Showing papers on "Methanosarcina barkeri published in 1988"

Structure and comparative analysis of the genes encoding component C of methyl coenzyme M reductase in the extremely thermophilic archaebacterium Methanothermus fervidus.

Abstract: A 6-kilobase-pair (kbp) region of the genome of the extremely thermophilic arachaebacterium Methanothermus fervidus which encodes the alpha, beta, and gamma subunit polypeptides of component C of methyl coenzyme M reductase was cloned and sequenced. Genes encoding the beta (mcrB) and gamma (mcrG) subunits were separated by two open reading frames (designated mcrC and mcrD) which encode unknown gene products. The M. fervidus genes were preceded by ribosome-binding sites, separated by short A + T-rich intergenic regions, contained unexpectedly few NNC codons, and exhibited inflexible codon usage at some locations. Sites of transcription initiation and termination flanking the mcrBDCGA cluster of genes in M. fervidus were identified. The sequences of the genes, the encoded polypeptides, and transcription regulatory signals in M. fervidus were compared with the functionally equivalent sequences from two mesophilic methanogens (Methanococcus vannielii and Methanosarcina barkeri) and from a moderate thermophile (Methanobacterium thermoautotrophicum Marburg). The amino acid sequences of the polypeptides encoded by the mcrBCGA genes in the two thermophiles were approximately 80% identical, whereas all other pairs of these gene products contained between 50 and 60% identical amino acid residues. The mcrD gene products have diverged more than the products of the other mcr genes. Identification of highly conserved regions within mcrA and mcrB suggested oligonucleotide sequences which might be developed as hybridization probes which could be used for identifying and quantifying all methanogens.

Electron-transport-driven sodium extrusion during methanogenesis from formaldehyde and molecular hydrogen by Methanosarcina barkeri

Abstract: Methanogenesis from formaldehyde or formaldehyde + H2, as carried out by Methanosarcina barkeri, was strictly dependent on sodium ions whereas methane formation from methanol + H2 or methanol + formaldehyde was Na+-independent. This indicates that the reduction of formaldehyde to the formal redox level of methanol exhibits a Na+ requirement. During methanogenesis from formaldehyde, a ΔpNa in the range of -62 mV to -80 mV was generated by means of a primary, electron-transport-driven sodium pump. This could be concluded from the following results obtained on cell suspensions of M. barkeri. 1 The addition of proton conductors or inhibitors of the Na+/H+ antiporter had no effect on sodium extrusion. 2 During methanogenesis from formaldehyde + H2 a ΔΨ of -60 mV to -70 mV was generated even in the presence of proton conductors. 3 ATPase inhibitors, applied in the presence of proton conductors, had no effect on primary sodium extrusion or generation of a ΔΨ. Evidence for a Na+-translocating ATPase could not be obtained.

Comparative analysis of genes encoding methyl coenzyme M reductase in methanogenic bacteria.

Abstract: The sequence of the gene cluster encoding the methyl coenzyme M reductase (MCR) in Methanococcus voltae was determined. It contains five open reading frames (ORF), three of which encode the known enzyme subunits. Putative ribosome binding sites were found in front of all ORFs. They differ in their degrees of complementarity to the 3′ end of the 16 S rRNA, which is discussed in terms of different translation efficiencies of the respective genes. The codon usage bias is different in the subunit encoding genes compared with the two other ORFs in the cluster and two other known genes of Mc. voltae. This is interpreted in terms of increased translational accuracy of the highly expressed MCR subunit genes. The derived polypeptide sequences encoded by the five ORFs of the MCR cluster were compared to those of the respective genes in Methanobacterium thermoautotrophicum Marburg and Methanosarcina barkeri. Conserved regions were detected in the enzyme subunits, which are candidates for factor binding domains. Conserved hydrophobic sequences found in the α and β subunits are discussed with respect to the membrane association of the enzyme.

The transmembrane electrochemical gradient of Na+ as driving force for methanol oxidation in Methanosarcina barkeri.

Abstract: A sodium ion gradient (inside low) across the cytoplasmic membrane of Methanosarcina barkeri was required for methanogenesis from methanol. This could be concluded from the following results. (a) Inhibition of the Na+/H+ antiporter by K+ or amiloride led to an inhibition of methanogenesis from methanol. (b) Upon addition of the sodium ionophore monensin the Na+ gradient was abolished and at the same time methanogenesis from methanol was inhibited. (c) Methanogenesis was impaired when the Na+ gradient had the opposite orientation (inside high). All these inhibitory effects were not observed when H2 was present in addition to methanol indicating that the oxidation of methanol to CO2 was driven by a sodium-motive force. In accordance with this, a methanol-dependent influx of Na+ and a corresponding decrease of the membrane potential could be observed, when the Na+/H+ antiporter was inhibited by amiloride. This influx was indicative of the presence of a Na+ transport system which was functional when the oxidation of methanol had to be driven, but was not functional when H2 was present for reduction of methanol to methane.

Diazotrophy and Nitrogenase Activity in the Archaebacterium Methanosarcina barkeri 227.

Abstract: Nitrogen fixation (diazotrophy) has recently been demonstrated in several methanogenic archaebacteria. To compare the process in an archaebacterium with that in eubacteria, we examined the properties of diazotrophic growth and nitrogenase activity in Methanosarcina barkeri 227. Growth yields with methanol or acetate as a growth substrate were significantly lower in N2-grown cultures than in NH4+-grown cultures, and the culture doubling times were increased, indicating that diazotrophy was energetically costly, as it is in eubacteria. Growth of nitrogen-fixing cells was inhibited when molybdenum was omitted from the medium; addition of 10 nM molybdate stimulated growth, while 1 μM molybdate restored maximum diazotrophic growth. Omission of molybdenum did not inhibit growth of ammonia-grown cells. Tungstate (100 μM) strongly inhibited growth of molybdenum-deficient diazotrophic cells, while ammonia-grown cells were unaffected. The addition of 100 nM vanadate or chromate did not stimulate diazotrophic growth of molybdenum-starved cells. These results are consistent with the presence of a molybdenum-containing nitrogenase in M. barkeri. Acetylene, the usual substrate for assaying nitrogenase activity, inhibited methanogenesis by M. barkeri and consequently needed to be used at a low partial pressure (0.3% of the headspace) when acetylene reduction by whole cells was assayed. Whole cells reduced 0.3% acetylene to ethylene at a very low rate (1 to 2 nmol h−1 mg of protein−1), and they “switched off” acetylene reduction in response to added ammonia or glutamine. Crude extracts from diazotrophic cells reduced 10% acetylene at a rate of 4 to 5 nmol of C2H4 formed h−1 mg of protein−1 when supplied with ATP and reducing power, while extracts of Klebsiella pneumoniae prepared by the same procedures had rates 100-fold higher. Acetylene reduction by extracts required ATP and was completely inhibited by 1 mM ADP in the presence of 5 mM ATP. The low rates of C2H2 reduction could be due to improper assay conditions, to switched-off enzyme, or to the nitrogenase's having lower activity towards acetylene than towards dinitrogen.

Coumarin and quinolone action in archaebacteria: evidence for the presence of a DNA gyrase-like enzyme.

Abstract: The action of novobiocin and coumermycin (two coumarins which interact with the gyrB subunit of eubacterial DNA gyrase) and ciprofloxacin (a fluoroquinolone which interacts with the gyrA subunit of DNA gyrase) was tested on several archaebacteria, including five methanogens, two halobacteria, and a thermoacidophile Most strains were sensitive to doses of coumarins (002 to 10 micrograms/ml) which specifically inhibit DNA gyrase in eubacteria Ciprofloxacin inhibited growth of the haloalkaliphilic strain Natronobacterium gregoryi and of the methanogen Methanosarcina barkeri In addition, ciprofloxacin partly relieved the sensitivity to coumarins (and vice versa) Novobiocin inhibited DNA replication in Halobacterium halobium rapidly and specifically Topological analysis has shown that the 17-kilobase plasmid from Halobacterium sp strain GRB is negatively supercoiled; this plasmid was relaxed after novobiocin treatment These results support the existence in archaebacteria of a coumarin and quinolone target related to eubacterial DNA gyrase

Isolation and characterization of methanogenic bacteria from landfills.

Abstract: Methanogenic bacteria were isolated from landfill sites in the United Kingdom. Strains of Methanobacterium formicicum, Methanosarcina barkeri, several different immunotypes of Methanobacterium bryantii, and a coccoid methanogen distinct from the reference immunotypes were identified.

Relationship of Intracellular Coenzyme F(420) Content to Growth and Metabolic Activity of Methanobacterium bryantii and Methanosarcina barkeri.

Abstract: The use of F(420) as a parameter for growth or metabolic activity of methanogenic bacteria was investigated. Two representative species of methanogens were grown in batch culture: Methanobacterium bryantii (strain M.o.H.G.) on H(2) and CO(2), and Methanosarcina barkeri (strain Fusaro) on methanol or acetate. The total intracellular content of coenzyme F(420) was followed by high-resolution fluorescence spectroscopy. F(420) concentration in M. bryantii ranged from 1.84 to 3.65 mumol . g of protein; and in M. barkeri grown with methanol it ranged from 0.84 to 1.54 mumol . g depending on growth conditions. The content of F(420) in M. barkeri was influenced by a factor of 2 depending on the composition of the medium (minimal or complex) and by a factor of 3 to 4 depending on whether methanol or acetate was used as the carbon source. A comparison of F(420) content with protein, cell dry weight, optical density, and specific methane production rate showed that the intracellular content of F(420) approximately followed the increase in biomass in both strains. In contrast, no correlation was found between specific methane production rate and intracellular F(420) content. However, qCH(4)(F(420)), calculated by dividing the methane production rate by the coenzyme F(420) concentration, almost paralleled qCH(4)(protein). These results suggest that F(420) may be used as a specific parameter for estimating the biomass, but not the metabolic activity, of methanogens; hence qCH(4)(F(420)) determined in mixed populations with complex carbon substrates must be considered as measure of the actual methanogenic activity and not as a measure of potential activity.

Bioenergetics of methanogenesis from acetate by Methanosarcina barkeri.

Abstract: Methane formation from acetate by resting cells of Methanosarcina barkeri was accompanied by an increase in the intracellular ATP content from 0.9 to 4.0 nmol/mg of protein. Correspondingly, the proton motive force increased to a steady-state level of -120mV. The transmembrane pH gradient, however, was reversed under these conditions and amounted to +20mV. The addition of the protonophore 3,5,3',4'-tetrachlorosalicylanilide led to a drastic decrease in the proton motive force and in the intracellular ATP content and to an inhibition of methane formation. The ATPase inhibitor N,N'-dicyclohexylcarbodiimide stopped methanogenesis, and the intracellular ATP content decreased. The proton motive force decreased also under these conditions, indicating that the proton motive force could not be generated from acetate without ATP. The overall process of methane formation from acetate was dependent on the presence of sodium ions; upon addition of acetate to cell suspensions of M. barkeri, a transmembrane Na/sup +/ gradient in the range of 4:1 (Na/sup +//sub out//Na/sup +//sub in/) was established. Possible sites of involvement of the Na/sup +/ gradient in the conversion of acetate to methane and carbon dioxide are discussed. Na/sup +/ is not involved in the CO dehydrogenase reaction.

Direct characterization of methanogens in two high-rate anaerobic biological reactors.

Abstract: The methanogenic flora from two types of turbulent, high-rate reactors was studied by immunologic methods as well as by phase-contrast, fluorescence, and scanning electron microscopy. The reactors were a fluidized sand-bed biofilm ANITRON reactor and an ultrafiltration membrane-associated suspended growth MARS reactor (both trademarks of Air Products and Chemicals, Inc., Allentown, Pa.). Conventional microscopic methods revealed complex mixtures of microbes of a range of sizes and shapes, among which morphotypes resembling Methanothrix spp. and Methanosarcina spp. were noticed. Precise identification of these and other methanogens was accomplished by antigenic fingerprinting with a comprehensive panel of calibrated antibody probes of predefined specificity spectra. The methanogens identified showed morphotypes and antigenic fingerprints indicating their close similarity with the following reference organisms: Methanobacterium formicicum MF and Methanosarcina barkeri W in the ANITRON reactor only; Methanosarcina barkeri R1M3, M. mazei S6, Methanogenium cariaci JR1, and Methanobrevibacter arboriphilus AZ in the MARS reactor only; and Methanobrevibacter smithii ALI and Methanothrix soehngenii Opfikon in both reactors. Species diversity and distribution appeared to be, at least in part, dependent on the degree of turbulence inside the reactor.

Involvement of a corrinoid enzyme in methanogenesis from acetate in Methanosarcina barkeri

Abstract: Extracts of acetate-grown Methanosarcina barkeri strain Fusaro formed methane from acetate plus ATP and form acetyl phosphate under H 2 . Coenzyme A (CoA) is stimulatory. Inhibitors of methanogenesis are cyanide, propyliodide and bromoethanesulfonic acid. In cofactor-free extracts methanogenic activity from acetate was restored by addition of ATP, CoA, coenzyme M and 7-mercaptoheptanoylthreonine phosphate. An enzyme-bound corrinoid was found to be involved in methanogenesis from acetate.

Interactions between the methanogen Methanosarcina barkeri and rumen holotrich ciliate protozoa

Abstract: Interspecies hydrogen transfer between rumen holotrich ciliate protoza and methanogenic bacteria has been demonstrated. As a result of the metabolic interaction with Methanosarcina barkeri, the metabolite profile of Isotricha spp. was altered and the production of butyrate and lactate was suppressed in the presence of the methanogen.Use of membrane‐inlet mass spectrometry confirmed that the presence of rumen holotrich ciliates reduced the apparent sensitivity of methanogenesis to the inhibitory effects of oxygen; a gas phase concentration of 7·4 kPa oxygen was required to inhibit methanogenesis in the presence of protozoa, while in pure cultures of M. barkeri, methanogenesis was inhibited by a gas phase oxygen concentration of 1·0 kPa.

Conservation of structure in the human gene encoding argininosuccinate synthetase and the argG genes of the archaebacteria Methanosarcina barkeri MS and Methanococcus vannielii.

Abstract: The DNA sequences of the argG genes of Methanosarcina barkeri MS and Methanococcus vannielii were determined. The polypeptide products of these methanogen genes have amino acid sequences which are 50% identical to each other and 38% identical to the amino acid sequence encoded by the exons of the human argininosuccinate synthetase gene. Introns in the human chromosomal gene separate regions which encode amino acids conserved in both the archaebacterial and human gene products. An open reading frame immediately upstream of argG in Methanosarcina barkeri MS codes for an amino acid sequence which is 45 and 31% identical to the sequences of the large subunits of carbamyl phosphate synthetase in Escherichia coli and Saccharomyces cerevisiae, respectively. If this gene encodes carbamyl phosphate synthetase in Methanosarcina barkeri, this is the first example, in an archaebacterium, of physical linkage of genes that encode enzymes which catalyze reactions in the same amino acid biosynthetic pathway.

A Simple Chromatographic Procedure for the Detection of Cyclized Archaebacterial Glycerol-Bisdiphytanyl-Glycerol Tetraether Core Lipids

Abstract: Archaebacterial glycerol-bisdiphytanyl-glycerol tetraether core lipids containing from one to eight cyclopentane rings could be resolved from each other and from the parent uncyclized C40,C40 lipid by TLC. The core lipids of examples from the genera Methanobacterium, Methanobrevibacter, Methanogenium and Methanoplanus did not contain cyclized forms of glycerol-bisdiphytanyl-glycerol tetraethers, whereas the core lipids of Methanosarcina barkeri contained glycerol-bisdiphytanyl-glycerol tetraethers with from one to three cyclopentane rings in each C40 isopranoid chain.

Ethane production by Methanosarcina barkeri during growth in ethanol supplemented medium

Abstract: Methanosarcina barkeri strain 227 produced ethane during growth on H2/CO2 when ethanol was added to the medium in concentrations of 89–974 mM; ethane production varied from 14 to 38 nmoles per tube (20 ml gas phase, 5.7 ml liquid) with increasing ethanol concentrations. Cells grown to mid-logarithmic phase (A600 ∼0.46, protein = 64 μg/ml) on H2/CO2, thoroughly flushed with H2/CO2, then exposed to ethanol, produced maximal ethane levels (at 585 and 974 mM ethanol) of about 215 nmoles per tube, with an ethane/methane ratio of 1×10-3. Mid-logarithmic-phase cultures of Methanosarcina barkeri strain Fusaro also produced ethane (up to 20 nmoles per tube) when exposed to ethanol. Cultures of strain 227 growing on methanol in the absence of H2 produced ≦6 nmoles per tube of ethane when supplemented with ethanol whereas those lacking ethanol but containing H2 and/or methanol produced ≦1.6 nmoles per tube. Cultures of Methanococcus deltae strains ΔLH and ΔRC, Methanospirillum hungatei or Methanobacterium thermoautotrophicum produced ≦5 nmoles ethane per tube when grown in medium containing ethanol. Ethanol concentrations of 177–886 mM were inhibitory to growth of all methanogens examined. Production of ethane by Methanosarcina was inhibited by >62 mM methanol, and both methanogenic inhibitors tested, CCl4 and Br−CH2−CH2−SO inf3 sup- , inhibited ethane and methane production concurrently. The data suggest that ethanol is converted to ethane by Methanosarcina species using the terminal portion of the methanol-to-methane pathway.

The deoxyribonucleoprotein complex from the archaebacterium Methanosarcina barkeri: characterization and ultrastructural localization of the chromosomal protein MC1

Abstract: The Methanosarcina barkeri protein, methanogen chromosomal protein 1 (MC1), a basic polypeptide of 93 amino acid residues, has been localized in the DNA-rich areas on immunolabelled bacterial cryos...

Cloning and physical mapping of RNA polymerase genes from Methanobacterium thermoautotrophicum and comparison of homologies and gene orders with those of RNA polymerase genes from other methanogenic archaebacteria.

Abstract: The structural genes encoding the four largest subunits of RNA polymerase, A, B', B", and C, were physically mapped in Methanobacterium thermoautotrophicum Winter. The genes formed a cluster in the order B", B', A, C and had a common orientation. DNA hybridization experiments yielded different degrees of homology between RNA polymerase gene sequences of different species of Methanobacterium and Methanococcus voltae. No homology was detectable between Methanobacterium thermoautotrophicum and Methanosarcina barkeri. From Southern hybridization experiments in which probes of the four genes from Methanobacterium thermoautotrophicum Winter and restriction digests of the genomic DNAs of the different methanogens were used, a common gene order of the RNA polymerase genes could be deduced.

Interconversion of F430 derivatives of methanogenic bacteria.

Abstract: F430 is the prosthetic group of the methylcoenzyme M reductase of methanogenic bacteria. The compound isolated from Methanosarcina barkeri appears to be identical to the one obtained from the only distinctly related Methanobacterium thermoautotrophicum. F430 is thermolabile and in the presence of acetonitrile or C10in4sup-two epimerization products are obtained upon heating; in the absence of these compounds F430 is oxidized to 12, 13-didehydro-F430. The latter is stereoselectively reduced under H2 atmosphere to F430 by cell-free extracts of M. barkeri or M. thermoautotrophicum. H2 may be replaced by the reduced methanogenic electron carrier coenzyme F420.

Isolation of subunits from Methanosarcina barkeri ATPase: nucleotide-binding site in the alpha subunit.

Abstract: The alpha (62,000-dalton) and beta (49,000-dalton) subunits of Methanosarcina barkeri ATPase were purified to homogeneity. The subunits and ATPase complex were trypsinized in the presence of various nucleotides. ATP and ADP changed the trypsin sensitivity of the alpha subunit in the complex and isolated forms, suggesting the presence of a nucleotide-binding site in the alpha subunit.

Presence of an unusual methanogenic bacterium in coal gasification waste.

Abstract: Methanogenic bacteria growing on a pilot-scale, anaerobic filter processing coal gasification waste were enriched in a mineral salts medium containing hydrogen and acetate as potential energy sources. Transfer of the enrichments to methanol medium resulted in the initial growth of a strain of Methanosarcina barkeri, but eventually small cocci became dominant. The cocci growing on methanol produced methane and exhibited the typical fluorescence of methanogenic bacteria. They grew in the presence of the cell wall synthesis-inhibiting antibiotics d-cycloserine, fosfomycin, penicillin G, and vancomycin as well as in the presence of kanamycin, an inhibitor of protein synthesis in eubacteria. The optimal growth temperature was 37°C, and the doubling time was 7.5 h. The strain lysed after reaching stationary phase. The bacterium grew poorly with hydrogen as the energy source and failed to grow on acetate. Morphologically, the coccus shared similarities with Methanosarcina sp. Cells were 1 μm wide, exhibited the typical thick cell wall and cross-wall formation, and formed tetrads. Packets and cysts were not formed. Images

Different effects of 5-fluorouracil on Methanosarcina barkeri and on Methanobacterium thermoautotrophicum

Abstract: Growth of Methanosarcina barkeri (strain Fusaro) was found to be inhibited by 5-fluorouracil (FU) only at relatively high concentrations (>50 μg/ml). Inhibition could not be relieved by uracil. Therefore, FU probably did not exert its effect via inhibition of DNA synthesis as is the case in other organisms. Control experiments with Methanobacterium thermoautotrophicum (strain Marburg) on the other hand revealed that the effect of FU on this archaebacterium is probably exerted at the level of nucleic acid synthesis. The M. thermoautotrophicum cultures rapidly acquired resistance towards the pyramidine analog.