Cataloging Essential Genes for Natural Transformation and Expanding Genetic Tools for Study of Archaea

Abstract

Horizontal gene transfer is a near ubiquitous way for organisms to acquire new geneticinformation. One mechanism of horizontal gene transfer is natural transformation, the uptake/incorporation of DNA from the environment into the genome. In Bacteria, DNA uptake has been well documented in numerous phylogenetically diverse species. Across all of these organisms, four operational steps have to occur. First, the pathway must be induced, entering cells into a state known as competence. Extracellular appendages on the cell then reach out from the cell surface and adsorb DNA. DNA is subsequently translocated through a transporter ComEC and bound by intracellular single-strand binding proteins for integration into the genome. In Archaea, no mechanism of natural transformation has been previously identifiedand homologs of bacterial competence genes are sparse. Presented here is the identification of the process of natural transformation in two distinct members of the Archaea. Using a variety of genetic techniques, I identify several components that are essential to the natural transformation pathway. This includes Type IV-like pili, putative membrane-bound substrate transporters, proteins predicted to bind DNA, as well as several hypothetical proteins. While this thesis provides the first catalog of genes essential to natural transformation, the exact mechanism that underlies this process is still elusive. The later chapters of this thesis will discuss preliminary approaches to determine the mechanisms of natural transformation mediated DNA uptake.