Phenoxyacetic acid degradation by the 2,4-dichlorophenoxyacetic acid (TFD) pathway of plasmid pJP4: mapping and characterization of the TFD regulatory gene, tfdR

Abstract

Plasmid pJP4 enables Alcaligenes eutrophus JMP134 to degrade 3-chlorobenzoate and 2,4-dichlorophenoxyacetic acid (TFD). Plasmid pRO101 is a derivative of pJP4 obtained by insertion of Tn1721 into a nonessential region of pJP4. Plasmid pRO101 was transferred by conjugation to several Pseudomonas strains and to A. eutrophus AEO106, a cured isolate of JMP134. AEO106(pRO101) 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 PPO300(pRO101), grew on PAA as the sole carbon source in the absence of inducer. This isolate carried a mutant plasmid, designated pRO103, derived from pRO101 through the deletion of a 3.9-kilobase DNA fragment. Plasmid pRO103 constitutively expressed the TFD pathway, and this allowed the metabolism of PAA in the absence of the inducer, TFD. Complementation of pRO103 in trans by a DNA fragment corresponding to the fragment deleted in pRO101 indicates that a negative control-regulatory gene (tfdR) is located on the BamHI E fragment of pRO101. Other subcloning experiments resulted in the cloning of the tfdA monooxygenase gene on a 3.5-kilobase fragment derived from pRO101. This subclone, in the absence of other pRO101 DNA, constitutively expressed the tfdA gene and allowed PPO300 to grow on PAA. Preliminary evidence suggests that the monooxygenase activity encoded by this DNA fragment is feedback-inhibited by phenols.

Characterization of a new 2,4-dichlorophenoxyacetic acid degrading plasmid pEST4011: physical map and localization of catabolic genes

“…The size (70 kbp) and restriction pattern of the derived plasmid pEST4011 (Fig. 1) differed considerably from those of pJP4 previously reported in Alcaligenes eutrophus JMP134 (Don & Pemberton, 1985;Harker et al, 1989).…”

“…The 2,4-D degradative plasmid pJP4 from Alcaligenes eutrophus JMP134 (isolated in Australia) has become a model for the investigation of 2,4-D degradation (Don & Pemberton, 1985;Ghosal & You, 1988Harker et al, 1989;Perkins et al, 1990). A second plasmid, pRC 10, present in a 2,4-D-degrading Flavobacteriurn sp.…”

Organization and sequence analysis of the 2,4-dichlorophenol hydroxylase and dichlorocatechol oxidative operons of plasmid pJP4

“…similar to a proposed u70-binding site of TTGGAC-18 bp-CAATCCT for catB (2,9). We note this because constitutive expression of tfdC is repressed by tfdR (19). The c70-binding site from tfdCDEF is overlapped by the conserved region and represents a potential site of action for negative regulation of tfdC expression by TfdR.…”

“…To complete the sequence of the tfdB and tfdCDEF operons, a 6.2-kb HindIII-SstI fragment of pEP102 was sequenced and joined to that of the 1.5-kb HindIII fragment by sequencing of a 312-base-pair (bp) HindIII fragment from pRD25A. (11,19,30,38). The open arrow depicts the position and direction of transcription of the tfdCDEF operon; the solid arrow indicates the location and orientation of the tfdB operon.…”

Nucleotide sequence and initial functional characterization of the clcR gene encoding a LysR family activator of the clcABD chlorocatechol operon in Pseudomonas putida

“…There is evidence that TfdR and TfdS are encoded by identical open reading frames and are the result of a gene duplication (55). However, other reports suggest differing repressing and/or activating functions and differing sites of actions for TfdR and TfdS (23,27,28 (40) indicate that the tfdCDEF promoter region bears strong similarity to the known binding site for CatR at the catBC promoter and to the promoter region of clcABD (39), suggesting that a LysR family member, possibly TfdS, does regulate tfdCDEF.…”