Unveiling hidden microbial diversity in Mars 2020 mission assembly cleanrooms with molecular insights into the persistence and perseverance of novel species defying metagenome sequencing.

NASA cleanrooms, which are critical for assembling space mission components, are maintained under stringent decontamination protocols to minimize biological contamination. These environments are characterized by nutrient-poor and oligotrophic conditions, leading to low microbial loads. Despite extensive cleaning, oligotrophs capable of surviving in such conditions continue to persist, often remaining undetected due to their low abundance, resistance to environmental stresses, and difficulties in biomolecule extraction. Even with shotgun metagenome sequencing technologies, these microbes may go undetected or be underrepresented due to their robust cell walls and the absence of reference genomes in publicly available databases. Over a 6-month study of Mars 2020 mission cleanrooms, 182 bacterial strains belonging to 19 families were identified using a whole-genome sequencing (WGS) approach. Among these, 14 novel Gram-positive species were discovered, including eight spore formers. Though the novel species comprised only 0.001% of the sequencing data, their successful cultivation allowed for functional characterization. Through WGS data mining, genomic traits associated with resilience in extreme conditions were revealed. These species were found to be involved in nitrogen cycling, carbohydrate metabolism, and radiation resistance, traits essential for survival in extreme environments. Furthermore, 12 biosynthetic gene clusters were identified, including those linked to ectoine and [Formula: see text]-poly-L-lysine production, suggesting potential biotechnological applications. These findings highlight the hidden microbial diversity within cleanrooms and emphasize the necessity of advanced detection strategies. A better understanding of these microbes will provide insights into extremophiles with applications in biotechnology, medical research, and life support systems for future space exploration missions.IMPORTANCEDespite strict decontamination protocols, NASA cleanrooms harbor low-biomass microbial communities adapted to nutrient-poor environments. These oligotrophic microbes often go undetected in shotgun metagenomics methods due to their low abundance, resistance to lysis, and lack of reference genomes. Standard shotgun metagenome sequencing methods fail to retrieve them, as dominant microbial DNA overshadows rare species. Over 6 months of monitoring Mars 2020 mission cleanrooms, 182 bacterial strains from 19 families were identified, including 14 novel Gram-positive species, 8 of which were spore formers. Though present at 0.001% abundance in sequencing data, we successfully cultured them, enabling functional characterization. These microbes exhibited roles in nitrogen cycling, carbohydrate metabolism, and radiation resistance, with 12 biosynthetic gene clusters linked to ectoine and [Formula: see text]-poly-L-lysine production. These findings highlight the previously underestimated microbial diversity in cleanrooms and emphasize the need for advanced detection strategies to explore extremophiles with applications in biotechnology and space exploration.