Cloning and characterization of a chromosomal DNA region required for growth on 2,4,5-T by Pseudomonas cepacia AC1100

Abstract

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 this 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 this 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 this 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. This 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. Copies of the insertions sequence element IS931 were identified either adjacent to or within this tft gene region in the genomes of two independent wild-type AC1100 isolates. Preliminary evidence suggests that these sequences either facilitate or are required for growth on 2,4,5-T and hence may be implicated in the genetic evolution of the 2,4,5-T metabolic pathway.