Construction of derivatives of the F plasmid pOX-tra715: characterization of traY and traD mutants that can be complemented in trans.

A derivative of the F plasmid, pOX38-tra715, expresses the entire F tra operon from a foreign promoter (PT7) derived from phage T7. A series of plasmids related to pOX38-tra715 were constructed which carry either deletion mutations or point mutations in traY. When the PT7 promoter was induced, these plasmids expressed the F pilus but were transfer deficient unless TraY was supplied In trans from compatible plasmids. Insertion of a kanamycin-resistance cassette in the traY gene of the pOX38 plasmid, which contains the wild-type PY promoter, resulted in loss of F piliation and transfer ability. Introduction of TraY in trans partially restored piliation and transfer suggesting that TraY has a role in positively regulating the PY promoter, pOX38-tra719-traD411, which contains a chloramphenicol-resistance cassette in place of the kanamycin-resistance cassette in pOX38-tra715 and a mutation in traD, was constructed to demonstrate the utility of this series of plasmids in studying the long (30 kb) F tra operon.