Construction and Use of an ipb DNA Module To Generate Pseudomonas Strains with Constitutive Trichloroethene and Isopropylbenzene Oxidation Activity

Abstract

ABSTRACT Pseudomonas sp. strain JR1 exhibits trichloroethene (TCE) oxidation activity with isopropylbenzene (IPB) as the inducer substrate. We previously reported the genes encoding the first three enzymes of the IPB-degradative pathway ( ipbA1 , ipbA2 , ipbA3 , ipbA4 , ipbB , and ipbC ) and identified the initial IPB dioxygenase (IpbA1A2A3A4) as responsible for TCE cooxidation (U. Pflugmacher, B. Averhoff, and G. Gottschalk, Appl. Environ. Microbiol. 62:3967–3977, 1996). Primer extension analyses revealed multiple transcriptional start points located upstream of the translational initiation codon of ipbA1 . The transcription from these start sites was found to be IPB dependent. Thirty-one base pairs upstream of the first transcriptional start point tandemly repeated DNA sequences overlapping the −35 region of a putative ς 70 promoter were found. These repeats exhibit significant sequence similarity to the operator-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 transcription of the IPB dioxygenase genes is modulated by a regulatory protein of the XylS/AraC family. The construction of an ipb DNA module 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 activities. Constitutive TCE oxidation of two such Pseudomonas hybrid strains, 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.