Detection of methanotroph diversity on roots of submerged rice plants by molecular retrieval of pmoA, mmoX, mxaF, and 16S rRNA and ribosomal DNA, including pmoA-based terminal restriction fragment length polymorphism profiling

The diversity of methanotrophic bacteria associated with roots of submerged rice plants was assessed using cultivation-independent techniques. The research focused mainly on the retrieval of pmoA, which encodes the alpha subunit of the particulate methane monooxygenase. A novel methanotroph-specific community-profiling method was established using the terminal restriction fragment length polymorphism (T-RFLP) technique. The T-RFLP profiles clearly revealed a more complex root-associated methanotrophic community than did banding patterns obtained by pmoA-based denaturing gradient gel electrophoresis. The comparison of pmoA-based T-RFLP profiles obtained from rice roots and bulk soil of flooded rice microcosms suggested that there was a substantially higher abundance of type I methanotrophs on rice roots than in the bulk soil. These were affiliated to the genera Methylomonas, Methylobacter, Methylococcus, and to a novel type I methanotroph sublineage. By contrast, type II methanotrophs of the Methylocystis-Methylosinus group could be detected with high relative signal intensity in both soil and root compartments. Phylogenetic treeing analyses and a set of substrate-diagnostic amino acid residues provided evidence that a novel pmoA lineage was detected. This branched distinctly from all currently known methanotrophs. To examine whether the retrieval of pmoA provided a complete view of root-associated methanotroph diversity, we also assessed the diversity detectable by recovery of genes coding for subunits of soluble methane monooxygenase (mmoX) and methanol dehydrogenase (mxaF). In addition, both 16S rRNA and 16S ribosomal DNA (rDNA) were retrieved using a PCR primer set specific to type I methanotrophs. The overall methanotroph diversity detected by recovery of mmoX, mxaF, and 16S rRNA and 16S rDNA corresponded well to the diversity detectable by retrieval of pmoA.     

The Methanol Dehydrogenase Gene,mxaF, as a Functional and Phylogenetic Marker for Proteobacterial Methanotrophs in Natural Environments

The mxaF gene, coding for the large (α) subunit of methanol dehydrogenase, is highly conserved among distantly related methylotrophic species in the Alpha-, Beta- and Gammaproteobacteria. It is ubiquitous in methanotrophs, in contrast to other methanotroph-specific genes such as the pmoA and mmoX genes, which are absent in some methanotrophic proteobacterial genera. This study examined the potential for using the mxaF gene as a functional and phylogenetic marker for methanotrophs. mxaF and 16S rRNA gene phylogenies were constructed based on over 100 database sequences of known proteobacterial methanotrophs and other methylotrophs to assess their evolutionary histories. Topology tests revealed that mxaF and 16S rDNA genes of methanotrophs do not show congruent evolutionary histories, with incongruencies in methanotrophic taxa in the Methylococcaceae, Methylocystaceae, and Beijerinckiacea. However, known methanotrophs generally formed coherent clades based on mxaF gene sequences, allowing for phylogenetic discrimination of major taxa. This feature highlights the mxaF gene’s usefulness as a biomarker in studying the molecular diversity of proteobacterial methanotrophs in nature. To verify this, PCR-directed assays targeting this gene were used to detect novel methanotrophs from diverse environments including soil, peatland, hydrothermal vent mussel tissues, and methanotroph isolates. The placement of the majority of environmental mxaF gene sequences in distinct methanotroph-specific clades (Methylocystaceae and Methylococcaceae) detected in this study supports the use of mxaF as a biomarker for methanotrophic proteobacteria.     

Rice roots select for type I methanotrophs in rice field soil

Methanotrophs are an important regulator for reducing methane (CH₄) emissions from rice field soils. The type I group of the proteobacterial methanotrophs are generally favored at low CH₄ concentration and high O₂ availability, while the type II group lives better under high CH₄ and limiting O₂ conditions. Such physiological differences are possibly reflected in their ecological preferences. In the present study, methanotrophic compositions were compared between rice-planted soil and non-planted soil and between the rhizosphere and rice roots by using terminal restriction fragment length polymorphism (T-RFLP) analysis of particulate methane monooxygenase (pmoA) genes. In addition, the effects of rice variety and nitrogen fertilizer were evaluated. The results showed that the terminal restriction fragments (T-RFs), which were characteristic for type I methanotrophs, substantially increased in the rhizosphere and on the roots compared with non-planted soils. Furthermore, the relative abundances of the type I methanotroph T-RFs were greater on roots than in the rhizosphere. Of type I methanotrophs, the 79 bp T-RF, which was characteristic for an unknown group or Methylococcus/Methylocaldum, markedly increased in field samples, while the 437 bp, which possibly represented Methylomonas, dominated in microcosm samples. These results suggested that type I methanotrophs were enriched or selected for by rice roots compared to type II methanotrophs. However, the members of type I methanotrophs are dynamic and sensitive to environmental change. Rice planting appeared to increase the copy number of pmoA genes relative to the non-planted soils. However, neither the rice variety nor the N fertilizer significantly influenced the dynamics of the methanotrophic community.     

Methanotrophic Communities in the Soils of the Russian Northern Taiga and Subarctic Tundra

The PCR analysis of DNA extracted from soil samples taken in the Russian northern taiga and subarctic tundra showed that the DNA extracts contain genes specific to methanotrophic bacteria, i.e., the mmoX gene encoding the conserved -subunit of the hydroxylase component of soluble methane monooxygenase, the pmoA gene encoding the -subunit of particulate methane monooxygenase, and the mxaFgene encoding the -subunit of methanol dehydrogenase. PCR analysis with group-specific primers also showed that methanotrophic bacteria in the northern taiga and subarctic tundra soils are essentially represented by the type I genera Methylobacter, Methylomonas, Methylosphaera, and Methylomicrobium and that some soil samples contain type II methanotrophs close to members of the genera Methylosinus and Methylocystis. The electron microscopic examination of enrichment cultures obtained from the soil samples confirmed the presence of methanotrophic bacteria in the ecosystems studied and showed that the methanotrophs contain only small amounts of intracytoplasmic membranes.     

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.     

Comparison of Aerobic Methanotrophic Communities in Littoral and Profundal Sediments of Lake Constance by a Molecular Approach

Analysis of pmoA and 16S rRNA gene clone libraries of methanotrophic bacteria in Lake Constance revealed an overall dominance of type I methanotrophs in both littoral and profundal sediments. The sediments exhibited minor differences in their methanotrophic community structures. Type X methanotrophs made up a significant part of the clone libraries only in the profundal sediment and were also found only there as a prominent peak by T-RFLP analyses.     

A novel methanotroph in the genus <Emphasis Type="Italic">Methylomonas</Emphasis> that contains a distinct clade of soluble methane monooxygenase

Aerobic methane oxidation is a key process in the global carbon cycle that acts as a major sink of methane. In this study, we describe a novel methanotroph designated EMGL16-1 that was isolated from a freshwater lake using the floating filter culture technique. Based on a phylogenetic analysis of 16S rRNA gene sequences, the isolate was found to be closely related to the genus Methylomonas in the family Methylococcaceae of the class Gammaproteobacteria with 94.2–97.4% 16S rRNA gene similarity to Methylomonas type strains. Comparison of chemotaxonomic and physiological properties further suggested that strain EMGL16-1 was taxonomically distinct from other species in the genus Methylomonas. The isolate was versatile in utilizing nitrogen sources such as molecular nitrogen, nitrate, nitrite, urea, and ammonium. The genes coding for subunit of the particulate form methane monooxygenase (pmoA), soluble methane monooxygenase (mmoX), and methanol dehydrogenase (mxaF) were detected in strain EMGL16-1. Phylogenetic analysis of mmoX indicated that mmoX of strain EMGL16-1 is distinct from those of other strains in the genus Methylomonas. This isolate probably represents a novel species in the genus. Our study provides new insights into the diversity of species in the genus Methylomonas and their environmental adaptations.     

Molecular Characterization of Functional and Phylogenetic Genes from Natural Populations of Methanotrophs in Lake Sediments

The 16S rRNA and pmoA genes from natural populations of methane-oxidizing bacteria (methanotrophs) were PCR amplified from total community DNA extracted from Lake Washington sediments obtained from the area where peak methane oxidation occurred. Clone libraries were constructed for each of the genes, and approximately 200 clones from each library were analyzed by using restriction fragment length polymorphism (RFLP) and the tetrameric restriction enzymes MspI, HaeIII, and HhaI. The PCR products were grouped based on their RFLP patterns, and representatives of each group were sequenced and analyzed. Studies of the 16S rRNA data obtained indicated that the existing primers did not reveal the total methanotrophic diversity present when these data were compared with pure-culture data obtained from the same environment. New primers specific for methanotrophs belonging to the genera Methylomonas, Methylosinus, and Methylocystis were developed and used to construct more complete clone libraries. Furthermore, a new primer was designed for one of the genes of the particulate methane monooxygenase in methanotrophs, pmoA. Phylogenetic analyses of both the 16S rRNA and pmoA gene sequences indicated that the new primers should detect these genes over the known diversity in methanotrophs. In addition to these findings, 16S rRNA data obtained in this study were combined with previously described phylogenetic data in order to identify operational taxonomic units that can be used to identify methanotrophs at the genus level.     

Molecular Analysis of Deep-Sea Hydrothermal Vent Aerobic Methanotrophs by Targeting Genes of 16S rRNA and Particulate Methane Monooxygenase

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.     

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.     

Improved Method for Detection of Methanotrophic Bacteria in Forest Soils by PCR

A primer set was designed for the specific detection of methanotrophic bacteria in forest soils by PCR. The primer sequences were derived from highly conservative regions of the pmoA gene, encoding the α-subunit of the particulate methane monooxygenase present in all methanotrophs. In control experiments with genomic DNA from a collection of different type I, II, and X methanotrophs, it could be demonstrated that the new primers were specific for members of the genera Methylosinus, Methylocystis, Methylomonas, Methylobacter, and Methylococcus. To test the suitability of the new primers for the detection of particulate methane monooxygenase (pMMO) containing methanotrophs in environmental samples we used DNA extracts from an acid spruce forest soil. For simple and rapid purification of the DNA extracts, the samples were separated by electrophoresis on a low-melting-point agarose gel. This allowed us to efficiently separate the DNA from coextracted humic acids. The DNA from the melted agarose gel was ready for use in PCR reactions. In PCR reactions with DNA from the Ah soil layer, products of the correct size were amplified by PCR by use of the new primers. By sequencing of cloned PCR products, it could be confirmed that the PCR products represented partial sequences with strong similarity to the pmoA gene. The sequence was most related to the pmoA sequence of a type II methanotroph strain isolated from the Ah layer of the investigated soils. Received: 1 September 2000 / Accepted: 2 October 2000     

Characterization of C1-Metabolizing Prokaryotic Communities in Methane Seep Habitats at the Kuroshima Knoll, Southern Ryukyu Arc, by Analyzing pmoA, mmoX, mxaF, mcrA, and 16S rRNA Genes

Samples from three submerged sites (MC, a core obtained in the methane seep area; MR, a reference core obtained at a distance from the methane seep; and HC, a gas-bubbling carbonate sample) at the Kuroshima Knoll in the southern Ryuku arc were analyzed to gain insight into the organisms present and the processes involved in this oxic-anoxic methane seep environment. 16S rRNA gene analyses by quantitative real-time PCR and clone library sequencing revealed that the MC core sediments contained abundant archaea (~34% of the total prokaryotes), including both mesophilic methanogens related to the genus Methanolobus and ANME-2 members of the Methanosarcinales, as well as members of the δ-Proteobacteria, suggesting that both anaerobic methane oxidation and methanogenesis occurred at this site. In addition, several functional genes connected with methane metabolism were analyzed by quantitative competitive-PCR, including the genes encoding particulate methane monooxygenase (pmoA), soluble methane monooxygenase (mmoX), methanol dehydrogenese (mxaF), and methyl coenzyme M reductase (mcrA). In the MC core sediments, the most abundant gene was mcrA (2.5 × 10⁶ copies/g [wet weight]), while the pmoA gene of the type I methanotrophs (5.9 × 10⁶ copies/g [wet weight]) was most abundant at the surface of the MC core. These results indicate that there is a very complex environment in which methane production, anaerobic methane oxidation, and aerobic methane oxidation all occur in close proximity. The HC carbonate site was rich in γ-Proteobacteria and had a high copy number of mxaF (7.1 × 10⁶ copies/g [wet weight]) and a much lower copy number of the pmoA gene (3.2 × 10² copies/g [wet weight]). The mmoX gene was never detected. In contrast, the reference core contained familiar sequences of marine sedimentary archaeal and bacterial groups but not groups specific to C₁ metabolism. Geochemical characterization of the amounts and isotopic composition of pore water methane and sulfate strongly supported the notion that in this zone both aerobic methane oxidation and anaerobic methane oxidation, as well as methanogenesis, occur.     

Activity and diversity of methanotrophs in the soil–water interface and rhizospheric soil from a flooded temperate rice field

Aims:  To combine molecular and cultivation techniques to characterize the methanotrophic community in the soil–water interface (SWI) and rhizospheric soil from flooded rice fields in Uruguay, a temperate region in South America.
Methods and Results:  A novel type I, related to the genus Methylococcus , and three type II methanotrophs were isolated from the highest positive dilution steps from the most probable number (MPN) counts. Potential methane oxidation activities measured in slurried samples were higher in the rhizospheric soil compared to the SWI and were stimulated by N-fertilization. PmoA (particulate methane monooxygenase) clone libraries were constructed for both rice microsites. SWI clones clustered in six groups related to cultivated and uncultivated members from different ecosystems of the genera Methylobacter , Methylomonas , Methylococcus and a novel type I sublineage while cultivation and T-RFLP (terminal restriction fragment length polymorphism) analysis confirmed the presence of type II methanotrophs.
Conclusions:  Cultivation techniques, cloning analysis and T-RFLP fingerprinting of the pmoA gene revealed a diverse methanotrophic community in the rice rhizospheric soil and SWI.
Significance and Impact of the Study:  This study reports, for the first time, the analysis of the methanotrophic diversity in rice SWI and this diversity may be exploited in reducing methane emissions.     

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.     

Diversity of the particulate methane monooxygenase gene in methanotrophic samples from different rice field soils in China and the Philippines

Methanotrophic bacteria play a crucial role in regulating the emission of CH4 from rice fields into the atmosphere. We investigated the CH4 oxidation activity together with the diversity of methanotrophic bacteria in ten rice field soils from different geographic locations. Upon incubation of aerated soil slurries under 7% CH4, rates of CH4 oxidation increased after a lag phase of 1-4 days and reached values of 3-10 micromol d(-1) g-dw(-1) soil. The methanotrophic community was assayed by retrieval of the pmoA gene which encodes the a subunit of the particulate methane monooxygenase. After extraction of DNA from actively CH4-oxidizing soil samples and PCR-amplification of the pmoA, the community was analyzed by Denaturant Gradient Gel Electrophoresis (DGGE) and Terminal Restriction Fragment Length Polymorphism (T-RFLP). DGGE bands were excised, the pmoA re-amplified, sequenced and the encoded amino acid sequence comparatively analyzed by phylogenetic treeing. The analyses allowed the detection of pmoA sequences related to the following methanotrophic genera: the type-I methanotrophs Methylobacter, Methylomicrobium, Methylococcus and Methylocaldum, and the type-II methanotrophs Methylocystis and Methylosinus. T-RFLP analysis detected a similar diversity, but type-II pmoA more frequently than DGGE. All soils but one contained type-II in addition to type-I methanotrophs. Type-I Methylomonas was not detected at all. Different combinations of methanotrophic genera were detected in the different soils. However, there was no obvious geographic pattern of the distribution of methanotrophs.     

Analysis of Methane Monooxygenase Genes in Mono Lake Suggests That Increased Methane Oxidation Activity May Correlate with a Change in Methanotroph Community Structure

Mono Lake is an alkaline hypersaline lake that supports high methane oxidation rates. Retrieved pmoA sequences showed a broad diversity of aerobic methane oxidizers including the type I methanotrophs Methylobacter (the dominant genus), Methylomicrobium, and Methylothermus, and the type II methanotroph Methylocystis. Stratification of Mono Lake resulted in variation of aerobic methane oxidation rates with depth. Methanotroph diversity as determined by analysis of pmoA using new denaturing gradient gel electrophoresis primers suggested that variations in methane oxidation activity may correlate with changes in methanotroph community composition.     

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.