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PubMed2005

Insights into the genomes of archaea mediating the anaerobic oxidation of methane.

The anaerobic oxidation of methane is a globally significant process which is mediated by consortia of yet uncultivated methanotrophic archaea (ANME) and sulfate-reducing bacteria. In order to gain deeper insights into genome characteristics of the different ANME groups, large-insert genomic libraries were constructed using DNA extracted from a methanotrophic microbial mat growing in the anoxic part of the Black Sea, and from sediments above gas hydrates at the Hydrate Ridge off the coast of Oregon. Analysis of these fosmid libraries with respect to archaeal 16S rRNA gene diversity revealed a single ANME-1b ribotype for the Black Sea libraries, whereas the sequences derived from the Hydrate Ridge library phylogenetically affiliated with the ANME-2a, ANME-2c and ANME-3 group. Genome walking for ANME-1b resulted in a contiguous 155 kb composite genome fragment. The comparison of a set of four genomic fragments belonging to the different ANME groups revealed differences in the rRNA operon structure and the average G+C content, with the ANME-2c contig showing the highest divergence within the set. A detailed analysis of the ANME contigs with respect to genes putatively involved in the anaerobic oxidation of methane led to the identification of: (i) a putative N5,N10-methenyltetrahydromethanopterin cyclohydrolase gene, (ii) a gene cluster supposedly encoding a novel type of heterodisulfide reductase/dehydrogenase complex and (iii) a gene cluster putatively encoding a new type of CO dehydrogenase/acetyl-CoA synthase enzyme complex.

Amino Acid Sequence↗
PubMed2006

Quantification of mcrA by quantitative fluorescent PCR in sediments from methane seep of the Nankai Trough.

A quantitative fluorogenic PCR method for group-specific methyl coenzyme M reductase subunit A genes (mcrA) from methanotrophic archaea was established and applied to the characterization of microbial communities in anoxic methane seep sediments at the accretionary prism of the Nankai Trough. All of the previously identified subgroups of anaerobic methanotroph (ANME) mcrA genes were detected in the cores up to 25 cm below the seafloor, but distributional patterns of mcrA genes were found to differ according to depth. These findings suggest a distinct distribution of phylogenetically and physiologically diverse methanotrophic archaea that mediate methane oxidation in the anoxic sediments. This quantification method will contribute to future investigations of methanotrophic microbial ecosystems in anoxic marine sediments.

Anaerobiosis↗
PubMed2006

Functional expression of the particulate methane mono-oxygenase gene in recombinant Rhodococcus erythropolis.

In order to construct an expression system for the particulate methane mono-oxygenase (pMMO) gene (pmo), the structural gene cluster pmoCAB amplified from Methylosinus trichosporium OB3b was inserted into a shuttle vector pBS305 under the control of a dsz promoter and transformed into Rhodococcus erythropolis LSSE8-1. A stable transformant was successfully obtained using ethane as the sole carbon source. Fluorescence in situ hybridization results showed that the dsz promoter allowed the pmo genes to be transcribed in the recombinant strain. The effects of Cu2+ and Zn2+ concentrations on cell growth and pMMO activity in ethane-containing medium were examined. It was discovered that 7.5 microM Cu2+ and 1.8 microM Zn2+ were suitable to achieve high cell concentration and pMMO activity, but the amount of methanol accumulated during methane oxidation by the recombinant strain was still low.

DNA, Bacterial↗
PubMed1985

Effect of a homologous series of halogenated methanes on pulmonary uptake of 5-hydroxytryptamine in isolated perfused rat lung.

The potency of halogenated methanes to inhibit uptake of 5-hydroxytryptamine (5-HT) from the pulmonary circulation was studied using an isolated, perfused and ventilated rat lung preparation. The agents were vaporized and mixed with the inlet air. The results indicate that the degree of chlorination is the most important factor for potency of the methanes to inhibit lung uptake of 5-HT. When hydrogen was substituted with fluorine the potency was decreased dramatically. Bromine seemed to have the opposite effect. The data also suggested that the degree of chlorination was more important rather than hydrophobic/hydrophilic balance of the solvent molecule. These effects seem to be correlated with the narcotic effects of the substances studied.

Animals↗
PubMed1982

Methane and hydrogen production by human intestinal anaerobic bacteria.

The gas above liquid cultures of a variety of human intestinal anaerobic bacteria was sampled and analysed by headspace gas chromatography. Hydrogen production was greatest with strains of the genus Clostridium, intermediate with anaerobic cocci and least with Bacteroides sp. Very few strains produced methane although small amounts were detected with one strain of B. thetaiotaomicron, C. perfringens and C. histolyticum. There may be a relationship between these anaerobic bacteria and several gastrointestinal disorders in which there is a build up of hydrogen or methane in the intestines.

Bacteroides↗
PubMed2004

Methanobactin, a copper-acquisition compound from methane-oxidizing bacteria.

Siderophores are extracellular iron-binding compounds that mediate iron transport into many cells. We present evidence of analogous molecules for copper transport from methane-oxidizing bacteria, represented here by a small fluorescent chromopeptide (C45N12O14H62Cu, 1216 daltons) produced by Methylosinus trichosporium OB3b. The crystal structure of this compound, methanobactin, was resolved to 1.15 angstroms. It is composed of a tetrapeptide, a tripeptide, and several unusual moieties, including two 4-thionyl-5-hydroxy-imidazole chromophores that coordinate the copper, a pyrrolidine that confers a bend in the overall chain, and an amino-terminal isopropylester group. The copper coordination environment includes a dual nitrogen- and sulfur-donating system derived from the thionyl imidazolate moieties. Structural elucidation of this molecule has broad implications in terms of organo-copper chemistry, biological methane oxidation, and global carbon cycling.

Amino Acids↗
PubMed1997

Dissociation of methane into hydrocarbons at extreme (planetary) pressure and temperature.

Constant-pressure, first-principles molecular dynamic simulations were used to investigate the behavior of methane at high pressure and temperature. Contrary to the current interpretation of shock-wave experiments, the simulations suggest that, below 100 gigapascals, methane dissociates into a mixture of hydrocarbons, and it separates into hydrogen and carbon only above 300 gigapascals. The simulation conditions (100 to 300 gigapascals; 4000 to 5000 kelvin) were chosen to follow the isentrope in the middle ice layers of Neptune and Uranus. Implications on the physics of these planets are discussed.

Atmosphere↗
PubMed1982

Role of vitamin B12 in methyl transfer for methane biosynthesis by Methanosarcina barkeri.

When Methanosarcina barkeri is grown on methanol as the sole carbon source, a B12-containing protein is synthesized by this organism. This B12 protein contains bound aquocobalamin, and when this cofactor is reduced and methylated with [14C]methyl iodide, the resultant [14C]methyl B12 protein is extremely active in the biosynthesis of 14C-labeled methane. These findings indicate that a B12-dependent system is operative in the biological formation of methane in addition to other systems that are B12-independent.

Bacterial Proteins↗
PubMed1995

Condensation of methane, ammonia, and water and the inhibition of convection in giant planets.

The condensation of chemical species of high molecular mass such as methane, ammonia, and water can inhibit convection in the hydrogen-helium atmospheres of the giant planets. Convection is inhibited in Uranus and Neptune when methane reaches an abundance of about 15 times the solar value and in Jupiter and Saturn if the abundance of water is more than about five times the solar value. The temperature gradient consequently becomes superadiabatic, which is observed in temperature profiles inferred from radio-occultation measurements. The planetary heat flux is then likely to be transported by another mechanism, possibly radiation in Uranus, or diffusive convection.

Ammonia↗
PubMed2006

Mixed pollutant degradation by Methylosinus trichosporium OB3b expressing either soluble or particulate methane monooxygenase: can the tortoise beat the hare?

Methanotrophs have been widely investigated for in situ bioremediation due to their ubiquity and their ability to degrade halogenated hydrocarbons through the activity of methane monooxygenase (MMO). It has been speculated that cells expressing the soluble form of MMO (sMMO) are more efficient in cleaning up sites polluted with halogenated hydrocarbons due to its broader substrate range and relatively fast degradation rates compared cells expressing the other form of MMO, the particulate MMO (pMMO). To examine this issue, the biodegradation of mixtures of chlorinated solvents, i.e., trichloroethylene (TCE), trans-dichloroethylene (t-DCE), and vinyl chloride (VC), by Methylosinus trichosporium OB3b in the presence of methane using either form of MMO was investigated over longer time frames than those commonly used, i.e., days instead of hours. Growth of M. trichosporium OB3b along with pollutant degradation were monitored and analyzed using a simple comparative model developed from the Omega model created for analysis of the competitive binding of oxygen and carbon dioxide by ribulose bisphosphate carboxylase. From these findings, it appears that at concentrations of VC, t-DCE, and TCE greater than 10 microM each, methanotrophs expressing pMMO have a competitive advantage over cells expressing sMMO due to higher growth rates. Despite such an apparent growth advantage, pMMO-expressing cells degraded less of these substrates at these concentrations than sMMO-expressing cells during active growth. If the concentrations were increased to 100 muM, however, not only did pMMO-expressing cells grow faster, they degraded more of these pollutants and did so in a shorter amount of time. These findings suggest that the relative rates of growth substrate and pollutant degradation are important factors in determining which form of MMO should be considered for pollutant degradation.

Biodegradation, Environmental↗
PubMed1978

Effect of sulfur-containing compounds on anaerobic degradation of cellulose to methane by mixed cultures obtained from sewage sludge.

Tests were made to determine the effects of inorganic and organic sulfur sources on the degradation of cellulose to methane in a chemically defined medium with sulfur-poor inoculum prepared from sewage sludge. The results show that a sulfur source of about a 0.85 mM concentration is essential for the degradation of cellulose to CH4. However, the production of CH4 from CO2 and H2 provided in the headspace occurred with 0.1 mM sulfate or sulfide. At a 9 mM concentration, all inorganic sulfur compounds other than sulfate inhibited both cellulose degradation and methane formation, and this inhibition increased in the order thiosulfate less than sulfite less than sulfide less than H2S. It appears that the degradation of cellulose to CH4 in a sulfate-free medium by inoculum maintained in a low-sulfur medium is inhibited because of the lack of availability of sulfur for growth of bacteria and synthesis of cell materials and sulfur-containing cofactors involved in cellulose degradation and methanogenesis. The reduction of methanogenesis by higher levels of sulfate probably occurs as a result of stimulation of reactions converting acetate and H2 to end products other than CH4.

Anaerobiosis↗
PubMed1979

Anaerobic metabolism of immediate methane precursors in Lake Mendota.

Lake Mendota sediments and the immediate overlying water column were studied to better understand the metabolism of the methanogenic precursors H2/CO2 and acetate in nature. The pool size of acetate (3.5 microns M) was very small, and the acetate turnover time (0.22h) was very rapid. The dissolved inorganic carbon pool was shown to be large (6.4 to 8.3 mM), and the turnover time was slow (111 H.). CO2 was shown to account for 41 +/- 5.5% of the methane produced in sediment. Acetate and H2/CO2 were simultaneously converted to CH4. The addition of H2 to sediments resulted in an increase specific activity of CH4 from H(14)CO3- and a decrease in specific activity of CH4 from [2-14C]acetate. Acetate addition resulted in a decrease in specific activity of CH4 from H(14)CO3-. The metabolism of H(14)CO3- or [2-14C]acetate to 14CH4 was not inhibited by addition of acetate or H2. After greater than 99% of added [2-14C]acetate had been turned over, 42% of the label was recovered as 14CH4 20% was recovered as 14CO2 and 38% was incorporated into sediment. Inhibitor studies of [2-14C]acetate metabolism in sediments demonstrated that CHCl3 completely inhibited CH4 formation, but not CO2 production. Air and nitrate addition inhibited CH4 formation and stimulated CO2 production, whereas fluoroacetate addition totally inhibited acetate metabolism. The oxidation of [2-14C]acetate to 14CO2 was shown to decrease with time when sediment was incubated before the addition of label, suggesting depletion of low levels of an endogenous sediment electron acceptor. Acetate metabolism varied seasonally and was related to the concentration of sulfate in the lake and interstitial water. Methanogenesis occurred in the sediment and in the water immediately overlying the sediment during period of lake stratification and several centimeters below the sediment-water interface during lake turnovers. These data indicate that methanogenesis in Lake Mendota sediments was limited by "immediate" methane precursor availability (i.e., acetate and H2), by competition for these substrates by nonmethanogens, and by seasonal variations which altered sediment and water chemistry.

Acetates↗
PubMed1989

Mass-spectrometric studies of the interrelations among hydrogenase, carbon monoxide dehydrogenase, and methane-forming activities in pure and mixed cultures of Desulfovibrio vulgaris, Desulfovibrio desulfuricans, and Methanosarcina barkeri.

The activities of pure and mixed cultures of Desulfovibrio vulgaris and Methanosarcina barkeri in the exponential growth phase were monitored by measuring changes in dissolved-gas concentration by membrane-inlet mass spectrometry. M. barkeri grown under H2-CO2 or methanol produced limited amounts of methane and practically no hydrogen from either substrate. The addition of CO resulted in a transient H2 production concomitant with CO consumption. Hydrogen was then taken up, and CH4 production increased. All these events were suppressed by KCN, which inhibited carbon monoxide dehydrogenase activity. Therefore, with both substrates, H2 appeared to be an intermediate in CO reduction to CH4. The cells grown on H2-CO2 consumed 4 mol of CO and produced 1 mol of CH4. Methanol-grown cells reduced CH3OH with H2 resulting from carbon monoxide dehydrogenase activity, and the ratio was then 1 mol of CH4 to 1 mol of CO. Only 12CH4 and no 13CH4 was obtained from 13CO, indicating that CO could not be the direct precursor of CH4. In mixed cultures of D. vulgaris and M. barkeri on lactate, an initial burst of H2 was observed, followed by a lower level of production, whereas methane synthesis was linear with time. Addition of CO to the mixed culture also resulted in transient extra H2 production but had no inhibitory effect upon CH4 formation, even when the sulfate reducer was D. vulgaris Hildenborough, whose periplasmic iron hydrogenase is very sensitive to CO. The hydrogen transfer is therefore probably mediated by a less CO-sensitive nickel-iron hydrogenase from either of both species.

Aldehyde Oxidoreductases↗
PubMed1999

Nitrous oxide production and methane oxidation by different ammonia-oxidizing bacteria.

Ammonia-oxidizing bacteria (AOB) are thought to contribute significantly to N2O production and methane oxidation in soils. Most of our knowledge derives from experiments with Nitrosomonas europaea, which appears to be of minor importance in most soils compared to Nitrosospira spp. We have conducted a comparative study of levels of aerobic N2O production in six phylogenetically different Nitrosospira strains newly isolated from soils and in two N. europaea and Nitrosospira multiformis type strains. The fraction of oxidized ammonium released as N2O during aerobic growth was remarkably constant (0.07 to 0.1%) for all the Nitrosospira strains, irrespective of the substrate supply (urea versus ammonium), the pH, or substrate limitation. N. europaea and Nitrosospira multiformis released similar fractions of N2O when they were supplied with ample amounts of substrates, but the fractions rose sharply (to 1 to 5%) when they were restricted by a low pH or substrate limitation. Phosphate buffer (versus HEPES) doubled the N2O release for all types of AOB. No detectable oxidation of atmospheric methane was detected. Calculations based on detection limits as well as data in the literature on CH4 oxidation by AOB bacteria prove that none of the tested strains contribute significantly to the oxidation of atmospheric CH4 in soils.

Ammonia↗
PubMed2003

Biogeochemical evidence that thermophilic archaea mediate the anaerobic oxidation of methane.

Distributions and isotopic analyses of lipids from sediment cores at a hydrothermally active site in the Guaymas Basin with a steep sedimentary temperature gradient revealed the presence of archaea that oxidize methane anaerobically. The presence of strongly (13)C-depleted lipids at greater depths in the sediments suggests that microbes involved in anaerobic oxidation of methane are present and presumably active at environmental temperatures of >30 degrees C, indicating that this process can occur not only at cold seeps but also at hydrothermal sites. The distribution of the membrane tetraether lipids of the methanotrophic archaea shows that these organisms have adapted their membrane composition to these high environmental temperatures.

Anaerobiosis↗
PubMed2005

First genome data from uncultured upland soil cluster alpha methanotrophs provide further evidence for a close phylogenetic relationship to Methylocapsa acidiphila B2 and for high-affinity methanotrophy involving particulate methane monooxygenase.

Members of upland soil cluster alpha (USC alpha) are assumed to be methanotrophic bacteria (MB) adapted to the trace level of atmospheric methane. So far, these MB have eluded all cultivation attempts. While the 16S rRNA phylogeny of USC alpha members is still not known, phylogenies constructed for the active-site polypeptide (encoded by pmoA) of particulate methane monooxygenase (pMMO) placed USC alpha next to the alphaproteobacterial Methylocapsa acidiphila B2. To assess whether the pmoA tree reflects the evolutionary identity of USC alpha, a 42-kb genomic contig of a USC alpha representative was obtained from acidic forest soil by screening a metagenomic fosmid library of 250,000 clones using pmoA-targeted PCR. For comparison, a 101-kb genomic contig from M. acidiphila was analyzed, including the pmo operon. The following three lines of evidence confirmed a close phylogenetic relationship between USC alpha and M. acidiphila: (i) tetranucleotide frequency patterns of 5-kb genomic subfragments, (ii) annotation and comparative analysis of the genomic fragments against all completely sequenced genomes available in public domain databases, and (iii) three single gene phylogenies constructed using the deduced amino acid sequences of a putative prephenate dehydratase, a staphylococcal-like nuclease, and a putative zinc metalloprotease. A comparative analysis of the pmo operons of USC alpha and M. acidiphila corroborated previous reports that both the pmo operon structure and the predicted secondary structure of deduced pMMO are highly conserved among all MB.

Bacteria↗
PubMed2005

Dual symbiosis in a Bathymodiolus sp. mussel from a methane seep on the Gabon continental margin (Southeast Atlantic): 16S rRNA phylogeny and distribution of the symbionts in gills.

Deep-sea mussels of the genus Bathymodiolus (Bivalvia: Mytilidae) harbor symbiotic bacteria in their gills and are among the dominant invertebrate species at cold seeps and hydrothermal vents. An undescribed Bathymodiolus species was collected at a depth of 3,150 m in a newly discovered cold seep area on the southeast Atlantic margin, close to the Zaire channel. Transmission electron microscopy, comparative 16S rRNA analysis, and fluorescence in situ hybridization indicated that this Bathymodiolus sp. lives in a dual symbiosis with sulfide- and methane-oxidizing bacteria. A distinct distribution pattern of the symbiotic bacteria in the gill epithelium was observed, with the thiotrophic symbiont dominating the apical region and the methanotrophic symbiont more abundant in the basal region of the bacteriocytes. No variations in this distribution pattern or in the relative abundances of the two symbionts were observed in mussels collected from three different mussel beds with methane concentrations ranging from 0.7 to 33.7 microM. The 16S rRNA sequence of the methanotrophic symbiont is most closely related to those of known methanotrophic symbionts from other bathymodiolid mussels. Surprisingly, the thiotrophic Bathymodiolus sp. 16S rRNA sequence does not fall into the monophyletic group of sequences from thiotrophic symbionts of all other Bathymodiolus hosts. While these mussel species all come from vents, this study describes the first thiotrophic sequence from a seep mussel and shows that it is most closely related (99% sequence identity) to an environmental clone sequence obtained from a hydrothermal plume near Japan.

Animals↗