Trace-gas metabolic versatility of the facultative methanotroph Methylocella silvestris

Crombie, Andrew T.; Murrell, J. Colin

doi:10.1038/nature13192

Cited by 85 publications

(88 citation statements)

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“…However, they differ with regard to their substrate utilization patterns. Methylocella species are facultative methanotrophs, which, in addition to C 1 compounds, utilize acetate and several other organic acids, ethanol, and some short-chain alkanes (2–4). In contrast, M.…”

Section: Genome Announcementmentioning

confidence: 99%

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Draft Genome Sequence of Methyloferula stellata AR4, an Obligate Methanotroph Possessing Only a Soluble Methane Monooxygenase

Dedysh¹,

Naumoff²,

Vorobev

et al. 2015

Genome Announc

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Section: Genome Announcementmentioning

confidence: 99%

“…An additional soluble diiron monooxygenase, i.e., propane monooxygenase, which is present in Methylocella silvestris BL2 (3, 11), is lacking in M. stellata.…”

Section: Genome Announcementmentioning

confidence: 99%

Draft Genome Sequence of Methyloferula stellata AR4, an Obligate Methanotroph Possessing Only a Soluble Methane Monooxygenase

Dedysh¹,

Naumoff²,

Vorobev

et al. 2015

Genome Announc

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“…Genetic manipulation of methanotrophic bacteria is often a slow process with marker exchange mutagenesis typically taking weeks per mutation, making it difficult to construct multiple mutations in a timely manner (9,10). Counterselection protocols can accelerate the process of creating multiple mutations in a strain by eliminating the need for antibiotic marker recycling and can enable other unmarked genomic manipulations such as point mutations and promoter swaps.…”

mentioning

confidence: 99%

Genetic Tools for the Industrially Promising Methanotroph Methylomicrobium buryatense

Puri

Owen

Chu

et al. 2015

Appl Environ Microbiol

161

173

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c Aerobic methanotrophs oxidize methane at ambient temperatures and pressures and are therefore attractive systems for methane-based bioconversions. In this work, we developed and validated genetic tools for Methylomicrobium buryatense, a haloalkaliphilic gammaproteobacterial (type I) methanotroph. M. buryatense was isolated directly on natural gas and grows robustly in pure culture with a 3-h doubling time, enabling rapid genetic manipulation compared to many other methanotrophic species. As a proof of concept, we used a sucrose counterselection system to eliminate glycogen production in M. buryatense by constructing unmarked deletions in two redundant glycogen synthase genes. We also selected for a more genetically tractable variant strain that can be conjugated with small incompatibility group P (IncP)-based broad-host-range vectors and determined that this capability is due to loss of the native plasmid. These tools make M. buryatense a promising model system for studying aerobic methanotroph physiology and enable metabolic engineering in this bacterium for industrial biocatalysis of methane. Methane, the principal component of natural gas, is a promising feedstock for industrial biotechnology. Domestic natural gas supplies are increasing, and natural gas prices have remained lower than oil prices over the last decade (1, 2). Methane has also been identified as a prime mitigation target for reducing greenhouse gas emissions (3). Aerobic methanotrophs oxidize methane under ambient temperatures and pressures and are genetically tractable, making these bacteria potentially useful biocatalysts for converting natural gas into chemicals and liquid fuels via metabolic engineering (1, 4).Previous industrial interest in aerobic methanotrophs has included attempts to produce biomass for use as single-cell protein or carotenoids in animal feed (5, 6). However, production of biomass from natural gas is complicated by the fact that nonmethane components of natural gas can be toxic to methanotrophs and can also provide substrates for growth of contaminating nonmethanotrophic bacterial species (5). Methylomicrobium buryatense is a gammaproteobacterial (type I) methanotroph that was isolated directly on natural gas from a soda lake in the Transbaikal region of Russia, a harsh environment with fluctuating temperatures, salinity, and pH levels (7). M. buryatense is naturally adapted to this environment and moderately haloalkaliphilic and consequently grows robustly in pure culture on natural gas, with increased resistance to contamination. The genome of this species was also recently sequenced and annotated by the Joint Genome Institute (8), enabling genomic manipulation for metabolic engineering. These characteristics make M. buryatense a strain of interest for industrial biotechnology.Genetic manipulation of methanotrophic bacteria is often a slow process with marker exchange mutagenesis typically taking weeks per mutation, making it difficult to construct multiple mutations in a timely manner (9, 10). Counterselection prot...

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“…Methanotrophs are widespread in the environment, found in diverse locations such as landfill cover, forest, agricultural, and volcanic soils; freshwater and marine sediments; and sewage sludge (6,10,11). Many are amenable to genetic manipulation (12)(13)(14)(15)(16)(17)(18).…”

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confidence: 99%

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

Kalidass

Haque

Baral

et al. 2015

Appl Environ Microbiol

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It 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 in Methylosinus trichosporium OB3b, 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 by M. trichosporium OB3b, 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 (RTqPCR) 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 active in 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.T he increased availability of methane through industrial practices such as hydraulic fracturing has enhanced interest in using methanotrophs, prokaryotes that thrive on methane as their sole source of carbon and energy, to convert methane to more valuable products, e.g., liquid biofuels, plastics, and protein to supplement animal feed (1-7). Methanotrophs have also received increasing attention given that methane is a very potent greenhouse gas, with a global warming potential 28 to 34 times that of carbon dioxide over a 100-year period (8), and methanotrophs are estimated to remove up to 90% of the methane produced in anaerobic soils (9). Methanotrophs are widespread in the environment, found in diverse locations such as landfill cover, forest, agricultural, and volcanic soils; freshwater and marine sediments; and sewage sludge (6, 10, 11). Many are amenable to genetic manipulation (12-18).An important issue, however, in the use of methanotrophy for any purpose is that the first step in methane oxidation, the conversion of methane to methanol, is performed by two different forms of methane monooxygenase (MMO) with different oxidation kinetics and affinities for methane. One form, the soluble methane monooxygenase (sMMO), is found in the cytoplasm and has relatively high turnover (maximal whole-cell methane oxidation rate [V max ] of ϳ730 nmol · min Ϫ...

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Trace-gas metabolic versatility of the facultative methanotroph Methylocella silvestris

Cited by 85 publications

References 42 publications

Draft Genome Sequence of Methyloferula stellata AR4, an Obligate Methanotroph Possessing Only a Soluble Methane Monooxygenase

Draft Genome Sequence of Methyloferula stellata AR4, an Obligate Methanotroph Possessing Only a Soluble Methane Monooxygenase

Genetic Tools for the Industrially Promising Methanotroph Methylomicrobium buryatense

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

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