volume 13 Number 22 1985 Nucleic Acids Research Regulation of the trfA and trfB promoters of broad host range plasmld RK2: Identification of sequences essential for regulation by trfBfkorA/korD Bimal D.M.Theophilus, Michael A.Cross, Christopher A.Smith and Christopher M.Thomas* Department of Genetics, University of Birmingham, P.O. Box 363, Birmingham B15 2TT, UK Received 4 October 1985; Accepted 30 October 1985 ABSTRACT Using a plasmld containing a transcriptional fusion in which the E.coll galX gene is expressed from the trfB promoter of broad host range plasmid RK2 we show that transcription from the trfB promoter is repressed by the products of both the trfB and korB genes as we have previously predicted from the sequence homology of the trfA and trfB promoters and the fact that the trfA promoter is regulated by trfB and korB. These loci, trfB and korB are normally transcribed from the trfB promoter. Thus the trfB lncC korB operon of RK2 is doubly autogeneously regulated. In addition, we describe the isolation and characterization of a mutant trfA promoter which has become insensitive to repression by trfB as a result of a point mutation within the inverted repeat sequence previously predicted to be the trfB protein binding site. These results provide strong evidence for our previously proposed model for control of transcription from the trfA and trfB promoters. INTRODUCTION The plasmid RK2, which is indistinguishable from plasmids RP4, RP1, R68, R18 (1,2) belongs to the Escherichia coli incompatibility group IncP and to the Pseudomonas species group IncP-1. The molecular genetics of these indistinguishable plasmids has been investigated (for a review, see ref. 3), because of their ability to transfer themselves between and maintain themselves in almost all Gram negative bacterial species, a property which is of both fundamental and applied interest. Examination of the physical and genetic map of RX2 (Fig. 1) reveals that if the blocks of known transposable elements and antibiotic resistance genes are ignored then the plasmid genome consists of a large block of conjugal transfer functions and a second block of functions which Include the origin of vegetative replication orlv^^ (9,10,11,12) and the trfA gene, which provides a trans-acting protein or proteins which activates oriV RK2 (13,10,14,15). The other functions in this second block include kil genes, which if cloned on their own are either lethal to their bacterial host (kilA, B_ and C; 13,16) or Inhibit plasmld maintenance (kllD 17; also e IRL Press Limited, Oxford, England. 8129