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

134heterotrimeric, Rrp4- and Csl4-containing capsin vivo. Furthermore, weobserved increased amounts of soluble, Rrp4-containing exosome in thestationary phase, when the vast majority of the DnaG-Csl4-exosomeremains non-soluble. Our data strongly suggest that temporal and spatialchanges in the localization of the exosome are based on changes in thecomposition of the RNA-binding cap and its interaction with DnaG.1. Evguenieva-Hackenberg, E., Walter, P., Hochleitner, E., Lottspeich, F., Klug, G. (2003) An exosome-likecomplex inSulfolobus solfataricus.EMBOreports4: 889-893.2. Evguenieva-Hackenberg, E. and Klug, G. (2009) RNA degradation in Archaea and Gram-negativebacteria different fromEscherichia coli.Progress in Molecular Biology and Translational Science85: 275-317.3. Roppelt, V., Klug, G., Evguenieva-Hackenberg, E. (2010) The evolutionarily conserved subunits Rrp4and Csl4 confer different substrate specificities to the archaeal exosome.FEBS Lett.584: 2931-2936.4. Walter, P., Klein, F., Lorentzen, E., Ilchmann, A,. Klug, G., Evguenieva-Hackenberg, E. (2006).Characterisation of native and reconstituted exosome complexes from the hyperthermophilicarchaeonSulfolobus solfataricus.Mol. Microbiol.62: 1076-1089.5. Roppelt, V., Hobel, C., Albers, S. V., Lassek, C., Schwarz, H., Klug, G., Evguenieva-Hackenberg, E.(2010) The archaeal exosome localizes to the membrane.FEBS Lett.584:2791-2795.OTV016Freshwater Actinobacteria acI as revealed by single-cellgenomicsS.L. Garcia* 1 , A. Srivastava 2 , H.-P. Grossart 2 , T. McMahon 3 , R. Stepanauskas 4 ,A. Sczyrba 5,6 , T. Woyke 5 , S. Barchmann 1 , F. Warnecke 11 Friedrich Schiller University, Jena School for Microbial Communication,Jena, Germany2 Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Department forLimnology Of Stratified Lakes, Neuglobsow, Germany3 University of Wisconsin – Madison, Department of Civil & EnvironmentalEngineering, Madison, United States4 Bigelow Laboratory for Ocean Sciences, Single Cell Genomics Center, WestBoothbay Harbor, United States5 DOE Joint Genome Institute, Microbial Program, Creek, United States6 University of Bielefeld, Department for Computational Metagenomics,Bielefeld, United StatesActinobacteria of the acI clade are often numerically dominatingfreshwater ecosystems where they can contribute >50% of the bacteria inthe surface water. However and as often with environmentally importantspecies they are uncultured to date. That is why we set out to study theirgenomic information in order to learn about their physiology andecological niche. We used a single cell genomics approach whichconsisted of the following steps: (1) single cell sorting by Fluorescenceactivatedcell sorting (FACS), (2) whole genome amplification (WGA)using Phi29 DNA polymerase, (3) screening of SAG (Single cell amplifiedgenome) DNA by 16S rRNA sequencing, (4) shotgun genomic sequencingfollowed by (5) genome assembly, annotation and data analysis using TheJoint Genome Institute’s (JGI) Integrated Microbial Genomes (IMG)analysis platform. We obtained a draft genomic sequence in 75 largercontigs (sum = 1.16 Mbp) and with an unusual low genomic G+C mol%(i.e. ~42%). Single copy gene analysis suggests an almost completegenome recovery. We also noticed a rather low percentage of genes withno predicted functions (i.e. ~15%) as compared to other cultured andgenome-sequenced microbial species. Our metabolic reconstruction hintsat the degradation of pentoses (e.g. xylose) instead of hexoses. We alsofound an actinorhodopsin gene that may contribute to energy conservationunder unfavorable conditions. This project reveals the possibilities andlimitations of single cell genomics for microbial species that defycultivation to date.OTV017Carbon and hydrogen isotope fractionation during nitritedependentanaerobic methane oxidation by MethylomirabilisoxyferaO. Rasigraf* 1 , C. Vogt 2 , H.-H. Richnow 2 , M.S.M. Jetten 1 , K.F. Ettwig 11 Radboud Universiteit Nijmegen, Microbiology, Nijmegen, Netherlands2 Helmholtz Centre for Environmental Research – UFZ, IsotopeBiogeochemistry, Leipzig, GermanyAnaerobic oxidation of methane coupled to nitrite reduction is a recentlydiscovered methane sink of as yet unknown global significance. Thebacteria that have been identified to carry out this process, CandidatusMethylomirabilis oxyfera, oxidize methane via the known aerobic pathwayinvolving the monooxygenase reaction [1]. In contrast to aerobicmethanotrophs, oxygen is produced intracellularly and used for theactivation of methane by a phylogenetically distinct particulate methanemonooxygenase (pMMO) [1]. Here we report the fractionation factors forcarbon and hydrogen during methane degradation by an enrichment cultureof M. oxyfera bacteria. In two separate batch incubation experiments withdifferent absolute biomass and methane contents, the specificmethanotrophic activity was similar and the progressive isotopeenrichment identical. The enrichment factors determined by Rayleighapproach were in the upper range of values reported so far for aerobicmethanotrophs. In addition, two-dimensional specific isotope analysis ( =( H -1 -1)/( C -1 -1)) was performed and also the determined value waswithin the range determined for other aerobic and anaerobicmethanotrophs. The results showed that in contrast to abiotic processesbiological methane oxidation exhibits a narrow range of fractionationfactors for carbon and hydrogen irrespective of the underlying biochemicalmechanisms. In contrast to aerobic proteobacterial methanotrophs, M.oxyfera does not assimilate its cell carbon from methane. Instead, only theCalvin-Benson-Bassham cycle of autotrophic carbon dioxide fixation wasshown to be complete in the genome, as well as transcribed and expressed[2]. Further experiments are conducted in order to experimentally validatethe proposed incorporation of carbon dioxide into cell biomass.[1] Ettwig et al. (2010) Nitrite-driven anaerobic methane oxidation by oxygenic bacteria. Nature 464, 543-548.[2] Wu et al. (2011) A new intra-aerobic metabolism in the nitrite-dependent anaerobic methane-oxidizingbacterium Candidatus 'Methylomirabilis oxyfera'. Biochemical Society Transactions 39, 243-248.OTV018Characterization of Novel Bacterial Alcohol DehydrogenasesCapable of Oxydizing 1,3-propanediolS. Elleuche*, B. Klippel, G. AntranikianTechnische Universität Hamburg-Harburg, Technische Mikrobiologie,Hamburg, Germany1,3-propanediole (1,3-PD) is a valuable compound for textile fiber, filmand plastic industry. It is chemically produced from acrolein or ethyleneoxide via 3-hydroxypropionaldehyde (3-HPA). Since the chemicalproduction of 1,3-PD is expensive and goes along with the formation oftoxic side products, much effort has been taken to establish amicrobiological production system. Facultative anaerobic microorganismshave been investigated with regard to their capability to produce 1,3-PDfrom glycerol. In a 2-step reaction, glycerol is converted to 3-HPA and thelatter is finally reduced to 1,3-PD by a 1,3-propanediol oxidoreductase(PDOR). The second reaction has been shown to be catalyzed by nonspecificalcohol dehydrogenases (ADH) as well. Since only a few PDORhave been investigated in detail, an approach to identify and characterizeADH with novel properties for the production of 1,3-PD has beenestablished. BLAST searches were performed using the sequences ofPDOR and related ADH with known activity towards 3-HPA or 1,3-PDfrom species of the genera Citrobacter, Clostridium, Klebsiella, andEscherichia coli. Putative homologues were identified in the genome ofthe bacterial species Oenococcus oeni, Dickeya zeae, Pectobacteriumatrosepticum, Pelobacter carbinolicus and from sequenced metagenomesderived from uncultivated bacteria living in deep sea-sediments. A total of10 different open reading frames were cloned into pQE30 expressionvectors and were purified after heterologous production in E. coli. Resultson the evolutionary relationships and biochemical properties of theenzymes will be presented.OTV019Bacterial CYP153 monooxygenases as biocatalysts for thesynthesis of -hydroxy fatty acidsS. Honda*, D. Scheps, L. Kühnel, B. Nestl, B. HauerUniversität Stuttgart, Institut für Technische Biochemie, Stuttgart,Germany-Hydroxy fatty acids (-OHFAs) and ,-dicarboxylic acids (,-DCAs) are multifunctional compounds useful for the production ofpolymers, lubricants, cosmetics and pharmaceuticals. Recently, mediumtolong-chain saturated -OHFAs have attracted considerable attention fortheir use as precursors of poly(-hydroxy fatty acids) [1]. These polymersexhibit similar or even superior physicochemical properties compared topolyethylene and other bioplastics. Long-chain cis-monounsaturated -OHFAs and ,-DCAs are also valuable because they yield polymers thatcan be cross-linked or chemically modified at their double bond sites [2].Cytochrome P450 monooxygenases (CYPs) are enzymes that usemolecular oxygen to insert one oxygen atom into non-activatedhydrocarbons. During the last two decades several eukaryotic CYPs havebeen isolated and engineered for the yeast-based production of -OHFAsand ,-DCAs [3]. Bacterial CYP153A enzymes are soluble alkane -hydroxylases [4] whose activity towards fatty acids has not been reportedyet. As certain CYP153A convert primary alcohols to ,-diols [5,6], wepresumed they -hydroxylated fatty acids as well.We functionally expressed CYP153A from Polaromonas sp.,Mycobacterium marinum and Marinobacter aquaeolei in E. coli toinvestigate their in vitro fatty acid oxidation profiles. Here we demonstratefor the first time that CYP153A enzymes oxidize fatty acids to -OHFAsand, sometimes, further to ,-DCAs. CYP153A from M. aquaeolei wasidentified as a fatty acid -hydroxylase with a broad substrate range. Thisbiocatalyst produced -OHFAs from medium-chain saturated and longchaincis/trans-monounsaturated fatty acids with 64 - 93% conversion and>95% -regioselectivity. Our study gives further insight into thephysiology of -oxidizing bacteria and provides the basis for thedevelopment of a recombinant E. coli system to synthesize -OHFAs fromrenewable feedstocks.We acknowledge financial support from the German Federal Ministry ofEducation and Research (BMBF) in the frame of the “Systems Biology inPseudomonas for Industrial Biocatalysis” project as well as the EuropeanBIOspektrum | Tagungsband 2012