Spacecraft Assembly Facility Research Articles

Whole-genome sequencing of Paenibacillus phoenicis isolated from the Phoenix Mars Lander spacecraft assembly facility.

The genome of Paenibacillus phoenicis, a spore-forming bacterium isolated from the spacecraft assembly facility of the Phoenix mission, was generated via hybrid assembly by merging short and long reads. Examining this genome may shed light on strategies to minimize the risk of contaminating extraterrestrial environments with Earth-based microorganisms.

Genomic and morphological characterization of Knufia obscura isolated from the Mars 2020 spacecraft assembly facility

Members of the family Trichomeriaceae, belonging to the Chaetothyriales order and the Ascomycota phylum, are known for their capability to inhabit hostile environments characterized by extreme temperatures, oligotrophic conditions, drought, or presence of toxic compounds. The genus Knufia encompasses many polyextremophilic species. In this report, the genomic and morphological features of the strain FJI-L2-BK-P2 presented, which was isolated from the Mars 2020 mission spacecraft assembly facility located at the Jet Propulsion Laboratory in Pasadena, California. The identification is based on sequence alignment for marker genes, multi-locus sequence analysis, and whole genome sequence phylogeny. The morphological features were studied using a diverse range of microscopic techniques (bright field, phase contrast, differential interference contrast and scanning electron microscopy). The phylogenetic marker genes of the strain FJI-L2-BK-P2 exhibited highest similarities with type strain of Knufia obscura (CBS 148926T) that was isolated from the gas tank of a car in Italy. To validate the species identity, whole genomes of both strains (FJI-L2-BK-P2 and CBS 148926T) were sequenced, annotated, and strain FJI-L2-BK-P2 was confirmed as K. obscura. The morphological analysis and description of the genomic characteristics of K. obscura FJI-L2-BK-P2 may contribute to refining the taxonomy of Knufia species. Key morphological features are reported in this K. obscura strain, resembling microsclerotia and chlamydospore-like propagules. These features known to be characteristic features in black fungi which could potentially facilitate their adaptation to harsh environments.

Susceptibility of extremophiles to far-UVC light for bioburden reduction in spacecraft assembly facilities

The prevention and reduction of microbial species entering and leaving Earth's biosphere is a critical aspect of planetary protection research. While various decontamination methods exist and are currently utilized for planetary protection purposes, the use of far-UVC light (200–230 nm) as a means for microbial reduction remains underexplored. Unlike conventional germicidal ultraviolet at 254 nm, which can pose a health risk to humans even with small exposure doses, far-UVC light poses minimal health hazard making it a suitable candidate for implementation in occupied areas of spacecraft assembly facilities. This study investigates the efficacy of far-UVC 222-nm light to inactivate bacteria using microbial species which are relevant to planetary protection either in vegetative cell or spore form. All the tested vegetative cells demonstrated susceptibility to 222-nm exposure, although susceptibility varied among the tested species. Notably, Deinococcus radiodurans, a species highly tolerant to extreme environmental conditions, exhibited the most resistance to far-UVC exposure with a dose of 112 mJ/cm2 required for a 1-log reduction in survival. While spore susceptibility was similar across the species tested, Bacillus pumilus spores were the most resistant of the tested spores when analyzed with a bi-exponential cell killing model (D90 of 6.8 mJ/cm2). Overall, these results demonstrate the efficacy of far-UVC light for reducing microbial bioburden to help ensure the success and safety of future space exploration missions.

Succession of the fungal community of a spacecraft assembly clean room when enriched in brines relevant to Mars

Abstract Spacecraft can carry microbial contaminants from spacecraft assembly facilities (SAFs) to the cold arid surface of Mars that may confound life detection missions or disrupt native ecosystems. Dry hygroscopic sulphate and (per)chlorate salts on Mars may absorb atmospheric humidity and deliquesce at certain times to produce dense brines, potential sources of liquid water. Microbial growth is generally prohibited under the non-permissive condition of extremely low water activity in the frigid potential brines on Mars. Here we challenged the microbial community from samples of the Jet Propulsion Laboratory SAF with the extreme chemical conditions of brines relevant to Mars. Enrichment cultures in SP medium supplemented with 50% MgSO4 or 20% NaClO3 were inoculated from washes of SAF floor wipes. Samples were taken for each of the first four weeks and then at six months after inoculation to follow changes in the SAF microbial community under high salinity for long periods. Metagenomic DNA extracts of community samples were examined by Illumina sequencing of 18S rRNA gene sequences using fungal primers. The fungal assemblage during the first month of enrichment was predominantly common Ascomycetes, primarily Saccharomyete yeasts. Basidiomycetes were detected, mainly in the Microbotryomycetes and Tremellomycetes. Fungi were much less abundant in enrichment cultures at 50% MgSO4 than at 20% NaClO3. After 6 months of enrichment, few fungi remained. Microbes persisting from the JPL SAF microbial community in aged cultures enriched at extreme salinities might be the most capable of subsequently surviving and proliferating at the near surface of Mars. The SAF fungal assemblage did not survive and proliferate as well as the SAF bacterial community.

Succession of the bacterial community from a spacecraft assembly clean room when enriched in brines relevant to Mars

Abstract Interplanetary spacecraft are built in a spacecraft assembly facility (SAF), a clean room designed to reduce microbial contamination that could confound life detection missions or influence native ecosystems. The frigid hyperarid near-surface environment of Mars has ample hygroscopic Mg and Na salts of chloride, (per)chlorate and sulphate that may deliquesce to form dense brines, liquids with low water activity, and freezing points <0°C. The current study sought to define the climax microbial community after 6 mo of enrichment of SAF floor wipe samples in salt plains medium supplemented with 50% (w/v; ~2 M; aw = 0.94) MgSO4 or 20% (w/v; ~1.9 M; aw = 0.91) NaClO3. After 1 wk, 4 wk and 6 mo of incubation, metagenomic DNA extracts of the enriched SAF microbial community were used for high-throughput sequencing of 16S rRNA genes and subsequent phylogenetic analyses. Additionally, dozens of bacterial strains were isolated by repetitive streak-plating from the climax community after 6 mo of enrichment. Early in the enrichment, staphylococci greatly dominated and then remained abundant members of the community. However, actinobacteria succeeded the staphylococci as the dominant taxa as the cultures matured, including Arthrobacter, Brachybacterium and Brevibacterium. A diverse assemblage of bacilli was present, with Oceanobacillus being especially abundant. The SAF culture collection included representatives of Brachybacterium conglomeratum, Brevibacterium sediminis, Oceanobacillus picturae and Staphylococcus sciuri. These were characterized with biochemical and physiological tests, revealing their high salinotolerance. Shannon diversity indices were generally near 2, reflecting modest diversity at several levels of identity and the community structures were uneven throughout. However, minor members of the community seem capable of the ecosystem functions required for biogeochemical cycling. For instance, organisms capable of all the functions of the N cycle were detected. The microbial assemblage in SAFs is the most likely to be transported by spacecraft to another world. While individual microbial populations may exhibit the qualities needed for survival at the near-surface of Mars, certainly entire communities with the capacity for complete biogeochemical cycling, would have a greater chance of survival and proliferation.

Profiling toxin genes and antibiotic resistance in Bacillus cereus isolated from pre-launch spacecraft.

Characterization of the microbiomes of pre-launch spacecraft in spacecraft assembly facilities is an important step in keeping crews healthy during journeys that can last several hundred days in small artificial environments in space. Bacillus cereus, a foodborne pathogenic bacterium, has the potential to be a significant source of food contamination in such environments. This bacterium is a spore-forming bacteria that resists different antimicrobial treatments in cleanrooms where spacecraft are assembled. This study evaluated 41 B. cereus isolates from four pre-launch spacecraft in spacecraft assembly facilities for their toxin gene profile and antibiotic resistance. Four enterotoxin genes (hlbC, cytK, nheA, and entFM) and two emetic toxin genes (ces and CER) were targeted for chromosomal DNA and plasmid DNA. Results showed 31.7, 7.3, 85, and 41.5% of isolates contained hblC, cytK, nheA, and entFM, respectively, in chromosomal or plasmid DNA. Overall, 37 isolates (90.2%) showed at least one enterotoxin gene. The emetic toxin gene, ces, was detected in the plasmid DNA of three isolates (7.3%). The antibiotic resistance of isolates was evaluated by the Kirby-Bauer disk diffusion procedure. All the isolates exhibited 100% susceptibility to gentamicin, 97% were susceptible to clindamycin, and 95% to chloramphenicol, imipenem, tetracycline, and vancomycin. The overall susceptibility average is 51%. However, 98% of the isolates were resistant to β-lactam antibiotics, 97.5% were resistant to sulfamethoxazole/trimethoprim, and 80% were resistant to rifampin. This study provides important information on B. cereus isolates from spacecraft assembly facilities for use in microbial monitoring programs of spacecraft.

Genomic characterization and radiation tolerance of Naganishia kalamii sp. nov. and Cystobasidium onofrii sp. nov. from Mars 2020 mission assembly facilities

During the construction and assembly of the Mars 2020 mission components at two different NASA cleanrooms, several fungal strains were isolated. Based on their colony morphology, two strains that showed yeast-like appearance were further characterized for their phylogenetic position. The species-level classification of these two novel strains, using traditional colony and cell morphology methods combined with the phylogenetic reconstructions using multi-locus sequence analysis (MLSA) based on several gene loci (ITS, LSU, SSU, RPB1, RPB2, CYTB and TEF1), and whole genome sequencing (WGS) was carried out. This polyphasic taxonomic approach supported the conclusion that the two basidiomycetous yeasts belong to hitherto undescribed species. The strain FJI-L2-BK-P3T, isolated from the Jet Propulsion Laboratory Spacecraft Assembly Facility, was placed in the Naganishia albida clade (Filobasidiales, Tremellomycetes), but is genetically and physiologically different from other members of the clade. Another yeast strain FKI-L6-BK-PAB1T, isolated from the Kennedy Space Center Payload Hazardous and Servicing Facility, was placed in the genus Cystobasidium (Cystobasidiales, Cystobasidiomycetes) and is distantly related to C. benthicum. Here we propose two novel species with the type strains, Naganishia kalamii sp. nov. (FJI-L2-BK-P3T = NRRL 64466 = DSM 115730) and Cystobasidium onofrii sp. nov. (FKI-L6-BK-PAB1T = NRRL 64426 = DSM 114625). The phylogenetic analyses revealed that single gene phylogenies (ITS or LSU) were not conclusive, and MLSA and WGS-based phylogenies were more advantageous for species discrimination in the two genera. The genomic analysis predicted proteins associated with dehydration and desiccation stress-response and the presence of genes that are directly related to osmotolerance and psychrotolerance in both novel yeasts described. Cells of these two newly-described yeasts were exposed to UV-C radiation and compared with N. onofrii, an extremophilic UV-C resistant cold-adapted Alpine yeast. Both novel species were UV resistant, emphasizing the need for collecting and characterizing extremotolerant microbes, including yeasts, to improve microbial reduction techniques used in NASA planetary protection programs.

Real-Time Quantification of Size-Resolved Bioaerosols and Inert Particles in Spacecraft Assembly Facilities at the NASA Jet Propulsion Laboratory.

Abstract Responsible space exploration is a cornerstone of planetary protection, particularly at sites in the Solar System with a high potential for the existence of extant life. To limit bioburden, spacecraft assembly occurs in cleanroom facilities. Cleanroom levels are established through air particulate counters that can assess particle size distribution and concentration but cannot detect bioaerosols. Additionally, these devices do not detect in real-time, which poses a risk to critical flight hardware assemblies or even mission timelines. A first-of-its-kind study was conducted to simultaneously detect bioaerosols, inert particles, and their size distribution in real-time in operational spacecraft assembly cleanrooms at NASA's Jet Propulsion Laboratory in Pasadena, CA, USA, using the BioVigilant IMD-A® 350 (Azbil Corporation, Tucson, AZ, USA). The IMD-350A continuously sampled during operations and no-operation 6 h intervals in two facilities per cleanroom class: ISO 6, ISO 7, and ISO 8. A positive correlation was established between human presence in the cleanroom and elevated bioaerosol counts. Smaller particles of sizes 0.5 and 1 μm constituted an average ∼91% of the total bioaerosols detected in At Work intervals across all ISO classes observed. The results of this study were used to establish bioburden particulate thresholds for the most stringent JPL cleanrooms used in the assembly of the Sample Caching System for the Mars 2020 Perseverance rover.

Multi-faceted metagenomic analysis of spacecraft associated surfaces reveal planetary protection relevant microbial composition.

The National Aeronautics and Space Administration (NASA) has been monitoring the microbial burden of spacecraft since the 1970's Viking missions. Originally culture-based and then focused 16S sequencing techniques were used, but we have now applied whole metagenomic sequencing to a variety of cleanroom samples at the Jet Propulsion Lab (JPL), including the Spacecraft Assembly Facility (SAF) with the goals of taxonomic identification and for functional assignment. Our samples included facility pre-filters, cleanroom vacuum debris, and surface wipes. The taxonomic composition was carried out by three different analysis tools to contrast marker, k-mer, and true alignment approaches. Hierarchical clustering analysis of the data separated vacuum particles from other SAF DNA samples. Vacuum particle samples were the most diverse while DNA samples from the ISO (International Standards Organization) compliant facilities and the SAF were the least diverse; all three were dominated by Proteobacteria. Wipe samples had higher diversity and were predominated by Actinobacteria, including human commensals Cutibacterium acnes and Corynebacterium spp. Taxa identified by the three methods were not identical, supporting the use of multiple methods for metagenome characterization. Likewise, functional annotation was performed using multiple methods. Vacuum particles and SAF samples contained strong signals of the tricarboxylic acid cycle and of amino acid biosynthesis, suggesting that many of the identified microorganisms have the ability to grow in nutrient-limited environments. In total, 18 samples generated high quality metagenome assembled genomes (MAG), which were dominated by Moraxella osloensis or Malassezia restricta. One M. osloensis MAG was assembled into a single circular scaffold and gene annotated. This study includes a rigorous quantitative determination of microbial loads and a qualitative dissection of microbial composition. Assembly of multiple specimens led to greater confidence for the identification of particular species and their predicted functional roles.

Description and Genome Characterization of Three Novel Fungal Strains Isolated from Mars 2020 Mission-Associated Spacecraft Assembly Facility Surfaces-Recommendations for Two New Genera and One Species.

National Aeronautics and Space Administration's (NASA) spacecraft assembly facilities are monitored for the presence of any bacteria or fungi that might conceivably survive a transfer to an extraterrestrial environment. Fungi present a broad and diverse range of phenotypic and functional traits to adapt to extreme conditions, hence the detection of fungi and subsequent eradication of them are needed to prevent forward contamination for future NASA missions. During the construction and assembly for the Mars 2020 mission, three fungal strains with unique morphological and phylogenetic properties were isolated from spacecraft assembly facilities. The reconstruction of phylogenetic trees based on several gene loci (ITS, LSU, SSU, RPB, TUB, TEF1) using multi-locus sequence typing (MLST) and whole genome sequencing (WGS) analyses supported the hypothesis that these were novel species. Here we report the genus or species-level classification of these three novel strains via a polyphasic approach using phylogenetic analysis, colony and cell morphology, and comparative analysis of WGS. The strain FJI-L9-BK-P1 isolated from the Jet Propulsion Laboratory Spacecraft Assembly Facility (JPL-SAF) exhibited a putative phylogenetic relationship with the strain Aaosphaeria arxii CBS175.79 but showed distinct morphology and microscopic features. Another JPL-SAF strain, FJII-L3-CM-DR1, was phylogenetically distinct from members of the family Trichomeriaceae and exhibited morphologically different features from the genera Lithohypha and Strelitziana. The strain FKI-L1-BK-DR1 isolated from the Kennedy Space Center facility was identified as a member of Dothideomycetes incertae sedis and is closely related to the family Kirschsteiniotheliaceae according to a phylogenetic analysis. The polyphasic taxonomic approach supported the recommendation for establishing two novel genera and one novel species. The names Aaosphaeria pasadenensis (FJI-L9-BK-P1 = NRRL 64424 = DSM 114621), Pasadenomyces melaninifex (FJII-L3-CM-DR1 = NRRL 64433 = DSM 114623), and Floridaphiala radiotolerans (FKI-L1-BK-DR1 = NRRL 64434 = DSM 114624) are proposed as type species. Furthermore, resistance to ultraviolet-C and presence of specific biosynthetic gene cluster(s) coding for metabolically active compounds are unique to these strains.

Draft Genome Sequences of Fungi Isolated from Mars 2020 Spacecraft Assembly Facilities.

During the Mars 2020 mission, several fungal strains were isolated from surfaces where spacecraft components were assembled. Draft genome sequencing and characterization will help identify the genes responsible for radiation resistance, supporting the development of countermeasures to prevent fungal contamination of extraterrestrial environments.

The Isolation and Characterization of Rare Mycobiome Associated With Spacecraft Assembly Cleanrooms.

Ensuring biological cleanliness while assembling and launching spacecraft is critical for robotic exploration of the solar system. To date, when preventing forward contamination of other celestial bodies, NASA Planetary Protection policies have focused on endospore-forming bacteria while fungi were neglected. In this study, for the first time the mycobiome of two spacecraft assembly facilities at Jet Propulsion Laboratory (JPL) and Kennedy Space Center (KSC) was assessed using both cultivation and sequencing techniques. To facilitate enumeration of viable fungal populations and downstream molecular analyses, collected samples were first treated with chloramphenicol for 24 h and then with propidium monoazide (PMA). Among cultivable fungi, 28 distinct species were observed, 16 at JPL and 16 at KSC facilities, while 13 isolates were potentially novel species. Only four isolated species Aureobasidium melanogenum, Penicillium fuscoglaucum, Penicillium decumbens, and Zalaria obscura were present in both cleanroom facilities, which suggests that mycobiomes differ significantly between distant locations. To better visualize the biogeography of all isolated strains the network analysis was undertaken and confirmed higher abundance of Malassezia globosa and Cyberlindnera jadinii. When amplicon sequencing was performed, JPL-SAF and KSC-PHSF showed differing mycobiomes. Metagenomic fungal reads were dominated by Ascomycota (91%) and Basidiomycota (7.15%). Similar to amplicon sequencing, the number of fungal reads changed following antibiotic treatment in both cleanrooms; however, the opposite trends were observed. Alas, treatment with the antibiotic did not allow for definitive ascribing changes observed in fungal populations between treated and untreated samples in both cleanrooms. Rather, these substantial differences in fungal abundance might be attributed to several factors, including the geographical location, climate and the in-house cleaning procedures used to maintain the cleanrooms. This study is a first step in characterizing cultivable and viable fungal populations in cleanrooms to assess fungal potential as biocontaminants during interplanetary explorations. The outcomes of this and future studies could be implemented in other cleanrooms that require to reduce microbial burden, like intensive care units, operating rooms, or cleanrooms in the semiconducting and pharmaceutical industries.

Genomic Characterization of Parengyodontium torokii sp. nov., a Biofilm-Forming Fungus Isolated from Mars 2020 Assembly Facility.

A fungal strain (FJII-L10-SW-P1) was isolated from the Mars 2020 spacecraft assembly facility and exhibited biofilm formation on spacecraft-qualified Teflon surfaces. The reconstruction of a six-loci gene tree (ITS, LSU, SSU, RPB1 and RPB2, and TEF1) using multi-locus sequence typing (MLST) analyses of the strain FJII-L10-SW-P1 supported a close relationship to other known Parengyodontium album subclade 3 isolates while being phylogenetically distinct from subclade 1 strains. The zig-zag rachides morphology of the conidiogenous cells and spindle-shaped conidia were the distinct morphological characteristics of the P. album subclade 3 strains. The MLST data and morphological analysis supported the conclusion that the P. album subclade 3 strains could be classified as a new species of the genus Parengyodontium and placed in the family Cordycipitaceae. The name Parengyodontium torokii sp. nov. is proposed to accommodate the strain, with FJII-L10-SW-P1 as the holotype. The genome of the FJII-L10-SW-P1 strain was sequenced, annotated, and the secondary metabolite clusters were identified. Genes predicted to be responsible for biofilm formation and adhesion to surfaces were identified. Homology-based assignment of gene ontologies to the predicted proteome of P. torokii revealed the presence of gene clusters responsible for synthesizing several metabolic compounds, including a cytochalasin that was also verified using traditional metabolomic analysis.

Clean room microbiome complexity impacts planetary protection bioburden

BackgroundThe Spacecraft Assembly Facility (SAF) at the NASA’s Jet Propulsion Laboratory is the primary cleanroom facility used in the construction of some of the planetary protection (PP)-sensitive missions developed by NASA, including the Mars 2020 Perseverance Rover that launched in July 2020. SAF floor samples (n=98) were collected, over a 6-month period in 2016 prior to the construction of the Mars rover subsystems, to better understand the temporal and spatial distribution of bacterial populations (total, viable, cultivable, and spore) in this unique cleanroom.ResultsCleanroom samples were examined for total (living and dead) and viable (living only) microbial populations using molecular approaches and cultured isolates employing the traditional NASA standard spore assay (NSA), which predominantly isolated spores. The 130 NSA isolates were represented by 16 bacterial genera, of which 97% were identified as spore-formers via Sanger sequencing. The most spatially abundant isolate was Bacillus subtilis, and the most temporally abundant spore-former was Virgibacillus panthothenticus. The 16S rRNA gene-targeted amplicon sequencing detected 51 additional genera not found in the NSA method. The amplicon sequencing of the samples treated with propidium monoazide (PMA), which would differentiate between viable and dead organisms, revealed a total of 54 genera: 46 viable non-spore forming genera and 8 viable spore forming genera in these samples. The microbial diversity generated by the amplicon sequencing corresponded to ~86% non-spore-formers and ~14% spore-formers. The most common spatially distributed genera were Sphinigobium, Geobacillus, and Bacillus whereas temporally distributed common genera were Acinetobacter, Geobacilllus, and Bacillus. Single-cell genomics detected 6 genera in the sample analyzed, with the most prominent being Acinetobacter.ConclusionThis study clearly established that detecting spores via NSA does not provide a complete assessment for the cleanliness of spacecraft-associated environments since it failed to detect several PP-relevant genera that were only recovered via molecular methods. This highlights the importance of a methodological paradigm shift to appropriately monitor bioburden in cleanrooms for not only the aeronautical industry but also for pharmaceutical, medical industries, etc., and the need to employ molecular sequencing to complement traditional culture-based assays.-vsg9JJhywtr3shQUX5nF9Video abstract

Performance of Multiple Metagenomics Pipelines in Understanding Microbial Diversity of a Low-Biomass Spacecraft Assembly Facility.

NASA planetary protection (PP) requires an assessment of the biological contamination of the potential microbial burden on spacecraft destined to explore planetary bodies that may harbor signs of life, like Mars and Europa. To help meet these goals, the performance of multiple metagenomic pipelines were compared and assessed for their ability to detect microbial diversity of a low-biomass clean room environment used to build spacecraft destined to these planetary bodies. Four vendors were chosen to implement their own metagenomic analysis pipeline on the shotgun sequences retrieved from environmental surfaces in the relevant environments at NASA’s Jet Propulsion Laboratory. None of the vendors showed the same microbial profile patterns when analyzing same raw dataset since each vendor used different pipelines, which begs the question of the validity of a single pipeline to be recommended for future NASA missions. All four vendors detected species of interest, including spore-forming and extremotolerant bacteria, that have the potential to hitch-hike on spacecraft and contaminate the planetary bodies explored. Some vendors demonstrated through functional analysis of the metagenomes that the molecular mechanisms for spore-formation and extremotolerance were represented in the data. However, relative abundances of these microorganisms varied drastically between vendor analyses, questioning the ability of these pipelines to quantify the number of PP-relevant microorganisms on a spacecraft surface. Metagenomics offers tantalizing access to the genetic and functional potential of a microbial community that may offer NASA a viable method for microbial burden assays for planetary protection purposes. However, future development of technologies such as streamlining the processing of shotgun metagenome sequence data, long read sequencing, and all-inclusive larger curated and annotated microbial genome databases will be required to validate and translate relative abundances into an actionable assessment of PP-related microbes of interest. Additionally, the future development of machine learning and artificial intelligence techniques could help enhance the quality of these metagenomic analyses by providing more accurate identification of the genetic and functional potential of a microbial community.

Draft Genome Sequences of Heat Shock-Tolerant Microbes Isolated from a Spacecraft Assembly Facility.

ABSTRACTHeat shock-tolerant microorganisms belonging to the orders Bacillales and Micrococcales were isolated from the Spacecraft Assembly Facility at the Jet Propulsion Laboratory, and 63 draft genome sequences were assembled and identified. Further analyses of these genomes can provide insight into methods for preventing forward contamination.

Alteration of Proteomes in First-Generation Cultures of Bacillus pumilus Spores Exposed to Outer Space.

Bacillus pumilus SAFR-032 was originally isolated from the Jet Propulsion Lab Spacecraft Assembly Facility and thoroughly characterized for its enhanced resistance to UV irradiation and oxidative stress. This unusual resistance of SAFR-032 is of particular concern in the context of planetary protection and calls for development of novel disinfection techniques to prevent extraterrestrial contamination. Previously, spores of SAFR-032 were exposed for 18 months to a variety of space conditions on board the International Space Station to investigate their resistance to Mars-like conditions and space travel. Here, proteomic characterization of vegetative SAFR-032 cells from space-surviving spores is presented in comparison to a ground control. Vegetative cells of the first passage were processed and subjected to quantitative proteomics using tandem mass tags. Approximately 60% of all proteins encoded by SAFR-032 were identified, and 301 proteins were differentially expressed among the strains. We found that proteins predicted to be involved in carbohydrate transport/metabolism and energy production/conversion had lower abundance than those of the ground control. For three proteins, we showed that the expected metabolic activities were decreased, as expected with direct enzymatic assays. This was consistent with a decrease of ATP production in the space-surviving strains. The same space-surviving strains showed increased abundance of proteins related to survival, growth advantage, and stress response. Such alterations in the proteomes provide insights into possible molecular mechanisms of B. pumilus SAFR-032 to adapt to and resist extreme extraterrestrial environments.IMPORTANCE Spore-forming bacteria are well known for their resistance to harsh environments and are of concern for spreading contamination to extraterrestrial bodies during future life detection missions. Bacillus pumilus has been regularly isolated from spacecraft-associated surfaces and exhibited unusual resistance to ultraviolet light and other sterilization techniques. A better understanding of the mechanisms of microbial survival and enhanced resistance is essential for developing novel disinfection protocols for the purpose of planetary protection. While genomic analyses did not reveal the unique characteristics that explain elevated UV resistance of space-exposed B. pumilus, the proteomics study presented here provided intriguing insight on key metabolic changes. The observed proteomics aberrations reveal a complex biological phenomenon that plays a role in bacterial survival and adaptation under long-term exposure to outer space. This adaptive ability of microorganisms needs to be considered by those tasked with eliminating forward contamination.

Metabolism and Biodegradation of Spacecraft Cleaning Reagents by Strains of Spacecraft-Associated Acinetobacter.

Spacecraft assembly facilities are oligotrophic and low-humidity environments, which are routinely cleaned using alcohol wipes for benchtops and spacecraft materials, and alkaline detergents for floors. Despite these cleaning protocols, spacecraft assembly facilities possess a persistent, diverse, dynamic, and low abundant core microbiome, where the Acinetobacter are among the dominant members of the community. In this report, we show that several spacecraft-associated Acinetobacter metabolize or biodegrade the spacecraft cleaning reagents of ethanol (ethyl alcohol), 2-propanol (isopropyl alcohol), and Kleenol 30 (floor detergent) under ultraminimal conditions. Using cultivation and stable isotope labeling studies, we show that ethanol is a sole carbon source when cultivating in 0.2 × M9 minimal medium containing 26 μM Fe(NH4)2(SO4)2. Although cultures expectedly did not grow solely on 2-propanol, cultivations on mixtures of ethanol and 2-propanol exhibited enhanced plate counts at mole ratios of ≤0.50. In support, enzymology experiments on cellular extracts were consistent with oxidation of ethanol and 2-propanol by a membrane-bound alcohol dehydrogenase. In the presence of Kleenol 30, untargeted metabolite profiling on ultraminimal cultures of Acinetobacter radioresistens 50v1 indicated (1) biodegradation of Kleenol 30 into products including ethylene glycols, (2) the potential metabolism of decanoate (formed during incubation of Kleenol 30 in 0.2 × M9), and (3) decreases in the abundances of several hydroxy- and ketoacids in the extracellular metabolome. In ultraminimal medium (when using ethanol as a sole carbon source), A. radioresistens 50v1 also exhibits a remarkable survival against hydrogen peroxide (∼1.5-log loss, ∼108 colony forming units (cfu)/mL, 10 mM H2O2), indicating a considerable tolerance toward oxidative stress under nutrient-restricted conditions. Together, these results suggest that the spacecraft cleaning reagents may (1) serve as nutrient sources under oligotrophic conditions and (2) sustain extremotolerances against the oxidative stresses associated with low-humidity environments. In perspective, this study provides a plausible biochemical rationale to the observed microbial ecology dynamics of spacecraft-associated environments.

Bacillus safensis FO-36b and Bacillus pumilus SAFR-032: a whole genome comparison of two spacecraft assembly facility isolates

BackgroundBacillus strains producing highly resistant spores have been isolated from cleanrooms and space craft assembly facilities. Organisms that can survive such conditions merit planetary protection concern and if that resistance can be transferred to other organisms, a health concern too. To further efforts to understand these resistances, the complete genome of Bacillus safensis strain FO-36b, which produces spores resistant to peroxide and radiation was determined. The genome was compared to the complete genome of B. pumilus SAFR-032, and the draft genomes of B. safensis JPL-MERTA-8-2 and the type strain B. pumilus ATCC7061T. Additional comparisons were made to 61 draft genomes that have been mostly identified as strains of B. pumilus or B. safensis.ResultsThe FO-36b gene order is essentially the same as that in SAFR-032 and other B. pumilus strains. The annotated genome has 3850 open reading frames and 40 noncoding RNAs and riboswitches. Of these, 307 are not shared by SAFR-032, and 65 are also not shared by MERTA and ATCC7061T. The FO-36b genome has ten unique open reading frames and two phage-like regions, homologous to the Bacillus bacteriophage SPP1 and Brevibacillus phage Jimmer1. Differing remnants of the Jimmer1 phage are found in essentially all B. safensis / B. pumilus strains. Seven unique genes are part of these phage elements. Whole Genome Phylogenetic Analysis of the B. pumilus, B. safensis and other Firmicutes genomes, separate them into three distinct clusters. Two clusters are subgroups of B. pumilus while one houses all the B. safensis strains. The Genome-genome distance analysis and a phylogenetic analysis of gyrA sequences corroborated these results.ConclusionsIt is not immediately obvious that the presence or absence of any specific gene or combination of genes is responsible for the variations in resistance seen. It is quite possible that distinctions in gene regulation can alter the expression levels of key proteins thereby changing the organism’s resistance properties without gain or loss of a particular gene. What is clear is that phage elements contribute significantly to genome variability. Multiple genome comparison indicates that many strains named as B. pumilus likely belong to the B. safensis group.

KatharoSeq Enables High-Throughput Microbiome Analysis from Low-Biomass Samples.

Microbiome analyses of low-biomass samples are challenging because of contamination and inefficiencies, leading many investigators to employ low-throughput methods with minimal controls. We developed a new automated protocol, KatharoSeq (from the Greek katharos [clean]), that outperforms single-tube extractions while processing at least five times as fast. KatharoSeq incorporates positive and negative controls to reveal the whole bacterial community from inputs of as few as 50 cells and correctly identifies 90.6% (standard error, 0.013%) of the reads from 500 cells. To demonstrate the broad utility of KatharoSeq, we performed 16S rRNA amplicon and shotgun metagenome analyses of the Jet Propulsion Laboratory spacecraft assembly facility (SAF; n = 192, 96), 52 rooms of a neonatal intensive care unit (NICU; n = 388, 337), and an endangered-abalone-rearing facility (n = 192, 123), obtaining spatially resolved, unique microbiomes reproducible across hundreds of samples. The SAF, our primary focus, contains 32 sOTUs (sub-OTUs, defined as exact sequence matches) and their inferred variants identified by the deblur algorithm, with four (Acinetobacter lwoffii, Paracoccus marcusii, Mycobacterium sp., and Novosphingobium) being present in >75% of the samples. According to microbial spatial topography, the most abundant cleanroom contaminant, A.lwoffii, is related to human foot traffic exposure. In the NICU, we have been able to discriminate environmental exposure related to patient infectious disease, and in the abalone facility, we show that microbial communities reflect the marine environment rather than human input. Consequently, we demonstrate the feasibility and utility of large-scale, low-biomass metagenomic analyses using the KatharoSeq protocol. IMPORTANCE Various indoor, outdoor, and host-associated environments contain small quantities of microbial biomass and represent a niche that is often understudied because of technical constraints. Many studies that attempt to evaluate these low-biomass microbiome samples are riddled with erroneous results that are typically false positive signals obtained during the sampling process. We have investigated various low-biomass kits and methods to determine the limit of detection of these pipelines. Here we present KatharoSeq, a high-throughput protocol combining laboratory and bioinformatic methods that can differentiate a true positive signal in samples with as few as 50 to 500 cells. We demonstrate the application of this method in three unique low-biomass environments, including a SAF, a hospital NICU, and an abalone-rearing facility.