The current rate of acquired antibiotic resistance (AR) in bacterial pathogens is projected to cause 10 million deaths per year and result in a global cost of up to 100 trillion USD by 2050. It is therefore urgent that we develop new therapies that target pathogen-specific virulence factors (i.e.-microbial genes required to cause disease). However, our limited understanding of how these virulence factors are regulated is a major obstacle in the field, and studies exploring how these pathogens coordinate expression of virulence genes are desperately needed in general. Many Gram negative pathogens harbor large extrachromosomal plasmids encoding genes important for virulence and/or antimicrobial resistance, many of which belong to the IncF family of plasmids. Understanding how these plasmids are regulated and maintained is critical for addressing the threat posed by AR pathogens, and yet the underlying biology regulating many of these plasmids remains poorly understood. Using the Gram negative enteric pathogen Y. pseudotuberculosis as a model organism, the aim of this research is to broaden our understanding of how facultative pathogens like Yersinia regulate virulence gene expression at the level of virulence plasmid copy number (PCN) and plasmid maintenance. Y. pseudotuberculosis is closely related to Y. pestis, the causative agent of the plague. Both Y. pseudotuberculosis and Y. pestis harbor an IncFII, ~70kb virulence plasmid known as pYV that encodes a major Yersinia virulence factor known as the type III secretion system (T3SS). The T3SS is a specialized injectisome used by many species of Gram negative pathogens to inject effector proteins into the host cell cytosol, interfering with host innate immunity signaling pathways and ultimately dampening host defense mechanisms. Despite being critical for Yersinia virulence, expression of the T3SS is energetically costly and must be tightly controlled to achieve optimal fitness, and so expression and activity of the T3SS is restricted to host temperatures of 37°C where it is needed for virulence. It was recently discovered that expression and activity of the T3SS is dynamically regulated via changes in PCN, or the number of pYV plasmids per Y. pseudotuberculosis cell. At environmental temperatures of 26°C, where the T3SS is not needed, pYV PCN is kept low to about one copy per cell. However, upon entering the mammalian host (37°C), pYV PCN is quickly and specifically increased to 5-12 copies per cell. This increase in pYV PCN is required for Y. pseudotuberculosis virulence in mice, and yet the mechanism by which pYV PCN is kept low at 26°C and rapidly increased at 37°C is completely unknown. The first chapter of this dissertation reviews how the Yersinia T3SS has served as a tool for discovering and elucidating host immune pathways that have evolved through extensive host-pathogen interactions with T3SS-utilizing bacteria. The rest of this work focuses on the discovery of two factors that impact pYV PCN and/or maintenance in Y. pseudotuberculosis. In chapter 2, we demonstrate that a transposon (Tn) insertion in a previously unannotated locus near the pYV IncF replicon drives abnormally high pYV PCN at environmental temperatures of 26°C, resulting in a hyperactive T3SS phenotype at 37°C and a severe growth defect under T3SS-inducing conditions. This locus, which we will call plasmid copy number inhibitory locus or pil, encodes a predicted ORF that is conserved in some other species of Gram negative bacteria. By taking advantage of the serve growth defect of this strain, we further demonstrate that pil insertion mutants can be used as genetic tools for identifying novel factors affecting pYV PCN, T3SS activity, and Yersinia growth in a suppressor screen. A pilot suppressor screen identified specific residues in factors that impacts pYV PCN and T3SS activity in various ways, and we propose that pil insertion mutants will serve as a powerful tool for identifying additional factors in future studies. Chapter 3 of this dissertation focuses on one of the hits arising from the pil suppressor screen, which identified the pcnB gene encoding the polyadenylase poly (A) polymerase I (PAP I) as a novel regulator of pYV maintenance in pathogenic Yersinia. We show that PAP I is essential for normal pYV PCN, T3SS activity, and Yersinia virulence in a mouse infection model. In addition, we report that PAP I is required for Y. pseudotuberculosis maintenance of sRNA-regulated plasmids that harbor AR genes in general, suggesting that PAP I is important for the maintenance of both virulence and AR plasmids by pathogenic bacteria. PAP I homologs are widespread in bacteria but have only been studied in a small handful of species to date. Importantly, this is the first time PAP I has been linked to regulation of a virulence plasmid in the natural pathogenic host for that plasmid. To our knowledge, this is also the first direct evidence that a bacterial polyadenylase impacts virulence of a bacterial pathogen. Overall, this work broadens our understanding of how IncF plasmids are regulated to balance virulence and bacterial fitness by pathogens like Y. pseudotuberculosis. Given that PAP I homologs are widespread and many AR plasmids and nearly all virulence plasmids belong to the IncF family, this work likely applies to other species of Gram negative pathogens as well. Finally, this work highlights that there are many additional levels of gene expression regulation to consider when it comes to addressing the threat posed by bacterial pathogens, including changes in gene dosage via changes in PCN of extrachromosomal plasmids and the potential contribution of polyadenylation to regulation of virulence gene expression.