Transcription initiation at multiple promoters of the pfl gene by E sigma 70-dependent transcription in vitro and heterologous expression in Pseudomonas putida in vivo

Abstract

In vitro transcription experiments were used to provide further evidence that the gene encoding pyruvate formate-lyase (EC 2.3.1.54) from Escherichia coli is transcribed from seven promoters which cover a region of 1.2 kilobase pairs of DNA (G. Sawers and A. Böck, J. Bacteriol., 171:2485-2498, 1989). The results demonstrated that all promoters were recognized by the major RNA polymerase holoenzyme species E sigma 70 in vitro. Further corroboration for multiple functional promoters came from heterologous expression of the pfl operon in the obligate aerobe Pseudomonas putida. An immunological analysis indicated that the pyruvate formate-lyase protein was synthesized from a multicopy plasmid in P. putida, and S1 nuclease protection of RNA transcripts confirmed that all the pfl promoters on the plasmid were recognized by the host RNA polymerase. Transcription initiated at the same sites in P. putida and in E. coli for all the transcripts that were analyzed.