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PubMed1991

Heat shock response of the archaebacterium Methanococcus voltae.

The general properties of the heat shock response of the archaebacterium Methanococcus voltae were characterized. The induction of 11 heat shock proteins, with apparent molecular weights ranging from 18,000 to 90,000, occurred optimally at 40 to 50 degrees C. Some of the heat shock proteins were preferentially enriched in either the soluble (cytoplasm) or particulate (membrane) fraction. Alternative stresses (ethanol, hydrogen peroxide, NaCl) stimulated the synthesis of subsets of the heat shock proteins as well as unique proteins. Western blot (immunoblot) analysis, in which antisera to Escherichia coli heat shock proteins (DnaK and GroEL) were used, did not detect any immunologically cross-reactive proteins. In addition, Southern blot analysis did not reveal any homology between M. voltae and four highly conserved heat shock genes, mopB and dnaK from E. coli and hsp70 genes from Drosophila species and Saccharomyces cerevisiae.

Archaea↗
PubMed1991

Isolation of a coenzyme M-auxotrophic mutant and transformation by electroporation in Methanococcus voltae.

An auxotrophic mutant of Methanococcus voltae was isolated that required coenzyme M (CoM) for growth. With the mutant as a recipient, conditions were developed that allowed the introduction of wild-type CoM+ DNA into the mutant methanogen via electroporation. This method also allowed the rescue of both a histidine and purine auxotroph as well as the introduction of DNA determining resistance to the CoM analog 2-bromoethanesulfonic acid. Electroporation of the CoM(+)-determining DNA was 50- to 80-fold more efficient than natural transformation.

Euryarchaeota↗
PubMed1991

Proportions of diether, macrocyclic diether, and tetraether lipids in Methanococcus jannaschii grown at different temperatures.

Growth of Methanococcus jannaschii over a wide temperature range (47 to 75 degrees C) is correlated with an ability to alter dramatically the proportions of three ether lipid cores. These lipids shifted from predominantly diether (2,3-di-O-phytanyl-sn-glycerol) at the lower growth temperatures to macrocyclic diether and tetraether at near optimal growth temperatures. Lipid head groups varied as well, especially with respect to an increase in phosphate at the higher temperatures.

Autoradiography↗
PubMed1989

Comparative studies of ribosomal proteins and their genes from Methanococcus vannielii and other organisms.

Using data from a partial protein sequence analysis of ribosomal proteins derived from the archaebacterium Methanococcus vannielii, oligonucleotide probes were synthesized. The probes enabled us to localize several ribosomal protein genes and to determine their nucleotide sequences. The amino acid sequences that were deduced from the genes correspond to proteins L12 and L10 from the rif operon, according to the genome organization in Escherichia coli, and to proteins L23 and L2, which have comparable locations, as in the Escherichia coli S10 operon. Various degrees of similarity were found when the four proteins were compared with the corresponding ribosomal proteins of prokaryotic or eukaryotic organisms. The highest sequence homology was found in counterparts from other archaebacteria, such as Halobacterium marismortui, Halobacterium halobium, or Sulfolobus. In general, the M. vannielii protein sequences were more related to the eukaryotic kingdom than to the Gram-positive or Gram-negative eubacteria. On the other hand, the organization of the ribosomal protein genes clearly follows the operon structure of the Escherichia coli genome and is different from the monocistronic eukaryotic gene arrangements. The protein coding regions were not interrupted by introns. Furthermore, the Shine-Dalgarno type sequences of methanogenic bacteria are homologous with those of eubacteria, and also their terminator regions are similar.

Amino Acid Sequence↗
PubMed1990

Structures of polar lipids from the thermophilic, deep-sea archaeobacterium Methanococcus jannaschii.

Cells of Methanococcus jannaschii, grown at 65 degrees C in a defined medium, contained 7% of lipid composed of 87% polar and 13% neutral components. Within the polar fraction 16 lipids were resolved by thin-layer chromatography, 4 of which were present in trace amounts. Staining reactions demonstrated that the more abundant lipids were glycolipids, aminophospholipids, and an aminophosphoglycolipid. Most of the polar fraction (82%) consisted of five diether lipids, which were purified and their structures were resolved largely through nuclear magnetic resonance, mass spectrometry, and optical rotation methods. Macrocyclic diethers had the head groups phosphoethanolamine-(1----6)-beta-D-glucopyranose, beta-D-glucopyranose, and beta-D-glucopyranosyl-(1----6)-beta-D-glucopyranose. Phosphoethanolamine was identified as a head group for both the noncyclized and macrocylic diether core lipids. The neutral lipids were mainly acyclic C30 isoprenoids, predominantly dihydro-, hexahydro, and octahydro-squalenes.

Carbohydrate Conformation↗
PubMed1989

Characterization of a P-type ATPase of the archaebacterium Methanococcus voltae.

The vanadate-sensitive ATPase of Methanococcus voltae has been purified by a procedure which includes, purification of the cytoplasmic membrane by sucrose gradient centrifugation, solubilization with Triton X-100, and DEAE-Sephadex and Sephacryl S-300 chromatography. While the DEAE-Sephadex step provided a preparation consisting of two polypeptides (74 and 52 kDa), the Sephacryl S-300 step yields a product with a subunit of 74 kDa. Incubation of either membranes or purified ATPase with [gamma-32P]ATP followed by acidic (pH 2.4) lithium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the vanadate-sensitive labeling of a 74-kDa acyl phosphate intermediate. These results indicate that the M. voltae ATPase is of the P-type.

Adenosine Triphosphatases↗
PubMed1988

Primary structure of the archaebacterial Methanococcus vannielii ribosomal protein L12. Amino acid sequence determination, oligonucleotide hybridization, and sequencing of the gene.

The primary structure of ribosomal protein L12 from Methanococcus vannielii has been determined by direct amino acid sequence analysis with automated liquid phase Edman degradation of the entire protein and manual 4-N,N'-dimethylaminoazobenzene-4'-isothiocyanate/phenylisothiocyanate sequencing of fragments obtained by enzymatic digestion and by partial acid hydrolysis. The knowledge of the amino acid sequences of these various fragments allowed the synthesis of two oligonucleotide probes complementary to the 5'- and the 3'-end of the gene, and they were used for hybridization with digested M. vannielii chromosomal DNA. Both oligonucleotide probes gave similar and clear hybridization signals. The plasmid pMvaX1 containing the entire gene of protein L12 was obtained. The nucleotide sequence complemented the partial amino acid sequence, and it is in full agreement with the protein sequence and the amino acid analysis. Comparison of secondary structural elements and hydrophobicity plots of the M. vannielii protein L12 with the known L12 sequences derived from other archaebacterial and eukaryotic sources show strong homologies among these sequences. They contain an exceptional highly conserved hydrophilic sequence area in the C-terminal part of the proteins. In comparison with eubacterial L12 proteins, the conservation is reduced to single amino acid residues. However, the eubacterial L12 proteins have hydrophilic regions similar to those of L12 from M. vannielii. These regions are predicted to be located at the surface of the proteins, as has been proven to be the case in crystallized Escherichia coli L12 protein. It is possible that the strongly conserved hydrophilic sequence regions form part of the factor-binding domain.

Amino Acid Sequence↗
PubMed1986

Structural characterization of the lipids of Methanococcus voltae, including a novel N-acetylglucosamine 1-phosphate diether.

The lipids of a moderately halophilic methanogen, Methanococcus voltae, accounted for 5.1% of the cell dry weight and consisted of 91% polar lipids and 9% neutral lipids. Twelve polar lipids were detected, three of which, all derivatives of 2,3-di-O-phytanyl-sn-glycerol, were identified as: 2,3-di-O-phytanyl-1-O-[beta-D-glucopyranosyl-(1-6)-beta-D- glucopyranosyl]-sn-glycerol, 2,3-di-O-phytanyl-1-O-[beta-D-glucopyranosyl]-sn-glycerol and a novel NAc-glucosamine 1-phosphate diether, and 2,3-di-O-phytanyl-1-[phosphoryl-2-acetamido-2-deoxy-beta-D- glucopyranosyl]-sn-glycerol. The neutral lipids consisted mainly of squalenes: squalene, dihydrosqualene, tetrahydrosqualene, hexahydrosqualene, and unidentified squalenes.

Chromatography, Thin Layer↗
PubMed1984

Six antigenic determinants in the surface layer of the archaebacterium Methanococcus vannielii revealed by monoclonal antibodies.

The immunogenicity and antigenic characteristics of the unique surface layer (S layer) of Methanococcus vanielii was studied with a panel of six monoclonal antibodies. Six surface determinants were identified for the first time, each recognized by one antibody exclusively. The determinants are proteins, located in the S layer, and accessible to antibody in whole, unfixed, as well as formalinized bacteria. Hence the six antigens and antibodies reported here should be useful for rapid identification of new isolates and for taxonomy of methanogens, notably Methanococcaceae. In this connection two novel applications of the slide immunoenzymatic assay were developed for analyses of monoclonal antibodies and their complementary sites in the bacterial envelope.

Animals↗
PubMed1982

A selenium-containing hydrogenase from Methanococcus vannielii. Identification of the selenium moiety as a selenocysteine residue.

A 75Se-labeled hydrogenase was purified to near homogeneity from extracts of Methanococcus vannielii cells grown in the presence of [75Se]selenite. The molecular weight of the enzyme was estimated as 340,000 by gel filtration. The enzyme tends to aggregate and occurs also as a larger protein species (Mr = 1.3 x 10(6)). The same phenomenon was observed on native gel electrophoretic analysis. Hydrogenase activity exhibited by these two protein bands was proportional to protein and 75Se content. Both molecular species reduce the natural cofactor, 8-hydroxy-5-deazaflavin, and tetrazolium dyes with molecular hydrogen. Sodium dodecyl sulfate-gel electrophoresis of 75Se-labeled enzyme showed that 75Se is present exclusively in an Mr = 42,000 subunit. A value of 3.8 g atoms of selenium/mol of enzyme (Mr = 340,000) was determined by atomic absorption analysis. The chemical form of selenium in the enzyme was shown to be selenocysteine. This was identified as the [75Se]carboxymethyl and [75Se]carboxyethyl derivatives in acid hydrolysates of alkylated 75Se-labeled protein. The hydrogenase is extremely oxygen-sensitive but can be reactivated by incubation with molecular hydrogen and dithiothreitol.

Cysteine↗
PubMed1984

Structural elucidation of a unique macrocyclic membrane lipid from a new, extremely thermophilic, deep-sea hydrothermal vent archaebacterium, Methanococcus jannaschii.

The membrane lipid of a new deep-sea hydrothermal vent methanogen, Methanococcus jannaschii, was isolated, purified, and structurally characterized. The total lipid extract, amounting to 32.2 micrograms/mg, dry cell weight, was fractionated on silica gel into a neutral lipid and polar lipid fraction. The neutral lipid fraction consisted of a series of isoprenoid hydrocarbons and free (nonphospholipid) alkylglycerol ethers. The polar phospholipid and glycolipid fraction (8.44 micrograms/mg, dry cell weight) was hydrolyzed with methanolic HCl, and the resulting alkylglycerol ethers were analyzed by a combination of chemical and spectroscopic techniques. The hydrolyzed polar lipid was primarily (95%) a unique, macrocyclic glycerol diether, heretofore unknown. High-field (250 MHz) proton nuclear magnetic resonance and infrared spectra of this novel macrocyclic compound are nearly identical and overlapping those of the known bis-(phytanyl)glycerol diether and bis-(diphytanyl)diglycerol tetraether. A field desorption mass spectrum revealed a molecular weight of 650 for the macrocyclic glycerol diether, 2 mass units less than that of bis-(phytanyl)glycerol diether. Degradation of the macrocyclic ether with boron tribromide resulted in diphytanyl dibromide, and further reaction of this dibromide with lithium aluminium hydride resulted in diphytane as determined by gas chromatography-mass spectrometry. The significance of the predominance of this structure in M. jannaschii is discussed. A survey of selected methanogenic Archaebacteria, including three thermophiles, failed to indicate the presence of the macrocyclic glycerol diether in any other microorganism, including two species of order Methanococcales, one species of Methanobacteriales, and three strains belonging to the order Methanomicrobiales.

Chemical Phenomena↗
PubMed1980

Reconstitution of a formate-NADP+ oxidoreductase from formate dehydrogenase and a 5-deazaflavin-linked NADP+ reductase isolated from Methanococcus vannielii.

The formate-dependent reduction of NADP+ by extracts of Methanococcus vannielii is catalyzed by a coupled system consisting of formate dehydrogenase, a 5-deazaflavin cofactor, and 5-deazaflavin-dependent NADP+ reductase. All three components were purified from crude extracts of M. vannielii. Recombination of these components reconstituted the formate-NADP+ oxidoreductase system. The formate dehydrogenase also can utilize FAD, FMN, and a number of artificial dyes as electron acceptors, but these do not replace the 5-deazaflavin cofactor in the coupled enzyme system. The reduced form of 5-deazaflavin binds readily to the NADP+ reductase apoprotein and is not dissociated by ammonium sulfate treatment at neutral pH under anaerobic conditions. This electron transfer cofactor from M. vannielii is identical in many of its properties to the 5-deazaflavin isolated from other methane-producing bacteria.

Aldehyde Oxidoreductases↗
PubMed1980

Purification and properties of 8-hydroxy-5-deazaflavin-dependent NADP+ reductase from Methanococcus vannielii.

The 8-hydroxy-5-deazaflavin-dependent NADP+ reductase component of the formate NADP+ oxidoreductase system of Methanococcus vannielii has been purified to homogeneity. The enzyme is specific for NADP+ and 8-hydroxy-5-deazaflavin. It catalyzes the reaction: 1,5-Dihydro-8-hydroxy-5-deazaflavin anion + NADP+ in equilibrium 8-hydroxy-5-deazaflavin + NADPH. The apparent molecular weight of the native enzyme is 85,000. A subunit molecular weight of 43,000 determined by sodium dodecyl sulfate gel electrophoresis indicates that the native enzyme is a dimer. The optimal temperature for catalytic activity is 17-20 degrees C and the pH maxima are 7.9 and 4.8 for the forward and reverse reactions, respectively. The kcat value of the forward reaction is 24 times greater than that of the reverse reaction, thus the production of NADPH at pH 7.0 is more favorable than its consumption. The reductase contains one or more sulfhydryl groups which are essential for catalytic activity.

Chemical Phenomena↗
PubMed1980

Stereochemical studies of 8-hydroxy-5-deazaflavin-dependent NADP+ reductase from Methanococcus vannielii.

The purified 8-hydroxy-5-deazaflavin-dependent NADP+ reductase from Methanococcus vannielii catalyzes an oxidation-reduction reaction between a novel 8-hydroxy-5-deazaflavin cofactor and nicotinamide adenine dinucleotide phosphate. The reaction was shown to be a direct hydride transfer process. Using stereospecifically 3H-labeled substrates, the steric course of this process was established to be S-specific with respect to the nicotinamide nucleotide. The 8-hydroxy-5-deazaflavin-dependent NADP+ reductase from M. vannielii and the hydrogenase system in the cell-free extracts of Methanobacterium thermoautotrophicum recognize the same side, designated as A side, with respect to the prochiral center at C-5 of the dihydro-8-hydroxy-5-deazaflavin cofactor.

Euryarchaeota↗
PubMed1981

Selenium-dependent and selenium-independent formate dehydrogenases of Methanococcus vannielii. Separation of the two forms and characterization of the purified selenium-independent form.

Anaerobic oxidation of formate by Methanococcus vannielii is catalyzed by two readily separable formate dehydrogenases. One of these is a 105,000-dalton protein that contains molybdenum, iron, and acid-labile sulfide, but not selenium. The other is a high molecular weight complex composed of selenoporotein and molybdo-iron sulfur protein subunits. Selenium occurs in this selenoenzyme in the chemical form of selenocysteine residues. M. vannielii cells from selenium-deficient media contain the 105,000-dalton formate dehydrogenase. Marked stimulation of growth by selenite supplementation is correlated with the simultaneous appearance in the cells of the high molecular weight selenoprotein . enzyme complex. The latter is the predominant form in cells from media additionally supplemented with tungstate. Under these conditions partial replacement of molybdenum with tungsten appears to occur. Both formate dehydrogenases are maximally active at pH 8.5 to 9.2 and at 60 degrees C and are extremely oxygen-sensitive. They utilize as electron acceptors 8-hydroxy-5-deazaflavin, FMN, FAD, and viologen and tetrazolium dyes.

Aldehyde Oxidoreductases↗
PubMed2000

Characterization of native and recombinant peptidyl prolyl cis-trans isomerases derived from Methanococcus thermolithotrophicus based on cDNA sequence.

It is important to establish whether a recombinant protein is an authentic copy of the predicted cDNA sequence. In this study, recombinant protein for native peptidyl prolyl cis-trans isomerase (N-PPIase) and double-labeled (13C- and 15N-) protein (DL-PPIase) appeared on the sodium dodecyl sulfate (SDS) electropherograms as two bands for N-PPIase and four bands for DL-PPIase. Since the N-terminal amino acid residues of all bands were the same, we characterized these bands using the peptide mapping method and amino acid composition analysis. Peptide mapping of the proteins seemed to be almost identical but they could not reflect the whole amino acid sequences of the protein. The bands on the polyvinylidene difluoride (PVDF) membrane, electroblotted after SDS-polyacrylamide gel electrophoresis (SDS-PAGE), were hydrolyzed and their amino acid composition was analyzed using a highly sensitive 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) amino acid analysis and compared with the cDNA sequences for proteins. The matching score (sigma(T%-E%)2) for similarity of proteins was calculated by summation of the square difference between the theoretical (T%) and the experimental (E%) amino acid composition of the recombinant protein. The amino acid composition of all bands of both proteins showed more than 93% of the theoretical values. The major molecular weights of both proteins were 16812 and 17694 by electrospray ionization (ESI)-mass spectrometry. However, the purified proteins also contained minor compounds with Mr of 3721 for N-PPIase and 5285 for DL-PPIase. These compounds were considered to be nonpeptidyl products that comigrated with the protein. Similarities of the amino acid composition of the four bands were more than 98%. Our results indicate that AQC amino acid analysis is the most suitable method for characterization of a recombinant protein.

Amino Acids↗