Abstract
Methylococcus capsulatus (Bath) is a methanotroph that possesses both a membrane‐embedded (pMMO) and a soluble methane monooxygenase (sMMO). The expression of these two MMO's is tightly controlled by the availability of copper in the growth medium, but the underlying mechanisms and the number of genes involved in this switch in methane oxidation is not yet fully elucidated. Microarray analyses were used to assess the transcriptome in cells producing either pMMO or sMMO. A total of 137 genes were differentially expressed, with 87 genes showing a significant up‐regulation during sMMO production. The majority of the differentially expressed genes could be assigned to functional roles in the energy metabolism and transport. Furthermore, three copper responding gene clusters were discovered, including an extended cluster that also harbors the genes for sMMO. Our data also indicates that major changes takes place in the respiratory chain between pMMO‐ and sMMO‐producing cells, and that quinone are predominantly used as the electron donors for methane oxidation by pMMO. Intriguingly, a large proportion of the differentially expressed genes between pMMO‐ and sMMO‐producing cells encode c‐type cytochromes. By combining microarray‐ and mass spectrometry data, a total of 35 c‐type cytochromes are apparently expressed in M. capsulatus when grown in nitrate mineral salt medium with methane as sole energy and carbon source, and the expression of 21 of these respond to the availability of copper. Interestingly, several of these c‐type cytochromes are recovered from the cell surface, suggesting that extracellular electron transfers may occur in M. capsulatus.
Highlights
Methylococcus capsulatus (Bath) is an obligate aerobic methanotroph that utilize methane or methanol as sole carbon and energy source (Anthony 1982). It belongs to the gamma- proteobacteria and is similar to type I methanotrophs by having an incomplete TCA cycle and stacks of internal membranes where the enzyme for methane oxidation, the particulate methane monooxygenase, is located (Hanson and Hanson 1996; Semrau et al 2010). pMMO is a copper-containing enzyme, and is dependent on both Cu(I) and Cu(II) for its catalytic activity (Lieberman and Rosenzweig 2005)
Prior to the microarray analyses, cultures were screened for soluble and cytoplasmic methane monooxygenase (sMMO) activity with the naphthalene assay, and as expected, sMMO was only produced in the cultures with no added copper (Brusseau et al 1990)
Six and eight genes are related to the fatty acid metabolism and regulation of transcription, respectively, which most likely are functionally linked to the large morphological difference between pMMO-and sMMO-producing M. capsulatus (Prior and Dalton 1985)
Summary
Methylococcus capsulatus (Bath) is an obligate aerobic methanotroph that utilize methane or methanol as sole carbon and energy source (Anthony 1982) It belongs to the gamma- proteobacteria and is similar to type I methanotrophs by having an incomplete TCA cycle and stacks of internal membranes where the enzyme for methane oxidation, the particulate methane monooxygenase (pMMO), is located (Hanson and Hanson 1996; Semrau et al 2010). In contrast to pMMO, sMMO is an Fe-containing enzyme, and is not dependent on copper for methane oxidation (for a review on sMMO, see Merkx et al 2001) This change in how methane is oxidized, in addition to the morphological changes that occurs in response to the bioavailability of copper, are known as the “copper switch”, and has gained considerable interest (Murrell et al 2000; Hakemian and Rosenzweig 2007; Semrau et al 2010). The underlying mechanisms and the number of genes involved in the “copper switch” are not yet fully elucidated
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