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Many species of pathogenic bacteria harbor critical plasmid-encoded virulence factors, and yet the regulation of plasmid replication is often poorly understood despite playing a critical role in plasmid-encoded gene expression. Human pathogenic , including the plague agent and its close relative , require the type III secretion system (T3SS) virulence factor to subvert host defense mechanisms and colonize host tissues. The T3SS is encoded on the IncFII plasmid for virulence (pYV). Several layers of gene regulation enables a large increase in expression of T3SS genes at mammalian body temperature. Surprisingly, T3SS expression is also controlled at the level of gene dosage. The number of pYV molecules relative to the number of chromosomes per cell, referred to as plasmid copy number, increases with temperature. The ability to increase and maintain elevated pYV plasmid copy number, and therefore T3SS gene dosage, at 37°C is important for virulence. In addition, pYV is highly stable in at all temperatures, despite being dispensable for growth outside the host. Yet how reinforces elevated plasmid replication and plasmid stability remains unclear. In this study, we show that the chromosomal gene encoding the polyadenylase PAP I is required for regulation of pYV plasmid copy number (PCN), maintenance of pYV in the bacterial population outside the host, robust T3SS activity, and virulence in a mouse infection model. Likewise, /PAP I is also required for robust expression of the virulence plasmid-encoded T3SS. Furthermore, and /PAP I is required for maintaining normal PCN of model antimicrobial resistance (AMR) plasmids whose replication is regulated by sRNA, thereby increasing antibiotic resistance by ten-fold. These data suggest that /PAP I contributes to the spread and stabilization of virulence and AMR plasmids in bacterial pathogens, and is essential in maintaining the gene dosage required to mediate plasmid-encoded traits. Importantly /PAP I has been bioinformatically identified in many species of bacteria despite being studied in only a few species to date. Our work highlights the potential importance of /PAP I in antibiotic resistance, and shows for the first time that /PAP I reinforces PCN and virulence plasmid stability in natural pathogenic hosts with a direct impact on bacterial virulence.