SITE-SPECIFIC MUTATIONS IN THE TRAI RELAXASE AND UPSTREAM REGION OF PLASMID RP4

The relaxase of RP4 nicks the double-stranded plasmid at the oriT site and binds covalently to DNA at the 5' end of the nick. The 80-kDa rel axase (TraI) is encoded on an operon with several overlapping open rea ding frames (ORFs). The importance in conjugation of a short ORF (traX ) with a start site overlapping the 5' terminus of traI was investigat ed, as well as the effects of specific mutations in the relaxase. Elim ination of TraX reduced the transfer efficiency by approximately 50% i n several intergeneric matings, especially when Escherichia coli was t he donor. While TraI was essential for transfer to occur, deletion of the C-terminus of TraI decreased, but did not eliminate plasmid transf er. Mutation of the active site tyrosine resulted in residual transfer associated with amino acid misincorporation. (C) 1995 Academic Press, Inc.