Long-term partners uncoupled Methane-munching archaea in marine sediments live closely coupled to sulfate-reducing bacteria in a syntrophic relationship. Surprisingly, however, these archaea do not necessarily need their bacterial partners to survive or grow. Scheller et al. performed stable isotope incubation experiments with deep-sea methane seep sediments (see the Perspective by Rotaru and Thamdrup). Several groups of methane-oxidizing archaea could use a range of soluble electron acceptors instead of coupling to active bacterial sulfate reduction. This decoupled pathway shows that methane-oxidizing archaea transfer electrons extracellularly and may even possess the capacity to respire iron and manganese minerals that are abundant in seafloor sediments. Science , this issue p. 703 ; see also p. 658

Large amounts (estimates range from 70 Tg per year to 300 Tg per year) of the potent greenhouse gas methane are oxidized to carbon dioxide in marine sediments by communities of methanotrophic archaea and sulphate-reducing bacteria, and thus are prevented from escaping into the atmosphere. Indirect evidence indicates that the anaerobic oxidation of methane might proceed as the reverse of archaeal methanogenesis from carbon dioxide with the nickel-containing methyl-coenzyme M reductase (MCR) as the methane-activating enzyme. However, experiments showing that MCR can catalyse the endergonic back reaction have been lacking. Here we report that purified MCR from Methanothermobacter marburgensis converts methane into methyl-coenzyme M under equilibrium conditions with apparent V(max) (maximum rate) and K(m) (Michaelis constant) values consistent with the observed in vivo kinetics of the anaerobic oxidation of methane with sulphate. This result supports the hypothesis of 'reverse methanogenesis' and is paramount to understanding the still-unknown mechanism of the last step of methanogenesis. The ability of MCR to cleave the particularly strong C-H bond of methane without the involvement of highly reactive oxygen-derived intermediates is directly relevant to catalytic C-H activation, currently an area of great interest in chemistry.

Here we describe the application of a new click chemistry method for fluorescent tracking of protein synthesis in individual microorganisms within environmental samples. This technique, termed bioorthogonal non-canonical amino acid tagging (BONCAT), is based on the in vivo incorporation of the non-canonical amino acid L-azidohomoalanine (AHA), a surrogate for l-methionine, followed by fluorescent labelling of AHA-containing cellular proteins by azide-alkyne click chemistry. BONCAT was evaluated with a range of phylogenetically and physiologically diverse archaeal and bacterial pure cultures and enrichments, and used to visualize translationally active cells within complex environmental samples including an oral biofilm, freshwater and anoxic sediment. We also developed combined assays that couple BONCAT with ribosomal RNA (rRNA)-targeted fluorescence in situ hybridization (FISH), enabling a direct link between taxonomic identity and translational activity. Using a methanotrophic enrichment culture incubated under different conditions, we demonstrate the potential of BONCAT-FISH to study microbial physiology in situ. A direct comparison of anabolic activity using BONCAT and stable isotope labelling by nano-scale secondary ion mass spectrometry (15NH3 assimilation) for individual cells within a sediment-sourced enrichment culture showed concordance between AHA-positive cells and 15N enrichment. BONCAT-FISH offers a fast, inexpensive and straightforward fluorescence microscopy method for studying the in situ activity of environmental microbes on a single-cell level.

The nickel enzyme methyl-coenzyme M reductase (MCR) catalyzes two important transformations in the global carbon cycle: methane formation and its reverse, the anaerobic oxidation of methane. MCR uses the methyl thioether methyl-coenzyme M (CH3-S-CH2CH2-SO3(-), Me-S-CoM) and the thiol coenzyme B (CoB-SH) as substrates and converts them reversibly to methane and the corresponding heterodisulfide (CoB-S-S-CoM). The catalytic mechanism is still unknown. Here, we present isotope effects for this reaction in both directions, catalyzed by the enzyme isolated from Methanothermobacter marburgensis . For methane formation, a carbon isotope effect ((12)CH3-S-CoM/(13)CH3-S-CoM) of 1.04 ± 0.01 was measured, showing that breaking of the C-S bond in the substrate Me-S-CoM is the rate-limiting step. A secondary isotope effect of 1.19 ± 0.01 per D in the methyl group of CD3-S-CoM indicates a geometric change of the methyl group from tetrahedral to trigonal planar upon going to the transition state of the rate-limiting step. This finding is consistent with an almost free methyl radical in the highest transition state. Methane activation proceeds with a primary isotope effect of 2.44 ± 0.22 for the C-H vs C-D bond breakage and a secondary isotope effect corresponding to 1.17 ± 0.05 per D. These values are consistent with isotope effects reported for oxidative cleavage/reductive coupling occurring at transition metal centers during C-H activation but are also in the range expected for the radical substitution mechanism proposed by Siegbahn et al. The isotope effects presented here constitute boundary conditions for any suggested or calculated mechanism.

Sulfate is the predominant electron acceptor for anaerobic oxidation of methane (AOM) in marine sediments. This process is carried out by a syntrophic consortium of anaerobic methanotrophic archaea (ANME) and sulfate reducing bacteria (SRB) through an energy conservation mechanism that is still poorly understood. It was previously hypothesized that ANME alone could couple methane oxidation to dissimilatory sulfate reduction, but a genetic and biochemical basis for this proposal has not been identified. Using comparative genomic and phylogenetic analyses, we found the genetic capacity in ANME and related methanogenic archaea for sulfate reduction, including sulfate adenylyltransferase, APS kinase, APS/PAPS reductase and two different sulfite reductases. Based on characterized homologs and the lack of associated energy conserving complexes, the sulfate reduction pathways in ANME are likely used for assimilation but not dissimilation of sulfate. Environmental metaproteomic analysis confirmed the expression of 6 proteins in the sulfate assimilation pathway of ANME. The highest expressed proteins related to sulfate assimilation were two sulfite reductases, namely assimilatory-type low-molecular-weight sulfite reductase (alSir) and a divergent group of coenzyme F420-dependent sulfite reductase (Group II Fsr). In methane seep sediment microcosm experiments, however, sulfite and zero-valent sulfur amendments were inhibitory to ANME-2a/2c while growth in their syntrophic SRB partner was not observed. Combined with our genomic and metaproteomic results, the passage of sulfur species by ANME as metabolic intermediates for their SRB partners is unlikely. Instead, our findings point to a possible niche for ANME to assimilate inorganic sulfur compounds more oxidized than sulfide in anoxic marine environments.