Analysis of the vegetative replication origin of broad-host-range plasmid RK2 by transposon mutagenesis

A range of Tn1723 transposon mutants of the oriV region of broad-host-range plasmid RK2 have been isolated, and the internal EcoRI fragment of the transposon has been deleted from each to reduce the insertion size from 9.6 kb (Tn1723) to 35 bp (delta Tn1723). Sequencing from the delta Tn1723-derived EcoRI site has allowed the precise mapping of these insertions to various points dispersed through the origin region. Using these mutants we have determined which regions of oriV RK2 are of functional importance to plasmid establishment following transformation of the host species Escherichia coli, Pseudomonas putida, and P. aeruginosa. Insertions into an A/T-rich region, and a region containing five direct repeat sequences prevented successful transformation of each host species tested, but the continuity of sequences adjacent to the five repeats were essential only in E. coli and P. putida. The establishment and maintenance in E. coli of a mini-RK2 replicon was found to be inhibited by transcription from an inducible promoter positioned to read into oriV RK2 against the direction of replication. Assays of transcription emerging from Tn1723 demonstrated significant levels from one end of the transposon only. Four mutants with insertions downstream of oriV RK2 were unable to become established in E. coli, and contained Tn1723 in the orientation which would supply transcription toward the oriV RK2 region. These results demonstrate both that the sequence requirements for oriV RK2 function differ between host bacterial species, and that origin function may be further influenced by the genetic environment in which it lies.