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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].sup.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 mmeG 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.sup.-] 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.sup.-] 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.

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 Iow copper-to-biomass ratios and pMMO at high copper-to-biomass ratios. Transcription of the smmo operon is initiated from a [[sigma].sup.N] promoter 5' of mmoX. In this study the genes encoding [[sigma].sup.N] (rpoN) and a typical [[sigma].sup.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].sup.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].sup.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.

Researchers created a PCR primer based on the msmA gene from Marinosulfonomonas methylotropha strain TR3 to amplify corresponding genes in Hyphomicrobium, Methylobacterium, and Pedomicrobium species. The msm genes code for a methanesulfonate monooxygenase, which oxidizes methanesulfonic acid to formaldehyde and sulfite.

A gene cluster in Methylocella silvestris BL2, a facultative methane-oxidizing bacterium belonging to the Alphaproteobacteria was characterized to understand the critical role of gmas bacterium for monomethylamine (MMA) metabolism. The eight-gene cluster within a putative [gamma]-glutamylamide (GMAS) gene homologue (gmas, Msil2635) along with kanamycin gene cassette which abolished growth of M. silvestris on MMA as either a sole carbon or a sole nitrogen source indicated the significance of GMAS in MMA metabolism by M. silvestris.

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 (y-glutamylmethylamide EGMA] 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 chroma- tography-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 methanol- grown cultures revealed that an eight-gene cluster (Msi12632 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 α-ketoglutarate. Marker exchange mutagenesis of a putative GMAS gene homologue (gmas, Msi12635) 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. [ABSTRACT FROM AUTHOR]

Methanotrophs oxidize methane to methanol using the enzyme methane monooxygenase. Methylosinus trichosporium OB3b has two such enzymes: a membrane-bound particulate methane monooxygenase (pMMO) and a soluble, cytoplasmic methane monooxygenase (sMMO). In methanotrophs possessing both enzymes, the expression of the genes encoding sMMO and pMMO is regulated by copper ions, with sMMO expressed solely when copper is limiting. Virtually nothing is known about the specific machinery involved in the copper-regulated transcription of mmo genes except the identification of two proteins necessary for the expression: a σ54-dependent transcriptional activator, MmoR, and a putative GroEL-like chaperone, MmoG. Genes encoding mmoR and mmoG are located immediately upstream of those encoding sMMO in the genome of M. trichosporium OB3b. Here, we use a green fluorescent protein promoter probe vector to show that nearly the complete intergenic DNA sequence between mmoG and mmoX is absolutely required for transcriptional activation. Furthermore, we used gel-shift assays to demonstrate that both MmoR and MmoG were required for protein binding to this region of DNA. [ABSTRACT FROM AUTHOR]