Abstract EANA2026-63

Tardigrades are among the most resilient organisms on Earth. These tiny animals (ranging from 0.1 mm to 1.2 mm in length) are particularly resistant due to their ability to enter a cryptobiotic, which enables survival under extreme desiccation, ionizing radiation, vacuum, and temperature fluctuations through metabolic suspension and cellular stabilization mechanisms, including trehalose accumulation and intrinsically disordered proteins. Here, we present the genomic characterization of a putative novel Paenibacillus lineage isolated from the microbiome of the eutardigrade Paramacrobiotus experimentalis (discovered by our team from Madagascar). Interestingly, Paenibacillus species are of astrobiological interest due to their ability to survive extreme conditions, form resistant endospores and biofilms, and serve as models for studying microbial survival, planetary protection, and the potential for life in extraterrestrial environments.

Whole-genome sequencing of three strains of new Penibacillus revealed highly conserved circular chromosomes (~6.95 Mb), with stable gene content (~6,600-6,700 predicted genes) and uniform GC content (46.52%), indicating a well-defined and conserved species-level core genome with minimal divergence among isolates. Comparative genomic analyses demonstrated that genetic variability is primarily associated with mobile genetic elements. All strains contained multiple prophage regions, comprising approximately 16-18% of the genome, indicative of extensive historical bacteriophage integration and suggesting a major role for phage-mediated genome evolution in adaptation to dynamic and extreme environments. Plasmid architecture exhibited a dual organization, consisting of conserved large plasmids shared across strains and smaller, mosaic/recombined plasmids. The latter partially resemble diverse environmental bacteria displaying signs of horizontal gene transfer.

Functional annotation revealed highly conserved metabolic capabilities, including carbohydrate-active enzymes (CAZymes), nutrient acquisition systems, and secretion-associated proteins. Notably, CAZyme profiles were identical across all strains, suggesting stable carbohydrate utilization strategies. Antimicrobial resistance determinants were limited and conserved, primarily involving glycopeptide resistance and efflux-associated systems. Additionally, all genomes encoded consistent sets of secondary metabolite biosynthetic gene clusters, including non-ribosomal peptide synthetases, ribosomally synthesized and post-translationally modified peptides, terpene pathways, and metallophore-associated clusters, indicating a conserved biosynthetic potential. Several plasmid-associated sequences and mobile elements showed similarity to bacteria from diverse and confined environments. While no direct ecological connection can be established, these similarities may reflect convergent adaptation to stressors such as desiccation. Importantly, the genomes encode conserved pathways related to stress response, dormancy, and biofilm formation, which may influence the microenvironment of host tardigrades. These features could contribute to microenvironmental stabilization during desiccation and potentially support bacterial persistence during host anhydrobiosis, although experimental validation is required.

Overall, the investigated Paenibacillus strains represent a genomically stable yet highly plastic lineage, characterized by a conserved core genome and a dynamic accessory genome shaped by phage integration and plasmid-mediated horizontal gene transfer, offering insights into microbial survival strategies in extreme, astrobiologically relevant microhabitats. Moreover, the Paenibacillus strains exhibit the highest genomic similarity to Paenibacillus vandeheii, a strain originally isolated from the International Space Station, while ANI values at the species delineation threshold (95–96%) suggest that they may represent a distinct, putative species-level lineage within the genus Paenibacillus.

This work was supported by grant no. UMO-2021/43/D/NZ8/00344 from the National Science Centre (Poland).