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

149transfer from the cluster to heme. Supplementation of HemW with an EPRactive Fe-Corrol revealed a 5x- and to a lesser extent 6x- coordinatedheme, the latter being an unusual form of coordination for heme.For further characterization of heme binding different spectroscopicmethods will be used (Raman resonance, Mössbauer, MCD, ITC) with thedetermination of the involved amino acid residues, the function of the ironsulphur cluster and SAM.To verify that HemW is truly a heme chaperon, heme-free Nitrate-Reductase and Cytochrome bd oxidase membrane vesicles will be testedfor heme transfer.OTP051Insights into the ecological distributions of the widely distributedDehalococcoides-related Chloroflexi in the marine subsurfaceK. Wasmund*, C. Algora, J. Müller, L. AdrianHelmholtz Centre for Environmental Research, Isotope Biogeochemistry,Leipzig, GermanyBacteria of the phylum Chloroflexi appear to be widely distributed andsometimes abundant in the marine subsurface. Most subsurfaceChloroflexi form a distinct ‘class level’ clade that are affiliated withorganohalide-respiring Dehalococcoides strains. Despite the apparentglobal ubiquity of these ‘Dehalococcoides-related Chloroflexi’ (DRC),little is known about their specific distributions and/or functionalproperties. In this research, specific PCR primers targeting 16S rRNAgenes of the DRC were designed and employed to study the distributionsof DRC in various subsurface environments. The assay proved highlyspecific and enabled the detection of a diverse range of DRC, oftenrevealing the co-existence of diverse DRC phylotypes even within singlesubsurface samples. Quantification of DRC in marine sediment cores froma collection of globally dispersed locations by real-time PCR suggeststhese bacteria are seemingly ubiquitous and establish highest numbers inthe shallow subsurface (i.e., in the upper meters), yet survive and persistwith burial. Pyrosequencing of DRC through various marine sedimentcores enabled high coverage of DRC diversity and therefore enabledpatterns of diversity through depth to be clearly distinguished. Thisapproach also revealed shifts in sub-groups of DRC through depth andsuggested different sub-groups within the DRC favor differentbiogeochemical conditions, and therefore these sub-groups likely utilizedifferent modes of metabolism.OTP052Bacteria from the Baltic Sea involved in the degradation ofterrestrial DOCJ. Simon*, J. OvermannLeibniz Institute DSMZ - German Collection of Microorganisms and CellCultures, Braunschweig, Department of Microbial Ecology and DiversityResearch, Braunschweig, GermanyPermafrost soils of the northern hemisphere store large amounts ofterrigenous dissolved organic carbon (tDOC). Climate change is expectedto result in a significantly increased transport of tDOC to marine habitats.In order to assess the role of increased tDOC mobilization for the globalcarbon budgets, the potential of tDOC degradation in the marineenvironment needs to be quantified. In the current study, key bacterialspecies involved in the degradation of tDOC in the Baltic Sea werestudied. Because of its unique salinity gradient that ranges from nearlylimnic to marine conditions and since it has been shown that the bacterialcommunity changes consistently along this salinity gradient, the Baltic Searepresents a suitable model system to study tDOC degradation underdifferent environmental conditions. Incubation experiments wereperformed in which Baltic Sea water was supplemented with fresh tDOCoriginating from the River Kalix (next to Överkalix, North Sweden). Highthroughput cultivation was used to recover relevant bacterial isolatesthrough the MultiDrop technique during different stages of tDOCdegradation. Six different growth media were designed that contain typicalconstituents of tDOC including a polymer mix and soluble and insolublehumic analogs. Changes in culturability were quantified through the mostprobable number technique. Community composition of culturablebacteria was assessed by DGGE-fingerprinting of 16S rRNA genes. Firstresults reveal specific changes in the community composition of bacteriathat lead to the dominance of different bacteria during the different stagesof the tDOC degradation.OTP053Acetone activation by strictly anaerobic bacteriaO.B. Gutiérrez Acosta*, B. SchinkKonstanz university, Biology, Konstanz, GermanyDegradation of acetone by strictly anaerobic bacteria is being investigatedwith the sulfate-reducing bacterium Desulfococcus biacutus. An initialATP-dependent carboxylation reaction has been proposed in the activationof acetone for aerobic and facultative anaerobic bacteria. In both types ofbacteria acetone is carboxylated to form acetoacetate as an intermediate.The mechanism proposed for those bacteria requires the investment of twoATP equivalents for the initial step in the activation of acetone. In the caseof sulfate-reducing bacteria, this carboxylation reaction is less likely tooccur. The extreme energy limitation of the degradation of acetonecoupled to sulfate reduction would not allow the sulfate reducers to apply acarboxylation reaction as the initial step. Therefore, we assumed thatsulfate-reducing bacteria use a different strategy in the activation ofacetone which is less energy expensive. A carbonylation reaction washypothesized for activation of acetone by D. biacutus. This carbonylationwould lead to 3-hydroxybutyrate or to an aldehyde derivative. Preliminarystudies of the proposed carbonylation suggest that this reaction could takeplace in the activation of acetone. The acetone degradation in cellsuspension experiments with D. biacutusshowed a sulfate-reducing activityfaster and higher in the presence of CO than in the presence of CO 2.Aldehyde dehydrogenase activity was detected specifically induced in cellextracts of acetone grown cells. This activity was enhanced by thepresence of ammonium in the test. Two dimensional electrophoresis withextracts ofD. biacutus showed different induced proteins in acetone growncells. MALDI-TOF-MS analysis of one of the acetone induced proteinsresulted in an unknown protein.OTP054Overexpression and purification of membrane proteins fromGluconobacter oxydansM. Meyer*, U. Deppenmeier, P. SchweigerInstitute for Microbiology and Biotechnology, University of Bonn, AppliedMicrobiology, Bonn, GermanyGluconobacter oxydans is a member of the Gram-negativeAcetobacteraceae that performs rapid incomplete oxidation of manysugars, sugar acids, polyols and alcohols. This feature has been exploitedin several biotechnological processes (e.g. production of vitamin C and theantidiabetic drug miglitol). The genome sequence of G. oxydans 621H isknown and it was found to contain over 70 uncharacterizedoxidoreductases. For industrial bioconversions, membrane-bounddehydrogenases are of major importance since the products are excretedinto the medium to almost quantitative yields. However, theoverexpression and purification of membrane-bound proteins is generallydifficult and time consuming. The membrane-bound glucosedehydrogenase, encoded by gox0265, was expressed from the previouslyconstructed plasmid pBBR1p452 1 in G. oxydans hsdR in an attempt toimprove the process of integral membrane protein purification. The vectorpBBR1p452 was constructed for gene expression in Gluconobacter spp.and its promoter displayed moderate strength. 1 Additionally, a C-terminalStrepTag was incorporated into the expression construct. Membranes ofthe overexpression strain had a specific activiy of 15 U/mg with glucose,which was seven-fold higher in comparison to the control strain. The rateof oxygen consumption of these membranes was very high (1100 nmol ½O 2 min -1 mg -1 ) and about three-times higher in comparison to the control.Glucose dehydrogenase was successfully purified from the membranes bysolubilisation with detergent and subsequent StrepTactin affinitychromatography. Purified mGDH had a specific activity of 150 U/mgusing D-glucose as substrate. Lower activities were also found with D-allose (43 % of activity compared to D-glucose), D-xylose (11 % ofactivity compared to D-glucose), D-galactose (7 % of activity compared toD-glucose) and D-gulose (4 % of activity compared to D-glucose). The K Mfor glucose was 3.4 mM and V max was 156 U/mg. These resultsdemonstrate, that the purification of active membrane proteins byStrepTactin affinity chromatography is possible and can be used for thecharacterization of novel dehydrogenases.1 Kallnik, V., Meyer, M., Deppenmeier, U., Schweiger, P. (2010). Construction of expressionvectors for protein production inGluconobacter oxydans. J. Biotechnol. 145, 260-265OTP055Inducible gene expression and protein production inMethanosarcina mazeiS. Mondorf*, C. Welte, U. DeppenmeierIfMB, Applied Microbiology, Bonn, GermanyThe methanogenic archaeon Methanosarcina mazei (Ms. mazei) is able toutilize different growth substrates such as H 2/CO 2, acetate, methylamines,and methanol. Many enzymes involved in the complex pathways ofmethanogenesis have been analyzed by heterologous overproduction in E.coli. However, for many methanogenic proteins this was not successfuldue to unusual prosthetic groups that will not correctly assemble in E. coli.Hence, a method for homologous production of proteins in Ms. mazei isdesirable.As a first step towards the production of complex proteins, the simplereporter protein -glucuronidase from E. coli was fused to the induciblepromoter p1687 from Ms. mazei using the shuttle vector pWM321 [1]. Inthe Ms. mazei genome, the p1687 promoter is located upstream of the genecluster mtt1/ mtb1 that is transcribed during growth on trimethylamine butdown-regulated by a factor of 200 when the cells grow on methanol [2].BIOspektrum | Tagungsband 2012