Plasmid pJP4 enables Alcaligenes eutrophus JMP134 to degrade 3-chlorobenzoate and 2,4-dichlorophenoxyacetic acid (TFD). Plasmid pR0101 is a derivative of pJP4 obtained by insertion of Tn1721 into a nonessential region of pJP4. Plasmid pR0101 was transferred by conjugation to several Pseudomonas strains and to A. eutrophus AEO106, a cured isolate of JMP134. AEO106(pR0101) and some Pseudomonas transconjugants grew on TFD. Transconjugants with a chromosomally encoded phenol hydroxylase also degraded phenoxyacetic acid (PAA) in the presence of an inducer of the TFD pathway, namely, TFD or 3-chlorobenzoate. A mutant of one such phenol-degrading strain, Pseudomonas putida PP0300(pR0101), grew on PAA as the sole carbon source in the absence of inducer. This isolate carried a mutant plasmid, designated pR-103, derived from pR0101 through the deletion of a 3.9-kilobase DNA fragment. Plasmid pR0103 constitutively expressed the TFD pathway, and this allowed the metabolism of PAA in the absence of the inducer, TFD. Complementation of pR0103 in trans by a DNA fragment corresponding to the fragment deleted in pR0101 indicates that a negative control-regulatory gene (tfdR) is located on the BamHI E fragment of pR0101. (Copyright (c) 1989, American Society for Microbiology.)