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

[3] Roppelt, V., Hobel, C., Albers, S. V., Lassek, C., Schwarz, H., Klug, G., and Evguenieva-Hackenberg, E. (2010) The archaeal exosome localizes to the membrane. FEBS Letters 584:2791-2795.RGP018Towards the composition of a regulatoryredox-networkfor photosynthetic gene expression in RhodospirillumrubrumA. Carius*, M. Henkel, H. GrammelSystems Biology, Max Planck Institute, Magdeburg, GermanyThe anoxygenic photosynthetic bacterium R. rubrum serves as a modelorganism for redox-controlled gene expression. The expression ofphotosynthetic membranes (PM) is a fair indicator for the redox states of thecells. Even if R. rubrum is grown in the dark, high levels of PM can bereached, depending not only on the oxygen supply, but also on thecomposition of the culture medium.The choice of carbon sources for example is of major importance: Succinatecombined with fructose will reach the highest PM levels under semiaerobicconditions. The addition of glutathione to the culture broth results insignificantly elevated PM-levels if an organic acid is used as carbon source.Glutathione can not be utilized as sole carbon source by R. rubrum.Supplementation with the amino acids contained in glutathione does notresult in elevated PM-levels. The influence of light on PM-expression isversatile.The control of PM expression in Rhodobacter species has been unravelled tosome extent, so we could identify some major dierences in comparisonwith R.rubrum.1. In Rhodobacter species, the PM-elevating eect of fructose is absent2. In R. rubrum, no homologue to the RegB/RegA two component redoxsensing system from Rhodobacter could be identified by BLAST analysis3. Active uptake of glutathione R. rubrum is possible, which then enhancesPM expression. In Rhodobacter glutathione causes no PM-elevating effect ifadded to the culture broth.In this work, we combine our results from dierent working approaches todefine a redox-regulated network for R. rubrum. It includes data from aligand-anity chromatography with coenzyme Q 10 as a ligand for thescreening of redox-sensitive histidine kinases, as well as intracellularglutathione data and information from deletion mutants. Additionally, thenecessary redoxpotential for the PpsR-switch was estimated and redoxsensitivestaining of reduced thiols was applied.RGP019Mechanism and Function of non standard CircadianClock Systems in CyanobacteriaA.K. Bäcker*, A. Wilde, J. Holtzendorff*Institute for Micro- and Molecular Biology, Justus-Liebig-University,Giessen, GermanyCircadian rhythms, oscillations with approximately 24 h periods concerningmany physiological activities, are found in most eukaryotes. Amongprokaryotes, exclusively cyanobacteria are known to harbour an internalclock. In the model strain Synechococcus elongatus PCC 7942, the kaiABCgene cluster is essential for the generation of circadian rhythms. It has beenshown by in vitro and in vivo experiments that the timing process itself isbased on rhythmic phosphorylation of KaiC hexamers, whereas ATPhydrolysis catalyzed by KaiC accounts for the reaction that defines the 24-hour period of the clock. In addition to the kaiABC gene cluster, the genomeof Synechocystis sp. PCC 6803 holds additional orphan kai genes located atdifferent sites on the chromosome which functions have not beeninvestigated. In contrast, different strains of the marine cyanobacteriumProchlorococcus are lacking the kaiA gene and components of the input andoutput pathways are missing or truncated. We aim to undeceive thephenomenon of multiple kai gene copies and of reduced kai operons incomparison to the well-studied protein clock of Synechococcus. The loss ofkaiA in Prochlorococcus transforms the circadian clock mechanism into thatof an hourglass. First analyses of Synechocystis kai knockout mutantsindicate that the deletion of the kaiABC cluster results in reduced fitnesscompared to the wild type, while deletion of kaiC2B2 is lethal. Furtherbiochemical characterization of the purified Synechocystis Kai proteins willyield insights into Kai protein complex formation, as well as ATPaseactivity and phosphorylation cycles of the three different KaiC proteins fromSynechocystis.RGP020Signal perception by the oxygen-sensing transcriptionalregulator Fnr of Bacillus subtilisE. Härtig* 1 , I. Gruner 2 , L. Böttger 3 , A.X. Trautwein 3 , D. Jahn 11 Department of Microbiology, University of Technology, Braunschweig,Germany2 DSM Nutritional Products, Kaiseraugst, Switzerland3 Institute of Physics, University of Lübeck, Lübeck, GermanyThe Bacillus subtilis redox regulator Fnr controls genes of the anaerobicmetabolism in response to low oxygen tension. Unlike its E. coli counterpartB. subtilis Fnr utilizes three cysteine residues and one unknown non-cysteineligand for the formation of the oxygen sensing [4Fe-4S] 2+ cluster. Using sitedirectedmutagenesis of fnr a variety of mutant proteins were created andactivity was tested in vivo using a fnr mutant complementation system withan Fnr-dependent narG-lacZ reporter gene fusion. Furthermore, recombinantanaerobically purified Fnr proteins were characterized by in vitro DNAbinding studies and transcription assays. An unusual structure for theoxygen-sensing [4Fe-4S] 2+ cluster was detected by a combination of geneticexperiments with UV/Vis and Mössbauer spectroscopy. Aspartate residue141 was identified as fourth iron-sulphur cluster ligand beside three cysteineresidues. Exchange of aspartate 141 to alanine abolished functional in vivocomplementation of an fnr knock out strain by the mutagenized fnr gene andin vitro DNA binding of the recombinant regulator FnrD141A. In contrast,substitution of aspartate 141 with cysteine preserved [4Fe-4S] 2+ structureand regulator function.J. Biol Chemistry in pressRGP021Quantitative analysis of the pmoA expression level in typeI and type II methanotrophsF. Brandt*, B. Pommerenke, M.G. DumontDepartment of Biogeochemistry, Max Planck Institute for TerrestrialMicrobiology, Marburg, GermanyMethane is a well-known greenhouse gas and the atmospheric concentrationhas increased dramatically over the last 250 years. Wetlands are a majorsource of methane where it is produced by methanogens and diffuses to theatmosphere. Aerobic methanotrophs are active at the oxic-anoxic interfaceof these environments and mitigate the release of methane to theatmosphere. Methanotrophs are unique in their ability to utilize methane astheir only carbon and energy source. On the basis of morphologicaldifferences like structure of intracytoplasmic membranes, physiologicalcharacteristics and phylogenetic placements, methanotrophs can beseparated into two groups: type I and type II. Type I methanotrophs belongto the family Methylococcaceae within the γ-subdivision of Proteobacteria,whereas type II methanotrophs belong to the family Methylocystaceae in theα-Proteobacteria. The first step and key reaction of methane oxidation is theintroduction of a hydroxyl group catalyzed by a methane monooxygenase(MMO) enzyme. The membrane bound form of this enzyme (pMMO) ispresent in almost all known methanotrophs and is found to be composed ofthree polypeptides: an α-subunit (PmoB), a β-subunit (PmoA) and a γ-subunit (PmoC). The genes encoding pMMO are encoding within thepmoCAB operon, which is regulated by a σ 70 promoter. The pmoA gene isfrequently used as a functional and phylogenetic marker for methanotrophs.A recent trend has been to use the relative abundance of pmoA mRNArecovered from environmental samples as a proxy for the relative activity ofdifferent methanotroph species. The objective of this study was toinvestigate how well pmoA transcript abundance correlates with the activityof various methanotroph species. Ten different methanotrophs, includingboth type I and II representatives, were grown in batch culture and theabundance of pmoA transcripts was determined by reverse transcription realtimePCR. Here we show the relationship between the absolute cell numberand the pmoA transcript levels of various species of type I and IImethanotrophs at different growth stages.spektrum | Tagungsband 2011