskens 2 , D. Schols 2 , E. Dittmann 11 Universität Potsdam, Mikrobiologie, Golm, Germany2 Katholieke Universiteit Leuven, Rega Institute for Medical Research, Leuven,BelgiumCarbohydrate binding agents such as lectins have proven to be valuablesource of anti-HIV therapeutics that may be applied as microbicides. It isknown that a variety of mannose-specific plant lectins that bindoligomannose glycans have strongin vitroanti- HIV-1 inhibitory activities,and therefore have been proposed as microbicide candidates for topicalprophylaxis of HIV-1 infection and as potential anti-HIV therapeutics.Here, we present the mannan-binding lectin microvirin (MVN) from thecyanobacterium Microcystis aeruginosa PCC 7806 that represents apromising new HIV microbicide candidate. The sugar specificity of theprotein was elucidated through carbohydrate microarrays, which revealedMVN to be selective for mannan-type oligosaccharides with terminal a(1-2)-mannose moieties. Compared to the related protein cyanovirin-n itexhibited comparable and broad anti-HIV-1 activity against all evaluatedHIV-1 virus strains and clinical isolates, but was much less (>500-fold)cytotoxic when evaluated in various human T cell lines and humanperipheral blood mononuclear cells (PBMC). In addition MVN was notmitogenic, did not induce cellular activation markers in PBMC and neverenhanced viral replication, as this was observed with cyanovirin inspecifically designed PBMC assays. The possible pathogenicconsequences associated with these side-effects have now raised the issueof safety of all other members of the antiviral class of lectins. We so farconclude that MVN has a superior safety profile in comparison with othermembers of the antiviral lectins that have been proposed as microbicidecandidates such as cyanovirin-n.Apart from its antiviral potential thein vivofunction of MVN wasextensively studied. Immunofluorescence microscopy (IFM) as well aslectin binding analysis (LBA) using FITC-labelled MVN were employedand confirmed that MVN is secreted from M. aeruginosa cells and binds toLPS on its cell surface. M. aeruginosa cells form large colonies and MVNis proposed to be involved in the cell-cell attachment. MVN orthologueswere identified in different cyanobacterial genera and are currently clonedand heterologously expressed in order to evaluate their antiviral activity.OTP134Engineering of Escherichia coli cells for the heterologousproduction of fucosylated human milk oligosaccharidesF. Baumgärtner*, L. Khan, G. Sprenger, C. AlbermannUniversity of Stuttgart, Institute of Microbiology, Stuttgart, GermanyAmong other biologically active substances, oligosaccharides represent afundamental component of human milk. They are known to showbeneficial effects for infants, such as inhibition of pathogenic infection bybinding pathogen receptors and growth promotion of bifidobacteria as keycommensals [1]. So far, investigation on the physiological function of milkoligosaccharides had only been accomplished by the use of singlecompounds or mixtures that were purified from breast milk.Comprehensive study or even clinical trials with single compoundsisolated from human milk were not possible, because major parts of theoligosaccharides in human milk are found only in small quantities.The work described here focuses on a novel method for the efficientsynthesis of oligosaccharides. The synthesis proceeds via a glycosylationreaction in recombinant Escherichia coli, which expresses suitableglycosyltransferases. The activated sugar precursors that are required forglycosyltransferase catalyzed reactions are generated by the metabolism ofthe organism. The principle possibility for a heterologous biosynthesis offucosylated oligosaccharides in E. coli was shown before [2, 3]. Here wepresent the construction of a plasmid-free strain for the heterologoussynthesis of 2’-fucosyllactose using the -Red recombineering technique[4]. After optimization of the heterologous gene expression, 2’-fucosyllactose was produced in a large scale fed-batch bioreactorcultivation using glycerol as carbon source and lactose as substrate.[1] Kunz et al. (2000) Oligosaccharides in Human Milk: Structural, Functional, and MetabolicAspects. Annual Review of Nutrition, 20:699-722[2] Albermann et al. (2001) Synthesis of the milk oligosaccharide 2’-fucosyllactose usingrecombinant bacterial enzymes. Carbohydrate Research, 334:97-103[3] Drouillard et al. (2006) Large-Scale Synthesis of H-Antigen Oligosaccharides by ExpressingHelicobacter pylori 1,2-Fucosyltransferase in Metabolically Engineered Escherichia coli Cells.Angewandte Chemie, 118:1810-1812[4] Albermann et al.(2010) A simple and reliable method to conduct and monitor expressioncassette integration into the Escherichia coli chromosome. Biotechnology Journal, 5:32-8OTP135Functional expression of the dirigent protein AtDIR6 in PichiapastorisC. Kazenwadel* 1 , J. Klebensberger 1 , A. Schaller 2 , B. Hauer 11 Universität Stuttgart, Institut für Technische Biochemie, Stuttgart, Germany2 Universität Hohenheim, Institut für Physiologie und Biotechnologie derPflanzen, Stuttgart, GermanyThe biosynthesis of lignans, a diverse class of secondary metabolites inplants, is initiated by an one-electron oxidation of monolignol substrates,followed by a phenoxy radical coupling reaction. In plants, this reactioncan occur in an enantioselective fashion.Interestingly, oxidases such aslaccases and peroxidases, which are essential to generate the initial radicalsfor the subsequent coupling reaction, do not exhibit any regio- orstereoselective control. The discovery of dirigent proteins fromForsythiaintermedia (FiDIR1) [1] andArabidopsis thaliana(AtDIR6) [2] mediatingthe stereoselective 8-8´coupling of coniferyl alcohol to either (+)- and (-)-pinoresinol, respectively, uncovered the nature of such an enantioselectivecontrol. The mode of mechanism is still elusive, however, it is suggestedthat dirigent proteins exist as homodimers lacking oxidative capacitythemselves. They rather capture and orientate free radicals generated fromoxidases in such a way that a specific coupling mode is favored, leading tothe formation of optically active compounds. In order to uncover theunderlying mechanism of this reaction, an effective protein expressionsystem based on a fermentation process would be highly beneficial.Therefore, we heterologously expressed the dirigent protein AtDIR6inEscherichia coli(E. coli) andPichia pastoris(P. pastoris). Whileexpression inE. colidid not yield a substantial amount of soluble protein indifferent strains and under various conditions, fed-batch fermentation ofP.pastoris resulted in 47 mg/L of glycosylated AtDIR6, which represents amore than 300 fold increase in yield compared to the expression with plantsuspension cultures. We found that the enantiomeric excess of (-)-pinoresinol in the phenoxy radical coupling of coniferyl alcohol using thepurified enzymein vitrowas comparable to the plant-derived enzyme.Further, we could demonstrate that the glycosylation ofP. pastoris-derivedAtDIR6 is essential for its dirigent activity. Taken together with the resultsobtained from CD-spectroscopy, our data strongly indicate that theglycosylation of AtDIR6 is critical for initial folding process as well as forthe conformational stability of the protein.BIOspektrum | Tagungsband 2012