INVIVO-GENERATED FUSION PROMOTERS IN PSEUDOMONAS-PUTIDA

Plasmid pEST1463 carrying the promoterless pheBA operon was cloned int o Pseudomonas putida PaW85, and phenol-utilizing colonies were isolate d on minimal plates containing phenol as the only carbon and energy so urce. In these clones, chromosomally located Tn4652 was transposed ups tream from the coding sequence of pheA (encoding phenol monooxygenase) . Sequence analysis together with mapping of the transcription start p oint of the pheBA operon in the recombinant plasmids revealed that fus ions of the -10 sequences present in the pheBA operon and -35 sequence located in the terminal inverted repeats of Tn4652 had generated func tional promoters under selective pressure in P. putida cells. These pr omoter sequences show similarity to the Escherichia coli RNA polymeras e sigma70 promoter consensus sequence. In three of the six fusion prom oters studied, the generation combined two distinct events: transposit ion of Tn4652 into DNA containing potential -10 sequences and point mu tations in these sequences. These mutations made the -10 sequences mor e like the sigma70 promoter consensus sequences.