scholarly journals Methane Monooxygenase Gene Transcripts as Quantitative Biomarkers of Methanotrophic Activity in Methylosinus trichosporium OB3b

2020 ◽  
Vol 86 (23) ◽  
Author(s):  
Egidio F. Tentori ◽  
Ruth E. Richardson
Keyword(s):  
Methane Oxidation ◽  
Methane Monooxygenase ◽  
Methane Emissions ◽  
Mrna Transcript ◽  
Methylosinus Trichosporium ◽  
Transcript Levels ◽  
Content Type ◽  
Oxidation Rates ◽  
Pure Cultures

ABSTRACT Methanotrophic microorganisms are characterized by their ability to oxidize methane. Globally they have a significant impact on methane emissions by attenuating net methane fluxes to the atmosphere in natural and engineered systems, though the populations are dynamic in their activity level in soils and waters. Methanotrophs oxidize methane using methane monooxygenase (MMO) enzymes, and selected subunit genes of the most common MMOs, specifically pmoA and mmoX, are used as biomarkers for the presence and abundance of populations of bacterial methanotrophs. The relative expression of these biomarker genes is dependent on copper-to-biomass ratios. Empirically derived quantitative relationships between methane oxidation biomarker transcript amounts and methanotrophic activity could facilitate determination of methane oxidation rates. In this study, pure cultures of a model type II methanotroph, Methylosinus trichosporium OB3b, were grown in hollow-fiber membrane bioreactors (HFMBR) under different steady-state methane oxidation conditions. Methanotroph biomass (DNA based) and methane oxidation biomarker mRNA transcript amounts were determined using quantitative PCR (qPCR) and reverse transcription-PCR (RT-qPCR), respectively. Under both copper-present and copper-limited conditions, per-cell pmoA mRNA transcript levels positively correlated with measured per-cell methane oxidation rates across 3 orders of magnitude. These correlations, if maintained across different methanotrophs, could prove valuable for inferring in situ oxidation rates of methanotrophs and understanding the dynamics of their impact on net methane emissions. IMPORTANCE Methanotrophs are naturally occurring microorganisms capable of oxidizing methane and have an impact on global net methane emissions. The genes pmoA and mmoX are used as biomarkers for bacterial methanotrophs. Quantitative relationships between transcript amounts of these genes and methane oxidation rates could facilitate estimation of methanotrophic activity. In this study, a strong correlation was observed between per-cell pmoA transcript levels and per-cell methane oxidation rates for pure cultures of the aerobic methanotroph M. trichosporium OB3b grown in bioreactors. If similar relationships exist across different methanotrophs, they could prove valuable for inferring in situ oxidation rates of methanotrophs and better understanding their impact on net methane emissions.

scholarly journals Methanobactin from Methylocystis sp. Strain SB2 Affects Gene Expression and Methane Monooxygenase Activity in Methylosinus trichosporium OB3b

2015 ◽  
Vol 81 (7) ◽  
pp. 2466-2473 ◽  
Author(s):  
Muhammad Farhan Ul-Haque ◽  
Bhagyalakshmi Kalidass ◽  
Alexey Vorobev ◽  
Bipin S. Baral ◽  
Alan A. DiSpirito ◽  
...  
Keyword(s):  
Gene Expression ◽  
Methane Monooxygenase ◽  
Particulate Methane Monooxygenase ◽  
Methylosinus Trichosporium Ob3b ◽  
Methylosinus Trichosporium ◽  
Monooxygenase Activity ◽  
Content Type ◽  
Soluble Methane Monooxygenase ◽  
A Subunit ◽  
Membrane Bound

ABSTRACTMethanotrophs can express a cytoplasmic (soluble) methane monooxygenase (sMMO) or membrane-bound (particulate) methane monooxygenase (pMMO). Expression of these MMOs is strongly regulated by the availability of copper. Many methanotrophs have been found to synthesize a novel compound, methanobactin (Mb), that is responsible for the uptake of copper, and methanobactin produced byMethylosinus trichosporiumOB3b plays a key role in controlling expression of MMO genes in this strain. As all known forms of methanobactin are structurally similar, it was hypothesized that methanobactin from one methanotroph may alter gene expression in another. WhenMethylosinus trichosporiumOB3b was grown in the presence of 1 μM CuCl2, expression ofmmoX, encoding a subunit of the hydroxylase component of sMMO, was very low.mmoXexpression increased, however, when methanobactin fromMethylocystissp. strain SB2 (SB2-Mb) was added, as did whole-cell sMMO activity, but there was no significant change in the amount of copper associated withM. trichosporiumOB3b. IfM. trichosporiumOB3b was grown in the absence of CuCl2, themmoXexpression level was high but decreased by several orders of magnitude if copper prebound to SB2-Mb (Cu-SB2-Mb) was added, and biomass-associated copper was increased. Exposure ofMethylosinus trichosporiumOB3b to SB2-Mb had no effect on expression ofmbnA, encoding the polypeptide precursor of methanobactin in either the presence or absence of CuCl2.mbnAexpression, however, was reduced when Cu-SB2-Mb was added in both the absence and presence of CuCl2. These data suggest that methanobactin acts as a general signaling molecule in methanotrophs and that methanobactin “piracy” may be commonplace.

scholarly journals Competition between Metals for Binding to Methanobactin Enables Expression of Soluble Methane Monooxygenase in the Presence of Copper

2014 ◽  
Vol 81 (3) ◽  
pp. 1024-1031 ◽  
Author(s):  
Bhagyalakshmi Kalidass ◽  
Muhammad Farhan Ul-Haque ◽  
Bipin S. Baral ◽  
Alan A. DiSpirito ◽  
Jeremy D. Semrau
Keyword(s):  
Methane Monooxygenase ◽  
High Specificity ◽  
Copper Uptake ◽  
Content Type ◽  
Soluble Methane Monooxygenase ◽  
Sds Page ◽  
Genes Encoding ◽  
Key Factor ◽  
Membrane Bound

ABSTRACTIt is well known that copper is a key factor regulating expression of the two forms of methane monooxygenase found in proteobacterial methanotrophs. Of these forms, the cytoplasmic, or soluble, methane monooxygenase (sMMO) is expressed only at low copper concentrations. The membrane-bound, or particulate, methane monooxygenase (pMMO) is constitutively expressed with respect to copper, and such expression increases with increasing copper. Recent findings have shown that copper uptake is mediated by a modified polypeptide, or chalkophore, termed methanobactin. Although methanobactin has high specificity for copper, it can bind other metals, e.g., gold. Here we show that inMethylosinus trichosporiumOB3b, sMMO is expressed and active in the presence of copper if gold is also simultaneously present. Such expression appears to be due to gold binding to methanobactin produced byM. trichosporiumOB3b, thereby limiting copper uptake. Such expression and activity, however, was significantly reduced if methanobactin preloaded with copper was also added. Further, quantitative reverse transcriptase PCR (RT-qPCR) of transcripts of genes encoding polypeptides of both forms of MMO and SDS-PAGE results indicate that both sMMO and pMMO can be expressed when copper and gold are present, as gold effectively competes with copper for binding to methanobactin. Such findings suggest that under certain geochemical conditions, both forms of MMO may be expressed and activein situ. Finally, these findings also suggest strategies whereby field sites can be manipulated to enhance sMMO expression, i.e., through the addition of a metal that can compete with copper for binding to methanobactin.

UK landfill methane emissions: Use of mobile plume measurements and carbon isotopic characterisation to reassess oxidation rates for open and closed sites 

2021 ◽  
Author(s):  
Semra Bakkaloglu ◽  
Dave Lowry ◽  
Rebecca Fisher ◽  
James France ◽  
Euan Nisbet
Keyword(s):  
Methane Oxidation ◽  
Mole Fraction ◽  
Oxidation Rate ◽  
Methane Emissions ◽  
Isotopic Signature ◽  
Oxidation Rates ◽  
Landfill Cover ◽  
Carbon Isotopic ◽  
Landfill Sites ◽  
The Impact

<p>Biological methane oxidation in landfill cover material can be characterised using stable isotopes. Methane oxidation fraction is calculated from the carbon isotopic signature of emitted CH<sub>4</sub>, with enhanced microbial consumption of methane in the aerobic portion of the landfill cover indicated by a shift to less depleted isotopic values in the residual methane emitted to air. This study was performed at four southwest England landfill sites. Mobile mole fraction measurement at the four sites was coupled with Flexfoil bag sampling of air for high-precision isotope analysis. Gas well samples collected from the pipeline systems and downwind plume air samples were utilized to estimate methane oxidation rate for whole sites. This work was designed to assess the impact on carbon isotopic signature and oxidation rate as UK landfill practice and waste streams have changed in recent years.</p><p>The landfill status such as closed and active, seasonal variation, cap stripping and site closure impact on landfill isotopic signature and oxidation rate were evaluated. The isotopic signature of <sup>13</sup>C-CH<sub>4</sub> values of emissions varied between -60 and -54‰, with an averaged value of -57 +- 2‰ for methane from closed and active landfill sites. Methane emissions from older, closed landfill sites were typically more enriched in <sup>13</sup>C than emissions from active sites. This study found that the isotopic signature of <sup>13</sup>C-CH<sub>4</sub> of fugitive methane did not show a seasonal trend, and there was no plume observed from a partial cap stripping process to assess changes in <sup>13</sup>C-CH<sub>4</sub>  isotopic signatures of emitted methane. Also, the closure of an active landfill cell caused a significant decrease in mole fraction of measured CH<sub>4</sub>, which was less depleted <sup>13</sup>C in the emitted plume due to a higher oxidation rate. Methane oxidation, estimated from the isotope fractionation, ranged from 3 to 27%, with mean values of 7% and 15% for active and closed landfills, respectively. These results indicate that the oxidation rate is highly site specific.</p><p> </p>

scholarly journals Abundance and Activity of Methanotrophic Bacteria in Littoral and Profundal Sediments of Lake Constance (Germany)

2008 ◽  
Vol 75 (1) ◽  
pp. 119-126 ◽  
Author(s):  
M. Rahalkar ◽  
J. Deutzmann ◽  
B. Schink ◽  
I. Bussmann
Keyword(s):  
Methane Oxidation ◽  
Lake Constance ◽  
Type I ◽  
Sediment Depth ◽  
Oxidation Activity ◽  
Oxidation Rates ◽  
Methane Oxidizing Bacteria ◽  
Littoral Sediment ◽  
Sediment Layers

ABSTRACT The abundances and activities of aerobic methane-oxidizing bacteria (MOB) were compared in depth profiles of littoral and profundal sediments of Lake Constance, Germany. Abundances were determined by quantitative PCR (qPCR) targeting the pmoA gene and by fluorescence in situ hybridization (FISH), and data were compared to methane oxidation rates calculated from high-resolution concentration profiles. qPCR using type I MOB-specific pmoA primers indicated that type I MOB represented a major proportion in both sediments at all depths. FISH indicated that in both sediments, type I MOB outnumbered type II MOB at least fourfold. Results obtained with both techniques indicated that in the littoral sediment, the highest numbers of methanotrophs were found at a depth of 2 to 3 cm, corresponding to the zone of highest methane oxidation activity, although no oxygen could be detected in this zone. In the profundal sediment, highest methane oxidation activities were found at a depth of 1 to 2 cm, while MOB abundance decreased gradually with sediment depth. In both sediments, MOB were also present at high numbers in deeper sediment layers where no methane oxidation activity could be observed.

scholarly journals Acetate Repression of Methane Oxidation by Supplemental Methylocella silvestris in a Peat Soil Microcosm

2011 ◽  
Vol 77 (12) ◽  
pp. 4234-4236 ◽  
Author(s):  
M. Tanvir Rahman ◽  
Andrew Crombie ◽  
Hélène Moussard ◽  
Yin Chen ◽  
J. Colin Murrell
Keyword(s):  
Stable Isotope ◽  
Methane Oxidation ◽  
Environmental Samples ◽  
Peat Soil ◽  
Methane Monooxygenase ◽  
Laboratory Culture ◽  
Stable Isotope Probing ◽  
Soil Microcosm ◽  
Content Type

ABSTRACTMethylocellaspp. are facultative methanotrophs that grow on methane and multicarbon substrates, such as acetate. Acetate represses transcription of methane monooxygenase ofMethylocella silvestrisin laboratory culture. DNA stable-isotope probing (DNA-SIP) using13C-methane and12C-acetate, carried out withMethylocella-spiked peat soil, showed that acetate also repressed methane oxidation byMethylocellain environmental samples.

scholarly journals Growth and Methane Oxidation Rates of Anaerobic Methanotrophic Archaea in a Continuous-Flow Bioreactor

2003 ◽  
Vol 69 (9) ◽  
pp. 5472-5482 ◽  
Author(s):  
Peter R. Girguis ◽  
Victoria J. Orphan ◽  
Steven J. Hallam ◽  
Edward F. DeLong
Keyword(s):  
Methane Oxidation ◽  
Continuous Flow ◽  
Molecular Techniques ◽  
Anaerobic Methane Oxidation ◽  
Cold Seep ◽  
Microbial Consortia ◽  
Sulfate Reducing ◽  
Oxidation Rates ◽  
In Situ Conditions ◽  
Pure Cultures

ABSTRACT Anaerobic methanotrophic archaea have recently been identified in anoxic marine sediments, but have not yet been recovered in pure culture. Physiological studies on freshly collected samples containing archaea and their sulfate-reducing syntrophic partners have been conducted, but sample availability and viability can limit the scope of these experiments. To better study microbial anaerobic methane oxidation, we developed a novel continuous-flow anaerobic methane incubation system (AMIS) that simulates the majority of in situ conditions and supports the metabolism and growth of anaerobic methanotrophic archaea. We incubated sediments collected from within and outside a methane cold seep in Monterey Canyon, Calif., for 24 weeks on the AMIS system. Anaerobic methane oxidation was measured in all sediments after incubation on AMIS, and quantitative molecular techniques verified the increases in methane-oxidizing archaeal populations in both seep and nonseep sediments. Our results demonstrate that the AMIS system stimulated the maintenance and growth of anaerobic methanotrophic archaea, and possibly their syntrophic, sulfate-reducing partners. Our data demonstrate the utility of combining physiological and molecular techniques to quantify the growth and metabolic activity of anaerobic microbial consortia. Further experiments with the AMIS system should provide a better understanding of the biological mechanisms of methane oxidation in anoxic marine environments. The AMIS may also enable the enrichment, purification, and isolation of methanotrophic archaea as pure cultures or defined syntrophic consortia.

scholarly journals Marker Exchange Mutagenesis ofmxaF, Encoding the Large Subunit of the Mxa Methanol Dehydrogenase, in Methylosinus trichosporium OB3b

2015 ◽  
Vol 82 (5) ◽  
pp. 1549-1555 ◽  
Author(s):  
Muhammad Farhan Ul Haque ◽  
Wenyu Gu ◽  
Alan A. DiSpirito ◽  
Jeremy D. Semrau
Keyword(s):  
Large Subunit ◽  
Methane Monooxygenase ◽  
Methanol Dehydrogenase ◽  
Methylosinus Trichosporium Ob3b ◽  
Initial Oxidation ◽  
Methylosinus Trichosporium ◽  
Content Type ◽  
Marker Exchange ◽  
Oxidation Of Methane ◽  
Soluble Methane Monooxygenase

ABSTRACTMethanotrophs have remarkable redundancy in multiple steps of the central pathway of methane oxidation to carbon dioxide. For example, it has been known for over 30 years that two forms of methane monooxygenase, responsible for oxidizing methane to methanol, exist in methanotrophs, i.e., soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO), and that expression of these two forms is controlled by the availability of copper. Specifically, sMMO expression occurs in the absence of copper, while pMMO expression increases with increasing copper concentrations. More recently, it was discovered that multiple forms of methanol dehydrogenase (MeDH), Mxa MeDH and Xox MeDH, also exist in methanotrophs and that the expression of these alternative forms is regulated by the availability of cerium. That is, expression of Xox MeDH increases in the presence of cerium, while Mxa MeDH expression decreases in the presence of cerium. As it had been earlier concluded that pMMO and Mxa MeDH form a supercomplex in which electrons from Mxa MeDH are back donated to pMMO to drive the initial oxidation of methane, we speculated that Mxa MeDH could be rendered inactive through marker-exchange mutagenesis but growth on methane could still be possible if cerium was added to increase the expression of Xox MeDH under sMMO-expressing conditions. Here we report thatmxaF, encoding the large subunit of Mxa MeDH, could indeed be knocked out inMethylosinus trichosporiumOB3b, yet growth on methane was still possible, so long as cerium was added. Interestingly, growth of this mutant occurred in both the presence and the absence of copper, suggesting that Xox MeDH can replace Mxa MeDH regardless of the form of MMO expressed.

scholarly journals Methanol Promotes Atmospheric Methane Oxidation by Methanotrophic Cultures and Soils

1998 ◽  
Vol 64 (3) ◽  
pp. 1091-1098 ◽  
Author(s):  
J. Benstead ◽  
G. M. King ◽  
H. G. Williams
Keyword(s):  
Methane Oxidation ◽  
Standard Errors ◽  
Atmospheric Methane ◽  
Methylosinus Trichosporium ◽  
Methane Consumption ◽  
Air Supply ◽  
Chemostat Cultures ◽  
Methane Uptake ◽  
Kinetics Of

ABSTRACT Two methanotrophic bacteria, Methylobacter albus BG8 and Methylosinus trichosporium OB3b, oxidized atmospheric methane during batch growth on methanol. Methane consumption was rapidly and substantially diminished (95% over 9 days) when washed cell suspensions were incubated without methanol in the presence of atmospheric methane (1.7 ppm). Methanotrophic activity was stimulated after methanol (10 mM) but not methane (1,000 ppm) addition. M. albus BG8 grown in continuous culture for 80 days with methanol retained the ability to oxidize atmospheric methane and oxidized methane in a chemostat air supply. Methane oxidation during growth on methanol was not affected by methane deprivation. Differences in the kinetics of methane uptake (apparent Km andV max) were observed between batch- and chemostat-grown cultures. The V max and apparent Km values (means ± standard errors) for methanol-limited chemostat cultures were 133 ± 46 nmol of methane 108 cells−1 h−1and 916 ± 235 ppm of methane (1.2 μM), respectively. These values were significantly lower than those determined with batch-grown cultures (V max of 648 ± 195 nmol of methane 108 cells−1 h−1 and apparent Km of 5,025 ± 1,234 ppm of methane [6.3 μM]). Methane consumption by soils was stimulated by the addition of methanol. These results suggest that methanol or other nonmethane substrates may promote atmospheric methane oxidation in situ.

scholarly journals A TonB-Dependent Transporter Is Responsible for Methanobactin Uptake by Methylosinus trichosporium OB3b

2016 ◽  
Vol 82 (6) ◽  
pp. 1917-1923 ◽  
Author(s):  
Wenyu Gu ◽  
Muhammad Farhan Ul Haque ◽  
Bipin S. Baral ◽  
Erick A. Turpin ◽  
Nathan L. Bandow ◽  
...  
Keyword(s):  
Methane Monooxygenase ◽  
Copper Uptake ◽  
Wild Type ◽  
Particulate Methane Monooxygenase ◽  
Methylosinus Trichosporium Ob3b ◽  
Gene Encoding ◽  
Methylosinus Trichosporium ◽  
Content Type ◽  
Soluble Methane Monooxygenase ◽  
Genes Encoding

ABSTRACTMethanobactin, a small modified polypeptide synthesized by methanotrophs for copper uptake, has been found to be chromosomally encoded. The gene encoding the polypeptide precursor of methanobactin,mbnA, is part of a gene cluster that also includes several genes encoding proteins of unknown function (but speculated to be involved in methanobactin formation) as well asmbnT, which encodes a TonB-dependent transporter hypothesized to be responsible for methanobactin uptake. To determine ifmbnTis truly responsible for methanobactin uptake, a knockout was constructed inMethylosinus trichosporiumOB3b using marker exchange mutagenesis. The resultingM. trichosporiummbnT::Gmrmutant was found to be able to produce methanobactin but was unable to internalize it. Further, if this mutant was grown in the presence of copper and exogenous methanobactin, copper uptake was significantly reduced. Expression ofmmoXandpmoA, encoding polypeptides of the soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO), respectively, also changed significantly when methanobactin was added, which indicates that the mutant was unable to collect copper under these conditions. Copper uptake and gene expression, however, were not affected in wild-typeM. trichosporiumOB3b, indicating that the TonB-dependent transporter encoded bymbnTis responsible for methanobactin uptake and that methanobactin is a key mechanism used by methanotrophs for copper uptake. When thembnT::Gmrmutant was grown under a range of copper concentrations in the absence of methanobactin, however, the phenotype of the mutant was indistinguishable from that of wild-typeM. trichosporiumOB3b, indicating that this methanotroph has multiple mechanisms for copper uptake.

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