title: Duplication of the mmoX gene in Methylosinus sporium: cloning, sequencing and mutational analysis.
abstract: The soluble methane monooxygenase (sMMO) is a key enzyme for methane oxidation, and is found in only some methanotrophs, including Methylosinus sporium 5. sMMO expression is regulated at the level of transcription from a sigma(54) promoter by a copper-switch, and is only expressed when the copper-to-biomass ratio during growth is low. Extensive phylogenetic and genetic analyses of sMMOs and other soluble di-iron monooxygenases reveal that these enzymes have only been acquired relatively recently through horizontal gene transfer. In this study, further evidence of horizontal gene transfer was obtained, through cloning and sequencing of the genes encoding the sMMO enzyme complex plus the regulatory genes mmoG and mmoR, and identification of a duplicate copy of the mmoX gene in Ms. sporium. mmoX encodes the alpha subunit of the hydroxylase of the sMMO enzyme, which constitutes the active site (Prior & Dalton, 1985). The mmoX genes were characterized at the molecular and biochemical levels. Although both copies were transcribed, only mmoX copy 1 was essential for sMMO activity. Construction of an sMMO(-) mutant by marker-exchange mutagenesis gave some possible insights into the role of the water-soluble pigment in siderophore-mediated iron acquisition. Finally, the amenability of Ms. sporium to genetic manipulation was demonstrated by complementing the sMMO(-) mutant by heterologous expression of sMMO genes from Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath), and it was shown that Ms. sporium could be used as an alternative model organism for molecular analysis of MMO regulation.
authors.affiliation: Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK.
authors.forename: Hanif
authors.initials: H
authors.lastname: Ali
authors.affiliation: Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK.
authors.forename: Julie
authors.initials: J
authors.lastname: Scanlan
authors.affiliation: Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK.
authors.forename: Marc G
authors.initials: MG
authors.lastname: Dumont
authors.affiliation: Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK.
authors.forename: J Colin
authors.initials: JC
authors.lastname: Murrell
mesh_terms: D001665:Binding Sites / Q000235:genetics; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D017353:Gene Deletion; D020440:Gene Duplication; D005798:Genes, Bacterial; D005809:Genes, Regulator; D005816:Genetic Complementation Test; D007501:Iron / Q000378:metabolism; D020581:Methylosinus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D009876:Operon; D010105:Oxygenases / Q000235:genetics*; D010802:Phylogeny; D010860:Pigments, Biological / Q000502:physiology; D021122:Protein Subunits / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D004269:DNA, Bacterial; D010860:Pigments, Biological; D021122:Protein Subunits; D007501:Iron; D010105:Oxygenases; C021555:methane monooxygenase