Acidic environments are widely distributed on Earth and are characterized by elevated concentrations of transition metals, metalloids, and other inorganic solutes (Quatrini and Johnson, 2018). Acidophiles inhabiting these environments exhibit unique physiological adaptations that enable them to tolerate extreme aquatic conditions. Although these adaptive traits have potential biotechnological applications such as the bioprocessing of acid mine drainage (Méndez-García et al., 2015), the taxonomic and functional diversity of acidophiles remains far from fully understood.

Archaeal lineages are also commonly detected in acidic environments. In particular, the order of Thermoplasmatales is recognized as one of the most common archaeal acidophiles (Baker and Banfield, 2003; Huber and Stetter, 2006). Over 700 draft genomes, representing 25 genus–level taxa, are preserved in the Genome Taxonomy Database (GTDB, R226). Despite its ubiquitous distribution in terrestrial acidic environments, microorganism under Thermoplasmatales remains difficult to cultivate, limiting our understanding of their ecological roles and metabolic capabilities. Therefore, expanding isolated acidophilic archaea is critical for linking genomic potential with physiological function in acidic ecosystems.

To date, in the order Thermoplasmatales, only four families—Ferroplasmaceae, Picrophilaceae, Thermoplasmataceae, and Cuniculiplasmataceae—and eight genera—Thermoplasma (Darland et al., 1970), Picrophilus (Schleper et al., 1995), Ferroplasma (Golyshina et al., 2000), Thermogymnomonas (Itoh et al., 2007), Acidiplasma (Golyshina et al., 2009), Cuniculiplasma (Golyshina et al., 2016), and Oxyplasma— (Golyshina et al., 2024) have been validly published and deposited in culture collections. All Thermoplasmatales isolates grow optimally under acidic conditions and have a pleomorphic cell shape, attributed to the lack of an intact cell wall, except for the genus Picrophilus, which has a S–layer cell wall. It has been reported that some Thermoplasmatales species serve as hosts for members of the phylum Microcaldota (formerly “Candidatus Micrarchaeota”, Golyshina et al., 2016, Golyshina et al., 2019; Krause et al., 2022), which are obligate symbiotic archaea belonging to the kingdom Nanobdellati (formerly DPANN, Kato et al., 2022). Thus, isolating Thermoplasmatales species can potentially impact our understanding of symbiotic interactions between Nanobdellati archaea and their hosts.

Here, we report the isolation and characterization of four strains, two representing a novel genus and species, and the other two representing novel species of the genus Cuniculiplasma, within the family Cuniculiplasmataceae.