Genetic rearrangements in plasmids specifying total degradation of chlorinated benzoic acids.

Abstract

Growth in a chemostat of the 3-chlorobenzoatepositive Pseudomonas putida cells harboring the plasmid pAC25, in presence of cells harboring the TOL plasmid, allows emergence of cells that can also utilize 4-chlorobenzoate (4Cba). Isolation of plasmid DNA from such cells demonstrate the deletion of a 11kb (Kilobase pair) EcoR1 fragment from the pAC25 plasmid; a portion of the TOL plasmid (41.5 kb TOL*) is also found to be transposed onto the chromosome of such cells. Further enrichment of the 4-chlorobenzoate-positive cells with 3,5-dichlorobenzoate (3,5-Dcb) as a sole carbon source has produced cells that can also slowly utilize 3,5-dichlorobenzoate. Isolation of plasmid DNA from such cells demonstrates the appearance of a second plasmid (pAC29). Restriction hybridization of pAC29 EcoRI fragments with pAC25 and TOL demonstrates that pAC29 is derived primarily by duplication of a segment of the pAC27 plasmid and a fragment from TOL, with further mutational divergence. Southern hybridization of the EcoRI-digested chromosomal DNA with 32P-labeled TOL, pTS11 and pTS71 plasmid DNAs demonstrates the presence of the TOL* transposon containing xylD, G, E and F genes in both 4Cba+ (pAC27+) and 3,5-DCb+ (pAC27+, pAC29+) cells. Isolation of plasmid DNA from 3,5-Dcb+ faster growing variants, obtained from slow-growing pAC27+ pAC29+ cells, demonstrates the presence of a single type of plasmid, with identical size and EcoRI digestion profile as pAC27. The implications of gene duplications and subsequent homologous recombination with regard to the biochemical pathway of 3,5-dichlorobenzoate degradation have been discussed.