Recent Evolution of Copper Tolerance of Spores of Bacillus subtilis

Astrobiology is the study of the origin, evolution, and future of life in the Universe. As our technology improves, we are better able to study other planets in the solar system and galaxy to look for life. However, there has not yet been a confirmed discovery of life anywhere other than Earth, so we must study the one available example of life that we currently have. Fortunately, there is a huge diversity of life on Earth – particularly in the form of microorganisms that inhabit extreme environments that can allow us to study the limits of tolerance and survival of life. One example of an extreme environment is in Death Valley, where microorganisms must survive high temperatures and prolonged periods without water. Also in Death Valley is a geochemical gradient from which bacterial strains of Bacillus species were collected. Bacillus species can form metabolically dormant endospores (spores) that are highly resistant to a variety of environmental stressors found on and off Earth. Because of this resistance, spores have been used in a variety of astrobiology studies looking at the hardiness of life. The experiments performed in this study used closely related strains of Bacillus subtilis from three putative ecotypes predicted by Ecotype Simulation to test for differences in spore tolerance to copper, a natural chemical toxin found in Death Valley soil. Ecotypes, by definition, are ecologically distinct with constrained diversity so that members in a given ecotype are ecologically homogeneous. It was originally hypothesized that the putative ecotypes, if true ecotypes, would exhibit differences in germination abilities (return from spore to vegetative state) in copper environments, but have strains within them that would respond homogeneously. Two sets of experiments were performed to test how the spores of the Bacillus strains would germinate in copper-infused versus non-copper growth medium environments. Strains from the three closely related putative ecotypes were collected and sporulation was induced for each experiment. Spores were harvested and checked for purity. The spores were grown on solid media plates that either contained a high concentration of copper or no copper. Fractions of viable germinating spores from the copper versus no copper conditions based on each strain's colony forming units allowed for the analysis of heterogeneity and/or homogeneity of ecotypes and strains. The second set of experiments used the Most Probable Number method for cell enumeration. This statistically-based method also used a comparison of germination of spores in high copper versus no copper to compare strains and ecotypes. Nested ANOVAs were performed to compare germination ratios of all ecotypes and strains. Both sets of experiments indicated that copper negatively impacted or delayed germination. Though there were large differences in putative ecotype averages in relative growth, the nested ANOVAs indicated that ecotypes were not significantly different from each other. However, strains within each ecotype, which are some of the closest relatives, exhibited significant variation in terms of spore germination in copper environments. The evidence of observable significant variation in copper tolerance among extremely closely relatives suggests that copper tolerance is easily, repeatedly, and convergently evolved. This facile evolution of tolerance to copper expands the potential environments of where to look for life as we continue our search in the Universe.