Methanotrophic bacteria convert methane to methanol using a methane monooxygenase enzyme (MMO). Two types of MMO exist: a membrane bound enzyme (PMMO) and a cytoplasmic enzyme (sMMO). A system for the site-directed mutagenesis of residues in the active site of sMMO has recently been developed that uses a sMMO-minus strain of Methylosinus trichosporium OB3b as the expression host (Smith et al., 2002. Appl. Environ. Microbiol. 68:5265-5273); this strain, designated Mutant F, was created by disrupting the mmoX gene by marker-exchange mutagenesis. In this study a Ms. trichosporium OB3b strain was created in which all the sMMO structural genes were deleted or disrupted. This mutant was designated Ms. trichosporium SMDM. The recombinant expression of sMMO was performed in Ms. trichosporium SMDM using the same sMMO expression plasmid used for Ms. trichosporium Mutant F. The effect of sMMO expression in the absence of the enigmatic mmoD gene was investigated. Preliminary results indicate that mmoD is required for active expression of sMMO. The sMMO genes from Methylocella silvestris BL2T were sequenced and conjugated into Ms. trichosporium SMDM on a broad host range plasmid. No expression of Methylocella silvestris BL2 T sMMO was detected in Ms. trichosporium SMDM. A new system for the mutagenesis of the Ms. trichosporium OB3b sMMO a-subunit was created. Chimaeric sMMO mutants were created by introducing gene sequence from the alkene monooxygenase enzyme of Rhodococcus corallin us into the mmoX. The chimaeric sMMO enzymes appeared to be unstable in Ms. trichosporium Mutant F. An attempt was made to improve the stability of sMMO mutants in Ms. trichosporium Mutant F by disrupting the gene encoding the Lon protease. The Ms. trichosporium OB3b Ion gene was cloned and sequenced and attempts were made to disrupt the Ms. trichosporium OB3b Ion by marker-exchange mutagenesis. A mutant was not obtained, suggesting that Lon may be essential for vegetative growth of Ms. trichosporium OB3b. The diversity of sMMO in several environmental samples was investigated using PCR. The objective was to isolate novel mmoX sequences from uncultivated methanotrophs that could be used to design sMMO mutagenesis experiments. New PCR primers targeting the mmoX were developed. The primers were used to generate libraries from a blanket bog peat (UK) and from cave water (Romania). A group of sequences that did not cluster with the mmoX of any cultivated methanotroph was obtained from the cave water. The use of a PCR independent approach to clone methanotroph genes from environmental samEles was also investigated. This was performed by developing a method to clone 1 C-DNA from a stable isotope probing experiment with 13CH4 into a BAC vector. A library of 2300 clones was generated. Greater than 95 % of plasmids analysed contained inserts, which ranged in size from approximately 10 - 30 kb. The library was screened for mxaF, mmoX and pmoA by colony hybridization. A clone (15 kb) containing pmoA was completely sequenced. Other genes encoding proteins with (potential) roles in methylotrophy were contained on the clone, includingpmoC, pmoB, folP, folK, mptG and moxF.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:414315 |
| Date | January 2004 |
| Creators | Dumont, Marc G. |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/67795/ |
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