Ulrich Ermler

Max Planck Institute of Biophysics, Frankfurt, Hesse, Germany

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Publications (120)690.29 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: NADH-dependent reduced ferredoxin: NADP oxidoreductase (NfnAB) is found in the cytoplasm of various anaerobic bacteria and archaea. The enzyme reversibly catalyzes the endergonic reduction of ferredoxin with NADPH driven by the exergonic transhydrogenation from NADPH onto NAD+. Coupling is most probably accomplished via the mechanism of flavin-based electron bifurcation. To understand this process on a structural basis we heterologously produced the NfnAB complex of Thermotoga maritima in E. coli, provided kinetic evidence for its bifurcating behavior and determined its X-ray structure in the absence and presence of NADH. The structure of NfnAB reveals an electron transfer route including the FAD (a-FAD) and the [2Fe-2S] cluster of NfnA and the FAD (b-FAD) and the two [4Fe-4S] clusters of NfnB. Ferredoxin is presumably docked onto NfnB close to the [4Fe-4S] cluster distal to b-FAD. NAD(H) binds to a-FAD and NADP(H) consequently to b-FAD which is positioned in the centre of the NfnAB complex and the site of electron bifurcation. Arg187 is hydrogen-bonded to N5 and O4 of the bifurcating b-FAD and might play a key role in adjusting a low redox potential of the FADH radical/FAD pair required for ferredoxin reduction. A mechanism of FAD-coupled electron bifurcation by NfnAB is proposed. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 07/2015; DOI:10.1074/jbc.M115.656520 · 4.57 Impact Factor
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    ABSTRACT: In chemical synthesis, the widely used Birch reduction of aromatic compounds to cyclic dienes requires alkali metals in ammonia as extremely low-potential electron donors. An analogous reaction is catalyzed by benzoyl-coenzyme A reductases (BCRs) that have a key role in the globally important bacterial degradation of aromatic compounds at anoxic sites. Because of the lack of structural information, the catalytic mechanism of enzymatic benzene ring reduction remained obscure. Here, we present the structural characterization of a dearomatizing BCR containing an unprecedented tungsten cofactor that transfers electrons to the benzene ring in an aprotic cavity. Substrate binding induces proton transfer from the bulk solvent to the active site by expelling a Zn(2+) that is crucial for active site encapsulation. Our results shed light on the structural basis of an electron transfer process at the negative redox potential limit in biology. They open the door for biological or biomimetic alternatives to a basic chemical synthetic tool.
    Nature Chemical Biology 06/2015; DOI:10.1038/nchembio.1849 · 13.22 Impact Factor
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    ABSTRACT: [Fe]-hydrogenase (Hmd), an enzyme of the methanogenic energy metabolism, harbors an iron-guanylylpyridinol (FeGP) cofactor used for H2 cleavage. The generated hydride is transferred to methenyl-tetrahydromethanopterin (methenyl-H4 MPT(+) ). Most hydrogenotrophic methanogens contain the hmd related genes hmdII and hmdIII. Their function is still elusive. We were able to reconstitute HmdII holoenzyme of Methanocaldococcus jannaschii with recombinantly produced apoenzyme and the FeGP cofactor, which is a prerequisite for an in vitro functional analysis. Infrared spectroscopic and X-ray structural data clearly indicated binding of the FeGP cofactor. Methylene-H4 MPT binding was detectable in the significantly altered infrared spectra of the HmdII holoenzyme and in the HmdII apoenzyme-methylene-H4 MPT complex structure. The related binding mode of the FeGP cofactor and methenyl-H4 MPT(+) compared to Hmd and their multiple contacts to the polypeptide highly suggest a biological role in HmdII. However, holo-HmdII did not catalyze the Hmd reaction, not even in a single turn-over process, as demonstrated by kinetic measurements. The found inactivity can be rationalized by an increased contact area between the C- and N-terminal folding units in HmdII compared to in Hmd that impairs the catalytically necessary open-to-close transition and by an exchange of a crucial histidine to a tyrosine. Mainly based on the presented data, a function of HmdII as Hmd isoenzyme, H2 sensor, FeGP-cofactor storage protein and scaffold protein for FeGP-cofactor biosynthesis could be excluded. Inspired by the recently found binding of HmdII to aminoacyl-tRNA synthases and tRNA, we tentatively consider HmdII as a regulatory protein for protein synthesis that senses the intracellular methylene-H4 MPT concentration. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    FEBS Journal 06/2015; DOI:10.1111/febs.13351 · 3.99 Impact Factor
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    ABSTRACT: The iron-guanylylpyridinol (FeGP) cofactor of [Fe]-hydrogenase contains a prominent iron centre with an acyl-Fe bond and is the only acyl-organometallic iron compound found in nature. Here, we identify the functions of HcgE and HcgF, involved in the biosynthesis of the FeGP cofactor using structure-to-function strategy. Analysis of the HcgE and HcgF crystal structures with and without bound substrates suggest that HcgE catalyses the adenylylation of the carboxy group of guanylylpyridinol (GP) to afford AMP-GP, and subsequently HcgF catalyses the transesterification of AMP-GP to afford a Cys (HcgF)-S-GP thioester. Both enzymatic reactions are confirmed by in vitro assays. The structural data also offer plausible catalytic mechanisms. This strategy of thioester activation corresponds to that used for ubiquitin activation, a key event in the regulation of multiple cellular processes. It further implicates a nucleophilic attack onto the acyl carbon presumably via an electron-rich Fe(0)- or Fe(I)-carbonyl complex in the Fe-acyl formation.
    Nature Communications 04/2015; 6:6895. DOI:10.1038/ncomms7895 · 10.74 Impact Factor
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    ABSTRACT: Isoprenoid biosynthesis in many bacteria, plant chloroplasts and parasitic protozoa but not in humans proceeds via the mevalonate independent 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. Its penultimate reaction step is catalyzed by (E)-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate (HMBPP) synthase (GcpE/IspG) which transforms 2-C-methyl-D-erythritol-2,4-cyclo-diphosphate (MEcPP) to HMBPP. In this report we present the structure of GcpE of Thermus thermophiles in complex with its product HMBPP at a resolution of 1.65 Å. The GcpE-HMBPP like the GcpE-MEcPP structure is found in a closed, the ligand-free GcpE structure in an open enzyme state. Imposed by the rigid protein scaffold inside the active site funnel, linear HMBPP and circular MEcPP adopt highly similar conformations. The confined space also determines the conformational freedom of transition state intermediates and the design of anti-infective drugs. The apical Fe of the [4Fe-4S] cluster is coordinated to MEcPP in the GcpE-MEcPP complex and to a hydroxyl/water ligand but not to HMBPP in the GcpE-HMBPP complex. The GcpE-HMBPP structure can be attributed to one step in the currently proposed GcpE reaction cycle. Copyright © 2015 Elsevier Inc. All rights reserved.
    Biochemical and Biophysical Research Communications 02/2015; 458(2). DOI:10.1016/j.bbrc.2015.01.088 · 2.28 Impact Factor
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    ABSTRACT: The molybdenum storage protein (MoSto) can store more than 100 Mo or W atoms as discrete polyoxometalate (POM) clusters. Here, we describe the three POM cluster sites along the threefold axis of the protein complex based on four X-ray structures with slightly different polyoxomolybdate compositions between 1.35 and 2 Å resolution. In contrast to the Moα-out binding site occupied by an Mo3 cluster, the Moα-in and Moβ binding sites contain rather weak and non-uniform electron density for the Mo atoms (but clearly identifiable by anomalous data), suggesting the presence of POM cluster ensembles and/or degradation products of larger aggregates. The “Moα-in cluster ensemble” was interpreted as an antiprism-like Mo6 species superimposed with an Mo7 pyramide and the “Moβ cluster ensemble” as an Mo13 cluster (present mostly in a degraded form) composed of a pyramidal Mo7 and a Mo3 building block linked by three spatially separated MoOx units. Inside the ball-shaped Mo13 cluster sits an occluded central atom, perhaps a metal ion. POM cluster formation at the Moα-in and Moβ sites appears to be driven by filtering out and binding/protecting self-assembled transient species complementary to the protein template.
    Journal of Inorganic Biochemistry 09/2014; 138. DOI:10.1016/j.jinorgbio.2014.05.009 · 3.27 Impact Factor
  • Biochimica et Biophysica Acta (BBA) - Bioenergetics 07/2014; 1837:e83. DOI:10.1016/j.bbabio.2014.05.087 · 4.83 Impact Factor
  • Takashi Fujishiro, Ulrich Ermler, Seigo Shima
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    ABSTRACT: HcgD, a homolog of the ubiquitous Nif3-like protein family, is found in a gene cluster involved in the biosynthesis of the iron-guanylylpyridinol (FeGP) cofactor of [Fe]-hydrogenase. The presented crystal structure and biochemical analyses indicated that HcgD has a dinuclear iron-center, which provides a pronounced binding site for anionic ligands. HcgD contains a stronger and a weaker bound iron; the latter being removable by chelating reagents preferentially in the oxidized state. Therefore, we propose HcgD as an iron chaperon in FeGP cofactor biosynthesis, which might also stimulate investigations on the functionally unknown but physiologically important eukaryotic Nif3-like protein family members.
    FEBS Letters 06/2014; 588(17). DOI:10.1016/j.febslet.2014.05.059 · 3.34 Impact Factor
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    ABSTRACT: The reversible redox reaction between coenzyme F420 and H2 to F420H2 is catalyzed by a F420-reducing [NiFe]-hydrogenase (FrhABG) which is an enzyme of the energy metabolism of methanogenic archaea. FrhABG is a group 3 [NiFe]-hydrogenase with a dodecameric quaternary structure of 1.25 MDa as recently revealed by high resolution cryo electron microscopy. We report on the crystal structure of FrhABG from Methanothermobacter marburgensis at 1.7Å resolution and compare it with the structures of group 1 [NiFe]-hydrogenases, the only group structurally characterized yet. FrhA is similar to the large subunit of group 1 [NiFe]-hydrogenases regarding its core structure and the embedded [NiFe]-center but different because of the truncation of ca. 160 residues which results in similar but modified H2- and proton- transport pathways and in suitable interfaces for oligomerization. The small subunit FrhG is composed of a N-terminal domain related to group 1 enzymes and a new C-terminal ferredoxin-like domain carrying the distal and medial [4Fe-4S] clusters. FrhB adopts a novel fold, binds one [4Fe-4S] cluster as well as one FAD in a U-shaped conformation and provides the F420-binding site at the Si-face of the isoalloxazine ring. Similar electrochemical potentials of both catalytic reactions and the electron-transferring [4Fe-4S] clusters, determined to be E°' ≈ -400mV, are in full agreement with the equalized forward and backward rates of the FrhABG reaction. The protein might contribute to balanced redox potentials by the aspartate coordination of the proximal [4Fe-4S] cluster, the new ferredoxin module and a rather negatively charged FAD surrounding.
    Journal of Molecular Biology 05/2014; 426(15). DOI:10.1016/j.jmb.2014.05.024 · 4.33 Impact Factor
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    ABSTRACT: The cbb3 cytochrome c oxidases are distant members of the superfamily of heme copper oxidases. These terminal oxidases couple O2 reduction with proton transport across the plasma membrane and, as a part of the respiratory chain, contribute to the generation of an electrochemical proton gradient. Compared with other structurally characterized members of the heme copper oxidases, the recently determined cbb3 oxidase structure at 3.2 Å resolution revealed significant differences in the electron supply system, the proton conducting pathways and the coupling of O2 reduction to proton translocation. In this paper, we present a detailed report on the key steps for structure determination. Improvement of the protein quality was achieved by optimization of the number of lipids attached to the protein as well as the separation of two cbb3 oxidase isoenzymes. The exchange of n-dodecyl-β-d-maltoside for a precisely defined mixture of two α-maltosides and decanoylsucrose as well as the choice of the crystallization method had a most profound impact on crystal quality. This report highlights problems frequently encountered in membrane protein crystallization and offers meaningful approaches to improve crystal quality. © 2014 The Protein Society.
    Protein Science 04/2014; 23(4). DOI:10.1002/pro.2423 · 2.85 Impact Factor
  • Takashi Fujishiro, Ulrich Ermler, Seigo Shima
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    ABSTRACT: HcgD and HcgD bind by X-ray crystallography (View interaction)
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    ABSTRACT: Electron bifurcation is a fundamental strategy of energy coupling originally discovered in the Q-cycle of many organisms. Recently a flavin-based electron bifurcation has been detected in anaerobes, first in clostridia and later in acetogens and methanogens. It enables anaerobic bacteria and archaea to reduce the two [4Fe-4S] cluster-containing ferredoxin, an energy rich compound that is used to conduct difficult reductions as well as to increase the efficiency of substrate level and electron transport phosphorylations (SLP and ETP). Here we characterize the bifurcating electron transferring flavoprotein (EtfAf) and butyryl-CoA dehydrogenase (BcdAf) from Acidaminococcus fermentans which couple the exergonic reduction of crotonyl-CoA to butyryl-CoA to the endergonic reduction of ferredoxin both with NADH. EtfAf contains one FAD (α-FAD) in subunit α and a second FAD (β-FAD) in subunit β. The distance between the two isoalloxazine rings is 18 Angstrom. The EtfAf-NAD+ complex structure revealed β-FAD as acceptor of the hydride of NADH. The formed β-FADH- is considered as the bifurcating electron donor. Due to a conformational change, α-FAD is able to approach β-FADH- by ca. 5 Angstrom and take up one electron yielding a stable anionic semiquinone, α-FAD-, which due to a second conformational change donates this electron further to FAD of BcdAf. The remaining non-stabilized neutral semiquinone, β-FADH, immediately reduces ferredoxin. Repetition of this process affords a second reduced ferredoxin and FADH- of BcdAf that converts crotonyl-CoA to butyryl-CoA.
    Journal of Biological Chemistry 12/2013; 289(8). DOI:10.1074/jbc.M113.521013 · 4.57 Impact Factor
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    ABSTRACT: Ein Reaktionsschritt der Biosynthese des [Fe]‐Hydrogenase‐Cofaktors wurde von S. Shima et al. in ihrer Zuschrift auf S. 12787 ff. aufgeklärt. Mithilfe struktureller Genomik in Kombination mit Modellreaktionen und gründlicher Produktanalyse durch Röntgenkristallographie von Protein‐Produkt‐Komplexen wurde gefunden, dass HcgB das Enzym ist, das die Guanylylpyridinolbildung aus einem 2,4‐Dihydroxypyridinderivat und Guanosintriphosphat katalysiert.
    Angewandte Chemie 11/2013; 125(48). DOI:10.1002/ange.201308951
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    ABSTRACT: [Fe]-Hydrogenase requires the iron guanylylpyridinol (FeGP) cofactor for activity. The function of HcgB, an enzyme in the biosynthesis of the FeGP cofactor, was predicted by structural genomics and confirmed by model reactions and various analytical methods: HcgB catalyzes the terminal guanylyltransferase reaction for the formation of guanylylpyridinol. GMP=guanosine monophosphate.
    Angewandte Chemie International Edition 11/2013; 52(48):12555-8. DOI:10.1002/anie.201306745 · 11.26 Impact Factor
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    ABSTRACT: One reaction step of the biosynthesis of [Fe]‐hydrogenase‐cofactor is elucidated by S. Shima et al. in their Communication on page 12555 ff. A structural genomics approach, in combination with model reactions and thorough product analysis by X‐ray crystallography of the protein–product complexes, revealed that HcgB is the enzyme that catalyzes guanylylpyridinol formation from a 2,4‐dihydroxypyridine derivative and guanosine triphosphate.
    Angewandte Chemie International Edition 11/2013; 52(48). DOI:10.1002/anie.201308951 · 11.26 Impact Factor
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    ABSTRACT: Terpenoid precursor biosynthesis occurs in human and many pathogenic organisms via the mevalonate and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways, respectively. We determined the X-ray structure of the Fe/S containing (E)-4-hydroxy-3-methyl-but-2-enyl-diphosphate reductase (LytB) of the pathogenic protozoa Plasmodium falciparum which catalyzes the terminal step of the MEP pathway. The cloverleaf fold and the active site of P. falciparum LytB corresponds to those of the Aquifex aeolicus and Escherichia coli enzymes. Its distinct electron donor [2Fe-2S] ferredoxin was modeled to its binding site by docking calculations. The presented structural data provide a platform for a rational search of anti-malarian drugs.
    FEBS letters 11/2013; 587(24). DOI:10.1016/j.febslet.2013.10.029 · 3.34 Impact Factor
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    ABSTRACT: ATP sulfurylase (ATPS) catalyzes a key reaction in the global sulfur cycle by reversibly converting inorganic sulfate (SO4 (2-)) with ATP to adenosine 5'-phosphosulfate (APS) and pyrophosphate (PPi). In this work we report on the sat encoded dissimilatory ATP sulfurylase from the sulfur-oxidizing purple sulfur bacterium Allochromatium vinosum. In this organism, the sat gene is located in one operon and co-transcribed with the aprMBA genes for membrane-bound APS reductase. Like APS reductase, Sat is dispensible for growth on reduced sulfur compounds due to the presence of an alternate, so far unidentified sulfite-oxidizing pathway in A. vinosum. Sulfate assimilation also proceeds independently of Sat by a separate pathway involving a cysDN-encoded assimilatory ATP sulfurylase. We produced the purple bacterial sat-encoded ATP sulfurylase as a recombinant protein in E. coli, determined crucial kinetic parameters and obtained a crystal structure in an open state with a ligand-free active site. By comparison with several known structures of the ATPS-APS complex in the closed state a scenario about substrate-induced conformational changes was worked out. Despite different kinetic properties ATPS involved in sulfur-oxidizing and sulfate-reducing processes are not distinguishable on a structural level presumably due to the interference between functional and evolutionary processes.
    PLoS ONE 10/2013; 8(9):e74707. DOI:10.1371/journal.pone.0074707 · 3.53 Impact Factor
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    ABSTRACT: Heterodisulfide reductase (Hdr) is a key enzyme in the energy metabolism of methanogenic archaea. The enzyme catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) to the thiol coenzymes M (CoM-SH) and B (CoB-SH). Cleavage of CoM-S-S-CoB at an unusual FeS cluster reveals unique substrate chemistry. The cluster is fixed by cysteines of two cysteine-rich CCG domain sequence motifs (CX31-39CCX35-36CXXC) of subunit HdrB of the Methanothermobacter marburgensis HdrABC complex. We report on Q-band (34 GHz) (57)Fe electron-nuclear double resonance (ENDOR) spectroscopic measurements on the oxidized form of the cluster found in HdrABC and in two other CCG-domain-containing proteins, recombinant HdrB of Hdr from M. marburgensis and recombinant SdhE of succinate: quinone reductase from Sulfolobus solfataricus P2. The spectra at 34 GHz show clearly improved resolution arising from the absence of proton resonances and polarization effects. Systematic spectral simulations of 34 GHz data combined with previous 9 GHz data allowed the unambiguous assignment of four (57)Fe hyperfine couplings to the cluster in all three proteins. (13)C Mims ENDOR spectra of labelled CoM-SH were consistent with the attachment of the substrate to the cluster in HdrABC, whereas in the other two proteins no substrate is present. (57)Fe resonances in all three systems revealed unusually large (57)Fe ENDOR hyperfine splitting as compared to known systems. The results infer that the cluster's unique magnetic properties arise from the CCG binding motif.
    European Journal of Biochemistry 09/2013; DOI:10.1007/s00775-013-1037-x · 3.16 Impact Factor
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    ABSTRACT: Inhibition mechanism: Isocyanides strongly inhibit [Fe]-hydrogenase. X-ray crystallography and X-ray absorption spectroscopy revealed that the isocyanide binds to the trans position, versus the acyl carbon of the Fe center, and is covalently bound to the pyridinol hydroxy oxygen. These results also indicated that the hydroxy group is essential for H2 activation.
    Angewandte Chemie International Edition 09/2013; 52(37). DOI:10.1002/anie.201305089 · 11.26 Impact Factor
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    ABSTRACT: Autotrophic members of the Sulfolobales (crenarchaeota) use the 3-hydroxypropionate/4-hydroxybutyrate cycle to assimilate CO2 into cell material. The product of the initial acetyl-CoA carboxylation with CO2, malonyl-CoA, is further reduced to malonic semialdehyde by an NADPH-dependent malonyl-CoA reductase (MCR); the enzyme also catalyzes the reduction of succinyl-CoA to succinic semialdehyde onwards in the cycle. Here, we present the crystal structure of Sulfolobus tokodaii malonyl-CoA reductase in the substrate-free state and in complex with NADP+ and CoA. Structural analysis revealed an unexpected reaction cycle in which NADP+ and CoA successively occupy identical binding sites. Both coenzymes are pressed into a S-shaped, nearly superimposable structure imposed by a fixed and preformed binding site. The template-governed cofactor shaping implicates the same binding site for the 3- and 2- ribose phosphate group of CoA and NADP+, respectively, but a different one for the common ADP part: the β-phosphate of CoA aligns with the α-phosphate of NADP+. Evolution from an NADP+ to a bispecific NADP+ and CoA binding site involves many amino acid exchanges within a complex process by which constraints of the CoA structure also influence NADP+ binding. Based on the paralogous aspartate-semialdehyde dehydrogenase structurally characterized with a covalent Cys-aspartyl adduct, a malonyl/succinyl group can be reliably modelled into MCR and discussed regarding its binding mode, the malonyl/succinyl specificity, and the catalyzed reaction. The modified polypeptide surrounding around the absent ammonium group in malonate/succinate compared to aspartate provides the structural basis for engineering a methylmalonyl-CoA reductase applied for biotechnical polyester building block synthesis.
    Journal of Biological Chemistry 01/2013; 288(9). DOI:10.1074/jbc.M112.421263 · 4.57 Impact Factor

Publication Stats

4k Citations
690.29 Total Impact Points

Institutions

  • 1995–2015
    • Max Planck Institute of Biophysics
      • Department of Molecular Membrane Biology
      Frankfurt, Hesse, Germany
  • 2012
    • French National Centre for Scientific Research
      Lutetia Parisorum, Île-de-France, France
  • 2011
    • University of Freiburg
      • Institute of Biology I
      Freiburg, Lower Saxony, Germany
  • 2010
    • Goethe-Universität Frankfurt am Main
      Frankfurt, Hesse, Germany
  • 2009
    • Max Planck Institute for Terrestrial Microbiology
      Marburg, Hesse, Germany
  • 2006
    • Universität Konstanz
      • Faculty of Sciences
      Konstanz, Baden-Wuerttemberg, Germany
  • 1997–2004
    • Philipps-Universität Marburg
      • Fachbereich Biologie
      Marburg an der Lahn, Hesse, Germany
  • 2001
    • Universität Regensburg
      • Lehrstuhl für Mikrobiologie
      Ratisbon, Bavaria, Germany