Chlorophenol hydroxylases encoded by plasmid pJP4 differentially contribute to chlorophenoxyacetic acid degradation

Abstract

Phenoxyalkanoic compounds are used worldwide as herbicides. Cupriavidus necator JMP134(pJP4) catabolizes 2,4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2-methylphenoxyacetate (MCPA), using tfd functions carried on plasmid pJP4. TfdA cleaves the ether bonds of these herbicides to produce 2,4-dichlorophenol (2,4-DCP) and 4-chloro-2-methylphenol (MCP), respectively. These intermediates can be degraded by two chlorophenol hydroxylases encoded by the tfdB(I) and tfdB(II) genes to produce the respective chlorocatechols. We studied the specific contribution of each of the TfdB enzymes to the 2,4-D/MCPA degradation pathway. To accomplish this, the tfdB(I) and tfdB(II) genes were independently inactivated, and growth on each chlorophenoxyacetate and total chlorophenol hydroxylase activity were measured for the mutant strains. The phenotype of these mutants shows that both TfdB enzymes are used for growth on 2,4-D or MCPA but that TfdB(I) contributes to a significantly higher extent than TfdB(II). Both enzymes showed similar specificity profiles, with 2,4-DCP, MCP, and 4-chlorophenol being the best substrates. An accumulation of chlorophenol was found to inhibit chlorophenoxyacetate degradation, and inactivation of the tfdB genes enhanced the toxic effect of 2,4-DCP on C. necator cells. Furthermore, increased chlorophenol production by overexpression of TfdA also had a negative effect on 2,4-D degradation by C. necator JMP134 and by a different host, Burkholderia xenovorans LB400, harboring plasmid pJP4. The results of this work indicate that codification and expression of the two tfdB genes in pJP4 are important to avoid toxic accumulations of chlorophenols during phenoxyacetic acid degradation and that a balance between chlorophenol-producing and chlorophenol-consuming reactions is necessary for growth on these compounds.

FIG. 1.

Genes involved in 2,4-D and MCPA degradation. (A) Chlorocatechol-producing peripheral reactions for 2,4-D and MCPA are carried out by enzymes encoded by the tfdA and tfdB genes in plasmid pJP4. (B) Chlorocatechol 1,2-dioxygenase (TfdC), chloromuconate cycloisomerase (TfdD), dienelactone hydrolase (TfdE), and maleylacetate reductase (TfdF) catalyze the conversion of chlorocatechols to chloromuconate, cis-dienelactone, maleylacetate, and β-ketoadipate, respectively. The arrow thicknesses indicate the relative specific activities of the enzymes encoded by each module (28, 30). (C) Organization of tfd genes in pJP4, including the tfdC_ID_IE_IF_IB_I and tfdD_IIC_IIE_IIF_IIB_II gene clusters. The diagram is not drawn to scale.

FIG. 2.

Growth of C. necator tfdB_I and tfdB_II mutants on chlorophenoxyacetates is differentially reduced. (A) Growth curves of strain JMP222(pJP4) (circles), the tfdB_I mutant (squares), and the tfdB_II mutant (triangles) on 2 mM 2,4-D (left) or MCPA (right). Representative OD₆₀₀ values are shown for at least three independent experiments. (B) Growth of C. necator derivatives with different 2,4-D or MCPA concentrations. Data for the tfdB_I mutant (pMLB1) constitutively expressing tfdB_I are included. OD₆₀₀ values were measured at stationary phase for all strains. Values shown are means of three independent experiments.

FIG. 3.

Intermediate accumulation during chlorophenoxyacetate degradation by C. necator JMP134 derivatives. Chlorinated intermediates were detected by HPLC, using samples of supernatants after incubation of preinduced cell suspensions (OD₆₀₀, 1.6) of strain JMP222(pJP4), the tfdB_I mutant, and the tfdB_II mutant with 1 mM 2,4-D (A to C) or MCPA (D to F). (G) Degradation of 2,4-D by strain JMP222(pJP4), as in panel A. 2,4-DCP (0.5 mM) was added to the cells after 20 min of incubation (+DCP). The discontinuous lines represent 2,4-D/2,4-DCP degradation in the absence of externally added 2,4-DCP. Symbols: diamonds, 2,4-D/MCPA; squares, 2,4-DCP/MCP; triangles, 2,4-dichloromuconate (2,4-DCM)/2-methyl-4-chloromuconate (MCM).

FIG. 4.

Effect of 2,4-DCP on cell viability of C. necator JMP134 derivatives. Cell suspensions of strain JMP222(pJP4) (triangles), the tfdB_I mutant (circles), or the tfdB_II mutant (squares) previously grown in 5 mM fructose were exposed to 1 mM (left) or 2 mM (right) 2,4-DCP, and samples were analyzed to determine the CFU at different times. Data for nonexposed tfdB_I mutant cells (open squares) are included in each panel as a control of viability during the time of the assay. The values are averages based on two replicates.