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1.
2.
Ralph W. Robinson 《Applied microbiology》1986,52(1):17-27
Methanosarcina mazei S6 and LYC were used to study the structure and differentiation of the aggregating methanogens. Cultures harvested under various conditions are described at the ultrastructural level. Cells of strain S6 are enclosed by a layer 12 nm thick in contact with the plasma membrane. In sarcinal colonies, cells are held in close association by a fibrous matrix up to 60 nm thick. Colony maturation was examined in strain S6 over a period of 1 year. Changes occurred in the shape and staining of individual cells. Also, various inclusion bodies were observed that either persist throughout colony maturation or are only found at certain growth stages. Two types of cores that are composed of double membranes in M. mazei S6 are described. One has a 90-nm diameter and contains electron-dense granules similar to those found in the cytoplasm. The other core type does not contain granules, is more numerous, and is found in older cultures. Two life cycles are described for M. mazei based on electron microscope examinations. A complex life cycle involving the release of single cells is described with two variations for strains S6 and LYC. When released cells of strain S6 are placed in fresh medium they can repeat the cycle. In addition, a limited cycle is described for both strains of M. mazei. This limited cycle contains the only sarcinal morphotypes observed in M. barkeri. When M. mazei S6 remains in the limited cycle and does not disaggregate in stationary phase, several types of possible resting forms are found. 相似文献
3.
At certain stages in its growth cycle, Methanosarcina mazei produces an enzyme (disaggregatase) that causes aggregates to separate into single cells. M. mazei S-6 and LYC both produce this enzymatic activity, although the specificities of activities differ. The disaggregatase of M. mazei S-6 had little effect on strain LYC cells, but the disaggregatase of M. mazei LYC disaggregated both strain LYC and strain S-6 cells. The disaggregatase of M. mazei LYC was purified by column chromatography, and it apparently consisted of two similar subunits with a combined molecular size of about 180,000 Da. Strain S-6 culture supernatants contained 14 U of activity per liter when activity was measured as uronic acids released from purified cell wall material. When the activity was quantified as the release of uronic acids from boiled M. mazei S-6 cells, the highest activity was found at pH 4.7 and at 35 degrees C. 相似文献
4.
Isolation and characterization of disaggregatase from Methanosarcina mazei LYC. 总被引:4,自引:2,他引:2
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At certain stages in its growth cycle, Methanosarcina mazei produces an enzyme (disaggregatase) that causes aggregates to separate into single cells. M. mazei S-6 and LYC both produce this enzymatic activity, although the specificities of activities differ. The disaggregatase of M. mazei S-6 had little effect on strain LYC cells, but the disaggregatase of M. mazei LYC disaggregated both strain LYC and strain S-6 cells. The disaggregatase of M. mazei LYC was purified by column chromatography, and it apparently consisted of two similar subunits with a combined molecular size of about 180,000 Da. Strain S-6 culture supernatants contained 14 U of activity per liter when activity was measured as uronic acids released from purified cell wall material. When the activity was quantified as the release of uronic acids from boiled M. mazei S-6 cells, the highest activity was found at pH 4.7 and at 35 degrees C. 相似文献
5.
I. Thomas H.-C. Dubourguier G. Prensier P. Debeire G. Albagnac 《Archives of microbiology》1987,148(3):193-201
Studies on immunological relationships among Methanosarcina genus using immunofluorescence and immunoprecipitation showed that a common antigen can be extracted by shaking in aqueous phase. This antigen was purified from Methanosarcina mazei. The protein had a molecular weight of 283400 daltons with three subunits, =68000, =43200 and =30500. It contained nickel, coenzyme M and F430. Its biochemical characteristics identified this antigen as the component C of the methyl CoM reductase complex. But EPR study showed that the nickel was Ni(II). Biological activity was detectable neither by heterologous in vitro assay nor by the DTT assay. Immunogold labelling showed that the component C was located randomly in the cytoplasm in Methanosarcina species and in Methanothrix soehngenii. In addition, specific labelling was also observed outside of the heteropolysaccharidic envelopes probably due to the absorption of component C released by the lysis of some cells in the clumps. 相似文献
6.
Ralph W. Robinson H. C. Aldrich Steven F. Hurst A. S. Bleiweis 《Applied microbiology》1985,49(2):321-327
Colonial aggregates of Methanosarcina (= Methanococcus) mazei were examined with scanning and transmission electron microscopy. Cells are irregular and grouped into multicellular sarcinal colonies, which may disaggregate in older cultures. The protoplast is bounded by a typical trilaminar plasma membrane, outside of which is a matrix of loose fibrils. The presence and compactness of matrix material are responsible for the close packing of cells, and colony disaggregation seems to be the result of matrix shedding and degradation. The cell envelope contains complex hetero polysaccharides of N-acetylgalactosamine and galacturonic and glucuronic acids. Polymers extruded by M. mazei are likely quite adhesive in nature, accounting for its strong adherence to surfaces and hardiness compared with many other methanogens. 相似文献
7.
M W Peck 《Applied and environmental microbiology》1989,55(4):940-945
Coenzyme F420 has been assayed by high-performance liquid chromatography with fluorimetric detection; this permits quantification of individual coenzyme F420 analogs whilst avoiding the inclusion of interfering material. The total intracellular coenzyme F420 content of Methanosarcina barkeri MS cultivated on methanol and on H2-CO2 and of Methanosarcina mazei S-6 cultured on methanol remained relatively constant during batch growth. The most abundant analogs in M. barkeri were coenzymes F420-2 and F420-4, whilst in M. mazei coenzymes F420-2 and F420-3 predominated. Significant changes in the relative proportions of the coenzyme F420 analogs were noted during batch growth, with coenzymes F420-2 and F420-4 showing opposite responses to each other and the same being also true for coenzymes F420-3 and F420-5. This suggests that an enzyme responsible for transferring pairs of glutamic acid residues may be active. The degradation fragment FO was also detected in cells in late exponential and stationary phase. Coenzyme F420 analogs were present in the culture supernatant of both methanogens, in similar proportions to that in the cells, except for FO which was principally located in the supernatant. 相似文献
8.
The Methanosarcina barkeri genome: comparative analysis with Methanosarcina acetivorans and Methanosarcina mazei reveals extensive rearrangement within methanosarcinal genomes 总被引:1,自引:0,他引:1
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Maeder DL Anderson I Brettin TS Bruce DC Gilna P Han CS Lapidus A Metcalf WW Saunders E Tapia R Sowers KR 《Journal of bacteriology》2006,188(22):7922-7931
We report here a comparative analysis of the genome sequence of Methanosarcina barkeri with those of Methanosarcina acetivorans and Methanosarcina mazei. The genome of M. barkeri is distinguished by having an organization that is well conserved with respect to the other Methanosarcina spp. in the region proximal to the origin of replication, with interspecies gene similarities as high as 95%. However, it is disordered and marked by increased transposase frequency and decreased gene synteny and gene density in the distal semigenome. Of the 3,680 open reading frames (ORFs) in M. barkeri, 746 had homologs with better than 80% identity to both M. acetivorans and M. mazei, while 128 nonhypothetical ORFs were unique (nonorthologous) among these species, including a complete formate dehydrogenase operon, genes required for N-acetylmuramic acid synthesis, a 14-gene gas vesicle cluster, and a bacterial-like P450-specific ferredoxin reductase cluster not previously observed or characterized for this genus. A cryptic 36-kbp plasmid sequence that contains an orc1 gene flanked by a presumptive origin of replication consisting of 38 tandem repeats of a 143-nucleotide motif was detected in M. barkeri. Three-way comparison of these genomes reveals differing mechanisms for the accrual of changes. Elongation of the relatively large M. acetivorans genome is the result of uniformly distributed multiple gene scale insertions and duplications, while the M. barkeri genome is characterized by localized inversions associated with the loss of gene content. In contrast, the short M. mazei genome most closely approximates the putative ancestral organizational state of these species. 相似文献
9.
The enzyme disaggregatase (Dag) from Methanosarcina mazei was studied immunochemically. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified Dag under reducing and nonreducing conditions revealed a single band with a 94-kDa molecular mass. Dag was found to be immunogenic in rabbits; a polyclonal antibody probe was prepared and used to detect the enzyme by slide immunoenzymatic assay, immunofluorescence, and immunoblotting in various species of Methanosarcina known to convert from packets to single cells, including M. mazei. The enzyme could not be detected in other members of the family Methanosarcinaceae that do not convert. By immunogold electron microscopy, Dag was mapped to the cell wall of packets and to the cell membrane of single cells of two M. mazei strains. 相似文献
10.
Primak YA Du M Miller MC Wells DH Nielsen AT Weyler W Beck ZQ 《Applied and environmental microbiology》2011,77(21):7772-7778
The mevalonate pathway is utilized for the biosynthesis of isoprenoids in many bacterial, eukaryotic, and archaeal organisms. Based on previous reports of its feedback inhibition, mevalonate kinase (MVK) may play an important regulatory role in the biosynthesis of mevalonate pathway-derived compounds. Here we report the purification, kinetic characterization, and inhibition analysis of the MVK from the archaeon Methanosarcina mazei. The inhibition of the M. mazei MVK by the following metabolites derived from the mevalonate pathway was explored: dimethylallyl diphosphate (DMAPP), geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), isopentenyl monophosphate (IP), and diphosphomevalonate. M. mazei MVK was not inhibited by DMAPP, GPP, FPP, diphosphomevalonate, or IP, a proposed intermediate in an alternative isoprenoid pathway present in archaea. Our findings suggest that the M. mazei MVK represents a distinct class of mevalonate kinases that can be differentiated from previously characterized MVKs based on its inhibition profile. 相似文献
11.
Methanosarcinae are the only archaeobacteria known to undergo major morphologic changes during growth involving unicellular and multicellular forms, and Methanosarcina mazei S-6 is the only strain for which three distinct forms, packets, single cells, and lamina, have so far been observed. It is reported that two pairs of these forms, either packets and single cells or single cells and lamina, grew and interconverted in medium with the same composition, Ca2+ and Mg2+ concentrations, and growth substrate, and that the two forms in each pair displayed distinctive differences revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting, the same growth medium-substrate notwithstanding. 相似文献
12.
Synthesis of cysteinyl-tRNA(Cys) in methanogenic archaea proceeds by a two-step pathway in which tRNA(Cys) is first aminoacylated with phosphoserine by phosphoseryl-tRNA synthetase (SepRS). Characterization of SepRS from the mesophile Methanosarcina mazei by gel filtration and nondenaturing mass spectrometry shows that the native enzyme exists as an alpha4 tetramer when expressed at high levels in Escherichia coli. However, active site titrations monitored by ATP/PP(i) burst kinetics, together with analysis of tRNA binding stoichiometry by fluorescence spectroscopy, show that the tetrameric enzyme binds two tRNAs and that only two of the four chemically equivalent subunits catalyze formation of phosphoseryl adenylate. Therefore, the phenomenon of half-of-the-sites activity, previously described for synthesis of 1 mol of tyrosyl adenylate by the dimeric class I tyrosyl-tRNA synthetase, operates as well in this homotetrameric class II tRNA synthetase. Analysis of cognate and noncognate reactions by ATP/PP(i) and aminoacylation kinetics strongly suggests that SepRS is able to discriminate against the noncognate amino acids glutamate, serine, and phosphothreonine without the need for a separate hydrolytic editing site. tRNA(Cys) binding to SepRS also enhances the capacity of the enzyme to discriminate among amino acids, indicating the existence of functional connectivity between the tRNA and amino acid binding sites of the enzyme. 相似文献
13.
Cornelia Welte Verena Kallnik Marcel Grapp Gunes Bender Steve Ragsdale Uwe Deppenmeier 《Journal of bacteriology》2010,192(3):674-678
Reduced ferredoxin is an intermediate in the methylotrophic and aceticlastic pathway of methanogenesis and donates electrons to membrane-integral proteins, which transfer electrons to the heterodisulfide reductase. A ferredoxin interaction has been observed previously for the Ech hydrogenase. Here we present a detailed analysis of a Methanosarcina mazei Δech mutant which shows decreased ferredoxin-dependent membrane-bound electron transport activity, a lower growth rate, and faster substrate consumption. Evidence is presented that a second protein whose identity is unknown oxidizes reduced ferredoxin, indicating an involvement in methanogenesis from methylated C1 compounds.The aceticlastic pathway of methanogenesis creates approximately 70% (10) of the biologically produced methane and is of great ecological importance, as methane is a potent greenhouse gas. Organisms using this pathway to convert acetate to methane belong exclusively to the genera Methanosarcina and Methanosaeta. The two carbon atoms of acetate have different fates in the pathway. The methyl moiety is converted to methane, whereas the carbonyl moiety is further oxidized to CO2 and the electrons derived from this oxidation step are used to reduce ferredoxin (Fd) (6). During methanogenesis from methylated C1 compounds (methanol and methylamines), one-quarter of the methyl groups are oxidized to obtain electrons for the reduction of heterodisulfide (27). A key enzyme in the oxidative part of methylotrophic methanogenesis is the formylmethanofuran dehydrogenase, which oxidizes the intermediate formylmethanofuran to CO2 (7). The electrons are transferred to Fd. It has been suggested that reduced ferredoxin (Fdred) donates electrons to the respiratory chain with the heterodisulfide (coenzyme M [CoM]-S-S-CoB) as the terminal electron acceptor and that the reaction is catalyzed by the Fdred:CoM-S-S-CoB oxidoreductase system (7, 24). The direct membrane-bound electron acceptor for Fdred is still a matter of debate; for the Ech hydrogenase, a reduced ferredoxin-accepting, H2-evolving activity has been observed for Methanosarcina barkeri (20), which implies that the H2:CoM-S-S-CoB oxidoreductase system is involved in electron transport (13). Direct electron flow from the Ech hydrogenase to the heterodisulfide reductase has not been shown to date (20, 21). In contrast to M. barkeri, Methanosarcina acetivorans lacks the Ech hydrogenase (11). It can nevertheless grow on acetate, which is why another complex present in this organism, the Rnf complex, is thought to be involved in the aceticlastic pathway of methanogenesis as an acceptor for Fdred (8, 10, 17). The Methanosarcina mazei genome, however, contains genes coding for the Ech hydrogenase, but this species lacks the Rnf complex (5).To investigate whether the Ech hydrogenase is the only means by which M. mazei channels electrons from Fdred into the respiratory chain, a mutant lacking the Ech hydrogenase (M. mazei Δech mutant) was constructed. Electron transport experiments using Fdred as the electron donor and CoM-S-S-CoB as the electron acceptor were conducted with wild-type and mutant membranes to gain deeper insight into the actual membrane-bound protein complexes that accept electrons from Fdred. Furthermore, an in-depth characterization of the growth and trimethylamine (TMA) consumption of the Δech mutant was performed, which provided insight into the in vivo role of Ech hydrogenase. 相似文献
14.
Spontaneous Disaggregation of Methanosarcina mazei S-6 and Its Use in the Development of Genetic Techniques for Methanosarcina spp 总被引:2,自引:4,他引:2
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Jane E. Harris 《Applied microbiology》1987,53(10):2500-2504
When monomethylamine was the growth substrate, spontaneous disaggregation of Methanosarcina mazei S-6 commenced at the mid-exponential phase and resulted in the formation of a suspension containing 108 to 109 free cells per ml. Free cells were osmotically fragile and amenable to extraction of DNA. Hypertonic media for the manipulation and regeneration of free cells into aggregates were developed, and plating efficiencies of 100% were achieved for M. mazei S-6 and LYC. Free cells of strain S-6 required MgCl2 (10 mM) for growth, whereas aggregates did not. Specific growth rates of strains S-6 and LYC were increased by MgCl2. Treatment with pronase caused sphere formation and removal of the protein wall of cells of strain S-6, but protoplasts could not be regenerated. The disaggregating enzyme produced by strain S-6 facilitated the preparation of suspensions of free cells of some strains of Methanosarcina barkeri. Although this provided a means of extracting high-molecular-weight DNA from M. barkeri, less than 0.1% of free cells were viable. 相似文献
15.
Changes in concentrations of coenzyme F420 analogs during batch growth of Methanosarcina barkeri and Methanosarcina mazei. 总被引:1,自引:0,他引:1
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M W Peck 《Applied microbiology》1989,55(4):940-945
Coenzyme F420 has been assayed by high-performance liquid chromatography with fluorimetric detection; this permits quantification of individual coenzyme F420 analogs whilst avoiding the inclusion of interfering material. The total intracellular coenzyme F420 content of Methanosarcina barkeri MS cultivated on methanol and on H2-CO2 and of Methanosarcina mazei S-6 cultured on methanol remained relatively constant during batch growth. The most abundant analogs in M. barkeri were coenzymes F420-2 and F420-4, whilst in M. mazei coenzymes F420-2 and F420-3 predominated. Significant changes in the relative proportions of the coenzyme F420 analogs were noted during batch growth, with coenzymes F420-2 and F420-4 showing opposite responses to each other and the same being also true for coenzymes F420-3 and F420-5. This suggests that an enzyme responsible for transferring pairs of glutamic acid residues may be active. The degradation fragment FO was also detected in cells in late exponential and stationary phase. Coenzyme F420 analogs were present in the culture supernatant of both methanogens, in similar proportions to that in the cells, except for FO which was principally located in the supernatant. 相似文献
16.
17.
Klunker D Haas B Hirtreiter A Figueiredo L Naylor DJ Pfeifer G Müller V Deppenmeier U Gottschalk G Hartl FU Hayer-Hartl M 《The Journal of biological chemistry》2003,278(35):33256-33267
Two distantly related classes of cylindrical chaperonin complexes assist in the folding of newly synthesized and stress-denatured proteins in an ATP-dependent manner. Group I chaperonins are thought to be restricted to the cytosol of bacteria and to mitochondria and chloroplasts, whereas the group II chaperonins are found in the archaeal and eukaryotic cytosol. Here we show that members of the archaeal genus Methanosarcina co-express both the complete group I (GroEL/GroES) and group II (thermosome/prefoldin) chaperonin systems in their cytosol. These mesophilic archaea have acquired between 20 and 35% of their genes by lateral gene transfer from bacteria. In Methanosarcina mazei G?1, both chaperonins are similarly abundant and are moderately induced under heat stress. The M. mazei GroEL/GroES proteins have the structural features of their bacterial counterparts. The thermosome contains three paralogous subunits, alpha, beta, and gamma, which assemble preferentially at a molar ratio of 2:1:1. As shown in vitro, the assembly reaction is dependent on ATP/Mg2+ or ADP/Mg2+ and the regulatory role of the beta subunit. The co-existence of both chaperonin systems in the same cellular compartment suggests the Methanosarcina species as useful model systems in studying the differential substrate specificity of the group I and II chaperonins and in elucidating how newly synthesized proteins are sorted from the ribosome to the proper chaperonin for folding. 相似文献
18.
A novel multicellular form of Methanosarcina mazei S-6 is described. It was termed lamina, and it formed during the exponential growth phase when packets or single cells were grown in 40 mM trimethylamine and a total concentration of 8.3 to 15.6 mM Ca2+ and/or Mg2+, in cultures that were not shaken. A distinct molecular event represented by the increment in expression and a spatial redistribution of an antigen during lamina formation is documented. 相似文献
19.
Medalia N Sharon M Martinez-Arias R Mihalache O Robinson CV Medalia O Zwickl P 《Journal of structural biology》2006,156(1):84-92
We have cloned the proteasome and the proteasome activating nucleotidase (PAN) genes from the mesophilic archaeon Methanosarcina mazei and produced the respective proteins in Escherichia coli cultures. The recombinant complexes were purified to homogeneity and characterized biochemically, structurally, and by mass spectrometry. We found that the degradation of Bodipy-casein by Methanosarcina proteasomes was activated by Methanosarcina PAN. Notably, the Methanosarcina PAN unfolded GFP-SsrA only in the presence of Methanosarcina proteasomes. Structural analysis by 2D averaging electron microscopy of negatively stained complexes displayed the typical structure for the proteasome, namely four-striped side-views and sevenfold-symmetric top-views, with 15 nm height and 11 nm diameter. The structural analysis of the PAN preparation revealed also four-striped side-views, albeit with a height of 18 nm and sixfold-symmetric top-views with a diameter of 15 nm, which corresponds most likely to a dimer of two hexameric complexes. Mass spectrometric analysis of both the Methanosarcina and the Methanocaldococcus PAN proteins indicated hexameric complexes. In summary, we performed a functional and structural characterization of the PAN and proteasome complexes from the archaeon M. mazei and described unique new structural and functional features. 相似文献
20.
Summary A co-culture of Clostridium formicoaceticum and Methanosarcina mazei converted lactate to methane and carbon dioxide at mesophilic temperatures and pH values near 7.0. Lactate was first converted to acetate by the homoacetogen, and then to CH4 and CO2 by the methanogen, with the second reaction as the rate-limiting step. The methane yield was about 1.45 mol/mol lactate. These two organisms formed a mutualistic association and may be useful together with the homolactic bacterium Stretococcus lactis to convert lactose to methane.
Offprint requests to: S. T. Yang 相似文献