Multispecies populations of methanotrophic
Methyloprofundus
and cultivation of a likely dominant species from the Iheya North deep-sea hydrothermal field
The Methyloprofundus clade is represented by uncultivated methanotrophic bacterial endosymbionts of deep-sea bathymodiolin mussels, but only a single free-living species has been cultivated to date. This study reveals the existence of free-living Methyloprofundus variants in the Iheya North deep-sea hydrothermal field in the mid-Okinawa Trough. A clade-targeted amplicon analysis of the particulate methane monooxygenase gene ( pmoA ) detected 647 amplicon sequence variants (ASVs) of the Methyloprofundus clade in microbial communities newly formed in in situ colonization systems. Such systems were deployed at colonies of bathymodiolin mussels and a galatheoid crab. These ASVs were classified into 161 species-like groups. The proportion of the species-like groups representing endosymbionts of mussels was unexpectedly low. A methanotrophic bacterium designated as INp10, a likely dominant species in the Methyloprofundus population in this field, was enriched in biofilm formed in a methane-fed cultivation system operated at 10°C. Genomic characterization with the gene transcription dataset of INp10 from biofilm suggested traits advantageous to niche competition in environments, such as mobility, chemotaxis, biofilm formation, offensive and defensive systems, and hypoxia tolerance. The notable metabolic traits INp10 shares with some Methyloprofundus members are the use of lanthanide-dependent XoxF as the sole methanol dehydrogenase due to the absence of the canonical MxaFI, the glycolytic pathway using fructose-6-phosphate aldolase instead of fructose-1,6-bisphosphate aldolase, and the potential to perform partial denitrification from nitrate under oxygen-limited conditions. These findings help better understand ecological strategies of this possibly widespread marine-specific methanotrophic clade. Importance The Iheya North deep-sea hydrothermal field in the mid-Okinawa Trough is characterized by abundant methane derived from organic-rich sediments and diverse chemosynthetic animal species, including those harboring methanotrophic bacterial symbionts such as bathymodiolin mussels Bathymodiolus japonicus and “ Bathymodiolus ” platifrons and a galatheoid crab Shinkaia crosnieri . Symbiotic methanotrophs have attracted significant attention, yet free-living methanotrophs in this environment have not been studied in detail. We focused on the free-living Methyloprofundus spp. that thrive in this hydrothermal field and identified an unexpectedly large number of species-like groups in this clade. Moreover, we enriched and characterized a methanotroph whose genome sequence indicated it corresponds to a new species in the genus Methyloprofundus . This species might be a dominant member of the indigenous Methyloprofundus population. New information on free-living Methyloprofundus spp. suggests that the hydrothermal field is a promising locale to investigate the adaptive capacity and associated genetic diversity of Methyloprofundus .
