The proposed research aims to understand the effects of the space environment on evolutionary processes in the bacterium Bacillus subtilis. Different mutant lines will be ‘raced’ along solid surfaces to allow continuous selection in the cultures and to maximize the number of generations possible. Deep sequencing of winners will identify evolutionary rates, mechanisms, and targets of selection. We propose printing wax barriers to make paths along a growth surface (agar, membranes) and spotting each starting position of each path with dormant spores of the experimental bacteria to ‘race’ different mutants. Once on orbit, the material is wetted with growth medium, allowing the individual spots of B. subtilis to grow along their determined paths. This approach provides an opportunity for exponential growth only along the propagating edges, generating continuous bottlenecking thus amplifying selective pressures on the experimental populations. By monitoring the respective growth rate of different mutant lines maintained in each of these experimental conditions, we can estimate relative fitness of the lines. Long-term changes in relative growth rate indicate adaptation. Deep-sequencing of DNA from adapted cells (‘winners’ at the end of runs) will identify genetic changes within the respective populations. We expect that rates of mutation will differ between microgravity, 1-g, and ground controls, and that the targets of these mutations will differ as the different populations of bacteria adapt to their respective conditions. This research will also utilize the native ability of B. subtilis to uptake foreign DNA. Information-rich environmental DNA is added into the growth medium, and the populations are raced as above. By sampling the winners, and identifying if/what foreign genes are assimilated in each treatment, this experiment will identify potential genes of interest for future studies of genetic adaptation to the space environment. Our approach maximizes the number of generations possible in the 60-day window for this call, and maximizes the potential for evolutionary processes to occur. By performing multi-generational experimental evolution on bacteria on the International Space Station, the work proposed here aims to advance understanding of the evolutionary processes and challenges facing biological systems in long-term space exploration and habitation.
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We propose to race wild-type against mutator strains along the paths using constant medium in microgravity and at 1-g (if possible) onboard ISS, and again on the ground. We propose a minimum n=12 for each treatment by strain pair, resulting in 60 total samples for this experiment. For the DNA uptake experiments, we propose to replicate the above experimental protocol, but split each n of 12 into two treatments of n=6. For these, the additional factor will be the incorporation of small quantities of DNA to the growth medium as a potential source of genetic source material. Equimolar quantities of similar sized fragments will be added to the two treatments- with one set using extracted environmental DNA from natural soils (DNA with high informational content from one of the native habitats of B. subtilis), and the other will be from low-complexity or low gene-content DNA such as digested pUC plasmids or synthesized nonsense DNA (this controls for the nutritional benefit of adding DNA to the media; the DNA free control can be interpreted from the samples of the first experiment as they will be otherwise identical). The original DNA and source bacterium would also be sequenced (in the case of B. subtilis sp. 168, the genome is already known). We then can identify what foreign DNA is incorporated in which environment, differentiate between uptake rates and type of genes between the treatments, thus obtaining a list of genes and pathways potentially ‘useful’ to the bacteria as they march onwards in their adaptation to the experiment. Replication will allow us to get a first glance of whether adaptation to space follows prescribed paths (the same genes are mutated, or taken up in the replicates) or if it occurs more randomly. In either case, linking the genetic changes that have facilitated the increases in fitness will provide targets for further study.
This investigation is currently in progress. Results will be available at the conclusion of the study.