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

1671. Davin LB, Wang H, Crowell AL, Bedgar DL, Martin DM, Sarkanen S, Lewis NG:StereoselectiveBimolecular Phenoxy Radical Coupling by an Auxiliary (Dirigent) Protein Without an ActiveCenter.Science1997,275(5298):362-3672. Pickel B, Constantin MA, Pfannstiel J, Conrad J, Beifuss U, Schaller A:An EnantiocomplementaryDirigent Protein for the Enantioselective Laccase-Catalyzed Oxidative Coupling of Phenols.AngewandteChemie-International Edition2010,49(1):202-204.OTP136The use of copper slag as armor stone in running waters - Howdoes rock chemistry effect natural biofilm formation?D. Mewes* 1 , W. Manz 1 , J. Koop 2 , C. Winkelmann 1 , J. Meier 11 University Koblenz-Landau, Institute for Integrated Natural Sciences, Biology,Koblenz, Germany2 German Federal Institute of Hydrology, Referat U4 - Tierökologie, Koblenz,GermanyBenthic biofilms are intimate associations of benthic algae andheterotrophic microbes within a matrix of extracellular polymericsubstances. They fulfill important ecosystem functions by providing basalenergy resources to higher trophic levels in lotic foodwebs and removingnutrients from the water column. They may also serve in the sequestrationof pollutants such as metall(oids), however, these may re-enter thefoodweb via grazing organisms. Copper slag, a by-product of oreprocessing, is a preferential constructing material in water ways due to itshigh mass density. Its use, however, may result in the release of ecotoxicologicallyrelevant metall(oids) (e.g. Cd, Zn, Cu). Hence, the aim of ajoint project with the German Federal Institute of Hydrology is toinvestigate the environmental impact of copper slag on benthic organismswith a special focus on the development of natural benthic biofilms in thepresent study. Six indoor stream mesocosms, each with a closed watercircuit, were set up and filled with sediment and water (625 l) of the riverRhine. Three of the six channels contained additionally rocks of copperslag, the other three contained basalt rocks as reference material. Biofilmswere sampled in 4-week intervals over a period of six months. In order todifferentiate between the effects of leached (dissolved) metall(oid)s andthose of the rock surface chemistry, biofilms were sampled from copperslag and basalt rocks as well as from rocks originating from river Rhinesediments. Biofilm samples will be characterized by determining totalorganic carbon (total biomass), Chla(autotrophic component),phospholipid-P (living biomass), and taxonomic composition. Thedetermination of both, total and living biomass, allows us to differentiatebetween mere biomass accumulation and actively growing or regeneratingbiomass. Preliminary results will be presented and discussed against thebackground of metall(oid) leaching and accumulation.OTP137Non-standard circadian clock systems in cyanobacteriaA. Wiegard*, L. Seeliger, I.M. AxmannCharité Universitätsmedizin und Humboldt-Universität zu Berlin, Institutfür Theoretische Biologie, Berlin, GermanyMany organisms adapted their biological activities to environmentalchanges associated with alternations of day and night. Most eukaryoteseven evolved internal timing systems to predict those day-night cycles.Among prokaryotes solely cyanobacteria are known to posses such acircadian clock. In the model strain Synechococcus elongatus PCC 7942 itconsists of just three proteins (KaiA, -B and -C) that display 24hroscillations in protein abundance, complex formation and posttranslationalmodification. KaiC as the core component undergoes rhythmicautophosphorylation and -dephosphorylation. These oscillations are aconsequence of KaiA sequestration by KaiC hexamers and KaiBCcomplexes (1).The number of kai-genes, however is not conserved among cyanobacterialspecies. Prochlorococcus has lost the kai gene and harbors a less robustclockwork based on KaiB and -C (2). In contrast, Synechocystis sp. PCC6803 expresses kaiAand even three homologs of both kaiB and-C.To gain insights into the non-standard circadian clock of Synechocystis weare characterizing its multiple Kai proteins in vitro and in vivo. Our in vitrodata suggest partial differences in their biochemical properties.Comparable to the well-studied Synechococcus counterpart,autophosphorylation of KaiC1 is enhanced by KaiA1, whereas thekinaseactivity of KaiC3 is independent of KaiA1. For in vivo analysesspecific antibodies against KaiA and the different KaiC proteins areavailable allowing us to investigate the putative dynamic behavior of theSynechocystis Kai proteins under different light/dark cycles as well asunder continuous conditions.Our findings suggest that the clockworks of cyanobacterial timing systemsdo not follow a universal blueprint. Further analyses will gain insights howthe composition of these clockworks contributes to their precision androbustness. Additionally our results might provide implications for theputative timing mechanisms of other bacterial species, such as purplebacteria, which encode KaiB and -C homologs but lack a kaiA relatedgene.(1) Brettschneider C, Rose RJ, Hertel S, Axmann IM, Heck AJ, Kollmann M. (2010) A sequestrationfeedback determines dynamics and temperature entrainment of the KaiABCcircadian clock. Mol SystBiol.6:389(2) AxmannIM, DühringU, SeeligerL, Arnold A, VanselowJT, Kramer A, and Wilde A (2009) Biochemicalevidence for a timing mechanism in prochlorococcus.J Bacteriol. 191(17):5342-7OTP138Phylogenetic analysis of -LactamesesO. Makarewicz, C. Stein*, M. PletzUniversitätsklinikum Jena, Sektion Klinische Infektiologie, Jena, GermanyObjectives: The number of annually identified b-lactamases with extendedactivity against cephalosporines (ESBL) increased during the last decadeindicating the need for appropriate deescalating antibiotic strategies. Thus,due to high recombinative genetic material of bacteria determination ofspecies remains not sufficient for clinical use. Based on amino acidsequence b-lactamases exhibit different substrate pattern allowingclassification into 16 functional groups [1]. Therefore, knowledge of thesequence is essential to identify ESBL variant and resistance properties.Although, some studies were performed to investigate substitutions ofTEM, SHV and CTX-M, SNP determination of other b-lactamases likeOXA or AmpC reminds more challenging due to the high sequencevariability. Here we present an overall phylogenetic update of b-lactamases based on amino acid (aa) sequences correlated to substrateinhibitorprofiles.Methods: We collected aa sequences of b-lactamases from NCBI database.In total, 643 sequences with at least 200 residues could be aligned andanalyzed using DS Gene 1.5 software (Accelrys Ltd) with the phylogeneticmethod by neighbor joininging. The resulting phylogenetic tree wascorrelated to the functional properties proposed by Bush and Jacoby in2010 [1] and analyzed in detail.Results: As expected, the alignment reflected the differences of thehydrolyzing mechanisms of the -lactamases. Closer relationships werefound for AmpC and OXA-type, whereas GES, CTX-M, IMI and KPCformed another phylogenetic group. Moreover, we could point outmutation hot spots, which are responsible for specific changes of thephenotype.Conclusion: Due to the expanding multi-resistance of pathogens, a fastidentification of the ESBL-variant is in focus of clinical interest and willallow the appropriate therapeutic intervention.We found evidence that some unique aa substitutions are sufficient tocause specific changes in the phenotype of TEM indicating that betterunderstanding of substitution’s dynamics within the types might simplifythe determination of the given b-lactamase by SNP typing. We focus onthe validation of such unique substitutions within the other molecularclasses that exhibit much higher sequence variation compared to TEM.1. Bush, K. and G.A. Jacoby,Updated functional classification of beta-lactamases.AntimicrobAgents Chemother, 2010.54(3): p. 969-76.OTP139Characterization of a Lipopeptide Biosurfactant Produced byBacteria Isolated from Petroleum-Polluted SoilW. El Moslimany* 1 , I. Al Rowaihi 1 , A. Humam 2 , A. Al Nayal 1 , R. Hamza 11 Arabian Gulf University, Biotechnology, Al Manama, Bahrain2 Saudi Aramco, Biotechnology, Dahran, Saudi ArabiaBiosurfactants are environmentally benign microbial products withtremendous environmental, industrial and biomedical applications. Thelarge scale production of biosurfactants has been hampered by their highproduction costs, poor strain productivity and the use of expensivesubstrates. Here, two bacterial strains were isolated from petroleumcontaminatedsoil via enrichment in rich medium (LB) and minimalmedium containing 2% Arabian light oil as the sole carbon source. Basedon 16S rDNA genes sequencing and phylogenetic analysis, the isolatedstrains could be affiliated to different species of the generaBacillus(strainI-15) andPseudomonas(strain I-19). Both strains emulsified crude oil inminimal medium within 2 to 7 days of incubation at 30 C. The oil dropletsof the produced emulsions had various sizes, indicating the production ofdifferent types of biosurfactants/ bioemulsifiers. Preliminary screeningassays such as oil displacement and droplet collapse, revealed the presenceof extracellular surface active agents (surfactants) in the cell free culturesupernatants.The I-19 isolate produced biosurfactant only when grown onhydrophobic substrates such as crude oil and diesel. Whereas the I-15strain produced biosurfactant when grown on crude oil or even watersoluble substrates such as glucose. Surface tension measurementsconfirmed biosurfactant production by the isolated bacteria. Glucosegrowncultures of the I-15 isolate reduced the surface tension of the growthmedium from 68 mN/m to ca 40 mN/m (ca 40 % reduction). Whereascrude oil-grown cultures of the I-19 strain brought about 20% reduction insurface tension as compared to that of the uninoculated medium. The CMCof the biosurfactant recovered from cultures of I-15 strain on glucose wasestimated to 200 mg/L. The biosurfactant caused a reversible inhibition ofthe growth of the I-15 strain on glucose. Fourier Transform InfraredSpectroscopy of the biosurfactant recovered from the I-15 cultures onglucose revealed functional groups that are typical of a lipopetideBIOspektrum | Tagungsband 2012