Fundamental Biological Features of Spaceflight: Advancing the Field to Enable Deep-Space Exploration.

Research on astronaut health and model organisms have revealed six features of spaceflight biology that guide our current understanding of fundamental molecular changes that occur during space travel. The features include oxidative stress, DNA damage, mitochondrial dysregulation, epigenetic changes (including gene regulation), telomere length alterations, and microbiome shifts. Here we review the known hazards of human spaceflight, how spaceflight affects living systems through these six fundamental features, and the associated health risks of space exploration. We also discuss the essential issues related to the health and safety of astronauts involved in future missions, especially planned long-duration and Martian missions.

Identification of the pigment and its role in UV resistance in Paecilomyces variotii, a Chernobyl isolate, using genetic manipulation strategies.

Fungi produce secondary metabolites that are not directly involved in their growth, but often contribute to their adaptation to extreme environmental stimuli and enable their survival. Conidial pigment or melanin is one of the secondary metabolites produced naturally by a polyketide synthesis (PKS) gene cluster in several filamentous fungi and is known to protect these fungi from extreme radiation conditions. Several pigmented or melanized fungi have been shown to grow under extreme radiation conditions at the Chernobyl nuclear accident site. Some of these fungi, including Paecilomyces variotii, were observed to grow towards the source of radiation. Therefore, in this study, we wanted to identify if the pigment produced by P. variotii, contributes to providing protection against radiation condition. We first identified the PKS gene responsible for synthesis of pigment in P. variotii and confirmed its role in providing protection against UV irradiation through CRISPR-Cas9 mediated gene deletion. This is the first report that describes the use of CRISPR methodology to create gene deletions in P. variotii. Further, we showed that the pigment produced by this fungus, was not inhibited by DHN-melanin pathway inhibitors, indicating that the fungus does not produce melanin. We then identified the pigment synthesized by the PKS gene of P. variotii, as a naptho-pyrone Ywa1, by heterologously expressing the gene in Aspergillus nidulans. The results obtained will further aid in understanding the mechanistic basis of radiation resistance.

Proteomic characterization of Aspergillus fumigatus isolated from air and surfaces of the International Space Station.

The on-going Microbial Observatory Experiments on the International Space Station (ISS) revealed the presence of various microorganisms that may be affected by the distinct environment of the ISS. The low-nutrient environment combined with enhanced irradiation and microgravity may trigger changes in the molecular suite of microorganisms leading to increased virulence and resistance of microbes. Proteomic characterization of two Aspergillus fumigatus strains, ISSFT-021 and IF1SW-F4, isolated from HEPA filter debris and cupola surface of the ISS, respectively, is presented, along with a comparison to well-studied clinical isolates Af293 and CEA10. In-depth analysis highlights variations in the proteome of both ISS-isolated strains when compared to the clinical strains. Proteins that showed increased abundance in ISS isolates were overall involved in stress responses, and carbohydrate and secondary metabolism. Among the most abundant proteins were Pst2 and ArtA involved in oxidative stress response, PdcA and AcuE responsible for ethanol fermentation and glyoxylate cycle, respectively, TpcA, TpcF, and TpcK that are part of trypacidin biosynthetic pathway, and a toxin Asp-hemolysin. This report provides insight into possible molecular adaptation of filamentous fungi to the unique ISS environment.

Correction to: Metagenome to phenome approach enables isolation and genomics characterization of Kalamiella piersonii gen. nov., sp. nov. from the International Space Station.

In our original published manuscript entitled "Metagenome to phenome approach enables isolation and genomics characterization of Kalamiella piersonii gen. nov., sp. nov. from the International Space Station" (Singh et al. 2019), we found a taxonomic description format error: As per the Rule 27.

Metagenome to phenome approach enables isolation and genomics characterization of Kalamiella piersonii gen. nov., sp. nov. from the International Space Station.

Several evolutionarily distinct, near full-length draft metagenome-resolved genomes (MRG), were assembled from sequences recovered from the International Space Station (ISS) environments. The retrieval of MRGs facilitated the exploration of a large collection of archived strains (~ 500 isolates) and assisted in isolating seven related strains. The whole genome sequences (WGS) of seven ISS strains exhibited 100% identity to the 4.85 × 106 bp of four MRGs. The "metagenome to phenome" approach led to the description of a novel bacterial genus from the ISS samples. The phylogenomics and traditional taxonomic approaches suggested that these seven ISS strains and four MRGs were not phylogenetically affiliated to any validly described genera of the family Erwiniaceae, but belong to a novel genus with the proposed name Kalamiella. Comparative genomic analyses of Kalamiella piersonii strains and MRGs showed genes associated with carbohydrate (348 genes), amino acid (384), RNA (59), and protein (214) metabolisms; membrane transport systems (108), pathways for biosynthesis of cofactors, vitamins, prosthetic groups, and pigments (179); as well as mechanisms for virulence, disease, and defense (50). Even though Kalamiella genome annotation and disc diffusion tests revealed multidrug resistance, the PathogenFinder algorithm predicted that K. piersonii strains are not human pathogens. This approach to isolating microbes allows for the characterization of functional pathways and their potential virulence properties that can directly affect human health. The isolation of novel strains from the ISS has broad applications in microbiology, not only because of concern for astronaut health but it might have a great potential for biotechnological relevance. The metagenome to phenome approach will help to improve our understanding of complex metabolic networks that control fundamental life processes under microgravity and in deep space.

International Space Station conditions alter genomics, proteomics, and metabolomics in Aspergillus nidulans.

The first global genomic, proteomic, and secondary metabolomic characterization of the filamentous fungus Aspergillus nidulans following growth onboard the International Space Station (ISS) is reported. The investigation included the A. nidulans wild-type and three mutant strains, two of which were genetically engineered to enhance secondary metabolite production. Whole genome sequencing revealed that ISS conditions altered the A. nidulans genome in specific regions. In strain CW12001, which features overexpression of the secondary metabolite global regulator laeA, ISS conditions induced the loss of the laeA stop codon. Differential expression of proteins involved in stress response, carbohydrate metabolic processes, and secondary metabolite biosynthesis was also observed. ISS conditions significantly decreased prenyl xanthone production in the wild-type strain and increased asperthecin production in LO1362 and CW12001, which are deficient in a major DNA repair mechanism. These data provide valuable insights into the adaptation mechanism of A. nidulans to spacecraft environments.

Multi-drug resistant Enterobacter bugandensis species isolated from the International Space Station and comparative genomic analyses with human pathogenic strains.

BACKGROUND: The antimicrobial resistance (AMR) phenotypic properties, multiple drug resistance (MDR) gene profiles, and genes related to potential virulence and pathogenic properties of five Enterobacter bugandensis strains isolated from the International Space Station (ISS) were carried out and compared with genomes of three clinical strains. Whole genome sequences of ISS strains were characterized using the hybrid de novo assembly of Nanopore and Illumina reads. In addition to traditional microbial taxonomic approaches, multilocus sequence typing (MLST) analysis was performed to classify the phylogenetic lineage. Agar diffusion discs assay was performed to test antibiotics susceptibility. The draft genomes after assembly and scaffolding were annotated with the Rapid Annotations using Subsystems Technology and RNAmmer servers for downstream analysis. RESULTS: Molecular phylogeny and whole genome analysis of the ISS strains with all publicly available Enterobacter genomes revealed that ISS strains were E. bugandensis and similar to the type strain EB-247T and two clinical isolates (153_ECLO and MBRL 1077). Comparative genomic analyses of all eight E. bungandensis strains showed, a total of 4733 genes were associated with carbohydrate metabolism (635 genes), amino acid and derivatives (496 genes), protein metabolism (291 genes), cofactors, vitamins, prosthetic groups, pigments (275 genes), membrane transport (247 genes), and RNA metabolism (239 genes). In addition, 112 genes identified in the ISS strains were involved in virulence, disease, and defense. Genes associated with resistance to antibiotics and toxic compounds, including the MDR tripartite system were also identified in the ISS strains. A multiple antibiotic resistance (MAR) locus or MAR operon encoding MarA, MarB, MarC, and MarR, which regulate more than 60 genes, including upregulation of drug efflux systems that have been reported in Escherichia coli K12, was also observed in the ISS strains. CONCLUSION: Given the MDR results for these ISS Enterobacter genomes and increased chance of pathogenicity (PathogenFinder algorithm with > 79% probability), these species pose important health considerations for future missions. Thorough genomic characterization of the strains isolated from ISS can help to understand the pathogenic potential, and inform future missions, but analyzing them in in-vivo systems is required to discern the influence of microgravity on their pathogenicity.

Pervasiveness of UVC254-resistant Geobacillus strains in extreme environments.

We have characterized a broad collection of extremophilic bacterial isolates from a deep subsurface mine, compost dumping sites, and several hot spring ecosystems. Spore-forming strains isolated from these environments comprised both obligate thermophiles/thermotolerant species (growing at > 55 °C; 240 strains) and mesophiles (growing at 15 to 40 °C; 12 strains). An overwhelming abundance of Geobacillus (81.3%) and Bacillus (18.3%) species was observed among the tested isolates. 16S rRNA sequence analysis documented the presence of 24 species among these isolates, but the 16S rRNA gene was shown to possess insufficient resolution to reliably discern Geobacillus phylogeny. gyrB-based phylogenetic analyses of nine strains revealed the presence of six known Geobacillus and one novel species. Multilocus sequence typing analyses based on seven different housekeeping genes deduced from whole genome sequencing of nine strains revealed the presence of three novel Geobacillus species. The vegetative cells of 41 Geobacillus strains were exposed to UVC254, and most (34 strains) survived 120 J/m2, while seven strains survived 300 J/m2, and cells of only one Geobacillus strain isolated from a compost facility survived 600 J/m2. Additionally, the UVC254 inactivation kinetics of spores from four Geobacillus strains isolated from three distinct geographical regions were evaluated and compared to that of a spacecraft assembly facility (SAF) clean room Geobacillus strain. The purified spores of the thermophilic SAF strain exhibited resistance to 2000 J/m2, whereas spores of two environmental Geobacillus strains showed resistance to 1000 J/m2. This study is the first to investigate UV resistance of environmental, obligately thermophilic Geobacillus strains, and also lays the foundation for advanced understanding of necessary sterilization protocols practiced in food, medical, pharmaceutical, and aerospace industries.

Solibacillus kalamii sp. nov., isolated from a high-efficiency particulate arrestance filter system used in the International Space Station.

A Gram-stain-positive, rod-shaped, endospore-forming, aerobic bacterial strain, designated ISSFR-015T, was isolated from a high-efficiency particulate arrestance filter in the International Space Station and was characterized by polyphasic taxonomy. A comparative analysis of the 16S rRNA gene sequence (1494 bp) of strain ISSFR-015T showed highest similarity to Solibacillus isronensis B3W22T (98.9 %), followed by Solibacillus silvestris HR3-23T (98.6 %) and Bacillus cecembensis PN5T (96.7 %). DNA-DNA hybridization analysis revealed that the DNA relatedness values of strain ISSFR-015T with other closely related species were in the range of 41-47 % [S. silvestrisMTCC 10789T (47 %), S. isronensis MTCC 7902T (41 %) and B. cecembensis MTCC 9127T (43 %)]. The DNA G+C content of strain ISSFR-015T was 45.4 mol%. The major fatty acids were iso-C15 : 0 (45.2 %) and C17 : 1ω10c (12.1 %). The polar lipid profile contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine and one unknown phospholipid. The isoprenoid quinones present in strain ISSFR-015T were MK-7 (86.8 %), MK-6 (11.6 %) and MK-8 (1.0 %). The peptidoglycan type of the cell wall was A4α l-Lys-d-Glu. Based on the phylogenetic analysis, strain ISSFR-015T belongs to the genus Solibacillus. The polyphasic taxonomic data, including low DNA-DNA hybridization values, and the chemotaxonomic analysis confirmed that strain ISSFR-015T represents a novel species, for which the name Solibacillus kalamii sp. nov. is proposed. The type strain for this proposed species is ISSFR-015T (=NRRL B-65388T=DSM 101595T).

Deinococcus phoenicis sp. nov., an extreme ionizing-radiation-resistant bacterium isolated from the Phoenix Lander assembly facility.

A bacterial strain, designated 1P10ME(T), which was resistant to extreme doses of ionizing radiation, pale-pink, non-motile, and a tetrad-forming coccoid was isolated from a cleanroom at the Kennedy Space Center, where the Phoenix spacecraft was assembled. Strain 1P10ME(T) showed optimum growth at 30 °C, with a pH range for growth of 6.5-9.0 and was highly sensitive to sodium chloride, growing only in medium with no added NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain 1P10ME(T) represents a novel member of the genus Deinococcus, with low sequence similarities (<93.5%) to recognized species of the genus Deinococcus. The predominant cellular fatty acid was C15:1ω6c. This novel strain exhibits extreme resistance to gamma radiation (D10 >8 kGy) and UV (D10 >1000 Jm(-2)). The results of our polyphasic taxonomic analyses suggest that strain 1P10ME(T) represents a novel species of the genus Deinococcus, for which the name Deinococcus phoenicis sp. nov. is proposed. The type strain is 1P10ME(T) ( = NRRL B-59546(T) = DSM 27173(T)).

International Space Station environmental microbiome - microbial inventories of ISS filter debris.

Despite an expanding array of molecular approaches for detecting microorganisms in a given sample, rapid and robust means of assessing the differential viability of the microbial cells, as a function of phylogenetic lineage, remain elusive. A propidium monoazide (PMA) treatment coupled with downstream quantitative polymerase chain reaction (qPCR) and pyrosequencing analyses was carried out to better understand the frequency, diversity, and distribution of viable microorganisms associated with debris collected from the crew quarters of the International Space Station (ISS). The cultured bacterial counts were more in the ISS samples than cultured fungal population. The rapid molecular analyses targeted to estimate viable population exhibited 5-fold increase in bacterial (qPCR-PMA assay) and 25-fold increase in microbial (adenosine triphosphate assay) burden than the cultured bacterial population. The ribosomal nucleic acid-based identification of cultivated strains revealed the presence of only four to eight bacterial species in the ISS samples, however, the viable bacterial diversity detected by the PMA-pyrosequencing method was far more diverse (12 to 23 bacterial taxa) with the majority consisting of members of actinobacterial genera (Propionibacterium, Corynebacterium) and Staphylococcus. Sample fractions not treated with PMA (inclusive of both live and dead cells) yielded a great abundance of highly diverse bacterial (94 to 118 taxa) and fungal lineages (41 taxa). Even though deep sequencing capability of the molecular analysis widened the understanding about the microbial diversity, the cultivation assay also proved to be essential since some of the spore-forming microorganisms were detected only by the culture-based method. Presented here are the findings of the first comprehensive effort to assess the viability of microbial cells associated with ISS surfaces, and correlate differential viability with phylogenetic affiliation.

Genomic, functional, and metabolic enhancements in multidrug-resistant Enterobacter bugandensis facilitating its persistence and succession in the International Space Station.

BACKGROUND: The International Space Station (ISS) stands as a testament to human achievement in space exploration. Despite its highly controlled environment, characterised by microgravity, increased CO 2 levels, and elevated solar radiation, microorganisms occupy a unique niche. These microbial inhabitants play a significant role in influencing the health and well-being of astronauts on board. One microorganism of particular interest in our study is Enterobacter bugandensis, primarily found in clinical specimens including the human gastrointestinal tract, and also reported to possess pathogenic traits, leading to a plethora of infections. RESULTS: Distinct from their Earth counterparts, ISS E. bugandensis strains have exhibited resistance mechanisms that categorise them within the ESKAPE pathogen group, a collection of pathogens recognised for their formidable resistance to antimicrobial treatments. During the 2-year Microbial Tracking 1 mission, 13 strains of multidrug-resistant E. bugandensis were isolated from various locations within the ISS. We have carried out a comprehensive study to understand the genomic intricacies of ISS-derived E. bugandensis in comparison to terrestrial strains, with a keen focus on those associated with clinical infections. We unravel the evolutionary trajectories of pivotal genes, especially those contributing to functional adaptations and potential antimicrobial resistance. A hypothesis central to our study was that the singular nature of the stresses of the space environment, distinct from any on Earth, could be driving these genomic adaptations. Extending our investigation, we meticulously mapped the prevalence and distribution of E. bugandensis across the ISS over time. This temporal analysis provided insights into the persistence, succession, and potential patterns of colonisation of E. bugandensis in space. Furthermore, by leveraging advanced analytical techniques, including metabolic modelling, we delved into the coexisting microbial communities alongside E. bugandensis in the ISS across multiple missions and spatial locations. This exploration revealed intricate microbial interactions, offering a window into the microbial ecosystem dynamics within the ISS. CONCLUSIONS: Our comprehensive analysis illuminated not only the ways these interactions sculpt microbial diversity but also the factors that might contribute to the potential dominance and succession of E. bugandensis within the ISS environment. The implications of these findings are twofold. Firstly, they shed light on microbial behaviour, adaptation, and evolution in extreme, isolated environments. Secondly, they underscore the need for robust preventive measures, ensuring the health and safety of astronauts by mitigating risks associated with potential pathogenic threats. Video Abstract.

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.

Novel spore-forming species exhibiting intrinsic resistance to third- and fourth-generation cephalosporins and description of Tigheibacillus jepli gen. nov., sp. nov.

A comprehensive microbial surveillance was conducted at NASA's Mars 2020 spacecraft assembly facility (SAF), where whole-genome sequencing (WGS) of 110 bacterial strains was performed. One isolate, designated 179-BFC-A-HST, exhibited less than 80% average nucleotide identity (ANI) to known species, suggesting a novel organism. This strain demonstrated high-level resistance [minimum inhibitory concentration (MIC) >256 mg/L] to third-generation cephalosporins, including ceftazidime, cefpodoxime, combination ceftazidime/avibactam, and the fourth-generation cephalosporin cefepime. The results of a comparative genomic analysis revealed that 179-BFC-A-HST is most closely related to Virgibacillus halophilus 5B73CT, sharing an ANI of 78.7% and a digital DNA-DNA hybridization (dDDH) value of 23.5%, while their 16S rRNA gene sequences shared 97.7% nucleotide identity. Based on these results and the recent recognition that the genus Virgibacillus is polyphyletic, strain 179-BFC-A-HST is proposed as a novel species of a novel genus, Tigheibacillus jepli gen. nov., sp. nov (type strain 179-BFC-A-HST = DSM 115946T = NRRL B-65666T), and its closest neighbor, V. halophilus, is proposed to be reassigned to this genus as Tigheibacillus halophilus comb. nov. (type strain 5B73CT = DSM 21623T = JCM 21758T = KCTC 13935T). It was also necessary to reclassify its second closest neighbor Virgibacillus soli, as a member of a novel genus Paracerasibacillus, reflecting its phylogenetic position relative to the genus Cerasibacillus, for which we propose Paracerasibacillus soli comb. nov. (type strain CC-YMP-6T = DSM 22952T = CCM 7714T). Within Amphibacillaceae (n = 64), P. soli exhibited 11 antibiotic resistance genes (ARG), while T. jepli encoded for 3, lacking any known ß-lactamases, suggesting resistance from variant penicillin-binding proteins, disrupting cephalosporin efficacy. P. soli was highly resistant to azithromycin (MIC >64 mg/L) yet susceptible to cephalosporins and penicillins. IMPORTANCE: The significance of this research extends to understanding microbial survival and adaptation in oligotrophic environments, such as those found in SAF. Whole-genome sequencing of several strains isolated from Mars 2020 mission assembly cleanroom facilities, including the discovery of the novel species Tigheibacillus jepli, highlights the resilience and antimicrobial resistance (AMR) in clinically relevant antibiotic classes of microbes in nutrient-scarce settings. The study also redefines the taxonomic classifications within the Amphibacillaceae family, aligning genetic identities with phylogenetic data. Investigating ARG and virulence factors (VF) across these strains illuminates the microbial capability for resistance under resource-limited conditions while emphasizing the role of human-associated VF in microbial survival, informing sterilization practices and microbial management in similar oligotrophic settings beyond spacecraft assembly cleanrooms such as pharmaceutical and medical industry cleanrooms.

Biochemical characterization of Fsa16295Glu from "Fervidibacter sacchari," the first hyperthermophilic GH50 with ß-1,3-endoglucanase activity and founding member of the subfamily GH50_3.

The aerobic hyperthermophile "Fervidibacter sacchari" catabolizes diverse polysaccharides and is the only cultivated member of the class "Fervidibacteria" within the phylum Armatimonadota. It encodes 117 putative glycoside hydrolases (GHs), including two from GH family 50 (GH50). In this study, we expressed, purified, and functionally characterized one of these GH50 enzymes, Fsa16295Glu. We show that Fsa16295Glu is a ß-1,3-endoglucanase with optimal activity on carboxymethyl curdlan (CM-curdlan) and only weak agarase activity, despite most GH50 enzymes being described as ß-agarases. The purified enzyme has a wide temperature range of 4-95°C (optimal 80°C), making it the first characterized hyperthermophilic representative of GH50. The enzyme is also active at a broad pH range of at least 5.5-11 (optimal 6.5-10). Fsa16295Glu possesses a relatively high kcat/KM of 1.82 × 107 s-1 M-1 with CM-curdlan and degrades CM-curdlan nearly completely to sugar monomers, indicating preferential hydrolysis of glucans containing ß-1,3 linkages. Finally, a phylogenetic analysis of Fsa16295Glu and all other GH50 enzymes revealed that Fsa16295Glu is distant from other characterized enzymes but phylogenetically related to enzymes from thermophilic archaea that were likely acquired horizontally from "Fervidibacteria." Given its functional and phylogenetic novelty, we propose that Fsa16295Glu represents a new enzyme subfamily, GH50_3.

Longitudinal multi-omics analysis of host microbiome architecture and immune responses during short-term spaceflight.

Maintenance of astronaut health during spaceflight will require monitoring and potentially modulating their microbiomes. However, documenting microbial shifts during spaceflight has been difficult due to mission constraints that lead to limited sampling and profiling. Here we executed a six-month longitudinal study to quantify the high-resolution human microbiome response to three days in orbit for four individuals. Using paired metagenomics and metatranscriptomics alongside single-nuclei immune cell profiling, we characterized time-dependent, multikingdom microbiome changes across 750 samples and 10 body sites before, during and after spaceflight at eight timepoints. We found that most alterations were transient across body sites; for example, viruses increased in skin sites mostly during flight. However, longer-term shifts were observed in the oral microbiome, including increased plaque-associated bacteria (for example, Fusobacteriota), which correlated with immune cell gene expression. Further, microbial genes associated with phage activity, toxin-antitoxin systems and stress response were enriched across multiple body sites. In total, this study reveals in-depth characterization of microbiome and immune response shifts experienced by astronauts during short-term spaceflight and the associated changes to the living environment, which can help guide future missions, spacecraft design and space habitat planning.

Planetary Protection Knowledge Gap Closure Enabling Crewed Missions to Mars.

As focus for exploration of Mars transitions from current robotic explorers to development of crewed missions, it remains important to protect the integrity of scientific investigations at Mars, as well as protect the Earth's biosphere from any potential harmful effects from returned martian material. This is the discipline of planetary protection, and the Committee on Space Research (COSPAR) maintains the consensus international policy and guidelines on how this is implemented. Based on National Aeronautics and Space Administration (NASA) and European Space Agency (ESA) studies that began in 2001, COSPAR adopted principles and guidelines for human missions to Mars in 2008. At that point, it was clear that to move from those qualitative provisions, a great deal of work and interaction with spacecraft designers would be necessary to generate meaningful quantitative recommendations that could embody the intent of the Outer Space Treaty (Article IX) in the design of such missions. Beginning in 2016, COSPAR then sponsored a multiyear interdisciplinary meeting series to address planetary protection "knowledge gaps" (KGs) with the intent of adapting and extending the current robotic mission-focused Planetary Protection Policy to support the design and implementation of crewed and hybrid exploration missions. This article describes the outcome of the interdisciplinary COSPAR meeting series, to describe and address these KGs, as well as identify potential paths to gap closure. It includes the background scientific basis for each topic area and knowledge updates since the meeting series ended. In particular, credible solutions for KG closure are described for the three topic areas of (1) microbial monitoring of spacecraft and crew health; (2) natural transport (and survival) of terrestrial microbial contamination at Mars, and (3) the technology and operation of spacecraft systems for contamination control. The article includes a KG data table on these topic areas, which is intended to be a point of departure for making future progress in developing an end-to-end planetary protection requirements implementation solution for a crewed mission to Mars. Overall, the workshop series has provided evidence of the feasibility of planetary protection implementation for a crewed Mars mission, given (1) the establishment of needed zoning, emission, transport, and survival parameters for terrestrial biological contamination and (2) the creation of an accepted risk-based compliance approach for adoption by spacefaring actors including national space agencies and commercial/nongovernment organizations.

Description of Tersicoccus phoenicis gen. nov., sp. nov. isolated from spacecraft assembly clean room environments.

Two strains of aerobic, non-motile, Gram-reaction-positive cocci were independently isolated from geographically distinct spacecraft assembly clean room facilities (Kennedy Space Center, Florida, USA and Centre Spatial Guyanais, Kourou, French Guiana). A polyphasic study was carried out to delineate the taxonomic identity of these two isolates (1P05MA(T) and KO_PS43). The 16S rRNA gene sequences exhibited a high similarity when compared to each other (100 %) and lower than 96.7 % relatedness with Arthrobacter crystallopoietes ATCC 15481(T), Arthrobacter luteolus ATCC BAA-272(T), Arthrobacter tumbae DSM 16406(T) and Arthrobacter subterraneus DSM 17585(T). In contrast with previously described Arthrobacter species, the novel isolates maintained their coccidal morphology throughout their growth and did not exhibit the rod-coccus life cycle typically observed in nearly all Arthrobacter species, except A. agilis. The distinct taxonomic identity of the novel isolates was confirmed based on their unique cell-wall peptidoglycan type (A.11.20; Lys-Ser-Ala2) and polar lipid profile (presence of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, an unknown phospholipid and two unknown glycolipids). The G+C content of the genomic DNA was 70.6 mol%. The novel strains revealed MK-9(H2) and MK-8(H2) as dominant menaquinones and exhibited fatty acid profiles consisting of major amounts of anteiso-C15 : 0 and anteiso-C17 : 0 and moderate amounts of iso-C15 : 0 discriminating them again from closely related Arthrobacter species. Based on these observations, the authors propose that strains 1P05MA(T) and KO_PS43 be assigned into a separate genus Tersicoccus gen. nov. For this new taxon, comprising strains 1P05MA(T) and KO_PS43, we propose the name Tersicoccus phoenicis gen. nov., sp. nov. (the type species of Tersicoccus), represented by the type strain Tersicoccus phoenicis 1P05MA(T) ( = NRRL B-59547(T) = DSM 30849(T)).