This study identifies genes that determine survival during a space flight, using the model eukaryotic organism, Saccharomyces cerevisiae. Select strains of a haploid yeast deletion series grew during storage in distilled water in space, but not in ground based static or clinorotation controls. The survival advantages in space in distilled water include a 133-fold advantage for the deletion of PEX19, a chaperone and import receptor for newly- synthesized class I peroxisomal membrane proteins, to 77–40 fold for deletion strains lacking elements of aerobic respiration, isocitrate metabolism, and mitochondrial electron transport. Following automated addition of rich growth media, the space flight was associated with a marked survival advantage of strains with deletions in catalytically active genes including hydrolases, oxidoreductases and transferases. When compared to static controls, space flight was associated with a marked survival disadvantage of deletion strains lacking transporter, antioxidant and catalytic activity. This study identifies yeast deletion strains with a survival advantage during storage in distilled water and space flight, and amplifies our understanding of the genes critical for survival in space.
Research Containing: Microbiology
Characterization of the Salmonella enterica serovar Typhimurium ydcI gene, which encodes a conserved DNA binding protein required for full acid stress resistance
Salmonella enterica serovar Typhimurium possesses a stimulon of genes that are differentially regulated in response to conditions of low fluid shear force that increase bacterial virulence and alter other phenotypes. In this study, we show that a previously uncharacterized member of this stimulon, ydcI or STM1625, encodes a highly conserved DNA binding protein with related homologs present in a range of gram-negative bacterial genera. Gene expression analysis shows that ydcI is expressed in different bacterial genera and is involved in its autoregulation in S. Typhimurium. We demonstrate that purified YdcI protein specifically binds a DNA probe consisting of its own promoter sequence. We constructed an S. Typhimurium DeltaydcI mutant strain and show that this strain is more sensitive to both organic and inorganic acid stress than is an isogenic WT strain, and this defect is complemented in trans. Moreover, our data indicate that ydcI is part of the rpoS regulon related to stress resistance. The S. Typhimurium DeltaydcI mutant was able to invade cultured cells to the same degree as the WT strain, but a strain in which ydcI expression is induced invaded cells at a level 2.8 times higher than that of the WT. In addition, induction of ydcI expression in S. Typhimurium resulted in the formation of a biofilm in stationary-phase cultures. These data indicate the ydcI gene encodes a conserved DNA binding protein involved with aspects of prokaryotic biology related to stress resistance and possibly virulence.
INTRODUCTION: Spaceflight aboard the International Space Station (ISS) involves stays of individual crewmembers for up to 6 mo during which they are exposed to a complex mixture of airborne pollutants. Methods to predict specific health effects from exposure to a mixture of air pollutants are not well developed. Herein, air monitoring data from the ISS are used to demonstrate a new method to estimate a threshold for possible health effects from exposure to mixtures. METHODS: An empirical, additive approach was developed to transform monthly air pollutant data, which had been obtained primarily by gas chromatography-mass spectrometry from samples of ISS air, to threshold (T) values for 16 adverse health effect groups. Spacecraft maximum allowable concentrations (SMACs), available for most spacecraft air pollutants, were used to form target-organ/effect groups, from which group T values were estimated. If T >1 for a group, then there is an unacceptable risk of the toxic effect. RESULTS: Samples of air taken from the ISS in 2010 revealed that all 16 toxicological groups were within safe limits. Highest T values were as follows: mucosal irritants (0.53 +/- 0.44), headache (0.52 +/- 0.06), central nervous system depression (0.25 +/- 0.06), and cardiac sensitization (0.13 +/- 0.04). DISCUSSION: The additive model is supported by limited inhalation data on rats in the literature. Our predictions of no adverse effect on crew health are useful as part of NASA's Lifetime Surveillance of Astronaut Health (LSAH). If one of the 16 levels had exceeded T=1, then standard surveillance could be supplemented to address this potential health risk.
Bacterial monitoring with adhesive sheet in the international space station-"Kibo", the Japanese experiment module
Microbiological monitoring is important to assure microbiological safety, especially in long-duration space habitation. We have been continuously monitoring the abundance and diversity of bacteria in the International Space Station (ISS)-"Kibo" module to accumulate knowledge on microbes in the ISS. In this study, we used a new sampling device, a microbe-collecting adhesive sheet developed in our laboratory. This adhesive sheet has high operability, needs no water for sampling, and is easy to transport and store. We first validated the adhesive sheet as a sampling device to be used in a space habitat with regard to the stability of the bacterial number on the sheet during prolonged storage of up to 12 months. Bacterial abundance on the surfaces in Kibo was then determined and was lower than on the surfaces in our laboratory (10(5) cells [cm(2)](-1)), except for the return air grill, and the bacteria detected in Kibo were human skin microflora. From these aspects of microbial abundance and their phylogenetic affiliation, we concluded that Kibo has been microbiologically well maintained; however, microbial abundance may increase with the prolonged stay of astronauts. To ensure crew safety and understand bacterial dynamics in space habitation environments, continuous bacterial monitoring in Kibo is required.
Effects of microgravity on the virulence of Listeria monocytogenes, Enterococcus faecalis, Candida albicans, and methicillin-resistant Staphylococcus aureus
To evaluate effects of microgravity on virulence, we studied the ability of four common clinical pathogens–Listeria monocytogenes, methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, and Candida albicans–to kill wild type Caenorhabditis elegans (C. elegans) nematodes at the larval and adult stages. Simultaneous studies were performed utilizing spaceflight, clinorotation in a 2-D clinorotation device, and static ground controls. The feeding rate of worms for killed E. coli was unaffected by spaceflight or clinorotation. Nematodes, microbes, and growth media were separated until exposed to true or modeled microgravity, then mixed and grown for 48 h. Experiments were terminated by paraformaldehyde fixation, and optical density measurements were used to assay residual microorganisms. Spaceflight was associated with reduced virulence for Listeria, Enterococcus, MRSA, and Candida for both larval and adult C. elegans. These are the first data acquired with a direct in vivo assay system in space to demonstrate virulence. Clinorotation reproduced the effects of spaceflight in some, but not all, virulence assays: Candida and Enterococcus were less virulent for larval worms but not adult worms, whereas virulence of MRSA and Listeria were unaffected by clinorotation in tests with both adult and larval worms. We conclude that four common clinical microorganisms are all less virulent in space.
Determination of colloidal and dissolved silver in water samples using colorimetric solid-phase extraction
The increase in bacterial resistance to antibiotics has led to resurgence in the use of silver as a biocidal agent in applications ranging from washing machine additives to the drinking water treatment system on the International Space Station (ISS). However, growing concerns about the possible toxicity of colloidal silver to bacteria, aquatic organisms and humans have led to recently issued regulations by the US EPA and FDA regarding the usage of silver. As part of an ongoing project, we have developed a rapid, simple method for determining total silver, both ionic (silver(I)) and colloidal, in 0.1–1 mg/L aqueous samples, which spans the ISS potable water target of 0.3–0.5 mg/L (total silver) and meets the US EPA limit of 0.1 mg/L in drinking water. The method is based on colorimetric solid-phase extraction (C-SPE) and involves the extraction of silver(I) from water samples by passage through a solid-phase membrane impregnated with the colorimetric reagent DMABR (5-[4-(dimethylamino)benzylidene]rhodanine). Silver(I) exhaustively reacts with impregnated DMABR to form a colored compound, which is quantified using a handheld diffuse reflectance spectrophotometer. Total silver is determined by first passing the sample through a cartridge containing Oxone, which exhaustively oxidizes colloidal silver to dissolved silver(I). The method, which takes less than 2 min to complete and requires only ∼1 mL of sample, has been validated through a series of tests, including a comparison with the ICP-MS analysis of a water sample from ISS that contained both silver(I) and colloidal silver. Potential earth-bound applications are also briefly discussed.
Cytogenetic analysis of peripheral blood lymphocytes is the most sensitive and reliable method currently available for in vivo assessment of the biological effects of exposure to radiation and provides the most informative measurement of radiation induced health risks. Data indicates that space missions of a few months or more can induce measureable increases in the yield of chromosome damage in the blood lymphocytes of astronauts that can be used to estimate an organ dose equivalent, and biodosimetry estimates lie within the range expected from physical dosimetry. Space biodosimetry poses some unique challenges compared to terrestrial biological assessments of radiation exposures, but data provides a direct measurement of space radiation damage, which takes into account individual radiosensitivity in the presence of confounding factors such as microgravity and other stress conditions. Moreover if chromosome damage persists in the blood for many years, results can be used for retrospective dose reconstruction. In contrast to physical measurements, which are external to body and require multiple devices to detect all radiation types all of which have poor sensitivity to neutrons, biodosimetry is internal and includes the effects of shielding provided by the body itself plus chromosome damage shows excellent sensitivity to protons, heavy ions, and neutrons. In addition, chromosome damage is reflective of cancer risk and biodosimetry values can therefore be used to validate and develop risk assessment models that can be used to characterize health risk incurred by crewmembers. The current paper presents a review of astronaut biodosimetry data, along with recently derived data on the relative cancer risk estimated using the quantitative approach derived from the European Study Group on Cytogenetic Biomarkers and Health database.
The effect of spaceflight on growth of Ulocladium chartarum colonies on the international space station
The objectives of this 14 days experiment were to investigate the effect of spaceflight on the growth of Ulocladium chartarum, to study the viability of the aerial and submerged mycelium and to put in evidence changes at the cellular level. U. chartarum was chosen for the spaceflight experiment because it is well known to be involved in biodeterioration of organic and inorganic substrates covered with organic deposits and expected to be a possible contaminant in Spaceships. Colonies grown on the International Space Station (ISS) and on Earth were analysed post-flight. This study clearly indicates that U. chartarum is able to grow under spaceflight conditions developing, as a response, a complex colony morphotype never mentioned previously. We observed that spaceflight reduced the rate of growth of aerial mycelium, but stimulated the growth of submerged mycelium and of new microcolonies. In Spaceships and Space Stations U. chartarum and other fungal species could find a favourable environment to grow invasively unnoticed in the depth of surfaces containing very small amount of substrate, posing a risk factor for biodegradation of structural components, as well as a direct threat for crew health. The colony growth cycle of U. chartarum provides a useful eukaryotic system for the study of fungal growth under spaceflight conditions.
Survival of microorganisms representing the three Domains of life inside the International Space Station
The present work was mainly focused to study the response of representative non pathogenic microorganisms to the environment inside the space vehicle at different mission stages (10, 56, and 226 days) within the frame of the Italian ENEIDE mission, from Feb to Oct 2005. Microorganisms were chosen according to their phylogenetic position and cell structures; they were representatives of the three taxonomic domains and belonged to different ecosystems (food, soil, intestinal tract, plants, deep-sea). They were the followings: Thermococcus guaymasensis (Domain Archaea); Saccharomyces cerevisiae (Domain Eucarya); Escherichia coli, Bacillus subtilis, Lactobacillus acidophilus, Enterococcus faecium, Pseudomonas fluorescens, and Rhizobium tropici (Domain Bacteria). As main environmental parameters we were interested in: a) space radiations; b) microgravity; c) temperature. The response of microorganisms was investigated in terms of survival rates, cell structure modifications, and genomic damages. The survival of cells was affected by both radiation doses and intrinsec cell features. As expected, only samples kept on the ISS for 226 days showed significant levels of mortality. Asfar as regard the effect on cell structures, these samples showed also remarkable morphological changes, particularly for Escherichia coli, Enterococcus faecium, and Saccharomyces cerevisiae. The data collected allowed to get new insights into the biological traits of microorganisms exposed to space environment during the flight on a spacecraft. Moreover, the result obtained may be important for the improvement of human conditions aboard space vehicles (nutraceuticals for astronauts and disinfections of ISS modules) and also for the potential development of closed systems devoted to vegetable productions and organic recycling.
Novel Sfp1 transcriptional regulation of Saccharomyces cerevisiae gene expression changes during spaceflight
This study identifies transcriptional regulation of stress response element (STRE) genes in space in the model eukaryotic organism, Saccharomyces cerevisiae. To determine transcription-factor dependence, gene expression changes in space were examined in strains bearing green fluorescent protein-tagged (GFP-tagged) reporters for YIL052C (Sfp1 dependent with stress), YST-2 (Sfp1/Rap1 dependent with stress), or SSA4 (Msn4 dependent with stress), along with strains of SSA4-GFP and YIL052C-GFP with individual deletions of the Msn4 or Sfp1. When compared to parallel ground controls, spaceflight induces significant gene expression changes in SSA4 (35% decrease) and YIL052C (45% decrease), while expression of YST-2 (0.08% decrease) did not change. In space, deletion of Sfp1 reversed the SSA4 gene expression effect (0.00% change), but Msn4 deletion yielded a similar decrease in SSA4 expression (34% change), which indicates that SSA4 gene expression is dependent on the Sfp1 transcription factor in space, unlike other stresses. For YIL052C, deletion of Sfp1 reversed the effect (0.01% change), and the Msn4 deletion maintained the decrease in expression (30% change), which indicates that expression of YIL052C is also dependent on Sfp1 in space. Spaceflight has selective and specific effects on SSA4 and YIL052C gene expression, indicated by novel dependence on Sfp1.