Eukaryotes’ closest relatives are internally simple syntrophic peptidotrophic archaea

Key Points:

• Biofilm samples containing diverse microorganisms including Promethearchaeota were collected from a gas production well in the Southern Kanto gas field, Japan, and cultured using specialized anaerobic media to isolate new strains.
• Growth conditions, substrate utilization, and temperature ranges for the cultured archaeal strains HC1 and SC1 were systematically evaluated using qPCR, iTag analysis, and microscopy techniques including epifluorescence, SEM, TEM, QFDE-EM, and cryo-EM.
• Comprehensive chemical analyses were conducted on culture media and cells to measure peptides, proteins, methane, haem B, nitrate, nitrite, and iron concentrations, providing insights into metabolic activities.
• Genomic, transcriptomic, proteomic, and glycoproteomic analyses were performed to assemble genomes, annotate genes including eukaryotic signature proteins (ESPs), and assess gene expression, supporting metabolic reconstruction and phylogenetic studies.
• Aerobic capacity of Promethearchaeota and related archaea was predicted using machine learning classifiers based on gene content related to oxygen metabolism, with phylogenetic analyses informing evolutionary relationships and metabolic strategies.