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.     

Identification of a complete methane monooxygenase operon from soil by combining stable isotope probing and metagenomic analysis

Stable isotope probing (SIP) allows the isolation of nucleic acids from targeted metabolically active organisms in environmental samples. In previous studies, DNA-SIP has been performed with the one-carbon growth substrates methane and methanol to study methylotrophic organisms. The methylotrophs that incorporated the labelled substrate were identified with polymerase chain reaction and sequencing of 16S rRNA and 'functional genes' for methanotrophs (mxaF, pmoA, mmoX). In this study, a SIP experiment was performed using a forest soil sample incubated with (13)CH(4), and the (13)C-DNA was purified and cloned into a bacterial artificial chromosome (BAC) plasmid. A library of 2300 clones was generated and most of the clones contained inserts between 10 and 30 kb. The library was probed for key methylotrophy genes and a 15.2 kb clone containing a pmoCAB operon, encoding particulate methane monooxygenase, was identified and sequenced. Analysis of the pmoA sequence suggested that the clone was most similar to that of a Methylocystis sp. previously detected in this forest soil. Twelve other open reading frames were identified on the clone, including the gene encoding beta-ribofuranosylaminobenzene 5'-phosphate synthase, which is involved in the biosynthesis of the 'archaeal' C(1)-carrier, tetrahydromethanopterin, which is also found in methylotrophs. This study demonstrates that relatively large DNA fragments from uncultivated organisms can be readily isolated using DNA-SIP, and cloned into a vector for metagenomic analysis.     

The soluble methane monooxygenase gene cluster of the trichloroethylene-degrading methanotroph Methylocystis sp. strain M.

In methanotrophic bacteria, methane is oxidized to methanol by the enzyme methane monooxygenase (MMO). The soluble MMO enzyme complex from Methylocystis sp. strain M also oxidizes a wide range of aliphatic and aromatic compounds, including trichloroethylene. In this study, heterologous DNA probes from the type II methanotroph Methylosinus trichosporium OB3b were used to isolate souble MMO (sMMO) genes from the type II methanotroph Methylocystis sp. strain M. sMMO genes from strain M are clustered on the chromosome and show a high degree of identity with the corresponding genes from Methylosinus trichosporium OB3b. Sequencing and phylogenetic analysis of the 16S rRNA gene from Methylocystis sp. strain M have confirmed that it is most closely related to the type II methanotroph Methylocystis parvus OBBP, which, unlike Methylocystis sp. strain M, does not possess an sMMO. A similar phylogenetic analysis using the pmoA gene, which encodes the 27-kDa polypeptide of the particulate MMO, also places Methylocystis sp. strain M firmly in the genus Methylocystis. This is the first report of isolation and characterization of methane oxidation genes from methanotrophs of the genus Methylocystis.     

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.     

Marine methylotrophs revealed by stable-isotope probing, multiple displacement amplification and metagenomics

The concentrations of one-carbon substrates that fuel methylotrophic microbial communities in the ocean are limited and the specialized guilds of bacteria that use these molecules may exist at low relative abundance. As a result, these organisms are difficult to identify and are often missed with existing cultivation and gene retrieval methods. Here, we demonstrate a novel proof of concept: using environmentally-relevant substrate concentrations in stable-isotope probing (SIP) incubations to yield sufficient DNA for large-insert metagenomic analysis through multiple displacement amplification (MDA). A marine surface-water sample was labelled sufficiently by incubation with near in situ concentrations of methanol. Picogram quantities of labelled (13)C-DNA were purified from caesium chloride gradients, amplified with MDA to produce microgram amounts of high-molecular-weight DNA ( 10 000 clones. Denaturing gradient gel electrophoresis (DGGE) demonstrated minimal bias associated with the MDA step and implicated Methylophaga-like phylotypes with the marine metabolism of methanol. Polymerase chain reaction screening of 1500 clones revealed a methanol dehydrogenase (MDH) containing insert and shotgun sequencing of this insert resulted in the assembly of a 9-kb fragment of DNA encoding a cluster of enzymes involved in MDH biosynthesis, regulation and assembly. This novel combination of methodology enables future structure-function studies of microbial communities to achieve the long-desired goal of identifying active microbial populations using in situ conditions and performing a directed metagenomic analysis for these ecologically relevant microorganisms.     

Detection, isolation, and characterization of acidophilic methanotrophs from Sphagnum mosses

Sphagnum peatlands are important ecosystems in the methane cycle. Methane-oxidizing bacteria in these ecosystems serve as a methane filter and limit methane emissions. Yet little is known about the diversity and identity of the methanotrophs present in and on Sphagnum mosses of peatlands, and only a few isolates are known. The methanotrophic community in Sphagnum mosses, originating from a Dutch peat bog, was investigated using a pmoA microarray. A high biodiversity of both gamma- and alphaproteobacterial methanotrophs was found. With Sphagnum mosses as the inoculum, alpha- and gammaproteobacterial acidophilic methanotrophs were isolated using established and newly designed media. The 16S rRNA, pmoA, pxmA, and mmoX gene sequences showed that the alphaproteobacterial isolates belonged to the Methylocystis and Methylosinus genera. The Methylosinus species isolated are the first acid-tolerant members of this genus. Of the acidophilic gammaproteobacterial strains isolated, strain M5 was affiliated with the Methylomonas genus, and the other strain, M200, may represent a novel genus, most closely related to the genera Methylosoma and Methylovulum. So far, no acidophilic or acid-tolerant methanotrophs in the Gammaproteobacteria class are known. All strains showed the typical features of either type I or II methanotrophs and are, to the best of our knowledge, the first isolated (acidophilic or acid-tolerant) methanotrophs from Sphagnum mosses.     

Identification of the functionally active methanotroph population in a peat soil microcosm by stable-isotope probing

The active population of low-affinity methanotrophs in a peat soil microcosm was characterized by stable-isotope probing. "Heavy" (13)C-labeled DNA, produced after microbial growth on (13)CH(4), was separated from naturally abundant (12)C-DNA by cesium chloride density gradient centrifugation and used as a template for the PCR. Amplification products of 16S rRNA genes and pmoA, mxaF, and mmoX, which encode key enzymes in the CH(4) oxidation pathway, were analyzed. Sequences related to extant type I and type II methanotrophs were identified, indicating that these methanotrophs were active in peat exposed to 8% (vol/vol) CH(4). The (13)C-DNA libraries also contained clones that were related to beta-subclass Proteobacteria, suggesting that novel groups of bacteria may also be involved in CH(4) cycling in this soil.     

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.     

Identification and quantification of uncultivated Proteobacteria associated with pyrene degradation in a bioreactor treating PAH-contaminated soil

Uncultivated bacteria associated with the degradation of pyrene in a bioreactor treating soil contaminated with polycyclic aromatic hydrocarbons (PAH) were identified by DNA-based stable-isotope probing (SIP) and quantified by real-time quantitative PCR. Most of the 16S rRNA gene sequences recovered from (13)C-enriched DNA fractions clustered phylogenetically within three separate groups of beta- and gamma-Proteobacteria unassociated with described genera and were designated "Pyrene Groups 1, 2 and 3". One recovered sequence was associated with the Sphingomonas genus. Pyrene Groups 1 and 3 were present in very low numbers in the bioreactor but represented 75% and 7%, respectively, of the sequences recovered from 16S rRNA gene clone libraries constructed from (13)C-enriched DNA. In a parallel time-course incubation with unlabelled pyrene, there was between a 2- and 4-order-of-magnitude increase in the abundance of 16S rRNA genes from Pyrene groups 1 and 3 and from targeted Sphingomonas spp. over a 10 day incubation. Sequences from Pyrene Group 2 were 11% of the SIP clone libraries but accounted for 14% of the total bacterial 16S rRNA genes in the bioreactor community. However, the abundance of this group did not increase significantly in response to pyrene disappearance. These data indicate that the primary pyrene degraders in the bioreactor were uncultivated, low-abundance beta- and gamma-Proteobacteria not previously associated with pyrene degradation.     

Identification of the bacterial community involved in methane-dependent denitrification in activated sludge using DNA stable-isotope probing

Methane is used as an alternative carbon source in the denitrification of wastewater lacking organic carbon sources because it is nontoxic and may be efficiently produced by anaerobic biological processes. Methane-dependent denitrification (MDD) in the presence of oxygen requires the co-occurrence of methanotrophy and denitrification. Activated sludge was incubated with 13C-labeled methane in either a nitrate-containing medium or a nitrate-free medium. Then, bacterial and methanotrophic populations were analyzed by cloning analysis and terminal restriction fragment length polymorphism analysis targeting 16S rRNA gene and cloning analysis targeting pmoA genes. DNA-based stable-isotope probing (DNA-SIP) analysis of the 16S rRNA gene revealed an association of the Methylococcaceae and the Hyphomicrobiaceae in a MDD ecosystem. Furthermore, supplementation of nitrate stimulated methane consumption and the activity of methanotrophic populations (i.e. the stimulation of uncultivated relatives of distinct groups of the Methylococcaceae). In particular, uncultured type-X methanotrophs of Gammaproteobacteria were dominant when nitrate was added, i.e. in the MDD incubations. On the other hand, most methanotrophs (types I, II, and X methanotrophs) were found to have been labeled with 13C under nitrate-free conditions. This DNA-SIP study identifies key bacterial populations involved in a MDD ecosystem.     

Diversity of soluble methane monooxygenase-containing methanotrophs isolated from polluted environments

Methanotrophs were enriched and isolated from polluted environments in Canada and Germany. Enrichments in low copper media were designed to specifically encourage growth of soluble methane monooxygenase (sMMO) containing organisms. The 10 isolates were characterized physiologically and genetically with one type I and nine type II methanotrophs being identified. Three key genes: 16S rRNA; pmoA and mmoX, encoding for the particulate and soluble methane monooxygenases respectively, were cloned from the isolates and sequenced. Phylogenetic analysis of these sequences identified strains, which were closely related to Methylococcus capsulatus, Methylocystis sp., Methylosinus sporium and Methylosinus trichosporium. Diversity of sMMO-containing methanotrophs detected in this and previous studies was rather narrow, both genetically and physiologically, suggesting possible constraints on genetic diversity of sMMO due to essential conservation of enzyme function.     

Molecular diversity of methanotrophs in Transbaikal soda lake sediments and identification of potentially active populations by stable isotope probing

Soda lakes are an environment with an unusually high pH and often high salinity. To identify the active methanotrophs in the Soda lake sediments, sediment slurries were incubated with a 10% (v/v) (13)CH(4) headspace and the (13)C-labelled DNA was subsequently extracted from these sediments following CsCl density gradient centrifugation. This DNA was then used as a template for PCR amplification of 16S rRNA genes and genes encoding PmoA and MmoX of methane monooxygenase, key enzymes in the methane oxidation pathway. Phylogenetic analysis of 16S rRNA genes, PmoA and MmoX identified that strains of Methylomicrobium, Methylobacter, Methylomonas and 'Methylothermus' had assimilated the (13)CH(4). Phylogenetic analysis of PmoA sequences amplified from DNA extracted from Soda lake sediments before Stable Isotope Probing (SIP) treatment showed that a much wider diversity of both type I and type II methanotroph sequences are present in this alkaline environment. The majority of methanotroph sequences detected in the (13)C-DNA studies were from type I methanotrophs, with 50% of 16S rRNA clones and 100% of pmoA clones from both Lake Suduntuiskii Torom and Lake Gorbunka suggesting that the type I methanotrophs are probably responsible for the majority of methane oxidation in this environment.     

Diversity of oxygenase genes from methane- and ammonia-oxidizing bacteria in the Eastern Snake River Plain aquifer

PCR amplification, restriction fragment length polymorphism, and phylogenetic analysis of oxygenase genes were used for the characterization of in situ methane- and ammonia-oxidizing bacteria from free-living and attached communities in the Eastern Snake River Plain aquifer. The following three methane monooxygenase (MMO) PCR primer sets were used: A189-A682, which amplifies an internal region of both the pmoA gene of the MMO particulate form and the amoA gene of ammonia monooxygenase; A189-mb661, which specifically targets the pmoA gene; and mmoXA-mmoXB, which amplifies the mmoX gene of the MMO soluble form (sMMO). Whole-genome amplification (WGA) was used to amplify metagenomic DNA from each community to assess its applicability for generating unbiased metagenomic template DNA. The majority of sequences in each archive were related to oxygenases of type II-like methanotrophs of the genus Methylocystis. A small subset of type I sequences found only in free-living communities possessed oxygenase genes that grouped nearest to Methylobacter and Methylomonas spp. Sequences similar to that of the amoA gene associated with ammonia-oxidizing bacteria (AOB) most closely matched a sequence from the uncultured bacterium BS870 but showed no substantial alignment to known cultured AOB. Based on these functional gene analyses, bacteria related to the type II methanotroph Methylocystis sp. were found to dominate both free-living and attached communities. Metagenomic DNA amplified by WGA showed characteristics similar to those of unamplified samples. Overall, numerous sMMO-like gene sequences that have been previously associated with high rates of trichloroethylene cometabolism were observed in both free-living and attached communities in this basaltic aquifer.     

Methane assimilation and trophic interactions with marine Methylomicrobium in deep-water coral reef sediment off the coast of Norway

Deep-water coral reefs are seafloor environments with diverse biological communities surrounded by cold permanent darkness. Sources of energy and carbon for the nourishment of these reefs are presently unclear. We investigated one aspect of the food web using DNA stable-isotope probing (DNA-SIP). Sediment from beneath a Lophelia pertusa reef off the coast of Norway was incubated until assimilation of 5 micromol 13CH4 g(-1) wet weight occurred. Extracted DNA was separated into 'light' and 'heavy' fractions for analysis of labelling. Bacterial community fingerprinting of PCR-amplified 16S rRNA gene fragments revealed two predominant 13C-specific bands. Sequencing of these bands indicated that carbon from 13CH4 had been assimilated by a Methylomicrobium and an uncultivated member of the Gammaproteobacteria. Cloning and sequencing of 16S rRNA genes from the heavy DNA, in addition to genes encoding particulate methane monooxygenase and methanol dehydrogenase, all linked Methylomicrobium with methane metabolism. Putative cross-feeders were affiliated with Methylophaga (Gammaproteobacteria), Hyphomicrobium (Alphaproteobacteria) and previously unrecognized methylotrophs of the Gammaproteobacteria, Alphaproteobacteria, Deferribacteres and Bacteroidetes. This first marine methane SIP study provides evidence for the presence of methylotrophs that participate in sediment food webs associated with deep-water coral reefs.     

Methanotroph diversity in landfill soil: isolation of novel type I and type II methanotrophs whose presence was suggested by culture-independent 16S ribosomal DNA analysis.

The diversity of the methanotrophic community in mildly acidic landfill cover soil was assessed by three methods: two culture-independent molecular approaches and a traditional culture-based approach. For the first of the molecular studies, two primer pairs specific for the 16S rRNA gene of validly published type I (including the former type X) and type II methanotrophs were identified and tested. These primers were used to amplify directly extracted soil DNA, and the products were used to construct type I and type II clone libraries. The second molecular approach, based on denaturing gradient gel electrophoresis (DGGE), provided profiles of the methanotrophic community members as distinguished by sequence differences in variable region 3 of the 16S ribosomal DNA. For the culturing studies, an extinction-dilution technique was employed to isolate slow-growing but numerically dominant strains. The key variables of the series of enrichment conditions were initial pH (4. 8 versus 6.8), air/CH(4)/CO(2) headspace ratio (50:45:5 versus 90:9:1), and concentration of the medium (1x nitrate minimal salts [NMS] versus 0.2x NMS). Screening of the isolates showed that the nutrient-rich 1x NMS selected for type I methanotrophs, while the nutrient-poor 0.2x NMS tended to enrich for type II methanotrophs. Partial sequencing of the 16S rRNA gene from selected clones and isolates revealed some of the same novel sequence types. Phylogenetic analysis of the type I clone library suggested the presence of a new phylotype related to the Methylobacter-Methylomicrobium group, and this was confirmed by isolating two members of this cluster. The type II clone library also suggested the existence of a novel group of related species distinct from the validly published Methylosinus and Methylocystis genera, and two members of this cluster were also successfully cultured. Partial sequencing of the pmoA gene, which codes for the 27-kDa polypeptide of the particulate methane monooxygenase, reaffirmed the phylogenetic placement of the four isolates. Finally, not all of the bands separated by DGGE could be accounted for by the clones and isolates. This polyphasic assessment of community structure demonstrates that much diversity among the obligate methane oxidizers has yet to be formally described.     

Detection and enumeration of methanotrophs in acidic Sphagnum peat by 16S rRNA fluorescence in situ hybridization, including the use of newly developed oligonucleotide probes for Methylocella palustris

Two 16S rRNA-targeted oligonucleotide probes, Mcell-1026 and Mcell-181, were developed for specific detection of the acidophilic methanotroph Methylocella palustris using fluorescence in situ hybridization (FISH). The fluorescence signal of probe Mcell-181 was enhanced by its combined application with the oligonucleotide helper probe H158. Mcell-1026 and Mcell-181, as well as 16S rRNA oligonucleotide probes with reported group specificity for either type I methanotrophs (probes M-84 and M-705) or the Methylosinus/Methylocystis group of type II methanotrophs (probes MA-221 and M-450), were used in FISH to determine the abundance of distinct methanotroph groups in a Sphagnum peat sample of pH 4.2. M. palustris was enumerated at greater than 10(6) cells per g of peat (wet weight), while the detectable population size of type I methanotrophs was three orders of magnitude below the population level of M. palustris. The cell counts with probe MA-221 suggested that only 10(4) type II methanotrophs per g of peat (wet weight) were present, while the use of probe M-450 revealed more than 10(6) type II methanotroph cells per g of the same samples. This discrepancy was due to the fact that probe M-450 targets almost all currently known strains of Methylosinus and Methylocystis, whereas probe MA-221, originally described as group specific, does not detect a large proportion of Methylocystis strains. The total number of methanotrophic bacteria detected by FISH was 3.0 (+/-0.2) x 10(6) cells per g (wet weight) of peat. This was about 0.8% of the total bacterial cell number. Thus, our study clearly suggests that M. palustris and a defined population of Methylocystis spp. were the predominant methanotrophs detectable by FISH in an acidic Sphagnum peat bog.