Nanosatellites for Biology in Space: In Situ Measurement of Bacillus subtilis Spore Germination and Growth after 6 Months in Low Earth Orbit on the O/OREOS Mission

We report here complete 6-month results from the orbiting Space Environment Survivability of Living Organisms (SESLO) experiment. The world’s first and only long-duration live-biology cubesat experiment, SESLO was executed by one of two 10-cm cube-format payloads aboard the 5.5-kg O/OREOS (Organism/Organic Exposure to Orbital Stresses) free-flying nanosatellite, which launched to a 72°-inclination, 650-km Earth orbit in 2010. The SESLO experiment measured the long-term survival, germination, metabolic, and growth responses of Bacillus subtilis spores exposed to microgravity and ionizing radiation including heavy-ion bombardment. A pair of radiation dosimeters (RadFETs, i.e., radiation-sensitive field-effect transistors) within the SESLO payload provided an in-situ dose rate estimate of 6–7.6 mGy/day throughout the mission. Microwells containing samples of dried spores of a wild-type B. subtilis strain and a radiation-sensitive mutant deficient in Non-Homologoous End Joining (NHEJ) were rehydrated after 14, 91, and 181 days in space with nutrient medium containing with the redox dye alamarBlue (aB), which changes color upon reaction with cellular metabolites. Three-color transmitted light intensity measurements of all microwells were telemetered to Earth within days of each 24-hour growth experiment. At 14 and 91 days, spaceflight samples germinated, grew, and metabolized significantly more slowly than matching ground-control samples, as measured both by aB reduction and optical density changes; these rate differences notwithstanding, the final optical density attained was the same in both flight and ground samples. After 181 days in space, spore germination and growth appeared hindered and abnormal. We attribute the differences not to an effect of the space environment per se, as both spaceflight and ground-control samples exhibited the same behavior, but to a pair of ~15-day thermal excursions, after the 91-day measurement and before the 181-day experiment, that peaked above 46 °C in the SESLO payload. Because the payload hardware operated nominally at 181 days, the growth issues point to heat damage, most likely to component(s) of the growth medium (RPMI 1640 containing aB) or to biocompatibility issues caused by heat-accelerated outgassing or leaching of harmful compounds from components of the SESLO hardware and electronics.

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Design and Validation of a Device for Mitigating Fluid Microgravity Effects in Biological Research in Canister Spaceflight Hardware

Frontiers in Space Technologies ◽

10.3389/frspt.2021.797518 ◽

2021 ◽

Vol 2 ◽

Author(s):

Wayne L. Nicholson ◽

Patricia Fajardo-Cavazos ◽

Caleb Turner ◽

Taylor M. Currie ◽

Geoffrey Gregory ◽

...

Keyword(s):

Bacillus Subtilis ◽

Space Station ◽

Petri Dish ◽

Microgravity Environment ◽

Direct Result ◽

Ground Control ◽

Biological Research ◽

Transcriptomic Response ◽

Liquid Cultures ◽

Control Samples

The major factor influencing the behavior of microbes growing in liquids in space is microgravity. We recently measured the transcriptomic response of the Gram-positive bacterium Bacillus subtilis to the microgravity environment inside the International Space Station (ISS) in spaceflight hardware called Biological Research in Canisters-Petri Dish Fixation Units (BRIC-PDFUs). In two separate experiments in the ISS, dubbed BRIC-21 and BRIC-23, we grew multiple replicates of the same B. subtilis strain in the same hardware, growth medium, and temperature with matching ground control samples (npj Micrograv. 5:1.2019, doi: 10.1038/s41526-018-0061-0). In both experiments we observed similar responses of the transcriptome to spaceflight. However, we also noted that the liquid cultures assumed a different configuration in microgravity (a toroidal shape) compared with the ground control samples (a flat disc shape), leading us to question whether the transcriptome differences we observed were a direct result of microgravity, or a secondary result of the different liquid geometries of the samples affecting, for example, oxygen availability. To mitigate the influence of microgravity on liquid geometry in BRIC canisters, we have designed an insert to replace the standard 60-mm Petri dish in BRIC-PDFU or BRIC-LED sample compartments. In this design, liquid cultures are expected to assume a more disk-like configuration regardless of gravity or its absence. We have: (i) constructed a prototype device by 3D printing; (ii) evaluated different starting materials, treatments, and coatings for their wettability (i.e., hydrophilicity) using contact angle measurements; (iii) confirmed that the device performs as designed by drop-tower testing and; (iv) performed material biocompatibility studies using liquid cultures of Bacillus subtilis and Staphylococcus aureus bacteria. Future microgravity testing of the device in the ISS is planned.

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Establishing Standard Protocols for Bacterial Culture in Biological Research in Canisters (BRIC) Hardware

Gravitational and Space Research ◽

10.2478/gsr-2016-0013 ◽

2020 ◽

Vol 4 (2) ◽

pp. 58-69 ◽

Cited By ~ 4

Author(s):

Patricia Fajardo-Cavazos ◽

Wayne L. Nicholson

Keyword(s):

Gene Expression ◽

Bacterial Culture ◽

Culture Conditions ◽

Media Culture ◽

Ground Control ◽

Biological Research ◽

Confounding Factors ◽

Cross Experiment ◽

Spaceflight Experiments ◽

Control Samples

AbstractThe NASA GeneLab Data System (GLDS) was recently developed to facilitate cross-experiment comparisons in order to understand the response of microorganisms to the human spaceflight environment. However, prior spaceflight experiments have been conducted using a wide variety of different hardware, media, culture conditions, and procedures. Such confounding factors could potentially mask true differences in gene expression between spaceflight and ground control samples. In an attempt to mitigate such confounding factors, we describe here the development of a standardized set of hardware, media, and protocols for liquid cultivation of microbes in Biological Research in Canisters (BRIC) spaceflight hardware, using the model bacteria Bacillus subtilis strain 168 and Staphylococcus aureus strain UAMS-1 as examples.

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The Effect of Low Earth Orbit Atomic Oxygen Exposure on Phenylphosphine Oxide-Containing Polymers

High Performance Polymers ◽

10.1088/0954-0083/12/1/304 ◽

2000 ◽

Vol 12 (1) ◽

pp. 43-52 ◽

Cited By ~ 30

Author(s):

John W Connell

Keyword(s):

Thin Film ◽

Atomic Oxygen ◽

Low Earth Orbit ◽

Space Environment ◽

Earth Orbit ◽

Cooperative Effort ◽

Flight Experiment ◽

Oxygen Exposure ◽

Atomic Oxygen Exposure ◽

Phenylphosphine Oxide

Thin films of phenylphosphine oxide-containing polymers were exposed to low Earth orbit aboard a space shuttle flight (STS-85) as part of flight experiment designated Evaluation of Space Environment and Effects on Materials (ESEM). This flight experiment was a cooperative effort between the NASA Langley Research Center (LaRC) and the National Space Development Agency of Japan (NASDA). The thin-film samples described herein were part of an atomic oxygen exposure (AOE) experiment and were exposed to primarily atomic oxygen (∼1×1019 atoms cm−2). The thin-film samples consisted of three phosphine oxide-containing polymers (arylene ether, benzimidazole and imide). Based on post-flight analyses using atomic force microscopy, x-ray photo-electron spectroscopy and weight loss data, it was found that the exposure of these materials to atomic oxygen (AO) produces a phosphorus oxide layer on the surface of the samples. Earlier work has shown that this layer provides a barrier towards further attack by AO. Consequently, these materials do not exhibit linear erosion rates which is in contrast with most organic polymers. Qualitatively, the results obtained from these analyses compare favourably with those obtained from samples exposed to AO and/or an oxygen plasma in ground-based exposure experiments. The results of the low Earth orbit AO exposure on these materials will be compared with those of ground-based exposure to AO.

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The affect of the space environment on the survival ofHalorubrum chaoviatorandSynechococcus(Nägeli): data from the Space Experiment OSMO on EXPOSE-R

International Journal of Astrobiology ◽

10.1017/s147355041400055x ◽

2014 ◽

Vol 14 (1) ◽

pp. 123-128 ◽

Cited By ~ 20

Author(s):

R. L. Mancinelli

Keyword(s):

Survival Rate ◽

Uv Radiation ◽

Molecular Probes ◽

Low Earth Orbit ◽

Space Environment ◽

Dust Particles ◽

Most Probable Number ◽

Earth Orbit ◽

Probable Number ◽

Space Vacuum

AbstractWe have shown using ESA's Biopan facility flown in Earth orbit that when exposed to the space environment for 2 weeks the survival rate ofSynechococcus(Nägeli), a halophilic cyanobacterium isolated from the evaporitic gypsum–halite crusts that form along the marine intertidal, andHalorubrum chaoviatora member of the Halobacteriaceae isolated from an evaporitic NaCl crystal obtained from a salt evaporation pond, were higher than all other test organisms exceptBacillusspores. These results led to the EXPOSE-R mission to extend and refine these experiments as part of the experimental package for the external platform space exposure facility on the ISS. The experiment was flown in February 2009 and the organisms were exposed to low-Earth orbit for nearly 2 years. Samples were either exposed to solar ultraviolet (UV)-radiation (λ > 110 nm or λ > 200 nm, cosmic radiation (dosage range 225–320 mGy), or kept in darkness shielded from solar UV-radiation. Half of each of the UV-radiation exposed samples and dark samples were exposed to space vacuum and half kept at 105pascals in argon. Duplicate samples were kept in the laboratory to serve as unexposed controls. Ground simulation control experiments were also performed. After retrieval, organism viability was tested using Molecular Probes Live–Dead Bac-Lite stain and by their reproduction capability. Samples kept in the dark, but exposed to space vacuum had a 90 ± 5% survival rate compared to the ground controls. Samples exposed to full UV-radiation for over a year were bleached and although results from Molecular Probes Live–Dead stain suggested ~10% survival, the data indicate that no survival was detected using cell growth and division using the most probable number method. Those samples exposed to attenuated UV-radiation exhibited limited survival. Results from of this study are relevant to understanding adaptation and evolution of life, the future of life beyond earth, the potential for interplanetary transfer of viable microbes via meteorites and dust particles as well as spacecraft, and the physiology of halophiles.

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Predicting the Structure and Dynamics of Membrane Protein GerAB from Bacillus subtilis

International Journal of Molecular Sciences ◽

10.3390/ijms22073793 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3793

Author(s):

Sophie Blinker ◽

Jocelyne Vreede ◽

Peter Setlow ◽

Stanley Brul

Keyword(s):

Bacillus Subtilis ◽

Membrane Protein ◽

Spore Germination ◽

Structural Information ◽

Transmembrane Protein ◽

Homology Modelling ◽

Water Channel ◽

Md Simulations ◽

Integral Membrane Protein ◽

Starting Point

Bacillus subtilis forms dormant spores upon nutrient depletion. Germinant receptors (GRs) in spore’s inner membrane respond to ligands such as L-alanine, and trigger spore germination. In B. subtilis spores, GerA is the major GR, and has three subunits, GerAA, GerAB, and GerAC. L-Alanine activation of GerA requires all three subunits, but which binds L-alanine is unknown. To date, how GRs trigger germination is unknown, in particular due to lack of detailed structural information about B subunits. Using homology modelling with molecular dynamics (MD) simulations, we present structural predictions for the integral membrane protein GerAB. These predictions indicate that GerAB is an α-helical transmembrane protein containing a water channel. The MD simulations with free L-alanine show that alanine binds transiently to specific sites on GerAB. These results provide a starting point for unraveling the mechanism of L-alanine mediated signaling by GerAB, which may facilitate early events in spore germination.

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Properties of spores of Bacillus subtilis with or without a transposon that decreases spore germination and increases spore wet heat resistance

Journal of Applied Microbiology ◽

10.1111/jam.15163 ◽

2021 ◽

Author(s):

Yannong Luo ◽

George Korza ◽

Angela M. DeMarco ◽

Oscar P. Kuipers ◽

Yong‐qing Li ◽

...

Keyword(s):

Bacillus Subtilis ◽

Heat Resistance ◽

Spore Germination ◽

Wet Heat

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Role of SpoVA Proteins in Release of Dipicolinic Acid during Germination of Bacillus subtilis Spores Triggered by Dodecylamine or Lysozyme

Journal of Bacteriology ◽

10.1128/jb.01613-06 ◽

2006 ◽

Vol 189 (5) ◽

pp. 1565-1572 ◽

Cited By ~ 82

Author(s):

Venkata Ramana Vepachedu ◽

Peter Setlow

Keyword(s):

Bacillus Subtilis ◽

Small Molecules ◽

Spore Germination ◽

Dipicolinic Acid ◽

Temperature Sensitive ◽

Wild Type ◽

Ph Optimum ◽

Inner Membrane ◽

Temperature Sensitive Mutants

ABSTRACT The release of dipicolinic acid (DPA) during the germination of Bacillus subtilis spores by the cationic surfactant dodecylamine exhibited a pH optimum of ∼9 and a temperature optimum of 60°C. DPA release during dodecylamine germination of B. subtilis spores with fourfold-elevated levels of the SpoVA proteins that have been suggested to be involved in the release of DPA during nutrient germination was about fourfold faster than DPA release during dodecylamine germination of wild-type spores and was inhibited by HgCl2. Spores carrying temperature-sensitive mutants in the spoVA operon were also temperature sensitive in DPA release during dodecylamine germination as well as in lysozyme germination of decoated spores. In addition to DPA, dodecylamine triggered the release of amounts of Ca2+ almost equivalent to those of DPA, and at least one other abundant spore small molecule, glutamic acid, was released in parallel with Ca2+ and DPA. These data indicate that (i) dodecylamine triggers spore germination by opening a channel in the inner membrane for Ca2+-DPA and other small molecules, (ii) this channel is composed at least in part of proteins, and (iii) SpoVA proteins are involved in the release of Ca2+-DPA and other small molecules during spore germination, perhaps by being a part of a channel in the spore's inner membrane.

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