STUDIES ON SPONTANEOUS PROMOTER-UP MUTATIONS IN THE TRANSCRIPTIONAL ACTIVATOR-ENCODING GENE PHLR AND THEIR EFFECTS ON THE DEGRADATION OF PHENOL IN ESCHERICHIA-COLI AND PSEUDOMONAS-PUTIDA

The activator-encoding gene phlR was identified upstream of the plasmi d-encoded operon for phenol degradation in Pseudomonas putida strain H by cassette mutagenesis and DNA sequence analysis. The deduced amino acid sequence of PHLR shows high homology to DmpR of P. putida sp. CF6 00 and to the chromosomally encoded PhhR of P. putida P35X reported pr eviously. Trans-activation of phenol degradation was observed when phl R was overexpressed in a phlR insertion mutant. Transconjugants of Esc herichia coli carrying pPGH 11, which contains the complete set of phl genes, are unable to grow on phenol as carbon source. However, two ty pes of mutants were selected for further characterization that were ab le to metabolize phenol as sole source of carbon and energy. In both t ypes of mutants enhanced expression of phlR is responsible for the Phl (+) phenotype. In type I (pPGH13) a deletion of 1 bp made the -35 regi on and the spacing between the -35 and -10 regions of the phlR promote r more similar to the consensus structure. In type II (pPGH14) a dupli cation of the phlR 5' region was identified that includes part of the -35 motif and reduces the spacing between the -35 and -10 regions. In addition, due to the duplication of part of phlR, the distance from th e phlR promoter to the catabolic ph( operon is increased. Different tr anscriptional start sites have been identified by primer extension ana lysis in clones harboring pPGH14 or the wild type phlR. Quantitative p rimer extension analysis revealed that the greatest amount of phlR tra nscript is expressed from the partial, phlR duplication. Growth on phe nol and phenol hydroxylase activity reflect the high level of phlR tra nscript in E. coli transconjugants. Overexpression of PhlR was also ob served when pPGH14 was transferred into P. putida, and results in earl ier induction of the phenol degradation operon relative to the wild-ty pe strain.