Efficient turnover of chlorocatechols is essential for growth of Ralstonia eutropha JMP134(pJP4) in 3-chlorobenzoic acid

Abstract

Ralstonia eutropha JMP134(pJP4) degrades 3-chlorobenzoate (3-CB) by using two not completely isofunctional, pJP4-encoded chlorocatechol degradation gene clusters, tfdC(I)D(I)E(I)F(I) and tfdD(II)C(II)E(II)F(II). Introduction of several copies of each gene cluster into R. eutropha JMP222, which lacks pJP4 and thus accumulates chlorocatechols from 3-CB, allows the derivatives to grow in this substrate. However, JMP222 derivatives containing one chromosomal copy of each cluster did not grow in 3-CB. The failure to grow in 3-CB was the result of accumulation of chlorocatechols due to the limiting activity of chlorocatechol 1,2-dioxygenase (TfdC), the first enzyme in the chlorocatechol degradation pathway. Micromolar concentrations of 3- and 4-chlorocatechol inhibited the growth of strains JMP134 and JMP222 in benzoate, and cells of strain JMP222 exposed to 3 mM 3-CB exhibited a 2-order-of-magnitude decrease in viability. This toxicity effect was not observed with strain JMP222 harboring multiple copies of the tfdC(I) gene, and the derivative of strain JMP222 containing tfdC(I)D(I)E(I)F(I) plus multiple copies of the tfdC(I) gene could efficiently grow in 3-CB. In addition, tfdC(I) and tfdC(II) gene mutants of strain JMP134 exhibited no growth and impaired growth in 3-CB, respectively. The introduction into strain JMP134 of the xylS-xylXYZL genes, encoding a broad-substrate-range benzoate 1,2-dioxygenase system and thus increasing the transformation of 3-CB into chlorocatechols, resulted in derivatives that exhibited a sharp decrease in the ability to grow in 3-CB. These observations indicate that the dosage of chlorocatechol-transforming genes is critical for growth in 3-CB. This effect depends on a delicate balance between chlorocatechol-producing and chlorocatechol-consuming reactions.

FIG. 1.

Genes involved in 3-CB degradation. (a) Chlorocatechol-producing peripheral reactions for 3-CB encoded in the chromosome of R. eutropha (ben genes), and the 1,2-toluate dioxygenase system (xyl genes) from pWW0. (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 thickness indicates the relative specific activity of the enzymes encoded by each module for intermediates of 3-CB metabolism (27, 30). (c) Organization of tfd genes in pJP4, including the tfdC_ID_IE_IF_I and tfdD_IIC_IIE_IIF_II gene clusters. The diagram is not to scale.

FIG. 2.

Growth with different 3-CB concentrations of R. eutropha derivatives harboring different copy numbers of ortho ring cleavage pathway tfd gene modules. Symbols: ▪, R. eutropha JMP222(pBBR1M-I); □, R. eutropha JMP222(pBBR1M-II); ▴, R. eutropha JMP222::R1TFD; ▵, R. eutropha JMP222::R2TFD; •, R. eutropha JMP222::R1TFD(pBBRCI); ○, R. eutropha JMP222::R1TFD(pBBRPI). OD₆₀₀ was measured at the stationary phase. The values are means based on triplicate experiments. The deviations (not shown for clarity) were less than 5 to 10%.

FIG. 3.

Accumulation of chlorocatechols and 2-chloromuconate from 3-CB. Chlorinated intermediates were detected by HPLC by using samples of supernatants after incubation of 0.2 mM 3-CB with preinduced cell suspensions (OD₆₀₀, 0.5) of strains JMP222 (a), JMP222::R1TFD (b), JMP222::R1TFD(pBBRCI) (c), JMP134(pJP4) (d), and JMP134::X(pJP4) (e). Symbols: •, 3-CB; □, 3-CC plus 4-CC; ▴, 2-chloromuconate.

FIG. 4.

Effect of 3-CB on cell viability of R. eutropha JMP134 derivatives. Cell suspensions of strain JMP222 (a) or JMP222(pBBRC1) (b) previously grown in 5 mM benzoate were exposed (□) or not exposed (▪) to 1 mM 3-CB, and samples were analyzed to determine the number of CFU at different times. The values are averages based on two replicates.

FIG. 5.

Growth with different 3-CB concentrations of R. eutropha derivatives harboring xyl genes or inactivated tfdC genes. Symbols: ⧫, R. eutropha JMP134(pJP4); ◊, R. eutropha JMP134::X(pJP4); •, R. eutropha JMP134(pJP4-ΔtfdC_I); ∗, R. eutropha JMP134(pJP4-ΔtfdC_II); ▴, R. eutropha JMP134::X(pJP4-F3). OD₆₀₀ was measured at the stationary phase. The values are means based on triplicate experiments. The deviations (not shown for clarity) were less than 5 to 10%.

FIG. 6.

Imbalance between chlorocatechol-producing and chlorocatechol-consuming reactions results in accumulation of toxic metabolites and in an inability to grow in 3-CB. The relative amounts of chlorocatechols, indicated by the different sizes of molecule diagrams, and the growth phenotypes in 3-CB are shown for strains JMP134(pJP4) (a), JMP134::X(pJP4) (b), JMP222::R1TFD (c), and JMP222::R1TFD(pBBRCI) (d). The arrow thickness indicates the relative specific activity of TfdC proteins.