VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

naphthalene and 2-methylnaphthalene were directly dissolved in artificialsea water medium (ASW) and inoculated with dense cell suspension of 2-methylnaphthalene-grown cultures. Depletion of naphthalene and 2-methylnaphthalene were monitored by HPLC. Under this condition all threestrains completely consumed 2-methylnaphthalene within 3-4 days;however, naphthalene degradation only started after about 10 daysadaptation time and took around 30-40 days for complete depletion. On theother hand, under the same experimental conditions, NaphS2 cells grownwith naphthalene were able to completely degrade naphthalene within 5 daysof incubation. Results showed that the capacity to degrade naphthalene wasnot preserved in 2-methylnaphthalene-grown cultures; however, it could beinduced. Based on these results proteins, specifically involved in anaerobicnaphthalene degradation could be identified via differential display twodimensionalgel electrophoresis protein analysis.[1] Musat, F. et al (2009): Anaerobic degradation of naphthalene and 2-methylnaphthalene by strainsof marine sulfate-reducing bacteria. Environmental Microbiology. 11: 209-219.AMP047A metabolomic view on the pathogenic bacteriumStaphylococcus aureusK. Dörries* 1 , M. Liebeke 2 , H. Meyer 1 , D. Zühlke 3 , S. Fuchs 3 , M. Hecker 3 ,M. Lalk 11 Institute of Pharmacy, Ernst-Moritz-Arndt-University, Greifswald,Germany2 Imperial College London, London, United Kingdom3 Institute for Microbiology, Ernst-Moritz-Arndt-University, Greifswald,GermanyStaphylococcus aureus as a facultative anaerobic bacterium is part of themammalian commensal flora. Nevertheless under specific conditions S.aureus causes strong infections and is able to invade tissues and cells. Withregard to its role as a leading nosocomial pathogen because of its increasingmultidrug resistance, investigations on S. aureus are of great interest.During host infection the bacterium has to cope with changing supply ofcarbon sources and varying oxygen availability up to anaerobic conditions.For a better understanding of its adaptive mechanisms and its regulatoryprocesses, S. aureus COL cells were cultivated under different growthconditions. By using 1 H-NMR, GC-MS and LC-MS we investigated theextra- and intracellular metabolome and observed distinct differencesbetween aerobically and anaerobically grown S. aureus COL cells.ARV001Replacing the Archaeal Path of SelenocysteineBiosynthesis with the BacterialM. Rother*, T. Stock, S. GoetzInstitute for Molecular Bio Science, Goethe-University, Frankfurt am Main,GermanyBiosynthesis of selenocysteine (sec), the 21st proteinogenic amino acid,occurs in a tRNA-bound fashion in all three domains of life. The secspecifictRNA (tRNA sec ) is mis-aminoacylated with serine (ser), which issubsequently converted to sec. While in Bacteria this conversion involves asingle step catalyzed by selenocysteine synthase (SelA), Archaea andEukarya phosphorylate ser-tRNA sec to O-phosphoseryl-(sep)-tRNA sec (usingsep-tRNA sec kinase, PSTK) which serves as substrate for sep-tRNA sec :secsynthase (SepSecS) to generate sec-tRNA sec . To investigate thephysiological role of sep-tRNA sec in Archaea, mutant Methanococcusmaripaludis strains lacking either PSTK or SepSecS were constructed andcomplemented with SelA from Escherichia coli. We could show that, bothPSTK and SepSecS are indispensable for selenoprotein synthesis in M.maripaludis, but also that the archaeal sec-synthesis pathway can be „shortcircuited”to the bacterial one. This finding rules out an essential role of thisaminoacyl-tRNA species in Archaea. Potential functions of sep-tRNA secother than as intermediate in sec synthesis are being addressed to eventuallyexplain why Archaea (and Eukarya) have evolved a three-step mechanismfor sec synthesis as compared to the two-step mechanism found in Bacteria.ARV002A heme-based redox sensor in the methanogenicarchaeon Methanosarcina acetivoransB. Molitor*, N. Frankenberg-DinkelDepartment for Biology and Biotechnology, Biology of Microorganisms,Ruhr Universität Bochum, Bochum, GermanyThe methanogenic archaeon Methanosarcina acetivorans C2A relies onmethanogenesis as the energy conserving mechanism. Therefore, it is able toutilize common methanogenic growth substrates such as methanol, acetateand different methylated compounds, but not CO 2/H 2. In additionM.acetivorans can use CO as a growth substrate. In contrast to other COutilizing organisms which produce H 2 during CO metabolism, M.acetivoransgenerates acetic acid, formic acid and methylated sulfides, besides methane,but not H 2.It was shown that three methyltransferase/corrinoid fusion proteins arerequired for generating dimethylsulfide (DMS) from CO and CH 4 fromDMS [1]. These proteins are each differentially regulated by a downstreamregulator protein [2]. MA4560, one of these regulators, is a putativeresponse regulator of a two component regulatory system together with themulti domain sensor histidine kinase MA4561. In order to learn more aboutthe sensor function of MA4561, the full-length protein consisting of twoconsecutive PAS and GAF domains joint to a histidine kinase domain washeterologously produced in Escherichia coli. In addition, different truncatedprotein variants were produced and purified using metal affinitychromatography. UV-vis spectrometry identified a redox-active hemecofactor in the second GAF domain of this multi domain protein. In contrastto many other known heme-based sensor proteins which bind the cofactornon-covalently, covalent attachement of heme could be demonstrated.Interestingly, autophosphorylation of the protein is highly dependent on theredox state of the central heme iron. Due to the involvement of thecorresponding response regulator MA4560 in regulating gene expression inresponse to CO and methylated sulfides, a potential role of the sensor kinaseMA4561 in redox or CO sensing via the heme cofactor is postulated.[1] Oelgeschläger, E., and M. Rother (2009): Mol Microbiol. 72(5), 1260-1272.[2] Bose, A. et al (2009): Mol Microbiol. 74(1), 227-238.ARV003Elucidation of the N-glycosylation pathway in thethermoacidophilic crenarchaeon SulfolobusacidocaldariusB. Meyer*, S.-V. AlbersMax Planck Institute for Molecular Biology of Archaea, Marburg, GermanyHistorically it was long been believed that glycosylation is a uniquephenomena restricted to Eukarya 1 , however, when in 1976 Mescher andStrominger purified the S-Layer protein from Halobacterium salinariumwhich contained glycans covalently linked to asparagine residues 2 , questionsevoked how N-glycosylation occurs in Bacteria and Archaea. Today N-glycosylation is thought to be conserved across all three major domains oflife. During the last years substantial progress in describing N-glycosylationpathways in three euryarchaeota 3-5 has been made. Although eukarya,bacteria, and archaea all seem to have certain characteristics of the N-glycosylation pathway in common, archaea displays a mosaic of featuresfrom the eukaryal and bacterial system. However, so far the N-glycosylationprocess in a crenarchaeota is still uncovered. Here we will report the firstresults elucidating the N-glycosylation pathway in the thermoacidophilicarchaeon Sulfolobus acidocaldarius. The N-glycosylation in S.acidocaldarius show same significant differences compared to these of theother archaea, e. g. scattered gene localization of glycosyltransferases (GT),challenging in identification of GT involved in the glycosylation processes.In contrast to the non essential N-glycosylation pathway in the studiedeuryarchaeota, the N-glycosylation pathway is essential for the survival of S.acidocaldarius. Further S. acidocaldarius exhibited a unique compositionand branched structure of the N-linked oligosaccharide, so far not found inother archaea.[1] Apweiler, R. et al (1999): On the frequency of protein glycosylation, as deduced from analysis ofthe SWISS-PROT database. Biochim Biophys Acta 1473, 4-8.[2] Mescher, M.F. & Strominger, J.L. Purification and characterization of a prokaryotic glycoproteinfrom cell-envelope of Halobacterium salinarium. J. Biol. Chem. 251, 2005-2014 (1976).[3] Chaban, B. et al (2009): AglC and AglK are involved in biosynthesis and attachment ofdiacetylated glucuronic acid to the N-glycan in Methanococcus voltae. J. Bacteriol. 191, 187-95.[4] Kelly, J. et al (2009): A novel N-linked flagellar glycan from Methanococcus maripaludis.Carbohydr. Res. 344, 648-53.[5] Yurist-Doutsch, S. et al (2010): N-glycosylation in Archaea: On the coordinated actions ofHaloferax volcanii AglF and AglM. Mol Microbiol.spektrum | Tagungsband 2011