Biological oxidation of methane to methanol by aerobic bacteria is catalysed by two different enzymes, the cytoplasmic or soluble methane monooxygenase (sMMO) and the membrane-bound or particulate methane monooxygenase (pMMO). Expression of MMOs is controlled by a 'copper-switch', i.e. sMMO is only expressed at very low copper : biomass ratios, while pMMO expression increases as this ratio increases. Methanotrophs synthesize a chalkophore, methanobactin, for the binding and import of copper. Previous work suggested that methanobactin was formed from a polypeptide precursor. Here we report that deletion of the gene suspected to encode for this precursor, mbnA, in Methylosinus trichosporium OB3b, abolishes methanobactin production. Further, gene expression assays indicate that methanobactin, together with another polypeptide of previously unknown function, MmoD, play key roles in regulating expression of MMOs. Based on these data, we propose a general model explaining how expression of the MMO operons is regulated by copper, methanobactin and MmoD. The basis of the 'copper-switch' is MmoD, and methanobactin amplifies the magnitude of the switch. Bioinformatic analysis of bacterial genomes indicates that the production of methanobactin-like compounds is not confined to methanotrophs, suggesting that its use as a metal-binding agent and/or role in gene regulation may be widespread in nature.

Methylocella silvestris BL2, a facultative methane utilizer, can grow on monomethylamine (MMA) as a sole carbon and nitrogen source. No activity of MMA dehydrogenase was detectable. Instead, this bacterium utilizes a methylated amino acid pathway (␥-glutamylmethylamide [GMA] and N-methylglutamate [NMG]) for MMA metabolism. The activities of the two key enzymes in this pathway, GMA synthetase and NMG dehydrogenase, were found when the bacterium was grown on MMA. GMA was detected by high-performance liquid chromatography-mass spectrometry only when the bacterium was grown on MMA but not when it was grown on methanol. Proteomic analysis of soluble and membrane fractions of the proteome from MMA-and methanolgrown cultures revealed that an eight-gene cluster (Msil2632 to Msil2639) was induced by MMA and cotranscribed as an operon, as shown by reverse transcription-PCR. GMA-dissimilating enzyme activity was also detected when it was grown on MMA. Formaldehyde and ammonium production from GMA was dependent on glutamate but not on ␣-ketoglutarate. Marker exchange mutagenesis of a putative GMAS gene homologue (gmas, Msil2635) within this eight-gene cluster, with a kanamycin gene cassette, abolished growth of M. silvestris on MMA as either a sole carbon or a sole nitrogen source. Overall, our results suggest that gmas is essential in MMA metabolism by M. silvestris.Monomethylamine (MMA) is ubiquitous in the environment. For example, putrefaction of proteins (14a, 17) and degradation of many nitrogen-containing pesticides and herbicides can release MMA (5, 16b, 18). In the marine environment, MMA is released from the degradation of quaternary amines, such as betaine, carnitine, choline, and trimethylamine N-oxide, which are used as osmolytes by many marine organisms (3, 6). Once released, MMA can be used by some microorganisms as a sole carbon and nitrogen source through different pathways. Methanogenic archaea, such as Methanosarcina and Methanomicrobium, can use MMA anaerobically as a substrate to produce methane via a methyltransferase system (28). Gram-positive bacteria, such as Arthrobacter, metabolize MMA aerobically via an oxidase, which breaks down MMA into formaldehyde and ammonium (39). Gram-negative bacteria such as Methylobacterium extorquens and Paracoccus denitrificans utilize MMA dehydrogenase, a multisubunit enzyme that generates formaldehyde and ammonium from MMA aerobically (9, 16). Many other Gram-negative bacteria, such as Aminobacter aminovorans (previously known as strain MA and strain MS), can use MMA as a sole carbon and nitrogen source aerobically; however, they lack MMA dehydrogenase. It has been shown that in these microorganisms, two unusual amino acids, ␥-glutamylmethylamide (GMA) and N-methylglutamate (NMG), are involved in MMA metabolism (1,23,33). In strain MA, an enzyme proposed as "NMG synthase" ("NMGS") converted MMA to NMG, which was subsequently oxidized to formaldehyde, regenerating glutamate (Glu) by a membrane-bounded particulate, NMG dehydrogenase (NMGDH) (33). The reactions carrie...

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 s N promoter 59 of mmoX. In this study the genes encoding s N (rpoN) and a typical s N -dependent transcriptional activator (mmoR) were cloned and sequenced. mmoR, a regulatory gene, and mmoG, a gene encoding a GroEL homologue, lie 59 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 s N plays a role in both nitrogen and carbon metabolism in Ms. trichosporiumOB3b. The data also indicate that mmoG is transcribed in a s 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.