Metals and Methanotrophs: 1. Genetic and Biochemical Characterization of the Uptake and Synthesis of Methanobactin; 2. Bioinformatic Analyses of the Effect of Rare Earth Elements on Gene Expression.

Abstract

Methanotrophs are a group of bacteria that use methane as their sole carbon and energy source. These microbes have various applications including methane removal, biodegradation of halogenated hydrocarbons, and valorization of methane to various products including biofuels, bioplastics, and single cell protein. Current obstacles for the application of aerobic methanotrophs include incomplete understanding of their metabolism and genetics. This work studies the methanotrophic response to metals, i.e., copper and rare earth elements, with the goal of achieving better control of methanotrophic activity. The expression and activities of alternative forms of methane monooxygenases in methanotrophs are regulated by the availability of copper. The genetic regulation by copper in methanotrophs involves in a copper-chelating molecule called methanobactin (mb) produced by methanotrophs. First, the uptake mechanism of mb was investigated. mbnT, encoding for a TonB-dependent transporter, was knocked-out in Methylosinus trichosporium OB3b. The mutant was able to synthesize and secrete mb but not take it up as evidenced by significant decrease in copper uptake when grown at presence of exogenous mb. The mutant was, however, still able to take up free copper, indicating that there is (are) alternative copper uptake pathway(s) in M. trichosporium OB3b. Second, the biosynthesitic pathway of mb was investigated. Specifically, mbnN, encoding for an aminotransferase, was disrupted in M. trichosporium OB3b. mb produced by this mutant has only one of the two oxazolone rings and the C-terminal methionine was missing. This study lays the foundation for achieving fine-tuning mb structure and for enhancing its production for potential applications. In addition to copper, it was found that cerium also regulates key enzymes in methanotrophs, i.e., alternative forms of methanol dehydrogenases (MeDHs). This finding was first extended to consider the effect of other rare earth elements (REEs). It was found that lanthanum, praseodymium, neodymium and samarium also regulate the expression of MeDHs in M. trichosporium OB3b. These effects, however, were only observed in the absence of copper, indicating cross-regulation by copper and REEs. Second, the whole transcriptomic response to copper and/or cerium in M. trichosporium OB3b was studied using transcriptomic analyses. Interestingly, the largest difference in gene expression was observed when both copper and cerium were present. Many genes of the central methane oxidation pathway, the serine cycle, and the ethylmalonyl-CoA pathway were upregulated, indicating more efficient carbon assimilation. Lastly, attempts were made to elucidate alternative mechanism(s) of copper uptake in M. trichosporium OB3b. Specifically, copCD, putatively encoding for a periplasmic copper-binding protein and an inner membrane protein, respectively, were knocked out in wildtype and a mb-defective mutant of M. trichosporium OB3b. Our results showed that these genes are not critical for copper uptake nor were they basis of copper-regulation in M. trichosporium OB3b.