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Localization of Methyl-Coenzyme M Reductase As Metabolic Marker for Diverse Methanogenic Archaea

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Localization of Methyl-Coenzyme M Reductase As Metabolic Marker for Diverse Methanogenic Archaea Hindawi Publishing Corporation Archaea Volume 2013, Article ID 920241, 7 pages http://dx.doi.org/10.1155/2013/920241 Research Article Localization of Methyl-Coenzyme M Reductase as Metabolic Marker for Diverse Methanogenic Archaea Christoph Wrede,1,2 Ulrike Walbaum,1,3 Andrea Ducki,1,4 Iris Heieren,1 and Michael Hoppert1,5 1 Institute of Microbiology and Genetics, Georg-August-Universitat¨ Gottingen,¨ Grisebachstraße 8, 37077 Gottingen,¨ Germany 2 Hannover Medical School, Institute of Functional and Applied Anatomy, Carl-Neuberg-Straße 1, 30625 Hannover, Germany 3 Behavioral Ecology and Sociobiology Unit, German Primate Center, Kellnerweg 4, 37077 Gottingen,¨ Germany 4 School of Veterinary and Biomedical Sciences, Murdoch University, 90 South Street Murdoch, WA 6150, Australia 5 Courant Centre Geobiology, Georg-August-Universitat¨ Gottingen,¨ Goldschmidtstraße 3, 37077 Gottingen,¨ Germany Correspondence should be addressed to Michael Hoppert; [email protected] Received 21 September 2012; Accepted 9 January 2013 Academic Editor: J. Reitner Copyright © 2013 Christoph Wrede et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Methyl-Coenzyme M reductase (MCR) as key enzyme for methanogenesis as well as for anaerobic oxidation of methane represents an important metabolic marker for both processes in microbial biofilms. Here, the potential of MCR-specific polyclonal antibodies as metabolic marker in various methanogenic Archaea is shown. For standard growth conditions in laboratory culture, the cytoplasmic localization of the enzyme in Methanothermobacter marburgensis, Methanothermobacter wolfei, Methanococcus maripaludis, Methanosarcina mazei, and in anaerobically methane-oxidizing biofilms is demonstrated. Under growth limiting conditions on nickel-depleted media, at low linear growth of cultures, a fraction of 50–70% of the enzyme was localized close to the cytoplasmic membrane, which implies “facultative” membrane association of the enzyme. This feature may be also useful for assessment of growth-limiting conditions in microbial biofilms. 1. Introduction HS-CoBisbroughtaboutbytheenzymeheterodisulfide reductase. For the regeneration of HS-CoB, reducing equiv- Methyl-coenzyme M reductase (MCR) is the key enzyme alents are needed, provided by hydrogenases and/or dehy- of the final, methane-forming step in methanogenesis. drogenases. The reducing equivalents are either guided via The enzyme catalyses the reductive cleavage of methyl- a membrane-bound electron transport chain to the enzyme coenzyme M (CoM-S-CH3) using coenzyme B (HS-CoB) or are directly transferred from the hydrogenase to the as reductant which results in the production of methane heterodisulfide reductase. The reactions are also coupled to and the heterodisulfide CoM-S-S-CoB. Though the involved chemiosmotic mechanisms, resulting in the generation of + enzymecomplexesaswellasthereactantsdifferbetween ATPviaaH -potential [4–6]. Like MtrE, the heterodisulfide Methanosarcinales, Methanobacteriales, and other groups of reductase is a part of a membrane-bound complex. The methanogens, the essential reaction steps are similar and methyl-coenzymeMreductasereactionstepitselfisnot require several membrane-dependent steps (see [1, 2]for membrane-dependent. The enzyme has been purified from review). The formation of methyl-coenzyme M is catalysed thecytoplasmicfractionsofmethanogenicArchaeaand by one subunit (MtrE) of a membrane-bound complex (the has been localized in the cytoplasm by immunoelectron 5 N -methyl-tetrahydromethanopterin:coenzyme M methyl- microscopy. The catalytic reaction does not depend on transferase) and is coupled with energy conservation via the addition of membrane preparations [7–11]. A number an electrochemical sodium potential across the cytoplasmic of experiments, however, indicate that there is a certain membrane (see [3] for review). Regeneration of the reductant affinity of the enzyme to the membrane [12, 13]. MCR 2 Archaea 94 Table 1: Partitioning of MCR as revealed by immunolocalization. 67 Approximate Concentration %markers Organism doubling of levulinic acid at the time (h) in the medium membrane 43 26 0.0 17 (kDa) Methanothermobacter marburgensis 35 0.05 36 30 38 0.2 70 Methanothermobacter 34 0.0 15 20 wolfei 42 0.1 52 ∗ Methanosarcina 20 0.0 32 Ms fr wo-Ni Mt wo Ms ma mazei (DSM 3647) Mt mb Mc ma Mt mb 34 0.05 60 Mt Ms ma-Ni Mt mb-Ni Mt Figure 1: Specificity of the polyclonal serum used for immunolo- calization. The slots depict crude extracts of the organisms after Western blotting of SDS gels and double-immunoperoxidase pre- frisia)andMethanosarcina mazei (DSM 3647) were grown cipitation. All slots show the typical pattern of MCR. For most heterotrophically [24, 25]. Nickel-limited media did not organisms (except Ms ma and Mt mb), only the two larger of contain nickel salts in trace element solutions and were the three MCR subunits are visible. When cells were grown on supplemented with up to 200 mM levulinic acid (cf. Table 1). nickel-depleted media, the respective slot is marked with -Ni. The For immunolocalization, cells were grown in batch cultures second slot (marked with an asterisk) shows a silver stained SDS- at linear growth rates with approximate doubling times polyacrylamide gel of the purified enzyme. Mt mb: Methanother- between 25 and 45 h (Table 1). Cell disruption was performed 2 mobacter marburgensis, Mt wo: Methanothermobacter wolfei, Mc with a French pressure cell operated at 1,500 lb/in and ∘ ma: Methanococcus maripaludis, Ms fr: Methanosarcina mazei (DSM subsequent centrifugation by 15,000 ×gfor25minat4Cin 3318, formerly Methanosarcina frisia), Ms ma: Methanosarcina mazei order to remove cell debris. The supernatant was used for (DSM 3647). Western-blotting (see below). For protein purification, cells of Methanothermobacter marburgensis were grown in 14 l- fermenters with a doubling time of 2.9 h in the exponential of Methanothermobacter marburgensis was located at the phase on mineral salt medium and continuous gassing with cytoplasmic membrane under nickel-depleted growth condi- H2/CO2 (80%/20%, v/v) as described [20]. Purification of tions. Also electron microscopy of vesicle preparations from MCR was performed according to [7]. The purified protein Methanobacteriales and Methanosarcina showed that at least a (MCR, i.e. the isoform I of methyl-coenzyme M reductase, fraction of MCR is membrane-associated. From these data, it Figure 1)wasusedforproductionofpolyclonalantisera[26]. was deduced that MCR might be part of a membrane-bound Proteinpurityandspecificityoftheantiserawastestedby multienzyme complex [14, 15]. SDS polyacrylamide gel electrophoresis and Western blotting For the reverse process, the anaerobic oxidation of [27–29] and by immunolocalization control experiments (see methane, a reverse operating methanogenic pathway has below, [30]). Protein assays were performed according to [31]. been postulated, with an MCR structurally very similar to the Samples of an environmental methane-oxidizing biofilms canonical enzyme [16–18].Inthepostulatedpathway,again, were obtained and processed as described [32, 33]. Microbial membrane binding is not necessarily required. However, as mat samples were collected in 2001 during a cruise with the in methanogenesis, membrane association might also be of Russian R/V “Professor Logachev” from the methane seep advantage, since the same membrane-dependent processes as arealocatedontheNW’Shelfregion(CrimeanShelf)in in methanogenesis are likely [17, 19]. the Black Sea. Material for transmission electron microscopy In Methanobacterium thermoautotrophicum,twodiffer- and immunofluorescence analyses was chemically fixed ina ∘ ent localizations of the MCR could already be shown [13]. In 4.0% (w/v) formaldehyde solution and kept at 4 Cin100mM ourstudy,weshowthattheseresultsarealsotrueforother PBS (phosphate-buffered saline, pH 7.0). The samples were methanogens, and we will discuss these results in view of washed several times in PBS and fixed in 0.3% (v/v) solution immunolocalization of the key enzyme MCR for studies in of glutardialdehyde and 0.5% (w/v) formaldehyde in PBS for ∘ environmental biofilms. 2hat4 C. The samples were then washed three times in PBS supplemented with 10 mM glycin. See below for subsequent 2. Materials and Methods dehydration and resin embedding. Active cultures were chemically fixed anaerobically by Methanothermobacter marburgensis (DSM 2133, formerly adding 0.2% (v/v) solution of glutardialdehyde and 0.3% Methanobacterium thermoautotrophicum, strain Marburg), (w/v) formaldehyde to the active culture under anaerobic ∘ Methanothermobacter wolfei (DSM 2970, formerly Metha- conditions. After incubation for 2 h at 4 C, the culture was nobacterium wolfei), and Methanococcus maripaludis (DSM centrifuged three times for 10 min at 9.000 ×gandresus- 2067) were grown autotrophically as described [20–23]. pended in PBS supplemented with 10 mM glycin. Molten ∘ Methanosarcina mazei (DSM 3318, formerly Methanosarcina agar (2%, w/v, 50 C)wasaddedtoanequalvolumeofthe Archaea 3 400 nm 400 nm (a) (b) 200 nm 200 nm (c) (d) Figure 2: Ultrathin sections of Methanosarcina mazei (DSM 3318, formerly Methanosarcina frisia (a, b) and Methanococcus maripaludis (c, d) grown on media without nickel depletion. Immunolabeled cells (b, d; a, c are negative controls) show cytoplasmic localization of MCR. The inset in (b)
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