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.

Are we There Yet? Understanding Interplanetary Microbial Hitchhikers using Molecular Methods.

Since the early time of space travel, planetary bodies undergoing chemical or biological evolution have been of particular interest for life detection missions. NASA's and ESA's Planetary Protection offices ensure responsible exploration of the solar system and aim at avoiding inadvertent contamination of celestial bodies with biomolecules or even living organisms. Life forms that have the potential to colonize foreign planetary bodies could be a threat to the integrity of science objectives of life detection missions. While standard requirements for assessing the cleanliness of spacecraft are still based on cultivation approaches, several molecular methods have been applied in the past to elucidate the full breadth of (micro)organisms that can be found on spacecraft and in cleanrooms, where the hardware is assembled. Here, we review molecular assays that have been applied in Planetary Protection research and list their significant advantages and disadvantages. By providing a comprehensive summary of the latest molecular methods yet to be applied in this research area, this article will not only aid in designing technological roadmaps for future Planetary Protection endeavors but also help other disciplines in environmental microbiology that deal with low biomass samples.

Bacillus glennii sp. nov. and Bacillus saganii sp. nov., isolated from the vehicle assembly building at Kennedy Space Center where the Viking spacecraft were assembled.

Two Gram-stain-positive, motile, endospore-forming, aerobic strains, designated V44-8T and V47-23aT, were isolated from environmental air sampling at the vehicle assembly building at Cape Canaveral, Florida, where the Viking spacecraft were assembled. Growth was observed at pH 7-9 (optimum, pH 9) for strain V44-8T, and pH 5-10 (pH 9) for strain V47-23aT. Both strains displayed growth in 0-5 % NaCl with an optimum at 1 % for strain V44-8T; 0 % for strain V47-23aT. Strains V44-8T and V47-23aT grew optimally at 32 °C, (15-32 °C) and 25 °C (20-45 °C), respectively. The cell wall of both strains contained meso-diaminopimelic acid as the diagnostic diamino acid. Both strains contained phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol. The predominant cellular fatty acids were anteiso-C15 : 0, iso-C14 : 0 and iso-C15 : 0. Strain V47.23aT shared its highest 16S rRNA sequence similarity with Bacillus cavernae DSM-105484T at 96.9%, and V44.8T with Bacillus zeae DSM-103964T at 96.6 %. Based on their phenotypic characteristics and phylogenetic position inferred from 16S rRNA gene sequence analyses, the isolates were identified as being a members of the genus Bacillus that forms a separate clade when compared to close relatives. Average nucleotide identity and average amino acid identity values between strains V44-8T and DSM-103964T were 72.1% and 67.5 %; V47-23aT and DSM-105484T were 62.4% and 69.1%, respectively. Based on the phenotypic, genomic and biochemical data, strains V44-8T and V47-23aT represent two novel species in the genus Bacillus for which the names Bacillus glennii sp. nov. [type strain, V44-8T (=ATCC BAA-2860T =DSM 105192T)], and Bacillus saganii sp. nov. [V47-23aT (=ATCC BAA-2861T=DSM 105190T)] are proposed.

Paenibacillus xerothermodurans sp. nov., an extremely dry heat resistant spore forming bacterium isolated from the soil of Cape Canaveral, Florida.

A Gram-stain-positive, motile, endospore-producing, facultative anaerobic bacterial strain, designated ATCC 27380T, was isolated from heat-stressed soil of Cape Canaveral, Florida, USA. Growth was observed at 20-42 °C (optimum, 37 °C), at pH 6.0-10.0 (optimum pH 7.0) and in the presence of 0.5-3 % NaCl (optimum 0.5 %). The cell wall contained meso-diaminopimelic acid as the diagnostic amino acid and the isoprenoid quinone was MK-7. The polar lipids present were phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol and one unknown phospholipid. The main fatty acids were iso-C15 : 0 and anteiso-C15 : 0. Phylogenetic analysis based on 16S rRNA gene sequencing affiliated strain ATCC 27380T to the genus Paenibacillus, and showed the highest sequence similarity to Paenibacillus rigui JCM 16352T (97.0 %). The other closely related type strains exhibited 16S rRNA gene sequence similarity values below 95.9 %. The draft genome of ATCC 27380T had a size of 4,361,187 bases, with a G+C content of 51.0 %. The average nucleotide identity and in silico DNA-DNA hybridization values between strain ATCC 27380T and P. rigui JCM 16352T were 72.5% and 18.5 %, respectively, which were below the threshold suggested for species differentiation (96% and 70 %, respectively). The average amino acid identity between strain ATCC 27380T and P. rigui JCM 16352T was 68.72 %, which was above the suggested genus level demarcation of 65 %. Based on phenotypic, genotypic and chemotaxonomic data, strain ATCC 27380T represents a novel species in the genus Paenibacillus, for which the name Paenibacillusxerothermodurans sp. nov. (=DSM 520T=NRRL NRS-1629T=ATCC 27380T) is proposed.

Mangrovibacter phragmitis sp. nov., an endophyte isolated from the roots of Phragmites karka.

A facultatively anaerobic, Gram-stain-negative, rod-shaped, nitrogen-fixing, endophytic bacterial strain designated MP23T was isolated from the roots of Phragmites karka growing in Chilika Lagoon, Odisha, India. Strain MP23T was slightly halophilic, and the optimal NaCl concentration and temperature for growth were 1 % and 30 °C, respectively. On the basis of 16S rRNA gene sequence similarities, strain MP23T was affiliated to the family Enterobacteriaceae and most closely related to Mangrovibacter yixingensis KCTC 42181T and Mangrovibacter plantisponsor DSM 19579T with 99.71 % similarity, followed by Salmonella enterica subsp. salamae DSM 9220T (97.22 %), Cronobacter condimenti LMG 26250T (97.14 %) and Salmonella enterica subsp. diarizonae DSM 14847T (97 %). Sequence analysis of 16S rRNA, hsp60, gyrB and rpoB genes showed that strain MP23T formed a phylogenetic cluster with M. yixingensis KCTC 42181T and M. plantisponsor DSM 19579T indicating that it belongs to the genus Mangrovibacter. The major cellular fatty acids were C16 : 0, C18 : 1ω6c and/or C18 : 1ω7c, C16 : 1ω6c and/or C16 : 1ω7c, C14 : 0, C14 : 0 3-OH and/or iso-C16 : 1 I and C17 : 0 cyclo. Polar lipids of strain MP23T consisted of phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine. The DNA G+C content was 50.3 mol%. Based on experimental DNA-DNA hybridization values and average nucleotide identity derived from in silico comparison of whole-genome sequences, strain MP23T could be distinguished from its closest neighbours. We therefore conclude that strain MP23T represents a novel species of the genus Mangrovibacter for which the name Mangrovibacter phragmitis sp. nov. is proposed. The type strain is MP23T (=DSM 100250T=KCTC 42580T).

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.

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)).

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.

Pyrosequencing-derived bacterial, archaeal, and fungal diversity of spacecraft hardware destined for Mars.

Spacecraft hardware and assembly cleanroom surfaces (233 m(2) in total) were sampled, total genomic DNA was extracted, hypervariable regions of the 16S rRNA gene (bacteria and archaea) and ribosomal internal transcribed spacer (ITS) region (fungi) were subjected to 454 tag-encoded pyrosequencing PCR amplification, and 203,852 resulting high-quality sequences were analyzed. Bioinformatic analyses revealed correlations between operational taxonomic unit (OTU) abundance and certain sample characteristics, such as source (cleanroom floor, ground support equipment [GSE], or spacecraft hardware), cleaning regimen applied, and location about the facility or spacecraft. National Aeronautics and Space Administration (NASA) cleanroom floor and GSE surfaces gave rise to a larger number of diverse bacterial communities (619 OTU; 20 m(2)) than colocated spacecraft hardware (187 OTU; 162 m(2)). In contrast to the results of bacterial pyrosequencing, where at least some sequences were generated from each of the 31 sample sets examined, only 13 and 18 of these sample sets gave rise to archaeal and fungal sequences, respectively. As was the case for bacteria, the abundance of fungal OTU in the GSE surface samples dramatically diminished (9× less) once cleaning protocols had been applied. The presence of OTU representative of actinobacteria, deinococci, acidobacteria, firmicutes, and proteobacteria on spacecraft surfaces suggests that certain bacterial lineages persist even following rigorous quality control and cleaning practices. The majority of bacterial OTU observed as being recurrent belonged to actinobacteria and alphaproteobacteria, supporting the hypothesis that the measures of cleanliness exerted in spacecraft assembly cleanrooms (SAC) inadvertently select for the organisms which are the most fit to survive long journeys in space.

Adaptation to Environmental Extremes Structures Functional Traits in Biological Soil Crust and Hypolithic Microbial Communities.

Biological soil crusts (biocrusts) are widespread in drylands and deserts. At the microhabitat scale, they also host hypolithic communities that live under semitranslucent stones. Both environmental niches experience exposure to extreme conditions such as high UV radiation, desiccation, temperature fluctuations, and resource limitation. However, hypolithic communities are somewhat protected from extremes relative to biocrust communities. Conditions are otherwise similar, so comparing them can answer outstanding questions regarding adaptations to environmental extremes. Using metagenomic sequencing, we assessed the functional potential of dryland soil communities and identified the functional underpinnings of ecological niche differentiation in biocrusts versus hypoliths. We also determined the effect of the anchoring photoautotroph (moss or cyanobacteria). Genes and pathways differing in abundance between biocrusts and hypoliths indicate that biocrust communities adapt to the higher levels of UV radiation, desiccation, and temperature extremes through an increased ability to repair damaged DNA, sense and respond to environmental stimuli, and interact with other community members and the environment. Intracellular competition appears to be crucial to both communities, with biocrust communities using the Type VI Secretion System (T6SS) and hypoliths favoring a diversity of antibiotics. The dominant primary producer had a reduced effect on community functional potential compared with niche, but an abundance of genes related to monosaccharide, amino acid, and osmoprotectant uptake in moss-dominated communities indicates reliance on resources provided to heterotrophs by mosses. Our findings indicate that functional traits in dryland communities are driven by adaptations to extremes and we identify strategies that likely enable survival in dryland ecosystems. IMPORTANCE Biocrusts serve as a keystone element of desert and dryland ecosystems, stabilizing soils, retaining moisture, and serving as a carbon and nitrogen source in oligotrophic environments. Biocrusts cover approximately 12% of the Earth's terrestrial surface but are threatened by climate change and anthropogenic disturbance. Given their keystone role in ecosystem functioning, loss will have wide-spread consequences. Biocrust microbial constituents must withstand polyextreme environmental conditions including high UV exposure, desiccation, oligotrophic conditions, and temperature fluctuations over short time scales. By comparing biocrust communities with co-occurring hypolithic communities (which inhabit the ventral sides of semitranslucent stones and are buffered from environmental extremes), we identified traits that are likely key adaptations to extreme conditions. These include DNA damage repair, environmental sensing and response, and intracellular competition. Comparison of the two niches, which differ primarily in exposure levels to extreme conditions, makes this system ideal for understanding how functional traits are structured by the environment.

Comparison of innovative molecular approaches and standard spore assays for assessment of surface cleanliness.

A bacterial spore assay and a molecular DNA microarray method were compared for their ability to assess relative cleanliness in the context of bacterial abundance and diversity on spacecraft surfaces. Colony counts derived from the NASA standard spore assay were extremely low for spacecraft surfaces. However, the PhyloChip generation 3 (G3) DNA microarray resolved the genetic signatures of a highly diverse suite of microorganisms in the very same sample set. Samples completely devoid of cultivable spores were shown to harbor the DNA of more than 100 distinct microbial phylotypes. Furthermore, samples with higher numbers of cultivable spores did not necessarily give rise to a greater microbial diversity upon analysis with the DNA microarray. The findings of this study clearly demonstrated that there is not a statistically significant correlation between the cultivable spore counts obtained from a sample and the degree of bacterial diversity present. Based on these results, it can be stated that validated state-of-the-art molecular techniques, such as DNA microarrays, can be utilized in parallel with classical culture-based methods to further describe the cleanliness of spacecraft surfaces.

Paenibacillus phoenicis sp. nov., isolated from the Phoenix Lander assembly facility and a subsurface molybdenum mine.

A novel Gram-positive, motile, endospore-forming, aerobic bacterium was isolated from the NASA Phoenix Lander assembly clean room that exhibits 100 % 16S rRNA gene sequence similarity to two strains isolated from a deep subsurface environment. All strains are rod-shaped, endospore-forming bacteria, whose endospores are resistant to UV radiation up to 500 J m(-2). A polyphasic taxonomic study including traditional phenotypic tests, fatty acid analysis, 16S rRNA gene sequencing and DNA-DNA hybridization analysis was performed to characterize these novel strains. The 16S rRNA gene sequencing convincingly grouped these novel strains within the genus Paenibacillus as a separate cluster from previously described species. The similarity of 16S rRNA gene sequences among the novel strains was identical but only 98.1 to 98.5 % with their nearest neighbours Paenibacillus barengoltzii ATCC BAA-1209(T) and Paenibacillus timonensis CIP 108005(T). The menaquinone MK-7 was dominant in these novel strains as shown in other species of the genus Paenibacillus. The DNA-DNA hybridization dissociation value was <45 % with the closest related species. The novel strains had DNA G+C contents of 51.9 to 52.8 mol%. Phenotypically, the novel strains can be readily differentiated from closely related species by the absence of urease and gelatinase and the production of acids from a variety of sugars including l-arabinose. The major fatty acid was anteiso-C(15 : 0) as seen in P. barengoltzii and P. timonensis whereas the proportion of C(16 : 0) was significantly different from the closely related species. Based on phylogenetic and phenotypic results, it was concluded that these strains represent a novel species of the genus Paenibacillus, for which the name Paenibacillus phoenicis sp. nov. is proposed. The type strain is 3PO2SA(T) ( = NRRL B-59348(T)  = NBRC 106274(T)).

STITCH: algorithm to splice, trim, identify, track, and capture the uniqueness of 16S rRNAs sequence pairs using public or in-house database.

A comparison of variable regions within the 16S rRNA gene is widely used to characterize relationships between bacteria and to identify phylogenetic affiliation of unknown bacteria. In environmental studies, polymerase chain reaction amplification of 16S rRNA followed by cloning and sequencing of numerous individual clones is an extensively used molecular method for elucidating microbial diversity. The sequencing process typically utilizes a forward and reverse primer pair to produce two partial reads (~700 to 800 base pairs each) that overlap and in total cover a large region of the full 16S rRNA sequence (~1.5 k base). In a typical application, this approach rapidly generates very large numbers of 16S rRNA datasets that can overwhelm manual processing efforts leading to both delays and errors. In particular, the approach presents two computational challenges: (1) the assembly of a composite sequence from the two partial reads and (2) the subsequent appropriate identification of the organism represented by the newly sequenced clones. Herein, we describe a software package, search, trim, identify, track, and capture the uniqueness of 16S rRNAs using public and in-house database (STITCH), which offers automated sequence pair splicing and genetic identification, thus simplifying the computationally intensive analysis of large sequencing libraries. The STITCH software is freely accessible over the Internet at: http://prion.bchs.uh.edu/stitch/.

Diversity of anaerobic microbes in spacecraft assembly clean rooms.

Although the cultivable and noncultivable microbial diversity of spacecraft assembly clean rooms has been previously documented using conventional and state-of-the-art molecular techniques, the occurrence of obligate anaerobes within these clean rooms is still uncertain. Therefore, anaerobic bacterial communities of three clean-room facilities were analyzed during assembly of the Mars Science Laboratory rover. Anaerobic bacteria were cultured on several media, and DNA was extracted from suitable anaerobic enrichments and examined with conventional 16S rRNA gene clone library, as well as high-density phylogenetic 16S rRNA gene microarray (PhyloChip) technologies. The culture-dependent analyses predominantly showed the presence of clostridial and propionibacterial strains. The 16S rRNA gene sequences retrieved from clone libraries revealed distinct microbial populations associated with each clean-room facility, clustered exclusively within gram-positive organisms. PhyloChip analysis detected a greater microbial diversity, spanning many phyla of bacteria, and provided a deeper insight into the microbial community structure of the clean-room facilities. This study presents an integrated approach for assessing the anaerobic microbial population within clean-room facilities, using both molecular and cultivation-based analyses. The results reveal that highly diverse anaerobic bacterial populations persist in the clean rooms even after the imposition of rigorous maintenance programs and will pose a challenge to planetary protection implementation activities.

Microbial diversity in uranium mining-impacted soils as revealed by high-density 16S microarray and clone library.

Microbial diversity was characterized in mining-impacted soils collected from two abandoned uranium mine sites, the Edgemont and the North Cave Hills, South Dakota, using a high-density 16S microarray (PhyloChip) and clone libraries. Characterization of the elemental compositions of soils by X-ray fluorescence spectroscopy revealed higher metal contamination including uranium at the Edgemont than at the North Cave Hills mine site. Microarray data demonstrated extensive phylogenetic diversity in soils and confirmed nearly all clone-detected taxonomic levels. Additionally, the microarray exhibited greater diversity than clone libraries at each taxonomic level at both the mine sites. Interestingly, the PhyloChip detected the largest number of taxa in Proteobacteria phylum for both the mine sites. However, clone libraries detected Acidobacteria and Bacteroidetes as the most numerically abundant phyla in the Edgemont and North Cave Hills mine sites, respectively. Several 16S rDNA signatures found in both the microarrays and clone libraries displayed sequence similarities with yet-uncultured bacteria representing a hitherto unidentified diversity. Results from this study demonstrated that highly diverse microbial populations were present in these uranium mine sites. Diversity indices indicated that microbial communities at the North Cave Hills mine site were much more diverse than those at the Edgemont mine site.

Microbial and mineralogical characterizations of soils collected from the deep biosphere of the former Homestake gold mine, South Dakota.

A microbial census on deep biosphere (1.34 km depth) microbial communities was performed in two soil samples collected from the Ross and number 6 Winze sites of the former Homestake gold mine, Lead, South Dakota using high-density 16S microarrays (PhyloChip). Soil mineralogical characterization was carried out using X-ray diffraction, X-ray photoelectron, and Mössbauer spectroscopic techniques which demonstrated silicates and iron minerals (phyllosilicates and clays) in both samples. Microarray data revealed extensive bacterial diversity in soils and detected the largest number of taxa in Proteobacteria phylum followed by Firmicutes and Actinobacteria. The archael communities in the deep gold mine environments were less diverse and belonged to phyla Euryarchaeota and Crenarchaeota. Both the samples showed remarkable similarities in microbial communities (1,360 common OTUs) despite distinct geochemical characteristics. Fifty-seven phylotypes could not be classified even at phylum level representing a hitherto unidentified diversity in deep biosphere. PhyloChip data also suggested considerable metabolic diversity by capturing several physiological groups such as sulfur-oxidizer, ammonia-oxidizers, iron-oxidizers, methane-oxidizers, and sulfate-reducers in both samples. High-density microarrays revealed the greatest prokaryotic diversity ever reported from deep subsurface habitat of gold mines.

End-to-End Protocol for the Detection of SARS-CoV-2 from Built Environments.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019, is a respiratory virus primarily transmitted person to person through inhalation of droplets or aerosols, laden with viral particles. However, as recent studies have shown, virions can remain infectious for up to 72 h on surfaces, which can lead to transmission through contact. Thus, a comprehensive study was conducted to determine the efficiency of protocols to recover SARS-CoV-2 from surfaces in built environments. This end-to-end (E2E) study showed that the effective combination for monitoring SARS-CoV-2 on surfaces includes using an Isohelix swab collection tool, DNA/RNA Shield as a preservative, an automated system for RNA extraction, and reverse transcriptase quantitative PCR (RT-qPCR) as the detection assay. Using this E2E approach, this study showed that, in some cases, noninfectious viral fragments of SARS-CoV-2 persisted on surfaces for as long as 8 days even after bleach treatment. Additionally, debris associated with specific built environment surfaces appeared to inhibit and negatively impact the recovery of RNA; Amerstat demonstrated the highest inhibition (>90%) when challenged with an inactivated viral control. Overall, it was determined that this E2E protocol required a minimum of 1,000 viral particles per 25 cm2 to successfully detect virus from test surfaces. Despite our findings of viral fragment longevity on surfaces, when this method was employed to evaluate 368 samples collected from various built environmental surfaces, all samples tested negative, indicating that the surfaces were either void of virus or below the detection limit of the assay.IMPORTANCE The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (the virus responsible for coronavirus disease 2019 [COVID-19]) pandemic has led to a global slowdown with far-reaching financial and social impacts. The SARS-CoV-2 respiratory virus is primarily transmitted from person to person through inhalation of infected droplets or aerosols. However, some studies have shown that virions can remain infectious on surfaces for days and can lead to human infection from contact with infected surfaces. Thus, a comprehensive study was conducted to determine the efficiency of protocols to recover SARS-CoV-2 from surfaces in built environments. This end-to-end study showed that the effective combination for monitoring SARS-CoV-2 on surfaces required a minimum of 1,000 viral particles per 25 cm2 to successfully detect virus from surfaces. This comprehensive study can provide valuable information regarding surface monitoring of various materials as well as the capacity to retain viral RNA and allow for effective disinfection.

Draft Genome Sequences of Bacillus glennii V44-8, Bacillus saganii V47-23a, Bacillus sp. Strain V59.32b, Bacillus sp. Strain MER_TA_151, and Paenibacillus sp. Strain MER_111, Isolated from Cleanrooms Where the Viking and Mars Exploration Rover Spacecraft Were Assembled.

We report the draft genome sequences of Bacillus glennii V44-8, Bacillus saganii V47-23a, and Bacillus sp. strain V59.32b, isolated from the Viking spacecraft assembly cleanroom, and Bacillus sp. strain MER_TA_151 and Paenibacillus sp. strain MER_111, isolated from the Mars Exploration Rover (MER) assembly cleanroom.