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

159specific nature. Lipid fractions from soil extracts were also analysed,presenting similar (although not identical) spectra to the studiedmethanogenic archaea. This fact points out to some archaeal lipids, such asarchaeol, as possible biosignatures.OTP098Novel fungal components for biofilm manipulationT. Kleintschek* 1 , H.-G. Lemaire 2 , U. Obst 1 , T. Schwartz 11 Karlsruhe Institute of Technology (KIT), Institute of FunctionalInterfaces, Karlsruhe, Germany2 BASF SE, Ludwigshafen, GermanyBiofouling presents a complex and a general problem in water-basedindustrial applications. For example, the bacterial attachment andsubsequent biofilm growth on reverse osmosis (RO) membranes arelargely responsible for the decline of the functional efficiency and the costeffectiveness.To date, for membrane cleaning mechanical or chemicalprocesses are commonly used. However, due to these treatments themembranes are often damaged which ultimately shortens the membranelife time. Therefore, several fungal supernatants are tested for activecomponents to achieve a careful and effective biofilm detachment ordestabilization from RO membranes. Fungi naturally produce a largenumber of metabolic products like exoenzymes. The fungal supernatants,produced by fermentation, are provided from an industrial type collection.To find novel fungal components 406 fungal supernatants were screened ina static high-throughput crystal violet assay with biofilms of singlebacterial species. The promising supernatants were subsequentlycharacterized with further methods, such as colorimetric assays andimmunofluorescence microscopy. To perform the testing of the promisingsupernatants closer to natural and technical environments, a microfluidichigh-throughput biofilm reactor will be developed and characterized.Denaturing gradient gel electrophoresis (DGGE) was used in order toanalyze the bacterial population of natural biofilms growing on ROmembranes.OTP099Development of a clean deletion and a transposon mutagenesisprocedure for Bacillus licheniformisM. Rachinger* 1 , M. Pfaffenhäuser 1 , M. Schwarzer 1 , B. Mühlthaler 1 ,J. Bongaerts 2 , S. Evers 2 , K.-H. Maurer 3 , W. Liebl 1 , A. Ehrenreich 11 TU München, Lehrstuhl für Mikrobiologie, Freising, Germany2 Henkel AG & Co. KGaA, Düsseldorf, Germany3 AB Enzymes GmbH, Darmstadt, GermanyBacillus licheniformis is an organism of great biotechnological potential.Based on its genome sequence a directed mutagenesis protocol enablesinvestigation of specific genes identified by sequence analysis whereasrandom mutagenesis is used for identification of unknown genes belongingto a defined function.For directed mutagenesis we established and developed a markerlessdeletion system in B. licheniformis. The resulting pKVM vector series canbe transferred by conjugation from E. coli and enables the construction ofdeletions up to 45 kbp. For a further improved and rapid procedure weused a nucleotide analogon for counter-selection without previousmodification of the initial strain. The pKVM vectors were exemplarilyused for deletion of genes involved in C2 metabolism and methylcitratecycle.For undirected mutagenesis we used the mariner based transposonTnYLB-1 which integrates at TA sites in the genome of B. licheniformis.The transposon system was transferred in a vector system capable forconjugative transfer and was subsequently used for construction of arandom transposon library. Transposition-rates up to 37 % were detectable.Transposon insertion sites were identified by vectorette-PCR and inverse-PCR. Finally, the library was screened for candidates involved inanaerobic growth and utilization of acetate.OTP100Unusual membrane dynamics of Ignicoccus: 3D ultrastructureanalyzed by serial sectioning and electron tomographyT. Heimerl* 1 , C. Meyer 2 , J. Flechsler 1 , U. Küper 1 , R. Wirth 1 , H. Huber 1 ,R. Rachel 11 Universität Regensburg, Lehrstuhl für Mikrobiologie, Regensburg, Germany2 Helmholtz Zentrum , Institute of Groundwater Ecology , München, GermanyThe hyperthermophilic chemolithoautotrophic Crenarchaeon Ignicoccushospitalisis an extraordinary organism concerning physiological features(e.g. CO 2 fixation), its ability to serve as host for Nanoarchaeum equitans,and also its ultrastructure [1, 2, 3]. In addition to its cytoplasmicmembrane, I. hospitalis has an outer membrane, and, in between bothmembranes, a large interspace with round and elongated membranesurroundedvesicles and tubes [1]. We are interested in analyzing thestructure and network of the vesicles, the unusual overall cell architectureof Ignicoccus hospitalis, and the contact site to N. equitans, by 3D electronmicroscopy.Cells were cultivated in cellulose capillaries, high-pressure frozen, freezesubstitutedand resin embedded. Serial 50 nm sections were imaged bytransmission electron microscopy, and data aligned and visualized as 3Dstacks. For obtaining a higher resolution in the z-axis, 200 nm sectionswere analyzed by electron tomography. The final models show that themembrane system of I. hospitalisis dynamic and complex: Thecytoplasmic membrane frequently forms offshoots and invaginations.Vesicles can be found that are released from or fuse with the cytoplasmicmembrane; these are either free or interconnected to other vesicles. Thephysiological role of this membrane vesicle system is yet unknown;however, it resembles the eukaryotic counterpart (like ER, Golgiapparatus, TGN), in structure and dynamics. In addition, the I. hospitalisgenome harbors seven proteins that are homologues to the Bet3 subunit ofthe eukaryotic vesicle tethering complex TRAPP I [4].Several macromolecules are part of the contact site: The N. equitans S-layer, and both, the inner and outer membrane of I. hospitalis. Accordingto labeling studies, N. equitans gains membrane lipids and amino acidsfrom its host. 2D and 3D immuno-localisation showed that the Ihomp1protein, the sulfur-H 2:oxidoreductase, and the A 1A OATP synthase arelocated in the outer membrane of I. hospitalis [5, 6], and are also part ofthe contact site. Biochemical studies helped to identify further proteinswhich might be relevant for cell-cell interaction and/or metabolitetransport, like components of ABC transporters [7]. They are in the focusof ongoing studies on the contact site.[1] W. Paper et al., Int J Syst Evol Biol 57 (2007), 803[2] U. Jahn et al., J Bacteriol 189 (2007), 4108[3] H. Huber et al., PNAS 105 (2008), 7851[4] M. Podar et al., Biol Direct, 3, (2008), 2[5] T. Burghardt et al., Mol Microbiol 63 (2007) 166[6] U. Küper et al., PNAS 107 (2010), 3152[7] T. Burghardt et al., Arch Microbiol 190, (2008), 379OTP101Characterisation of heat resistant spore formers isolated fromfoodsA. Rütschle* 1 , G. Lücking 1 , M. Ehling-Schulz 2 , S. Scherer 11 Technische Universität München, ZIEL, Abteilung Mikrobiologie, Freising,Germany2 Veterinärmedizinische Universität Wien, Institut für FunktionelleMikrobiologie, Wien, AustriaAerobic spore formers (in many cases Bacillus species) are consistentlydetected in sterilized food and display a real hazard for the food industryand the consumer. Especially dairy products like UHT-cream, UHT-milk,soft cheese or milk powder are often contaminated. In the context of aFEI/AiF research project (AiF 16012N) spore formers were isolated out ofdifferent foods (raw materials, pre and final products) and the foodprocessing environment. In total, 450 isolates were identified via FTIRspectroscopyor 16S-rRNA sequencing and the heat resistance of thespores was tested at 100°C for 20 min. It turned out that 97 of the 450isolates survived this thermal treatment. 29% of these heat resistantisolates were Bacillus subtilis, 17% Geobacillus stearothermophilus, 10%Bacillus amyloliquefaciens and 10% Bacillus licheniformis. B. subtilis wasthe most frequently detected heat resistant species. The heat resistanceproperties of these isolates were determined in more detail and thermalinactivation kinetics of 24 different B. subtilis strains at 95°C and 100°Cwere performed. The resulting D-values were strain-specific and rangedfrom 15 min to more than 180 min. Further genetic and phenotypicanalyses may provide new insights into the strongly varying heatresistance properties of the Bacillus species [1].1. This research project was supported by the German Ministry of Economics and Technology (viaAiF) and the FEI (Forschungskreis der Ernährungsindustrie e.V., Bonn). Project AiF 16012N.OTP102Thauera aromatica harbours a broad-host plasmid of the IncP-1plasmid that can be eliminated in a model constructed wetlandP.M. Martínez-Lavanchy*, V. Imparato, Z. Chen, U. Kappelmeyer,P. Kuschk, M. Kästner, H.J. Heipieper, J.A. MüllerHelmholtz Centre for Environmental Research, EnvironmentalBiotechnology, Leipzig, GermanyThe denitrifying ß-proteobacterium, Thauera aromatica, has served as amodel organism for biochemical research on anaerobic degradation ofaromatic compounds for many years. The genome of the type strain, T.aromatica DSM6984, has now been sequenced by us. As expected, thosetransformation capabilities are reflected in the 4.3 Mbp genome.Unexpected, however, was the presence of a novel broad host plasmid ofthe IncP-1 family, pKJK10, that confers resistance to several classes ofwidely used antibiotics. Plasmids of this particular family are beingconsidered as major vehicles for the spread of antibiotic resistance genes inclinical settings, thereby having a dramatic effect on medical treatmentoptions of microbial infections. To our knowledge, this is the first time thatan IncP-1 plasmid has been found in an environmental microbe. WeBIOspektrum | Tagungsband 2012