Use of Catabolic Mutants of Pseudomonas Cepacia AC1100 to Isolate Genetic Elements Responsible for 2,4,5-Trichlorophenoxyacetic Acid Degradation
Pseudomonas cepacia AC1100, constructed by plasmid-assisted molecular breeding, is a unique microorganism capable of complete mineralization of the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). 2,4,5-T is a major component of the defoliant Agent Orange. Due to its ability to remove over 95% of the pollutant from contaminated soils, AC1100 is considered a candidate for deliberate release into the environment. We have isolated a number of 2,4,5-T minus mutants which appear spontaneously after a few generations of growth on nonselective media. Some of the blocked mutants accumulated colored metabolites of 2,4,5-T. This genetic instability may be a reflection of the gene rearrangements involved in initial. evolution of AC1100 in the chemostat. To isolate the genetic elements contributing to 2,4,5-T degradation, we mobilized a cosmid-gene bank of AC1100 into several Tn5-induced blocked mutants. The partial diploids, resulting from complementation of insertion mutations, restored the 2,4,5-T degradative ability. A hybrid cosmid pUS1, carrying an AC1100 DNA fragment, complemented the Tn5-induced mutant PT88 and was stably maintained along with the indigenous plasmids in this strain. pUS1 complemented two other Tn5-induced mutants and also restored 2,4,5-T+ phenotype of SM9A, one of the spontaneous blocked mutants of AC1100. A 25-kb insert from pUS1 was mapped for its restriction endonuclease sites and was used for subcloning smaller fragments on a broad host-range plasmid.