Responses of various microorganisms to the spaceflight environment have been examined by numerous studies conducted either in space or on the ground in clinostats. The results clearly show that several microbial behaviors differ in space, but not in a consistent way. The underlying mechanism(s) involved are not explainable by a single model. Exposure to the spaceflight environment (e.g., microgravity, radiation) alters a microbial cell's immediate surroundings, lowering convective mass and heat transfer or reducing mechanical shear forces. Changes in such fundamental physical forces affect the rates at which gases, nutrients, signaling molecules, and waste products are exchanged between microbes and their surroundings. Microbes perceive these alterations as environmental stress (i.e., the "spaceflight syndrome") and mount a complex set of stress responses. One consequence of the spaceflight stress response is alteration in their susceptibility to antibiotics. This could be due to mechanisms such as: production of biofilms, alteration of cell surfaces, up-regulation of drug efflux systems, or an increased rate of mutation in genes encoding drug targets. All of these mechanisms have been well documented as outputs of global microbial stress responses. Of particular interest is oxidative stress, which has been shown both to trigger maladaptive responses in higher organisms including astronauts, and to increase bacterial resistance to antibiotics.
Central Hypothesis: Microorganisms subjected to the integrated spaceflight environment invoke a spectrum of stress responses, some leading to alterations in their antibiotic susceptibility. The underlying mechanisms can be identified using transcriptome profiling of model organisms.
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Specific Aims: We postulate that spaceflight stress responses vary depending on the organism studied because each species of microbe is endowed with its own unique collection of stress response systems. In order to probe the underlying mechanism(s) involved it will be necessary to study spaceflight stress response at the genomic level using model organisms. Specifically we plan to:
A. Expose the model bacteria Bacillus subtilis and Staphylococcus epidermidis to: (i) simulated microgravity in the laboratory and (ii) actual spaceflight in 2 parallel BRIC canisters (BRIC-A and BRIC-B) aboard the International Space Station (ISS), with appropriate ground controls. Cells cultivated in BRIC-A will be fixed rapidly with RNA Later and cells in BRIC-B will be frozen in a viable state for return to Earth.
B. Total RNA will be extracted from cells in BRIC-A and subjected to transcriptome analysis using Whole Transcriptome Shotgun Sequencing (a.k.a. RNA sequencing or "RNA-Seq") at the University of Florida Interdisciplinary Center for Biotechnology Research (UF-ICBR), to identify the suite of stress responses induced by exposure to spaceflight or simulated microgravity. Cells in BRIC-B will be thawed and immediately assayed for (i) viability, (ii) resistance levels to a battery of antibiotics in the Omnilog system, and (iii) rates of spontaneous mutation to antibiotic resistance.
Post-experiment sample processing: Frozen cell samples were transferred to the benchtop and partially thawed. The partially frozen cell slurry was transferred using a sterile disposable rubber spatulas into sterile 50-mL conical centrifuge tubes and placed immediately on ice. The culture volume recovered was recorded for later calculations. Samples were further processed for (i) viable counts; (ii) isolation of rifampicin-resistant (RFM^R) mutants; (iii) total RNA isolation; or (iv) Omnilog phenotype microarray profiling.
(i) For viable counts, cultures were diluted serially tenfold in TSY medium, dilutions plated on TSY, and colonies counted after incubation at 37°C for 24 hours.
(ii) To select for RFM^R mutants, cultures were concentrated by centrifugation, plated without dilution onto TSY+RFM (5 µg/mL) plates, and colonies counted after incubation at 37°C for 24 hours. The frequency of mutation to RFM^R was calculated by dividing the total number of RFM^R mutants by the total number of viable cells from each culture. Individual RFM^R mutants were streak-purified and saved as frozen -70°C glycerol stocks for further characterization by DNA sequencing.
(iii) For total RNA isolation, cells were recovered by centrifugation (10,000 rpm, 20 min, 4°C) in a benchtop centrifuge. Supernatants were discarded and cell pellets immediately processed on ice for total RNA extraction. (iv) For Omnilog phenotype microarray experiments, frozen samples were thawed, washed once in Phosphate-Buffered Saline, diluted to the appropriate cell concentration in Inoculating Fluid, and inoculated into PM-11, PM-12, and PM-13 plates containing various concentrations of 72 different antibiotics. Plates were incubated at 37°C in the Omnilog instrument (Biolog, Inc.) and plate images recorded at 15-min intervals for 24 hours. Antibiotic resistance profiles were compared between FL and GC samples for significant differences.