Analysis of transcription from the trfA promoter of broad host range plasmid RK2 in Escherichia coli, Pseudomonas putida, and Pseudomonas aeruginosa.

Reverse transcriptase mapping has been used to analyze transcription from the trfA promoter of broad host range plasmid RK2. The results show that trfA operon mRNA has the same 5' end in Pseudomonas aeruginosa, Pseudomonas putida, and Escherichia coli. The strengths of wild-type and mutant trfA promoters, which differ by defined base substitutions, have been compared and the positions of their transcriptional start sites determined. While these base substitutions do not alter the transcriptional start site, they do have marked effects on promoter strength which are broadly similar in each of the host species. A single base pair substitution, which lies in the region corresponding to the E. coli promoter consensus, brings about a large reduction in gene expression while the introduction of a second mutation, at a locus outside this region, has no further effect on promoter strength. The results indicate that these Pseudomonas species possess an RNA polymerase which recognizes the same region of the trfA promoter as that utilized by E. coli RNA polymerase. Within the limits of these observations it is clear that the trfA operon is transcribed from a single promoter which can function efficiently in diverse species, a property which may be important for its broad host range.