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The plasmid host range of the replicon, pPS10, from Pseudomonas syringae pv. savastanoi can be changed by a conservative amino acid substitution and different host growth temperatures. pPS10 can establish efficiently in Escherichia coli at below 31 degrees Celsius while at 37 degrees it only establishes efficiently in P. aeruginosa. Three mutants with changes in the amino-terminal region of the RepA replication protein also showed improved E. coli establishment without affecting P. aeruginosa establishment efficiency.

The RepA protein of plasmid R1 is rate-limiting for initiation of R1 replication. Its synthesis is mainly regulated by interactions of the antisense RNA, CopA, with the leader region of the RepA mRNA, CopT. This work describes the characterization of several mutants with sequence alterations in the intergenic region between the copA gene and the repA reading frame. The analysis showed that most of the mutations led both to a decrease in stability of maintenance of mini-R1 derivatives and to lowered repA expression assayed in translational repA-lacZ fusion constructs. Destruction of the copA gene and replacement of the upstream region by the tac promoter in the latter constructs indicated that these mutations per se alter the expression of repA. In addition, we show that particular mutations in this region can directly affect CopA-mediated control, either by changing the kinetics of interaction of CopA RNA with the RepA mRNA and/or by modifying the activity of the copA promoter. These data indicate the importance of the region analysed in the process that controls R1 replication. [ABSTRACT FROM AUTHOR]