Localization of the Natural Transformation Machinery and Mechanisms of DNA Selection in Campylobacter jejuni

Abstract

The human pathogen Campylobacter jejuni is naturally competent for transformation with its own DNA. In this thesis work, we investigated key gene products of the transformation apparatus as well as the mechanism by which C. jejuni selects DNA for uptake and recombination. We demonstrate that two NTPases, CtsP and CtsE, are required for transformation. Their localization to the membrane is through a mechanism not generally observed with other competence ATPases. Further, CtsP interacts with another competence protein, CtsX, a single-pass transmembrane protein lacking significant homology to other proteins. Also investigated was the mechanism of DNA selection in C. jejuni. This bacterium is very selective in the DNA it uses during transformation, only using self or DNA derived from closely related strains. We demonstrate that this selection is based on adenine-methylation within a specific sequence motif, RAATTY. This site is broadly conserved in Campylobacter species and is over-represented in the C. jejuni genome. Methylation at RAATTY is conferred by CtsM, an orphan DNA methyltransferase also highly conserved in Campylobacter. CtsM is dispensable for transformation, but genomic DNA from a ctsM mutant serves as a transformation substrate with efficiency several orders of magnitude below DNA from a ctsM+ strain. A single methylated site is sufficient to confer wild type transformability to otherwise untransformable DNA. We also demonstrate that DNA lacking RAATTY methylation is transported inside the cell, implying that discrimination of methylated versus unmethylated DNA does not occur on the outer membrane. Finally, this work provides a potentially powerful tool to carry out rapid genome editing of C. jejuni, requiring only that an incoming homologous DNA fragment be RATTY-methylated for uptake and recombination. This is the first example of a methylation dependent mechanism of DNA selection during transformation.