Site-directed mutations in the repA C-terminal region of plasmid Rts1: Pleiotropic effects on the replication and autorepressor functions

We induced site-directed mutations near the 3' terminus of the gene repA, which encodes the protein of 288 amino acid residues essential for plasmid Rts1 replications, and obtained seven repA mutants. Three of them contained small deletions at the 3' terminus. Mutant repAzΔC4, which encodes a RepA protein that lacks the C-terminal four amino acids, expressed a high-copy-number phenotype and had lost both autorepressor and incompatibility functions. Deletion of one additional amino acid residue to form the RepAzΔC5 protein caused restoration of the wild-type copy number and strong incompatibility. Studies of the remaining four repA mutants, each of which contained a single amino acid substitution near the RepA C terminus, suggested that Lys-268 is involved in both ori(Rts1) activation and autorepressor-incompatibility activities and that Arg-279 contributes to ori(Rts1) activation but not to incompatibility. Lys-268 is part of a dual-lysine sequence with Lys-267 and is located 21 amino acids upstream of the RepA C terminus. A dual-lysine sequence is also found at a similar position in both mini-F RepE and mini-P1 RepA proteins.