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.

Biochemical and Genetic Bases of Indole-3-Acetic Acid (Auxin Phytohormone) Degradation by the Plant-Growth-Promoting Rhizobacterium Paraburkholderia phytofirmans PsJN

“…The lag phase of strain PsJN for growth on IAA is also longer than that of P. putida 1290 (12), which may be explained by a tightly controlled iac gene induction to avoid formation of higher levels of toxic intermediates. In this context, accumulation of catechol, a toxic intermediate preventing bacterial growth (24)(25)(26), was reported by Scott et al (14) and also detected in this study (see below). Since the same concentrations of IAA (a 10-carbon-atom molecule) and Bz (a seven-carbon molecule) produce similar biomass yields (Fig.…”

Molecular and Population Analyses of a Recombination Event in the Catabolic Plasmid pJP4

“…5), indicating that strain JMP134(pJP4) is selected over strain JMP134(pJP4-FM), even though the latter harbors two copies of the tfdA gene. Gene dosage effects for growth on 2,4-D and MCPA have been recently demonstrated (16), which can be explained by the formation of the toxic compounds 2,4-dichlorophenol and 4-chloro-2-methylphenol as intermediates. The turnover of these chlorophenols depends on the activities of the TfdA and TfdB proteins (16,27).…”

“…Gene dosage effects for growth on 2,4-D and MCPA have been recently demonstrated (16), which can be explained by the formation of the toxic compounds 2,4-dichlorophenol and 4-chloro-2-methylphenol as intermediates. The turnover of these chlorophenols depends on the activities of the TfdA and TfdB proteins (16,27). The latter are encoded by the tfdB genes, which are located in the pJP4 plasmid and absent in pJP4-FM ( Fig.…”

“…This substituted phenol, like several other phenols, can then be transformed to a substituted catechol by the TfdBI and TfdBII proteins on plasmid pJP4. In the case of substitutions that are not halogens, such as in the case of 2-MPA, the compounds can be degraded by chromosomally encoded phenol hydroxylase functions and the chromosomal meta ring cleavage pathway encoded in C. necator JMP134 (16,27). Therefore, cells harboring pJP4-FM should have a selective advantage in culture media containing 2-MPA as the sole carbon and energy source.…”

“…Initial degradation of 2-MPA requires the tfdA gene, but subsequent catabolism needs functions that are encoded in the chromosome of the strain. In this case, the missing tfdB gene functions can be carried out by a chromosomally encoded phenol hydroxylase for nonhalogenated phenols (13,16) (Fig. 1B).…”