The development of genetic techniques for the obligate methanotroph, Methylococcus capsulatus (Bath)
In order to further the study of the molecular biology and biochemistry of the obligate methanotroph Methylococcus capsulatus (Bath) several genetic techniques where investigated. A system for transfer of plasmids from E.coli to M.capsulatus by conjugation using a filter-mating technique was developed, and a variety of broad-host-range plasmids were transferred. Several parameters which could effect the efficiency of plasmid transfer were assessed, including the possible presence of a restriction/modification system, and it was found that transfer was at its' most efficient when M.capsulatus was in early logarithmic growth, transfer took place over 24 hours at 37oC, and the ratio of donor to recipient was at least 1:5. It was also possible to transfer the plasmids RP4 and pULB113 from M.capsulatus to E.coli. The initial development of a vector for analysis of promoter expression in M.capsulatus was attempted. Several plasmids were also investigated for their ability to act as mutagenesis vectors in M.capsulatus. pSUP2021 was found to transfer into M.capsulatus and Tn5, present on the vector, was shown to be inserted into the chromosome. The entire pJFF350 vector was found to be transferred into the chromosome, including Omegon-kanamycin fragment, IS1 ends and RP4-mob fragment. This vector was used to produce novel plasmids containing pJFF350 DNA and M.capsulatus chromosomal DNA. Two vectors, developed for marker-exchange mutagenesis of glutamine synthetase, were tested and found to be unsuccessful. A further vector was developed from pJFF350 and was found to transfer into the chromosome, but as yet its exact position of insertion is unclear. Electroporation was investigated, however, despite the alteration of electrical parameters and the pretreatment of cells no success was achieved. Results appeared to indicate a problem with a restriction/modification system. The mobilization of the chromosome using RP4 prime plasmids and pULBl13 was attempted but was unsuccessful.