Methane monooxygenase gene expression mediated by methanobactin in the presence of mineral copper sources

Abstract

Methane is a major greenhouse gas linked to global warming; however, patterns of in situ methane oxidation by methane-oxidizing bacteria (methanotrophs), nature's main biological mechanism for methane suppression, are often inconsistent with laboratory predictions. For example, one would expect a strong relationship between methanotroph ecology and Cu level because methanotrophs require Cu to sustain particulate methane monooxygenase (pMMO), the most efficient enzyme for methane oxidation. However, no correlation has been observed in nature, which is surprising because methane monooxygenase (MMO) gene expression has been unequivocally linked to Cu availability. Here we provide a fundamental explanation for this lack of correlation. We propose that MMO expression in nature is largely controlled by solid-phase Cu geochemistry and the relative ability of Cu acquisition systems in methanotrophs, such as methanobactins (mb), to obtain Cu from mineral sources. To test this hypothesis, RT-PCR expression assays were developed for Methylosinus trichosporium OB3b (which produces mb) to quantify pMMO, soluble MMO (the alternate MMO expressed when Cu is "unavailable"), and 16S-rRNA gene expression under progressively more stringent Cu supply conditions. When Cu was provided as CuCl(2), pMMO transcript levels increased significantly consistent with laboratory work. However, when Cu was provided as Cu-doped iron oxide, pMMO transcript levels increased only when mb was also present. Finally, when Cu was provided as Cu-doped borosilicate glass, pMMO transcription patterns varied depending on the ambient mb:Cu supply ratio. Cu geochemistry clearly influences MMO expression in terrestrial systems, and, as such, local Cu mineralogy might provide an explanation for methane oxidation patterns in the natural environment.

Fig. 1.

pmoA (pMMO subunit), mmoX (sMMO subunit), and 16S-rRNA gene transcript levels in a M. trichosporium OB3b presented with different sudden copper exposures: copper-free NSM media (A–C), 5 μM as CuCl₂ (D–F), 5 μM copper provided as Cu-doped iron oxide (G–I), and 5 μM Cu supplied as Cu-borosilicate glass (J–I). Transcript levels were determined at three different levels of mb: no mb (●), 0.2:1 mb:Cu stoichiometry (○), and 2.0:1 mb:Cu stoichiometry (▾). Dashed lines represent RT-PCR detection limits for pmoA and mmoX.

Fig. 2.

16S-rRNA transcript levels normalized to initial 16S-rRNA levels in M. trichosporium OB3b exposed to increasing levels of CuCl₂: 0 μM CuCl₂ (A), 10 μM CuCl₂ (B), and 25 μM CuCl₂ (C).