Analysis of Mechanisms for Electron Uptake by Methanothrix harundinacea 6Ac During Direct Interspecies Electron Transfer.

Direct interspecies electron transfer (DIET) is a syntrophic metabolism wherein free electrons are directly transferred between microorganisms without the mediation of intermediates such as molecular hydrogen or formate. Previous research has demonstrated that Methanothrix harundinacea 6Ac is capable of reducing carbon dioxide through DIET. However, the mechanisms underlying electron uptake in M. harundinacea 6Ac during DIET remain poorly understood. This study aims to elucidate the electron and proton flux in M. harundinacea 6Ac during DIET and to propose a model for electron uptake in this organism, primarily based on the analysis of gene transcript levels, genomic characteristics of M. harundinacea 6Ac, and the pathways generating fully reduced ferridoxin (Fdred2-), reduced coenzyme F420 (F420H2), coenzyme M (CoM-SH), and coenzyme B (CoB-SH) during DIET. The findings suggest that membrane-bound heterodisulfide reductase (HdrED), F420H2-dehydrogenase lacking subunit F (Fpo-), and cytoplasmic heterodisulfide reductase (HdrABC)-subunit B of F420-reducing hydrogenase (FrhB) complex play critical roles in electron uptake in M. harundinacea 6Ac during DIET. Specifically, Fpo- is responsible for generating Fdred2- with reduced methanophenazine (MPH2), driven by a proton motive force, while HdrED facilitates the reduction of heterodisulfide of coenzyme M and coenzyme B (CoM-S-S-CoB) to CoM-SH and CoB-SH using MPH2. Additionally, cytoplasmic heterodisulfide reductase HdrABC and subunit B of coenzyme F420-hydrogenase complex (HdrABC-FrhB complex) catalyzes the reduction of oxidized coenzyme F420 (F420) to F420H2, utilizing CoM-SH, CoB-SH, and Fdred2-. This study represents the first genetics-based functional characterization of electron and proton flux in M. harundinacea 6Ac during DIET, providing a model for further investigation of electron uptake in Methanosaeta species. Furthermore, it deepens our understanding of the mechanisms underlying electron uptake in methanogens during DIET.