Investigating the gene regulation and physiology of methoxydotrophic archaea

Lignin, one of Earth's most abundant organic polymers, is a significant source of methoxylated aromatic compounds (MACs). While bacteria-mediated MAC conversion is well-documented, archaeal degradation of MACs has only recently been discovered. Methermicoccus shengliensis, a methanogenic archaeon, is the first archaeon identified to convert MACs, a process termed methoxydotrophy [1]. Recent studies reveal that M. shengliensis uses an O-demethylase system for MAC conversion, resembling methyltransferase systems of acetogenic bacteria [2]. Comparative transcriptomics identified an operon, the mto operon, within M. shengliensis that encodes essential MAC-converting proteins, with homologous genes found across diverse archaeal taxa. While the roles of most mto proteins in M. shengliensis have been elucidated, two hypothetical proteins with DNA-binding structural motifs encoded within this operon remain uncharacterized. We aim to elucidate mto gene regulation and characterize these putative DNA-binding proteins by confirming their DNA-binding abilities through EMSA (Electrophoretic Mobility Shift Assay) and performing a promoter pull-down assay with M. shengliensis cell extract, followed by MALDI-TOF analysis. Additionally, we seek to characterize the O-demethylase system of further archaea and understand their role within the metabolism of these organisms. Methanolacinia petrolearia is a hydrogenotrophic methanogen that primarily uses H₂ and CO₂ for CH₄ production and encodes the Mto proteins, but no MAC transporters. Growth experiments with M. petrolearia revealed tolerance to toxic methyl halides (e.g., chloromethane), suggesting these compounds might be converted/detoxified by the Mto system. We will determine the substrate spectrum of this Mto system by conducting enzyme activity assays while using methyl halides such as chloromethane and various MACs as substrates. Additionally, gene expression analysis (RT-qPCR) will be conducted to assess mto gene expression in response to chloromethane or MAC exposure. This research enhances our understanding of archaeal methoxydotrophy by studying the regulation of the mto genes and by characterizing Mto systems of further archaea alongside their role within the metabolism.

[1] Mayumi et al. (2016) Science 354: 222. [2] Kurth et al. (2021) ISME J 15: 3549.