Characterization of the Molecular Basis of Regulation of Gene Expression by Metals in Methanotrophs

Abstract

Methane-oxidizing bacteria, or methanotrophs, can use methane as their sole carbon and energy source, and have a wide range of applications including: (1) methane removal from the atmosphere, (2) pollutant degradation, and (3) valorization of methane. Such applications are strongly dependent on copper and rare earth elements (REEs) due to their central role in regulating the metabolism of methanotrophs. To fully utilize these intriguing microbes for various applications, the genetics and biochemistry of metal uptake systems in methanotrophs must thus be identified and characterized. To address this general goal, this work first sought to characterize the evolution of methanotrophs to glean insights into potential uptake systems of copper and REEs, and to develop strategies to optimize their production for industrial and medical applications. Bioinformatic analyses suggested that methylotrophs with preexisting copper uptake system(s) may have evolved into methanotrophs with the lateral gene transfer of methane monooxygenase, the critical enzyme that oxidizes methane to methanol in methanotrophs. In addition, lateral gene transfer events of methanol dehydrogenase (MeDH) with a REE active site (Xox-MeDH) were more prevalent than those of MeDH with Ca(II). This may be attributable to the higher catalytic efficiency of the former MeDH, which consequently increased the fitness of methanotrophs with multiple copies. Second, competition between methanotrophs for copper was investigated to identify “cheating” behavior amongst methanotrophs and determine how the collective activity of methanotrophs is affected. Some methanotrophs produce methanobactin (MB), a chalkophore that can strongly bind and deliver copper. It was found that methanotrophs Methylomicrobium album BG8 and Methylocystis sp. strain Rockwell, both non-MB producers, can take up MB, while Methylococcus capsulatus Bath cannot. In addition, Mmb. album BG8 was found to produce a novel chalkophore yet to be characterized. Moreover, Mmb. album BG8 and Methylocystis sp. strain Rockwell could also take up methylmercury-MB (MeHg-MB) complex and subsequently demethylate MeHg into the less toxic inorganic mercury. The results of this study provide insight into copper competition between methanotrophs and potential applications exploiting such interaction, such as MeHg remediation. Finally, the mechanism of methanotrophic-mediated MeHg demethylation was investigated. It has been found that MB serves as a delivery mechanism for MeHg into the cell of methanotrophs, where Xox-MeDH contributes to demethylating MeHg. The genes encoding for organoarsenical lyase (ArsI), responsible for cleaving the carbon-arsenic bond, and lanmodulin (LanM), a periplasmic REE-binding protein, were each knocked out in wildtype Methylosinus trichosporium OB3b. Deletion of arsI did not affect MeHg degradation in the Msn. trichosporium OB3b delarsI mutant. However, the dellanM mutant was unable to degrade MeHg under all conditions tested, suggesting lanM to be critical for MeHg degradation. In addition, a spheroplast prepared from Msn. trichosporium OB3b delmbnT mutant exhibited decreased MeHg degradation, whereas greater MeHg degradation was observed in the extract containing the periplasm and outer membrane debris. These results suggest that MeHg degradation occurs in the periplasm, where both Xox-MeDH and LanM reside. The results of this study are anticipated to contribute to our ability to utilize methanotrophy for a wide range of applications.