Cloning and Characterization of a Chromosomal DNA Region Required for Growth on 2,4,5-T by 'Pseudomonas cepacia' AC1100

摘要

A series of spontaneous 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) nonmetabolizing mutants of Pseudomonas cepacia AC1100 were characterized to be defective in either 2,4,5-T uptake or conversion of the compound to 2,4,5-trichlorophenol (2,4,5-TCP). Two of these mutants, RHC22 and RHC23, were complemented for growth on 2,4,5-T using an AC1100 genomic library constructed in the cosmid vector pCP13. Recombinant cosmids isolated from the complemented mutants contained a 27.5-kb insert which frequently underwent various-sized deletions in Escherichia coli. Hybridization studies showed the DNA to be of chromosomal origin and totally deleted in RHC22, RHC23 and other similar mutants. Complementation analyses of RHC22 with a series of subcloned fragments and spontaneously deleted derivatives of the recombinant cosmid pRHC21 showed the 2,4,5-T(tft) genes to occur within an 8.9-kb region. Pseudomonas aeruginosa cells transformed with the DNA acquired the ability to convert 2,4,5-T to 2,4,5-TCP. The genetic determinant for this function was further localized within a 3.7-kb region. The DNA, in the absence of other sequences from the 8.9-kb tft gene region allowed RHC22 cells to metabolize 2,4,5-T, but at low rates which were insufficient to support growth. (Copyright (c) 1991 Elsevier Science Publishers B.V.)