In-vivo studies on the cis-acting replication initiator protein of IncFII plasmid NR1.

Using segment-directed mutagenesis, a temperature-sensitive mutant of the gene that encodes the cis-acting RepA1 initiation protein of the IncFII plasmid NR1 was isolated. The mutant protein was unable to promote initiation of plasmid replication in vivo at 42 degrees C. Both the wild-type and the mutant repA1 genes were cloned separately into the high-expression vector plasmid pAS1. In these pAS1-repA1 derivatives, the transcription of the repA1 gene was under the control of the lambda PL promoter, which was regulated by the temperature-sensitive lambda cI857 repressor protein. The translation initiation of the repA1 mRNA from these derivatives was mediated by the lambda cII Shine-Dalgarno sequence and initiation codon. The yield of 33,000 Mr RepA1 protein detected on SDS/polyacrylamide gels from Escherichia coli cells containing the pAS1-repA1 derivatives was dependent upon whether the newly synthesized RepA1 was capable of interacting in cis with the downstream NR1 replication origin on the cloned DNA fragment. Mutations in the repA1 gene or deletions of the cis origin region dramatically increased the detectable yield of RepA1 protein. Deletion of the NR1 origin region from the pAS1 derivative containing the wild-type repA1 gene enabled the cis-acting RepA1 protein to complement partially the temperature-sensitive repA1 mutant in trans, to increase the copy number in trans of plasmids that contained the NR1 replicon, and to help NR1 derivatives overcome plasmid incompatibility. The trans effects of RepA1 provided by the pAS1-repA1 derivatives that retained the origin in cis were much less significant. RepA1 provided in trans also stimulated the replication of plasmids carrying cloned copies of the NR1 replication origin region regardless of whether the origin was transcribed from an upstream promoter.