Use of Catabolic Mutants of Pseudomonas Cepacia AC1100 to Isolate Genetic Elements Responsible for 2,4,5-Trichlorophenoxyacetic Acid Degradation

  • U. M. X. Sangodkar
  • P. J. Chapman
  • R. A. Haugland
  • A. M. Chakrabarty
Part of the Basic Life Sciences book series (BLSC, volume 45)

Abstract

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.

Keywords

Methyl Ethyl Ketone Deliberate Release Degradative Ability Minus Mutant Indigenous Plasmid 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Copyright information

© Springer Science+Business Media New York 1988

Authors and Affiliations

  • U. M. X. Sangodkar
    • 1
  • P. J. Chapman
    • 2
  • R. A. Haugland
    • 3
  • A. M. Chakrabarty
    • 4
  1. 1.U.S. Environmental Protection AgencyGulf BreezeUSA
  2. 2.University of MinnesotaMinneapolisUSA
  3. 3.U.S. Environmental Protection AgencyCincinnatiUSA
  4. 4.University of Illinois at ChicagoChicagoUSA

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