Molecular insights into atmospheric methane-oxidizing USCγ from desert grassland soil based on metagenome-assembled genome analysis.

Upland Soil Cluster Gamma (USCγ) is a key high-affinity aerobic methanotroph driving atmospheric methane oxidation in grassland soils; however, it has never been obtained in pure culture, and its metabolic processes remain largely unknown. Here, we reconstructed a USCγ metagenome-assembled genome (MAG) containing the complete pmoA gene from desert grassland soil in northwestern China, designated USC_AKS. At the site, USCγ accounted for 9.83% of the microbial community in the 10-20 cm layer. BLASTn of its 16S rRNA gene against the NCBI database (excluding uncultured/environmental sequences) showed 93.03% similarity to the non-methanotroph Thioalkalivibrio sulfidiphilus HL-EbGr7 (order Chromatiales). The closest match among named species was an uncultured bacterium (JN672117) at 97.86% similarity. Its pmoA shares 96.18% similarity with the original USCγ-defining sequence. Phylogenomic analysis placed USC_AKS and seven other USCγ MAGs into a monophyletic group of three subclades, distantly related to culturable Type I methanotrophs. Their genomic average nucleotide identity values are all below 95%, confirming eight distinct species. Like other USCγ MAGs, USC_AKS encodes a complete pmoCAB operon, an XoxF-type methanol dehydrogenase, and enzymes for formaldehyde oxidation to CO2. However, it lacks key ribulose monophosphate (RuMP) cycle genes encoding 3-hexulose-6-phosphate synthase (hps) and 6-phospho-3-hexulose isomerase (phi). The serine cycle also appears incomplete, as these MAGs lack hpr, the gene encoding hydroxypyruvate reductase. Moreover, none encode Rubisco, ruling out the Calvin-Benson-Bassham CO2-fixation pathway. Consequently, the metabolic characteristics of USCγ-particularly its carbon assimilation pathway-remain enigmatic, and obtaining pure cultures or enriched consortia is likely the only route to resolving this mystery.