First isolation of a methanotrophic Mycobacterium reveals ammonia- and pH-tolerant methane oxidation

Methanotrophs facilitate a key step in the global carbon cycle-oxidation of natural and anthropogenic methane-and comprehension of their physiology is critical in unraveling the ecology revolving around microbial methane sinks. Recent literature on aerobic methanotroph physiology focuses on two phyla (Pseudomonadota and Verrucomicrobiota) containing methane-oxidizing isolates; however, such studies no longer discuss a group of gram-positive methanotrophs proposed to belong to Actinomycetota (formerly Actinobacteria). Despite their identification four decades ago, cultures have been lost, leaving their biology and sequenced-based taxonomy enigmatic. Here, we report isolation and characterization of a gram-positive methanotroph, strain MM-1, belonging to Mycobacterium of the phylum Actinomycetota. This marks Actinomycetota as the third phylum (and the only phylum of the kingdom Bacillati) containing isolated aerobic methanotrophs. Unlike conventional methanotrophs, MM-1 demonstrated a remarkably broad pH tolerance and high resistance to a ubiquitous competitive inhibitor of methanotrophy, ammonia. MM-1 possesses a canonical soluble methane monooxygenase and lacks known genes for the degradation of hydroxylamine (a toxic byproduct of ammonia oxidation), suggesting a novel NH₃ tolerance mechanism. The unique tolerance to ammonia would allow Actinomycetota methanotrophs to serve as methane sinks in various settings where conventional methanotrophs would be challenged. A 16S rRNA gene-based survey showed that sequences similar to MM-1 were detected in various environments, including drinking water systems, where the ammonia-to-methane ratio is high, posing challenges for conventional methanotrophs. Through the first isolation of Actinomycetota methanotrophs, our study broadens the recognized physiological range of methanotrophs and suggests the existence of overlooked ecological niches tied to methane oxidation.

Methane is a significant contributor to climate change (27 times more potent as a greenhouse gas than carbon dioxide), and the largest biological sink is methane-oxidizing bacteria: methanotrophs. Although such organisms are thought to be phylogenetically diverse, so far, physiological characterization has been limited to a few clades for which isolates have been captured. Here we successfully obtain the first aerobic methanotrophic isolate outside the phyla Pseudomonadota and Verrucomicrobiota, from the genus Mycobacterium of the phylum Actinomycetota. This isolate exhibited remarkable tolerance to a universal inhibitor of methanotrophy, ammonia, well above concentrations where conventional methanotrophy stops. This expands our understanding of the ecological significance of Mycobacterium, which is generally associated with the medical field for pathogenicity. Our discovery shows that methanotrophy may contribute to mitigating methane emissions in a wider range of environmental conditions than previously thought, providing critical insight for comprehensive evaluation of methanotrophy's contributions to the global methane cycle.