Genetic evidence for the direction of transcription of the trfA gene of broad host range plasmid RK2

Abstract

For replication of broad host range plasmid RK2 in Escherichia coli two regions of the plasmid genome are essential, oriV RK2 between 12.0 and 12.7 kb on the genome (defined clockwise from the unique EcoRI site) and trfA, located between 16.0 and 17.4 kb, which provides a trans-acting product necessary for oriV RK2 function. The properties of an insertion mutant of a mini-RK2/ColE1 hybrid plasmid suggest that the trfA promoter lies clockwise from the 17.4 kb RK2 coordinate. Fusion of the trfA gene lacking its normal promoter to the E. coli trpE gene in a hybrid plasmid confirmed that trfA is transcribed anticlockwise, towards the Tcr gene of RK2. Repression of trpE promoter activity in these fusions showed that replication of an RK2 replicon can be regulated by varying the level of trfA gene expression. The results also indicate the presence of a transcription unit running anticlockwise through the trfB region located between 54.0 and 56.0 kb on the RK2 genome.