| dc.description.abstract | DNA replication and transcription occur in all living cells across all domains of life. Both essential processes occur simultaneously, on the same template, leading to conflicts between their macromolecular machines. Head-on conflicts disrupt replication progression, cause breaks in the DNA, and ultimately promote mutagenesis, which can impact adaptive evolution. The prevalence of mutagenic head-on conflicts in pathogenic bacteria species could be a novel mechanism by which bacterial pathogens facilitate adaptation and evolve their own virulence specifically during infection. However, how replication and transcription interfere with one another in the context of infection, and any consequences thereof, remains unknown.
In this dissertation, I present post infection chromatin immunoprecipitation followed by deep sequencing (PIC-seq) as a technique capable of testing this model. PIC-seq is sensitive enough to selectively isolate bacterial protein-chromatin complexes specifically while the pathogenic cells reside within eukaryotic host cells. In using this method to map RNA polymerase occupancy genome-wide in Salmonella enterica, I discovered that infection of a host leads to drastic changes in RNA polymerase association patterns with DNA. Notably, backtracking, a stable state in which RNA polymerase is stalled, is more prevalent in pathogenic cells during infection than the same cells grown in broth culture. This backtracking downregulates gene expression and negatively affects S. enterica pathogenesis, if unresolved. Conflicts between replication and transcription, which threaten genome stability, are likely worsened under these conditions due to pervasive RNA polymerase backtracking during infection. Further characterizing changes to molecular transactions within bacterial pathogens during infection remains a key future direction, enabled by the development of PIC-seq. Overall, the work described herein is an important step forward in our understanding of host-pathogen interactions with respect to adaptation and evolution. | |
