Showing papers on "Methanosarcina barkeri published in 1992"

Salt dependence, kinetic properties and catalytic mechanism of N-formylmethanofuran:tetrahydromethanopterin formyltransferase from the extreme thermophile Methanopyrus kandleri.

Abstract: N-Formylmethanofuran(CHO-MFR): tetrahydromethanopterin(H4MPT) formyltransferase (for-myltransferase) from the extremely thermophilic Methanopyrus kandleri was purified over 100-fold to apparent homogeneity with a 54% yield. The monomeric enzyme had an apparent molecular mass of 35 kDa. The N-terminal amino acid sequence of the polypeptide was determined. The formyltransferase was found to be absolutely dependent on the presence of phosphate or sulfate salts for activity. The ability of salts to activate the enzyme decreased in the order K2HPO4 > (NH4)2SO4 > K2SO4 > Na2SO4 > Na2HPO4. The salts KCl, NaCl and NH4Cl did not activate the enzyme. The dependence of activity on salt concentration showed a sigmoidal curve. For half-maximal activity, 1 M K2HPO4 and 1.2 M (NH4)2SO4 were required. A detailed kinetic analysis revcaled that phosphates and sulfates both affected the Vmax rather than the Km for CHO-MFR and H4MPT. At the optimal salt concentration and at 65°C, the Vmax was 2700 U/mg (1 U = 1 μmol/min), the Km for CHO-MFR was 50 μM and the Km for H4MPT was 100 μM. At 90°C, the temperature optimum of the enzyme, the Vmax was about 2.5-fold higher than at 65°C. Thermostability as well as activity of formyltransferase was dramatically increased in the presence of salts, 1.5 M being required for optimal stabilization. The efficiency of salts in protecting formyltransferase from heat inactivation at 90°C decreased in the order K2HPO4= (NH4)2SO4≫ KCI = NH4Cl = NaCl ≫ Na2SO4 > Na2HPO4. The catalytic mechanism of formyltransferase was determined to be of the ternary-complex type. The properties of the enzyme from M. kandleri are compared with those of formyltransferase from Methanobacterium thermoautotrophicum, Methanosarcina barkeri and Archaeoglobus fulgidus.

Evidence for the involvement of corrinoids and factor F430 in the reductive dechlorination of 1,2-dichloroethane by Methanosarcina barkeri.

Abstract: Cobalamin and the native and diepimeric forms of factor F430 catalyzed the reductive dechlorination of 1,2-dichloroethane (1,2-DCA) to ethylene or chloroethane (CA) in a buffer with Ti(III) citrate as the electron donor. Ethylene was the major product in the cobalamin-catalyzed transformation, and the ratio of ethylene to CA formed was 25:1. Native F430 and 12,13-di-epi-F430 produced ethylene and CA in ratios of about 2:1 and 1:1, respectively. Cobalamin dechlorinated 1,2-DCA much faster than did factor F430. Dechlorination rates by all three catalysts showed a distinct pH dependence, correlated in a linear manner with the catalyst concentration and doubled with a temperature increase of 10 degrees C. Crude and boiled cell extracts of Methanosarcina barkeri also dechlorinated 1,2-DCA to ethylene and CA with Ti(III) citrate as the reductant. The catalytic components in boiled extracts were heat and oxygen stable and had low molecular masses. Fractionation of boiled extracts by a hydrophobic interaction column revealed that part of the dechlorinating components had a hydrophilic and part had a hydrophobic character. These chemical properties of the dechlorinating components and spectral analysis of boiled extracts indicated that corrinoids or factor F430 was responsible for the dechlorinations. The ratios of 3:1 to 7:1 of ethylene and CA formed by cell extracts suggested that both cofactors were concomitantly active.

N5-methyltetrahydromethanopterin: coenzyme M methyltransferase in methanogenic archaebacteria is a membrane protein.

Abstract: An assay is described that allows the direct measurement of the enzyme activity catalyzing the transfer of the methyl group from N5-methyltetrahydromethanopterin (CH3−H4MPT) to coenzyme M (H−S−CoM) in methanogenic archaebacteria. With this method the topology, the partial purification, and the catalytic properties of the methyltransferase in methanol- and acetate-grown Methanosarcina barkeri and in H2/CO2-grown Methanobacterium thermoautotrophicum were studied. The enzyme activity was found to be associated almost completely with the membrane fraction and to require detergents for solubilization. The transferase activity in methanol-grown M. barkeri was studied in detail. The membrane fraction exhibited a specific activity of CH3−S−CoM formation from CH3−H4MPT (apparent Km=50 μM) and H−S−CoM (apparent Km=250 μM) of approximately 0.6 μmol·min-1·mg protein-1. For activity the presence of Ti(III) citrate (apparent Km=15 μM) and of ATP (apparent Km=30 μM) were required in catalytic amounts. Ti(III) could be substituted by reduced ferredoxin. ATP could not be substituted by AMP, CTP, GTP, S-adenosylmethionine, or by ATP analogues. The membrane fraction was methylated by CH3−H4MPT in the absence of H−S−CoM. This methylation was dependent on Ti(III) and ATP. The methylated membrane fraction catalyzed the methyltransfer from CH3−H4MPT to H−S−CoM in the absence of ATP and Ti(III). Demethylation in the presence of H−S−CoM also did not require Ti(III) or ATP. Based on these findings a mechanism for the methyltransfer reaction and for the activation of the enzyme is proposed.

Effect of coculture of anaerobic fungi isolated from ruminants and non-ruminants with methanogenic bacteria on cellulolytic and xylanolytic enzyme activities.

Abstract: Neocallimastix strain N1, an isolate from a ruminant (sheep), was cocultured with three Methanobacterium formicicum strains, Methanosarcina barkeri, and Methanobrevibacter smithii. The coculture with Methanobacterium formicicum strains resulted in the highest production of cellulolytic and xylanolytic enzymes. Subsequently four anaerobic fungi, two Neocallimastix strains (N1 and N2) from a ruminant and two Piromyces species from non-ruminants (E2 and R1), were grown in coculture with Methanobacterium formicicum DSM 3637 on filter paper cellulose and monitored over a 7-day period for substrate utilisation, fermentation products, and secretion of cellulolytic and xylanolytic enzymes. Methanogens caused a shift in fermentation products to more acetate and less ethanol, lactate and succinate. Furthermore the cellulose digestion rate increased by coculture. For cocultures of Neocallimastix strains with Methanobacterium formicicum strains the cellulolytic and xylanolytic enzyme production increased. Avicelase, CMCase and xylanase were almost completely secreted into the medium, while 40-60% of the beta-glucosidase was found to be cell bound. Coculture had no significant effect on the location of cellulolytic and xylanolytic enzymes.

Dehalogenation of trichlorofluoromethane (CFC‐11) by Methanosarcina barkeri

Abstract: Methanobacterium barkeri was found to catalyze the reductive dehalogenation of trichlorofluoromethane (CFC-11), also known as FREON 11 Products detected were CHFCI2, CH2FCI, CO and fluoride

Taxonomy and Halotolerance of Mesophilic Methanosarcina Strains, Assignment of Strains to Species, and Synonymy of Methanosarcina mazei and Methanosarcina frisia

Abstract: We examined 22 previously described and newly isolated Methanosarcina strains by performing denaturing gel electrophoresis of whole-cell proteins and assigned these strains to previously described species. Methanosarcina mazei S-6T (T = type strain) and Methanosarcina frisia C 16T were very similar in terms of the electrophoresis patterns of their proteins and in their DNA sequences (the results of reassociation experiments indicated that there was 77% sequence similarity). Thus, M. frisia is a junior subjective synonym of M. mazei, and strain C 16 is a reference strain of M. mazei. M. mazei C 16 was similar to M. mazei in other characteristics that have not been reported previously, including the ability to catabolize acetate and a lack of halophily. All of the Methanosarcina strains examined, including the marine strains M. mazei C 16 (= M. frisia C 16T) and Methanosarcina acetivorans C2AT, were slightly halotolerant (rather than halophilic, as originally described). Methanosarcina sp. strain FR-1, which has gas vesicles, was more similar to Methanosarcina barkeri MST than to Methanosarcina vacuolata Z-761T in both its protein patterns and its DNA sequence (80% similarity to M. barkeri MST and 38% similarity to M. vacuolata Z-761T). Thus, the presence of gas vesicles is not an adequate taxonomic characteristic for assigning Methanosarcina strains to M. vacuolata.

Microbial dehalogenation using methanosarcina

Abstract: A method for bioremediation of hazardous wastes is disclosed. The method can be used for anaerobic treatment of a liquid or slurry hazardous waste stream (e.g., industrial wastewater or sludge) or for treatment of contaminated groundwater. Removal of halogenated (e.g., chlorinated) hydrocarbons, such as tetrachloroethylene, trichloroethylene, 1,1,1-trichloroethane and similar xenobiotics is possible. The method involves biotransformation of (dehalogenation) halogenated hydrocarbons by means of natural methanogenic bacteria, Methanosarcina barkeri strain 227 and/or Methanosarcina vacuolata. These methanogens can accomplish cometabolism of chlorinated hydrocarbons during metabolism of a primary substrate such as hydrogen-carbon dioxide, methanol, methylamine, dimethylamine, trimethylamine and acetate. Reductive dechlorination, energy conservation and control of air pollution are accomplished.

Effect of magnesium on methanogenic subpopulations in a thermophilic acetate-degrading granular consortium.

Abstract: The effects of Mg2+ on thermophilic (55 degrees C) granules grown on acetate in 0.2-liter upflow anaerobic sludge blanket reactors were studied. The methanogens in the granules were identified and counted by using antibody probes and the antigenic fingerprinting method. Packets of large coccoidal cells antigenically related to Methanosarcina thermophila TM-1 were scarce in the absence of Mg2+ but increased with increasing Mg2+ concentrations up to 30 mM; Methanosarcina packets immunologically related to Methanosarcina barkeri R1M3 showed a similar trend, and their numbers increased up to 100 mM Mg2+. The number of single cells antigenically related to TM-1, R1M3, and Methanosarcina mazei S-6 were scarce at low Mg2+ concentrations but increased drastically at 30 and 100 mM Mg2+. The number of rod-shaped bacteria antigenically related to Methanobacterium thermoautotrophicum GC1 and delta H was highest with no Mg2+ present, and their numbers decreased with increasing concentrations of the cation. These quantitative data, obtained by counting cells in suspensions made from disrupted granules, were confirmed by microscopic observation of the methanogenic subpopulations in thin histologic sections of the granules. Images

Identification of degradation artifacts formed upon treatment of hydroxydiether lipids from methanogens with methanolic HCl

Abstract: Treatment of purified 3-hydroxydiether lipid from Methanosarcina barkeri by standard conditions for head-group removal (2.5% methanolic HCl, 70 °C) resulted in conversion to six identifiable degradation products. The four most abundant products were identified by mass spectrometry and nuclear magnetic resonance as monophytanylglycerol, 3-methoxydiether, and cis–trans isomers of a diether unsaturated between carbons 3 and 4. The latter lipid products may be mistaken for 2,3-di-O-phytanyl-sn-glycerol (standard diether) and 2-O-sesterterpanyl-3-O-phytanyl-sn-glycerol when separated by thin-layer chromatography. Key words: hydroxydiether lipids, degradation artifacts, methanolic HCl, Methanosarcina barkeri.

Purification and enzymic characterization of the cytoplasmic pyrophosphatase from the thermoacidophilic archaebacterium Thermoplasma acidophilum

Abstract: Cytoplasmic pyrophosphatase has been isolated from the thermoacidophilic archaebacterium Thermoplasma acidophilum. The enzyme was purified to electrophoretic homogeneity by combining ion-exchange and affinity-chromatographic separations. This soluble pyrophosphatase probably consists of six identical subunits, since SDS/PAGE gave an estimate of about 22 kDa for a single subunit and size-exclusion chromatography under non-denaturing conditions indicates a molecular mass of 110 ± 5 kDa. The two most prominent catalytic features of this enzyme are the absolute requirement for divalent cations for catalytic action, Mg2+ conferring the highest activity, and the pronounced specificity for PPi. The catalytic behavior apparently follows simple Michaelis-Menten kinetics with a Km of about 7 μM for PPi and a specific acitivity of about 1200 U/mg at 56°C. Surprisingly, maximum activity could be observed at 85°C which is more than 20°C above the temperature for optimal growth. Several cytoplasmic extracts of eubacteria and archaebacteria have been probed with a polyclonal antiserum raised against the purified archaebacterial protein. The only noticeable cross-reactivity could be detected with an extract from the methanogen Methanosarcina barkeri although this probably does not reflect the inferred phylogenetic relationship between methanogens and Thermoplasma acidophilum.

Properties Of The Plasma Membrane Atpases Of The Halophilic Archaebacteria Haloferax Mediterranei And Haloferax Volcanii

Abstract: Both, Haloferax mediterranei and Haloferax volcanii membranes contain ATPases which are capable of hydrolyzing ATP in presence of Mg²⁺ or Mn²⁺. The ATPases require high concentrations of NaCl, a pH value of 9, and high temperatures up to 60 °C. Free manganese ions inhibited the enzyme activity of either ATPase. The ATPases of Hf. mediterranei and Hf. volcanii, respectively, show different sensitivities to inhibitors of ATP hydrolysis. ATP hydrolysis of isolated Hf. mediterranei ATPase was inhibited by NaN₃, which was reported to be specific for F-ATPases, by nitrate and N-ethylmaleimide (NEM), which are specific inhibitors of V-ATPases. ATP hydrolysis of Haloferax mediterranei membranes was not inhibited by DCCD , but [¹⁴C]DCCD was bound to a 14 kDa peptide of the isolated, partially purified enzyme. Furthermore, the ATPase was inactivated by preincubation with 7-chloro-4-nitrobenzofurazan (NBD-Cl). The ATPase activity of Hf. volcanii membranes was inhibited by NEM but not by nitrate and NaN₃. SDS gel electrophoresis of the partially purified enzyme of Haloferax mediterranei showed putative ATPase subunits of 53.5, 49, 42, 22, 21, 14, 12, and 7.5 kDa. Immunoblots showed cross reactivity between a 53 kDa peptide and anti-β (chloroplast F₁), as well as between 53, 50 and 47 kDa peptides and an ATPase antibody of Methanosarcina barkeri. The results will be discussed in context with the placement of the archaebacterial ATPases (A-ATPases) between F- and V-ATPases

5-Formyl-5,6,7,8-tetrahydromethanopterin is the intermediate in the process of methanogenesis in Methanosarcina barkeri.

Abstract: Formylmethanofuran:tetrahydromethanopterin (H4MPT) formyltransferase and 5,10-methenyl-H4MPT cyclohydrolase purified from Methanosarcina barkeri catalyze a formyl group transfer and the hydrolysis of the methenyl function, respectively. The results from UV spectroscopy and HPLC analyses, and comparison with results obtained with the enzymes isolated from Methanobacterium thermoautotrophicum showed 5-formyl-H4MPT to be the product of the formyltransferase and cyclohydrolase reactions in M. barkeri. The findings disagree with an earlier report in which 10-formyl-H4MPT was identified as the product of the cyclohydrolase in the latter organism. In addition, it was observed that 10-formyl-H4MPT, which is non-enzymically formed from 5,10-methenyl-H4MPT at alkaline pH, becomes rapidly converted into the 5-formyl derivative. The latter finding explains why the nature of the formyl species previously had been improperly assigned.