Genome-scale revealing the central metabolic network of the fast growing methanotroph Methylomonas sp. ZR1.

Methylomonas sp. ZR1 was an isolated new methanotrophs that could utilize methane and methanol growing fast and synthesizing value added compounds such as lycopene. In this study, the genomic study integrated with the comparative transcriptome analysis were taken to understanding the metabolic characteristic of ZR1 grown on methane and methanol at normal and high temperature regime. Complete Embden-Meyerhof-Parnas pathway (EMP), Entner-Doudoroff pathway (ED), Pentose Phosphate Pathway (PP) and Tricarboxy Acid Cycle (TCA) were found to be operated in ZR1. In addition, the energy saving ppi-dependent EMP enzyme, coupled with the complete and efficient central carbon metabolic network might be responsible for its fast growing nature. Transcript level analysis of the central carbon metabolism indicated that formaldehyde metabolism was a key nod that may be in charge of the carbon conversion efficiency (CCE) divergent of ZR1 grown on methanol and methane. Flexible nitrogen and carotene metabolism pattern were also investigated in ZR1. Nitrogenase genes in ZR1 were found to be highly expressed with methane even in the presence of sufficient nitrate. It appears that, higher lycopene production in ZR1 grown on methane might be attributed to the higher proportion of transcript level of C40 to C30 metabolic gene. Higher transcript level of exopolysaccharides metabolic gene and stress responding proteins indicated that ZR1 was confronted with severer growth stress with methanol than with methane. Additionally, lower transcript level of the TCA cycle, the dramatic high expression level of the nitric oxide reductase and stress responding protein, revealed the imbalance of the central carbon and nitrogen metabolic status, which would result in the worse growth of ZR1 with methanol at 30 °C.

Isolation of a methane-oxidizing bacterium that bioremediates hexavalent chromium from a formerly industrialized Suburban River.

Sediment samples were taken from sediment adjacent to a suburban river in Sheffield in Northern England that had suffered heavy metal pollution due to previous activity of the steel industry (between the 17th and 19th centuries). The most abundant heavy metals found in the samples were lead, chromium, nickel, arsenic and cobalt, with maximum concentrations of 412·80, 25·232, 25·196, 8·123 and 7·66 mg kg-1 , respectively. Enrichment cultures were set up using methane as carbon and energy source, as a result of which a strain of methanotroph was isolated that was shown via 16S rRNA gene sequencing to be a strain Methylomonas koyamae and given the designation SHU1. M. koyamae SHU1 removed hexavalent chromium from an initial concentration of 10 ppm, which was inhibited by the metabolic inhibitor sodium azide or the methane monooxygenase inhibitor phenylacetylene. To the authors' knowledge, this is the first description of a strain of the widely environmentally distributed genus Methylomonas that is capable of remediating hexavalent chromium. SIGNIFICANCE AND IMPACT OF THE STUDY: Aerobic methanotrophic bacteria are known for bioremediation of an increasing range of organic and inorganic pollutants, using methane as carbon and energy source. Previously, one laboratory methanotroph strain, Methylococcus capsulatus Bath, was known to bioremediate toxic chromium (VI) by reducing it to chromium (III). Here, a newly isolated methanotroph strain, Methylomonas koyamae SHU1, has been shown able to remediate chromium (VI). This indicates that chromium (VI) bioremediation is not unique to M. capsulatus and moreover adds weight to the suggestion that methanotrophs may contribute directly to chromium (VI) detoxification in nature and in polymicrobial bioremediation fed with methane.

A novel methanotroph in the genus Methylomonas 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.

Synthesizing value-added products from methane by a new Methylomonas.

AIMS: Methane and methanol are potential carbon sources of industrial micro-organisms in addition to crop-derived bio-carbon sources. Methanotrophs that can utilize these simple, stable and large amounts chemicals are expected to be developed into 'cell factories' for the production of specific chemicals. In this study, a methanotroph that can synthesize lycopene, C30 carotenoid and exopolysaccharides (EPS) with relative better performances from C1 substrates was isolated, and its performances were evaluated. METHODS AND RESULTS: The isolated strain was identified as Methylomonas sp. ZR1 based on 16S rRNA sequence analysis. Its maximum specific growth rate achieved 0·200 h-1 under flask culture conditions, and 0·386 h-1 in bubble column reactors. ZR1 was able to utilize 35 g l-1 of methanol and even exhibited slight growth in the presence of 40 g l-1 of methanol. Furthermore, ZR1 was proved to synthesize lycopene (C40 carotenoids) besides the C30 carotenoids through HPLC-DAD and HPLC-MS/MS analysis methods. Its carotenoid extracts exhibited excellent antioxidative activities measured by the ABTS+ method. Plenty of polysaccharides were also synthesized by ZR1, the components of the polysaccharides were identified as glucose, mannose and galactose with a proportion of 1 : 2 : 1 by GC-MS, and its yield achieved 0·13 g g-1 cell dry weight. CONCLUSIONS: The isolated strain has great potential for the production of value-added bioproducts from C1 compounds because of its excellent C1 substrate utilizing abilities and its abilities to naturally synthesize lycopene, C30 carotenoids and EPS. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we isolated a fast-growing methanotroph, its C1 carbon substrate utilizing ability is excellent in comparison with reported methanotrophs. Furthermore, besides polysaccharides and C30 carotenoids which were commonly synthesized by methanotrophs, our findings suggested that C40 lycopene could also be naturally synthesized from methane by methanotrophs.

Enrichment culture and identification of endophytic methanotrophs isolated from peatland plants.

Aerobic methane-oxidizing bacteria (MOB) are an environmentally significant group of microorganisms due to their role in the global carbon cycle. Research conducted over the past few decades has increased the interest in discovering novel genera of methane-degrading bacteria, which efficiently utilize methane and decrease the global warming effect. Moreover, methanotrophs have more promising applications in environmental bioengineering, biotechnology, and pharmacy. The investigations were undertaken to recognize the variety of endophytic methanotrophic bacteria associated with Carex nigra, Vaccinium oxycoccus, and Eriophorum vaginatum originating from Moszne peatland (East Poland). Methanotrophic bacteria were isolated from plants by adding sterile fragments of different parts of plants (roots and stems) to agar mineral medium (nitrate mineral salts (NMS)) and incubated at different methane values (1-20% CH4). Single colonies were streaked on new NMS agar media and, after incubation, transferred to liquid NMS medium. Bacterial growth dynamics in the culture solution was studied by optical density-OD600 and methane consumption. Changes in the methane concentration during incubation were controlled by the gas chromatography technique. Characterization of methanotrophs was made by fluorescence in situ hybridization (FISH) with Mg705 and Mg84 for type I methanotrophs and Ma450 for type II methanotrophs. Identification of endophytes was performed after 16S ribosomal RNA (rRNA) and mmoX gene amplification. Our study confirmed the presence of both types of methanotrophic bacteria (types I and II) with the predominance of type I methanotrophs. Among cultivable methanotrophs, there were different strains of the genus Methylomonas and Methylosinus. Furthermore, we determined the potential of the examined bacteria for methane oxidation, which ranged from 0.463 ± 0.067 to 5.928 ± 0.169 µmol/L CH4/mL/day.

Methane oxidation coupled to nitrate reduction under hypoxia by the Gammaproteobacterium Methylomonas denitrificans, sp. nov. type strain FJG1.

Obligate methanotrophs belonging to the Phyla Proteobacteria and Verrucomicrobia require oxygen for respiration and methane oxidation; nevertheless, aerobic methanotrophs are abundant and active in low oxygen environments. While genomes of some aerobic methanotrophs encode putative nitrogen oxide reductases, it is not understood whether these metabolic modules are used for NOx detoxification, denitrification or other purposes. Here we demonstrate using microsensor measurements that a gammaproteobacterial methanotroph Methylomonas denitrificans sp. nov. strain FJG1(T) couples methane oxidation to nitrate reduction under oxygen limitation, releasing nitrous oxide as a terminal product. Illumina RNA-Seq data revealed differential expression of genes encoding a denitrification pathway previously unknown to methanotrophs as well as the pxmABC operon in M. denitrificans sp. nov. strain FJG1(T) in response to hypoxia. Physiological and transcriptome data indicate that genetic inventory encoding the denitrification pathway is upregulated only upon availability of nitrate under oxygen limitation. In addition, quantitation of ATP levels demonstrates that the denitrification pathway employs inventory such as nitrate reductase NarGH serving M. denitrificans sp. nov. strain FJG1(T) to conserve energy during oxygen limitation. This study unravelled an unexpected metabolic flexibility of aerobic methanotrophs, thereby assigning these bacteria a new role at the metabolic intersection of the carbon and nitrogen cycles.

Field-scale tracking of active methane-oxidizing communities in a landfill cover soil reveals spatial and seasonal variability.

Aerobic methane-oxidizing bacteria (MOB) in soils mitigate methane (CH4 ) emissions. We assessed spatial and seasonal differences in active MOB communities in a landfill cover soil characterized by highly variable environmental conditions. Field-based measurements of CH4 oxidation activity and stable-isotope probing of polar lipid-derived fatty acids (PLFA-SIP) were complemented by microarray analysis of pmoA genes and transcripts, linking diversity and function at the field scale. In situ CH4 oxidation rates varied between sites and were generally one order of magnitude lower in winter compared with summer. Results from PLFA-SIP and pmoA transcripts were largely congruent, revealing distinct spatial and seasonal clustering. Overall, active MOB communities were highly diverse. Type Ia MOB, specifically Methylomonas and Methylobacter, were key drivers for CH4 oxidation, particularly at a high-activity site. Type II MOB were mainly active at a site showing substantial fluctuations in CH4 loading and soil moisture content. Notably, Upland Soil Cluster-gamma-related pmoA transcripts were also detected, indicating concurrent oxidation of atmospheric CH4 . Spatial separation was less distinct in winter, with Methylobacter and uncultured MOB mediating CH4 oxidation. We propose that high diversity of active MOB communities in this soil is promoted by high variability in environmental conditions, facilitating substantial removal of CH4 generated in the waste body.

Niche differentiation in nitrogen metabolism among methanotrophs within an operational taxonomic unit.

BACKGROUND: The currently accepted thesis on nitrogenous fertilizer additions on methane oxidation activity assumes niche partitioning among methanotrophic species, with activity responses to changes in nitrogen content being dependent on the in situ methanotrophic community structure Unfortunately, widely applied tools for microbial community assessment only have a limited phylogenetic resolution mostly restricted to genus level diversity, and not to species level as often mistakenly assumed. As a consequence, intragenus or intraspecies metabolic versatility in nitrogen metabolism was never evaluated nor considered among methanotrophic bacteria as a source of differential responses of methane oxidation to nitrogen amendments. RESULTS: We demonstrated that fourteen genotypically different Methylomonas strains, thus distinct below the level at which most techniques assign operational taxonomic units (OTU), show a versatile physiology in their nitrogen metabolism. Differential responses, even among strains with identical 16S rRNA or pmoA gene sequences, were observed for production of nitrite and nitrous oxide from nitrate or ammonium, nitrogen fixation and tolerance to high levels of ammonium, nitrate, and hydroxylamine. Overall, reduction of nitrate to nitrite, nitrogen fixation, higher tolerance to ammonium than nitrate and tolerance and assimilation of nitrite were general features. CONCLUSIONS: Differential responses among closely related methanotrophic strains to overcome inhibition and toxicity from high nitrogen loads and assimilation of various nitrogen sources yield competitive fitness advantages to individual methane-oxidizing bacteria. Our observations proved that community structure at the deepest phylogenetic resolution potentially influences in situ functioning.

Methylomonas lenta sp. nov., a methanotroph isolated from manure and a denitrification tank.

Two methanotrophic bacteria, strains R-45377(T) and R-45370, were respectively isolated from a slurry pit of a cow stable and from a denitrification tank of a wastewater treatment plant in Belgium. The strains showed 99.9 % 16S rRNA gene sequence similarity. Cells were Gram-negative, motile rods containing type I methanotroph intracytoplasmic membranes. Colonies and liquid cultures appeared white to pale pink. The pmoA gene encoding particulate methane monooxygenase (pMMO) and the nifH gene encoding nitrogenase were present. Soluble methane monooxygenase (sMMO) activity, the presence of the mmoX gene encoding sMMO and the presence of the pxmA gene encoding a sequence-divergent pMMO were not detected. Methane and methanol were utilized as sole carbon sources. The strains grew optimally at 20 °C (range 15-28 °C) and at pH 6.8-7.3 (range pH 6.3-7.8). The strains grew in media supplemented with up to 1.2 % NaCl. The major cellular fatty acids were C16 : 1ω8c, C16 : 1ω5c, C16 : 1ω7c, C14 : 0, C15 : 0 and C16 : 0 and the DNA G+C content was 47 mol%. 16S rRNA gene- and pmoA-based phylogenetic analyses showed that the isolates cluster among members of the genus Methylomonas within the class Gammaproteobacteria, with pairwise 16S rRNA gene sequence similarities of 97.5 and 97.2 % between R-45377(T) and the closest related type strains, Methylomonas scandinavica SR5(T) and Methylomonas paludis MG30(T), respectively. Based on phenotypic characterization of strains R-45377(T) and R-45370, their low 16S rRNA gene sequence similarities and the formation of a separate phylogenetic lineage compared with existing species of the genus Methylomonas, we propose to classify these strains in a novel species, Methylomonas lenta sp. nov., with R-45377(T) ( = LMG 26260(T) = JCM 19378(T)) as the type strain.

Methylomonas paludis sp. nov., the first acid-tolerant member of the genus Methylomonas, from an acidic wetland.

An aerobic methanotrophic bacterium was isolated from an acidic (pH 3.9) Sphagnum peat bog in north-eastern Russia and designated strain MG30(T). Cells of this strain were Gram-negative, pale pink-pigmented, non-motile, thick rods that were covered by large polysaccharide capsules and contained an intracytoplasmic membrane system typical of type I methanotrophs. They possessed a particulate methane monooxygenase enzyme (pMMO) and utilized only methane and methanol. Carbon was assimilated via the ribulose-monophosphate pathway; nitrogen was fixed via an oxygen-sensitive nitrogenase. Strain MG30(T) was able to grow at a pH range of 3.8-7.3 (optimum pH 5.8-6.4) and at temperatures between 8 and 30 °C (optimum 20-25 °C). The major cellular fatty acids were C16:1ω5t, C16:1ω8c, C16:1ω7c and C14:0; the DNA G+C content was 48.5 mol%. The isolate belongs to the family Methylococcaceae of the class Gammaproteobacteria and displayed 94.7-96.9% 16S rRNA gene sequence similarity to members of the genus Methylomonas. However, strain MG30(T) differed from all taxonomically characterized members of this genus by the absence of motility, the ability to grow in acidic conditions and low DNA G+C content. Therefore, we propose to classify this strain as representing a novel, acid-tolerant species of the genus Methylomonas, Methylomonas paludis sp. nov. Strain MG30(T) (=DSM 24973(T)=VKM B-2745(T)) is the type strain.

Distribution of methanotrophs in the phyllosphere.

Plants have been reported to emit methane as well as methanol originating in their cell-wall constituents. We investigated methanotrophs in the phyllosphere by the enrichment culture method with methane as sole carbon source. We enriched methanotrophs from the leaves, flowers, bark, and roots of various plants. Analysis of the pmoA and mxaF genes retrieved from the enrichment cultures revealed that methanotrophs closely related to the genera Methylomonas, Methylosinus, and Methylocystis inhabit not only the rhizosphere but also the phyllosphere, together with methanol-utilizing bacteria.

Methylomonas koyamae sp. nov., a type I methane-oxidizing bacterium from floodwater of a rice paddy field.

A novel methane-oxidizing bacterium, strain Fw12E-Y(T), was isolated from floodwater of a rice paddy field in Japan. Cells of strain Fw12E-Y(T) were Gram-negative, motile rods with a single polar flagellum and type I intracytoplasmic membrane arrangement. The strain grew only on methane or methanol as sole carbon and energy source. It was able to grow at 10-40 °C (optimum 30 °C), at pH 5.5-7.0 (optimum 6.5) and with 0-0.1% (w/w) NaCl (no growth above 0.5% NaCl). 16S rRNA gene sequence analysis showed that strain Fw12E-Y(T) is related most closely to members of the genus Methylomonas, but at low levels of similarity (95.0-95.4%). Phylogenetic analysis of pmoA and mxaF genes indicated that the strain belongs to the genus Methylomonas (97 and 92 % deduced amino acid sequence identities to Methylomonas methanica S1(T), respectively). The DNA G+C content of strain Fw12E-Y(T) was 57.1 mol%. Chemotaxonomic data regarding the major quinone (MQ-8) and major fatty acids (C(16:1) and C(14:0)) also supported its affiliation to the genus Methylomonas. Based on phenotypic, genomic and phylogenetic data, strain Fw12E-Y(T) is considered to represent a novel species of the genus Methylomonas, for which the name Methylomonas koyamae sp. nov. is proposed. The type strain is Fw12E-Y(T) ( = JCM 16701(T) = NBRC 105905(T) = NCIMB 14606(T)).

[Sequence analysis of 16S rDNA and genes of soluble methane monooxygenase from Methylomonas sp. GYJ3].

Soluble methane monooxygenase (MMO) from methanotrophs is a member of binuclear iron-containing multicomponent oxygenases, which can catalyze bioconversion of methane to methanol at ambient temperature and regulate methane recycle in nature. The research focused mainly on the sequence analysis of 16S rDNA and sMMO genes from Methylomonas sp. GYJ3. With the aid of the information from GenBank, the PCR primers and the sequence primers were designed, obtained a 5690bp of sMMO fragment and a 1280bp of 16S rDNA. Sequence comparison for MMOX with counterpart of other five strains showed that from 78% to 99% identity in protein level and from 71 % to 97% identity in gene level, in the separate comparison of six components, only orfY component had a lower identical. The multiple alignment of MMOX amino acid sequence with other four strains showed that there is a high conservation, especially in two Fe binding regions. 16S rDNA phylogenetic analysis demonstrated that Methylomonas sp. GYJ3 is relative with gamma proteobacteria. Phylogenetic analysis of MMOX amino acid sequence showed that Methylomonas sp. GYJ3 is closer to Methylomonas sp. KSW III of type I methanotrophs. It was concluded that Methylomonas sp. GYJ3 is belong to the genus of type I methanotroph Methylomonas, and the result was a direct evidence for the sMMO can be expressed in type I methanotrophs. The theoretical pI of hydroxylase was 6.28 and the theoretical MW of hydroxylase was 248874.41Da.

[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.

Estimating high-affinity methanotrophic bacterial biomass, growth, and turnover in soil by phospholipid fatty acid 13C labeling.

A time series phospholipid fatty acid (PLFA) 13C-labeling study was undertaken to determine methanotrophic taxon, calculate methanotrophic biomass, and assess carbon recycling in an upland brown earth soil from Bronydd Mawr (Wales, United Kingdom). Laboratory incubations of soils were performed at ambient CH4 concentrations using synthetic air containing 2 parts per million of volume of 13CH4. Flowthrough chambers maintained a stable CH4 concentration throughout the 11-week incubation. Soils were analyzed at weekly intervals by gas chromatography (GC), GC-mass spectrometry, and GC-combustion-isotope ratio mass spectrometry to identify and quantify individual PLFAs and trace the incorporation of 13C label into the microbial biomass. Incorporation of the 13C label was seen throughout the experiment, with the rate of incorporation decreasing after 9 weeks. The delta13C values of individual PLFAs showed that 13C label was incorporated into different components to various extents and at various rates, reflecting the diversity of PLFA sources. Quantitative assessments of 13C-labeled PLFAs showed that the methanotrophic population was of constant structure throughout the experiment. The dominant 13C-labeled PLFA was 18:1omega7c, with 16:1omega5 present at lower abundance, suggesting the presence of novel type II methanotrophs. The biomass of methane-oxidizing bacteria at optimum labeling was estimated to be about 7.2 x 10(6) cells g(-1) of soil (dry weight). While recycling of 13C label from the methanotrophic biomass must occur, it is a slower process than initial 13CH4 incorporation, with only about 5 to 10% of 13C-labeled PLFAs reflecting this process. Thus, 13C-labeled PLFA distributions determined at any time point during 13CH4 incubation can be used for chemotaxonomic assessments, although extended incubations are required to achieve optimum 13C labeling for methanotrophic biomass determinations.

[Classification of Methylomonas rubra sp. nov].

Strain Methylomonas rubra 15sh(T), deposited in several collections of microorganisms (NCIMB 11913(T) = UCM B-3075(T) = ACM 3303(T)), is the subject of numerous studies. However, the name of this strain is not valid, primarily due to the phenotypic similarity of the species M. rubra to the species M. methanica. The results of the present study and data available in the literature indicate that M. rubra deserves the status of a separate species. Strains of M. rubra differ from strains of M. methanica in a number of properties, such as the ability to reduce nitrates to nitrites, the structure of intracytoplasmic membranes, and the presence of a new coenzyme Q. The distinctions between the species M. rubra and M. methanica were confirmed by comparison of electrophoretic patterns of their cellular proteins, by results of DNA-DNA hybridization, and by the data from 16S rRNA gene sequencing (the level of phylogenetic homology between these two species was below 95%). Phylogenetic and phenotypic analyses showed that strains of M. rubra cannot be assigned to any species of the genus Methylomonas. Results of polyphasic analysis suggest an independent taxonomic status of strain Methylomonas rubra 15sh(T). This paper contains description of Methylomonas rubra sp. nov. with the type strain 15sh(T) = NCIMB 11913T = UCM B-3075(T) = ACM 3303(T). The nucleotide sequence of the 16S rRNA gene of strain 15sh(T) has been deposited with the GenBank database under the accession number AY995198.

Analysis of fae and fhcD genes in Mono Lake, California.

Genes for two enzymes of the tetrahydromethanopterin-linked C(1) transfer pathway (fae and fhcD) were detected in hypersaline, hyperalkaline Mono Lake (California), via PCR amplification and analysis. Low diversity for fae and fhcD was noted, in contrast to the diversity previously detected in a freshwater lake, Lake Washington (Washington).

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.

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.