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)


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.


Methyl Ethyl Ketone Deliberate Release Degradative Ability Minus Mutant Indigenous Plasmid 
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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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