A Study of Nucleotide Sequences of nifH Genes of Some 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 the remoteness of nifH gene sequences of methanotroph types I and II. At the same time, a close relationship was found between nifH of type I methanotrophs and representatives of -proteobacteria and between nifH genes of type II methanotrophs and representatives of -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 being 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.     

Comparative characterization of cultured methane-oxidizing bacteria by serological and molecular methods

Three stable methane-oxidizing enrichment cultures, SB26, SB31, and SB31A were analyzed by transmission electron microscopy and by serological and molecular techniques. Electron microscopy revealed the presence of both type I and type II methanotrophs in SB31 and SB31A enrichments; only type II methanotrophs were found in SB26 enrichment. Methylosinus trichosporium was detected in all three enrichments by the application of species-specific antibodies. Additionally, Methylocystis echinoides was found in SB26 culture; Methylococcus capsulatus, in SB31 and SB31A; and Methylomonas methanica, in SB31. The analysis with pmoA and nifH gene sequences as phylogenetic markers revealed the presence of Methylosinus/Methylocystis group in all communities. Moreover, the analysis of pmoA sequences revealed the presence of Methylomonas in SB31. Methylocella was detected in SB31 and SB31A enrichments only by nifH analysis. It was concluded that the simultaneous application of different approaches reveals more reliable information on the diversity of methanotrophs.     

nifH sequences and nitrogen fixation in type I and type II methanotrophs

Some methane-oxidizing bacteria (methanotrophs) are known to be capable of expressing nitrogenase and utilizing N2 as a nitrogen source. However, no sequences are available for nif genes in these strains, and the known nitrogen-fixing methanotrophs are confined mainly to a few genera. The purpose of this work was to assess the nitrogen-fixing capabilities of a variety of methanotroph strains. nifH gene fragments from four type I methanotrophs and seven type II methanotrophs were PCR amplified and sequenced. Nitrogenase activity was confirmed in selected type I and type II strains by acetylene reduction. Activities ranged from 0.4 to 3.3 nmol/min/mg of protein. Sequence analysis shows that the nifH sequences from the type I and type II strains cluster with nifH sequences from other gamma proteobacteria and alpha proteobacteria, respectively. The translated nifH sequences from three Methylomonas strains show high identity (95 to 99%) to several published translated environmental nifH sequences PCR amplified from rice roots and a freshwater lake. The translated nifH sequences from the type II strains show high identity (94 to 99%) to published translated nifH sequences from a variety of environments, including rice roots, a freshwater lake, an oligotrophic ocean, and forest soil. These results provide evidence for nitrogen fixation in a broad range of methanotrophs and suggest that nitrogen-fixing methanotrophs may be widespread and important in the nitrogen cycling of many environments.     

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.     

The active methanotrophic community in hydromorphic soils changes in response to changing methane concentration

Methanotrophic communities were studied in several periodically water-saturated gleyic soils. When sampled, each soil had an oxic upper layer and consumed methane from the atmosphere (at 1.75 ppmv). In most gleyic soils the K(m(app)) values for methane were between 70 and 800 ppmv. These are higher than most values observed in dry upland soils, but lower than those measured in wetlands. Based on cultivation-independent retrieval of the pmoA-gene and quantification of partial pmoA gene sequences, type II (Alphaproteobacteria) methanotrophs of the genus Methylocystis spp. were abundant (> 10(7) pmoA target molecules per gram of dry soil). Type I (Gammaproteobacteria) methanotrophs related to the genera Methylobacter and Methylocaldum/Methylococcus were detected in some soils. Six pmoA sequence types not closely related to sequences from cultivated methanotrophs were detected as well, indicating that diverse uncultivated methanotrophs were present. Three Gleysols were incubated under different mixing ratios of (13)C-labelled methane to examine (13)C incorporation into phospholipid fatty acids (PLFAs). Phospholipid fatty acids typical of type II methanotrophs, 16:0 and 18:1omega7c, were labelled with (13)C in all soils after incubation under an atmosphere containing 30 ppmv of methane. Incubation under 500 ppmv of methane resulted in labelling of additional PLFAs besides 16:0 and 18:1omega7c, suggesting that the composition of the active methanotrophic community changed in response to increased methane supply. In two soils, 16:1 PLFAs typical of type I methanotrophs were strongly labelled after incubation under the high methane mixing ratio only. Type II methanotrophs are most likely responsible for atmospheric methane uptake in these soils, while type I methanotrophs become active when methane is produced in the soil.     

Characterization of Root-Associated Methanotrophs from Three Freshwater Macrophytes: Pontederia cordata, Sparganium eurycarpum, and Sagittaria latifolia

Root-associated methanotrophic bacteria were enriched from three common aquatic macrophytes: Pontederia cordata, Sparganium eurycarpum, and Sagittaria latifolia. At least seven distinct taxa belonging to groups I and II were identified and presumptively assigned to the genera Methylosinus, Methylocystis, Methylomonas, and Methylococcus. Four of these strains appeared to be novel on the basis of partial 16S ribosomal DNA sequence analysis. The root-methanotroph association did not appear to be highly specific, since multiple methanotrophs were isolated from each of the three plant species. Group II methanotrophs were isolated most frequently; though less common, group I isolates accounted for three of the seven distinct methanotrophs. Apparent K(m) values for methane uptake by representative cultures ranged from 3 to >17 muM; for five of the eight cultures examined, apparent K(m) values agreed well with apparent K(m) estimates for plant roots, suggesting that these strains may be representative of those active in situ.     

Family- and genus-level 16S rRNA-targeted oligonucleotide probes for ecological studies of methanotrophic bacteria

Methanotrophic bacteria play a major role in the global carbon cycle, degrade xenobiotic pollutants, and have the potential for a variety of biotechnological applications. To facilitate ecological studies of these important organisms, we developed a suite of oligonucleotide probes for quantitative analysis of methanotroph-specific 16S rRNA from environmental samples. Two probes target methanotrophs in the family Methylocystaceae (type II methanotrophs) as a group. No oligonucleotide signatures that distinguish between the two genera in this family, Methylocystis and Methylosinus, were identified. Two other probes target, as a single group, a majority of the known methanotrophs belonging to the family Methylococcaceae (type I/X methanotrophs). The remaining probes target members of individual genera of the Methylococcaceae, including Methylobacter, Methylomonas, Methylomicrobium, Methylococcus, and Methylocaldum. One of the family-level probes also covers all methanotrophic endosymbionts of marine mollusks for which 16S rRNA sequences have been published. The two known species of the newly described genus Methylosarcina gen. nov. are covered by a probe that otherwise targets only members of the closely related genus Methylomicrobium. None of the probes covers strains of the newly proposed genera Methylocella and "Methylothermus," which are polyphyletic with respect to the recognized methanotrophic families. Empirically determined midpoint dissociation temperatures were 49 to 57 degrees C for all probes. In dot blot screening against RNA from positive- and negative-control strains, the probes were specific to their intended targets. The broad coverage and high degree of specificity of this new suite of probes will provide more detailed, quantitative information about the community structure of methanotrophs in environmental samples than was previously available.     

Molecular analyses of the methane-oxidizing microbial community in rice field soil by targeting the genes of the 16S rRNA, particulate methane monooxygenase, and methanol dehydrogenase

Rice field soil with a nonsaturated water content induced CH4 consumption activity when it was supplemented with 5% CH4. After a lag phase of 3 days, CH4 was consumed rapidly until the concentration was less than 1.8 parts per million by volume (ppmv). However, the soil was not able to maintain the oxidation activity at near-atmospheric CH4 mixing ratios (i.e., 5 ppmv). The soil microbial community was monitored by performing denaturing gradient gel electrophoresis (DGGE) during the oxidation process with different PCR primer sets based on the 16S rRNA gene and on functional genes. A universal small-subunit (SSU) ribosomal DNA (rDNA) primer set and 16S rDNA primer sets specifically targeting type I methylotrophs (members of the gamma subdivision of the class Proteobacteria [gamma-Proteobacteria]) and type II methylotrophs (members of the alpha-Proteobacteria) were used. Functional PCR primers targeted the genes for particulate methane monooxygenase (pmoA) and methanol dehydrogenase (mxaF), which code for key enzymes in the catabolism of all methanotrophs. The yield of PCR products amplified from DNA in soil that oxidized CH4 was the same as the yield of PCR products amplified from control soil when the universal SSU rDNA primer set was used but was significantly greater when primer sets specific for methanotrophs were used. The DGGE patterns and the sequences of major DGGE bands obtained with the universal SSU rDNA primer set showed that the community structure was dominated by nonmethanotrophic populations related to the genera Flavobacterium and Bacillus and was not influenced by CH4. The structure of the methylotroph community as determined with the specific primer sets was less complex; this community consisted of both type I and type II methanotrophs related to the genera Methylobacter, Methylococcus, and Methylocystis. DGGE profiles of PCR products amplified with functional gene primer sets that targeted the mxaF and pmoA genes revealed that there were pronounced community shifts when CH4 oxidation began. High CH4 concentrations stimulated both type I and II methanotrophs in rice field soil with a nonsaturated water content, as determined with both ribosomal and functional gene markers.     

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.     

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.     

Biochemical and molecular characterization of methanotrophs in soil from a pristine New Zealand beech forest

Methane (CH4) oxidation and the methanotrophic community structure of a pristine New Zealand beech forest were investigated using biochemical and molecular methods. Phospholipid-fatty acid-stable-isotope probing (PLFA-SIP) was used to identify the active population of methanotrophs in soil beneath the forest floor, while terminal-restriction fragment length polymorphism (T-RFLP) and cloning and sequencing of the pmoA gene were used to characterize the methanotrophic community. PLFA-SIP suggested that type II methanotrophs were the predominant active group. T-RFLP and cloning and sequencing of the pmoA genes revealed that the methanotrophic community was diverse, and a slightly higher number of type II methanotrophs were detected in the clone library. Most of the clones from type II methanotrophs were related to uncultured pmoA genes obtained directly from environmental samples, while clones from type I were distantly related to Methylococcus capsulatus. A combined data analysis suggested that the type II methanotrophs may be mainly responsible for atmospheric CH4 consumption. Further sequence analysis suggested that most of the methanotrophs detected shared their phylogeny with methanotrophs reported from soils in the Northern Hemisphere. However, some of the pmoA sequences obtained from this forest had comparatively low similarity (<97%) to known sequences available in public databases, suggesting that they may belong to novel groups of methanotrophic bacteria. Different methods of methanotrophic community analysis were also compared, and it is suggested that a combination of molecular methods with PLFA-SIP can address several shortcomings of stable isotope probing.     

Soluble methane monooxygenase gene clusters from trichloroethylene-degrading Methylomonas sp. strains and detection of methanotrophs during in situ bioremediation

The soluble MMO (sMMO) gene clusters from group I methanotrophs were characterized. An 8.1-kb KpnI fragment from Methylomonas sp. strain KSWIII and a 7.5-kb SalI fragment from Methylomonas sp. strain KSPIII which contained the sMMO gene clusters were cloned and sequenced. The sequences of these two fragments were almost identical. The sMMO gene clusters in the fragment consisted of six open reading frames which were 52 to 79% similar to the corresponding genes of previously described sMMO gene clusters of the group II and group X methanotrophs. The phylogenetic analysis of the predicted amino acid sequences of sMMO demonstrated that the sMMOs from these strains were closer to that from M. capsulatus Bath in the group X methanotrophs than to those from Methylosinus trichosporium OB3b and Methylocystis sp. strain M in the group II methanotrophs. Based on the sequence data of sMMO genes of our strains and other methanotrophs, we designed a new PCR primer to amplify sMMO gene fragments of all the known methanotrophs harboring the mmoX gene. The primer set was successfully used for detecting methanotrophs in the groundwater of trichloroethylene-contaminated sites during in situ-biostimulation treatments.     

Comparative characterization of methanotrophic enrichments by serological and molecular methods

Three stable methane-oxidizing enrichment cultures, SB26, SB31, and SB31A, were analyzed by transmission electron microscopy and by serological and molecular techniques. Electron microscopy revealed the presence of both type I and type II methanotrophs in SB31 and SB31A enrichments; only type II methanotrophs were found in SB26 enrichment. Methylosinus trichosporium was detected in all three enrichments by the application of species-specific antibodies. Additionally, Methylocystis echinoides was found in SB26 culture; Methylococcus capsulatus, in SB31 and SB31A; and Methylomonas methanica, in SB31. The analysis with pmoA and nifH gene sequences as phylogenetic markers revealed the presence of Methylosinus/Methylocystis group in all communities. Moreover, the analysis of pmoA sequences revealed the presence of Methylomonas in SB31. Methylocella was detected in SB31 and SB31A enrichments only by nifH analysis. It was concluded that the simultaneous application of different approaches reveals more reliable information on the diversity of methanotrophs.     

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.     

Methanotrophic communities in the soils of Russian northern taiga and subarctic tundra

The PCR analysis of DNA extracted from soil samples taken in 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 alpha-subunit of the hydroxylase component of soluble methane monooxygenase, the pmoA gene encoding the alpha-subunit of particulate methane monooxygenase, and the mxaF gene encoding the alpha-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.     

nifH Sequences and Nitrogen Fixation in Type I and Type II Methanotrophs

Some methane-oxidizing bacteria (methanotrophs) are known to be capable of expressing nitrogenase and utilizing N₂ as a nitrogen source. However, no sequences are available for nif genes in these strains, and the known nitrogen-fixing methanotrophs are confined mainly to a few genera. The purpose of this work was to assess the nitrogen-fixing capabilities of a variety of methanotroph strains. nifH gene fragments from four type I methanotrophs and seven type II methanotrophs were PCR amplified and sequenced. Nitrogenase activity was confirmed in selected type I and type II strains by acetylene reduction. Activities ranged from 0.4 to 3.3 nmol/min/mg of protein. Sequence analysis shows that the nifH sequences from the type I and type II strains cluster with nifH sequences from other gamma proteobacteria and alpha proteobacteria, respectively. The translated nifH sequences from three Methylomonas strains show high identity (95 to 99%) to several published translated environmental nifH sequences PCR amplified from rice roots and a freshwater lake. The translated nifH sequences from the type II strains show high identity (94 to 99%) to published translated nifH sequences from a variety of environments, including rice roots, a freshwater lake, an oligotrophic ocean, and forest soil. These results provide evidence for nitrogen fixation in a broad range of methanotrophs and suggest that nitrogen-fixing methanotrophs may be widespread and important in the nitrogen cycling of many environments.     

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.