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Stratosphere Conditions Inactivate Bacterial Endospores from a Mars Spacecraft Assembly Facility.

ABSTRACT: Every spacecraft sent to Mars is allowed to land viable microbial bioburden, including hardy endospore-forming bacteria resistant to environmental extremes. Earth's stratosphere is severely cold, dry, irradiated, and oligotrophic; it can be used as a stand-in location for predicting how stowaway microbes might respond to the martian surface. We launched E-MIST, a high-altitude NASA balloon payload on 10 October 2015 carrying known quantities of viable Bacillus pumilus SAFR-032 (4.07?×?107 spores per sample), a radiation-tolerant strain collected from a spacecraft assembly facility. The payload spent 8?h at ?31?km above sea level, exposing bacterial spores to the stratosphere. We found that within 120 and 240?min, spore viability was significantly reduced by 2 and 4 orders of magnitude, respectively. By 480?min, <0.001% of spores carried to the stratosphere remained viable. Our balloon flight results predict that most terrestrial bacteria would be inactivated within the first sol on Mars if contaminated spacecraft surfaces receive direct sunlight. Unfortunately, an instrument malfunction prevented the acquisition of UV light measurements during our balloon mission. To make up for the absence of radiometer data, we calculated a stratosphere UV model and conducted ground tests with a 271.1?nm UVC light source (0.5 W/m2), observing a similarly rapid inactivation rate when using a lower number of contaminants (640 spores per sample). The starting concentration of spores and microconfiguration on hardware surfaces appeared to influence survivability outcomes in both experiments. With the relatively few spores that survived the stratosphere, we performed a resequencing analysis and identified three single nucleotide polymorphisms compared to unexposed controls. It is therefore plausible that bacteria enduring radiation-rich environments (e.g., Earth's upper atmosphere, interplanetary space, or the surface of Mars) may be pushed in evolutionarily consequential directions. Key Words: Planetary protection-Stratosphere-Balloon-Mars analog environment-E-MIST payload-Bacillus pumilus SAFR-032. Astrobiology 17, 337-350.

SUBMITTER: Khodadad CL

PROVIDER: S-EPMC5399745 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Stratosphere Conditions Inactivate Bacterial Endospores from a Mars Spacecraft Assembly Facility.

Khodadad Christina L CL Wong Gregory M GM James Leandro M LM Thakrar Prital J PJ Lane Michael A MA Catechis John A JA Smith David J DJ

Astrobiology 20170321 4

Every spacecraft sent to Mars is allowed to land viable microbial bioburden, including hardy endospore-forming bacteria resistant to environmental extremes. Earth's stratosphere is severely cold, dry, irradiated, and oligotrophic; it can be used as a stand-in location for predicting how stowaway microbes might respond to the martian surface. We launched E-MIST, a high-altitude NASA balloon payload on 10 October 2015 carrying known quantities of viable Bacillus pumilus SAFR-032 (4.07 × 10<sup>7</ ...[more]

PMID: 28323456

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Project description: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,&nbsp;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&nbsp;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.

Project description:A Gram-positive, coccoid, motile, aerobic bacterium, designated strain B12T was isolated from a Jet Propulsion Laboratory spacecraft assembly cleanroom, Pasadena, CA, United States. Strain B12T was resistant to chloramphenicol (100 ?g/mL), and is a relatively slow grower (3-5 days optimal). Strain B12T was found to grow optimally at 28 to 32°C, pH 7 to 8, and 0.5% NaCl. Fatty acid methyl ester analysis showed that the major fatty acid of the strain B12T was anteiso C15 : 0 (66.3%), which is also produced by other Kineococcus species. However, arachidonic acid (C20 : 4 ?6,9,12,16c) was present in strain B12T and Kineococcus glutinatus YIM 75677T but absent in all other Kineococcus species. 16S rRNA analysis revealed that strain B12T was 97.9% similar to Kineococcus radiotolerans and falls within the Kineococcus clade. Low 16S rRNA gene sequence similarities (<94%) with other genera in the family Kineosporiaceae, including Angustibacter (93%), Kineosporia (94% to 95%), Pseudokineococcus (93%), Quadrisphaera (93%), and Thalassiella (94%) demonstrated that the strain B12T does not belong to these genera. Phylogenetic analysis of the gyrB gene show that all known Kineococcus species exhibited <86% sequence similarity with B12T. Multi-locus sequence and whole genome sequence analyses confirmed that B12T clades with other Kineococcus species. Average nucleotide identity of strain B12T were 75-78% with other Kineococcus species, while values ranged from 72-75% with species from other genera within family Kineosporiaceae. Average amino-acid identities were 66-72% with other Kineococcus species, while they ranged from 50-58% with species from other genera. The dDDH comparison of strain B12T genome with members of genera Kineococcus showed 20-22% similarity, again demonstrating that B12T is distantly related to other members of the genus. Furthermore, analysis of whole proteome deduced from WGS places strain B12T in order Kineosporiales, confirming that strain B12T is a novel member of family Kineosporiaceae. Based on these analyses and other genome characteristics, strain B12T is assigned to a novel species within the genus Kineococcus, and the name Kineococcus rubinsiae sp. nov., is proposed. The type strain is B12T (=FJII-L1-CM-PAB2T; NRRL B-65556T, DSM 110506T).

Project description: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.

Project description: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.

Dataset Information

Stratosphere Conditions Inactivate Bacterial Endospores from a Mars Spacecraft Assembly Facility.

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Stratosphere Conditions Inactivate Bacterial Endospores from a Mars Spacecraft Assembly Facility.

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