Alan I. Majerník

The Methanothermobacter thermautotrophicus ExoIII homologue Mth212 is a DNA uridine endonuclease

The genome of Methanothermobacter thermautotrophicus, as a hitherto unique case, is apparently devoid of genes coding for general uracil DNA glycosylases, the universal mediators of base excision repair following hydrolytic deamination of DNA cytosine residues. We have now identified protein Mth212, a member of the ExoIII family of nucleases, as a possible initiator of DNA uracil repair in this organism. This enzyme, in addition to bearing all the enzymological hallmarks of an ExoIII homologue, is a DNA uridine endonuclease (U-endo) that nicks double-stranded DNA at the 5′-side of a 2′-d-uridine residue, irrespective of the nature of the opposing nucleotide. This type of activity has not been described before; it is absent from the ExoIII homologues of Escherichia coli, Homo sapiens and Methanosarcina mazei, all of which are equipped with uracil DNA repair glycosylases. The U-endo activity of Mth212 is served by the same catalytic center as its AP-endo activity.

DNA Content and Nucleoid Distribution in Methanothermobacter thermautotrophicus

Flow cytometry and epifluorescence microscopy results for the euryarchaeon Methanothermobacter thermautotrophicus were consistent with filaments containing multiple cells. Filaments of one to four cells contained two to eight nucleoids. Single chromosome-containing cells were not observed. Filaments containing multiple genome copies displayed synchronous DNA replication initiation. Chromosome segregation occurred during replication or rapidly after replication termination.

The presence of H+ and Na+‐translocating ATPases in Methanobacterium thermoautotrophicum and their possible function under alkaline conditions

Two ATPases with different apparent molecular masses of approx. 500 kDa and 400 kDa were identified in the EDTA extract of the cell membranes of Methanobacterium thermoautotrophicum. Western blotting with polyclonal antiserum reactive with β‐subunit of mitochondrial ATPase from rat liver and yeast was used for further analysis of these ATPases. A strong crossreactivity with a single protein band with an apparent molecular weight of about 53 kDa (similar to β‐subunit of F‐type ATPase from other sources) was found in protein extracts of whole cells of Methanobacterium thermoautotrophicum strains ΔH and Marburg, as well as of Methanospirillum hungatei. This indicates the presence of F‐type ATPase in methanogens. ATP synthesis driven by membrane potential which was generated by artificially‐imposed ΔpH in the presence of protonophorous uncoupler and sodium ions was stimulated by bafilomycin A1, an inhibitor of V‐ and A‐type ATPases, as well as by harmaline, an inhibitor of Na+/H+ antiporter. These results indicate that cells of Methanobacterium thermoautotrophicum strain ΔH contain the F‐type ATP synthase which is Na+‐translocating in addition to V‐ or A‐type ATP synthase which is H+‐translocating.

Na(+)-driven ATP synthesis in Methanobacterium thermoautotrophicum and its differentiation from H(+)-driven ATP synthesis by rhodamine 6G.

Rhodamine 6G (3 μM) effectively inhibited ΔpH‐driven ATP synthesis in Methanobacterium thermoautotrophicum while ΔpNa‐driven ATP synthesis was not affected by it. Rhodamine 6G inhibited Mg2+‐stimulated ATPase activity of membrane vesicles prepared from these cells but the ATPase catalytic sector detached from the membrane was insensitive to this inhibitor. Methanogenesis‐driven ATP synthesis at pH 6.8 of cells grown in the presence of 50 mM NaCl was inhibited by rhodamine 6G both in the presence of 5 mM and 50 mM NaCl. On the other hand, the methanogenesis‐driven ATP synthesis at pH 8.0 of cells grown in the presence of 50 mM NaCl was slightly inhibited by rhodamine 6G in the presence of 5 mM NaCl and was not inhibited at all in the presence of 50 mM NaCl. The growth experiments have shown that cells of Methanobacterium thermoautotrophicum can grow under alkaline conditions even in the presence of rhodamine 6G and of high NaCl concentration when the growth media were inoculated with the cells which had been grown in the presence of 50 mM NaCl. These results indicate that sodium‐motive force‐driven ATP synthase in Methanobacterium thermoautotrophicum operates effectively in alkaline conditions and it might be the sole ATP synthesizing system when the proton‐motive force‐supported ATP synthesis is inhibited by rhodamine 6G.

Isolation and characterization of a neomycin-resistant mutant of Methanobacterium thermoautotrophicum with a lesion in Na+-translocating ATPase (synthase)

A mutant of Methanobacterium thermoautotrophicum with a lesion in membrane Na+‐translocating ATPase (synthase) was isolated. The total ATPase activity in permeabilized cells of this mutant was elevated three‐fold as compared with the wild‐type strain. In contrast to wild‐type cells, mutant ATPase was neither inhibited by DCCD nor stimulated by Na+ ions. The methane formation rate of the mutant cells at pH 7.5 under non‐growing conditions was nearly twice that of the wild‐type strain and was stimulated by sodium ions. On the other hand, the ATP synthesis driven by methanogenesis under the same conditions was lower that of the wild‐type under the same conditions, and contrary to the wild‐type was not stimulated by Na+ ions. ATP synthesis driven by a potassium diffusion potential in the presence of sodium ions was markedly diminished in the mutant cells. The membrane potential values of the wild‐type and the mutant cells in the presence of 10 mM NaCl at pH 7.0 were comparable at energized conditions (−223 mV and −230 mV respectively). The Mg2+‐dependent ATPase activity of the 105×g supernatant of broken cells from the mutant cells was 30% higher than in the wild‐type. On the other hand, two bands with Mg2+‐dependent ATPase activity were identified by native PAGE in this fraction in both wild‐type as well as in mutant. These data suggest that the binding of Na+‐translocating ATPase (synthase) to the membrane spanning part is changed in the mutant strain.

Biochemical characteristics of a mutant of the methanoarchaeonMethanothermobacter thermautotrophicus resistant to the protonophoric uncoupler TCS

In an attempt to more closely define a protein basis of differences in ATPase and ATP synthase activities in a mutant of the methanoarchaeonMethanothermobacter thermautotrophicus resistant to the protonophoric uncoupler TCS (3,3′,4′,5-tetrachlorosalicylanilide), the composition of membrane associated proteins from the wild-type and mutant strains has been compared. The uncoupler-resistance in the mutant strain was not accompanied by changes in a protein size or changes in the level of subunits A, B andc (proteolipid) of the A1A0-type ATPase-synthase. On the other hand, we revealed a 670-kDa membrane-associated protein complex that is abundantly present only in the mutant strain; it is composed of at least 5 different subunits of 95, 52, 42, 29 and 22 kDa.

A study of the Na /H antiport in the archaeon Methanobacterium thermoautotrophicum strain ΔH

The ability of the cells of Mb. thermoautotrophicum strain ΔH to generate a proton gradient (driven by a concentration gradient of sodium ions) at pH 6.8 as well as at pH 8 was demonstrated. The electrogenic Na+/H+ antiport responsible for this process was shown to be inhibited by EIPA and also by DCCD. Artificially increasing of intracellular concentration of Ca2+ in these cells enhanced the Na+/H+ antiport activity, while the lowering of external Ca2+ by EGTA significantly decreased this activity. The apparent Km values for Na+ about 14 and 3 mM at pH 6.8 and 8, respectively, and Vmax about 214 (pH 6.8) and 155 (pH 8) ΔQ/min per mg of cell proteins, respectively, were calculated. It is concluded that the described processes are mediated by the Na+/H+ antiporter which might be a clue to the adaptive bioenergetic behaviour of the cells of Mb. thermoautotrophicum strain ΔH under the different physiological conditions.

Amiloride resistance in the methanoarcheonMethanothermobacter thermoautotrophicus: Characterization of membrane-associated proteins

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.

Membrane proteins and squalene-hydrosqualene profile in methanoarchaeon Methanothermobacter thermautotrophicus resistant to N,N'-dicyclohexylcarbodiimide.

The biochemical basis of a defective bioenergetic system was attempted to be determined in N,N′-dicyclohexylcarbodiimide (DCCD)-resistant mutant of Methanothermobacter thermautotrophicus. Components participating in the maintenance of methanoarchaeal membrane structure and function, such as the composition of the mixture of squalene and its hydrosqualene derivatives and also properties of membrane-associated proteins were compared in wild-type and mutant cells. The impairment of the bioenergetic system in DCCD-resistant mutant was detectable in the membrane-protein profile; it was also accompanied by changes in proportions of squalene-hydrosqualenes.

Isolation and characterization of a N,N′-dicyclohexylcarbodiimide-resistant mutant of Methanothermobacter thermautotrophicus with alterations to the ATP synthesis machinery

A spontaneous mutant of Methanothermobacter thermautotrophicus resistant toward the ATP-synthase inhibitor N,N′-dicyclohexylcarbodiimide (DCCD) was isolated. DCCD normally inhibits methanogenic electron-transport-driven ATP synthesis, however, the DCCD-resistant strain exhibited methanogenesis in the presence of 300 μmol/L DCCD. Total ATP synthesis was shown to be higher in the mutant strain, both in the presence and absence of DCCD. These results suggested a modification in the ATP-synthesizing system of the mutant strain. Using Blue Native PAGE combined with MALDI TOF/TOF mass spectrometry, increased concentrations of both the A1 and Ao subcomplexes of the A1Ao-type synthase were identified in the mutant strain. However, no alterations were found in the structural genes (atp) for the A1Ao ATP synthase. The results imply that DCCD resistance is a consequence of increased A1Ao ATP synthase expression, and suggest that genes involved in regulating synthase expression are responsible for DCCD resistance.