Aerobic methanotrophic bacteria are capable of utilizing methane as their sole energy source. They are commonly found at the oxic/anoxic interfaces of environments such as wetlands, aquatic sediments, and landfills, where they feed on methane produced in anoxic zones of these environments. Until recently, all known species of aerobic methanotrophs belonged to the phylum Proteobacteria, in the classes Gammaproteobacteria and Alphaproteobacteria. However, in 2007-2008 three research groups independently described the isolation of thermoacidophilic methanotrophs that represented a distinct lineage within the bacterial phylum Verrucomicrobia. Isolates were obtained from geothermal areas in Italy, New Zealand and Russia. They are by far the most acidophilic methanotrophs known, with a lower growth limit below pH 1. Here we summarize the properties of these novel methanotrophic Verrucomicrobia, compare them with the proteobacterial methanotrophs, propose a unified taxonomic framework for them and speculate on their potential environmental significance. New genomic and physiological data are combined with existing information to allow detailed comparison of the three strains. We propose the new genus Methylacidiphilum to encompass all three newly discovered bacteria.

Methanotrophic bacteria constitute a ubiquitous group of microorganisms playing an important role in the biogeochemical carbon cycle and in control of global warming through natural reduction of methane emission. These bacteria share the unique ability of using methane as a sole carbon and energy source and have been found in a great variety of habitats. Phylogenetically, known methanotrophs constitute a rather limited group and have so far only been affiliated with the Proteobacteria. Here, we report the isolation and initial characterization of a nonproteobacterial obligately methanotrophic bacterium. The isolate, designated Kam1, was recovered from an acidic hot spring in Kamchatka, Russia, and is more thermoacidophilic than any other known methanotroph, with optimal growth at Ϸ55°C and pH 3.5. Kam1 is only distantly related to all previously known methanotrophs and belongs to the Verrucomicrobia lineage of evolution. Genes for methane monooxygenases, essential for initiation of methane oxidation, could not be detected by using standard primers in PCR amplification and Southern blot analysis, suggesting the presence of a different methane oxidation enzyme. Kam1 also lacks the well developed intracellular membrane systems typical for other methanotrophs. The isolate represents a previously unrecognized biological methane sink, and, due to its unusual phylogenetic affiliation, it will shed important light on the origin, evolution, and diversity of biological methane oxidation and on the adaptation of this process to extreme habitats. Furthermore, Kam1 will add to our knowledge of the metabolic traits and biogeochemical roles of the widespread but poorly understood Verrucomicrobia phylum.extremophile ͉ methanotroph

Molecular and culture-based methods were used to investigate the microbial diversity in produced water obtained from the high-temperature Troll oil formation in the North Sea. 16S rRNA gene libraries were generated from total community DNA, using universal archaeal or bacterial oligonucleotide primer sets. Sequence analysis of 88 clones in the bacterial library indicated that they originated from members of Firmicutes (48 sequences), Bacteroidetes (17 sequences), delta-Proteobacteria (15 sequences), Spirochaetes (5 sequences), Thermotogales (2 sequences) and gamma-Proteobacteria (1 sequence). Twenty-two sequences in the archaeal library were close relatives to members of the genera Methanococcus (18 sequences), Methanolobus (3 sequences) and Thermococcus (1 sequence). Most of the bacterial sequences shared less than 95% identity with their closest match in GenBank, indicating that the produced water harbours a unique community of novel bacterial species or genera. Members of the thermophilic genera Thermosipho, Thermotoga, Anaerophaga and Thermovirga were isolated. The Troll formations are not injected with sea water. Thus, dramatic changes of the in situ conditions have been avoided, and a common source of continuous contamination from injection water can be excluded. However, the majority of the organisms detected in the gene libraries were most closely related to cultivated organisms with optimum temperatures for growth well below the in situ reservoir temperature (70 degrees C), indicating that produced water from the Troll platform harbours a substantial amount of non-indigenous organisms. This was confirmed by the isolation of a number of mesophilic and moderately thermophilic organisms that were unable to grow at reservoir temperature.

With the aim of gaining insight into the molecular and phylogenetic relationships of isocitrate dehydrogenase (IDH) from hyperthermophiles, we carried out a comparative study of putative IDHs identified in the genomes of the eubacterium Thermotoga maritima and the archaea Aeropyrum pernix and Pyrococcus furiosus. An optimum for activity at 90°C or above was found for each IDH. PfIDH and ApIDH were the most thermostable with a melting temperature of 103.7 and 109.9°C, respectively, compared with 98.3 and 98.5°C for TmIDH and AfIDH, respectively. Analytical ultracentrifugation revealed a tetrameric oligomeric state for TmIDH and a homodimeric state for ApIDH and PfIDH. TmIDH and ApIDH were NADP-dependent (K m(NADP) of 55.2 and 44.4 M, respectively) whereas PfIDH was NAD-dependent (K m(NAD) of 68.3 M). These data document that TmIDH represents a novel tetrameric NADP-dependent form of IDH and that PfIDH is a homodimeric NAD-dependent IDH not previously found among the archaea. The homodimeric NADP-IDH present in A. pernix is the most common form of IDH known so far. The evolutionary relationships of ApIDH, PfIDH, and TmIDH with all of the available amino acid sequences of di-and multimeric IDHs are described and discussed.Isocitrate dehydrogenases (IDHs) 1 are a broadly distributed and well characterized group of enzymes that catalyze the oxidative decarboxylation of D-isocitrate to 2-oxoglutarate and CO 2 with NAD ϩ (EC 1.1.1.41) or NADP ϩ (EC 1.1.1.42) as cofactor. IDH from Escherichia coli has been studied extensively with regard to its catalytic mechanism, kinetics, and regulation, and so far the three-dimensional structure is available only for IDH from E. coli and Bacillus subtilis (1-11). Resolution of the structure of NAD-dependent homodimeric isopropylmalate dehydrogenase (IMDH) from Thermus thermophilus revealed that this enzyme, together with EcIDH, represents a unique class of metal-dependent decarboxylating dehydrogenases that lack the ␤␣␤␣␤ motif characteristic of the nucleotide-binding Rossman fold (12, 13). IDH and IMDH are specific for structurally similar substrates of the form HOOC(OH)CHCH(X), in which X represents CH 2 COOH for IDH and CH(CH 3 ) 2 for IMDH (14, 15). However, EcIDH and T. thermophilus IMDH exhibit strong preference for their natural substrate, and the structural determinants for substrate and cofactor specificity have been identified (16 -20) Recently, tartrate dehydrogenase (TDH) and homoisocitrate dehydrogenase (HDH) have been suggested as members of the metal-dependent decarboxylating dehydrogenases (21-23).The IDHs comprise a diverse enzyme family with regard to cofactor specificity, primary structure, and oligomeric state. The archaea Archaeoglobus fulgidus, Caldococcus noboribetus, Haloferax volcanii, Sulfolobus solfataricus, and Sulfolobus acidocaldarius contain a single homodimeric IDH that is NADP-dependent or shows dual coenzyme specificity (24 -27). A homodimeric NAD-IDH is present in Methylophilus methylotrophus (28). However, most bacteria contain a single homodime...

Hypoliths (cryptic microbial assemblages that develop on the undersides of translucent rocks) are significant contributors to regional C and N budgets in both hot and cold deserts. Previous studies in the Dry Valleys of Eastern Antarctica have reported three morphologically distinct hypolithic community types: cyanobacteria dominated (type I), fungus dominated (type II) and moss dominated (type III). Here we present terminal-restriction fragment length polymorphism analyses to elucidate the bacterial community structure in hypolithons and the surrounding soils. We show clear and robust distinction in bacterial composition between bulk surface soils and hypolithons. Moreover, the bacterial assemblages were similar in types II and III hypolithons and clearly distinct from those found in type I. Through 16S rRNA gene 454 pyrosequencing, we show that Proteobacteria dominated all three types of hypolithic communities. As expected, Cyanobacteria were more abundant in type I hypolithons, whereas Actinobacteria were relatively more abundant in types II and III hypolithons, and were the dominant group in soils. Using a probabilistic dissimilarity metric and random sampling, we demonstrate that deterministic processes are more important in shaping the structure of the bacterial community found in types II and III hypolithons. Most notably, the data presented in this study suggest that hypolithic bacterial communities establish via a successional model, with the type I hypolithons acting as the basal development state.