Metabolism of methane and propane and the role of the glyoxylate bypass enzymes in Methylocella silvestris BL2 (2011)

Methylocella silvestris BL2 is a moderately acidophilic facultative methanotroph&#13;\nisolated from forest soil in 2003. Uniquely, it has the ability to grow on a wide range&#13;\nof multi-carbon compounds in addition to methane. An analysis of growth conditions&#13;\nidentified the requirements for robust and predictable growth on a wide range of&#13;\nsubstrates. A simple and effective method of targeted mutagenesis was developed,&#13;\nwhich relies on electroporation with a linear DNA fragment, and several strains with&#13;\ndeletions of key enzymes were constructed using this method. Deletion of isocitrate&#13;\nlyase demonstrated that this enzyme is required for growth on both one-carbon and&#13;\ntwo-carbon compounds. The second enzyme of the glyoxylate cycle, malate synthase,&#13;\nwas shown to be essential for growth on two-carbon compounds. However,&#13;\nsurprisingly, deletion of glyoxylate cycle enzymes had a dramatic effect on&#13;\nexpression of methanol dehydrogenase. Possible causes of this effect are discussed.&#13;\nSurprisingly, M. silvestris was able to grow on propane and the presence and&#13;\nexpression of a gene cluster encoding a putative propane monooxygenase was&#13;\nconfirmed. This enzyme was found to be a second soluble diiron monooxygenase&#13;\n(SDIMO) with homology to the propane monooxygenase from Gordonia TY5,&#13;\nidentifying M. silvestris as the first known methanotroph to contain SDIMOs from&#13;\nmore than one group. Deletion of these enzymes in turn was used to determine the&#13;\nrequirement for each during growth on methane or propane. The soluble methane&#13;\nmonooxygenase (sMMO) was found to be capable of oxidising propane, whereas the&#13;\npropane monooxygenase (PrMO) was unable to oxidise methane. However, although&#13;\na strain lacking the PrMO was capable of growth on 2.5% (v/v) propane, it was&#13;\nunable to grow on this gas at 20% (v/v), and at 2.5%, assimilation into biomass was&#13;\nless efficient in comparison to the wild-type. Evidence is presented that products of&#13;\noxidation of propane by the sMMO may be toxic to the cell or inhibitory to growth in&#13;\nthe absence of the PrMO. Both the sMMO and the PrMO were found to be capable of&#13;\noxidation of a wide range of aliphatic and aromatic compounds, including&#13;\nxenobiotics, suggesting a possible role in bioremediation. M. silvestris BL2 was&#13;\nfound to oxidise propane at both terminal and sub-terminal positions, resulting in 1-&#13;\npropanol and 2-propanol respectively, and biochemical methods were used to assay&#13;\nthe enzymes of terminal and sub-terminal pathways. Assimilation of 1-propanol was&#13;\nfound to be by the methylmalonyl-CoA pathway, and the data suggested that 2-&#13;\npropanol was oxidised to acetone and acetol. The final gene of the PrMO genecluster,&#13;\npredicted to encode a flavin adenine dinucleotide (FAD)-containing enzyme&#13;\nwith homology to characterised membrane-bound D-gluconate dehydrogenase from&#13;\nGluconobacter spp., was found to be essential for growth on 2-propanol and acetone&#13;\nand may be involved in the oxidation of acetol during propane metabolism by the&#13;\nsub-terminal pathway.