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Recent literature on aerobic methanotroph physiology focuses on two phyla (\n                      <jats:italic toggle=\"yes\">Pseudomonadota<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">Verrucomicrobiota<\/jats:italic>\n                      ) containing methane-oxidizing isolates; however, such studies no longer discuss a group of gram-positive methanotrophs proposed to belong to\n                      <jats:italic toggle=\"yes\">Actinomycetota<\/jats:italic>\n                      (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\n                      <jats:italic toggle=\"yes\">Mycobacterium<\/jats:italic>\n                      of the phylum\n                      <jats:italic toggle=\"yes\">Actinomycetota<\/jats:italic>\n                      . This marks\n                      <jats:italic toggle=\"yes\">Actinomycetota<\/jats:italic>\n                      as the third phylum (and the only phylum of the kingdom\n                      <jats:italic toggle=\"yes\">Bacillati<\/jats:italic>\n                      ) 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 NH\n                      <jats:sub>3<\/jats:sub>\n                      tolerance mechanism. The unique tolerance to ammonia would allow\n                      <jats:italic toggle=\"yes\">Actinomycetota<\/jats:italic>\n                      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\n                      <jats:italic toggle=\"yes\">Actinomycetota<\/jats:italic>\n                      methanotrophs, our study broadens the recognized physiological range of methanotrophs and suggests the existence of overlooked ecological niches tied to methane oxidation.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      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\n                      <jats:italic toggle=\"yes\">Pseudomonadota<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">Verrucomicrobiota<\/jats:italic>\n                      , from the genus\n                      <jats:italic toggle=\"yes\">Mycobacterium<\/jats:italic>\n                      of the phylum\n                      <jats:italic toggle=\"yes\">Actinomycetota<\/jats:italic>\n                      . 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\n                      <jats:italic toggle=\"yes\">Mycobacterium<\/jats:italic>\n                      , 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\u2019s contributions to the global methane cycle.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/aem.00796-25","type":"journal-article","created":{"date-parts":[[2025,7,31]],"date-time":"2025-07-31T13:00:13Z","timestamp":1753966813000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":15,"title":["First isolation of a methanotrophic\n                    <i>Mycobacterium<\/i>\n                    reveals ammonia- and pH-tolerant methane 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