Methanotrophic bacteria in cold seeps of the floodplains of northern rivers

Small mud volcanoes (cold seeps), which are common in the floodplains of northern rivers, are potentially important (although poorly studied) sources of atmospheric methane. Field research on the cold seeps of the Mukhrina River (Khanty-Mansiysk Autonomous okrug, Russia) revealed methane fluxes from these structures to be orders of magnitude higher than from equivalent areas of the mid-taiga bogs. Microbial communities developing around the seeps were formed under conditions of high methane concentrations, low temperatures (3–5°C), and near-neutral pH. Molecular identification of methane-oxidizing bacteria from this community by analysis of the pmoA gene encoding particulate methane monooxygenase revealed both type I and type II methanotrophs (classes Gammaproteobacteria and Alphaproteobacteria, respectively), with prevalence of type I methanotrophs. Among the latter, microorganisms related to Methylobacter psychrophilus and Methylobacter tundripaludum, Crenothrix polyspora (a stagnant water dweller), and a number of methanotrophs belonging to unknown taxa were detected. Growth characteristics of two methanotrophic isolates were determined. Methylobacter sp. CMS7 exhibited active growth at 4–10°C, while Methylocystis sp. SB12 grew better at 20°C. Experimental results confirmed the major role of methanotrophic gammaproteobacteria in controlling the methane emission from cold river seeps.     

Stable Isotope Probing Analysis of the Diversity and Activity of Methanotrophic Bacteria in Soils from the Canadian High Arctic

The melting of permafrost and its potential impact on CH₄ emissions are major concerns in the context of global warming. Methanotrophic bacteria have the capacity to mitigate CH₄ emissions from melting permafrost. Here, we used quantitative PCR (qPCR), stable isotope probing (SIP) of DNA, denaturing gradient gel electrophoresis (DGGE) fingerprinting, and sequencing of the 16S rRNA and pmoA genes to study the activity and diversity of methanotrophic bacteria in active-layer soils from Ellesmere Island in the Canadian high Arctic. Results showed that most of the soils had the capacity to oxidize CH₄ at 4°C and at room temperature (RT), but the oxidation rates were greater at RT than at 4°C and were significantly enhanced by nutrient amendment. The DGGE banding patterns associated with active methanotrophic bacterial populations were also different depending on the temperature of incubation and the addition of nutrients. Sequencing of the 16S rRNA and pmoA genes indicated a low diversity of the active methanotrophic bacteria, with all methanotroph 16S rRNA and pmoA gene sequences being related to type I methanotrophs from Methylobacter and Methylosarcina. The dominance of type I methanotrophs over type II methanotrophs in the native soil samples was confirmed by qPCR of the 16S rRNA gene with primers specific for these two groups of bacteria. The 16S rRNA and pmoA gene sequences related to those of Methylobacter tundripaludum were found in all soils, regardless of the incubation conditions, and they might therefore play a role in CH₄ degradation in situ. This work is providing new information supporting the potential importance of Methylobacter spp. in Arctic soils found in previous studies and contributes to the limited body of knowledge on methanotrophic activity and diversity in this extreme environment.Permafrost regions occupy approximately 22% of the exposed land area of the Northern Hemisphere (63). In the past 100 years, the average temperatures in the arctic regions have increased at almost twice the average global rate (25). The melting of permafrost is one of the most important impacts of global warming on these high-latitude environments, and theoretical modeling suggests that as much as 90% of the permafrost could thaw by the end of the 21st century (29). While it has been generally reported that 15% of the total soil organic carbon is stocked in permafrost (42), a recent estimate indicates that it contains as much as 50% of the global belowground organic carbon pool (53). Carbon stocked in permafrost is now regarded as one of the most important carbon-climate feedbacks because of the size of the carbon pool and the intensity of climate change at high latitudes (46, 47). The presence of these large amounts of carbon in permafrost is raising serious concerns whether melting permafrost, and the resulting increase in microbial activity, might be a source of extensive emissions of the greenhouse gases carbon dioxide and methane (CH₄) to the atmosphere. The actual emissions of CH₄ from soils of high latitudes have been estimated to represent about 25% of the emissions from natural sources (19). Methane, which is 25 times more potent than carbon dioxide as a greenhouse gas (25), is produced by methanogenic archaea under anaerobic conditions. These microorganisms are known to inhabit permafrost environments (44, 49), and their capacity to produce methane at cold temperatures has been reported (20, 35, 44, 56). Their methanogenic activity is expected to increase as permafrost soil temperature increases (20). Moreover, large amounts of methane are stocked as methane hydrates in permafrost at an average depth of several hundred meters (33). Methane is also found in permafrost layers near the surface and could potentially be liberated to the atmosphere as permafrost melts (44).Methane can be oxidized in aerobic zones by aerobic methanotrophic bacteria or in anaerobic zones by anaerobic methanotrophic archaea (for a recent review, see reference 27). Anaerobic methane oxidizers were not covered in the context of this study, which focused exclusively on aerobic methanotrophs. These bacteria utilize methane as the sole carbon and energy source through the activity of the enzyme methane monooxygenase (MMO). Most known aerobic methanotrophs are divided into two major groups (type I and type II) based on phylogeny and carbon assimilation pathways (5). Type I methanotrophs, also known as Gammaproteobacteria methanotrophs (6) belong to the family Methylococcaceae within the Gammaproteobacteria subdivision, while type II methanotrophs (Alphaproteobacteria methanotrophs) belong to the family Methylocystaceae in the Alphaproteobacteria subdivision (5). Because of their capacity to oxidize methane, aerobic methanotrophs can significantly reduce methane emissions to the atmosphere and play an important role in the global methane cycle (12, 22). Methanotrophic activity has been observed in cold environments, and methanotrophs might contribute to the reduction of methane emissions from melting permafrost. Aerobic methanotrophic bacteria from cold environments have been reviewed in detail elsewhere (54).Most studies addressing methanotrophs from cold environments were conducted on soils from very few sites located in Northern Europe and Siberia (14, 30, 31, 40, 56-58), while methanotrophic bacterial populations in soils from the Canadian high Arctic remain mostly unexplored (41). In addition, most of these studies were conducted at low latitudes, and the pool of knowledge concerning the activity and diversity of methanotrophic bacterial populations in high Arctic soils is limited. The question being addressed in this study is whether there are active methanotrophs in the active-layer soil in the high Arctic. Therefore, the present work had two objectives: (i) to evaluate the methane oxidation capacity of three active-layer soils from the Canadian high Arctic under various incubation conditions and (ii) to identify and characterize the diversity of the active methanotrophs in these soils using stable isotope probing (SIP) of DNA (DNA-SIP) and sequencing of the 16S rRNA and pmoA genes. With this work, we identify for the first time active methanotrophs in high Arctic soils through the use of DNA-SIP.     

Culturable psychrotolerant methanotrophic bacteria in landfill cover soil

Methanotrophs closely related to psychrotolerant members of the genera Methylobacter and Methylocella were identified in cultures enriched at 10°C from landfill cover soil samples collected in the period from April to November. Mesophilic methanotrophs of the genera Methylobacter and Methylosinus were found in cultures enriched at 20°C from the same cover soil samples. A thermotolerant methanotroph related to Methylocaldum gracile was identified in the culture enriched at 40°C from a sample collected in May (the temperature of the cover soil was 11.5–12.5°C). In addition to methanotrophs, methylobacteria of the genera Methylotenera and Methylovorus and members of the genera Verrucomicrobium, Pseudomonas, Pseudoxanthomonas, Dokdonella, Candidatus Protochlamydia, and Thiorhodospira were also identified in the enrichment cultures. A methanotroph closely related to the psychrotolerant species Methylobacter tundripaludum (98% sequence identity of 16S rRNA genes with the type strain SV96^T) was isolated in pure culture. The introduction of a mixture of the methanotrophic enrichments, grown at 15°C, into the landfill cover soil resulted in a decrease in methane emission from the landfill surface in autumn (October, November). The inoculum used was demonstrated to contain methanotrophs closely related to Methylobacter tundripaludum SV96.     

Spatiotemporal variations in an assemblage of closely related planktonic aerobic methanotrophs

1. The assemblage of aerobic methane‐oxidising bacteria (MOB) was investigated in different seasons in the water column of a stratified freshwater lake. Species composition was analysed by performing denaturing gradient gel electrophoresis (DGGE) of the 16S rRNA genes and cloning analysis of the pmoA gene, which encodes the α‐subunit of particulate methane monooxygenase. The relative abundance of MOB to total bacteria was deduced from the copy number of the pmoA gene and 16S rRNA gene using real‐time polymerase chain reaction. 2. The profiles of the DGGE banding patterns changed with water depth, and these changes correlated with oxygen concentration and water temperature. The sequences of the DGGE bands obtained were all associated with the genus Methylobacter. During the analysis of pmoA gene, all clones sequenced were that of the Methylobacter/Methylosarcina group. The relative abundances of pmoA gene peaked around the oxycline, and small peaks of pmoA gene were also observed near the surface when peaks of methane were observed at the corresponding depth. 3. Profiles of the DGGE banding patterns suggested that ecophysiological characteristics differ among members of the genus Methylobacter; this indicates the importance of investigating the MOB assemblage at the species level or lower. Planktonic MOB seemed to be abundant around oxycline.     

Microbial diversity and activity through a permafrost/ground ice core profile from the Canadian high Arctic

Culture‐dependent and culture‐independent methods were used in an investigation of the microbial diversity in a permafrost/massive ground ice core from the Canadian high Arctic. Denaturing gradient gel electrophoresis as well as Bacteria and Archaea 16S rRNA gene clone libraries showed differences in the composition of the microbial communities in the distinct core horizons. Microbial diversity was similar in the active layer (surface) soil, permafrost table and permafrost horizons while the ground ice microbial community showed low diversity. Bacteria and Archaea sequences related to the Actinobacteria (54%) and Crenarchaeota (100%) respectively were predominant in the active layer while the majority of sequences in the permafrost were related to the Proteobacteria (57%) and Euryarchaeota (76%). The most abundant phyla in the ground ice clone libraries were the Firmicutes (59%) and Crenarchaeota (82%). Isolates from the permafrost were both less abundant and diverse than in the active layer soil, while no culturable cells were recovered from the ground ice. Mineralization of [1‐¹⁴C] acetic acid and [2‐¹⁴C] glucose was used to detect microbial activity in the different horizons in the core. Mineralization was detected at near ambient permafrost temperatures (?15°C), indicating that permafrost may harbour an active microbial population, while the low microbial diversity, abundance and activity in ground ice suggests a less hospitable microbial habitat.     

Microbial diversity of active layer and permafrost in an acidic wetland from the Canadian High Arctic

The abundance and structure of archaeal and bacterial communities from the active layer and the associated permafrost of a moderately acidic (pH < 5.0) High Arctic wetland (Axel Heiberg Island, Nunavut, Canada) were investigated using culture- and molecular-based methods. Aerobic viable cell counts from the active layer were ～100-fold greater than those from the permafrost (2.5 × 10(5) CFU·(g soil dry mass)(-1)); however, a greater diversity of isolates were cultured from permafrost, as determined by 16S rRNA gene sequencing. Isolates from both layers demonstrated growth characteristics of a psychrotolerant, halotolerant, and acidotolerant community. Archaea constituted 0.1% of the total 16S rRNA gene copy number and, in the 16S rRNA gene clone library, predominantly (71% and 95%) consisted of Crenarchaeota related to Group I. 1b. In contrast, bacterial communities were diverse (Shannon's diversity index, H = ～4), with Acidobacteria constituting the largest division of active layer clones (30%) and Actinobacteria most abundant in permafrost (28%). Direct comparisons of 16S rRNA gene sequence data highlighted significant differences between the bacterial communities of each layer, with the greatest differences occurring within Actinobacteria. Comparisons of 16S rRNA gene sequences with those from other Arctic permafrost and cold-temperature wetlands revealed commonly occurring taxa within the phyla Chloroflexi, Acidobacteria, and Actinobacteria (families Intrasporangiaceae and Rubrobacteraceae).     

Activity, distribution, and abundance of methane-oxidizing bacteria in the near surface soils of onshore oil and gas fields

Methane-oxidizing bacteria (MOB) have long been used as an important biological indicator for oil and gas prospecting, but the ecological characteristics of MOB in hydrocarbon microseep systems are still poorly understood. In this study, the activity, distribution, and abundance of aerobic methanotrophic communities in the surface soils underlying an oil and gas field were investigated using biogeochemical and molecular ecological techniques. Measurements of potential methane oxidation rates and pmoA gene copy numbers showed that soils inside an oil and gas field are hot spots of methane oxidation and MOB abundance. Correspondingly, terminal restriction fragment length polymorphism analyses in combination with cloning and sequencing of pmoA genes also revealed considerable differences in the methanotrophic community composition between oil and gas fields and the surrounding soils. Principal component analysis ordination furthermore indicated a coincidence between elevated CH₄ oxidation activity and the methanotrophic community structure with type I methanotrophic Methylococcus and Methylobacter, in particular, as indicator species of oil and gas fields. Collectively, our results show that trace methane migrated from oil and gas reservoirs can considerably influence not only the quantity but also the structure of the methanotrophic community.     

Structural and functional features of methanotrophs from hypersaline and alkaline lakes

Ten strains of aerobic methanotrophic bacteria represented by halophilic neutrophiles or halotolerant alkaliphiles were isolated from saline and alkaline lakes of southeast Siberia, Mongolia, Africa, and North America. Based on analysis of the nucleotide sequences of 16S rRNA gene and the pmoA gene encoding particulate methane monooxygenase, the isolates were classified as Methylomicrobium alcaliphilum, Methylomicrobium buryatense, and Methylobacter marinus. All strains of the genus Methylomicrobium were shown to synthesize glycoprotein S-layers located on the cell surface with hexagonal symmetry (p6) as a monolayer of cup-shaped structures or fine “inverted” conical structures and as plates consisting of protein subunits with inclined (p2) symmetry. During adaptation to the high salinity of the medium, isolated methanotrophs synthesize osmoprotectants: ectoine, sucrose, and glutamate. The ectC gene encoding ectoine synthase (EctC) was identified in six methanotrophic strains. Phylogenetic analysis of translated amino acid sequence of the ectC gene fragment suggests lateral transfer of the genes of ectoine synthesis as the most probable way for methanotrophs to acquire resistance to high external salinity.     

Methanotrophic diversity in an agricultural soil as evaluated by denaturing gradient gel electrophoresis profiles of pmoA, mxaF and 16S rDNA sequences

Molecular methods were used to characterize the diversity of a methanotrophic population in an agricultural soil. For this purpose we have used DGGE analysis of functional and phylogenetic markers. Functional markers utilised comprised the pmoA-gene coding for the -subunit of the particulate methane monooxygenase (pMMO) present in all known methanotrophs and the mxaF-gene coding for the -subunit of methanol dehydrogenase (MDH) present in all Gram-negative methylotrophs. In addition, we have used 16S rDNA as a phylogenetic marker. DGGE patterns of an enrichment culture, and sequencing of major DGGE bands obtained with the bacterial specific primers showed that the community structure was dominated by methanotrophic populations related to Methylobacter sp. and Methylomicrobium sp. The PCR products amplified with the functional primer sets were related to both type I and type II methanotrophs. We also designed a new pmoA-targeting primer set which could be used in a nested protocol to amplify PCR-products from DNA extracted directly from the soil.     

Methanotrophic Activity,Abundance, and Diversity in Forested Swamp Pools: Spatiotemporal Dynamics and Influences on Methane Fluxes

Methane oxidation (methanotrophy) in the water column and sediments of forested swamp pools likely control seasonal and annual emission of CH₄ from these systems, but the methanotrophic microbial communities, their activities, locations, and overall impact, is poorly understood. Several techniques including ¹⁴CH₄ oxidation assays, culture-based most probable number (MPN) estimates of methane-oxidizing bacteria (MOB) and protozoan abundance, MOB strain isolation and characterization, and PCR techniques were used to investigate methanotrophy at a forested swamp near Ithaca, New York. The greatest methanotrophic activity and largest numbers of MOB occurred predominantly at the low oxygen sediment/water interface in the water column. Seasonally, methanotrophic activity was very dynamic, ranging from 0.1 to 61.9 μ moles CH₄ d^?1 g^?1 dry sediment, and correlated most strongly with dissolved inorganic carbon (r = 0.896). Incorporation of methanotrophic variables (abundance and activity) into existing CH₄ flux regression models improved model fit, particularly during mid summer when CH₄ fluxes were most dynamic. Annually integrated methane flux and methanotrophic activity measurements indicate that differences in methanotrophic activity at the sediment/water interface likely accounted for differences in the annual CH₄ emission from the field site. Direct isolations of MOB resulted in the repeated isolation of organisms most closely related to Methylomonas methanica S1. A single acidophilic, type II MOB related to Methylocella palustris K was also isolated. Using a PCR-based MPN method and 16S rRNA genome copy number from isolates and control strains, type I and type II MOB were enumerated and revealed type I dominance of the sediment-associated MOB community.     

Microbial communities of the discharge zone of oil- and gas-bearing fluids in low-mineral Lake Baikal

At the site of natural ingress of oil, microbial diversity in the Central Baikal bottom sediments differing in the chemical composition of pore waters was studied by molecular biological techniques. The sediments saturated with oil and methane were found to contain members of 10 bacterial and 2 archaeal phyla. The oxidized sediment layer contained methanotrophic bacteria belonging to the Alphaproteobacteria, which had a specific structure of the pmoA gene and clustered together with uncultured methanotrophs from cold ecosystems. The upper sediment layer also contained oil-oxidizing bacteria and the alkB genes most closely related to those of Rhodococcus. The microbial community of reduced sediments exhibited lower diversity and was represented mostly by the organisms involved in hydrocarbon biodegradation.     

Molecular biologic techniques applied to the microbial prospecting of oil and gas in the Ban 876 gas and oil field in China

Currently, molecular biologic techniques achieve a great development in studies of soil samples. The objective of this research is to improve methods for microbial prospecting of oil and gas by applying culture-independent techniques to soil sampled from above a known oil and gas field. Firstly, the community structure of soil bacteria above the Ban 876 Gas and Oil Field was analyzed based on 16S rRNA gene clone libraries. The soil bacteria communities were consistently different along the depth; however, Chloroflexi and Gemmatimonadetes were predominant and methanotrophs were minor in both bacteria libraries (DGS1 and DGS2). Secondly, the numbers of methane-oxidizing bacteria, quantified using a culture-dependent procedure and culture-independent group-specific real-time PCR (RT-PCR), respectively, were inconsistent with a quantify variance of one or two orders of magnitude. Special emphasis was given to the counting advantages of RT-PCR based on the methanotrophic pmoA gene. Finally, the diversity and distribution of methanotrophic communities in the soil samples were analyzed by constructing clone libraries of functional gene. All 508-bp inserts in clones phylogenetically belonged to the methanotrophic pmoA gene with similarities from 83% to 100%. However, most of the similarities were below 96%. Five clone libraries of methanotrophs clearly showed that the anomalous methanotrophs (Methylosinus and Methylocystis) occupy the studied area.     

Bacterial Community Structure in Two Permafrost Wetlands on the Tibetan Plateau and Sanjiang Plain,China

Permafrost wetlands are important methane emission sources and fragile ecosystems sensitive to climate change. Presently, there remains a lack of knowledge regarding bacterial communities, especially methanotrophs in vast areas of permafrost on the Tibetan Plateau in Northwest China and the Sanjiang Plain (SJ) in Northeast China. In this study, 16S rRNA-based quantitative PCR (qPCR) and 454 pyrosequencing were used to identify bacterial communities in soils sampled from a littoral wetland of Lake Namco on the Tibetan Plateau (NMC) and an alluvial wetland on the SJ. Additionally, methanotroph-specific primers targeting particulate methane monooxygenase subunit A gene (pmoA) were used for qPCR and pyrosequencing analysis of methanotrophic community structure in NMC soils. qPCR analysis revealed the presence of 10¹⁰ 16S rRNA gene copies per gram of wet soil in both wetlands, with 10⁸ pmoA copies per gram of wet soil in NMC. The two permafrost wetlands showed similar bacterial community compositions, which differed from those reported in other cold environments. Proteobacteria, Actinobacteria , and Chloroflexi were the most abundant phyla in both wetlands, whereas Acidobacteria was prevalent in the acidic wetland SJ only. These four phyla constituted more than 80 % of total bacterial community diversity in permafrost wetland soils, and Methylobacter of type I methanotrophs was overwhelmingly dominant in NMC soils. This study is the first major bacterial sequencing effort of permafrost in the NMC and SJ wetlands, which provides fundamental data for further studies of microbial function in extreme ecosystems under climate change scenarios.     

Genome sequence of the Arctic methanotroph Methylobacter tundripaludum SV96

Methylobacter tundripaludum SV96^T (ATCC BAA-1195) is a psychrotolerant aerobic methane-oxidizing gammaproteobacterium (Methylococcales, Methylococcaceae) living in High Arctic wetland soil. The strain was isolated from soil harvested in July 1996 close to the settlement Ny-Ålesund, Svalbard, Norway (78°56′N, 11°53′E), and described as a novel species in 2006. The genome includes pmo and pxm operons encoding copper membrane monooxygenases (Cu-MMOs), genes required for nitrogen fixation, and the nirS gene implicated in dissimilatory nitrite reduction to NO but no identifiable inventory for further processing of nitrogen oxides. These genome data provide the basis to investigate M. tundripaludum SV96, identified as a major player in the biogeochemistry of Arctic environments.     

Molecular Analyses of Novel Methanotrophic Communities in Forest Soil That Oxidize Atmospheric Methane

Forest and other upland soils are important sinks for atmospheric CH₄, consuming 20 to 60 Tg of CH₄ per year. Consumption of atmospheric CH₄ by soil is a microbiological process. However, little is known about the methanotrophic bacterial community in forest soils. We measured vertical profiles of atmospheric CH₄ oxidation rates in a German forest soil and characterized the methanotrophic populations by PCR and denaturing gradient gel electrophoresis (DGGE) with primer sets targeting the pmoA gene, coding for the α subunit of the particulate methane monooxygenase, and the small-subunit rRNA gene (SSU rDNA) of all life. The forest soil was a sink for atmospheric CH₄ in situ and in vitro at all times. In winter, atmospheric CH₄ was oxidized in a well-defined subsurface soil layer (6 to 14 cm deep), whereas in summer, the complete soil core was active (0 cm to 26 cm deep). The content of total extractable DNA was about 10-fold higher in summer than in winter. It decreased with soil depth (0 to 28 cm deep) from about 40 to 1 μg DNA per g (dry weight) of soil. The PCR product concentration of SSU rDNA of all life was constant both in winter and in summer. However, the PCR product concentration of pmoA changed with depth and season. pmoA was detected only in soil layers with active CH₄ oxidation, i.e., 6 to 16 cm deep in winter and throughout the soil core in summer. The same methanotrophic populations were present in winter and summer. Layers with high CH₄ consumption rates also exhibited more bands of pmoA in DGGE, indicating that high CH₄ oxidation activity was positively correlated with the number of methanotrophic populations present. The pmoA sequences derived from excised DGGE bands were only distantly related to those of known methanotrophs, indicating the existence of unknown methanotrophs involved in atmospheric CH₄ consumption.     

Biogeography of wetland rice methanotrophs

We focused on the functional guild of methane oxidizing bacteria (MOB) as model organisms to get deeper insights into microbial biogeography. The pmoA gene was used as a functional and phylogenetic marker for MOB in two approaches: (i) a pmoA database (> 4000 sequences) was evaluated to obtain insights into MOB diversity in Italian rice paddies, and paddy fields worldwide. The results show a wide geographical distribution of pmoA genotypes that seem to be specifically adapted to paddy fields (e.g. Rice Paddy Cluster 1 and Rice Paddy Cluster 2). (ii) On the smaller geographical scale, we designed a factorial experiment including three different locations, two rice varieties and two habitats (soil and roots) within each of three rice fields. Multivariate analysis of terminal restriction fragment analysis profiles revealed different community patterns at the three field sites, located 10–20 km apart. Root samples were characterized by high abundance of type I MOB whereas the rice variety had no effect. With the agronomical practice being nearly identical, historical contingencies might be responsible for the field site differences. Considering a large reservoir of viable yet inactive MOB cells acting as a microbial seed bank, environmental conditions might have selected and activated a different subset at a time thereby shaping the community.     

pmoA-Based Analysis of Methanotrophs in a Littoral Lake Sediment Reveals a Diverse and Stable Community in a Dynamic Environment

Diversity and community structure of aerobic methane-oxidizing bacteria in the littoral sediment of Lake Constance was investigated by cloning analysis and terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of the pmoA gene. Phylogenetic analysis revealed a high diversity of type I and type II methanotrophs in the oxygenated uppermost centimeter of the sediment. T-RFLP profiles indicated a high similarity between the active methanotrophic community in the oxic layer and the inactive community in an anoxic sediment layer at a 10-cm depth. There were also no major changes in community structure between littoral sediment cores sampled in summer and winter. By contrast, the fingerprint patterns showed substantial differences between the methanotrophic communities of littoral and profundal sediments.     

Molecular Characterization and In Situ Localization of Endosymbiotic 16S Ribosomal RNA and RuBisCO Genes in the Pogonophoran Tissue

Gutless pogonophorans are generally thought to live in symbiosis with methane-oxidizing bacteria (methanotrophs). We identified a 16S ribosomal RNA gene (rDNA) and a ribulose-1,5-bisphosphate carboxlase/oxygenase (RuBisCO, E.C.4.1.1.39) gene that encode the form I large subunit (cbbL) from symbiont-bearing tissue of the pogonophoran Oligobrachia mashikoi. Phylogenetic analysis of the 16S rDNA sequence suggested that the pogonophoran endosymbiont belonged to the -subdivision of Proteobacteria. The endosymbiont was most closely related to an uncultured bacterium from a hydrocarbon seep, forming a unique clade adjacent to the known methanotrophic 16S rDNA cluster. The RuBisCO gene from the pogonophoran tissue was closely related to those of the chemoautotrophic genera Thiobacillus and Hydrogenovibrio. Presence of the RuBisCO gene suggested a methanotrophic symbiosis because some methanotrophic bacteria are known to be capable of autotrophy via the Calvin cycle. In contrast, particulate and soluble methane monooxygenase genes (pmoA and mmoX) and the methanol dehydrogenase gene (mxaF), which are indicators for methanotrophs or methylotrophs, were not detected by repeated trial of polymerase chain reaction. For 16S rRNA and RuBisCO genes, endosymbiotic localizations were confirmed by in situ hybridization. These results support the possibilities that the pogonophoran host has a novel endosymbiont which belongs to the -subdivision of Proteobacteria, and that the endosymbiont has the gene of the autotrophic enzyme RuBisCO.