mmoX Gene Research Articles

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.

Methane-Oxidizing Bacteria in a Finnish Raised Mire Complex: Effects of Site Fertility and Drainage

Methane-oxidizing bacteria (MOB) are the only biological sinks for methane (CH4). Drainage of peatlands is known to decrease overall CH4 emission, but the effect on MOB is unknown. The objective of this work was to characterize the MOB community and activity in two ecohydrologically different pristine peatland ecosystems, a fen and a bog, and their counterparts that were drained in 1961. Oligotrophic fens are groundwater-fed peatlands, but ombrotrophic bogs receive additional water and nutrients only from rainwater. The sites were sampled in August 2003 down to 10 cm below the water table (WT), and cores were divided into 10-cm subsamples. CH4 oxidation was measured by gas chromatography (GC) to characterize MOB activity. The MOB community structure was characterized by polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) and sequencing methods using partial pmoA and mmoX genes. The highest CH4 oxidation rates were measured from the subsamples 20-30 and 30-40 cm above WT at the pristine oligotrophic fen (12.7 and 10.5 micromol CH4 dm-3 h-1, respectively), but the rates decreased to almost zero in the vicinity of WT. In the pristine ombrotrophic bog, the highest oxidation rate at 0-10 cm was lower than in the fen (8.10 micromol CH4 dm-3 h-1), but in contrast to the fen, oxidation rates of 4.5 micromol CH4 dm-3 h-1 were observed at WT and 10 cm below WT. Drainage reduced the CH4 oxidation rates to maximum values of 1.67 and 5.77 micromol CH4 dm-3 h-1 at 30-40 and 20-30 cm of the fen and bog site, respectively. From the total of 13 pmoA-derived DGGE bands found in the study, 11, 3, 6, and 2 were observed in the pristine fen and bog and their drained counterparts, respectively. According to the nonmetric multidimensional scaling of the DGGE banding pattern, the MOB community of the pristine fen differed from the other sites. The majority of partial pmoA sequences belonged to type I MOB, whereas the partial mmoX bands that were observed only in the bog sites formed a distinct group relating more to type II MOB. This study indicates that fen and bog ecosystems differ in MOB activity and community structure, and both these factors are affected by drainage.

Facultative Methanotrophs Revisited

Facultative Methanotrophs Revisited

Abundance and activity of uncultured methanotrophic bacteria involved in the consumption of atmospheric methane in two forest soils

The activity and abundance of methanotrophic bacteria were measured in an acidic and a neutral forest soil. The soils exhibited high uptake rates (>30 microg CH4 m(-2) h(-1)) of atmospheric CH4 at all measurement times throughout the vegetation period. The abundances of various phylogenetic groups of methanotrophs, including some uncultured putative ones, were measured using real-time polymerase chain reaction assays. Each assay specifically targeted the pmoA gene or mmoX gene of a particular group of methanotrophs, or the amoA gene of ammonia-oxidizing bacteria. As yet uncultured methanotrophs of a group previously named 'forest soil cluster' or 'USC alpha' were numerically dominant in the acidic soil, while cultured but taxonomically uncharacterized methanotrophs of a group 'Cluster I' were dominant in the neutral soil. Each group was detected in numbers equivalent to about 10(6) pmoA gene copies per gram dry weight of soil and comprised >90% of the detectable methanotrophic bacteria in the respective soil. As the numbers of ammonia-oxidizing bacteria were similar but not higher, they could not have accounted for the observed CH4 uptake rates due to their low cell-specific CH4 oxidation activity. Based on CH4 flux and bacterial abundance data, estimated cell-specific CH4 oxidation rates of the detected methanotrophic bacteria were 540-800 x 10(-18) mol cell(-1) h(-1), which is high compared with literature values of cultured methanotrophic bacteria. These estimated cell-specific CH4 oxidation rates are sufficiently high to allow not only maintenance but even growth on atmospheric CH4 alone. Transcripts of mRNA of the pmoA gene were detected in the acidic soil, demonstrating that USC alpha methanotrophs expressed pmoA under ambient methane mixing ratios. On the other hand, pmoA transcripts of Cluster I or of other methanotrophic groups were not detectable. Our study suggests that USC alpha and Cluster I methanotrophs are adapted to the low concentration of methane in forest soils by possessing high cell-specific CH4 oxidation activities.

Diversity of methanotroph communities in a basalt aquifer

Methanotrophic bacteria play an important role in global cycling of carbon and co-metabolism of contaminants. Methanotrophs from pristine regions of the Snake River Plain Aquifer (SRPA; Idaho, USA) were studied in order to gain insight into the native groundwater communities' genetic potential to carry out TCE co-metabolism. Wells were selected that were proximal to a TCE plume believed to be undergoing natural attenuation. Methane concentrations ranged from 1 to >1000 nM. Carbon isotope ratios and diversity data together suggest that the SRPA contains active communities of methanotrophs that oxidize microbially produced methane. Microorganisms removed from groundwater by filtration were used as inocula for enrichments or frozen immediately and DNA was subsequently extracted for molecular characterization. Primers that specifically target methanotroph 16S rRNA genes or genes that code for subunits of soluble or particulate methane monooxygenase, mmoX and pmoA, respectively, were used to characterize the indigenous methanotrophs via PCR, cloning, RFLP analysis, and sequencing. Type I methanotroph clones aligned with Methylomonas, Methylocaldum, and Methylobacter sequences and a distinct 16S rRNA phylogenetic lineage grouped near Methylobacter. The majority of clone sequences in type II methanotroph 16S rRNA, pmoA, and mmoX gene libraries grouped closely with sequences in the Methylocystis genus. A subset of the type II methanotroph clones from the aquifer had sequences that aligned most closely to Methylosinus trichosporium OB3b and Methylocystis spp., known TCE-co-metabolizing methanotrophs.

Molecular phylogeny of type II methane-oxidizing bacteria isolated from various environments.

Type II methane-oxidizing bacteria (MOB) were isolated from diverse environments, including rice paddies, pristine and polluted freshwaters and sediments, mangrove roots, upland soils, brackish water ecosystems, moors, oil wells, water purification systems and livestock manure. Isolates were identified based on morphological traits as either Methylocystis spp., Methylosinus sporium or Methylosinus trichosporium. Molecular phylogenies were constructed based on nearly complete 16S rRNA gene sequences, and on partial sequences of genes encoding PmoA (a subunit of particulate methane monooxygenase), MxaF (a subunit of methanol dehydrogenase) and MmoX (a subunit of soluble methane monooxygenase). The maximum pairwise 16S rDNA difference between isolates was 4.2%, and considerable variability was evident within the Methylocystis (maximum difference 3.6%). Due to this variability, some of the published 'specific' oligonucleotide primers for type II MOB exhibit multiple mismatches with gene sequences from some isolates. The phylogenetic tree constructed from pmoA gene sequences closely mirrored that constructed from 16S rDNA sequences, and both supported the presently accepted taxonomy of type II MOB. Contrary to previously published phylogenetic trees, morphologically distinguishable species were generally monophyletic based on pmoA or 16S rRNA gene sequences. This was not true for phylogenies constructed from mmoX and mxaF gene sequences. The phylogeny of mxaF gene sequences suggested that horizontal transfer of this gene may have occurred across type II MOB species. Soluble methane monooxygenase could not be detected in many Methylocystis strains either by an enzyme activity test (oxidation of naphthalene) or by PCR-based amplification of an mmoX gene.

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.

Monitoring impact of in situ biostimulation treatment on groundwater bacterial community by DGGE.

Changes in bacterial diversity during the field experiment on biostimulation were monitored by denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rDNA fragments. The results revealed that the bacterial community was disturbed after the start of treatment, continued to change for 45 days or 60 days and then formed a relatively stable community different from the original community structure. DGGE analysis of soluble methane monooxygenase (sMMO) hydroxylase gene fragments, mmoX, was performed to monitor the shifts in the numerically dominant sMMO-containing methanotrophs during the field experiment. Sequence analysis on the mmoX gene fragments from the DGGE bands implied that the biostimulation treatment caused a shift of potential dominant sMMO-containing methanotrophs from type I methanotrophs to type II methanotrophs.

Monitoring impact of in situ biostimulation treatment on groundwater bacterial community by DGGE

Changes in bacterial diversity during the field experiment on biostimulation were monitored by denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rDNA fragments. The results revealed that the bacterial community was disturbed after the start of treatment, continued to change for 45 days or 60 days and then formed a relatively stable community different from the original community structure. DGGE analysis of soluble methane monooxygenase (sMMO) hydroxylase gene fragments, mmoX, was performed to monitor the shifts in the numerically dominant sMMO-containing methanotrophs during the field experiment. Sequence analysis on the mmoX gene fragments from the DGGE bands implied that the biostimulation treatment caused a shift of potential dominant sMMO-containing methanotrophs from type I methanotrophs to type II methanotrophs.

Homologous expression of soluble methane monooxygenase genes in Methylosinus trichosporium OB3b.

An homologous expression system has been developed for soluble methane monooxygenase (sMMO) genes from Methylosinus trichosporium OB3b. sMMO-minus mutants were previously obtained after marker-exchange mutagenesis, by the insertion of a kanamycin-resistance cassette into the mmoX gene of the sMMO operon. Complementation of the sMMO-minus genotype was achieved by conjugation with broad-host-range plasmids containing the native promoter and sMMO operon from Ms. trichosporium OB3b (pVK100Sc and pHM2). In wild-type methanotrophs, copper ions present in the growth medium at concentrations greater than 0.25 microM inhibit transcription of sMMO genes. The stable maintenance of pVK100Sc resulted in transconjugant methanotrophs with a decreased sensitivity to copper, since expression of sMMO occurred at copper sulphate concentrations of 7.5 microM. sMMO activity was only detected in soluble extracts after the addition of purified sMMO reductase component, which is inhibited by copper ions in vitro. This phenomenon could have arisen due to the increased number of sMMO gene copies (derived from pVK100Sc) in the cell. Transconjugants obtained from conjugations with pHM2 expressed sMMO at copper concentrations of 0-2.5 microM only and sMMO activity was not restored by the addition of purified reductase component at copper concentrations higher than 2.5 microM. Southern hybridization showed that the plasmid had integrated into the chromosome, probably by a single homologous recombination event. This is the first report of homologous sMMO expression in a methanotroph with enzyme activities that are comparable to the activity reported in wild-type strains. This expression system will be useful for site-directed mutagenesis of active-site residues of sMMO from Ms. trichosporium OB3b.

Oxidation of trichloroethylene and dimethyl sulfide by a marine Methylomicrobium strain containing soluble methane monooxygenase.

Sixteen marine methanotrophic bacteria were isolated and 14 marine methanotrophic mixed cultures were obtained. They were assayed for soluble methane monooxygenase (sMMO) by naphthalene oxidation and only one isolate (strain NI) was positive. Strain NI degraded trichloroehylene (TCE) more efficiently than other methanotrophic isolates containing no sMMO only under copper limiting conditions. Dimethyl sulfide (DMS), one of the radiatively important trace gases released from the sea, was transformed to dimethyl sulfoxide (DMSO) by methanotrophs and the efficiency for the transformation of DMS to DMSO was not as much affected by the presence of sMMO as that of TCE. The taxonomical properties of strain NI and phylogenetic analysis based on 16S rDNA genes indicated that strain NI was a type I methanotroph belonging to the genus Methylomicrobium, and closely related to Methylomicrobium pelagicum. The partial mmoX gene of strain NI was amplified by the primers common to three other mmoX genes and its 270 bp were sequenced. 77 residues out of the 89 amino acids derived from the sequences were common among the four mmoX genes.

Detection of methanotrophic bacteria in environmental samples with the PCR.

We designed PCR primers by using the DNA sequences of the soluble methane monooxygenase gene clusters of Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath), and these primers were found to be specific for four of the five structural genes in the soluble methane monooxygenase gene clusters of several methanotrophs. We also designed primers for the gram-negative methylotroph-specific methanol dehydrogenase gene moxF. The specificity of these primers was confirmed by hybridizing and sequencing the PCR products obtained. The primers were then used to amplify methanotroph DNAs in samples obtained from various aquatic and terrestrial environments. Our sequencing data suggest that a large number of different methanotrophs are present in peat samples and also that there is a high level of variability in the mmoC gene, which codes for the reductase component of the soluble methane monooxygenase, while the mmoX gene, which codes for the alpha subunit of the hydroxylase component of this enzyme complex, appears to be highly conserved in methanotrophs.

Molecular genetics of methane oxidation.

Biological methane oxidation is carried out by methanotrophs, bacteria that utilize methane as their sole carbon and energy source. The enzyme they contain that is responsible for methane oxidation is methane monooxygenase, the most well studied being the soluble methane monooxygenase enzyme complexes from Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b. In both organisms, the genes encoding soluble methane monooxygenase have been found to be clustered on the chromosome in the order mmoX, mmoY, mmoB, mmoZ, orfY and mmoC. These genes encode the alpha and beta subunits of Protein A, Protein B, the gamma subunit of Protein A, a protein of unknown function and Protein C respectively of the soluble methane monooxygenase complex. The complete DNA sequences of both gene clusters have been determined and they show considerable homology. Expression of soluble methane monooxygenase genes occurs under growth conditions where the copper-to-biomass ratio is low. Transcriptional regulation of the gene cluster from Methylosinus occurred at an RpoN-like promoter, 5' of the mmoX gene. mmoB and mmoC of Methylococcus have been expressed in E. coli and the proteins obtained were functionally active. Soluble methane monooxygenase mutants have been constructed by marker-exchange mutagenesis. They were found to be more stable than those generated using the suicide substrate dichloromethane. Soluble methane monooxygenase probes have been used to detect both methane monooxygenase gene-specific DNA and methanotrophs in natural environmental samples.