Lang, Kevin2015-10-132015-10-132015-05https://hdl.handle.net/11299/174866University of Minnesota Ph.D. dissertation. May 2015. Major: Comparative and Molecular Biosciences. Advisors: Timothy Johnson, Randall Singer. 1 computer file (PDF); viii, 121 pages.Plasmids are extrachromosomal DNA elements that often carry beneficial phenotypes for the bacterial host. Incompatibility type A/C (IncA/C) plasmids are large (~100 ~ 200 kilobases (kb)), conjugative plasmids that are carried by Gram-negative bacteria. IncA/C plasmids often carry numerous genes that confer resistance to antimicrobials and have been isolated from many types of bacteria that pose significant risks in both human and animal medicine. Although IncA/C plasmids have been described in the literature for many years, little is known about their basic biology. For the past decade, many fully sequenced IncA/C variants have been described. There has been a lack of work concerning core functions of these plasmids, such as, replication, conjugative transfer and maintenance. This dissertation focuses on how these plasmids are regulated on a transcriptional level. We used pAR060302, a prototypical IncA/C plasmid, to conduct several experiments investigating exactly what genes are transcribed and how different conditions affect their transcriptional landscape. RNA-Seq was used to understand how antimicrobial exposure can affect the way genes are expressed on IncA/C plasmids. We found that, under the conditions we tested, antimicrobials have little effect on the transcription of genes on pAR060302. However, this initial study was the first to explore genes carried IncA/C plasmids in terms of their expression. Further RNA-Seq experiments were carried out in several different bacterial genera all carrying the same IncA/C plasmid, pAR060302. These experiments attempted to characterize how a broad host-range plasmid, such as IncA/C, might be differentially regulated in different hosts. We found that only subtle changes occur in the expression of plasmid genes. Carriage of IncA/C plasmids was found to have diverse effects on chromosomal gene expression. Genes involved in 2-carbon metabolism in Escherichia coli are up-regulated due to plasmid carriage. Our results suggest that plasmid encoded factors might serve varying levels of importance depending on the host chromosomal background. Experiments were carried out on E. coli carrying plasmids with mutations in a group of predicted transcriptional regulators to determine their function. We identified the positive regulators of conjugative transfer in IncA/C plasmids, acaD and acaC. We also found a repressor of transfer, acr2, which encodes an H-NS-like protein. We further show that acr2 might regulate genes beyond those that are involved in conjugative transfer. This dissertation builds on our understanding on what mechanisms are important for the maintenance of large plasmids in Gram-negative bacteria. Understanding how plasmids might specifically tune host metabolism to improve competitive fitness would impact what evolutionary processes were involved in their emergence. Characterization of regulatory networks that govern core plasmid processes, such as conjugation, might assist in the development of new models of how these plasmids disseminate throughout bacterial populations.enantibiotic resistancegene regulationPlasmidsThesis or Dissertation