Essentiality of the three carboxyl-terminal amino acids of the plasmid RK2 replication initiation protein TrfA for DNA binding and replication activity in gram-negative bacteria.

In a previous study of mutations in trfA, the gene encoding the replication initiation protein of the broad host-range plasmid RK2, a carboxyl-terminal deletion of 3 amino acids of the TrfA protein was found to be completely nonfunctional for RK2 replication in Escherichia coli and other Gram-negative bacteria. In this work site-directed mutagenesis of the trfA gene was carried out to construct TrfA proteins altered in the three carboxyl-terminal positions. Specifically, TrfA proteins with deletions or substitutions of the terminal cysteine, lysine, and arginine (codons 380-382, respectively) were constructed and characterized for their ability to initiate replication from an RK2 origin in vivo in E. coli, Azotobacter vinelandii, Pseudomonas putida, and Agrobacterium tumefaciens and for binding activity to the iterons at the replication origin. Substitutions of the cysteine at position 380 with a glycine or an arginine resulted in a TrfA protein defective in binding to the RK2 origin and, therefore, defective in replication initiation activity in all four Gram-negative bacteria. Substitution of a serine at that position preserved limited function in replication and DNA binding. The lysine at position 381 could be changed to a glutamine without any obvious change in TrfA function. Deletion of the terminal arginine at position 382 did not affect the ability of TrfA to bind to origin iterons but caused a complete loss of replication activity in all four bacteria. Substitution of this terminal arginine with alanine, serine, or glutamic acid also produced replication-defective TrfA protein in all four bacterial hosts while not affecting iteron binding activity. However, substitution of this arginine with a lysine resulted in a loss of replication activity in E. coli and A. vinelandii but had no effect in P. putida and A. tumefaciens. These observations suggest that the terminal arginine plays an essential role in the activity of the TrfA protein, possibly interaction with host proteins, which can be separated from its iteron binding activity.