Methylococcus capsulatus (Bath) is a Gram-negative, spherical-shaped bacterium that gains its needs of carbon and energy via oxidation of methane, a potent greenhouse gas, thus alleviating global warming. This bacterium oxidises methane to methanol using a membrane-bound particulate methane monooxygenase (pMMO) or a soluble, cytoplasmic methane monooxygenases (sMMO). Copper-tobiomass- ratios significantly affect the expression and activity of both enzymes; the biosynthesis of sMMO is switched on when copper-to-biomass ratios are low, while pMMO is up-regulated when they are high. The exact mechanisms by which copper regulates the switching between sMMO and pMMO are not fully elucidated. Therefore, the main aim of this study was to shed some light on this copper switch, taking the advantage of the availability of the genome sequence of this organism, together with mutagenesis and transcriptional regulation studies. Three potential copper transport Mc. capsulatus mutants; ΔcopA1, ΔcopA2 and ΔcopA3 were generated. The genes inactivated encode three different P-type ATPase homologs. This revealed that CopA1, CopA2 and CopA3 have roles in copper homeostasis, although disruption of genes encoding these proteins individually did not result in constitutive sMMO expression. In addition, three mutants; ΔnrpS-1, ΔnrpS-2 and pkS were constructed. pkS encodes for a polyketide synthase, nrpS-1 and nrpS-2 encode for two non-ribosomal peptide synthetases. The products of these genes were proposed to be involved in biosynthesis of methanobactin, a short peptide that scavenges copper when it is limited. Results suggested that nrpS-2 and pkS might be involved in production of a functional methanobactin. Putative coding sequences predicted to be involved in methanobactin biosynthesis in another methane-oxidising bacterium, Methylosinus trichosporium (OB3b), were also mutated. The mutant was unable to produce methanobactin, could not express sMMO, and was copper resistant compared to the wild-type organism. Therefore, methanobactin is ribosomally- produced in Ms. trichosporium. Corresponding genes were not identified in the genome of Mc. capsulatus. A microarray-based comparative expression profiling study of whole-genome transcriptomics of Mc. capsulatus expressing sMMO versus pMMO was carried out to identify genes involved in regulation of MMO by copper. This identified 53 genes that were differentially expressed and hence promising candidate genes for future studies of MMO regulation. For example, tetR, a down-regulated gene, encodes a putative transcriptional regulator and tonB, an up-regulated gene, which encodes a protein that is a part of a membrane transporter. Interestingly, a cluster of six genes 5’ of sMMO was up-regulated; five of them were found to be co-transcribed. A mutant was made in an up-regulated gene encoding ScO protein (synthesis of cytochrome c). The mutant could tolerate high concentrations of copper compared to the wild-type strain. The work presented in this study is considered a step forward towards understanding the regulatory mechanisms of the copper switch in methanotrophs and provided the basis for new lines of future research to fully understand this phenomenon.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:560328 |
| Date | January 2012 |
| Creators | Khalifa, Ashraf |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/49461/ |
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