Transcriptomic profiling ofMethylococcus capsulatus(Bath) during growth with two different methane monooxygenases

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.

Chalkophores

“…It also does not bind heparin, commonly used as a proxy for DNA binding (71). Despite reports to the contrary (62), mmoD is downregulated in response to copper in at least two species (71, 73), which would be unexpected for a copper-binding repressor. Finally, in the obligate sMMO-producing Methylocella silvestris BL2, which has MmoD but not the pMMO or Mbn operons, copper addition does not repress sMMO (131).…”

“…Interestingly, genes encoding regulatory proteins in both operons, including mbnI and mbnR in the mbn operon and mmoR, the recently identified mmoF (71, 73), and mmoG in the sMMO operon (Figure 2), exhibit similar patterns. All five genes are almost completely downregulated within 15 minutes of copper addition, while the transcription levels of the remaining genes in the two operons drop primarily over 5- and 24-hour time periods (71).…”

“…However, neither Mbn nor sMMO operons are necessary for fully triggering the copper switch. Species lacking Mbn operons can still switch between sMMO and pMMO production (35, 73), indicating that Mbn is not primarily responsible for copper-dependent repression of the sMMO operon. Moreover, species lacking either the sMMO operon (and thus MmoD) or both the sMMO and Mbn operons still exhibit copper-responsive phenotypes, including decreased Mbn production and intracytoplasmic membrane formation (77, 129, 130).…”

Methanobactins: Maintaining copper homeostasis in methanotrophs and beyond

“…There is also no evidence that MmoD binds DNA, copper, or Mbn/CuMbn (71,80). Despite claims to the contrary (47), MmoD transcription is strongly copper-repressed in several species (70,71,76), which is incompatible with several regulatory schemes. Finally, the overlap between species producing sMMO, pMMO, and Mbn is quite small (20).…”

Metabolic engineering of methanotrophs and its application to production of chemicals and biofuels from methane

“…Microarray analyses have been used to assess the transcriptome in cells of Methylococcus capsulatus (Bath) during growth with two different methane monooxygenases, either pMMO or sMMO. Quinone was predominantly employed as the electron donor for methane oxidation in pMMO‐producing cells in the presence of copper. In addition, the expression of 21 of 35 genes encoding c‐type cytochromes were dependent on the availability of copper …”