| Abstract: | Products from the transfer region of the F plasmid are involved in the conjugation process. Although 21 tra gene products have been characterized into 5 different functional groups, a number of genes in the tra region have not been functionally characterized due to the lack of mutations; such genes include traR, traU, traX, trbC, trbJ, and trbH. I constructed kanamycin insertion mutations in these gene sequences and crossed each mutation into the complete transfer operon of a transmissible F plasmid derivative. I then tested the effect of these mutations on plasmid transfer and F-specific bacteriophage infection. My results showed that traR, trbH, trbJ, and traX are not required for either phage infection or transfer processes, but that trbC is required for pilus assembly. One of the non-essential genes, traX, turned out to be required for acetylation of F-pilus subunit. Since I detected two traX products and both migrated faster on SDS-PAGE than expected from the DNA sequence, I constructed a series of clones designed to determine the nature and origin of the products and their pilin acetylation activity. My studies also showed that traQ and traX are the only tra genes required for F-pilin maturation. I performed pulse-chase experiments with cells carrying combinations of traA, traQ, and traX, and suggest a pathway that describes the steps in F-pilin maturation. In addition, a new system for studying the F transfer system was developed by replacing the major tra promoter with the T7 late gene promoter. With this T7 promoter derivative of F, expression of the entire transfer region could be manipulated by regulating T7 RNA polymerase expression. The plasmid is as transmissible and confers the same sensitivity to F-pilus-specific phages as the wild-type F, and all of its tra proteins can be labeled specifically in the presence of rifampicin. Using this plasmid, mutations in several additional tra genes were constructed and analyzed. The phenotype of traJ mutant derivatives showed that tra J is not required for F conjugation except to enhance transcription. Mutation of traY did not affect F-piliation or F-specific phage sensitivity in this system but did affect transfer. The mutant properties of the trbC derivatives were consistent with those of my other trbC mutants, suggesting one can take advantage of the T7 promoter system in studies of any tra gene. |
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