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Duplication of the mmoX gene in Methylosinus sporium: cloning, sequencing and mutational analysis

Duplication of the mmoX gene in Methylosinus sporium: cloning, sequencing and mutational analysis
Duplication of the mmoX gene in Methylosinus sporium: cloning, sequencing and mutational analysis
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 ? 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 ? 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
2931-2942
Ali, Hanif
cabd35ca-19d9-4d77-8767-4cf04aaf6cb6
Scanlan, Julie
76e5fb31-0ca6-4980-8b6e-20d5ee1ca27d
Dumont, Marc
afd9f08f-bdbb-4cee-b792-1a7f000ee511
Murrell, J. Colin
22f4dcaa-2fab-4b88-9365-0418bae53ec9
Ali, Hanif
cabd35ca-19d9-4d77-8767-4cf04aaf6cb6
Scanlan, Julie
76e5fb31-0ca6-4980-8b6e-20d5ee1ca27d
Dumont, Marc
afd9f08f-bdbb-4cee-b792-1a7f000ee511
Murrell, J. Colin
22f4dcaa-2fab-4b88-9365-0418bae53ec9

Ali, Hanif, Scanlan, Julie, Dumont, Marc and Murrell, J. Colin (2006) Duplication of the mmoX gene in Methylosinus sporium: cloning, sequencing and mutational analysis. Microbiology, 152 (10), 2931-2942. (doi:10.1099/mic.0.29031-0). (PMID:17005974)

Record type: Article

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 ? 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 ? 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

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Accepted/In Press date: 14 June 2006
Published date: October 2006
Organisations: Centre for Biological Sciences

Identifiers

Local EPrints ID: 387950
URI: https://eprints.soton.ac.uk/id/eprint/387950
PURE UUID: 8134946c-bbda-46fa-962e-188d3ebeb3ec
ORCID for Marc Dumont: ORCID iD orcid.org/0000-0002-7347-8668

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Date deposited: 20 Jun 2016 12:46
Last modified: 06 Jun 2018 12:20

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