Metabolism of methane and propane and the role of the glyoxylate bypass enzymes in Methylocella silvestris BL2

Methylocella silvestris BL2 is a moderately acidophilic facultative methanotroph isolated from forest soil in 2003. Uniquely, it has the ability to grow on a wide range of multi-carbon compounds in addition to methane. An analysis of growth conditions identified the requirements for robust and predictable growth on a wide range of substrates. A simple and effective method of targeted mutagenesis was developed, which relies on electroporation with a linear DNA fragment, and several strains with deletions of key enzymes were constructed using this method. Deletion of isocitrate lyase demonstrated that this enzyme is required for growth on both one-carbon and two-carbon compounds. The second enzyme of the glyoxylate cycle, malate synthase, was shown to be essential for growth on two-carbon compounds. However, surprisingly, deletion of glyoxylate cycle enzymes had a dramatic effect on expression of methanol dehydrogenase. Possible causes of this effect are discussed. Surprisingly, M. silvestris was able to grow on propane and the presence and expression of a gene cluster encoding a putative propane monooxygenase was confirmed. This enzyme was found to be a second soluble diiron monooxygenase (SDIMO) with homology to the propane monooxygenase from Gordonia TY5, identifying M. silvestris as the first known methanotroph to contain SDIMOs from more than one group. Deletion of these enzymes in turn was used to determine the requirement for each during growth on methane or propane. The soluble methane monooxygenase (sMMO) was found to be capable of oxidising propane, whereas the propane monooxygenase (PrMO) was unable to oxidise methane. However, although a strain lacking the PrMO was capable of growth on 2.5% (v/v) propane, it was unable to grow on this gas at 20% (v/v), and at 2.5%, assimilation into biomass was less efficient in comparison to the wild-type. Evidence is presented that products of oxidation of propane by the sMMO may be toxic to the cell or inhibitory to growth in the absence of the PrMO. Both the sMMO and the PrMO were found to be capable of oxidation of a wide range of aliphatic and aromatic compounds, including xenobiotics, suggesting a possible role in bioremediation. M. silvestris BL2 was found to oxidise propane at both terminal and sub-terminal positions, resulting in 1- propanol and 2-propanol respectively, and biochemical methods were used to assay the enzymes of terminal and sub-terminal pathways. Assimilation of 1-propanol was found to be by the methylmalonyl-CoA pathway, and the data suggested that 2- propanol was oxidised to acetone and acetol. The final gene of the PrMO genecluster, predicted to encode a flavin adenine dinucleotide (FAD)-containing enzyme with homology to characterised membrane-bound D-gluconate dehydrogenase from Gluconobacter spp., was found to be essential for growth on 2-propanol and acetone and may be involved in the oxidation of acetol during propane metabolism by the sub-terminal pathway

Metabolism of methane and propane and the role of the glyoxylate bypass enzymes in Methylocella silvestris BL2

Methylocella silvestris BL2 is a moderately acidophilic facultative methanotroph isolated from forest soil in 2003. Uniquely, it has the ability to grow on a wide range of multi-carbon compounds in addition to methane. An analysis of growth conditions identified the requirements for robust and predictable growth on a wide range of substrates. A simple and effective method of targeted mutagenesis was developed, which relies on electroporation with a linear DNA fragment, and several strains with deletions of key enzymes were constructed using this method. Deletion of isocitrate lyase demonstrated that this enzyme is required for growth on both one-carbon and two-carbon compounds. The second enzyme of the glyoxylate cycle, malate synthase, was shown to be essential for growth on two-carbon compounds. However, surprisingly, deletion of glyoxylate cycle enzymes had a dramatic effect on expression of methanol dehydrogenase. Possible causes of this effect are discussed. Surprisingly, M. silvestris was able to grow on propane and the presence and expression of a gene cluster encoding a putative propane monooxygenase was confirmed. This enzyme was found to be a second soluble diiron monooxygenase (SDIMO) with homology to the propane monooxygenase from Gordonia TY5, identifying M. silvestris as the first known methanotroph to contain SDIMOs from more than one group. Deletion of these enzymes in turn was used to determine the requirement for each during growth on methane or propane. The soluble methane monooxygenase (sMMO) was found to be capable of oxidising propane, whereas the propane monooxygenase (PrMO) was unable to oxidise methane. However, although a strain lacking the PrMO was capable of growth on 2.5% (v/v) propane, it was unable to grow on this gas at 20% (v/v), and at 2.5%, assimilation into biomass was less efficient in comparison to the wild-type. Evidence is presented that products of oxidation of propane by the sMMO may be toxic to the cell or inhibitory to growth in the absence of the PrMO. Both the sMMO and the PrMO were found to be capable of oxidation of a wide range of aliphatic and aromatic compounds, including xenobiotics, suggesting a possible role in bioremediation. M. silvestris BL2 was found to oxidise propane at both terminal and sub-terminal positions, resulting in 1- propanol and 2-propanol respectively, and biochemical methods were used to assay the enzymes of terminal and sub-terminal pathways. Assimilation of 1-propanol was found to be by the methylmalonyl-CoA pathway, and the data suggested that 2- propanol was oxidised to acetone and acetol. The final gene of the PrMO genecluster, predicted to encode a flavin adenine dinucleotide (FAD)-containing enzyme with homology to characterised membrane-bound D-gluconate dehydrogenase from Gluconobacter spp., was found to be essential for growth on 2-propanol and acetone and may be involved in the oxidation of acetol during propane metabolism by the sub-terminal pathway.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

The effect of lanthanum on growth and gene expression in a facultative methanotroph

The biological importance of lanthanides has only recently been identified, initially as the active site metal of the alternative methanol dehydrogenase (MDH) Xox-MDH. So far, the effect of lanthanide (Ln) has only been studied in relatively few organisms. This work investigated the effects of Ln on gene transcription and protein expression in the facultative methanotroph Methylocella silvestris BL2, a widely distributed methane-oxidizing bacterium with the unique ability to grow not just on methane but also on other typical components of natural gas, ethane and propane. Expression of calcium- or Ln-dependent MDH was controlled by Ln (the lanthanide switch) during growth on one-, two- or three-carbon substrates, and Ln imparted a considerable advantage during growth on propane, a novel result extending the importance of Ln to consumers of this component of natural gas. Two Xox-MDHs were expressed and regulated by Ln in M. silvestris, but interestingly Ln repressed rather than induced expression of the second Xox-MDH. Despite the metabolic versatility of M. silvestris, no other alcohol dehydrogenases were expressed, and in double-mutant strains lacking genes encoding both Ca- and Ln-dependent MDHs (mxaF and xoxF5 or xoxF1), growth on methanol and ethanol appeared to be enabled by expression of the soluble methane monooxygenase

Isoprene oxidation by the gram-negative model bacterium variovorax sp. WS11

Plant-produced isoprene (2-methyl-1,3-butadiene) represents a significant portion of global volatile organic compound production, equaled only by methane. A metabolic pathway for the degradation of isoprene was first described for the Gram-positive bacterium Rhodococcus sp. AD45, and an alternative model organism has yet to be characterised. Here, we report the characterisation of a novel Gram-negative isoprene-degrading bacterium, Variovorax sp. WS11. Isoprene metabolism in this bacterium involves a plasmid-encoded iso metabolic gene cluster which differs from that found in Rhodococcus sp. AD45 in terms of organisation and regulation. Expression of iso metabolic genes is significantly upregulated by both isoprene and epoxyisoprene. The enzyme responsible for the initial oxidation of isoprene, isoprene monooxygenase, oxidises a wide range of alkene substrates in a manner which is strongly influenced by the presence of alkyl side-chains and differs from other well-characterised soluble diiron monooxygenases according to its response to alkyne inhibitors. This study presents Variovorax sp. WS11 as both a comparative and contrasting model organism for the study of isoprene metabolism in bacteria, aiding our understanding of the conservation of this biochemical pathway across diverse ecological niches

Microbial metabolism of isoprene: a much-neglected climate-active gas

The climate-active gas isoprene is the major volatile produced by a variety of trees and is released into the atmosphere in enormous quantities, on a par with global emissions of methane. While isoprene production in plants and its effect on atmospheric chemistry have received considerable attention, research into the biological isoprene sink has been neglected until recently. Here, we review current knowledge on the sources and sinks of isoprene and outline its environmental effects. Focusing on degradation by microbes, many of which are able to use isoprene as the sole source of carbon and energy, we review recent studies characterizing novel isoprene degraders isolated from soils, marine sediments and in association with plants. We describe the development and use of molecular methods to identify, quantify and genetically characterize isoprene-degrading strains in environmental samples. Finally, this review identifies research imperatives for the further study of the environmental impact, ecology, regulation and biochemistry of this interesting group of microbes

Genome Scale Metabolic Model of the versatile methanotroph Methylocella silvestris

BACKGROUND: Methylocella silvestris is a facultative aerobic methanotrophic bacterium which uses not only methane, but also other alkanes such as ethane and propane, as carbon and energy sources. Its high metabolic versatility, together with the availability of tools for its genetic engineering, make it a very promising platform for metabolic engineering and industrial biotechnology using natural gas as substrate. RESULTS: The first Genome Scale Metabolic Model for M. silvestris is presented. The model has been used to predict the ability of M. silvestris to grow on 12 different substrates, the growth phenotype of two deletion mutants (ΔICL and ΔMS), and biomass yield on methane and ethanol. The model, together with phenotypic characterization of the deletion mutants, revealed that M. silvestris uses the glyoxylate shuttle for the assimilation of C1 and C2 substrates, which is unique in contrast to published reports of other methanotrophs. Two alternative pathways for propane metabolism have been identified and validated experimentally using enzyme activity tests and constructing a deletion mutant (Δ1641), which enabled the identification of acetol as one of the intermediates of propane assimilation via 2-propanol. The model was also used to integrate proteomic data and to identify key enzymes responsible for the adaptation of M. silvestris to different substrates. CONCLUSIONS: The model has been used to elucidate key metabolic features of M. silvestris, such as its use of the glyoxylate shuttle for the assimilation of one and two carbon compounds and the existence of two parallel metabolic pathways for propane assimilation. This model, together with the fact that tools for its genetic engineering already exist, paves the way for the use of M. silvestris as a platform for metabolic engineering and industrial exploitation of methanotrophs

Draft Genome Sequences of Obligate Methylotrophs <i>Methylovorus</i> sp. Strain MM2 and <i>Methylobacillus</i> sp. Strain MM3, Isolated from Grassland Soil

Methylotrophs of the family Methylophilaceae were isolated from grassland soil. Here, we report the draft genome sequences of two obligate methylotrophs, Methylovorus sp. strain MM2 and Methylobacillus sp. strain MM3. These genome sequences provide further insights into the genetic and metabolic diversity of the Methylophilaceae

Facultative methanotrophs - diversity, genetics, molecular ecology and biotechnological potential:a mini-review

Methane-oxidizing bacteria (methanotrophs) play a vital role in reducing atmospheric methane emissions, and hence mitigating their potent global warming effects. A significant proportion of the methane released is thermogenic natural gas, containing associated short-chain alkanes as well as methane. It was one hundred years following the description of methanotrophs that facultative strains were discovered and validly described. These can use some multi-carbon compounds in addition to methane, often small organic acids, such as acetate, or ethanol, although Methylocella strains can also use short-chain alkanes, presumably deriving a competitive advantage from this metabolic versatility. Here, we review the diversity and molecular ecology of facultative methanotrophs. We discuss the genetic potential of the known strains and outline the consequent benefits they may obtain. Finally, we review the biotechnological promise of these fascinating microbes