Our long-term objective is to understand the genetic, structural, and regulatory bases for conjugal DNA donor activity of F+ strains of Escherichia coli. A critical and largely unexplored aspect of the problem is how the activities of the plasmid and chromosomal genomes of donor strains are coordinated in the formation of donor-specific surface structures. It now appears that these morphogenetic events are controlled by a cell-plasmid regulatory network that includes the F plasmid TraJ protein and the chromosomal cpx and sfrA gene products. Using in vivo and in vitro approaches, including purification of the native TraJ protein, we will determine whehter or not the TraJ protein acts directly to regulate expression of the other F plasmid tra genes. We will determine whether the TraJ protein acts exclusively to regulate tra gene expression or whether it functions at subsequent stages of donor-specific morphogenesis as well. In the course of these experiments, we will determine the quantitative relationship between tra gene expression the formation of donor structures, and the cellular level of the TraJ protein, controlled as the independent variable by use of a lac-traJ operon fusion. We will also construct a plasmid that expresses all of the F plasmid genes required for the formation of F-pili independently of the TraJ protein. That plasmid will be used to construct strains that overproduce F-pili for structural and biochemical studies. Using Mu(amp-lac) fusions, we will ask whether any chromosomal genes are regulated by F plasmid gene products. Using genetic and quantitative physiological approaches, we will determine how the chromosmal cpx and afrA gene products regulate the TraJ protein level and whether or not that is their only contribution to donor-specific morphogenesis. These studies directly address the problem of bacterial cell-plasmid interactions. Insofar as plasmids contribute to bacterial pathogenicity, virulence, and antibiotic resistance, studies of these interactions are directly related to human health.
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