For replication of broad host range plasmid RK2 in two regions of the plasmid genome are essential, between 12·0 and 12·7 kb on the genome (defined clockwise from the unique RI site) and , located between 16·0 and 17·4 kb, which provides a -acting product necessary for function. The properties of an insertion mutant of a mini-RK2/ColEl hybrid plasmid suggest that the promoter lies clockwise from the 17·4 kb RK2 coordinate. Fusion of the gene lacking its normal promoter to the gene in a hybrid plasmid confirmed that is transcribed anticlockwise, towards the Tc gene of RK2. Repression of promoter activity in these fusions showed that replication of an RK2 replicon can be regulated by varying the level of gene expression. The results also indicate the presence of a transcription unit running anticlockwise through the region located between 54·0 and 56·0 kb on the RK2 genome.


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