Publication Date

April 2016

Advisor(s)

Frederick Cohan

Major

Biology (BIOL)

Language

English (United States)

Abstract

Speciation in the bacterial world is still not well defined, and members of the same taxonomically described species can have high variance in ecological parameters. Twenty-seven strains of the Bacillus subtilis-licheniformis clade of bacteria were isolated from an area adjacent to a saltpan in Death Valley in the Great Basin Desert of Eastern California. The study site has a discerned gradient of salinity and ion concentrations. The strains used in this thesis have previously been demarcated into 9 different putative ecotypes (PEs) using gyrA sequences (Kopac, 2014). The strains were tested against high levels of copper (0.8 mMol), boron (60 mMol), zinc (1 mMol), and manganese (6.4 mMol) ions to establish their tolerance to each element. Tolerance was determined by the ability of endospores to germinate and grow in media with each element compared to paired tests in media with no stressor. It was expected that tolerance would be homogenous within PEs, as ecotypes are predicted to be coherent and ecologically homogenous groups. It was also expected that tolerance may be heterogeneous between PEs, as varying amounts of metallic and metalloid ions could result in varying selective pressures for evolution of tolerance. However, these predictions were not supported by the results of this study. It appears that PE has no effect on the overall variety of tolerance, and strains within PEs can have great heterogeneity. This was true for all 4 elements. These results suggest that single gene sequences are not sufficient for demarcating ecotypes in Bacillus from this sample site, and that the strains used are likely evolving under a Recurrent Niche Invasion, Speedy Speciation, or Speciesless model. It was also shown that tolerance of copper and zinc are highly correlated (p < 0.001), and this correlation is phylogenetically independent (p = 0.0001). This could be because there are 2 separate genes that allow for tolerance of copper and zinc that are frequently inherited on a single plasmid, or a common mechanism or transcription regulator is responsible for tolerance to both divalent metallic cations. Furthermore, it was shown that high levels of toxic elements repress germination and/or outgrowth to a greater degree after 36-48 hours than after 84-96 hours. The results of this thesis, and the future directions presented, could have importance in a number of fields. The great heterogeneity among strains within PEs in terms of their metal and metalloid tolerance, is important for the study of microbial evolution and speciation, as it shows that even the most closely related bacteria can have extreme ecological differences, and speciation (i.e. the formation of ecologically distinct groups) may occur much faster than is commonly accepted. Tolerance to extreme conditions, such as those near Death Valley saltpans, is important to the study of astrobiology, as examining how life adapts to seemingly lethal environments on Earth could be helpful in discovering and understanding life elsewhere. Finally, the biosorption capabilities of many toxin-tolerant bacteria is a popularly proposed mechanism for cleaning up anthropogenically contaminated soils and water systems, and the bacteria used in this study may be possible candidates for this kind of bioremediation.

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