Development of genetic tools in methanotrophs and the molecular regulation of methane monooxygenase

The oxidation of methane to methanol by methanotrophs is catalysed by the enzyme methane monooxygenase (MMO). In some methanotrophs two distinct MMOs are found; a membrane bound particulate (pMMO) and a cytoplasmic soluble (sMMO) methane monooxygenase. The intracellular location of MMO is dependent on the copper-to-biomass ratio. pMMO is expressed when the copper-to-biomass ratio is high (>0.25 IlM) and sMMO is expressed when the copper-to-biomass ratio is low «0.25 IlM). Although a great deal of information is known about the expression of MMO with respect to copper, the molecular mechanism regulating this 'copper-switch' is unknown. The aim of this study was to gain further insights into the regulation of MMO expression by copper ions. The complete sMMO operon, including the regulatory genes, mmoR and mmoG were cloned and sequenced from Methylosinus sporium. The genes encoding sMMO are present as single copy in methanotrophs. In this study, duplicate copies of the mmoX gene encoding for the a-subunit of the hydroxylase were characterised at the molecular and biochemical level. Mutational analysis indicated that the second copy was not essential for sMMO expression and also gave insights into the role of the water soluble pigment in siderophore-mediated iron-acquisition. sMMO-minus mutants were complemented by heterologous expression of sMMO genes from Methylosinus trichosporium and Methylococcus capsulatus. These experiments demonstrated the amenability of Ms. sporium to genetic manipulations facilitating its use as an alternative model organism for molecular analysis of MMO regulation. To aid transcriptional analysis of the MMO operons, a series of integrative and broad-host range promoter probe vectors, containing g{p, xylE, kmR or lacZ, were constructed and tested with the copper repressible sMMO 0'54 promoter. The usefulness of these reporter genes for the high-throughput detection of sMMO mutants was also assessed. The expression of LacZ in Mc. capsulatus via the sMMO 0'54 promoter yielded a powerful genetic screen for sMMO mutants. This system was coupled with transposon mutagenesis for surveying the genome on a global scale for sMMO regulatory genes and served as an alternative assay system for detecting sMMO expression. This assay system had the specific advantage in that it was more sensitive and in this context, it was selective for sMMO-minus mutants defective only at the transcriptional level. In collaboration with Robert Csaki (University of Szeged), a transposon mutagenesis protocol was established from which a number of sMMOminus mutants were identified. Genetic tools developed in this study were used to investigate copper mediated transcriptional regulation of the pMMO 0'70 promoter, sMMO 0'54 promoter and its regulatory genes, mmoR and mmoG. Transcription of the pMMO operon was shown to be constitutive. The sMMO 0'54 promoter was reconfirmed to be copper repressible and mmoR and mmoG were shown to be essential for transcription initiation from the sMMO 0'54 promoter, but were not regulated themselves by copper at the transcriptional level. All of these data were confirmed by constructing chromosomal gene fusions with various reporter genes, RT-PCR and RNA dot-blotting. The availability of the Mc. capsulatus genome sequence during this study allowed targeted mutagenesis to be carried out on copper targets responsible for copper transport. 'Knock-out' mutants of a copper transporting gene and a non-ribosomal peptide synthetase gene responsible for the putative biosynthesis of methanobactin, which is believed to be involved in delivering copper to pMMO, were constructed. The phenotypes of these mutants with regards to MMO expression are yet to be analysed.