rpoN, mmoR and mmoG, genes involved in regulating the expression of soluble methane monooxygenase in Methylosinus trichosporium OB3b

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Abstract
The methanotrophic bacteriumMethylosinus trichosporiumOB3b converts methane to methanol using two distinct forms of methane monooxygenase (MMO) enzyme: a cytoplasmic soluble form (sMMO) and a membrane-bound form (pMMO). The transcription of these two operons is known to proceed in a reciprocal fashion with sMMO expressed at low copper-to-biomass ratios and pMMO at high copper-to-biomass ratios. Transcription of thesmmooperon is initiated from aσNpromoter 5′ ofmmoX. In this study the genes encodingσN(rpoN) and a typicalσN-dependent transcriptional activator (mmoR) were cloned and sequenced.mmoR, a regulatory gene, andmmoG, a gene encoding a GroEL homologue, lie 5′ of the structural genes for the sMMO enzyme. Subsequent mutation ofrpoNandmmoRby marker-exchange mutagenesis resulted in strains Gm1 and JS1, which were unable to express functional sMMO or initiate transcription ofmmoX. AnrpoNmutant was also unable to fix nitrogen or use nitrate as sole nitrogen source, indicating thatσNplays a role in both nitrogen and carbon metabolism inMs. trichosporiumOB3b. The data also indicate thatmmoGis transcribed in aσN- and MmoR-independent manner. Marker-exchange mutagenesis ofmmoGrevealed that MmoG is necessary forsmmogene transcription and activity and may be an MmoR-specific chaperone required for functional assembly of transcriptionally competent MmoRin vivo. The data presented allow the proposal of a more complete model for copper-mediated regulation ofsmmogene expression.