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PubMed1989

Genes encoding 5S rRNA and tRNAs in the extremely thermophilic archaebacterium Methanothermus fervidus.

Methanothermus fervidus was shown to have two 5S rRNA-encoding genes linked in rRNA operons to 16S and 23S rRNA-encoding genes. Sequencing of a cloned 5S rRNA gene confirmed that M. fervidus is a member of the Methanobacteriales, although its 5S rRNA is also similar in both primary sequence and predicted secondary structure to the 5S rRNA of the non-methanogenic, but also extremely thermophilic archaebacterium, Thermococcus celer. Two clusters of tRNA genes have also been cloned and sequenced form M. fervidus. The smaller cluster, cloned in pET5401, is composed of 5'-tRNA(UGUThr)-tRNA(UGGPro)-tRNA(GUCAsp)-tRNA(UUUL ys)-3' and the larger cluster, cloned in pET5475, is composed of 5'-tRNA(GUUAsn)-tRNA(CAUMet)-tRNA(UUCGlu)-tRNA(UAGL eu)-tRNA(GUGHis)-3'. The encoded tRNAs, with the exception of the tRNA(Leu), translate abundant codons in M. fervidus. The tRNA genes do not contain introns or encode 3'-terminal CCA residues. Homologous clusters of tRNA genes have been sequenced from Methanococcus vannielii and Methanococcus voltae, so that comparisons of transcription signals, gene organizations and primary sequences can be made and features possibly related to thermostability identified. During evolution, a 5S rRNA gene appears to have been incorporated into the cluster of tRNA genes in the methanococci but not in M. fervidus.

Amino Acid Sequence↗
PubMed2004

Construction of a hybrid quadrupole/Fourier transform ion cyclotron resonance mass spectrometer for versatile MS/MS above 10 kDa.

Technological advancements including an open-cylindrical Penning trap with capacitively coupled ICR cell, selective ion accumulation with a resolving quadrupole, and a voltage gradient used during ion extraction from an octopole ion trap, have individually improved dynamic range and sensitivity in Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS). Documented here is a new instrument utilizing these technologies toward the robust detection and fragmentation of biomolecules >10 kDa. Up to 55-fold enhancement in ion population by selective ion accumulation combined with 10- to 20- fold signal-to-noise improvement by application of a DC voltage gradient to an accumulation octopole during the ion transfer event offers improved signal-to-noise (or speed) of MS/MS experiments, for proteins from Methanococcus jannaschii and Saccharomyces cerevisiae whole cell lysates. After external quadrupole filtering with a 40 m/z window, three proteins were fragmented (and identified) in parallel from the database of Methanococcus jannaschii. Electron capture dissociation (ECD) of an intact yeast protein provides extensive sequence information resulting in a high degree of localization for an N-terminal acetylation. Hybrid fragmentation, infrared multiphoton dissociation (IRMPD) followed by low energy electrons (ECD), with the electron source located laterally off the z-axis and external to the magnet bore, presents a strategy for identification of proteins by means of the sequence tag approach. Automated implementation of diverse MS(n) approaches in a Q-FTMS instrument promises to help realize "top-down" proteomics in the future.

Bacterial Proteins↗
PubMed1985

Structure and sequence divergence of two archaebacterial genes.

The DNA sequences of a region that includes the hisA gene of two related methanogenic archaebacteria, Methanococcus voltae and Methanococcus vannielii, have been compared. Both organisms show a similar genome organization in this region, displaying three open reading frames (ORFs) separated by regions of very high A + T content. Two of the ORFs, including ORFHisA, show significant DNA sequence homology. As might be expected for organisms having a genome that is A + T-rich, there is a high preference for A and U as the third base in codons. Although the regions upstream of the structural genes contain prokaryotic-like promoter sequences, it is not known whether they are recognized as promoters in these archaebacterial cells. A ribosome binding site, G-G-T-G, is located 6 base pairs preceding the ATG translation initiation sequence of both hisA genes. The sequences upstream of the two hisA genes show only limited sequence homology. The M. voltae intergenic region contains four tandemly arranged repetitions of an 11-base-pair sequence, whereas the M. vannielii sequence contains both direct and inverted repetitive sequences. Based on the degree of hisA sequence homology, we conclude that M. voltae and M. vannielii are less closely related taxonomically than are members of the enteric group of eubacteria.

Archaea↗
PubMed1987

Putative promoter elements for the ribosomal RNA genes of the thermoacidophilic archaebacterium Sulfolobus sp. strain B12.

In Sulfolobus sp. strain B12, single-copy genes encode the three ribosomal RNAs. The genes for the 16S rRNA and for the 23S rRNA are closely linked but separated from the 5S rRNA gene. Transcription of the 16S/23S rRNA gene cluster starts 139 nucleotides upstream of the 5'-end of mature 16S rRNA. For the 5S rRNA gene the point of transcription initiation coincides with the 5'-end of mature 5S rRNA. The comparison of the upstream regions for these transcriptional start sites shows the presence of a completely conserved trinucleotide sequence around the point of transcription initiation and a completely conserved octanucleotide sequence about 22 nucleotides upstream of it. These sequences are only moderately homologous to putative promoter elements for stable RNA genes in the closely related archaebacterium Thermoproteus tenax (1), but they are very similar to corresponding sequences in the distantly related archaebacterium Methanococcus vannielii (2). The consensus sequence found for Sulfolobus and Methanococcus could therefore constitute the archetype of an archaebacterial promoter for stable RNA genes.

Archaea↗
PubMed1991

Pattern of organotin inhibition of methanogenic bacteria.

Seven organotin compounds and tin chloride were tested for their effects on the methanogenic bacteria Methanococcus thermolithotrophicus, Methanococcus deltae delta LH, and Methanosarcina barkeri 227. The methanogens were strongly inhibited by triethyltin, tripropyltin, and monophenyltin compounds, generally at concentrations below 0.05 mM. Less inhibition by tributyltin and diphenyltin was observed at levels below 0.1 mM, but complete inhibition was observed at a 1 mM concentration. Tin chloride inhibited all methanogens, with nearly complete inhibition at a 1 mM concentration. There was no inhibition by tetra-n-butyltin and triphenyltin compounds even at 2 mM, the highest concentration tested. The 50 and 100% inhibitory concentrations of all compounds were estimated; these values varied with both the compound tested and the bacterium tested. The 50% inhibitory concentration estimate generally decreased (i.e., giving a higher toxicity) as the total surface area of the alkyltin molecules decreased. These results differ considerably from those reported previously for aerobic microorganisms (G. Eng, E. J. Tierney, J. M. Bellama, and F. E. Brinckman, Appl. Organometallic Chem. 2:171-175, 1988), where a clear correlation between increasing total molecular surface area and increasing toxicity was documented with a variety of organisms. Using the same procedures as for the methanogens, we examined the effects of organotin compounds on Escherichia coli growing aerobically or anaerobically. The E. coli inhibition pattern clearly resembled that seen in the data of Eng et al., under both aerobic and anaerobic conditions.

Escherichia coli↗
PubMed1991

Effect of pH on Anaerobic Mild Steel Corrosion by Methanogenic Bacteria.

Methanogens can use H(2) produced by cathodic depolarization-mediated oxidation of elemental iron to produce methane. Thermodynamic consideration of the cathodic depolarization mechanism predicts more oxidation of Fe at lower pH. Methanogenic responses to pH by Methanococcus deltae, Methanococcus thermolithotrophicus, and Methanosarcina barkeri were examined. When grown on H(2)-CO(2), these bacteria had pH optima from 6.2 to 7.0, but when all H(2) was supplied from Fe, methanogenic pH optima were lower, 5.4 to 6.5. Corrosion was monitored with and without cultures and at various pHs; more corrosion occurred when cultures were present, biologically induced corrosion was greatest at the pH optima for methanogenesis from Fe, and corrosion without cultures increased with a drop in pH.

Journal Article↗
PubMed1992

Formation of unilamellar liposomes from total polar lipid extracts of methanogens.

Unilamellar liposomes were formed by controlled detergent dialysis of mixed micelles consisting of acetone-insoluble total polar lipids extracted from various methanogens and the detergent n-octyl-beta-D-glucopyranoside. The final liposome populations were studied by dynamic light scattering and electron microscopy. Unilamellar liposomes with mean diameters smaller than 100 nm were obtained with lipid extracts of Methanococcus voltae, Methanosarcina mazei, Methanosaeta concilii, and Methanococcus jannaschii (grown at 50 degrees C), whereas larger (greater than 100-nm) unilamellar liposomes were obtained with lipid extracts of M. jannaschii grown at 65 degrees C. These liposomes were shown to be closed intact vesicles capable of retaining entrapped [14C]sucrose for extended periods of time. With the exception of Methanospirillum hungatei liposomes, all size distributions of the different liposome populations were fairly homogeneous.

Dialysis↗
PubMed1984

Formaldehyde oxidation and methanogenesis.

Formaldehyde oxidation by cell-free extracts of Methanobacterium thermoautotrophicum was shown to drive methanogenesis from CH3-S-coenzyme M or HCHO under a nonreductive atmosphere of N2. Under N2 when HCHO was the sole source of carbon and reducing equivalents in the reaction, it underwent oxidation and reduction events (disproportionation), the sum of the reactions being 3 HCHO + H2O----CH4 + 2 HCOO - + 2H+. This reaction predicts a CH4/HCHO ratio of 1/3, which is in agreement with the experimental finding of 1/2.9. In extracts of the mesophilic methanogen Methanococcus voltae and the extreme thermophile Methanococcus jannaschii , which exhibited formate dehydrogenase activity, the CH4/HCHO ratio was 1/2. NADPH stimulated methane formation from HCHO under N2. An unidentified, oxygen-labile cofactor, the formaldehyde activation factor, present in boiled-cell extract was discovered. Methanopterin , an oxygen-stable molecule, also substituted for boiled-cell extract.

Euryarchaeota↗
PubMed1987

Pathway of acetate assimilation in autotrophic and heterotrophic methanococci.

The autotroph Methanococcus maripaludis contained high levels of acetate-coenzyme A ligase, pyruvate synthase, pyruvate, water dikinase, pyruvate carboxylase, and the enzymes of the incomplete reductive tricarboxylic acid cycle. Phosphoenolpyruvate carboxykinase, citrate synthase, and isocitrate dehydrogenase were not detected. In contrast, the heterotroph Methanococcus sp. strain A3 contained acetate kinase, and acetate coenzyme A ligase was virtually absent.

Acetates↗
PubMed1993

Freeze-fracture planes of methanogen membranes correlate with the content of tetraether lipids.

Methanospirillum hungatei GP1 contained 50% of its ether core lipids (polar lipids less head groups) as tetraether lipids, and its plasma membrane failed to fracture along its hydrophobic domain during freeze-etching. The membrane of Methanosaeta ("Methanothrix") concilii did not contain tetraether lipids and easily fractured to reveal typical intramembranous particles. Methanococcus jannaschii grown at 50 degrees C contained 20% tetraether core lipids, which increased to 45% when cells were grown at 70 degrees C. The frequency of membrane fracture was reduced as the membrane-spanning tetraether lipids approached 45%. As the tetraether lipid content increased, and while fracture was still possible, the particle density in the membrane increased; these added particles could be tetraether lipid complexes torn from the opposing membrane face. The diether membrane (no tetraether lipid) of Methanococcus voltae easily fractured, and the intramembranous particle density was low. Protein-free liposomes containing tetraether core lipids (ca. 45%) also did not fracture, whereas those made up exclusively of diether lipids did split, indicating that tetraether lipids add considerable vertical stability to the membrane. At tetraether lipid concentrations below 45%, liposome bilayers fractured to reveal small intramembranous particles which we interpret to be tetraether lipid complexes.

Cell Membrane↗
PubMedDate not supplied

[Use of the level of G+C n DNA for studying the molecular phylogeny o f methanogenic archaebacteria].

A new method for theoretical analysis of the molecular phylogeny of bacteria, successfully applied earlier to nitrifying bacteria, was used to study the molecular phylogeny of methanogenic archaebacteria. The group studied included Methanococcus igneus, Methanococcus vannielii, Methanothermus fervidus, Methanolobus tindarius, Methanobacterium formicicum, Methanosarcina barkeri, Methanobacterium thermoformicicum, Methanoplanus limicola, Methanospirillum hungatei, and Methanobacterium thermoautotrophicum. Based on the hypothesis that direct linear regression always exists between evolutionary changes in the DNA G + C content and the primary structure of rRNA, the branching order of the phylogenetic tree of methanogenic archaebacteria was determined. For this tree, the values of the evolutionary distance between 16S rRNA primary structures Ei and the values of the G + C evolutionary distance P(i) exhibited a correlation coefficient 0.78. Thus, the DNA G + C content is not only an important taxonomic characteristics but also provides information helpful for the determination of the branching order of phylogenetic trees constructed based on 16S rRNA primary structures.

Adenosine↗
PubMed1990

Overexpression of the methanococcal ribosomal protein L12 in Escherichia coli and its incorporation into halobacterial 50 S subunits yielding active ribosomes.

The gene for the ribosomal L12 protein from the archaebacterium Methanococcus vannielii was cloned into the expression vector pKK223-3. The protein was overexpressed and remained stable in Escherichia coli XL1 cells. Purification yielded a protein with the same amino acid composition and sequence as in Methanococcus but it was acetylated at the N terminus as in the case with the homologous protein of E. coli. The in vivo incorporation of the overexpressed protein into the E. coli ribosomes was not observed. The overexpressed M. vannielii protein MvaL12e was incorporated into halobacterial ribosomes, thereby displacing the corresponding halobacterial L12 protein. Intact 70 S ribosomes were reconstituted from halobacterial 50 S subunits carrying the MvaL12e protein. These ribosomes were as active as native halobacterial ribosomes in a poly(U) assay. On the other hand, our attempts to incorporate L12 proteins from Bacillus stearothermophilus and E. coli into halobacterial ribosomes were not successful. These results support the conclusion which is based on primary sequence and predicted secondary structure comparisons that there exist two distinct L12 protein families, namely the eubacterial L12 protein family and the eukaryotic/archaebacterial L12 protein family.

Amino Acids↗
PubMed2000

Membrane properties of archaeal macrocyclic diether phospholipids.

Several biophysical properties of four synthetic archaeal phospholipids [one polyprenyl macrocyclic lipid A and three polyprenyl double-chain lipids (B, C, D) bearing zero, one or four double bonds in each chain] were studied using differential scanning calorimetry, electron and optical microscopies, stopped-flow/light scattering and solid-state 2H-NMR techniques. These phospholipids gave a variety of self-organized structures in water, in particular vesicles and tubules. These assemblies change in response to simple thermal convection. Some specific membrane properties of these archaeal phospholipids were observed: They are in a liquid-crystalline state over a wide temperature range; the dynamics of their polyprenyl chains is higher than that of n-acyl chains; the water permeability of the membranes is lower than that of n-acyl phospholipid membranes. It was also found that macrocyclization remarkably improves the barrier properties to water and the membrane stability. This may be related to the adaptation of Methanococcus jannaschii to the extreme conditions of the deep-sea hydrothermal vents.

Cryoelectron Microscopy↗
PubMed2002

Fourier-transform mass spectrometry for automated fragmentation and identification of 5-20 kDa proteins in mixtures.

When presented with a mixture of intact proteins, electrospray ionization with Fourier-transform mass spectrometry (ESI-FTMS) has the capability to obtain direct fragmentation information from isolated ions. However, the automation of this capability has not been achieved to date. We have developed software for unattended acquisition of protein tandem mass spectrometry (MS/MS) data and batch processing of the resulting files for identification of whole proteins. Mixtures of both protein standards (8-29 kDa) and Methanococcus jannaschii cytosolic proteins (up to six components + 20 kDa) were infused via an autosampler, and MS/MS data were acquired without human intervention. The acquisition software recognizes ESI charge state patterns, generates protein-specific isolation waveforms on-the-fly, and fragments ions using two different infrared laser times. In addition to protein standards, five wild-type proteins (7-14 kDa) were identified automatically with 100% sequence coverage from the M. jannaschii database. The software underpins a measurement platform for sample-dependent acquisition of MS/MS data for whole proteins, a critical step to realize proteomics with 100% sequence coverage in a higher throughput setting.

Archaeal Proteins↗
PubMed2001

Selenoprotein synthesis in archaea.

The availability of the genome sequences from several archaea has facilitated the identification of the encoded selenoproteins and also of most of the components of the machinery for selenocysteine biosynthesis and insertion. Until now, selenoproteins have been identified solely in species of the genera Methanococcus (M.) and Methanopyrus. Apart from selenophosphate synthetase, they include only enzymes with a function in energy metabolism. Like in bacteria and eukarya, selenocysteine insertion is directed by a UGA codon in the mRNA and involves the action of a specific tRNA and of selenophosphate as the selenium donor. Major differences to the bacterial system, however, are that no homolog for the bacterial selenocysteine synthase was found and, especially, that the SECIS element of the mRNA is positioned in the 3' nontranslated region. The characterisation of a homolog for the bacterial SelB protein showed that it does not bind to the SECIS element necessitating the activity of at least a second protein. The use of the genetic system of M. maripaludis allowed the heterologous expression of a selenoprotein gene from M. jannaschii and will facilitate the elucidation of the mechanism of the selenocysteine insertion process in the future.

Amino Acid Sequence↗
PubMed1997

Evaluation of algorithms used for cross-species proteome characterisation.

The ability to effectively search databases for the identification of protein spots from two-dimensional electrophoresis gels has become an essential step in the study of microbial proteomes. A variety of analytical techniques are currently being employed during protein characterisation. A number of algorithms used to search databases, accessible via the World Wide Web, depend upon information concerning N- and C-terminal microsequence, amino acid composition, and peptide-mass fingerprinting. The effectiveness of nine such algorithms, as well as COMBINED (software developed in this laboratory for identifying proteins across species boundaries) was examined. Fifty-four ribosomal proteins from the Mycoplasma genitalium genome, and 72 amino acyl tRNA synthetases from the Haemophilus influenzae, M. genitalium and Methanococcus jannaschii genomes were chosen for study. These proteins were selected because they represent a wide range of sequence identities across species boundaries (22.7-100% identity), as detected by standard sequence alignment tools. Such sequence variation allowed for a statistical comparison of algorithm success measured against published sequence identity. The ability of analytical techniques used in protein characterisation and associated database query programs to detect identity at the functional group level was examined for proteins with low levels of homology at the gene/protein sequence level. The significance of these theoretical data manipulations provided the means to predict the utility of data acquired experimentally for non-sequence-dependent software in proteome analysis. The data obtained also predicted that 'sequence tagging' of peptide fingerprints would need to be accompanied by at least 11-20 residues of amino acid sequence for it to be widely used for protein characterisation across species boundaries.

Algorithms↗
PubMed2006

Structural studies of MJ1529, an O6-methylguanine-DNA methyltransferase.

The structure of an O6-methylguanine-DNA methyltransferase (MGMT) from the thermophile Methanococcus jannaschii has been determined using multinuclear multidimensional NMR spectroscopy. The structure is similar to homologs from other organisms that have been determined by crystallography, with some variation in the N-terminal domain. The C-terminal domain is more highly conserved in both sequence and structure. Regions of the protein show broadening, reflecting conformational flexibility that is likely related to function.

Amino Acid Sequence↗
PubMed2003

ProteomeWeb: a web-based interface for the display and interrogation of proteomes.

The analysis of proteomes, i.e., the proteins expressed by biological organisms under a given set of conditions at a given time, requires separating complex protein mixtures into discrete protein components, measuring their relative abundances, and identifying the individual protein components. Many types of data are generated during the course of proteome analysis, including graphic images of the protein profiles, flat files containing numeric data, spreadsheets for assimilating numeric data, and relational database tables for integrating data from multiple experiments. As part of a project to describe the proteomes of microbes of interest to the U.S. Department of Energy, a World-Wide Web-based interface has been developed for the display of protein profiles generated by two-dimensional gel electrophoresis. The web interface is capable of obtaining protein identifications on the fly, interrogating the quantitative data in the context of available genome sequence information, and relating the proteome data to existing metabolic pathway databases. Analysis of protein expression profiles is expedited, providing the capability to efficiently determine the gene locations for proteins modulated in abundance in response to different growth conditions and to locate the positions of the proteins within specific metabolic pathways. The proteome of the archaeon Methanococcus jannaschii, a microbe for which the complete genome sequence is available, is used to demonstrate the capabilities of this evolving web interface (http://proteomeweb.anl.gov).

Amino Acid Sequence↗