Erickson, Rebecca2019-04-092019-04-092019-02https://hdl.handle.net/11299/202421University of Minnesota Ph.D. dissertation. February 2019. Major: Microbiology, Immunology and Cancer Biology. Advisors: Gary Dunny, Wei-Shou Hu. 1 computer file (PDF); xiv, 178 pages.Enterococcus faecalis is a commensal member of the gastrointestinal tract of animals including humans but is also an opportunistic pathogen and a major cause of healthcare-associated infections. Its pathogenicity is thought to arise in immunocompromised people and after infection, treatment is difficult due to antibiotic resistance. E. faecalis is particularly good at transferring antibiotic resistance by mechanisms like conjugation and conjugative transfer of plasmids can occur at a high frequency without antibiotic selection. Conjugative plasmid pCF10 encodes tetracycline resistance and transfer between E. faecalis cells is facilitated by cell-to-cell communication. This signaling triggers expression of genes from pCF10 that encode for transfer machinery. The response to signaling is robust and has been extensively studied at the population level. However, it has recently become apparent that there is response variation. Understanding the mechanisms that underlie variation in response initiation is important to preventing transfer. Studies presented in this dissertation adapt fluorescence in situ Hybridization Chain Reaction (HCR) for single cell analysis of transcripts and explore questions about the pCF10 conjugation system that would not have otherwise been possible. In chapter 3, variation in the signaling response was assessed and the response was shown to be very heterogenous. When the level of signal is low, (like what might occur naturally), a minority of cells respond. Although stochasticity in the system may give rise to such heterogeneity, work in chapter 4 investigates the response impact of a few specific mechanistic players (PrgX, C, and I). Changing the levels of these components was shown to change the outcome. Lastly, single cell analysis was used in chapter 5 to assess the expression of genes required for conjugative transfer. These results show that the few responding cells commit to expression of all the genes encoding for production of the conjugation machinery. Overall, these results suggest that the pCF10 system is evolutionarily tuned for specific levels of each component and poised to have response variation for a population of cells. Thus, a small percent of cells can respond and since the majority of responding cells are able to conjugate, plasmid transfer is highly efficient. These results also exemplify how small differences in two cells can precipitate different responses in otherwise identical cells exposed to very similar conditions. Information about variation in the initiation of the signaling response required for pCF10 transfer is important to understanding the general biology of gene transfer among bacteria. In the future, this information will be important for successful design of effective interventions to the transfer of genes conferring antibiotic resistance.enAntibiotic resistanceConjugationGene transferMicroscopySignalingSingle cell analysisThesis or Dissertation