Tentative Consensus Sequences Research Articles

Nucleotide sequence of a DNA segment promoting transcription in Pseudomonas putida.

A DNA segment that promotes gene expression in Pseudomonas putida was identified in pTN8, a mutant plasmid of an RP4-TOL recombinant. A promoter on the segment was cloned with a promoter-probe vector containing the xylE gene of the TOL plasmid. The xylE gene was expressed under the control of the promoter, and the gene product catechol 2,3-dioxygenase was constitutively synthesized. As analyzed by an S1 nuclease protection assay, the amount of mRNA produced in P. putida was more than that in Escherichia coli. Fine S1 nuclease mapping and reverse transcriptase mapping revealed three tandem transcription start sites in both P. putida and E. coli. The nucleotide sequence preceding the transcription start sites was determined; a part of this sequence contained a sequence homologous to E. coli promoter sequences. A tentative consensus sequence for P. putida constitutive promoters is proposed.

Transcription of the TOL plasmid toluate catabolic pathway operon of Pseudomonas putida is determined by a pair of co-ordinately and positively regulated overlapping promoters.

Expression of the meta-cleavage pathway operon of TOL plasmid pWW0 of Pseudomonas putida is positively regulated by the xylS gene product. We have sequenced the promoter region of this operon and localized the transcription initiation sites. Two overlapping promoters, designated Pm1 and Pm2, are responsible for the positively regulated expression of the meta-pathway operon. Mutants of P. putida were isolated that expressed the meta-cleavage pathway operon constitutively. Several plasmid-located mutations that led to constitutivity were characterized by sequencing and the transcription initiation sites on mutant plasmids localized. This resulted in the identification of newly created promoters whose functioning did not require the xylS product. Comparison of the promoter sequences obtained suggests a tentative consensus sequence for promoters of P. putida which is significantly different from that of E. coli.