Abstract
An amiloride-resistant mutant with diminished Na+/H+ antiporter activity was isolated fromMethanothermobacter thermoautotrophicus. To define the protein basis of amiloride resistance, the composition of membrane-associated proteins was partially characterized and compared with that of the wild type strain. An abundant 670-kDa membrane-associated protein that was present only in the mutant strain was analyzed by MALDI-TOF MS and identified as a coenzyme F420-reducing hydrogenase. The amiloride resistance was not accompanied by changes in protein size or changes in the level of subunits A or B of the A1Ao-type ATP synthase; on the other hand, the SDS-PAGE patterns of the chloroform-methanol extract of membranes from both strains were different. Two bands with calculated molecular mass 16 and 11 kDa were identified as MtrD and AtpK, respectively. The observed over-expression of a 22.7-kDa protein in the mutant cells may represent the multimeric form of the MtrD subunit. These results show that the impairment of the Na+/H+ antiporter system in the amiloride-resistant mutant ofMethanothermobacter thermoautotrophicus is accompanied by only small changes in a few membrane-associated proteins.
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References
Becher B., Müller V., Gottschalk G.: The methyl-tetrahydromethanopterin: coenzyme M methyltransferase ofMethanosarcina strain Göl is a primary sodium pump.FEMS Microbiol.Lett. 91, 239–224 (1992).
Čuboňová E., Majernik A., Šmigáň P.: Biochemical characteristics of a mutant of the methanoarchaeonMethanothermobacter thermoautotrophicus resistant to the protonophoric uncoupler TCS.Folia Microbiol. 49, 147–150 (2004).
Deppenmeier U., Muller V., Gottschalk G.: Pathways of energy conservation in methanogenic bacteria.Arch.Microbiol. 165, 149–163 (1996).
Kaesler B., Schönheit P.: The sodium cycle in methanogenesis: CO2 reduction to the formaldehyde level in methanogenic bacteria is driven by a primary electrochemical potential of Na+ generated by formaldehyde reduction to CH4.Eur.J.Biochem. 186, 309–316 (1989).
Kinclová-Zimmermannová O., Flegelová H., Sychrová H.: Rice Na+/H+-antiporter Nhx1 partially complements the alkali-metal-cation sensitivity of yeast strains lacking three sodium transporters.Folia Microbiol. 49, 519–526 (2004).
Laemmli U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227, 680–685 (1970).
Lienard T., Gottschalk G.: Cloning, sequencing and expression of the genes encoding the sodium translocatingN 5-methyl-tetrahydromethanopterin:coenzyme M methyltransferase of the archaeonMethanosarcina mazei Göl.FEBS Lett. 425, 204–208 (1998).
Majernik A., Šmigáň P., Greksák M.: A study of the Na+/H+ antiport in the archaconMethanobacterium thermoautotrophicum strain δH.Biochem.Mol.Biol.Internat. 43, 123–132 (1997).
Padan E., Schuldiner S.: Molecular physiology of Na+/H+ antiporters, key transporters in circulation of Na+ and H+ in cells.Biophys.Acta 1185, 129–151 (1994).
Perski H.J., Moll J., Thauer R.K.: Sodium dependence of growth and methane formation inMethanobacterium thermoautotrophicum.Arch.Microbiol. 130, 319–321 (1981).
Perski H.J., Schonheit P., Thauer R.K.: Sodium dependence of methane formation in methanogenic bacteria.Arch.Microbiol. 143, 323–326 (1982).
Ruppert C., Wimmers S., Lemker T., Müller V.: The AlAo ATPase fromMethanosarcina mazer: cloning of the 5′ end of theaha operon encoding the membrane domain and expression of the proteolipid in a membrane-bound form inEscherichia coli.J.Bacteriol. 180, 3448–3452 (1998).
Ruppert C., Schmid R., Hedderich R., Muller V.: Selective extraction of D of the Na+-translocating methyltransferase and subunitc of the AlAo ATPase from the cytoplasmic membrane of methanogenic archaea by chloroform/methanol and characterization of subunitc ofMethanothermobacter thermoautotrophics as a 16-kDa proteolipid.FEMS Microbiol.Lett. 195, 47–51 (2001).
Skulachev V.P.: The sodium cycle: a novel type of bacterial energetics.J.Bioenergetic.Biomembr. 21, 635–647 (1989).
Skulachev V.P.: The latest news from the sodium world.Biochim.Biophys.Acta 1187, 216–221 (1994).
Šmigaň P., Polák P., Majerník A., Greksák M.: Isolation and characterization of a neomycin-resistant mutant ofMethanobacterium thermoautotrophicum with a lesion in Na+-translocating ATPase (synthase).FEBS Lett. 420, 93–96 (1997).
Šurín S., Vidová M., Majerník A., McErmott P., Chong J.P.J., Šmigáň P.: The impact of proton/sodium ion antiport deficiency in amiloride-resistant mutant ofMethanothermobacter thermoautotrophicus on the cell physiology and bioenergetic functions, in press (2006).
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This work was supported in part by a research grants (VEGA project 2/3047/23) of theSlovak Academy of Sciences and theSlovak Science and Technology Assistance Agency (APVT 51-024 904).
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Šurín, S., Čuboňová, L., Majerník, A.I. et al. Amiloride resistance in the methanoarcheonMethanothermobacter thermoautotrophicus: Characterization of membrane-associated proteins. Folia Microbiol 51, 313–316 (2006). https://doi.org/10.1007/BF02931822
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DOI: https://doi.org/10.1007/BF02931822