ERP129115 PRJEB45004 ena-STUDY-KAUST-17-05-2021-08:19:26:392-212 A novel methanotrophic Mycobacterium species as 1 primary producer in a cave microbial community To date, only members of the bacterial phyla Proteobacteria and Verrucomicrobia are known to be methanotrophic. Here,we report that this metabolic trait has also evolved within the Actinobacteria. We isolated and cultivated a methanotrophicMycobacterium, which is closely related to Mycobacterium tuberculosis. It is the dominant species in an extremely acidic biofilmat a gaseous chemocline interface between ancient volcanic gases and the Earth's atmosphere inside Puturosu Mountain.Metagenomic and proteomic analyses on samples of the biofilm revealed that this novel Mycobacterium expresses a full suiteof enzymes required for methanotrophic growth, including a soluble methane monooxygenase for the oxygenation of methaneto methanol and a special Zn-dependent alcohol dehydrogenase to oxidize methanol into formaldehyde, and enzymes involvedin carbon fixation via the ribulose monophosphate pathway. Growth experiments and stable isotope probing with 13C-labelledmethane confirmed that this novel Mycobacterium can grow methanotrophically with methane as a carbon and energy source.Based on its dominance within the biofilm community, we hypothesize that M. methanotrophicum is the primary producer withinthe biofilm and that other members of the microbial community consume its exudates or necromass. Methanotrophic Mycobacterium species To date, only members of the bacterial phyla Proteobacteria and Verrucomicrobia are known to be methanotrophic. Here,we report that this metabolic trait has also evolved within the Actinobacteria. We isolated and cultivated a methanotrophicMycobacterium, which is closely related to Mycobacterium tuberculosis. It is the dominant species in an extremely acidic biofilmat a gaseous chemocline interface between ancient volcanic gases and the Earth's atmosphere inside Puturosu Mountain.Metagenomic and proteomic analyses on samples of the biofilm revealed that this novel Mycobacterium expresses a full suiteof enzymes required for methanotrophic growth, including a soluble methane monooxygenase for the oxygenation of methaneto methanol and a special Zn-dependent alcohol dehydrogenase to oxidize methanol into formaldehyde, and enzymes involvedin carbon fixation via the ribulose monophosphate pathway. Growth experiments and stable isotope probing with 13C-labelledmethane confirmed that this novel Mycobacterium can grow methanotrophically with methane as a carbon and energy source.Based on its dominance within the biofilm community, we hypothesize that M. methanotrophicum is the primary producer withinthe biofilm and that other members of the microbial community consume its exudates or necromass. ENA-FIRST-PUBLIC 2021-07-16 ENA-LAST-UPDATE 2021-07-16