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.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:550192 |
| Date | January 2011 |
| Creators | Crombie, Andrew |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/45158/ |
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