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3Methane oxidation can occur in both aerobic and anaerobic environments; however, these are completely different processes involving different groups of prokaryotes. Aerobic methane oxidation is carried out by aerobic methanotrophs, and anaerobic methane oxidizers, discovered recently, thrive under anaerobic conditions and use sulfate or nitrate as electron donors for methane oxidation (11,104). This review will focus on the aerobic oxidation of methane.Aerobic methanotrophs are a unique group of methylotrophic bacteria that utilize methane as a sole carbon and energy source (52). Based on their cell morphology, ultrastructure, phylogeny, and metabolic pathways, methanotrophs can be divided into two taxonomic groups: type I and type II. Type I methanotrophs include the genera Methylobacter, Methylomicrobium, Methylomonas, Methylocaldum, Methylosphaera, Methylothermus, Methylosarcina, Methylohalobius, Methylosoma, and Methylococcus, which belong to the gamma subdivision of the Proteobacteria (Fig. 1). The type II methanotrophs Methylocystis, Methylosinus, Methylocella, and Methylocapsa are in the alpha subdivision of the Proteobacteria (52) (Fig. 1). Recently, two filamentous methane oxidizers have been described, Crenothrix polyspora (113), which has a novel pmoA, and Clonothrix fusca (125), which has a conventional pmoA. Both are gammaproteobacteria and are closely related to the type I methanotrophs. Most extant methanotrophs are cultured at 20 to 45°C and neutral pH but have also recently been isolated from extreme environments (reviewed in reference 122).The first step in the oxidation of methane to CO 2 is the conversion of methane to methanol by the enzyme methane monooxygenase. There are two forms of this enzyme: a particulate membrane bound form (pMMO) and a soluble cytoplasmic form (sMMO). The pMMO has been reported in all methanotrophs except for the genus Methylocella (121), whereas the sMMO is present only in certain methanotroph strains (94). The pMMO is a membrane bound copper and iron containing enzyme (reviewed in reference 49). The structural genes for this enzyme have been cloned and sequenced from Methylococcus capsulatus Bath (107, 114), Methylocystis sp. strain M, and Methylosinus trichosporium OB3b (45). They lie in a three-gene operon, pmoCAB, which code for three integral membrane polypeptides of approximately 23, 27, and 45 kDa, respectively. These operons are present in duplicate copies in all three organisms. These duplicate copies of pmoCAB are virtually identical and are transcribed from 70 -type promoters found upstream of the pmoC gene (45, 110).The sMMO is a cytoplasmic enzyme containing a unique di-iron site at its catalytic center. It has a broad substrate range, including trichloroethylene, alkanes, alkenes, and aromatic compounds. The biochemistry of the sMMO has been studied in detail (reviewed in reference 75). It consists of three components: a hydroxylase, which is a dimer of three subunits, (␣␤␥) 2 ; a regulatory protein (protein B); and a reductase (protein C). It is e...

Recent applications of molecular genetics to edaphic microbial communities of the McMurdo Dry Valleys and elsewhere have rejected a long-held belief that Antarctic soils contain extremely limited microbial diversity. The Inter-Valley Soil Comparative Survey aims to elucidate the factors shaping these unique microbial communities and their biogeography by integrating molecular genetic approaches with biogeochemical analyses. Although the microbial communities of Dry Valley soils may be complex, there is little doubt that the ecosystem's food web is relatively simple, and evidence suggests that physicochemical conditions may have the dominant role in shaping microbial communities. To examine this hypothesis, bacterial communities from representative soil samples collected in four geographically disparate Dry Valleys were analyzed using molecular genetic tools, including pyrosequencing of 16S rRNA gene PCR amplicons. Results show that the four communities are structurally and phylogenetically distinct, and possess significantly different levels of diversity. Strikingly, only 2 of 214 phylotypes were found in all four valleys, challenging a widespread assumption that the microbiota of the Dry Valleys is composed of a few cosmopolitan species. Analysis of soil geochemical properties indicated that salt content, alongside altitude and Cu2+, was significantly correlated with differences in microbial communities. Our results indicate that the microbial ecology of Dry Valley soils is highly localized and that physicochemical factors potentially have major roles in shaping the microbiology of ice-free areas of Antarctica. These findings hint at links between Dry Valley glacial geomorphology and microbial ecology, and raise previously unrecognized issues related to environmental management of this unique ecosystem.

The metabolism of one-carbon (C 1 ) compounds in the marine environment affects global warming, seawater ecology and atmospheric chemistry. Despite their global significance, marine microorganisms that consume C 1 compounds in situ remain poorly characterized. Stable-isotope probing (SIP) is an ideal tool for linking the function and phylogeny of methylotrophic organisms by the metabolism and incorporation of stable-isotope-labelled substrates into nucleic acids. By combining DNA-SIP and time-series sampling, we characterized the organisms involved in the assimilation of methanol and methylamine in coastal sea water (Plymouth, UK). Labelled nucleic acids were analysed by denaturing gradient gel electrophoresis (DGGE) and clone libraries of 16S rRNA genes. In addition, we characterized the functional gene complement of labelled nucleic acids with an improved primer set targeting methanol dehydrogenase (mxaF) and newly designed primers for methylamine dehydrogenase (mauA). Predominant DGGE phylotypes, 16S rRNA, methanol and methylamine dehydrogenase gene sequences, and cultured isolates all implicated Methylophaga spp, moderately halophilic marine methylotrophs, in the consumption of both methanol and methylamine. Additionally, an mxaF sequence obtained from DNA extracted from sea water clustered with those detected in 13 C-DNA, suggesting a predominance of Methylophaga spp among marine methylotrophs. Unexpectedly, most predominant 16S rRNA and functional gene sequences from 13 C-DNA were clustered in distinct substrate-specific clades, with 16S rRNA genes clustering with sequences from the Gammaproteobacteria. These clades have no cultured representatives and reveal an ecological adaptation of particular uncultured methylotrophs to specific C 1 compounds in the coastal marine environment.

Movile Cave is an unusual groundwater ecosystem that is supported by in situ chemoautotrophic production. The cave atmosphere contains 1-2% methane (CH4), although much higher concentrations are found in gas bubbles that keep microbial mats afloat on the water surface. As previous analyses of stable carbon isotope ratios have suggested that methane oxidation occurs in this environment, we hypothesized that aerobic methane-oxidizing bacteria (methanotrophs) are active in Movile Cave. To identify the active methanotrophs in the water and mat material from Movile Cave, a microcosm was incubated with a 10%13CH4 headspace in a DNA-based stable isotope probing (DNA-SIP) experiment. Using improved centrifugation conditions, a 13C-labelled DNA fraction was collected and used as a template for polymerase chain reaction amplification. Analysis of genes encoding the small-subunit rRNA and key enzymes in the methane oxidation pathway of methanotrophs identified that strains of Methylomonas, Methylococcus and Methylocystis/Methylosinus had assimilated the 13CH4, and that these methanotrophs contain genes encoding both known types of methane monooxygenase (MMO). Sequences of non-methanotrophic bacteria and an alga provided evidence for turnover of CH4 due to possible cross-feeding on 13C-labelled metabolites or biomass. Our results suggest that aerobic methanotrophs actively convert CH4 into complex organic compounds in Movile Cave and thus help to sustain a diverse community of microorganisms in this closed ecosystem.

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Three particulate methane monooxygenase PCR primer sets (A189-A682, A189-A650, and A189-mb661) were investigated for their ability to assess methanotroph diversity in soils from three sites, i.e., heath, oak, and sitka, each of which was capable of oxidizing atmospheric concentrations of methane. Each PCR primer set was used to construct a library containing 50 clones from each soil type. The clones from each library were grouped by restriction fragment length polymorphism, and representatives from each group were sequenced and analyzed. Libraries constructed with the A189-A682 PCR primer set were dominated by amoA-related sequences or nonspecific PCR products with nonsense open reading frames. The primer set could not be used to assess methanotroph diversity in these soils. A new pmoA-specific primer, A650, was designed in this study. The A189-A650 primer set demonstrated distinct biases both in clone library analysis and when incorporated into denaturing gradient gel electrophoresis analysis. The A189-mb661 PCR primer set demonstrated the largest retrieval of methanotroph diversity of all of the primer sets. However, this primer set did not retrieve sequences linked with novel high-affinity methane oxidizers from the soil libraries, which were detected using the A189-A650 primer set. A combination of all three primer sets appears to be required to examine both methanotroph diversity and the presence of novel methane monooxygenase sequences.