The class Mollicutes within the phylum Mycoplasmatota (“Tenericutes”) accommodates tiny wall-less prokaryotes with small genomes. The vast majority of Mollicutes species are commensals or parasites of humans, animals, insects, or plants (Brown et al., 2018). The taxonomic status of Mollicutes is currently uncertain. At the time of writing, Genome Taxonomy Database (GTDB) placed most wall-less bacteria into separate phylum comprising a single class that includes orders Mycoplasmatales, Acholeplasmatales, Izemoplasmatales and Erysipelotrichales, and several order-level unclassified lineages (Parks et al., 2022). The NCBI taxonomy database considers Mollicutes and Candidatus Izemoplasma as separate classes of Mycoplasmatota (Schoch et al., 2020). The order Izemoplasmatales has been proposed based on description of a type genus “Candidatus Izemoplasma” (Skennerton et al., 2016; Zheng et al., 2021). Currently, “Izemoplasmatales” includes two species of two genera: “Xianfuyuplasma coldseepsis” and “Hujiaoplasma nucleasis”, the names of which have not been validly published (Zheng et al., 2023, 2021).

It has been hypothesized that wall-less bacteria evolved from Gram-positive bacteria by reductive evolution, and during this process, they lost much of their ancestral genome but retained genes essential for life. The availability of nutrients from the hosts resulted in the loss of many genes required for assimilative processes (Razin et al., 1998). A consequence of the dependence on nutrient supply is the inability of many Mollicutes to grow in axenic cultures. In spite of a wide distribution of Mycoplasmatota in diverse environments, the number of described species of free-living wall-less bacteria are very limited (Wang et al., 2020). In addition to two cultivated representatives of “Izemoplasmatales”, isolated from cold marine seeps, member of the order Haloplasmatales – Haloplasma contractile was isolated from marine hypersaline brine-sediment interface and two members of the order Acholeplasmatales – Mariniplasma anaerobium and Peloplasma aerotolerans were isolated from brackish lake and terrestrial mud volcano (Antunes et al., 2008; Khomyakova et al., 2024; Watanabe et al., 2021; Zheng et al., 2023, 2021). It is noteworthy that all these free-living mollicutes are obligate or aerotolerant anaerobes.

One of the natural sources of methane emission into the atmosphere is mud volcanism (Mazzini and Etiope, 2017). Terrestrial mud volcanoes (TMVs) are geologically connected with deep subsurface petroleum and natural gas reservoirs, supplying prokaryotic communities with various organic and inorganic compounds, which can be used in microbial metabolism. The Taman Peninsula at the south of Russia is among the regions with the most pronounced mud volcanism; the Kerch–Taman mud volcano province includes over 100 active TMVs (Kikvadze et al., 2014). Representatives of the сlass Mollicutes have been detected by molecular methods based on microbial community analysis in several TMVs (Hsu et al., 2021; Tu et al., 2022), however to date only one cultivated Mollicutes from mud volcanoes – Peloplasma aerotolerans – is known (Khomyakova et al., 2024). In this study we isolated from TMV a free-living, cell wall-less obligately anaerobic strain, described its phenotypic and genomic properties and propose to assign it to a novel genus and species, a novel family and a novel order of the class Mollicutes, for which the names Liberiplasma polymorphum gen. nov., sp. nov., Liberiplasmataceae fam. nov. and Liberiplasmatales ord. nov. are proposed.