Date of Completion

8-5-2019

Embargo Period

7-28-2019

Keywords

restriction-modification, methylation, DNA methylation, Halobacteria, Haloarchaea

Major Advisor

R. Thane Papke

Associate Advisor

J. Peter Gogarten

Associate Advisor

Daniel Gage

Associate Advisor

Kenneth Noll

Associate Advisor

Michael O'Neill

Field of Study

Molecular and Cell Biology

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

The Halobacteria are a class of archaeal organisms which are obligate halophiles. Several studies have described the Halobacteria as highly recombinogenic, yet members even within the same geographic location cluster into distinct phylogroups, suggesting that barriers exist which limit recombination. Barriers to recombination in the Halobacteria include CRISPRs, glycosylation, and archaeosortases. Another possible barrier which could limit gene transfer might be restriction-modification (RM) systems, which consist of restriction endonucleases (REases) and DNA methyltransferases (MTases) that both target the same sequence of DNA. The REase cleaves the target sequence, whereas the MTase methylates the site and protects it from cleavage. This dissertation examines the role of DNA methylation and RM systems in the Halobacteria and their impact on halobacterial speciation and genetic recombination. Using the model haloarchaeon, Haloferax volcanii, the genomic DNA methylation patterns (methylome) of an archaeal organism was characterized for the first time. Further investigations via gene deletion were used to identify the DNA methyltransferases responsible for the detected methylation patterns in H. volcanii, and experiments were conducted to determine the impact of RM systems on recombination via cell-to-cell mating. The distribution of MTase and RM system genes throughout the Halobacteria was also examined using a bioinformatics approach. The results indicated that the methylome of H. volcanii consists of two methylated motifs: GCAm6BNNNNNNVTGC, methylated by the Type I RM system RmeRMS, and Cm4TAG, methylated by the orphan MTase HVO_0794. Three other putative MTase genes (HVO_C0040, HVO_A0079, and HVO_A0237) were also identified, but were not observed to contribute to the methylome. Results from mating experiments indicated that RM systems could potentially limit recombination via cell-to-cell mating in H. volcanii. Furthermore, RM system genes were observed to be patchily distributed among the Halobacteria, whereas orphan MTase gene families were more widespread and well-conserved. The results of these studies provide insight into the life cycle of RM systems in the Halobacteria and how they might contribute to halobacterial diversification and speciation. The RM deletion mutants of H. volcanii also have the potential to be useful in future work examining the role of RM systems and DNA methylation in the Halobacteria.

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