Molecular characterization of methanotrophic isolates from freshwater lake sediment

Profiles of dissolved O(2) and methane with increasing depth were generated for Lake Washington sediment, which suggested the zone of methane oxidation is limited to the top 0.8 cm of the sediment. Methane oxidation potentials were measured for 0.5-cm layers down to 1.5 cm and found to be relatively constant at 270 to 350 micromol/liter of sediment/h. Approximately 65% of the methane was oxidized to cell material or metabolites, a signature suggestive of type I methanotrophs. Eleven methanotroph strains were isolated from the lake sediment and analyzed. Five of these strains classed as type I, while six were classed as type II strains by 16S rRNA gene sequence analysis. Southern hybridization analysis with oligonucleotide probes detected, on average, one to two copies of pmoA and one to three copies of 16S rRNA genes. Only one restriction length polymorphism pattern was shown for pmoA genes in each isolate, and in cases where, sequencing was done, the pmoA copies were found to be almost identical. PCR primers were developed for mmoX which amplified 1.2-kb regions from all six strains that tested positive for cytoplasmic soluble methane mono-oxygenase (sMMO) activity. Phylogenetic analysis of the translated PCR products with published mmoX sequences showed that MmoX falls into two distinct clusters, one containing the orthologs from type I strains and another containing the orthologs from type II strains. The presence of sMMO-containing Methylomonas strains in a pristine freshwater lake environment suggests that these methanotrophs are more widespread than has been previously thought.     

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.     

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.     

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.     

Shifts in identity and activity of methanotrophs in arctic lake sediments in response to temperature changes

Methane (CH(4)) flux to the atmosphere is mitigated via microbial CH(4) oxidation in sediments and water. As arctic temperatures increase, understanding the effects of temperature on the activity and identity of methanotrophs in arctic lake sediments is important to predicting future CH(4) emissions. We used DNA-based stable-isotope probing (SIP), quantitative PCR (Q-PCR), and pyrosequencing analyses to identify and characterize methanotrophic communities active at a range of temperatures (4°C, 10°C, and 21°C) in sediments (to a depth of 25 cm) sampled from Lake Qalluuraq on the North Slope of Alaska. CH(4) oxidation activity was measured in microcosm incubations containing sediments at all temperatures, with the highest CH(4) oxidation potential of 37.5 μmol g(-1) day(-1) in the uppermost (depth, 0 to 1 cm) sediment at 21°C after 2 to 5 days of incubation. Q-PCR of pmoA and of the 16S rRNA genes of type I and type II methanotrophs, and pyrosequencing of 16S rRNA genes in (13)C-labeled DNA obtained by SIP demonstrated that the type I methanotrophs Methylobacter, Methylomonas, and Methylosoma dominated carbon acquisition from CH(4) in the sediments. The identity and relative abundance of active methanotrophs differed with the incubation temperature. Methylotrophs were also abundant in the microbial community that derived carbon from CH(4), especially in the deeper sediments (depth, 15 to 20 cm) at low temperatures (4°C and 10°C), and showed a good linear relationship (R = 0.82) with the relative abundances of methanotrophs in pyrosequencing reads. This study describes for the first time how methanotrophic communities in arctic lake sediments respond to temperature variations.     

Microbial diversity in sediments associated with a shallow methane seep in the tropical Timor Sea of Australia reveals a novel aerobic methanotroph diversity

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 α-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.     

A novel pmoA lineage represented by the acidophilic methanotrophic bacterium Methylocapsa acidophila B2

A fragment of the functional gene pmoA, which encodes the active-site polypeptide of particulate methane monooxygenase (pMMO), was PCR-amplified from DNA of the recently described acidophilic methanotrophic bacterium Methylocapsa acidiphila [corrected] B2. This methanotroph was isolated from an acidic Sphagnum peat bog and possesses a novel type III arrangement of intracytoplasmic membranes. Comparative sequence analysis revealed that the inferred peptide sequence of pmoA of Methylocapsa acidiphila [corrected] B2 belongs to a novel PmoA lineage. This lineage was only distantly related to the PmoA sequence cluster of type II methanotrophs, with identity values between 69.5% and 72%. The identity values between the PmoA of Methylocapsa acidiphila [corrected] B2 and PmoA sequences of type I methanotrophs ranged from 55.5 to 68%. However, the PmoA of this acidophilic methanotroph was more closely affiliated with the inferred peptide sequences of pmoA clones retrieved from various acidic upland soils showing atmospheric methane consumption. The PmoA identity values with these clones were 79.5-81%. Nonetheless, the apparent affinity for methane exhibited by Methylocapsa acidiphila [corrected] B2 was 1-2 microM, which is similar to values measured in other methanotrophic bacteria. This finding suggests that the pMMO of Methylocapsa acidiphila [corrected] B2 is not a novel enzyme specialized to have a high affinity for methane and that apparent "high-affinity" methane uptake is either the result of particular culture conditions or is performed by another pMMO type.     

Diversity and activity of methanotrophs in alkaline soil from a Chinese coal mine

Culture-independent molecular biological techniques, including 16S rRNA gene and functional gene clone libraries and microarray analyses using pmoA (encoding a key subunit of particulate methane monooxygenase), were applied to investigate the methanotroph community structure in alkaline soil from a Chinese coal mine. This environment contained a high diversity of methanotrophs, including the type II methanotrophs Methylosinus / Methylocystis , type I methanotrophs related to Methylobacter / Methylosoma and Methylococcus , and a number of as yet uncultivated methanotrophs. In order to identify the metabolically active methane-oxidizing bacteria from this alkaline environment, DNA stable isotope probing (DNA-SIP) experiments using ¹³CH₄ were carried out. This showed that both type I and type II methanotrophs were active, together with methanotrophs related to Methylocella , which had previously been found only in acidic environments. Methylotrophs, including Methylopila and Hyphomicrobium , were also detected in soil DNA and after DNA-SIP experiments. DNA sequence information on the most abundant, active methanotrophs in this alkaline soil will facilitate the design of oligonucleotide probes to monitor enrichment cultures when isolating key alkaliphilic methanotrophs from such environments.     

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 α 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.     

Nutrient amendments in soil DNA stable isotope probing experiments reduce the observed methanotroph diversity

Stable isotope probing (SIP) can be used to analyze the active bacterial populations involved in a process by incorporating 13C-labeled substrate into cellular components such as DNA. Relatively long incubation times are often used with laboratory microcosms in order to incorporate sufficient 13C into the DNA of the target organisms. Addition of nutrients can be used to accelerate the processes. However, unnatural concentrations of nutrients may artificially change bacterial diversity and activity. In this study, methanotroph activity and diversity in soil was examined during the consumption of 13CH4 with three DNA-SIP experiments, using microcosms with natural field soil water conditions, the addition of water, and the addition of mineral salts solution. Methanotroph population diversity was studied by targeting 16S rRNA and pmoA genes. Clone library analyses, denaturing gradient gel electrophoresis fingerprinting, and pmoA microarray hybridization analyses were carried out. Most methanotroph diversity (type I and type II methanotrophs) was observed in non-amended SIP microcosms. Although this treatment probably best reflected the in situ environmental conditions, one major disadvantage of this incubation was that the incorporation of 13CH4 was slow and some cross-feeding of 13C occurred, thereby leading to labeling of nonmethanotroph microorganisms. Conversely, microcosms supplemented with mineral salts medium exhibited rapid consumption of 13CH4, resulting in the labeling of a less diverse population of only type I methanotrophs. DNA-SIP incubations using water-amended microcosms yielded faster incorporation of 13C into active methanotrophs while avoiding the cross-feeding of 13C.     

Identification of functionally active aerobic methanotrophs in sediments from an arctic lake using stable isotope probing

Arctic lakes are a significant source of the greenhouse gas methane (CH₄), but the role that methane oxidizing bacteria (methanotrophs) play in limiting the overall CH₄ flux is poorly understood. Here, we used stable isotope probing (SIP) techniques to identify the metabolically active aerobic methanotrophs in upper sediments (0–1 cm) from an arctic lake in northern Alaska sampled during ice‐free summer conditions. The highest CH₄ oxidation potential was observed in the upper sediment (0–1 cm depth) with 1.59 µmol g wet weight^?1 day^?1 compared with the deeper sediment samples (1–3 cm, 3–5 cm and 5–10 cm), which exhibited CH₄ oxidation potentials below 0.4 µmol g wet weight^?1 day^?1. Both type I and type II methanotrophs were directly detected in the upper sediment total communities using targeted primer sets based on 16S rRNA genes. Sequencing of 16S rRNA genes and functional genes (pmoA and mxaF) in the ¹³C‐DNA from the upper sediment indicated that type I methanotrophs, mainly Methylobacter, Methylosoma, Methylomonas and Methylovulum miyakonense, dominated the assimilation of CH₄. Methylotrophs, including the genera Methylophilus and/or Methylotenera, were also abundant in the ¹³C‐DNA. Our results show that a diverse microbial consortium acquired carbon from CH₄ in the sediments of this arctic lake.     

Study of nucleotide sequences of nifH genes in methanotrophic bacteria

Using a previously developed primer system, nifH gene fragments 450 nucleotides long were amplified, cloned, and sequenced for representatives of nitrogen-fixing methanotrophic bacteria of the genera Methylococcus, Methylocystis and Methylosinus. Fragments of nifH genes were also detected and sequenced in representatives of the genera Methylomonas and Methylobacter, which were not considered diazotrophs until recently. Phylogenetic analysis revealed remoteness of nifH genes sequences of methanotroph types I and II. At the same time, close relationship was found between nifH of type I methanotrophs and representatives of gamma-proteobacteria and between nifH genes of type II methanotrophs and representatives of alpha-proteobacteria. The results obtained in this study are in good accordance with the data of phylogenetic analysis based on 16S rRNA sequence comparison with the only exception of Methylococcus capsulatus strains, whose nifH genes proved to be closely related to nifH genes of Methylocystis and Methylosinus representatives. Our findings extend the database of primary sequences of nifH genes and allow the contribution of methanotrophs to the process of nitrogen fixation to be estimated.     

The particulate methane monooxygenase gene pmoA and its use as a functional gene probe for methanotrophs

The particulate methane monooxygenase gene pmoA, encoding the 27 kDa polypeptide of the membrane-bound particulate methane monooxygenase, was amplified by PCR from DNA isolated from a blanket peat bog and from enrichment cultures established, from the same environment, using methane as sole carbon and energy source. The resulting 525 bp PCR products were cloned and a representative number of clones were sequenced. Phylogenetic analysis of the derived amino acid sequences of the pmoA clones retrieved directly from environmental DNA samples revealed that they form a distinct cluster within representative PmoA sequences from type II methanotrophs and may originate from a novel group of acidophilic methanotrophs. The study also demonstrated the utility of the pmoA gene as a phylogenetic marker for identifying methanotroph-specific DNA sequences in the environment.     

Fluorescent oligonucleotide rDNA probes for specific detection of methane oxidising bacteria

Oligonucleotide probes targeting the 16S rRNA of distinct phylogenetic groups of methanotrophs were designed for the in situ detection of these organisms. A probe, MG-64, detected specifically type I methanotrophs, while probes MA-221 and MA-621, detected type II methanotrophs in whole cell hybridisations. A probe Mc1029 was also designed which targeted only organisms from the Methylococcus genus after whole cell hybridisations. All probes were labelled with the fluorochrome Cy3 and optimum conditions for hybridisation were determined. Non-specific target sites of the type I (MG-64) and type II (MA-621) probes to non-methanotrophic organisms are highlighted. The probes are however used in studying enrichment cultures and environments where selective pressure favours the growth of methanotrophs over other organisms. The application of these probes was demonstrated in the detection of type I methanotrophs with the MG-64 probe in an enrichment culture from an estuarine sample demonstrating methane oxidation. The detection of type I methanotrophs was confirmed by a 16S rDNA molecular analysis of the estuarine enrichment culture which demonstrated that the most abundant bacterial clone type in the 16S rDNA library was most closely related to Methylobacter sp. strain BB5.1, a type I methanotroph also isolated from an estuarine environment.     

Sources for sedimentary bacteriohopanepolyols as revealed by 16S rDNA stratigraphy

Bacteriohopanoids are widespread lipid biomarkers in the sedimentary record. Many aerobic and anaerobic bacteria are potential sources of these lipids which sometimes complicates the use of these biomarkers as proxies for ecological and environmental changes. Therefore, we applied preserved 16S ribosomal RNA genes to identify likely Holocene biological sources of bacteriohopanepolyols (BHPs) in the sulfidic sediments of the permanently stratified postglacial Ace Lake, Antarctica. A suite of intact BHPs were identified, which revealed a variety of structural forms whose composition differed through the sediment core reflecting changes in bacterial populations induced by large changes in lake salinity. Stable isotopic compositions of the hopanols formed from periodic acid-cleaved BHPs, showed that some were substantially depleted in ¹³C, indicative of their methanotrophic origin. Using sensitive molecular tools, we found that Type I and II methanotrophic bacteria (respectively Methylomonas and Methylocystis ) were unique to the oldest lacustrine sediments (> 9400 years BP), but quantification of fossil DNA revealed that the Type I methanotrophs, including methanotrophs related to methanotrophic gill symbionts of deep-sea cold-seep mussels, were the main precursors of the 35-amino BHPs (i.e. aminopentol, -tetrol and -triols). After isolation of the lake ∼3000 years ago, one Type I methanotroph of the 'methanotrophic gill symbionts cluster' remained the most obvious source of aminotetrol and -triol. We, furthermore, identified a Synechococcus phylotype related to pelagic freshwater strains in the oldest lacustrine sediments as a putative source of 2-methylbacteriohopanetetrol (2-Me BHT). This combined application of advanced geochemical and paleogenomical tools further refined our knowledge about Holocene biogeochemical processes in Ace Lake.     

Identity of active methanotrophs in landfill cover soil as revealed by DNA-stable isotope probing

A considerable amount of methane produced during decomposition of landfill waste can be oxidized in landfill cover soil by methane-oxidizing bacteria (methanotrophs) thus reducing greenhouse gas emissions to the atmosphere. The identity of active methanotrophs in Roscommon landfill cover soil, a slightly acidic peat soil, was assessed by DNA-stable isotope probing (SIP). Landfill cover soil slurries were incubated with (13)C-labelled methane and under either nutrient-rich nitrate mineral salt medium or water. The identity of active methanotrophs was revealed by analysis of (13)C-labelled DNA fractions. The diversity of functional genes (pmoA and mmoX) and 16S rRNA genes was analyzed using clone libraries, microarrays and denaturing gradient gel electrophoresis. 16S rRNA gene analysis revealed that the cover soil was mainly dominated by Type II methanotrophs closely related to the genera Methylocella and Methylocapsa and to Methylocystis species. These results were supported by analysis of mmoX genes in (13)C-DNA. Analysis of pmoA gene diversity indicated that a significant proportion of active bacteria were also closely related to the Type I methanotrophs, Methylobacter and Methylomonas species. Environmental conditions in the slightly acidic peat soil from Roscommon landfill cover allow establishment of both Type I and Type II methanotrophs.