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

[3] Yuzenkova. Y. and N. Zenkin (2010): Central role of the RNA polymerase trigger loop in intrinsicRNA hydrolysis. Proc. Natl. Acad. Sci. USA, 107, 10878-10883.ARP012In vivo analysis of archaeal transcription machinery byChIPR. Reichelt*, M. ThommDepartment of Mikrobiology & Archaea Center, University of Regensburg,Regensburg, GermanyGene expression in archaea is mediated by an eukaryotic-like transcriptionalmachinery and promoter elements. The minimal transcription apparatus ofthe hyperthermophilic euryarchaeon Pyrococcus furiosus consists of an 11subunit RNA polymerase (RNAP) and the two general transcription factorsTATA-binding protein (TBP) and transcription factor B (TFB). Thepresence of these two factors is sufficient for initiation and basaltranscription in vitro. A third factor, transcription factor E (TFE), which ishomologous to the N-terminus of the alpha subunit of the eukaryotictranscription factor IIE (TFIIE), seems to play an important role in opencomplex formation. Moreover, in vitro crosslinking data revealed anunexpected presence of archaeal TFE in elongation complexes, which wasnot observed for eukaryotic TFIIE.To investigate the presence of archaeal TFE in elongation complexes in vivo,we applied the method of Chromatin Immunoprecipitation (ChIP) to variouscomponents of the archaeal transcription apparatus at selected genes (eg.gdh, pfk and hsp20). As expected, the presence of TBP and TFB is restrictedto promoter regions and RNAP was located both in promoter and transcribedregions of these genes. Currently, we are applying this approach to study theoccupancy and distribution of TFE at selected genes. These studies willcontribute to a better understanding of the role of TFE in archaealtranscription elongation in vivo. Furthermore, combining ChIP with nextgenerationsequencing technologies (ChIP-Seq) will help to elucidate thegeneral role of TFE in archaeal transcription.ARP014Functional expression of an archaeal A 1 A O ATP synthasein a bacterial hostC. Gloger*, V. MüllerInstitute for Molecular Bio Science, Department of Molecular Microbiologyand Bioenergetics, Goethe-University, Frankfurt am Main, GermanyThe class of archaeal A 1A O ATP synthases is the least understood class ofATP synthases. Structural information was obtained in recent years for theA 1A O ATP synthase purified from Methanocaldococcus jannaschii (1) andPyroccoccus furiosus (2) and some subunits have been overproduced in E.coli, purified and their structure has been determined. In contrast, still littleis known about their function. This is mainly due to the poor growth and thenon-availability of a genetic system for most archaea. To overcome thisshortcoming, we have cloned the A 1A O ATP synthase operon from themesophile Methanosarcina mazei in an expression vector and expressed thegenes in a F 1F O ATP synthase negative mutant of E. coli. This recombinantstrain expresses a functional ATP synthase, as demonstrated by DpH-drivenATP synthesis in cell suspensions (3). Next, we prepared inverted vesiclesfrom the recombinant strain. These coupled NADH oxidation to thesynthesis of ATP that was inhibited by DCCD and DES. ATP synthesis wasinhibited by protonophores as well as sodium ionophores, indicating DY asdriving force for ATP synthesis. These data demonstrate, for the first time,the production of a functional archaeal A 1A O ATP synthase in a bacterialhost and pave the road for a molecular analysis of the class of archaeal A 1A OATP synthases.(1) Ünal Coskün, Yuriy L. Chaban, Astrid Lingl, Volker Müller, Wilko Keegstra, Egbert J. Boekemaand Gerhard Grüber (2004): Structure and Subunit Arrangement of the A-type ATP SynthaseComplex from the Archaeon Methanococcus jannaschii Visualized by Electron Microscopy. J. Biol.Chem. 279, 38644-38648.(2) Janet Vonck, Kim Y. Pisa, Nina Morgner, Bernhard Brutschy and Volker Müller (2009): ThreedimensionalStructure of A1Ao ATP Synthase from the Hyperthermophilic Archaeon Pyrococcusfuriosus by Electron Microscopy. J. Biol. Chem. 284, 10110-10119.(3) Kim Y. Pisa, Claudia Weidner, Heiko Maischak, Holger Kavermann, Volker Müller (2007): Thecoupling ion in the methanoarchaeal ATP synthases: H + vs. Na + in the A1Ao ATP synthase from thearchaeon Methanosarcina mazei Gö1. FEMS Microbiol. Lett. 227, 56-63.ARP013GvpD-mediated reduction of the transcription activatorGvpE of Halobacterium salinarum using GFP fusions asreporterI. Schmidt*, F. PfeiferInsitute of Microbiology and Genetics, University of Technology,Darmstadt, GermanyFourteen gvp genes are involved in gas vesicle formation in Halobacteriumsalinarum that are arranged in two oppositely orientated gene clusters,gvpACNO and gvpDEFGHIJKLM. The products of gvpE and gvpD areinvolved in the regulation of gas vesicle formation. GvpE is a transcriptionalactivator enhancing the transcription at the two promoters P A and P D,whereas GvpD is involved in repression. The presence of GvpD leads to theabsence of GvpE in Haloferax volcanii transformants [1, 2]. To investigatewhether the reduction of the amount of GvpE in the presence of GvpD is dueto proteolytic degradation of GvpE, N- and C-terminal fusions of GFP wereconstructed. The function of these fusion proteins was studied in P A-bgaHtransformants where the β-galactosidase activities were very similar usingwild-type GvpE, GvpE-GFP or GFP-GvpE. The proteolytic reduction ofGvpE was quantified in the respective H. volcanii transformants carryingGvpD in addition to the GvpE fusion proteins. GvpD wild-type and twoGvpD mutants were used, GvpD Mut6 lacking the repressing function and thesuperrepressor GvpD 3-AAA. Both GFP fusion proteins showed similar effectsin the presence of different GvpD variants, i.e. the presence of defectiveGvpD Mut6 did not alter the fluorescence, whereas wild-type GvpD andsuperrepressor GvpD 3-AAA reduced fluorescence. The effect was muchstronger when GFP-GvpE was used with an observed reduction of 70% forwild-type GvpD and below 10% for superrepressor GvpD 3-AAA. This GFP-GvpE fusion will be used as a valuable reporter system to study effects offurther GvpE mutations and the interaction of GvpE and GvpD.[1] Zimmermann and Pfeifer (2003): Mol. Microbiol. 49(3): 783-794.[2] Scheuch et al (2008): Arch. Microbiol. 190(3):333-339.ARP015Presence of a Na + -translocating ATP synthase in themethanogenic archaeon Methanosarcina acetivoransK. Schlegel*, V. MüllerMolecular Microbiology and Bioenergetics, Institute für Molecular BioScience, Goethe-University, Frankfurt am Main, GermanyDuring metabolism methanogenic archaea can build up a proton- as well as aNa + -gradient [1]. The ATP synthase of methanogenic archaea has aconserved sodium ion binding motif in the membrane-embedded rotorsubunit c but evidence for Na + driven ATP synthesis in methanogens islacking [2]. To address this question, we have established an invertedmembrane vesicle (IMV) system of Methanosarcina acetivorans. IMVscatalyzed ATP hydrolysis with a rate of 35 nmol/min*mg. ATP hydrolysiswas accompanied with the transport of 22 Na + into the lumen of the IMVs.Na + -transport was inhibited by sodium ionophores but not by protonophoresindicating a direct coupling. Furthermore, ATP synthesis as well as Na + -transport was inhibited by the ATP synthase directed inhibitor DCCD. Thesedata demonstrate that the A 1A O ATP synthase can use Na + as coupling ion.[1] Deppenmeier, U. and V. Müller (2008): Life close to the thermodynamic limit: how methanogenicarchaea conserve energy. Results Probl. Cell. Differ. 45: 123-152.[2] Pisa, K.Y. et al (2007): The coupling ion in methanoarchaeal ATP synthases: H + versus Na + in theA1AO ATP synthase from the archaeon Methanosarcina mazei Gö1. FEMS Microbiol. Lett.277(1):56-63.ARP016In vivo analysis of gas vesicle-coding proteins ofHalobacterium salinarum PHH1S. Tavlaridou*, F. PfeiferInstitute for Microbiology & Genetics, University of Technology, Darmstadt,GermanyHalobacterium salinarium PHH1 is a gas vesicle-producing organism. Thegas vesicle formation is encoded by the p-vac region, consisting of 14 gasvesicle protein (gvp) genes, located in two clusters, p-gvpACNO and p-gvpDEFGHIJKL. GvpA and GvpC are structural proteins, whereas GvpEand GvpD are involved in regulation. The functions of other Gvp proteinsspektrum | Tagungsband 2011