In-vivo-generated fusion promoters in Pseudomonas putida.
Plasmid pEST1463 carrying the promoterless pheBA operon was cloned into Pseudomonas putida PaW85, and phenol-utilizing colonies were isolated on minimal plates containing phenol as the only carbon and energy source. In these clones, chromosomally located Tn4652 was transposed upstream from the coding sequencing of pheA (encoding phenol monooxygenase). Sequence analysis together with mapping of the transcription start point of the pheBA operon in the recombinant plasmids revealed that fusions of the -10 sequences present in the pheBA operon and -35 sequence located in the terminal inverted repeats of Tn4652 had generated functional promoters under selective pressure in P. putida cells. These promoter sequences show similarity to the Escherichia coli RNA polymerase sigma 70 promoter consensus sequence. In three of the six fusion promoters studied, the generation combined two distinct events: transposition of Tn4652 into DNA containing potential -10 sequences and point mutations in these sequences. These mutations made the -10 sequences more like the sigma 70 promoter consensus sequences.
Institute of Molecular and Cell Biology, Estonian Biocentre, Tartu.
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Nurk
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Tamm
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Hôrak
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Kivisaar
D001483:Base Sequence; D050548:Catechol 1,2-Dioxygenase; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D049308:Dioxygenases; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006899:Mixed Function Oxygenases / Q000235:genetics*; D008969:Molecular Sequence Data; D009876:Operon; D010105:Oxygenases / Q000235:genetics*; D011401:Promoter Regions, Genetic; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics*; D015183:Restriction Mapping; D014158:Transcription, Genetic
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D006899:Mixed Function Oxygenases; D010105:Oxygenases; D049308:Dioxygenases; D050548:Catechol 1,2-Dioxygenase; C022336:phenol 2-monooxygenase