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

132understand the exact role of FlaH in the assembly and function of thecrenarchaeal flagellum.OTV007Subcellular positioning of a DNA-binding protein throughconstraint movementL. Simon* 1 , M. Ulbrich 2 , J. Ries 3 , H. Ewers 3 , P.L. Graumann 11 University of Freiburg, Mikrobiologie, Freiburg, Germany2 University of Freiburg, Bioss (Centre for Biological signalling studies)and Department for Medicine, Institute of Physiology, Freiburg, Germany3 ETH Zürich, Laboratory of Physical Chemistry, Zürich, SwitzerlandMany protein complexes localize to defined regions within cells. Thebacterial SMC complex consists of a central SMC dimer and twoaccessory factors, ScpA and ScpB. SMC binds non-specifically to DNA invitro, while ScpA and ScpB appear to confer a regulatory function. Thecomplex plays an important role in chromosome condensation andsegregation during the bacterial cell cycle, and forms two discretesubcellular centres, one in each cell half, when imaged with conventionalepi-fluorescence microscopy. Using single molecule microscopy andtracking we show that localization is achieved through limited yet rapidmovement of the SMC subunits through a cell half, while the accessoryScpAB subunits mediate temporal arrest of a subset of SMC molecules atthe centre of a cell half. Thus, specific localization is achieved bymovement through the nucleoid and transient arrest at the nucleoid centre.FRAP studies show that the SMC pool has a high turnover within a cellhalf and is also replenished through de novo protein synthesis, yielding anadditional level of protein dynamics. Diffusion/movement within a limitedcompartment and transient arrest may be a general means to accumulateproteins within non-compartmentalized cells.OTV008Protein complexes involved in the electron transport chain ofanammox bacteriaN. de Almeida* 1 , H. Wessels 2 , W. Maalcke 1 , J. Keltjens 1 , M. Jetten 1 , B. Kartal 11 Radboud University Nijmegen , Microbiology, Nijmegen, Netherlands2 Radboud University Nijmegen Medical Centre, Department of Pediatrics,Nijmegen, NetherlandsAnammox bacteria combine ammonia with nitrite to dinitrogen gas withnitric oxide and hydrazine as intermediates (1). Oxidation of the latteryields low-redox-potential electrons, which can be used for CO 2 fixation.We hypothesize that these are replenished through the oxidation of nitriteto nitrate by a nitrite oxidizing system (NAR) (2). As nitrite is a relativelypoor reductant, the electrons have to be energized to enter the bc 1-complexor to feed a quinone pool, which implies reverse electron transport.The gene cluster that contains the catalytic subunits of nitrite oxidizing(narGH) system covers almost the full natural repertoire of electroncarriers. This includes genes encoding six putative heme-containingproteins and two putative blue-copper proteins and a putative anchor to themembrane showing homology to a cytochrome bd oxidase subunit (2).Furthermore, the genome of the anammox bacterium Candidatus Kueneniastuttgartiensis shows a high redundancy of respiratory genes, suggesting anintricate cellular electron transport system. Interestingly, the three operonsencoding for the bc 1 complexes, complex III in the respiratory chain, alldiffer in their subunit composition from the canonical bc 1 complexes inother microorganisms. One operon consists only of a heme b /c fusionprotein and the Rieske protein. The other two operons encode for multiheme c containing genes, NAD(P) oxidoreductase subunits and,intriguingly one of them contains a hydroxylamine oxidoreductase subunit.The combination of these subunits strongly suggests that electrons derivedfrom different oxidation reactions could be wired to different electronacceptors, once entering the bc 1 complexes.The whole protein complement of K. stuttgartiensis membranes wasdetermined with protein correlation profiling using LC-MS/MS data fromconsecutive Blue Native (BN) gel slices (3). The detection of differentcomplexes was coupled to in-gel activities of the respiratory complexes inBN gels. Further, the catalytic subunit of the nitrite oxidizing system of K.stuttgartiensis was purified.1) Kartal B, et al (2011): Molecular mechanism of anaerobic ammonium Oxidation. Nature 479: 127-130.2) de Almeida NM, et al (2011): Proteins and protein complexes involved in the biochemical reactions ofanaerobic ammonium-oxidizing bacteria. Biochemical Society Transactions. 39: 303-308.3)Wessels JCT, et al (2009) LC-MS/MS as an alternative for SDS-PAGE in blue native analysis of proteincomplexes. Proteomics 17: 4221-4228.OTV009Replication fork movement and methylation governs SeqAbinding to the Escherichia coli chromosomeT. Waldminghaus* 1 , C. Weigel 2 , K. Skarstad 31 LOEWE-Zentrum für Synthetische Mikrobiologie, Philipps-Universität,Marburg, Germany2 HTW, Department of Life Science Engineering, Berlin, Germany3 Norwegian Institute for Cancer Research, Cell Biology, Oslo, NorwayChromosomes are composed of enormously long DNA molecules whichmust be distributed correctly as the cells grow and divide. In Escherichiacoli the SeqA protein might be involved in organization of new DNAbehind the replication forks. SeqA binds specific to GATC sequenceswhich are methylated on the A of the old strand but not on the new strand.Such hemi-methylated DNA is produced by progression of the replicationforks and lasts until Dam methyltransferase methylates the new strand. It istherefore believed that a region of hemi-methylated DNA covered bySeqA follows the replication fork. We show that this is indeed the case byusing global ChIP on Chip analysis of SeqA in cells synchronizedregarding DNA replication. To assess hemi-methylation we developed thefirst genome wide method for methylation analysis in bacteria. Acomparison of rapid and slow growth conditions showed that in cells withmultiple replication forks per chromosome, the old forks bind little SeqA.Analysis of strains with strong SeqA binding sites at differentchromosomal loci supported this finding. The results indicate that a reorganizationof the chromosome occurs at a timepoint coinciding with theend of SeqA dependent origin sequestration. We suggest that areorganization event occurs resulting in both origin desequestration andloss of old replication forks from the SeqA structures.Waldminghaus, T. and Skarstad, K. (2009) The Escherichia coli SeqA protein. Plasmid, 61, 141-150.Waldminghaus, T. and Skarstad, K. (2010) ChIP on Chip: surprising results are often artifacts. BMCGenomics. 11, 414.OTV010Translocation of sodium ions by the ND5 subunit ofmitochondrial complex I from the yeast Yarrowia lipolyticaH. Grönheim*, W. Steffen, J. SteuberUniversität Hohenheim, Mikrobiologie FG Zelluläre Mikrobiologie,Stuttgart, GermanyMitochondrial complex I (NADH:ubiquinone oxidoreductase), localized inthe inner mitochondrial membrane, is the first enzyme of the electrontransport chain of the oxidative phosphorylation system. The L-shapedcomplex is partitioned into a peripheral arm and a membrane-bounddomain.In the peripheral arm electrons from NADH are transferred to ubiquinonevia iron sulfur clusters, using FMN as cofactor. This process is coupled, byconformational changes as structural data indicates, with the translocationof protons by the membrane-bound domain (Brandt 2006; Efremov,Baradaran et al. 2010; Efremov and Sazanov 2011). Here we focus on theND5 subunit of the membrane-bound domain of the mammalian complexwhich is considered to be involved in the translocation of protons.Previous studies showed that the ND5 homologue NuoL from E. colicomplex I transports sodium ions across the membrane (Gemperli,Schaffitzel et al. 2007). We also observed that ND5 from human complexI, when inserted into the inner mitochondrial membrane of S. cerevisiae,leads to an increased salt sensitivity of the yeast cells, suggesting that ND5promotes the leakage of cations across the mitochondrial membrane(Steffen, Gemperli et al. 2010). Here, we investigate the cation transportactivity of the ND5 homologue from the yeast Y. lipolytica produced asGFP-ND5 fusion protein in ER vesicles from S. cerevisiae. The topologyof ND5 in the vesicles was analyzed by limited proteolysis. The N-terminal GFP fusion to ND5 was oriented towards the external lumen ofER vesicles. This uniform orientation of ND5 in vesicles was theprerequisite for cation transport studies where a Na + concentration gradientwas applied. ER vesicles containing GFP-ND5 exhibited a significantlyhigher Na + uptake activity than control vesicles without ND5. Thisindicates that the individual ND5 protein which is highly related tosecondary Na + /H + antiporters contains a channel for Na + .Brandt, U. (2006). "Energy converting NADH:quinone oxidoreductase (complex I)."Annu Rev Biochem75:69-92.Efremov, R. G., R. Baradaran, et al. (2010). "The architecture of respiratory complex I."Nature465(7297):441-445.Efremov, R. G. and L. A. Sazanov (2011). "Structure of the membrane domain of respiratory complexI."Nature476(7361): 414-420.Gemperli, A. C., C. Schaffitzel, et al. (2007). "Transport of Na + and K + by an antiporter-related subunit fromthe Escherichia coli NADH dehydrogenase I produced in Saccharomyces cerevisiae."Arch Microbiol188(5):509-521.Steffen, W., A. C. Gemperli, et al. (2010). "Organelle-specific expression of subunit ND5 of human complexI (NADH dehydrogenase) alters cation homeostasis in Saccharomyces cerevisiae."FEMS Yeast Res10(6):648-659.BIOspektrum | Tagungsband 2012