CONSTRUCTION AND USE OF AN IPB DNA MODULE TO GENERATE PSEUDOMONAS STRAINS WITH CONSTITUTIVE TRICHLOROETHENE AND ISOPROPYLBENZENE OXIDATION ACTIVITY

Pseudomonas sp. strain JR1 exhibits trichloroethene (TCE) oxidation ac tivity with isopropylbenzene (IPB) as the inducer substrate. We previo usly reported the genes encoding the first three enzymes of the IPB-de gradative pathway (ipbA1, ipbA2, ipbA3, ipbA4, ipbB, and ipbC) and ide ntified the initial IPB dioxygenase (IpbA1 A2A3A4) as responsible for TCE cooxidation (U, Pflugmacher, B, Averhoff, and G, Gottschalk, Appl. Environ, Microbiol. 62:3967-3977, 1996), Primer extension analyses re vealed multiple transcriptional start points located upstream of the t ranslational initiation codon of ipbA1, The transcription from these s tart sites was found to be IPB dependent. Thirty-one base pairs upstre am of the first transcriptional start point tandemly repeated DNA sequ ences overlapping the -35 region of a putative sigma(70) promoter were found. These repeats exhibit significant sequence similarity to the o perator-promoter region of the xyl meta operon in Pseudomonas putida, which is required for the binding of XylS, a regulatory protein of the XylS (also called AraC) family. These similarities suggest that the t ranscription of the IPB dioxygenase genes is modulated by a regulatory protein of the XylS/AraC family. The construction of an ipb DNA modul e devoid of this ipb operator-promoter region and the stable insertion of this DNA module into the genomes of different Pseudomonas strains resulted in pseudomonads with constitutive IPB and TCE oxidation activ ities. Constitutive TCE oxidation of two such Pseudomonas hybrid strai ns, JR1A::ipb and CBS-3::ipb, was found to be stable for more than 120 generations in antibiotic-free medium. Evaluation of constitutive TCE degradation rates revealed that continuous cultivation of strain JR1A ::ipb resulted in a significant increase in rates of TCE degradation.