Mutations in the gene encoding the replication-initiation protein of plasmid RK2 produce elevated copy numbers of RK2 derivatives in Escherichia coli and distantly related bacteria.

Mini-replicons of the broad-host-range plasmid RK2 with increased copy number (cn) due to mutations in the gene encoding the essential replication initiation protein TrfA are described. The cn of these derivatives have been determined in Escherichia coli, Pseudomonas aeruginosa and Agrobacterium tumefaciens and were found to be elevated in all three bacterial hosts. One of the cn mutations was introduced into the intact 60-kb RK2 plasmid by homologous recombination in vivo, resulting in an approximately twofold cn increase. The expression of trfA from this mutant RK2 plasmid did not respond to the cn change as predicted by a simple transcription rate-limitation, replication control model. Implications for the model of RK2 replication control and the potential use of mutant RK2 mini-replicons as high-copy broad-host-range gene cloning vectors are discussed.