CONTROL OF F PLASMID TRANSFER IN E. COLI (CONJUGATION, BACTERIAL GENETICS, TRA-J, ESCHERICHIA)

Abstract

Conjugal DNA donor and related activities of Escherichia coli F('+) and Hfr strains require at least 25 plasmid and five chromosomal gene products. Most of the F plasmid DNA transfer (tra) genes are organized into a co-regulated gene block (traY-Z operon) that is controlled by a complex regulatory mechanism. The first part of this thesis concerns two chromosomal genes, cpxA and cpxB, that are required for expression of F plasmid tra functions. Mutations of both genes prevents transcription of the traY-Z operon of F. This defect is attributed to the failure of cpxA cpxB mutant cells to accumulate the traJ gene product, a protein required for expression of the other tra genes. TraJ protein accumulation was assayed by using UV-irradiated cells infected with a (lamda)traJ transducing bacteriophage carrying an intact traJ gene and its normal control sequences. Although mutant cells failed to accumulate TraJ protein, both mutant and wild-type cells accumulated comparable amounts of traJ mRNA. In addition, a plasmid, pLS200, was constructed which carried a traJ-lacZ protein fusion placing (beta)-galactosidase synthesis under traJ transcription and translation control signals. (beta)-Galactosidase levels were comparable in cpx mutant and wild-type cells carrying pLS200, suggesting that the effect of the cpx mutations on TraJ accumulation is post-transcriptional and perhaps post-translational.;The second part of this thesis examines the plasmid-specific FinO-FinP genetic regulatory mechanism. The finP gene product from F (finP(,F)) acts in conjunction with the finO product encoded by certain R plasmids to repress TraJ synthesis. R plasmids produce their own plasmid-specific FinP. We devised an in vivo assay for both FinP and FinO using pLS200. R100, a FinO('+) plasmid, but not R100drd1, a finO mutant, reduced the level of (beta)-galactosidase in cells containing pLS200, indicating that this plasmid carries an active finP locus. Moreover, R100 transfer from cells containing pLS200 was derepressed to the level of R100drd1 transfer, suggesting that FinP(,F) was over-produced sufficiently from pLS200 to titrate all the FinO in the cell. A potential finP transcript was detected in cells carrying pLS200 that correlated with R100 derepression. The transcript of approximately 100 bases was coded from within the traJ leader region and anti-sense to traJ mRNA. We postulate that this transcript is the finP gene product and that the RNA molecule itself is the active repressor.