The methanotrophic bacterium Methylosinus trichosporium OB3b 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 the smmo operon is initiated from a sigma(N) promoter 5' of mmoX. In this study the genes encoding sigma(N) (rpoN) and a typical sigma(N)-dependent transcriptional activator (mmoR) were cloned and sequenced. mmoR, a regulatory gene, and mmoG, a gene encoding a GroEL homologue, lie 5' of the structural genes for the sMMO enzyme. Subsequent mutation of rpoN and mmoR by marker-exchange mutagenesis resulted in strains Gm1 and JS1, which were unable to express functional sMMO or initiate transcription of mmoX. An rpoN mutant was also unable to fix nitrogen or use nitrate as sole nitrogen source, indicating that sigma(N) plays a role in both nitrogen and carbon metabolism in Ms. trichosporium OB3b. The data also indicate that mmoG is transcribed in a sigma(N)- and MmoR-independent manner. Marker-exchange mutagenesis of mmoG revealed that MmoG is necessary for smmo gene transcription and activity and may be an MmoR-specific chaperone required for functional assembly of transcriptionally competent MmoR in vivo. The data presented allow the proposal of a more complete model for copper-mediated regulation of smmo gene expression.