GENOME-INFORMED FUNCTIONAL TRAIT CATALOGS AND COMMUNITY MODELING IDENTIFY PLANETARY-PROTECTION RISKS IN SPACECRAFT CLEANROOM MICROBIOMES

Kurzfassung

Introduction: Spacecraft assembly cleanrooms are among the most stringently controlled built environments but nonetheless host microbial communities able to withstand extreme physicochemical stressors relevant to space missions. Characterizing the functional potential and interaction dynamics of these resilient taxa is essential for improving contamination control and refining planetary protection assessments. Methods: We performed deep metagenomic sequencing of microbial assemblages from ESA cleanrooms and recovered metagenome-assembled genomes (MAGs) using genome-centric metagenomics and ensemble binning. Functional profiling combined flexible genome-scale phenotype prediction frameworks, supervised machine-learning models, and large phenotypic-trait and metabolic-interaction resources of 1,868 genomes/bins. We also used community-scale metabolic modeling of 1,261 representative strains, including flux-balance simulations, to infermetabolite exchanges, minimal growth requirements, and generalist–specialist strategies across 414 environmental gradients, including conditions relevant to potential extraterrestrial analogs. Results: Metagenome-assembled genomes (MAGs) from ESA’s Kourou spaceport showed similar proportions of Planetary Protection relevant traits compared with ESA cleanroom isolates, ISS genome sets, and the human skin genome catalog. Genomic signatures indicate anaerobic capacity (cofactor biosynthesis, redox sensors, peptide/arginine catabolism) and potential autotrophy (maturation systems and enzymes for H2 utilization and CO2 fixation). Cold adaptation was reflected by ribosomal and protein-folding support, membrane lipid remodeling, and compatible-solute systems. Desiccation tolerance featured antioxidant defenses, osmoprotectant transporters, cell-wall strengthening enzymes, and DNA-repair functions. Salt tolerance and resilience were associated with ion-homeostasis machinery, Na+-coupled transporters, and compatible-solute uptake/synthesis. Sporulation potential appeared in regulatory networks, proteases, and transport systems required for spore formation and maturation. Genomes from the ISS were significantly enriched for anaerobic lifestyles, while ensemble-binned MAGs showed overrepresentation of cryotolerance and ESA’s isolate collection was enriched for desiccation tolerance, haloresilience, and sporulation (all chi-square p 0.001). The JUICE dataset exhibited higher proportions of autotrophy, nitrite-oxidizing bacteria (NOB), nif clusters, saccharolytic metabolism, phototrophy, and succinate metabolism (many comparisons p 0.001), with smaller or non-significant increases for halophily, symbiosis, and thermophily. Within Staphylococcus, genomes from the Kourou cleanroom (during JUICE sampling) showed an elevated representation of autotrophy, cryotolerance and a halophilic phenotype, though trait repertoires varied substantially at the subspecies/genome level. Discussion: Integrating phenotype prediction with metabolic-interaction modeling reveals distinct functional signatures linked to desiccation tolerance, oxidative-stress mitigation, nutrient-limited growth, and potential replication in extraterrestrial environments. Genome-level heterogeneity—especially within genera such as Staphylococcus—emphasizes the need for whole-genome predictions rather than genus-level generalizations. Identified most predictive features (enzymes, transporters, regulators, membrane and respiratory components) provide mechanistic hypotheses for stress resistance. Conclusion and Importance: This integrative approach produces functional genome catalogs that complement spore-based assays, enabling more mechanistic evaluations of microbial persistence and evidence-based planetary protection. Genome-informed trait assessments support targeted functional screenings in future sampling campaigns and improve forward-contamination risk estimates for Mars and icy worlds.