Lignin is one of the most prevalent organic polymers found on Earth, being a major source of methoxylated aromatics. While bacteria-mediated conversion of these compounds has been extensively documented, the archaeal degradation of methoxylated aromatics has only recently come to light. Methermicoccus shengliensis, a methanogenic archaeon, was identified as the pioneering archaeon capable of converting methoxylated aromatics into methane, also called methoxydotrophic methanogenesis [1]. A recent study revealed that M. shengliensis has an O-demethylase system for conversion of methoxylated aromatics that shares greater similarity with methyltransferase systems of acetogenic bacteria than the respective systems of methylotrophic archaea [2]. By using comparative transcriptomics, an operon, referred to as the mto operon, was found to be upregulated under methoxydotrophic growth in M. shengliensis [2]. This operon encodes the proteins required for the conversion of methoxylated aromatics. While the roles of most proteins encoded by this operon have been elucidated, two hypothetical proteins that have structural motifs of DNA-binding domains remain uncharacterized. Our goal is to uncover the regulation of the mto genes and the function of the aforesaid hypothetical proteins. To validate the DNA-binding proficiency of the putative regulators, Electrophoretic Mobility Shift Assay trials were initiated. We focused on identifying specific non-coding DNA regions within or adjacent to the mto operon that potentially serve as binding sites for these hypothetical proteins. In addition, we will screen the transcriptional regulators of the whole mto operon by using the promoter region of the mto gene cluster together with M. shengliensis cell extract and magnetic separation technology. We aim to identify the proteins that are binding to the promotor of this gene cluster. This study will help us grasp how methoxydotrophy is controlled on gene level in archaea and how specific methanogens like M. shengliensis regulate conversion of a variety of methylated and methoxylated compounds.

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