Genomic insights into novel extremotolerant bacteria isolated from the NASA Phoenix mission spacecraft assembly cleanrooms

Abstract

Background: Human-designed oligotrophic environments, such as cleanrooms, harbor unique microbial communities shaped by selective pressures like temperature, humidity, nutrient availability, cleaning reagents, and radiation. Maintaining the biological cleanliness of NASA's mission-associated cleanrooms, where spacecraft are assembled and tested, is critical for planetary protection. Even with stringent controls such as regulated airflow, temperature management, and rigorous cleaning, resilient microorganisms can persist in these environments, posing potential risks for space missions.

Results: During the Phoenix spacecraft mission, genomes of 215 bacterial isolates were sequenced and based on overall genome-related indices, 53 strains belonging to 26 novel species were recognized. Metagenome mapping indicated less than 0.1% of the reads associated with novel species, suggesting their rarity. Genes responsible for biofilm formation, such as BolA (COG0271) and CvpA (COG1286), were predominantly found in proteobacterial members but were absent in other non-spore-forming and spore-forming species. YqgA (COG1811) was detected in most spore-forming members but was absent in Paenibacillus and non-spore-forming species. Cell fate regulators, COG1774 (YaaT), COG3679 (YlbF, YheA/YmcA), and COG4550 (YmcA, YheA/YmcA), controlling sporulation, competence, and biofilm development processes, were observed in all spore-formers but were missing in non-spore-forming species. COG analyses further revealed resistance-conferring proteins in all spore-formers (n = 13 species) and eight actinobacterial species, responsible for enhanced membrane transport and signaling under radiation (COG3253), transcription regulation under radiation stress (COG1108), and DNA repair and stress responses (COG2318). Additional functional analysis revealed that Agrococcus phoenicis, Microbacterium canaveralium, and Microbacterium jpeli contained biosynthetic gene clusters (BGCs) for ε-poly-L-lysine, beneficial in food preservation and biomedical applications. Two novel Sphingomonas species exhibited for zeaxanthin, an antioxidant beneficial for eye health. Paenibacillus canaveralius harbored genes for bacillibactin, crucial for iron acquisition. Georgenia phoenicis had BGCs for alkylresorcinols, compounds with antimicrobial and anticancer properties used in food preservation and pharmaceuticals.

Conclusion: Despite stringent decontamination and controlled environmental conditions, cleanrooms harbor unique bacterial species that form biofilms, resist various stressors, and produce valuable biotechnological compounds. The reduced microbial competition in these environments enhances the discovery of novel microbial diversity, contributing to the mitigation of microbial contamination and fostering biotechnological innovation. Video Abstract.

Keywords: BGCs; Cleanroom; Extreme environment; Novel species; Phoenix mission; Phylogenomics.

Fig. 1

Phylogenetic tree of non-spore-forming bacteria (n = 38) spanning into nine genera from Phoenix spacecraft mission. Novel species are highlighted in red, and their corresponding NCBI accessions are provided. Bootstrap values (expressed as percentages) are indicated near the branches

Fig. 2

Phylogenetic tree of 15 novel strains of spore-forming bacteria from Bacillaceae family isolated from Phoenix spacecraft mission. Novel species are highlighted in red, and their corresponding NCBI accessions are provided. Bootstrap values (expressed as percentages) are indicated near the branches

Fig. 3

Scanning electron microscopy of the novel species isolated from the Phoenix spacecraft assembly cleanroom

Fig. 4

Metagenomic read mapping to novel isolates from NASA cleanrooms, highlighting temporal and spatial dynamics. A Spatial distribution of mapped reads across 26 novel species, showing distinct signatures between spore-forming and non-spore-forming bacteria in different NASA cleanroom locations. B Box plots illustrating the breadth of coverage (> 1%) of consensus genomes constructed from mapped reads aligned to 23 novel species (out of 26). Reads were collected from cleanrooms at SAF JPL and KSC-PHSF in 2016 (red) and KSC-PHSF in 2018 (blue)

Fig. 5

Functional insights into novel species from NASA cleanrooms. A Presence of radiation resistance COGs (from Pal et al. [21]) in the 26 novel species, revealing their genetic potential for radiation resilience. B Presence of biofilm-associated COGs in the novel bacterial species. C. Presence of antimicrobial-resistance genes and similarity with the drug class

Fig. 6

Comparative analysis of ε-poly-L-lysine synthetase (epls) in novel species. A ε-poly-L-lysine gene cluster comparison in Epichloe festucae (fungal producer), Corynebacterium variabile (bacterial producer), and three novel species from this study (Agrococcus phoenicis, Microbacterium canaveralium, Microbacterium jepli) and Leifsonia virtsii (isolated from ISS) show conserved gene cluster architecture. B Protein sequence alignment of ε-poly-L-lysine synthetase enzymes from these organisms exhibits conserved domains, including NRPS adenylation (A), thiolation (T), transmembrane (TM), and C-terminal tandem domains (C1, C2, C3)