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

PSP022Genome analysis and heterologous expression of acetateactivatingenzymes in the anammox bacterium KueneniastuttgartiensisL. Russ*, H.R. Harhangi, J. Schellekens, B. Kartal, H.J.M. Op den Camp,M.S.M. JettenIWWR, Microbiology, Nijmegen, NetherlandsBacteria capable of anaerobic ammonium oxidation (anammox) derive theirenergy for growth from the conversion of ammonium and nitrite intodinitrogen gas, thereby constituting a significant sink for fixed nitrogenunder anoxic conditions. Cellular carbon is hypothesized to be fixed via theacetyl-CoA pathway, suggesting a chemolithoautotrophic lifestyle.However, it was shown that anammox bacteria have a more versatilemetabolism than previously assumed: several genera have been shown to useorganic compounds i.e. acetate as electron donors to reduce nitrate andnitrite to dinitrogen gas via ammonium. Acetate is an environmentallyrelevant organic acid that has to be activated to acetyl-CoA prior to itsutilization in metabolism. One of the key enzymes catalyzing the directformation of acetyl-CoA from acetate is AMP-forming acetyl-CoAsynthetase (ACS). In prokaryotes it is known to operate in an assimilatoryroute during growth on low acetate concentrations.The present study focuses on the functional expression of the most highlyexpressed acetate-activating enzyme of K. stuttgartiensis, a putative acsgene. An ackA-pta-acs triple mutant of E. coli was complemented with theK. stuttgartiensis acs gene resulting in recovery of growth on acetate. Thepurified enzyme showed activity towards several short chain organic acidswith the highest conversion rates for acetate. The specific activity withpropionate and formate was reduced by 1.2 and 1.5-fold respectively;whereas butyrate and isobutyrate were converted at even lower rates. Thebroad substrate specificity might be established by a substitution in one offour conserved residues in the acetate-binding pocket that determinesspecificity of the acyl-substrate as has been shown previously.Here we could demonstrate that acetate could be activated by an acs-likeprotein of K. stuttgartiensis. This is a first indication about the mechanismof acetate utilization in anammox, although the incorporation of acetatederivedcarbon into cellular biomass could not be detected so far.PSP023The CoxD protein, a novel AAA+ ATPase involved inmetal cluster assembly: hydrolysis of nucleotidetriphosphatesand oligomerizationT. Maisel* 1 , T. Mielke 2 , J. Bürger 2 , O. Meyer 11 Chair of Microbiology, University of Bayreuth, Bayreuth, Germany2 Max Planck Institute for Molecular Genetics, Berlin, GermanyThe CoxD protein from the aerobic CO-utilizing, chemolithoautotrophic α-proteobacterium Oligotropha carboxidovorans is involved in theposttranslational biosynthesis of [CuSMoO 2] active site of COdehydrogenase [1]. CoxD is predicted as a MoxR-like AAA+ ATPasechaperone related to the hexameric, ring-shaped BchI component of Mg 2+ -chelatases [1,2]. Because it was not possible to purify homologous CoxD inan active state from cytoplasmic membranes its role as an AAA+ ATPasewas mainly confined to the knowledge of its primary sequence. Here weshow the recombinant production of functional CoxD protein from inclusionbodies produced in E. coli and present direct evidence which establishesCoxD as an AAA+ ATPase.Recombinant CoxD protein was expressed in inclusion bodies at a level of38 % of the total cell protein and was purified to 95 % homogeneity. TheCoxD inclusion bodies were solubilized employing elevated concentrationsof urea, and CoxD was refolded by pulsed ultradilution ( ~ 50-fold). Uv-visand circular dichroism spectroscopy indicated that refolded CoxD is stablysoluble and contains secondary structural elements. Refolded CoxD proteinwas shown to hydrolyze ATP in a Mg 2+ depending reaction yieldinginorganic phosphate (P i) and ADP in equimolar amounts. V max of MgATPhydrolysis was 8.86 nmol P i min -1 mg -1 with a K M of 0.58 mM MgATP.Hydrolysis of MgATP was hampered by MgATPγS but not affected byMgGTP. Sucrose density gradient centrifugation suggested that CoxDoligomerizes as a hexamer, and direct evidence for the oligomerization ofCoxD was obtained from electron microscopy of negatively stained (uranylacetate) samples. With the BchI subunit of Mg-chelatase as template, a 3Dstructure prediction of CoxD was generated.[1] Pelzmann, A. et al (2009): J. Biol. Chem. 284 (14), 9578-9586.[2] Lundqvist, J. et al (2010) Structure 18, 354-365.PSP024Denitrification is linked to magnetite biomineralization inMagnetosprillum gryphiswaldenseY. Li*, E. Katzmann, D. SchülerDepartment of Biology I, Microbiology, Technical University, Munich,GermanyMagnetospirillum gryphiswaldense is an aquatic microorganism, which cansynthesize intracellular magnetic particles referred to as bacterial magneticparticles or magnetosomes. M. gryphiswaldense is also capable ofdissimilatory nitrate reduction. The magnetite synthesis is only inducedwhen the oxygen concentration is below a threshold value [1] , and it has beensuggested that NirS protein had a novel function, Fe (II): nitriteoxidoreductase in vitro [2] . However, the relationship between denitrificationand magnetite biomineralization is poorly understood.Metabolic reconstruction from M. gryphiswaldense genome data revealed acomplete pathway of denitrification, including genes for nitrate reductase(nap), nitrite reductase (nirS), nitric oxide reductase (norCB) and nitrousoxide reductase (nosZ).A Δnap deletion mutant had no obvious effect on growth and magnetosomeformation. A ΔnirS mutant in aerobic culture showed a similar growth rateas wild type. However, ΔnirS was clearly impacted on growth andmagnetism under micro- and anaerobic conditions in the present of nitrate.Smaller, misshapen and misaligned magnetite crystals were formed in ΔnirSmutant. In addition NirS protein was upregulated by nitrate anddownregulated by nitrite. ΔnorCB could not grow under micro- andanaerobic conditions, but had a lower magnetism and poor growth whenhigher oxygen was supplied. ΔnosZ did not affect magnetosome formation,but only showed a lower growth under anaerobic conditions, which might beresulted from less energy supply. Our data indicate the denitrification geneshave effects on growth and magnetosome formation in M. gryphiswaldense.The effects of denitrification, in particular nirS, are consistent with formersuggestion. NirS protein might participate in magnetosome formation duringdenitrification by oxidation of ferrous to ferric formation of mixed-valenceFe 3O 4 under anaerobic conditions.[1] Heyen U, Schüler D (2003) Growth and magnetosome formation by microaerophilicMagnetosprillum strains in an oxygen-controlled fermentor. Appl Microbiol Biotechnol 61:536-544[2] Yamazaki T, Oyanagi H, Fujuwara T, Fukumori Y (1995) Nitrite reductase from the magnetotacticbacterium Magnetospirillum magnetotacticum; a novel cytochrome cd1 with Fe (II): nitriteoxidoreductase activity. Eur J Biochem 233:655-671PSP025Biosynthesis of (Bacterio)chlorophylls: ATP-DependentTransient Subunit Interaction and Electron Transfer ofDark Operative Protochlorophyllide OxidoreductaseJ. Moser* 1 , M. Bröcker 2 , F. Lendzian 3 , H. Scheer 4 , W.-D. Schubert 5 ,D. Jahn 11 Institute for Microbiology, University of Technology, Braunschweig,Germany2 Department of Molecular Biophysics and Biochemistry , Yale University,New Haven, USA3 Institute for Chemistry, Institute of Technology, Berlin, Germany4 Department of Biology I, Technical University, Munich, Germany5 Department of Biotechnology, University of the Western Cape, Cape Town,South AfricaDark operative protochlorophyllide oxidoreductase (DPOR) catalyzes thetwo electron reduction of protochlorophyllide a to form chlorophyllide a, thelast common precursor of chlorophyll a and bacteriochlorophyll abiosynthesis. Although DPOR shares significant amino acid sequencehomologies to nitrogenase only the initial catalytic steps resemblenitrogenase catalysis. During ATP-dependent DPOR catalysis thehomodimeric ChlL 2 subunit carrying a [4Fe-4S] cluster, transfers electronsto the corresponding heterotetrameric subunit (ChlN/ChlB) 2 which alsopossesses a redox active [4Fe-4S] cluster. To investigate the transientinteraction of both subcomplexes and the resulting electron transferreactions, the ternary DPOR enzyme holocomplex comprising subunitsChlN, ChlB and ChlL was trapped as an octameric (ChlN/ChlB) 2(ChlL 2) 2complex after incubation with the non hydrolyzable ATP analogs adenosine-5´(γ-thio)-triphosphate, adenosine-5´(βγ-imido)-triphosphate or MgADP incombination with AlF 4 - . Additionally, a mutant ChlL 2 protein, with a deletedLeucin 153 in the switch-II region also allowed for the formation of a stableoctameric complex. Electron paramagnetic resonance spectroscopy ofternary DPOR complexes revealed a reduced [4Fe-4S] cluster located onChlL 2, indicating that complete ATP hydrolysis is a prerequisite forspektrum | Tagungsband 2011