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 Methyl-coenzyme M reductase (MCR) is the key enzyme of the final, methane-forming step in methanogenesis. The enzyme catalyses the reductive cleavage of methyl-coenzyme M (CoM-S-CH3) using coenzyme B (HS-CoB) as reductant which results in the production of methane and the heterodisulfide CoM-S-S-CoB. Though the involved enzyme complexes as well as the reactants differ between Methanosarcinales, Methanobacteriales, and other groups of methanogens, the essential reaction steps are similar and require several membrane-dependent steps (see [1, 2] for review). The formation of methyl-coenzyme M is catalysed by one subunit (MtrE) of a membrane-bound complex (the N5-methyl-tetrahydromethanopterin:coenzyme M methyltransferase) and is coupled with energy conservation via an electrochemical sodium potential across the cytoplasmic membrane (see [3] for review). Regeneration of the reductant HS-CoB is brought about by the enzyme heterodisulfide reductase. For the regeneration of HS-CoB, reducing equivalents are needed, provided by hydrogenases and/or dehydrogenases. The reducing equivalents are either guided via a membrane-bound electron transport chain to the enzyme or are directly transferred from the hydrogenase to the heterodisulfide reductase. The reactions are also coupled to chemiosmotic mechanisms, resulting in the generation of ATP via a H+-potential [4–6]. Like MtrE, the heterodisulfide reductase is a part of a membrane-bound complex. The methyl-coenzyme M reductase reaction step itself is not membrane-dependent. The enzyme has been purified from the cytoplasmic fractions of methanogenic Archaea and has been localized in the
References
[1]
R. K. Thauer, “Biochemistry of methanogenesis: a tribute to Marjory Stephenson,” Microbiology, vol. 144, no. 9, pp. 2377–2406, 1998.
[2]
R. K. Thauer, A. K. Kaster, H. Seedorf, W. Buckel, and R. Hedderich, “Methanogenic archaea: ecologically relevant differences in energy conservation,” Nature Reviews Microbiology, vol. 6, no. 8, pp. 579–591, 2008.
[3]
G. Gottschalk and R. K. Thauer, “The Na+-translocating methyltransferase complex from methanogenic archaea,” Biochimica et Biophysica Acta, vol. 1505, no. 1, pp. 28–36, 2001.
[4]
E. Stupperich, A. Juza, M. Hoppert, and F. Mayer, “Cloning, sequencing and immunological characterization of the corrinoid-containing subunit of the N5-methyltetrahydromethanopterin:coenzyme-M methyltransferase from Methanobacterium thermoautotrophicum,” European Journal of Biochemistry, vol. 217, no. 1, pp. 115–121, 1993.
[5]
U. Deppenmeier, “Redox-driven proton translocation in methanogenic archaea,” Cellular and Molecular Life Sciences, vol. 59, no. 9, pp. 1513–1533, 2002.
[6]
A. Stojanowic, G. J. Mander, E. C. Duin, and R. Hedderich, “Physiological role of the F420-non-reducing hydrogenase (MvH) from Methanothermobacter marburgensis,” Archives of Microbiology, vol. 180, no. 3, pp. 194–203, 2003.
[7]
R. P. Hausinger, W. H. Orme-Johnson, and C. Walsh, “Nickel tetrapyrrole cofactor F430: comparison of the forms bound to methyl coenzyme M reductase and protein free in cells of Methanobacterium thermoautotrophicum ΔH,” Biochemistry, vol. 23, no. 5, pp. 801–804, 1984.
[8]
I. Thomas, H.-C. Dubourguier, G. Prensier, P. Debeire, and G. Albagnac, “Purification of component C from Methanosarcina mazei and immunolocalization in Methanosarcinaceae,” Archives of Microbiology, vol. 148, no. 3, pp. 193–201, 1987.
[9]
J. Ellermann, R. Hedderich, R. B？cher, and R. K. Thauer, “The final step in methane formation. Investigations with highly purified methyl-CoM reductase (component C) from Methanobacterium thermoautotrophicum (strain Marburg),” European Journal of Biochemistry, vol. 172, no. 3, pp. 669–677, 1988.
[10]
J. Ellermann, S. Rospert, R. K. Thauer et al., “Methyl-coenzyme-M reductase from Methanobacterium thermoautotrophicum (strain Marburg)-purity, activity and novel inhibitors,” European Journal of Biochemistry, vol. 184, no. 1, pp. 63–68, 1989.
[11]
L. G. Bonacker, S. Baudner, and R. K. Thauer, “Differential expression of the two methyl-coenzyme M reductases in Methanobacterium thermoautotrophicum as determined immunochemically via isoenzyme-speficic antisera,” European Journal of Biochemistry, vol. 206, no. 1, pp. 87–92, 1992.
[12]
R. Ossmer, T. Mund, and P. L. Hartzell, “Immunocytochemical localization of component C of the methylreductase system in Methanococcus voltae and Methanobacterium thermoautotrophicum,” Proceedings of the National Academy of Sciences of the United States of America, vol. 83, no. 16, pp. 5789–5792, 1986.
[13]
H. C. Aldrich, D. B. Beimborn, M. Bokranz, and P. Sch？nheit, “Immunocytochemical localization of methyl-coenzyme M reductase in Methanobacterium thermoautotrophicum,” Archives of Microbiology, vol. 147, no. 2, pp. 190–194, 1987.
[14]
F. Mayer, M. Rohde, M. Salzmann, A. Jussofie, and G. Gottschalk, “The methanoreductosome: a high-molecular-weight enzyme complex in the methanogenic bacterium strain G？1 that contains components of the methylreductase system,” Journal of Bacteriology, vol. 170, no. 4, pp. 1438–1444, 1988.
[15]
M. Hoppert and F. Mayer, “Electron microscopy of native and artificial methylreductase high-molecular-weight complexes in strain G？ 1 and Methanococcus voltae,” FEBS Letters, vol. 267, no. 1, pp. 33–37, 1990.
[16]
M. Krüger, A. Meyerdierks, F. O. Gl？ckner et al., “A conspicuous nickel protein in microbial mats that oxidize methane anaerobically,” Nature, vol. 426, no. 6968, pp. 878–881, 2003.
[17]
S. J. Hallam, N. Putnam, C. M. Preston et al., “Reverse methanogenesis: testing the hypothesis with environmental genomics,” Science, vol. 305, no. 5689, pp. 1457–1462, 2004.
[18]
S. Shima, M. Krüger, T. Weinert et al., “Structure of a methyl-coenzyme M reductase from Black Sea mats that oxidize methane anaerobically,” Nature, vol. 481, no. 7379, pp. 98–101, 2011.
[19]
A. Meyerdierks, M. Kube, I. Kostadinov et al., “Metagenome and mRNA expression analyses of anaerobic methanotrophic archaea of the ANME-1 group,” Environmental Microbiology, vol. 12, no. 2, pp. 422–439, 2010.
[20]
P. Sch？nheit, J. Moll, and R. K. Thauer, “Growth parameters (K(s), μ(max), Y(s)) of Methanobacterium thermoautotrophicum,” Archives of Microbiology, vol. 127, no. 1, pp. 59–65, 1980.
[21]
H. Huber, M. Thomm, H. Konig, G. Thies, and K. O. Stetter, “Methanococcus thermolithotrophicus, a novel thermophilic lithotrophic methanogen,” Archives of Microbiology, vol. 132, no. 1, pp. 47–50, 1982.
[22]
N. Belay, R. Sparling, and L. Daniels, “Dinitrogen fixation by a thermophilic methanogenic bacterium,” Nature, vol. 312, no. 5991, pp. 286–288, 1984.
[23]
J. Winter, C. Lerp, and H. P. Zabel, “Methanobacterium wolfei, sp. nov., a new tungsten-requiring, thermophilic, autotrophic methanogen,” Systematic and Applied Microbiology, vol. 5, no. 4, pp. 457–466, 1984.
[24]
H. Hippe, D. Caspari, K. Fiebig, and G. Gottschalk, “Utilization of trimethylamine and other N methyl compounds for growth and methane formation by Methanosarcina barkeri,” Proceedings of the National Academy of Sciences of the United States of America, vol. 76, no. 1, pp. 494–498, 1979.
[25]
H. K？nig and K. O. Stetter, “Isolation and characterization of Methanolobus tindarius, sp. nov., a coccoid methanogen growing only on methanol and methylamines,” Zentralblatt für Bakteriologie Mikrobiologie und Hygiene, vol. 3, no. 4, pp. 478–490, 1982.
[26]
E. Harlow and D. Lane, Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory, New York, NY, USA, 1988.
[27]
U. K. Laemmli, “Cleavage of structural proteins during the assembly of the head of bacteriophage T4,” Nature, vol. 227, no. 5259, pp. 680–685, 1970.
[28]
H. Towbin, T. Staehelin, and J. Gordon, “Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications,” Proceedings of the National Academy of Sciences of the United States of America, vol. 76, no. 9, pp. 4350–4354, 1979.
[29]
H. Blum, H. Beier, and J. H. Gross, “Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels,” Electrophoresis, vol. 8, no. 2, pp. 93–99, 1987.
[30]
M. Hoppert and A. Holzenburg, Electron Microscopy in Microbiology, Bios Scientific, Oxford, UK, 1998.
[31]
M. M. Bradford, “A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding,” Analytical Biochemistry, vol. 72, no. 1-2, pp. 248–254, 1976.
[32]
J. Reitner, J. Peckmann, M. Blumenberg, W. Michaelis, A. Reimer, and V. Thiel, “Concretionary methane-seep carbonates and associated microbial communities in Black Sea sediments,” Palaeogeography, Palaeoclimatology, Palaeoecology, vol. 227, no. 1–3, pp. 18–30, 2005.
[33]
C. Heller, M. Hoppert, and J. Reitner, “Immunological localization of coenzyme M reductase in anaerobic methane-oxidizing archaea of ANME 1 and ANME 2 type,” Geomicrobiology Journal, vol. 25, no. 3-4, pp. 149–156, 2008.
[34]
M. K？mper, S. Vetterkind, R. Berker, and M. Hoppert, “Methods for in situ detection and characterization of extracellular polymers in biofilms by electron microscopy,” Journal of Microbiological Methods, vol. 57, no. 1, pp. 55–64, 2004.
[35]
L. G. Bonacker, S. Baudner, E. M？rschel, R. B？cher, and R. K. Thauer, “Properties of the two isoenzymes of methyl-coenzyme M reductase in Methanobacterium thermoautotrophicum,” European Journal of Biochemistry, vol. 217, no. 2, pp. 587–595, 1993.
[36]
C. Wrede, V. Krukenberg, A. Dreier, J. Reitner, C. Heller, and M. Hoppert, “Detection of metabolic key enzymes of methane turnover processes in cold seep microbial biofilms,” Geomicrobiology Journal, vol. 30, no. 3, pp. 214–227, 2013.
[37]
S. Rospert, D. Linder, J. Ellermann, and R. K. Thauer, “Two genetically distinct methyl-coenzyme M reductases in Methanobacterium thermoautotrophicum strain Marburg and delta H,” European Journal of Biochemistry, vol. 194, no. 3, pp. 871–877, 1990.
[38]
A. Lehmacher and H. P. Klenk, “Characterization and phylogeny of mcrII, a gene cluster encoding an isoenzyme of methyl coenzyme M reductase from hyperthermophilic Methanothermus fervidus,” Molecular and General Genetics, vol. 243, no. 2, pp. 198–206, 1994.
[39]
C. J. Bult, O. White, G. J. Olsen et al., “Complete genome sequence of the Methanogenic archaeon, Methanococcus jannaschii,” Science, vol. 273, no. 5278, pp. 1058–1073, 1996.
[40]
E. Springer, M. S. Sachs, C. R. Woese, and D. R. Boone, “Partial gene sequences for the A subunit of methyl-coenzyme M reductase (mcrI) as a phylogenetic tool for the family Methanosarcinaceae,” International Journal of Systematic Bacteriology, vol. 45, no. 3, pp. 554–559, 1995.
[41]
R. Jaenchen, H. H. Gilles, and R. K. Thauer, “Inhibition of factor F430 synthesis by levulinic acid in Methanobacterium thermoautotrophicum,” FEMS Microbiology Letters, vol. 12, no. 2, pp. 167–170, 1981.
[42]
K. U. Hinrichs, J. M. Hayes, S. P. Sylva, P. G. Brewert, and E. F. DeLong, “Methane-consuming archaebacteria in marine sediments,” Nature, vol. 398, no. 6730, pp. 802–805, 1999.
[43]
V. J. Orphan, C. H. House, K. U. Hinrichs, K. D. McKeegan, and E. F. DeLong, “Methane-consuming archaea revealed by directly coupled isotopic and phylogenetic analysis,” Science, vol. 293, no. 5529, pp. 484–487, 2001.
[44]
M. Hoppert and F. Mayer, “Principles of macromolecular organization and cell function in bacteria and archaea,” Cell Biochemistry and Biophysics, vol. 31, no. 3, pp. 247–284, 1999.
