Aerobic methanotrophic communities at the Red Sea brine-seawater interface

The central rift of the Red Sea contains 25 brine pools with different physicochemical conditions, dictating the diversity and abundance of the microbial community. Three of these pools, the Atlantis II, Kebrit and Discovery Deeps, are uniquely characterized by a high concentration of hydrocarbons. The brine-seawater interface, described as an anoxic-oxic (brine-seawater) boundary, is characterized by a high methane concentration, thus favoring aerobic methane oxidation. The current study analyzed the aerobic free–living methane-oxidizing bacterial communities that potentially contribute to methane oxidation at the brine-seawater interfaces of the three aforementioned brine pools, using metagenomic pyrosequencing, 16S rRNA pyrotags and pmoA library constructs. The sequencing of 16S rRNA pyrotags revealed that these interfaces are characterized by high microbial community diversity. Signatures of aerobic methane-oxidizing bacteria were detected in the Atlantis II Interface (ATII-I) and the Kebrit Deep Upper (KB-U) and Lower (KB-L) brine-seawater interfaces. Through phylogenetic analysis of pmoA, we further demonstrated that the ATII-I aerobic methanotroph community is highly diverse. We propose four ATII-I pmoA clusters. Most importantly, cluster 2 groups with marine methane seep methanotrophs, and cluster 4 represent a unique lineage of an uncultured bacterium with divergent alkane monooxygenases. Moreover, non-metric multidimensional scaling (NMDS) based on the ordination of putative enzymes involved in methane metabolism showed that the Kebrit interface layers were distinct from the ATII-I and DD-I brine-seawater interfaces.

Related papers:

title: Genome Sequence of Haloplasma contractile, an Unusual Contractile Bacterium from a Deep-Sea Anoxic Brine Lake
abstract: We present the draft genome of Haloplasma contractile, isolated from a deep-sea brine and representing a new order between Firmicutesand Mollicutes. Its complex morphology with contractile protrusions might be strongly influenced by the presence of seven MreB/Mbl homologs, which appears to be the highest copy number ever reported.

title: Microbiology of the Red Sea (and other) deep-sea anoxic brine lakes.
abstract: The Red Sea harbours approximately 25 deep-sea anoxic brine pools. They constitute extremely unique and complex habitats with the conjugation of several extreme physicochemical parameters rendering them some of the most inhospitable environments on Earth. After 50 years of research mostly driven by chemists, geophysicists and geologists, the microbiology of the brines has been receiving increased interest in the last decade. Recent molecular and cultivation-based studies have provided us with a first glimpse on the enormous biodiversity of the local microbial communities, the identification of several new taxonomic groups, and the isolation of novel extremophiles that thrive in these environments. This review presents a general overview of these unusual biotopes and compares them with other similar environments in the Mediterranean Sea and the Gulf of Mexico, with a focus on their microbial ecology.

title: Vertical, horizontal, and temporal changes in temperature in the Atlantis II and Discovery hot brine pools, Red Sea
abstract: In October 2008, we measured temperature and salinity in hot, hypersaline brine filling the Atlantis II and Discovery Deeps on the Red Sea spreading center west of Jeddah, Saudi Arabia. In agreement with previous observations in the Atlantis II Deep, we found a stack of four convective layers with vertically uniform temperature profiles separated by thin interfaces with high vertical temperature gradients. Temperature in the thick lower convective layer in the Atlantis II Deep continued to slowly increase at 0.1 °C/year since the last observations in 1997. Previously published data show that the temperature of all four convective layers increased since the 1960s at the same rate, from which we infer that diffusive vertical heat flux between convective layers is rapid on time scales of 3–5 years and, thus, heat is lost from the brine pools to overlying Red Sea Deep Water. Heat budgets suggest that the heat flux from hydrothermal venting has decreased from 0.54 GW to 0.18 GW since 1966. A tow-yo survey found that temperature in the upper convective layers changes about 0.2 °C over 5–6 km but the temperature in the lower brine layer remains constant. Temperature in the lower convective layer in the Discovery Deep remains unchanged at 48 °C. To explain these results, we hypothesize that heat flux from a hydrothermal vent in the floor of the Discovery Deep has been stable for 40 years, whereas temperature of the brine in the Atlantis II Deep is adjusting to the change in hydrothermal heat flux from the vent in the Southwest Basin. We found no changes in the upper transition layer at 1900–1990 m depth that appeared between 1976 and 1992 and suggest that this layer originated from the seafloor elsewhere in the rift.

title: Methane in Red Sea brines
abstract: Concentrations of hydrocarbon gases and stable carbon isotope ratios of methane from the water column of Shaban, Kebrit, Atlantis II and Discovery deeps, Red Sea, have been determined. Methane concentrations (yield C1) range from < 50 nL/L (Red Sea deep water) to ca. 22 × 10−3 L/L (Kebrit brine). Stable carbon isotopes of methane are between −30 and 43‰. Hydrocarbon gases in the brines are originally of thermogenetic origin (Kebrit: C1/C2∼ 57; δ13C1 ∼ −30‰). Methane concentrations in the transition zones between brines and Red Sea deep water decreased, especially in the Atlantis II/Discovery deeps, associated with a strong shift of δ13C1 to positive values. This shift is related to bacterial oxidation of methane in the transition zone between brine layers and overlying Red Sea deep water. Oxidized methane mixes with Red Sea deep water methane. A connection between the Atlantis II and the Discovery brine is postulated on the basis of the geochemical data.

title: Hydrographic structure of brine-filled deeps in the Red Sea—new results from the Shaban, Kebrit, Atlantis II, and Discovery Deep
abstract: Hydrographic profiles with CTD and water samples from four brine-filled deeps in the Red Sea (Shaban, Kebrit, Atlantis II and Discovery Deep) were taken during the Meteor 31/2 expedition in February/March 1995. In the Shaban Deep area two smaller basins containing high saline brine were found in addition to the already known southern basins. Corresponding salinities (150.7%. Cl in the upper brine section) and near equal brine levels (around 1325 m water depth) suggest subbottom connections below the sills separating the northern and southern basins. An insignificant decrease in temperature (to about 24.1 †C) has occurred in the upper brine since detection of the Shaban Deep in 1981 and no changes were found for the brine-seawater level. No significant changes have been observed in the Kebrit Deep over the last 23 years in temperature (24.3 †C), chlorinity (154.0 ± 0.3%. Cl), and brine level (1466 ± 1 m). The transition from high saline brine to normal seawater salinity probably extends over a depth of only a few decimetres or less. Strong changes, however, were registered for the Atlantis II Deep. The temperature increase, known from numerous earlier investigations, continued. The gradient of increase was the steepest during the last 2.5 years and a temperature of 71.7 °C was reached in the lower brine of the SW basin. The structure of the lower transition zone, between about 1990 m water depth and high saline brine, has also changed significantly, now containing two convective layers with nearly constant temperatures (61 and 55 °C, respectively). The neighbouring Discovery Deep also shows evidence of increased hydrothermal activities. While the temperature was nearly constant (near 44.7 °C) until 1977, it has since increased to almost 50 °C and the brine level has risen by about 17 m.

title: High-resolution methane profiles across anoxic brine-seawater boundaries in the Atlantis-II, Discovery, and Kebrit Deeps (Red Sea)
abstract: A decrease in temperature (ΔT up to 45.5 °C) and chloride concentration (ΔCl up to 4.65 mol/l) characterises the brine–seawater boundary in the Atlantis-II, Discovery, and Kebrit Deeps of the Red Sea, where redox conditions change from anoxic to oxic over a boundary layer several meters thick. High-resolution (100 cm) profiles of the methane concentration, stable carbon isotope ratio of methane, and redox-sensitive tracers (O2, Mn4+/Mn2+, Fe3+/Fe2+, and SO42−) were measured across the brine–seawater boundary layer to investigate methane fluxes and secondary methane oxidation processes. 
 
Substantial amounts of thermogenic hydrocarbons are found in the deep brines (mostly methane, with a maximum concentration up to 4.8×105 nmol/l), and steep methane concentration gradients mainly controlled by diffusive flow characterize the brine–seawater boundary (maximum of 2×105 nmol/l/m in Kebrit Deep). However, locally the actual methane concentration profiles deviate from theoretical diffusion-controlled concentration profiles and extremely positive δ13C–CH4 values can be found (up to +49‰ PDB in the Discovery Deep). Both, the actual CH4 concentration profiles and the carbon-13 enrichment in the residual CH4 of the Atlantis-II and Discovery Deeps indicate consumption (oxidation) of 12C-rich CH4 under suboxic conditions (probably utilizing readily available—up to 2000 μmol/l—Mn(IV)-oxihydroxides as electron acceptor). Thus, a combined diffusion–oxidation model was used to calculate methane fluxes of 0.3–393 kg/year across the brine–seawater boundary layer. Assuming steady-state conditions, this slow loss of methane from the brines into the Red Sea bottom water reflects a low thermogenic hydrocarbon input into the deep brines.

title: Methanotrophic bacteria.
abstract: Methane-utilizing bacteria (methanotrophs) are a diverse group of gram-negative bacteria that are related to other members of the Proteobacteria. These bacteria are classified into three groups based on the pathways used for assimilation of formaldehyde, the major source of cell carbon, and other physiological and morphological features. The type I and type X methanotrophs are found within the gamma subdivision of the Proteobacteria and employ the ribulose monophosphate pathway for formaldehyde assimilation, whereas type II methanotrophs, which employ the serine pathway for formaldehyde assimilation, form a coherent cluster within the beta subdivision of the Proteobacteria. Methanotrophic bacteria are ubiquitous. The growth of type II bacteria appears to be favored in environments that contain relatively high levels of methane, low levels of dissolved oxygen, and limiting concentrations of combined nitrogen and/or copper. Type I methanotrophs appear to be dominant in environments in which methane is limiting and combined nitrogen and copper levels are relatively high. These bacteria serve as biofilters for the oxidation of methane produced in anaerobic environments, and when oxygen is present in soils, atmospheric methane is oxidized. Their activities in nature are greatly influenced by agricultural practices and other human activities. Recent evidence indicates that naturally occurring, uncultured methanotrophs represent new genera. Methanotrophs that are capable of oxidizing methane at atmospheric levels exhibit methane oxidation kinetics different from those of methanotrophs available in pure cultures. A limited number of methanotrophs have the genetic capacity to synthesize a soluble methane monooxygenase which catalyzes the rapid oxidation of environmental pollutants including trichloroethylene.

title: Molecular Ecology Techniques for the Study of Aerobic Methanotrophs
abstract: Methane 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

title: Methylocella Species Are Facultatively Methanotrophic
abstract: All aerobic methanotrophic bacteria described to date are unable to grow on substrates containing carbon-carbon bonds. Here we demonstrate that members of the recently discovered genus Methylocella are an exception to this. These bacteria are able to use as their sole energy source the one-carbon compounds methane and methanol, as well as the multicarbon compounds acetate, pyruvate, succinate, malate, and ethanol. To conclusively verify facultative growth, acetate and methane were used as model substrates in growth experiments with the type strain Methylocella silvestris BL2. Quantitative real-time PCR targeting the mmoX gene, which encodes a subunit of soluble methane monooxygenase, showed that copies of this gene increased in parallel with cell counts during growth on either acetate or methane as the sole substrate. This verified that cells possessing the genetic basis of methane oxidation grew on acetate as well as methane. Cloning of 16S rRNA genes and fluorescence in situ hybridization with strain-specific and genus-specific oligonucleotide probes detected no contaminants in cultures. The growth rate and carbon conversion efficiency were higher on acetate than on methane, and when both substrates were provided in excess, acetate was preferably used and methane oxidation was shut down. Our data demonstrate that not all methanotrophic bacteria are limited to growing on one-carbon compounds. This could have major implications for understanding the factors controlling methane fluxes in the environment.

title: Methyloferula stellata gen. nov., sp. nov., an acidophilic, obligately methanotrophic bacterium that possesses only a soluble methane monooxygenase.
abstract: Two strains of aerobic methanotrophic bacteria, AR4(T) and SOP9, were isolated from acidic (pH 3.8-4.0) Sphagnum peat bogs in Russia. Another phenotypically similar isolate, strain LAY, was obtained from an acidic (pH 4.0) forest soil in Germany. Cells of these strains were Gram-negative, non-pigmented, non-motile, thin rods that multiplied by irregular cell division and formed rosettes or amorphous cell conglomerates. Similar to Methylocella species, strains AR4(T), SOP9 and LAY possessed only a soluble form of methane monooxygenase (sMMO) and lacked intracytoplasmic membranes. Growth occurred only on methane and methanol; the latter was the preferred growth substrate. mRNA transcripts of sMMO were detectable in cells when either methane or both methane and methanol were available. Carbon was assimilated via the serine and ribulose-bisphosphate (RuBP) pathways; nitrogen was fixed via an oxygen-sensitive nitrogenase. Strains AR4(T), SOP9 and LAY were moderately acidophilic, mesophilic organisms capable of growth between pH 3.5 and 7.2 (optimum pH 4.8-5.2) and at 4-33 °C (optimum 20-23 °C). The major cellular fatty acid was 18 : 1ω7c and the quinone was Q-10. The DNA G+C content was 55.6-57.5 mol%. The isolates belonged to the family Beijerinckiaceae of the class Alphaproteobacteria and were most closely related to the sMMO-possessing methanotrophs of the genus Methylocella (96.4-97.0 % 16S rRNA gene sequence similarity), particulate MMO (pMMO)-possessing methanotrophs of the genus Methylocapsa (96.1-97.0 %), facultative methylotrophs of the genus Methylovirgula (96.1-96.3 %) and non-methanotrophic organotrophs of the genus Beijerinckia (96.5-97.0 %). Phenotypically, strains AR4(T), SOP9 and LAY were most similar to Methylocella species, but differed from members of this genus by cell morphology, greater tolerance of low pH, detectable activities of RuBP pathway enzymes and inability to grow on multicarbon compounds. Therefore, we propose a novel genus and species, Methyloferula stellata gen. nov., sp. nov., to accommodate strains AR4(T), SOP9 and LAY. Strain AR4(T) ( = DSM 22108(T)  = LMG 25277(T)  = VKM B-2543(T)) is the type strain of Methyloferula stellata.

title: Methylocapsa aurea sp. nov., a facultative methanotroph possessing a particulate methane monooxygenase, and emended description of the genus Methylocapsa.
abstract: An aerobic, methanotrophic bacterium, designated KYG(T), was isolated from a forest soil in Germany. Cells of strain KYG(T) were Gram-negative, non-motile, slightly curved rods that multiplied by binary fission and produced yellow colonies. The cells contained intracellular granules of poly-β-hydroxybutyrate at each cell pole, a particulate methane monooxygenase (pMMO) and stacks of intracytoplasmic membranes (ICMs) packed in parallel along one side of the cell envelope. Strain KYG(T) grew at pH 5.2-7.2 and 2-33 °C and could fix atmospheric nitrogen under reduced oxygen tension. The major cellular fatty acid was C(18 : 1)ω7c (81.5 %) and the DNA G+C content was 61.4 mol%. Strain KYG(T) belonged to the family Beijerinckiaceae of the class Alphaproteobacteria and was most closely related to the obligate methanotroph Methylocapsa acidiphila B2(T) (98.1 % 16S rRNA gene sequence similarity and 84.7 % pmoA sequence similarity). Unlike Methylocapsa acidiphila B2(T), which grows only on methane and methanol, strain KYG(T) was able to grow facultatively on acetate. Facultative acetate utilization is a characteristic of the methanotrophs of the genus Methylocella, but the genus Methylocella does not produce pMMO or ICMs. Strain KYG(T) differed from Methylocapsa acidiphila B2(T) on the basis of substrate utilization pattern, pigmentation, pH range, cell ultrastructure and efficiency of dinitrogen fixation. Therefore, we propose a novel species, Methylocapsa aurea sp. nov., to accommodate this bacterium. The type strain is KYG(T) (=DSM 22158(T) =VKM B-2544(T)).

title: Acetate utilization as a survival strategy of peat-inhabiting Methylocystis spp.
abstract: Representatives of the genus Methylocystis are traditionally considered to be obligately methanotrophic bacteria, which are incapable of growth on multicarbon substrates. Here, we describe a novel member of this genus, strain H2s, which represents a numerically abundant and ecologically important methanotroph population in northern Sphagnum-dominated wetlands. This isolate demonstrates a clear preference for growth on methane but is able to grow slowly on acetate in the absence of methane. Strain H2s possesses both forms of methane monooxygenase (particulate and soluble MMO) and a well-developed system of intracytoplasmic membranes (ICM). In cells grown for several transfers on acetate, these ICM are maintained, although in a reduced form, and mRNA transcripts of particulate MMO are detectable. These cells resume their growth on methane faster than those kept for the same period of time without any substrate. Growth on acetate leads to a major shift in the phospholipid fatty acid composition. The re-examination of all type strains of the validly described Methylocystis species showed that Methylocystis heyeri H2(T) and Methylocystis echinoides IMET10491(T) are also capable of slow growth on acetate. This capability might represent an important part of the survival strategy of Methylocystis spp. in environments where methane availability is variable or limited.

title: Characterization of a novel facultative Methylocystis species capable of growth on methane, acetate and ethanol.
abstract: A non-motile strain of Methylocystis, strain SB2, isolated from a spring bog in southeast Michigan, had a curved rod morphology with a typical type II intracytoplasmic membrane system. This organism expressed the membrane-bound or particulate methane monooxygenase (pMMO) as well as a chalkophore with high affinity for copper and did not express the cytoplasmic or soluble methane monooxygenase (sMMO). Strain SB2 was found to grow within the pH range of 6-9, with optimal growth at 6.8. Growth was observed at temperatures ranging between 10°C and 30°C, with no growth at 37°C. The DNA G+C content was 62.9 mol%. Predominant fatty acids were 18:1ω7c (72.7%) and 18:1ω9c (24%) when grown on methane. Phylogenetic comparisons based on both pmoA and 16S rRNA sequences indicated that this organism belonged to the Methylocystis genus, and was closely related to Methylocystis rosea SV97(T) and Methylocystis echinoides IMET10491(T) (98% 16S rRNA gene sequence similarity to both strains). DNA : DNA hybridizations indicated that strain SB2 had 70% similarity with M. rosea SV97(T) . Unlike M. rosea SV97(T) , strain SB2 was able to utilize not only methane for growth, but also ethanol and acetate. Furthermore, the predominant fatty acids in strain SB2 were different from those found in M. rosea SV97(T) , i.e. 54.2% and 39.7% of fatty acids are 18:1ω8 and 18:1ω7 in M. rosea SV97(T) , while 18:1ω8 is completely absent in strain SB2.

title: Cohn’s Crenothrix is a filamentous methane oxidizer with an unusual methane monooxygenase
abstract: 135 years ago Ferdinand Cohn, the founder of bacteriology, microscopically observed a conspicuous filamentous bacterium with a complex life cycle and described it as Crenothrix polyspora. This uncultured bacterium is infamous for mass developments in drinking water systems, but its phylogeny and physiology remained unknown. We show that C. polyspora is a gammaproteobacterium closely related to methanotrophs and capable of oxidizing methane. We discovered that C. polyspora encodes a phylogenetically very unusual particulate methane monooxygenase whose expression is strongly increased in the presence of methane. Our findings demonstrate a previously unrecognized complexity of the evolutionary history and cell biology of methane-oxidizing bacteria.

title: Clonothrix fusca Roze 1896, a filamentous, sheathed, methanotrophic gamma-proteobacterium.
abstract: Crenothrix polyspora Cohn 1870 and Clonothrix fusca Roze 1896 are two filamentous, sheathed microorganisms exhibiting complex morphological differentiation, whose phylogeny and physiology have been obscure for a long time due to the inability to cultivate them. Very recently, DNA sequencing data from uncultured C. polyspora-enriched material have suggested that Crenothrix is a methane-oxidizing gamma-proteobacterium (39). In contrast, the possible ecological function of C. fusca, originally considered a developmental stage of C. polyspora, is unknown. In this study, temporal succession of two filamentous, sheathed microorganisms resembling Cohn's Crenothrix and Roze's Clonothrix was observed by analyzing the microbial community of an artesian well by optical microscopy. Combined culture-based and culture-independent approaches enabled us to assign C. fusca to a novel subgroup of methane-oxidizing gamma-proteobacteria distinct from that of C. polyspora. This assignment was supported by (i) methane uptake and assimilation experiments, (ii) ultrastructural data showing the presence in C. fusca cytoplasm of an elaborate membrane system resembling that of methanotrophic gamma-proteobacteria, and (iii) sequencing data demonstrating the presence in its genome of a methanol dehydrogenase alpha subunit-encoding gene (mxaF) and a conventional particulate methane mono-oxygenase alpha subunit-encoding gene (pmoA) that is different from the unusual pmoA (u-pmoA) of C. polyspora.

title: Methane oxidation by an extremely acidophilic bacterium of the phylum Verrucomicrobia
abstract: Aerobic methanotrophic bacteria consume methane as it diffuses away from methanogenic zones of soil and sediment. They act as a biofilter to reduce methane emissions to the atmosphere, and they are therefore targets in strategies to combat global climate change. No cultured methanotroph grows optimally below pH 5, but some environments with active methane cycles are very acidic. Here we describe an extremely acidophilic methanotroph that grows optimally at pH 2.0–2.5. Unlike the known methanotrophs, it does not belong to the phylum Proteobacteria but rather to the Verrucomicrobia, a widespread and diverse bacterial phylum that primarily comprises uncultivated species with unknown genotypes. Analysis of its draft genome detected genes encoding particulate methane monooxygenase that were homologous to genes found in methanotrophic proteobacteria. However, known genetic modules for methanol and formaldehyde oxidation were incomplete or missing, suggesting that the bacterium uses some novel methylotrophic pathways. Phylogenetic analysis of its three pmoA genes (encoding a subunit of particulate methane monooxygenase) placed them into a distinct cluster from proteobacterial homologues. This indicates an ancient divergence of Verrucomicrobia and Proteobacteria methanotrophs rather than a recent horizontal gene transfer of methanotrophic ability. The findings show that methanotrophy in the Bacteria is more taxonomically, ecologically and genetically diverse than previously thought, and that previous studies have failed to assess the full diversity of methanotrophs in acidic environments.

title: Methanotrophy below pH 1 by a new Verrucomicrobia species
abstract: Mud volcanoes, mudpots and fumaroles are remarkable geological features characterized by the emission of gas, water and/or semi-liquid mud matrices with significant methane fluxes to the atmosphere (10-1 to 103 t y-1). Environmental conditions in these areas vary from ambient temperature and neutral pH to high temperatures and low pH. Although there are strong indications for biological methane consumption in mud volcanoes, no methanotrophic bacteria are known that would thrive in the hostile conditions of fumaroles (temperatures up to 70 °C and pH down to 1.8). The first step in aerobic methane oxidation is performed by a soluble or membrane-bound methane mono-oxygenase. Here we report that pmoA (encoding the β-subunit of membrane-bound methane mono-oxygenase) clone libraries, made by using DNA extracted from the Solfatara volcano mudpot and surrounding bare soil near the fumaroles, showed clusters of novel and distant pmoA genes. After methanotrophic enrichment at 50 °C and pH 2.0 the most distant cluster, sharing less than 50% identity with any other described pmoA gene, was represented in the culture. Finally we isolated an acidiphilic methanotrophic bacterium Acidimethylosilex fumarolicum SolV belonging to the Planctomycetes/Verrucomicrobia/Chlamydiae superphylum, ‘outside’ the subphyla of the Alpha- and Gammaproteobacteria containing the established methanotrophs. This bacterium grows under oxygen limitation on methane as the sole source of energy, down to pH 0.8—far below the pH optimum of any previously described methanotroph. A. fumarolicum SolV has three different pmoA genes, with two that are very similar to sequences retrieved from the mudpot. Highly homologous environmental 16S rRNA gene sequences from Yellowstone Park show that this new type of methanotrophic bacteria may be a common inhabitant of extreme environments. This is the first time that a representative of the widely distributed Verrucomicrobia phylum, of which most members remain uncultivated, is coupled to a geochemically relevant reaction.

title: Methane oxidation at 55°C and pH 2 by a thermoacidophilic bacterium belonging to the Verrucomicrobia phylum
abstract: 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 ≈55°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.

title: Molecular phylogeny of methylotrophs in a deep-sea sediment from a tropical west Pacific Warm Pool.
abstract: The presence and phylogeny of methylotrophs, including methanotrophs, in a deep-sea sediment of a tropical west Pacific Warm Pool site WP was investigated by molecular marker-based analysis of mxaF, pmoA and archaeal 16S rRNA genes. MxaF amino acid sequence analysis revealed that microbes belonging to the alpha-Proteobacteria and most related to Hyphomicrobium and Methylobacterium were the dominant aerobic methylotrophs in this deep-sea sediment; also, a small percentage of type II methanotrophs, closely related to Methylocystis and Methylosinus, were detected in this environment. On the other hand, the use of a pmoA gene marker could not demonstrate the presence of any methanotrophs in this environment, suggesting that the mxaF gene probe is a more suitable marker in this deep-sea sediment for the detection of methylotrophs (including methanotrophs). mxaF quantitative polymerase chain reaction results showed that the west Pacific WP sediment contained approximately 3x10(4-5) methylotrophs per gram sediment, 10-100 times more than the samples collected from several other deep-sea Pacific sediments, but, on the other hand, about 10 times less than the amounts present in samples collected from rice and flower garden soil. Archaeal diversity as analyzed by 16S rRNA gene sequences indicated that a non-thermophilic marine group I crenarchaeote was the major archaeal group present in the west Pacific WP.

title: Diversity of functional genes of methanogens, methanotrophs and sulfate reducers in deep-sea hydrothermal environments.
abstract: To contribute to the identification of methanogens, methanotrophs and sulfate-reducing bacteria (SRB) in microbial communities from the 13 degrees N (East Pacific Rise) and Rainbow (Mid-Atlantic Ridge) hydrothermal vent fields, we investigated the diversity of mcrA, pmoA and dsrAB genes sequences. Clone libraries were obtained using DNA isolated from fragments of diffuse vents, sediment and in situ samplers. The clones were categorized by restriction fragment length polymorphism, and representatives of each group were sequenced. Sequences were related to that of hyperthermophilic (order Methanopyrales and family Methanocaldococcaceae), thermophilic and mesophilic (family Methanococcaceae) methanogens, thermophilic (proposed genus 'Methylothermus') and mesophilic type I methanotrophs, and hyperthermophilic (order Archaeoglobales), thermophilic (order Thermodesulfobacteriales) and mesophilic (family Desulfobulbaceae) SRB. Several of the obtained sequences were distantly related to the genes of cultivated organisms, providing evidence of the existence of novel lineages in the three functional groups. This study provides for the first time an insight into the diversity of several functional genes of deep-sea hydrothermal system microorganisms.

title: Diversity of functional genes for methanotrophs in sediments associated with gas hydrates and hydrocarbon seeps in the Gulf of Mexico.
abstract: Methanotrophs are ubiquitous in soil, fresh water and the open ocean, but have not been well characterized in deep-sea hydrocarbon seeps and gas hydrates, where methane is unusually abundant. Here we report the presence of new functional genes for the aerobic oxidation of methane by methanotrophs in marine sediments associated with gas hydrates and hydrocarbon seeps in the Gulf of Mexico. Samples were collected from two hydrate locations (GC185 and GC234): one hydrocarbon-seep location at a brine pool (GC233) and one background-marine location about 1.2 miles north of the brine pool (NBP). Community DNA was extracted from each location to establish clone libraries for the pmoA functional gene using a PCR-based cloning approach. Three hundred and ninety clones were screened by sequencing and 46 operational taxonomic units were obtained. Eight operational taxonomic units were present in every sample; one of them was predominant and accounted for 22.8-25.3% of each clone library. Principal-component analysis indicated that samples GC185 and GC234 were closely related and, along with GC233, were significantly different from NBP. These results indicate that methanotrophic communities may be similarly impacted by hydrocarbons at the gas-hydrate and seep sites, and can be distinguished from methanotrophic communities in the normal marine sediment. Furthermore, cluster analysis showed that 84.8% of operational taxonomic units from all samples formed distinct clusters, which could not be grouped with any published pmoA sequences, indicating that a considerable number of novel methanotrophic species may exist in the Gulf of Mexico.

title: Planktonic and Sediment-Associated Aerobic Methanotrophs in Two Seep Systems along the North American Margin
abstract: Methane vents are of significant geochemical and ecological importance. Notable progress has been made toward understanding anaerobic methane oxidation in marine sediments; however, the diversity and distribution of aerobic methanotrophs in the water column are poorly characterized. Both environments play an essential role in regulating methane release from the oceans to the atmosphere. In this study, the diversity of particulate methane monooxygenase (pmoA) and 16S rRNA genes from two methane vent environments along the California continental margin was characterized. The pmoA phylotypes recovered from methane-rich sediments and the overlying water column differed. Sediments harbored the greatest number of unique pmoA phylotypes broadly affiliated with the Methylococcaceae family, whereas planktonic pmoA phylotypes formed three clades that were distinct from the sediment-hosted methanotrophs and distantly related to established methanotrophic clades. Water column-associated phylotypes were highly similar between field sites, suggesting that planktonic methanotroph diversity is controlled primarily by environmental factors rather than geographical proximity. Analysis of 16S rRNA genes from methane-rich waters did not readily recover known methanotrophic lineages, with only a few phylotypes demonstrating distant relatedness to Methylococcus. The development of new pmo primers increased the recovery of monooxygenase genes from the water column and led to the discovery of a highly diverged monooxygenase sequence which is phylogenetically intermediate to Amo and pMMO. This sequence potentiates insight into the amo/pmo superfamily. Together, these findings lend perspective into the diversity and segregation of aerobic methanotrophs within different methane-rich habitats in the marine environment.

title: Microbial communities in iron-silica-rich microbial mats at deep-sea hydrothermal fields of the Southern Mariana Trough.
abstract: The abundance, diversity and composition of bacterial and archaeal communities in the microbial mats at deep-sea hydrothermal fields were investigated, using culture-independent 16S rRNA and functional gene analyses combined with mineralogical analysis. Microbial mats were collected at two hydrothermal areas on the ridge of the back-arc spreading centre in the Southern Mariana Trough. Scanning electron microscope and energy dispersive X-ray spectroscopic (SEM-EDS) analyses revealed that the mats were mainly composed of amorphous silica and contained numerous filamentous structures of iron hydroxides. Direct cell counting with SYBR Green I staining showed that the prokaryotic cell densities were more than 10(8) cells g(-1). Quantitative polymerase chain reaction (Q-PCR) analysis revealed that Bacteria are more abundant than Archaea in the microbial communities. Furthermore, zetaproteobacterial cells accounted for 6% and 22% of the prokaryotic cells in each mat estimated by Q-PCR with newly designed primers and TaqMan probe. Phylotypes related to iron-oxidizers, methanotrophs/methylotrophs, ammonia-oxidizers and sulfate-reducers were found in the 16S rRNA gene clone libraries constructed from each mat sample. A variety of unique archaeal 16S rRNA gene phylotypes, several pmoA, dsrAB and archaeal amoA gene phylotypes were also recovered from the microbial mats. Our results provide insights into the diversity and abundance of microbial communities within microbial mats in deep-sea hydrothermal fields.

title: Identification of active methylotrophic bacteria inhabiting surface sediment of a marine estuary.
abstract: Methylotrophs play an essential role in the global carbon cycle due to their participation in methane oxidation and C1 metabolism. Despite this important biogeochemical role, marine and estuarine microorganisms that consume C1 compounds are poorly characterized. In this study, we investigated the diversity of active methylotrophs and methanotrophs in sediment from the Colne Estuary (Brightlingsea, UK). Aerobic surface sediment samples were examined for the presence of C1 -utilizing communities using DNA stable-isotope probing (DNA-SIP) with (13) C-labelled methane, methanol and monomethylamine. Active methylotrophic bacteria were confirmed after DNA-SIP and denaturing gradient gel electrophoresis analyses. Clone libraries of 16S rRNA gene amplicons revealed the presence of methylotrophic bacteria affiliated with Methylophaga spp. in methanol and monomethylamine incubations. The addition of marine ammonium mineral salts medium to the microcosms increased the rate of substrate metabolism in DNA-SIP incubations, although nutrient addition did not affect the active populations contributing (13) C-labelled DNA. The (13) CH4 SIP incubations indicated the predominant activity of type I methanotrophs and microarray hybridization of amplified particulate methane monooxygenase (pmoA) genes confirmed the role of type Ia methanotrophs in SIP incubations. Type II methanotrophs (i.e. Methylocystis and Methylosinus) were only detected in the original sediment and in the unlabelled DNA fractions, which indicated that type II methanotrophs were not actively involved in C1 compound assimilation in DNA-SIP incubations with estuarine surface sediment samples.

title: Phylogenetic diversity of methanogenic, sulfate-reducing and methanotrophic prokaryotes from deep-sea hydrothermal vents and cold seeps
abstract: Microbial communities of methanogenic, sulfate-reducing and methanotrophic prokaryotes from deep-sea environments were investigated by molecular phylogenetic analysis of the deduced amino acid sequences of the genes encoding for the methyl coenzyme M reductase (mcrA), dissimilatory sulfite reductase (dsrAB) and particulate methane monoxygenase (pmoA), respectively. Clone libraries of PCR amplified genes were constructed using DNA extracted from deep-sea vent chimneys (Rainbow and Logatchev hydrothermal vent fields, Mid-Atlantic Ridge, Atlantic Ocean; 9°N East Pacific Rise, Pacific Ocean) and from vertically subsampled sediment cores from cold-seep areas (Blake Ridge, western Atlantic Ocean; Florida Escarpment, Gulf of Mexico). Recombinant clones were screened by RFLP and representative dsrAB, mcrA and pmoA genes were sequenced. The dsrAB sequences grouped primarily within the orders Desulfobacterales, Syntrophobacterales and the Gram-positive order Clostridales. Cold-seep mcrA sequences were distributed among the ANME-2c, -2d and -2e groups, which were previously shown to be associated with the anaerobic oxidation of methane. This study also reports the first mcrA sequences from a high-temperature, black smoker chimney (Logatchev) to group within the ANME-2e subgroup. The majority of the remaining hydrothermal vent mcrA sequences were primarily related to thermophilic members of the anaerobic, methanogenic order Methanococcales. A shift in the dominant ANME-2 group with depth in the sediment for both Florida Escarpment and Blake Ridge mcrA libraries was detected. ANME-2d related clones were detected in the top zones of both cores, with the frequency of ANME-2e related clones increasing with depth. All pmoA sequences retrieved from the cold-seep sites were found to be related to Type I methanotrophic members of the γ-proteobacteria, and were primarily distributed among three major clusters of sequences. No Type II pmoA sequences related to methanotrophic members of the α-proteobacteria were detected, suggesting that the methanotrophic communities in these cold-seep areas are dominated by Type I γ-proteobacteria.

title: Microbial diversity in sediments associated with a shallow methane seep in the tropical Timor Sea of Australia reveals a novel aerobic methanotroph diversity.
abstract: This study examined the diversity of Bacteria, Archaea and in particular aerobic methanotrophs associated with a shallow (84 m) methane seep in the tropical Timor Sea, Australia. Seepage of thermogenic methane was associated with a large carbonate hardground covered in coarse carbonate-rich sediments and various benthic organisms such as solitary corals. The diversity of Bacteria and Archaea was studied by analysis of cloned 16S rRNA genes, while aerobic methanotrophic bacteria were quantified using real-time PCR targeting the alpha-subunit of particulate methane monooxygenase (pmoA) genes and diversity was studied by analysis of cloned pmoA genes. Phylogenetic analysis of bacterial and archaeal 16S rRNA genes revealed diverse and mostly novel phylotypes related to sequences previously recovered from marine sediments. A small number of bacterial 16S rRNA gene sequences were related to aerobic methanotrophs distantly related to the genera Methylococcus and Methylocaldum. Real-time PCR targeting pmoA genes showed that the highest numbers of methanotrophs were present in surface sediments associated with the seep area. Phylogenetic analysis of pmoA sequences revealed that all phylotypes were novel and fell into two large clusters comprised of only marine sequences distantly related to the genera Methylococcus and Methylocaldum that were clearly divergent from terrestrial phylotypes. This study provides evidence for the existence of a novel microbial diversity and diverse aerobic methanotrophs that appear to constitute marine specialized lineages.

title: Identification of Novel Methane-, Ethane-, and Propane-Oxidizing Bacteria at Marine Hydrocarbon Seeps by Stable Isotope Probing
abstract: Marine hydrocarbon seeps supply oil and gas to microorganisms in sediments and overlying water. We used stable isotope probing (SIP) to identify aerobic bacteria oxidizing gaseous hydrocarbons in surface sediment from the Coal Oil Point seep field located offshore of Santa Barbara, California. After incubating sediment with 13C-labeled methane, ethane, or propane, we confirmed the incorporation of 13C into fatty acids and DNA. Terminal restriction fragment length polymorphism (T-RFLP) analysis and sequencing of the 16S rRNA and particulate methane monooxygenase (pmoA) genes in 13C-DNA revealed groups of microbes not previously thought to contribute to methane, ethane, or propane oxidation. First, 13C methane was primarily assimilated by Gammaproteobacteria species from the family Methylococcaceae, Gammaproteobacteria related to Methylophaga, and Betaproteobacteria from the family Methylophilaceae. Species of the latter two genera have not been previously shown to oxidize methane and may have been cross-feeding on methanol, but species of both genera were heavily labeled after just 3 days. pmoA sequences were affiliated with species of Methylococcaceae, but most were not closely related to cultured methanotrophs. Second, 13C ethane was consumed by members of a novel group of Methylococcaceae. Growth with ethane as the major carbon source has not previously been observed in members of the Methylococcaceae; a highly divergent pmoA-like gene detected in the 13C-labeled DNA may encode an ethane monooxygenase. Third, 13C propane was consumed by members of a group of unclassified Gammaproteobacteria species not previously linked to propane oxidation. This study identifies several bacterial lineages as participants in the oxidation of gaseous hydrocarbons in marine seeps and supports the idea of an alternate function for some pmoA-like genes.

title: Molecular Analysis of Deep-Sea Hydrothermal Vent Aerobic Methanotrophs by Targeting Genes of 16S rRNA and Particulate Methane Monooxygenase
abstract: Molecular diversity of deep-sea hydrothermal vent aerobic methanotrophs was studied using both 16S ribosomalDNA and pmoA encoding the subunit A of particulate methane monooxygenase (pMOA). Hydrothermal vent plume and chimney samples were collected from back-arc vent at Mid-Okinawa Trough (MOT), Japan, and the Trans-Atlantic Geotraverse (TAG) site along Mid-Atlantic Ridge, respectively. The target genes were amplified by polymerase chain reaction from the bulk DNA using specific primers and cloned. Fifty clones from each clone library were directly sequenced. The 16S rDNA sequences were grouped into 3 operational taxonomic units (OTUs), 2 from MOT and 1 from TAG. Two OTUs (1 MOT and 1 TAG) were located within the branch of type I methanotrophic ?-Proteobacteria. Another MOT OTU formed a unique phylogenetic lineage related to type I methanotrophs. Direct sequencing of 50 clones each from the MOT and TAG samples yielded 17 and 4 operational pmoA units (OPUs), respectively. The phylogenetic tree based on the pMOA amino acid sequences deduced from OPUs formed diverse phylogenetic lineages within the branch of type I methanotrophs, except for the OPU MOT-pmoA-8 related to type X methanotrophs. The deduced pMOA topologies were similar to those of all known pMOA, which may suggest that the pmoA gene is conserved through evolution. Neither the 16S rDNA nor pmoA molecular analysis could detect type II methanotrophs, which suggests the absence of type II methanotrophs in the collected vent samples.

title: Abundance and distribution of diverse membrane-bound monooxygenase (Cu-MMO) genes within the Costa Rica oxygen minimum zone.
abstract: Diverse copper-containing membrane-bound monooxygenase-encoding sequences (Cu-MMOs) have recently been described from the marine environment, suggesting widespread potential for oxidation of reduced substrates. Here, we used the well-defined oxygen and methane gradients associated with the Costa Rican oxygen minimum zone (OMZ) to gain insight into the physico-chemical parameters influencing the distribution and abundance of Cu-MMO-encoding marine microorganisms. Two Methylococcales-related Cu-MMO-encoding lineages, termed groups OPU1 and OPU3, demonstrated differences in their relative abundance, with both pmoA and candidate 16S rRNA genes correlating significantly with reduced environmental oxygen concentrations and depth. In contrast, a newly identified Cu-MMO-encoding lineage, Group C, was primarily associated with the oxygenated euphotic zone. An updated phylogenetic analysis including these sequences, a marine pxmABC gene cluster, ethylene-utilizing Cu-MMO-encoding lineages and previously reported planktonic Cu-MMOs (Groups W, X, Z and O) demonstrates the breadth of diversity of Cu-MMO-encoding marine microorganisms. Groups C and X affiliated phylogenetically with ethane- and ethylene-oxidizing Cu-MMOs, Groups W and O affiliated phylogenetically with the recently described Cu-MMO 'pXMO', and Group Z clustered with Cu-MMOs recovered from soils. Collectively, these data demonstrate widespread genetic potential in ocean waters for the oxidation of small, reduced molecules and advance our understanding of the microorganisms involved in methane cycling in the OMZ environment.

title: Unique Prokaryotic Consortia in Geochemically Distinct Sediments from Red Sea Atlantis II and Discovery Deep Brine Pools
abstract: The seafloor is a unique environment, which allows insights into how geochemical processes affect the diversity of biological life. Among its diverse ecosystems are deep-sea brine pools - water bodies characterized by a unique combination of extreme conditions. The ‘polyextremophiles’ that constitute the microbial assemblage of these deep hot brines have not been comprehensively studied. We report a comparative taxonomic analysis of the prokaryotic communities of the sediments directly below the Red Sea brine pools, namely, Atlantis II, Discovery, Chain Deep, and an adjacent brine-influenced site. Analyses of sediment samples and high-throughput pyrosequencing of PCR-amplified environmental 16S ribosomal RNA genes (16S rDNA) revealed that one sulfur (S)-rich Atlantis II and one nitrogen (N)-rich Discovery Deep section contained distinct microbial populations that differed from those found in the other sediment samples examined. Proteobacteria, Actinobacteria, Cyanobacteria, Deferribacteres, and Euryarchaeota were the most abundant bacterial and archaeal phyla in both the S- and N-rich sections. Relative abundance-based hierarchical clustering of the 16S rDNA pyrotags assigned to major taxonomic groups allowed us to categorize the archaeal and bacterial communities into three major and distinct groups; group I was unique to the S-rich Atlantis II section (ATII-1), group II was characteristic for the N-rich Discovery sample (DD-1), and group III reflected the composition of the remaining sediments. Many of the groups detected in the S-rich Atlantis II section are likely to play a dominant role in the cycling of methane and sulfur due to their phylogenetic affiliations with bacteria and archaea involved in anaerobic methane oxidation and sulfate reduction.

title: Core Microbial Functional Activities in Ocean Environments Revealed by Global Metagenomic Profiling Analyses
abstract: Metagenomics-based functional profiling analysis is an effective means of gaining deeper insight into the composition of marine microbial populations and developing a better understanding of the interplay between the functional genome content of microbial communities and abiotic factors. Here we present a comprehensive analysis of 24 datasets covering surface and depth-related environments at 11 sites around the world's oceans. The complete datasets comprises approximately 12 million sequences, totaling 5,358 Mb. Based on profiling patterns of Clusters of Orthologous Groups (COGs) of proteins, a core set of reference photic and aphotic depth-related COGs, and a collection of COGs that are associated with extreme oxygen limitation were defined. Their inferred functions were utilized as indicators to characterize the distribution of light- and oxygen-related biological activities in marine environments. The results reveal that, while light level in the water column is a major determinant of phenotypic adaptation in marine microorganisms, oxygen concentration in the aphotic zone has a significant impact only in extremely hypoxic waters. Phylogenetic profiling of the reference photic/aphotic gene sets revealed a greater variety of source organisms in the aphotic zone, although the majority of individual photic and aphotic depth-related COGs are assigned to the same taxa across the different sites. This increase in phylogenetic and functional diversity of the core aphotic related COGs most probably reflects selection for the utilization of a broad range of alternate energy sources in the absence of light.

title: The Sorcerer II Global Ocean Sampling Expedition: Northwest Atlantic through Eastern Tropical Pacific
abstract: The world's oceans contain a complex mixture of micro-organisms that are for the most part, uncharacterized both genetically and biochemically. We report here a metagenomic study of the marine planktonic microbiota in which surface (mostly marine) water samples were analyzed as part of the Sorcerer II Global Ocean Sampling expedition. These samples, collected across a several-thousand km transect from the North Atlantic through the Panama Canal and ending in the South Pacific yielded an extensive dataset consisting of 7.7 million sequencing reads (6.3 billion bp). Though a few major microbial clades dominate the planktonic marine niche, the dataset contains great diversity with 85% of the assembled sequence and 57% of the unassembled data being unique at a 98% sequence identity cutoff. Using the metadata associated with each sample and sequencing library, we developed new comparative genomic and assembly methods. One comparative genomic method, termed “fragment recruitment,” addressed questions of genome structure, evolution, and taxonomic or phylogenetic diversity, as well as the biochemical diversity of genes and gene families. A second method, termed “extreme assembly,” made possible the assembly and reconstruction of large segments of abundant but clearly nonclonal organisms. Within all abundant populations analyzed, we found extensive intra-ribotype diversity in several forms: (1) extensive sequence variation within orthologous regions throughout a given genome; despite coverage of individual ribotypes approaching 500-fold, most individual sequencing reads are unique; (2) numerous changes in gene content some with direct adaptive implications; and (3) hypervariable genomic islands that are too variable to assemble. The intra-ribotype diversity is organized into genetically isolated populations that have overlapping but independent distributions, implying distinct environmental preference. We present novel methods for measuring the genomic similarity between metagenomic samples and show how they may be grouped into several community types. Specific functional adaptations can be identified both within individual ribotypes and across the entire community, including proteorhodopsin spectral tuning and the presence or absence of the phosphate-binding gene PstS.

title: DNA purification and isolation using a solid-phase.
abstract: A method of separating polynucleotides, such as DNA, RNA and PNA, from a solution containing polynucleotides by reversibly and non-specifically binding the polynucleotides to a solid surface, such as a magnetic microparticle, having a functional group-coated surface is disclosed. The salt and polyalkylene glycol concentration of the solution is adjusted to levels which result in polynucleotide binding to the magnetic microparticles. The magnetic microparticles with bound polynucleotides are separated from the solution and the polynucleotides are eluted from the magnetic microparticles.

title: Methane-Oxidizing Bacteria in a California Upland Grassland Soil: Diversity and Response to Simulated Global Change
abstract: We investigated the diversity of methane-oxidizing bacteria (i.e., methanotrophs) in an annual upland grassland in northern California, using comparative sequence analysis of the pmoA gene. In addition to identifying type II methanotrophs commonly found in soils, we discovered three novel pmoA lineages for which no cultivated members have been previously reported. These novel pmoA clades clustered together either with clone sequences related to “RA 14” or “WB5FH-A,” which both represent clusters of environmentally retrieved sequences of putative atmospheric methane oxidizers. Conservation of amino acid residues and rates of nonsynonymous versus synonymous nucleotide substitution in these novel lineages suggests that the pmoA genes in these clades code for functionally active methane monooxygenases. The novel clades responded to simulated global changes differently than the type II methanotrophs. We observed that the relative abundance of type II methanotrophs declined in response to increased precipitation and increased atmospheric temperature, with a significant antagonistic interaction between these factors such that the effect of both together was less than that expected from their individual effects. Two of the novel clades were not observed to respond significantly to these environmental changes, while one of the novel clades had an opposite response, increasing in relative abundance in response to increased precipitation and atmospheric temperature, with a significant antagonistic interaction between these factors.

title: Microbial diversity in the deep sea and the underexplored “rare biosphere”
abstract: The evolution of marine microbes over billions of years predicts that the composition of microbial communities should be much greater than the published estimates of a few thousand distinct kinds of microbes per liter of seawater. By adopting a massively parallel tag sequencing strategy, we show that bacterial communities of deep water masses of the North Atlantic and diffuse flow hydrothermal vents are one to two orders of magnitude more complex than previously reported for any microbial environment. A relatively small number of different populations dominate all samples, but thousands of low-abundance populations account for most of the observed phylogenetic diversity. This “rare biosphere” is very ancient and may represent a nearly inexhaustible source of genomic innovation. Members of the rare biosphere are highly divergent from each other and, at different times in earth's history, may have had a profound impact on shaping planetary processes.

title: VAMPS: a website for visualization and analysis of microbial population structures
abstract: BackgroundThe advent of next-generation DNA sequencing platforms has revolutionized molecular microbial ecology by making the detailed analysis of complex communities over time and space a tractable research pursuit for small research groups. However, the ability to generate 105–108 reads with relative ease brings with it many downstream complications. Beyond the computational resources and skills needed to process and analyze data, it is difficult to compare datasets in an intuitive and interactive manner that leads to hypothesis generation and testing.ResultsWe developed the free web service VAMPS (Visualization and Analysis of Microbial Population Structures, http://vamps.mbl.edu) to address these challenges and to facilitate research by individuals or collaborating groups working on projects with large-scale sequencing data. Users can upload marker gene sequences and associated metadata; reads are quality filtered and assigned to both taxonomic structures and to taxonomy-independent clusters. A simple point-and-click interface allows users to select for analysis any combination of their own or their collaborators’ private data and data from public projects, filter these by their choice of taxonomic and/or abundance criteria, and then explore these data using a wide range of analytic methods and visualizations. Each result is extensively hyperlinked to other analysis and visualization options, promoting data exploration and leading to a greater understanding of data relationships.ConclusionsVAMPS allows researchers using marker gene sequence data to analyze the diversity of microbial communities and the relationships between communities, to explore these analyses in an intuitive visual context, and to download data, results, and images for publication. VAMPS obviates the need for individual research groups to make the considerable investment in computational infrastructure and bioinformatic support otherwise necessary to process, analyze, and interpret massive amounts of next-generation sequence data. Any web-capable device can be used to upload, process, explore, and extract data and results from VAMPS. VAMPS encourages researchers to share sequence and metadata, and fosters collaboration between researchers of disparate biomes who recognize common patterns in shared data.

title: UCHIME improves sensitivity and speed of chimera detection
abstract: Chimeric DNA sequences often form during polymerase chain reaction amplification, especially when sequencing single regions (e.g. 16S rRNA or fungal Internal Transcribed Spacer) to assess diversity or compare populations. Undetected chimeras may be misinterpreted as novel species, causing inflated estimates of diversity and spurious inferences of differences between populations. Detection and removal of chimeras is therefore of critical importance in such experiments.  We describe UCHIME, a new program that detects chimeric sequences with two or more segments. UCHIME either uses a database of chimera-free sequences or detects chimeras de novo by exploiting abundance data. UCHIME has better sensitivity than ChimeraSlayer (previously the most sensitive database method), especially with short, noisy sequences. In testing on artificial bacterial communities with known composition, UCHIME de novo sensitivity is shown to be comparable to Perseus. UCHIME is >100× faster than Perseus and >1000× faster than ChimeraSlayer.  Source, binaries and data: http://drive5.com/uchime.

title: QIIME allows analysis of high-throughput community sequencing data

title: Exploring Microbial Diversity and Taxonomy Using SSU rRNA Hypervariable Tag Sequencing
abstract: Massively parallel pyrosequencing of hypervariable regions from small subunit ribosomal RNA (SSU rRNA) genes can sample a microbial community two or three orders of magnitude more deeply per dollar and per hour than capillary sequencing of full-length SSU rRNA. As with full-length rRNA surveys, each sequence read is a tag surrogate for a single microbe. However, rather than assigning taxonomy by creating gene trees de novo that include all experimental sequences and certain reference taxa, we compare the hypervariable region tags to an extensive database of rRNA sequences and assign taxonomy based on the best match in a Global Alignment for Sequence Taxonomy (GAST) process. The resulting taxonomic census provides information on both composition and diversity of the microbial community. To determine the effectiveness of using only hypervariable region tags for assessing microbial community membership, we compared the taxonomy assigned to the V3 and V6 hypervariable regions with the taxonomy assigned to full-length SSU rRNA sequences isolated from both the human gut and a deep-sea hydrothermal vent. The hypervariable region tags and full-length rRNA sequences provided equivalent taxonomy and measures of relative abundance of microbial communities, even for tags up to 15% divergent from their nearest reference match. The greater sampling depth per dollar afforded by massively parallel pyrosequencing reveals many more members of the “rare biosphere” than does capillary sequencing of the full-length gene. In addition, tag sequencing eliminates cloning bias and the sequences are short enough to be completely sequenced in a single read, maximizing the number of organisms sampled in a run while minimizing chimera formation. This technique allows the cost-effective exploration of changes in microbial community structure, including the rare biosphere, over space and time and can be applied immediately to initiatives, such as the Human Microbiome Project.

title: The SILVA ribosomal RNA gene database project: improved data processing and web-based tools
abstract: SILVA (from Latin silva, forest, http://www.arb-silva.de) is a comprehensive web resource for up to date, quality-controlled databases of aligned ribosomal RNA (rRNA) gene sequences from the Bacteria, Archaea and Eukaryota domains and supplementary online services. The referred database release 111 (July 2012) contains 3 194 778 small subunit and 288 717 large subunit rRNA gene sequences. Since the initial description of the project, substantial new features have been introduced, including advanced quality control procedures, an improved rRNA gene aligner, online tools for probe and primer evaluation and optimized browsing, searching and downloading on the website. Furthermore, the extensively curated SILVA taxonomy and the new non-redundant SILVA datasets provide an ideal reference for high-throughput classification of data from next-generation sequencing approaches.

title: Search and clustering orders of magnitude faster than BLAST
abstract: Biological sequence data is accumulating rapidly, motivating the development of improved high-throughput methods for sequence classification.



UBLAST and USEARCH are new algorithms enabling sensitive local and global search of large sequence databases at exceptionally high speeds. They are often orders of magnitude faster than BLAST in practical applications, though sensitivity to distant protein relationships is lower. UCLUST is a new clustering method that exploits USEARCH to assign sequences to clusters. UCLUST offers several advantages over the widely used program CD-HIT, including higher speed, lower memory use, improved sensitivity, clustering at lower identities and classification of much larger datasets.


AVAILABILITY
Binaries are available at no charge for non-commercial use at http://www.drive5.com/usearch.

title: MUSCLE: multiple sequence alignment with high accuracy and high throughput.
abstract: We describe MUSCLE, a new computer program for creating multiple alignments of protein sequences. Elements of the algorithm include fast distance estimation using kmer counting, progressive alignment using a new profile function we call the log-expectation score, and refinement using tree-dependent restricted partitioning. The speed and accuracy of MUSCLE are compared with T-Coffee, MAFFT and CLUSTALW on four test sets of reference alignments: BAliBASE, SABmark, SMART and a new benchmark, PREFAB. MUSCLE achieves the highest, or joint highest, rank in accuracy on each of these sets. Without refinement, MUSCLE achieves average accuracy statistically indistinguishable from T-Coffee and MAFFT, and is the fastest of the tested methods for large numbers of sequences, aligning 5000 sequences of average length 350 in 7 min on a current desktop computer. The MUSCLE program, source code and PREFAB test data are freely available at http://www.drive5. com/muscle.

title: FastTree: Computing Large Minimum Evolution Trees with Profiles instead of a Distance Matrix
abstract: Gene families are growing rapidly, but standard methods for inferring phylogenies do not scale to alignments with over 10,000 sequences. We present FastTree, a method for constructing large phylogenies and for estimating their reliability. Instead of storing a distance matrix, FastTree stores sequence profiles of internal nodes in the tree. FastTree uses these profiles to implement Neighbor-Joining and uses heuristics to quickly identify candidate joins. FastTree then uses nearest neighbor interchanges to reduce the length of the tree. For an alignment with N sequences, L sites, and a different characters, a distance matrix requires O(N2) space and O(N2L) time, but FastTree requires just O(NLa + N) memory and O(Nlog (N)La) time. To estimate the tree's reliability, FastTree uses local bootstrapping, which gives another 100-fold speedup over a distance matrix. For example, FastTree computed a tree and support values for 158,022 distinct 16S ribosomal RNAs in 17 h and 2.4 GB of memory. Just computing pairwise Jukes–Cantor distances and storing them, without inferring a tree or bootstrapping, would require 17 h and 50 GB of memory. In simulations, FastTree was slightly more accurate than Neighbor-Joining, BIONJ, or FastME; on genuine alignments, FastTree's topologies had higher likelihoods.

title: FragGeneScan: predicting genes in short and error-prone reads
abstract: The advances of next-generation sequencing technology have facilitated metagenomics research that attempts to determine directly the whole collection of genetic material within an environmental sample (i.e. the metagenome). Identification of genes directly from short reads has become an important yet challenging problem in annotating metagenomes, since the assembly of metagenomes is often not available. Gene predictors developed for whole genomes (e.g. Glimmer) and recently developed for metagenomic sequences (e.g. MetaGene) show a significant decrease in performance as the sequencing error rates increase, or as reads get shorter. We have developed a novel gene prediction method FragGeneScan, which combines sequencing error models and codon usages in a hidden Markov model to improve the prediction of protein-coding region in short reads. The performance of FragGeneScan was comparable to Glimmer and MetaGene for complete genomes. But for short reads, FragGeneScan consistently outperformed MetaGene (accuracy improved ∼62% for reads of 400 bases with 1% sequencing errors, and ∼18% for short reads of 100 bases that are error free). When applied to metagenomes, FragGeneScan recovered substantially more genes than MetaGene predicted (>90% of the genes identified by homology search), and many novel genes with no homologs in current protein sequence database.

title: Introducing mothur: Open-Source, Platform-Independent, Community-Supported Software for Describing and Comparing Microbial Communities
abstract: mothur aims to be a comprehensive software package that allows users to use a single piece of software to analyze community sequence data. It builds upon previous tools to provide a flexible and powerful software package for analyzing sequencing data. As a case study, we used mothur to trim, screen, and align sequences; calculate distances; assign sequences to operational taxonomic units; and describe the α and β diversity of eight marine samples previously characterized by pyrosequencing of 16S rRNA gene fragments. This analysis of more than 222,000 sequences was completed in less than 2 h with a laptop computer.

title: Molecular phylogeny of type II methane-oxidizing bacteria isolated from various environments.
abstract: Type II methane-oxidizing bacteria (MOB) were isolated from diverse environments, including rice paddies, pristine and polluted freshwaters and sediments, mangrove roots, upland soils, brackish water ecosystems, moors, oil wells, water purification systems and livestock manure. Isolates were identified based on morphological traits as either Methylocystis spp., Methylosinus sporium or Methylosinus trichosporium. Molecular phylogenies were constructed based on nearly complete 16S rRNA gene sequences, and on partial sequences of genes encoding PmoA (a subunit of particulate methane monooxygenase), MxaF (a subunit of methanol dehydrogenase) and MmoX (a subunit of soluble methane monooxygenase). The maximum pairwise 16S rDNA difference between isolates was 4.2%, and considerable variability was evident within the Methylocystis (maximum difference 3.6%). Due to this variability, some of the published 'specific' oligonucleotide primers for type II MOB exhibit multiple mismatches with gene sequences from some isolates. The phylogenetic tree constructed from pmoA gene sequences closely mirrored that constructed from 16S rDNA sequences, and both supported the presently accepted taxonomy of type II MOB. Contrary to previously published phylogenetic trees, morphologically distinguishable species were generally monophyletic based on pmoA or 16S rRNA gene sequences. This was not true for phylogenies constructed from mmoX and mxaF gene sequences. The phylogeny of mxaF gene sequences suggested that horizontal transfer of this gene may have occurred across type II MOB species. Soluble methane monooxygenase could not be detected in many Methylocystis strains either by an enzyme activity test (oxidation of naphthalene) or by PCR-based amplification of an mmoX gene.

title: A novel family of functional operons encoding methane/ammonia monooxygenase-related proteins in gammaproteobacterial methanotrophs.
abstract: Genomes of alphaproteobacterial and verrucomicrobial methane-oxidizing bacteria (MOB) encode sequence-divergent copies of particulate methane monooxygenase [pMMO = (PmoABC); pmoCAB]. In contrast, sequenced gammaproteobacterial MOB (Gamma-MOB) genomes contain single or multiple near-identical copies of pmoCAB operons. In betaproteobacterial ammonia-oxidizing bacteria (Beta-AOB), near-identical amoCAB operons encode ammonia monooxygenase (AMO), a homologue of pMMO. Here, we report that Gamma-MOB in the genera Methylomonas, Methylobacter and Methylomicrobium also encode a sequence-divergent particulate monooxygenase (pXMO). Whereas all known genes encoding pMMO or AMO cluster in the order 'CAB', the genes encoding pXMO are uniquely organized in the non-canonical form 'pxmABC.' Steady state pxm mRNA was detected in cultures of Methylomonas sp. as well as in freshwater creek sediment samples, demonstrating that pxm genes are expressed in culture and in situ. Inclusion of PxmA and PxmB proteins in phylogenetic analyses of the Pmo/Amo protein superfamilies created trifurcated trees with three major clades: (i) Pmo of Alpha- and Gamma-MOB and Amo of Gamma-AOB; (ii) Amo of Beta-AOB, Pmo of putative ethane-oxidizing Gamma-MOB and Pxm of Gamma-MOB; and (iii) verrucomicrobial Pmo and Amo of ammonia-oxidizing Archaea. These data support but do not prove the hypothesis that oxygen-dependent methane and ammonia monooxygenases evolved from a substrate-promiscuous ancestor after horizontal transfer while being integrated into the catabolic contexts of their extant hosts.

title: RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models
abstract: RAxML-VI-HPC (randomized axelerated maximum likelihood for high performance computing) is a sequential and parallel program for inference of large phylogenies with maximum likelihood (ML). Low-level technical optimizations, a modification of the search algorithm, and the use of the GTR+CAT approximation as replacement for GTR+Gamma yield a program that is between 2.7 and 52 times faster than the previous version of RAxML. A large-scale performance comparison with GARLI, PHYML, IQPNNI and MrBayes on real data containing 1000 up to 6722 taxa shows that RAxML requires at least 5.6 times less main memory and yields better trees in similar times than the best competing program (GARLI) on datasets up to 2500 taxa. On datasets > or =4000 taxa it also runs 2-3 times faster than GARLI. RAxML has been parallelized with MPI to conduct parallel multiple bootstraps and inferences on distinct starting trees. The program has been used to compute ML trees on two of the largest alignments to date containing 25,057 (1463 bp) and 2182 (51,089 bp) taxa, respectively.


AVAILABILITY
icwww.epfl.ch/~stamatak

title: New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0.
abstract: PhyML is a phylogeny software based on the maximum-likelihood principle. Early PhyML versions used a fast algorithm performing nearest neighbor interchanges to improve a reasonable starting tree topology. Since the original publication (Guindon S., Gascuel O. 2003. A simple, fast and accurate algorithm to estimate large phylogenies by maximum likelihood. ), PhyML has been widely used (>2500 citations in ISI Web of Science) because of its simplicity and a fair compromise between accuracy and speed. In the meantime, research around PhyML has continued, and this article describes the new algorithms and methods implemented in the program. First, we introduce a new algorithm to search the tree space with user-defined intensity using subtree pruning and regrafting topological moves. The parsimony criterion is used here to filter out the least promising topology modifications with respect to the likelihood function. The analysis of a large collection of real nucleotide and amino acid data sets of various sizes demonstrates the good performance of this method. Second, we describe a new test to assess the support of the data for internal branches of a phylogeny. This approach extends the recently proposed approximate likelihood-ratio test and relies on a nonparametric, Shimodaira-Hasegawa-like procedure. A detailed analysis of real alignments sheds light on the links between this new approach and the more classical nonparametric bootstrap method. Overall, our tests show that the last version (3.0) of PhyML is fast, accurate, stable, and ready to use. A Web server and binary files are available from http://www.atgc-montpellier.fr/phyml/.

title: phyloXML: XML for evolutionary biology and comparative genomics
abstract: BackgroundEvolutionary trees are central to a wide range of biological studies. In many of these studies, tree nodes and branches need to be associated (or annotated) with various attributes. For example, in studies concerned with organismal relationships, tree nodes are associated with taxonomic names, whereas tree branches have lengths and oftentimes support values. Gene trees used in comparative genomics or phylogenomics are usually annotated with taxonomic information, genome-related data, such as gene names and functional annotations, as well as events such as gene duplications, speciations, or exon shufflings, combined with information related to the evolutionary tree itself. The data standards currently used for evolutionary trees have limited capacities to incorporate such annotations of different data types.ResultsWe developed a XML language, named phyloXML, for describing evolutionary trees, as well as various associated data items. PhyloXML provides elements for commonly used items, such as branch lengths, support values, taxonomic names, and gene names and identifiers. By using "property" elements, phyloXML can be adapted to novel and unforeseen use cases. We also developed various software tools for reading, writing, conversion, and visualization of phyloXML formatted data.ConclusionPhyloXML is an XML language defined by a complete schema in XSD that allows storing and exchanging the structures of evolutionary trees as well as associated data. More information about phyloXML itself, the XSD schema, as well as tools implementing and supporting phyloXML, is available at http://www.phyloxml.org.

title: The metagenomics RAST server – a public resource for the automatic phylogenetic and functional analysis of metagenomes
abstract: AbstractBackgroundRandom community genomes (metagenomes) are now commonly used to study microbes in different environments. Over the past few years, the major challenge associated with metagenomics shifted from generating to analyzing sequences. High-throughput, low-cost next-generation sequencing has provided access to metagenomics to a wide range of researchers. high-throughput pipeline has been constructed to provide high-performance computing to all researchers interested in using metagenomics. The pipeline produces automated functional assignments of sequences in the metagenome by comparing both protein and nucleotide databases. Phylogenetic and functional summaries of the metagenomes are generated, and tools for comparative metagenomics are incorporated into the standard views. User access is controlled to ensure data privacy, but the collaborative environment underpinning the service provides a framework for sharing datasets between multiple users. In the metagenomics RAST, all users retain full control of their data, and everything is available for download in a variety of formats.ConclusionThe open-source metagenomics RAST service provides a new paradigm for the annotation and analysis of metagenomes. With built-in support for multiple data sources and a back end that houses abstract data types, the metagenomics RAST is stable, extensible, and freely available to all researchers. This service has removed one of the primary bottlenecks in metagenome sequence analysis – the availability of high-performance computing for annotating the data.
 http://metagenomics.nmpdr.org

title: Multivariate dispersion as a measure of beta diversity.
abstract: Beta diversity can be defined as the variability in species composition among sampling units for a given area. We propose that it can be measured as the average dissimilarity from individual observation units to their group centroid in multivariate space, using an appropriate dissimilarity measure. Differences in beta diversity among different areas or groups of samples can be tested using this approach. The choice of transformation and dissimilarity measure has important consequences for interpreting results. For kelp holdfast assemblages from New Zealand, variation in species composition was greater in smaller holdfasts, while variation in relative abundances was greater in larger holdasts. Variation in community structure of Norwegian continental shelf macrobenthic fauna increased with increases in environmental heterogeneity, regardless of the measure used. We propose a new dissimilarity measure which allows the relative weight placed on changes in composition vs. abundance to be specified explicitly.

title: Pvclust: an R package for assessing the uncertainty in hierarchical clustering
abstract: Pvclust is an add-on package for a statistical software R to assess the uncertainty in hierarchical cluster analysis. Pvclust can be used easily for general statistical problems, such as DNA microarray analysis, to perform the bootstrap analysis of clustering, which has been popular in phylogenetic analysis. Pvclust calculates probability values (p-values) for each cluster using bootstrap resampling techniques. Two types of p-values are available: approximately unbiased (AU) p-value and bootstrap probability (BP) value. Multiscale bootstrap resampling is used for the calculation of AU p-value, which has superiority in bias over BP value calculated by the ordinary bootstrap resampling. In addition the computation time can be enormously decreased with parallel computing option.

title: Finding Groups in Data: An Introduction to Chster Analysis
abstract: The Wiley-Interscience Paperback Series consists of selected books that have been made more accessible to consumers in an effort to increase In both the increasingly important and distribution we show how these methods. Our experiments demonstrate that together can deal with most applications technometrics. In an appropriate visualization technique is to these new. The well written and efficiently finds accurate clusters in data including. Of applied value for several preprocessing tasks discontinuity preserving smoothing feature clusters! However the model based notion of domain knowledge from real data repositories in data. Discusses various types of study the user set explicitly but also propose another. This book make understandable the cluster analysis is based notion of starsmodern treatment.

title: Methylomarinum vadi gen. nov., sp. nov., a methanotroph isolated from two distinct marine environments.
abstract: Two aerobic methane-oxidizing bacterial strains were isolated from distinct marine environments in Japan. Strains IT-4(T) and T2-1 were Gram-stain-negative, aerobic, motile, plump short rods or oval-shaped bacteria with a single polar flagellum and type I intracytoplasmic membranes. They were obligate methanotrophs that grew only on methane or methanol. Each strain possessed the particulate methane monooxygenase (pMMO). The ribulose monophosphate pathway was operative for carbon assimilation. The strains grew best at 37 °C, and did not grow at 45 °C. NaCl was required for growth within a concentration range of 1-8 % (w/v). The major phospholipid fatty acids were C16 : 0, C16 : 1ω7c, and C16 : 1ω5t. The major isoprenoid quinone was MQ-8. The DNA G+C content was 50.9-51.7 mol%. The 16S rRNA gene sequences of the strains showed 99.4 % similarity to each other, and DNA-DNA hybridization analysis indicated that the strains were representatives of the same species. The 16S rRNA gene sequences were highly similar to some marine environmental sequences (94.0-97.7 % similarity), but did not show similarities more than 94 % with sequences of members of other related genera, such as Methylomicrobium, Methylobacter, Methylomonas and Methylosarcina. Phylogenies based on 16S rRNA gene sequences and deduced partial PmoA sequences, and the physiological and chemotaxonomic characteristics revealed that strains IT-4(T) and T2-1 represent a novel species of a new genus in the family Methylococcaceae, for which the name Methylomarinum vadi gen. nov., sp. nov. is proposed. The type strain is IT-4(T) ( = JCM 13665(T) = DSM 18976(T)).

title: Analysis of a PAH-degrading bacterial population in subsurface sediments on the Mid-Atlantic Ridge
abstract: Little is known about the types and concentrations of polycyclic aromatic hydrocarbons (PAHs) existing in the deep-sea subsurface environment, which is believed to be cold, oligothrophic and of high static pressure. PAHs in the upper layers of the water column are unavoidably subjected to degradation while they are deposited to the sea floor and become embedded in the deep-sea sediment. In this report, a high concentration of PAHs was discovered in the sediment 2.7 m beneath the bottom surface at a water depth of 3962 m on the Mid-Atlantic Ridge (MAR). The total concentration of PAHs was 445 ng (g dry wt sediment) -1 . Among the seven detected PAHs, the concentrations of phenanthrene (222 ng g -1 ) and fluorene (79 ng g -1 ) were relatively high. In addition, PAH-degrading bacteria were found within the sediments. As in a previously detected site on the MAR, in the PAH-enriched region of this site, a bacterium of the genus Cycloclasticus was found to be the predominant isolate detected by PCR-DGGE analysis. In addition, bacteria of the Halomonas , Marinobacter , Alcanivorax , Thalassospira and Maricaulis genera, were also included in the PAH-degrading community. In summary, a high concentration of PAHs was detected in the subsurface of the deep-sea sediment, and once again, the Cycloclasticus bacterium was confirmed to be a ubiquitous marine PAH degrader even in the subsurface marine environment. Considering the abundance of PAHs therein, biodegradation is thus thought to be inactive, probably because of the low temperature, limited oxygen and/or limited nutrients.

title: Light hydrocarbons in Red Sea brines and sediments
abstract: Light hydrocarbon (C1-C3) concentrations in the water from four Red Sea brine basins (Atlantis II, Suakin, Nereus and Valdivia Deeps) and in sediment pore waters from two of these areas (Atlantis II and Suakin Deeps) are reported. The hydrocarbon gases in the Suakin Deep brine (T = ~ 25°C, Cl − = ~ 85‰ , CH 4 = ~ 71 1 ) are apparently of biogenic origin as evidenced by C 1 (C 2 + C 3 ) ratios of ~ 1000. Methane concentrations (6–8 μl/l) in Suakin Deep sediments are nearly equal to those in the brine, suggesting sedimentary interstitial waters may be the source of the brine and associated methane. The Atlantis II Deep has two brine layers with significantly different light hydrocarbon concentrations indicating separate sources. The upper brine (T = ~ 50°C, Cl − = ~ 73‰ , CH4 = ~ 155 μl/l) gas seems to be of biogenic origin [ C 1 (C 2 + C 3 ) = ~1100 ], whereas the lower brine (T = ~ 61°C, Cl − = ~ 155‰ , CH4 = ~ 120μl/l) gas is apparently of thermogenic origin [ C 1 (C 2 + C 3 ) = ~ 50 ]. The thermogenic gas resulting from thermal cracking of organic matter in the sedimentary column apparently migrates into the basin with the brine, whereas the biogenic gas is produced in situ or at the seawater-brine interface. Methane concentrations in Atlantis II interstitial waters underlying the lower brine are about one half brine concentrations; this difference possibly reflects the known temporal variations of hydrothermal activity in the basin.

title: Candidate OP Phyla: Importance, Ecology and Cultivation Prospects
abstract: AbsractOP phyla were created in the domain bacteria, based on the group of 16S rRNA gene sequences recovered from the Obsidian Pool. However, due to the lack of cultured representative it is referred to as candidate phyla. Wider ecological occurrence was predicted for the OP phyla, especially OP3, OP10 and OP11. Recently, members of phylum OP5 and OP10 were cultured, providing clues to their cultivation prospects. At last the bioprospecting potentials of the OP members are discussed herein.

title: Phylogenetic and Metabolic Diversity of Planctomycetes from Anaerobic, Sulfide- and Sulfur-Rich Zodletone Spring, Oklahoma
abstract: We investigated the phylogenetic diversity and metabolic capabilities of members of the phylum Planctomycetes in the anaerobic, sulfide-saturated sediments of a mesophilic spring (Zodletone Spring) in southwestern Oklahoma. Culture-independent analyses of 16S rRNA gene sequences generated using Planctomycetes-biased primer pairs suggested that an extremely diverse community of Planctomycetes is present at the spring. Although sequences that are phylogenetically affiliated with cultured heterotrophic Planctomycetes were identified, the majority of the sequences belonged to several globally distributed, as-yet-uncultured Planctomycetes lineages. Using complex organic media (aqueous extracts of the spring sediments and rumen fluid), we isolated two novel strains that belonged to the Pirellula-Rhodopirellula-Blastopirellula clade within the Planctomycetes. The two strains had identical 16S rRNA gene sequences, and their closest relatives were isolates from Kiel Fjord (Germany), Keauhou Beach (HI), a marine aquarium, and tissues of marine organisms (Aplysina sp. sponges and postlarvae of the giant tiger prawn Penaeus monodon). The closest recognized cultured relative of strain Zi62 was Blastopirellula marina (93.9% sequence similarity). Detailed characterization of strain Zi62 revealed its ability to reduce elemental sulfur to sulfide under anaerobic conditions, as well as its ability to produce acids from sugars; both characteristics may potentially allow strain Zi62 to survive and grow in the anaerobic, sulfide- and sulfur-rich environment at the spring source. Overall, this work indicates that anaerobic metabolic abilities are widely distributed among all major Planctomycetes lineages and suggests carbohydrate fermentation and sulfur reduction as possible mechanisms employed by heterotrophic Planctomycetes for growth and survival under anaerobic conditions.

title: SAR11 clade dominates ocean surface bacterioplankton communities
abstract: The most abundant class of bacterial ribosomal RNA genes detected in seawater DNA by gene cloning belongs to SAR11—an α-proteobacterial clade. Other than indications of their prevalence in seawater, little is known about these organisms. Here we report quantitative measurements of the cellular abundance of the SAR11 clade in northwestern Sargasso Sea waters to 3,000 m and in Oregon coastal surface waters. On average, the SAR11 clade accounts for a third of the cells present in surface waters and nearly a fifth of the cells present in the mesopelagic zone. In some regions, members of the SAR11 clade represent as much as 50% of the total surface microbial community and 25% of the subeuphotic microbial community. By extrapolation, we estimate that globally there are 2.4 × 1028 SAR11 cells in the oceans, half of which are located in the euphotic zone. Although the biogeochemical role of the SAR11 clade remains uncertain, these data support the conclusion that this microbial group is among the most successful organisms on Earth.

title: Cultivation of the ubiquitous SAR11 marine bacterioplankton clade
abstract: The α-proteobacterial lineage that contains SAR11 and related ribosomal RNA gene clones was among the first groups of organisms to be identified when cultivation-independent approaches based on rRNA gene cloning and sequencing were applied to survey microbial diversity in natural ecosystems. This group accounts for 26% of all ribosomal RNA genes that have been identified in sea water and has been found in nearly every pelagic marine bacterioplankton community studied by these methods. The SAR11 clade represents a pervasive problem in microbiology: despite its ubiquity, it has defied cultivation efforts. Genetic evidence suggests that diverse uncultivated microbial taxa dominate most natural ecosystems, which has prompted widespread efforts to elucidate the geochemical activities of these organisms without the benefit of cultures for study. Here we report the isolation of representatives of the SAR11 clade. Eighteen cultures were initially obtained by means of high-throughput procedures for isolating cell cultures through the dilution of natural microbial communities into very low nutrient media. Eleven of these cultures have been successfully passaged and cryopreserved for future study. The volume of these cells, about 0.01 µm3, places them among the smallest free-living cells in culture.

title: Distinctive Microbial Community Structure in Highly Stratified Deep-Sea Brine Water Columns
abstract: Atlantis II and Discovery are two hydrothermal and hypersaline deep-sea pools in the Red Sea rift that are characterized by strong thermohalo-stratification and temperatures steadily peaking near the bottom. We conducted comprehensive vertical profiling of the microbial populations in both pools and highlighted the influential environmental factors. Pyrosequencing of the 16S rRNA genes revealed shifts in community structures vis-à-vis depth. High diversity and low abundance were features of the deepest convective layers despite the low cell density. Surprisingly, the brine interfaces had significantly higher cell counts than the overlying deep-sea water, yet they were lowest in diversity. Vertical stratification of the bacterial populations was apparent as we moved from the Alphaproteobacteria-dominated deep sea to the Planctomycetaceae- or Deferribacteres-dominated interfaces to the Gammaproteobacteria-dominated brine layers. Archaeal marine group I was dominant in the deep-sea water and interfaces, while several euryarchaeotic groups increased in the brine. Across sites, microbial phylotypes and abundances varied substantially in the brine interface of Discovery compared with Atlantis II, despite the near-identical populations in the overlying deep-sea waters. The lowest convective layers harbored interestingly similar microbial communities, even though temperature and heavy metal concentrations were very different. Multivariate analysis indicated that temperature and salinity were the major influences shaping the communities. The harsh conditions and the low-abundance phylotypes could explain the observed correlation in the brine pools.

title: Dynamics of methanotrophic communities in tropical alkaline landfill upland soil
abstract: Landfill methanotrophs play an important role in greenhouse gas reduction. To understand the dynamics of methanotroph population, quantitative polymerase chain reaction methods (q-PCR) and terminal-restriction fragment length polymorphism (T-RFLP) analyses were used to explore the methanotrophic communities in tropical alkaline landfill soils at 20 and 40 cm depths with different vegetation during a year round study. The q-PCR revealed that the amount of methanotrophs were 1.25 × 10 3 to 1.93 × 10 6 pmo A-copy (g dry wt soil) −1 and were negatively correlated with accumulated precipitation before sampling. The pmo A-based T-RFLP analyses showed that Methylocaldum related populations were dominant for a long residence time, and Methylocystis related populations were dominant in the deeper sites of milk bush-covered region. Canonical correspondence analysis showed that vegetation, accumulated precipitation, temperature, sampling depth and soil pH could affect the methanotrophic community structures. The pmo A-based T-RFLP analysis used in this study provided good resolution within type I methanotrophs and could easily distinguish between type I and type II methanotrophs.

title: Isolation of Two Novel Marine Ethylene-Assimilating Bacteria, Haliea Species ETY-M and ETY-NAG, Containing Particulate Methane Monooxygenase-like Genes
abstract: Two novel ethylene-assimilating bacteria, strains ETY-M and ETY-NAG, were isolated from seawater around Japan. The characteristics of both strains were investigated, and phylogenetic analyses of their 16S rRNA gene sequences showed that they belonged to the genus Haliea. In C1–4 gaseous hydrocarbons, both strains grew only on ethylene, but degraded ethane, propylene, and propane in addition to ethylene. Methane, n-butane, and i-butane were not utilized or degraded by either strain. Soluble methane monooxygenase-type genes, which are ubiquitous in alkene-assimilating bacteria for initial oxidation of alkenes, were not detected in these strains, although genes similar to particulate methane monooxygenases (pMMO)/ammonia monooxygenases (AMO) were observed. The phylogenetic tree of the deduced amino acid sequences formed a new clade near the monooxygenases of ethane-assimilating bacteria similar to other clades of pMMOs in type I, type II, and Verrucomicrobia methanotrophs and AMOs in alpha and beta proteobacteria.

title: Microbial Diversity of the Brine-Seawater Interface of the Kebrit Deep, Red Sea, Studied via 16S rRNA Gene Sequences and Cultivation Methods
abstract: The brine-seawater interface of the Kebrit Deep, northern Red Sea, was investigated for the presence of microorganisms using phylogenetic analysis combined with cultivation methods. Under strictly anaerobic culture conditions, novel halophiles were isolated. The new rod-shaped isolates belong to the halophilic genus Halanaerobiumand are the first representatives of the genus obtained from deep-sea, anaerobic brine pools. Within the genus Halanaerobium, they represent new species which grow chemoorganotrophically at NaCl concentrations ranging from 5 to 34%. The cellular fatty acid compositions are consistent with those of otherHalanaerobium representatives, showing unusually large amounts of Δ7 and Δ11 16:1 fatty acids. Phylogenetic analysis of the brine-seawater interface sample revealed the presence of various bacterial 16S rRNA gene sequences dominated by cultivated members of the bacterial domain, with the majority affiliated with the genusHalanaerobium. The new Halanaerobium 16S rRNA clone sequences showed the highest similarity (99.9%) to the sequence of isolate KT-8-13 from the Kebrit Deep brine. In this initial survey, our polyphasic approach demonstrates that novel halophiles thrive in the anaerobic, deep-sea brine pool of the Kebrit Deep, Red Sea. They may contribute significantly to the anaerobic degradation of organic matter enriched at the brine-seawater interface.