Characterization of a Copper-Chelating Natural Product from the Methanotroph Methylosinus sp. LW3.

Methanobactins (Mbns) are ribosomally produced, post-translationally modified peptidic natural products that bind copper with high affinity. Methanotrophic bacteria use Mbns to acquire copper needed for enzymatic methane oxidation. Despite the presence of Mbn operons in a range of methanotroph and other bacterial genomes, few Mbns have been isolated and structurally characterized. Here we report the isolation of a novel Mbn from the methanotroph Methylosinus (Ms.) sp. LW3. Mass spectrometric and nuclear magnetic resonance spectroscopic data indicate that this Mbn, the largest characterized to date, consists of a 13-amino acid backbone modified to include pyrazinedione/oxazolone rings and neighboring thioamide groups derived from cysteine residues. The pyrazinedione ring is more stable to acid hydrolysis than the oxazolone ring and likely protects the Mbn from degradation. The structure corresponds exactly to that predicted on the basis of the Ms. sp. LW3 Mbn operon content, providing support for the proposed role of an uncharacterized biosynthetic enzyme, MbnF, and expanding the diversity of known Mbns.

Isolation and Genomic Characterization of a Proteobacterial Methanotroph Requiring Lanthanides.

Although the bioavailability of rare earth elements (REEs, including scandium, yttrium, and 15 lanthanides) has not yet been examined in detail, methane-oxidizing bacteria (methanotrophs) were recently shown to harbor specific types of methanol dehydrogenases (XoxF-MDHs) that contain lanthanides in their active site, whereas their well-characterized counterparts (MxaF-MDHs) were Ca2+-dependent. However, lanthanide dependency in methanotrophs has not been demonstrated, except in acidic environments in which the solubility of lanthanides is high. We herein report the isolation of a lanthanide-dependent methanotroph from a circumneutral environment in which lanthanides only slightly dissolved. Methanotrophs were enriched and isolated from pond sediment using mineral medium supplemented with CaCl2 or REE chlorides. A methanotroph isolated from the cerium (Ce) chloride-supplemented culture, Methylosinus sp. strain Ce-a6, was clearly dependent on lanthanide. Strain Ce-a6 only required approximately 30 nM lanthanide chloride for its optimal growth and exhibited the ability to utilize insoluble lanthanide oxides, which may enable survival in circumneutral environments. Genome and gene expression analyses revealed that strain Ce-a6 lost the ability to produce functional MxaF-MDH, and this may have been due to a large-scale deletion around the mxa gene cluster. The present results provide evidence for lanthanide dependency as a novel survival strategy by methanotrophs in circumneutral environments.

Characterization of Methanobactin from Methylosinus sp. LW4.

Methanobactins (Mbns) are a growing family of ribosomally produced, post-translationally modified natural products. Characteristic nitrogen-containing heterocycles and neighboring thioamides allow these compounds to bind copper with high affinity. Genome mining has enabled the identification of Mbn operons in bacterial genomes and the prediction of diverse Mbn structures from operon content and precursor peptide sequence. Here we report the characterization of Mbn from Methylosinus (Ms.) species (sp.) LW4. The peptide backbone is distinct from all previously characterized Mbns, and the post-translational modifications correspond precisely to those predicted on the basis of the Ms. sp. LW4 Mbn operon. Thus, prediction based on genome analysis combined with isolation and structural characterization represents a phylogenetic approach to finding diverse Mbns and elucidating their biosynthetic pathways.

[Culturable psychrotolerant methanotrophic bacteria in landfill cover soil].

Methanotrophs closely related to psychrotolerant members of the genera Methylobacter and Methylocella were identified in cultures enriched at 10@C from landfill cover soil samples collected in the period from April to November. Mesophilic methanotrophs of the genera Methylobacter and Methylosinus were found in cultures enriched at 20 degrees C from the same cover soil samples. A thermotolerant methanotroph related to Methylocaldum gracile was identified in the culture enriched at 40 degrees C from a sample collected in May (the temperature of the cover soil was 11.5-12.5 degrees C). In addition to methanotrophs, methylobacteria of the genera Methylotenera and Methylovorus and members of the genera Verrucomicrobium, Pseudomonas, Pseudoxanthomonas, Dokdonella, Candidatus Protochlamydia, and Thiorhodospira were also identified in the enrichment cultures. A methanotroph closely related to the psychrotolerant species Methylobacter tundripaludum (98% sequence identity of 16S r-RNA genes with the type strain SV96(T)) was isolated in pure culture. The introduction of a mixture of the methanotrophic enrichments, grown at 15 degrees C, into the landfill cover soil resulted in a decrease in methane emission from the landfill surface in autumn (October, November). The inoculum used was demonstrated to contain methanotrophs closely related to Methylobacter tundripaludum SV96.

Methane emission and dynamics of methanotrophic and methanogenic communities in a flooded rice field ecosystem.

Methane emissions, along with methanotrophs and methanogens and soil chemical properties, were investigated in a flooded rice ecosystem. Methane emission increased after rice transplantation (from 7.2 to 552 mg day(-1) m(-2) ) and was positively and significantly correlated with transcripts of pmoA and mcrA genes, transcript/gene ratios of mcrA, temperature and total organic carbon. Methane flux was negatively correlated with sulfate concentration. Methanotrophs represented only a small proportion (0.79-1.75%) of the total bacterial 16S rRNA gene reads: Methylocystis (type II methanotroph) decreased rapidly after rice transplantation, while Methylosinus and unclassified Methylocystaceae (type II) were relatively constant throughout rice cultivation. Methylocaldum, Methylobacter, Methylomonas and Methylosarcina (type I) were sparse during the early period, but they increased after 60 days, and their maximum abundances were observed at 90-120 days. Of 33 218 archaeal reads, 68.3-86.6% were classified as methanogens. Methanosaeta, Methanocella, Methanosarcina and Methanobacterium were dominant methanogens, and their maximum abundances were observed at days 60-90. Only four reads were characteristic of anaerobic methanotrophs, suggesting that anaerobic methane metabolism is negligible in this rice paddy system. After completing a multivariate canonical correspondence analysis of our integrated data set, we found normalized mcrA/pmoA transcript ratios to be a promising parameter for predicting net methane fluxes emitted from rice paddy soils.

Stress resistance and C1 metabolism involved in plant colonization of a methanotroph Methylosinus sp. B4S.

Methanotrophs are widespread and have been isolated from various environments including the phyllosphere. In this study, we characterized the plant colonization by Methylosinus sp. B4S, an α-proteobacterial methanotroph isolated from plant leaf. The gfp-tagged Methylosinus sp. B4S cells were observed to colonize Arabidopsis leaf surfaces by forming aggregates. We cloned and sequenced the general stress response genes, phyR, nepR and ecfG, from Methylosinus sp. B4S. In vitro analysis showed that the phyR expression level was increased after heat shock challenge, and phyR was shown to be involved in resistance to heat shock and UV light. In the phyllospheric condition, the gene expression level of phyR as well as mmoX and mxaF was found to be relatively high, compared with methane-grown liquid cultures. The phyR-deletion strain as well as the wild-type strain inoculated on Arabidopsis leaves proliferated at the initial phase and then gradually decreased during plant colonization. These results have shed light firstly on the importance of general stress resistance and C1 metabolism in methanotroph living in the phyllosphere.

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.

Analysis of methanotrophic communities in landfill biofilters using diagnostic microarray.

Biofilters operated for the microbial oxidation of landfill methane at two sites in Northern Germany were analysed for the composition of their methanotrophic community by means of diagnostic microarray targeting the pmoA gene of methanotrophs. The gas emitted from site Francop (FR) contained the typical principal components (CH4, CO2, N2) only, while the gas at the second site Müggenburger Strasse (MU) was additionally charged with non-methane volatile organic compounds (NMVOCs). Methane oxidation activity measured at 22 degrees C varied between 7 and 103 microg CH4 (g dw)(-1) h(-1) at site FR and between 0.9 and 21 microg CH4 (g dw)(-1) h(-1) at site MU, depending on the depth considered. The calculated size of the active methanotrophic population varied between 3 x 10(9) and 5 x 10(11) cells (g dw)(-1) for biofilter FR and 4 x 10(8) to 1 x 10(10) cells (g dw)(-1) for biofilter MU. The methanotrophic community in both biofilters as well as the methanotrophs present in the landfill gas at site FR was strongly dominated by type II organisms, presumably as a result of high methane loads, low copper concentration and low nitrogen availability. Within each biofilter, community composition differed markedly with depth, reflecting either the different conditions of diffusive oxygen supply or the properties of the two layers of materials used in the filters or both. The two biofilter communities differed significantly. Type I methanotrophs were detected in biofilter FR but not in biofilter MU. The type II community in biofilter FR was dominated by Methylocystis species, whereas the biofilter at site MU hosted a high abundance of Methylosinus species while showing less overall methanotroph diversity. It is speculated that the differing composition of the type II population at site MU is driven by the presence of NMVOCs in the landfill gas fed to the biofilter, selecting for organisms capable of co-oxidative degradation of these compounds.

Duplication of the mmoX gene in Methylosinus sporium: cloning, sequencing and mutational analysis.

The soluble methane monooxygenase (sMMO) is a key enzyme for methane oxidation, and is found in only some methanotrophs, including Methylosinus sporium 5. sMMO expression is regulated at the level of transcription from a sigma(54) promoter by a copper-switch, and is only expressed when the copper-to-biomass ratio during growth is low. Extensive phylogenetic and genetic analyses of sMMOs and other soluble di-iron monooxygenases reveal that these enzymes have only been acquired relatively recently through horizontal gene transfer. In this study, further evidence of horizontal gene transfer was obtained, through cloning and sequencing of the genes encoding the sMMO enzyme complex plus the regulatory genes mmoG and mmoR, and identification of a duplicate copy of the mmoX gene in Ms. sporium. mmoX encodes the alpha subunit of the hydroxylase of the sMMO enzyme, which constitutes the active site (Prior & Dalton, 1985). The mmoX genes were characterized at the molecular and biochemical levels. Although both copies were transcribed, only mmoX copy 1 was essential for sMMO activity. Construction of an sMMO(-) mutant by marker-exchange mutagenesis gave some possible insights into the role of the water-soluble pigment in siderophore-mediated iron acquisition. Finally, the amenability of Ms. sporium to genetic manipulation was demonstrated by complementing the sMMO(-) mutant by heterologous expression of sMMO genes from Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath), and it was shown that Ms. sporium could be used as an alternative model organism for molecular analysis of MMO regulation.

Functional expression of the particulate methane mono-oxygenase gene in recombinant Rhodococcus erythropolis.

In order to construct an expression system for the particulate methane mono-oxygenase (pMMO) gene (pmo), the structural gene cluster pmoCAB amplified from Methylosinus trichosporium OB3b was inserted into a shuttle vector pBS305 under the control of a dsz promoter and transformed into Rhodococcus erythropolis LSSE8-1. A stable transformant was successfully obtained using ethane as the sole carbon source. Fluorescence in situ hybridization results showed that the dsz promoter allowed the pmo genes to be transcribed in the recombinant strain. The effects of Cu2+ and Zn2+ concentrations on cell growth and pMMO activity in ethane-containing medium were examined. It was discovered that 7.5 microM Cu2+ and 1.8 microM Zn2+ were suitable to achieve high cell concentration and pMMO activity, but the amount of methanol accumulated during methane oxidation by the recombinant strain was still low.

Preferential cultivation of type II methanotrophic bacteria from littoral sediments (Lake Constance).

Most widely used medium for cultivation of methanotrophic bacteria from various environments is that proposed in 1970 by Whittenbury. In order to adapt and optimize medium for culturing of methanotrophs from freshwater sediment, media with varying concentrations of substrates, phosphate, nitrate, and other mineral salts were used to enumerate methanotrophs by the most probable number method. High concentrations (>1 mM) of magnesium and sulfate, and high concentrations of nitrate (>500 microM) significantly reduced the number of cultured methanotrophs, whereas phosphate in the range of 15-1500 microM had no influence. Also oxygen and carbon dioxide influenced the culturing efficiency, with an optimal mixing ratio of 17% O(2) and 3% CO(2); the mixing ratio of methane (6-32%) had no effect. A clone library of pmoA genes amplified by PCR from DNA extracted from sediment revealed the presence of both type I and type II methanotrophs. Nonetheless, the cultivation of methanotrophs, also with the improved medium, clearly favored growth of type II methanotrophs of the Methylosinus/Methylocystis group. Although significantly more methanotrophs could be cultured with the modified medium, their diversity did not mirror the diversity of methanotrophs in the sediment sample detected by molecular biology method.

Methanotrophic diversity in high arctic wetlands on the islands of Svalbard (Norway)--denaturing gradient gel electrophoresis analysis of soil DNA and enrichment cultures.

The methanotrophic community in arctic soil from the islands of Svalbard, Norway (78 degrees N) was analysed by combining group-specific PCR with PCR of the highly variable V3 region of the 16S rRNA gene and then by denaturing gradient gel electrophoresis (DGGE). Selected bands were sequenced for identification. The analyses were performed with DNA extracted directly from soil and from enrichment cultures at 10 and 20 degrees C. The two genera Methylobacter and Methylosinus were found in all localities studied. The DGGE band patterns were simple, and DNA fragments with single base differences were separated. The arctic tundra is a potential source of extensive methane emission due to climatic warming because of its large reservoirs of stored organic carbon. Higher temperatures due to climatic warming can cause increased methane production, and the abundance and activity of methane-oxidizing bacteria in the arctic soil may be important regulators for methane emission to the atmosphere.

[Methanotrophic communities in the soils of Russian northern taiga and subarctic tundra]. / Metanotrofnye soobshchestva pochv severnoi taigi i subarkticheskoi tundry Rossii.

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.

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.

Production and consumption of nitric oxide by three methanotrophic bacteria.

We studied nitrogen oxide production and consumption by methanotrophs Methylobacter luteus (group I), Methylosinus trichosporium OB3b (group II), and an isolate from a hardwood swamp soil, here identified by 16S ribosomal DNA sequencing as Methylobacter sp. strain T20 (group I). All could consume nitric oxide (nitrogen monoxide, NO), and produce small amounts of nitrous oxide (N(2)O). Only Methylobacter strain T20 produced large amounts of NO (>250 parts per million by volume [ppmv] in the headspace) at specific activities of up to 2.0 x 10(-17) mol of NO cell(-1) day(-1), mostly after a culture became O(2) limited. Production of NO by strain T20 occurred mostly in nitrate-containing medium under anaerobic or nearly anaerobic conditions, was inhibited by chlorate, tungstate, and O(2), and required CH(4). Denitrification (methanol-supported N(2)O production from nitrate in the presence of acetylene) could not be detected and thus did not appear to be involved in the production of NO. Furthermore, cd(1) and Cu nitrite reductases, NO reductase, and N(2)O reductase could not be detected by PCR amplification of the nirS, nirK, norB, and nosZ genes, respectively. M. luteus and M. trichosporium produced some NO in ammonium-containing medium under aerobic conditions, likely as a result of methanotrophic nitrification and chemical decomposition of nitrite. For Methylobacter strain T20, arginine did not stimulate NO production under aerobiosis, suggesting that NO synthase was not involved. We conclude that strain T20 causes assimilatory reduction of nitrate to nitrite, which then decomposes chemically to NO. The production of NO by methanotrophs such as Methylobacter strain T20 could be of ecological significance in habitats near aerobic-anaerobic interfaces where fluctuating O(2) and nitrate availability occur.

Molecular analysis of the pmo (particulate methane monooxygenase) operons from two type II methanotrophs.

The particulate methane monooxygenase gene clusters, pmoCAB, from two representative type II methanotrophs of the alpha-Proteobacteria, Methylosinus trichosporium OB3b and Methylocystis sp. strain M, have been cloned and sequenced. Primer extension experiments revealed that the pmo cluster is probably transcribed from a single transcriptional start site located 300 bp upstream of the start of the first gene, pmoC, for Methylocystis sp. strain M. Immediately upstream of the putative start site, consensus sequences for sigma(70) promoters were identified, suggesting that these pmo genes are recognized by sigma(70) and negatively regulated under low-copper conditions. The pmo genes were cloned in several overlapping fragments, since parts of these genes appeared to be toxic to the Escherichia coli host. Methanotrophs contain two virtually identical copies of pmo genes, and it was necessary to use Southern blotting and probing with pmo gene fragments in order to differentiate between the two pmoCAB clusters in both methanotrophs. The complete DNA sequence of one copy of pmo genes from each organism is reported here. The gene sequences are 84% similar to each other and 75% similar to that of a type I methanotroph of the gamma-Proteobacteria, Methylococcus capsulatus Bath. The derived proteins PmoC and PmoA are predicted to be highly hydrophobic and consist mainly of transmembrane-spanning regions, whereas PmoB has only two putative transmembrane-spanning helices. Hybridization experiments showed that there are two copies of pmoC in both M. trichosporium OB3b and Methylocystis sp. strain M, and not three copies as found in M. capsulatus Bath.

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.