Plasmids with mutations in tifA, the gene encoding the replication initiation protein of the broad-host-range plasmid RK2, were isolated and characterized. Mutants identified from a nitrosoguanidine bank were defective in supporting the replication of a wild-type RK2 origin in Escherichia coli. Most of the mutations were clustered in a region of tifA corresponding to the carboxy-terminal quarter of the TrfA protein. 5' and 3' deletion mutants of trfA were also constructed. A C-terminal deletion of three amino acids of the Tr A protein was completely nonfunctional for RK2 replication. However, a deletion of 25 amino acids from the start of the 33-kDa TrfA protein was still competent for replication. Further characterization of the point and deletion trfA mutants in vivo revealed that a subset was capable of supporting RK2 replication in other gram-negative bacteria, including Pseudononas putida, Agrobacterium tumefaciens, and Azotobacter vinelandii. Selected mutant TrfA proteins were partially purified and characterized in vitro. Velocity sedimentation analysis of these partially purified TrfA proteins indicated that the wild-type protein and all mutant TrfA proteins examined exist as dimers in solution. Results from in vitro replication assays corroborated the experimental findings in vivo. Gel retardation results clearly indicated that the point mutant TrfA-33:151S, which was completely defective in replication of an RK2 origin in all of the bacterial hosts tested in vivo, and a carboxy-terminal deletion mutant, TrfA-33:CA305, were not able to bind iterons in vitro. In addition to the proteins from mutants that were totally defective in DNA binding, several other mutant proteins were either partially defective or could not be distinguished from the wild-type protein in binding to the origin region. The mutant proteins with apparently normal DNA-binding activity in vitro either were inactive in all four gram-negative bacteria tested or exhibited differences in functionality depending on the host organism. These mutant TrfA proteins may be altered in the ability to interact with the replication proteins of the specific host bacterium.RK2 is a 60-kb self-transmissible plasmid that can be stably maintained in a wide variety of gram-negative hosts (9,21,27,32,33,40). It is a member of the IncP1 incompatibility group, and the number of copies of RK2 in Escherichia coli has been estimated as four to seven per chromosome (17). Two plasmid elements that are essential for RK2 plasmid replication are the cis-acting origin of replication, oriV, and the trans-acting replication initiation protein, TrfA (16,47,50,51). The origin of replication was originally defined as a 700-bp HaeII fragment. In addition to eight 17-bp direct repeats, or iterons, arranged in two clusters of five and three, the origin contains an A+T-and G+C-rich region and sequences homologous to DnaA boxes (43). In E. coli, a 393-bp HpaII fragment containing the five-iteron cluster, three of the putative DnaA boxes, and the A+T-and G+C-rich region is also ...
