SummaryThe molecular regulation of methane oxidation in the first fully authenticated facultative methanotroph Methylocella silvestris BL2 was assessed during growth on methane and acetate. Problems of poor growth of Methylocella spp. in small-scale batch culture were overcome by growth in fermentor culture. The genes encoding soluble methane monooxygenase were cloned and sequenced, which revealed that the structural genes for soluble methane monooxygenase, mmoXYBZDC , were adjacent to two genes, mmoR and mmoG , encoding a σ σ σ σ 54 transcriptional activator and a putative GroEL-like chaperone, located downstream (3 ′ ′ ′ ′ ) of mmoC . Transcriptional analysis revealed that the genes were all cotranscribed from a σ σ σ σ 54 -dependent promoter located upstream (5 ′ ′ ′ ′ ) of mmoX . The transcriptional start site was mapped. Transcriptional analysis of soluble methane monooxygenase genes and expression studies on fermentor grown cultures showed that acetate repressed transcription of sMMO in M. silvestris BL2. The possibility of the presence of a particulate, membrane-bound methane monooxygenase enzyme in M. silvestris BL2 and the copper-mediated regulation of soluble methane monooxygenase was investigated. Both were shown to be absent. A promoter probe vector was constructed and used to assay transcription of the promoter of the soluble methane monoxygenase genes of M. silvestris BL2 grown under various conditions and with different substrates. These data represent the first insights into the molecular physiology of a facultative methanotroph.

Methylocella silvestris BL2 is an aerobic methanotroph originally isolated from an acidic forest soil in Germany. It is the first fully authenticated facultative methanotroph. It grows not only on methane and other one-carbon (C1) substrates, but also on some compounds containing carbon-carbon bonds, such as acetate, pyruvate, propane, and succinate. Here we report the full genome sequence of this bacterium

Methane is one of the most important greenhouse gases, and its concentration in the atmosphere is increasing by approximately 1% per year (16). Methane-oxidizing bacteria, or methanotrophs, are a key group of bacteria involved in the global methane cycle and can be found in many environments. For example, they limit the efflux of methane produced in flooded soils and wetlands and consume atmospheric methane directly in aerated upland soils (2,14). Methanotrophs have generally been considered to be obligate in nature, i.e., growing only on methane as their sole source of carbon and energy. In this issue of the Journal of Bacteriology, Dedysh and colleagues provide the first unequivocal proof of a genus of methanotrophic bacteria (Methylocella) that are capable of growth on a number of multicarbon substrates, dispelling the notion that methanotrophy is an obligate phenomenon (6). In a century of research since the Dutch microbiologist Söhngen described the first methanotroph, Bacillus methanicus, in 1906 (25), significant advances in understanding the ecology and physiology of these organisms have contributed to our understanding of methane cycling in the environment. A brief history of the field will allow the reader to appreciate the nature of this highly controversial topic in the field of bacterial one-carbon metabolism.Claims for the existence of facultative methanotrophs (i.e., methanotrophs capable of growth on multicarbon as well as one-carbon substrates) have a long and somewhat checkered history dating back almost 30 years, when Patt et al. first isolated (22) and later described (23) Methylobacterium organophilum, which was able to grow on methane or glucose. This was followed by other reports of facultative methanotrophs, notably strain R6, Methylobacterium ethanolicum, and Methylomonas sp. strain 761M (18,21,32). In most cases, either the methane-oxidizing capacity was lost or the results were not substantiated in other laboratories. For example, for M. organophilum it was hypothesized that methane oxidation genes were plasmid encoded and that the loss of growth on methane was attributable to loss of a plasmid. In the case of M. ethanolicum, the culture was shown to be a very tight syntrophic association between a methanotroph and a Xanthobacter species (17). The controversy surrounding facultative methanotrophs was recently highlighted by the description of a "methane-oxidizing" methylotroph, Methylobacterium populi, and the rebuttal that followed (5, 28). Thankfully, doubts about the existence of facultative methanotrophs seem finally to have been dispelled by the meticulous experiments performed by Dedysh et al. (6). Quantitative real-time PCR targeting the mmoX gene, which encodes the hydroxylase component of the soluble methane monooxygenase (sMMO), the key enzyme for methane oxidation in Methylocella silvestris BL2, showed a parallel increase of copies of mmoX and microscopic cell counts in both methane-and acetate-grown cultures. In addition to fluorescence in situ hybridization using strain-an...