Genetic characterization of 2,4,6-trichlorophenol degradation in Cupriavidus necator JMP134

Abstract

The degradation pathway of 2,4,6-trichlorophenol (2,4,6-TCP), a hazardous pollutant, in the aerobic bacterium Cupriavidus necator JMP134(pJP4) (formerly Ralstonia eutropha JMP134) is encoded by the tcp genes. These genes are located in a genetic context, tcpRXABCYD, which resembles a putative catabolic operon. In this work, these gene sequences were individually disrupted and mutant strains were evaluated for their ability to grow on or degrade 2,4,6-TCP. The tcpX and tcpA mutants completely failed to degrade this compound. Although the tcpC mutant was also unable to grow on 2,4,6-TCP, it still transformed this chlorophenol to 6-chlorohydroquinol. In contrast, the tcpD mutant grew on 2,4,6-TCP, suggesting the presence of additional maleylacetate reductase-encoding genes. Five other open reading frames encoding maleylacetate reductases, in addition to the tcpD gene, were found in the genome of C. necator, and two of them provide this function in the tcpD mutant. The tcpR gene, encoding a putative LysR-type transcriptional regulator, was disrupted, and this mutant strain completely failed to grow on 2,4,6-TCP. Transcriptional fusion studies demonstrated that TcpR activates the expression of the tcp genes, responding specifically to 2,4,6-TCP. The transcriptional start of the tcp operon was mapped, and a putative sigma(70)-type promoter was identified.

FIG. 1.

(A) Degradative pathway proposed for 2,4,6-TCP in C. necator JMP134. 2,4,6-TCP-MO, HQDO, and MAR catalyze the conversion of 2,4,6-trichlorophenol to 2,6-dichlorohydroquinone, 6-chlorohydroxyquinol, 2-chloromaleylacetate, maleylacetate, and β-ketoadipate, respectively. (B) Genetic context of the tcpRXABCYD gene cluster. The bar represents 1 kb. The numbers in circles correspond to the primers described in Table 2: 1, FAD2; 2, tcpR2; 3, tcpX1; 4, tcpA3; 5, tcpAend; 6, tcpC2; 7, tcpC1; 8, tcpY2; 9, tcpY1; 10, tcpD2; 11, tcpD1; and 12, TCPout. (C) Schematic representation of the organization of the tcp promoter region. The arrows indicate the starts of transcription and translation. The transcriptional initiation nucleotide (+1) and the putative −35 and −10 motifs are underlined.

FIG. 2.

Degradation of 2,4,6-TCP by tcpX and tcpA mutant strains of C. necator JMP134 and some of its derivatives. (A) Removal of 2,4,6-TCP was detected by high-performance liquid chromatography using samples of supernatants after incubation of preinduced cell suspensions (OD₆₀₀ of 1.0). ▪, strain JMP134; ▴, JMP134 tcpX; ▾, JMP134 tcpX (S7X) constitutively expressing the tcpX gene; ▵, JMP134 tcpA; ▿, JMP134 tcpA (S7A) constitutively expressing the tcpA gene. (B) Removal of 2,4,6-TCP by strains constitutively expressing the tcpX, tcpA, or tcpB genes. ▪, strain JMP134; ▴, JMP134 tcpR (S7XA) constitutively expressing the tcpXA genes; ▾, JMP134 tcpR (S7A) constitutively expressing the tcpA gene; ⋄, JMP134 tcpR (S7AB) constitutively expressing the tcpAB genes; •, JMP134 tcpR. The values are averages from three or four replicates, with standard deviations of less than 5%.

FIG. 3.

Growth yields obtained after growing C. necator JMP134 and some of its derivatives on different 2,4,6-TCP concentrations: C. necator JMP134 (X), JMP134 tcpR (▪), JMP134 tcpR (pPRTCP) (□), JMP134 tcpC (▴), JMP134 tcpC (pS7C) constitutively expressing tcpC (▵) JMP134 tcpD (•), and JMP134 tcpD (pS7D) constitutively expressing the tcpD gene (○). Values shown are means of three or four independent experiments, with standard deviations of less than 5%. An OD₆₀₀ of 1.0 corresponds to about 5 × 10⁸ CFU per ml.

FIG. 4.

Involvement of MARs in 2,4,6-TCP degradation. (A) Growth yields of C. necator JMP134 derivatives at different concentrations of 2,4,6-TCP. Strain JMP134 and mutant strains (JMP134 tfdFII, JMP134 ReutB4694, and JMP134 ReutC5982) were without difference with respect to the wild type (X), JMP134 tfdFI (▴), JMP134 tcpD (▪), and JMP134 ReutB4129 (○). Values shown are means of three or four independent experiments, with standard deviations of less than 5%. An OD₆₀₀ of 1.0 corresponds to about 5 × 10⁸ CFU ml⁻¹.

FIG. 5.

β-Galactosidase activities from tcpR-tcpX′-lacZ fusions with different aromatic compounds. C. necator JMP134 tcpR (pPRTCP) was grown until the OD₆₀₀ was 0.5 and exposed to 0.1 mM aromatic compounds for 3 h. Abbreviations: SUC, succinate; 2,4,6-TBP, 2,4,6-tribromophenol; PCP, pentachlorophenol; TCHBQ, tetrachlorohydroxibenzoquinone; 2,4,5-T, 2,4,5-trichlorophenoxyacetic acid; 2,4,5-TCP, 2,4,5-trichlorophenol; 2,4,6-TCB, 2,4,6-trichlorobenzoate; 2,4,6-TCA, 2,4,6-trichloroanisole; 2,3-DCP, 2,3-dichlorophenol; 4-CP, 4-chlorophenol; and 3-NP, 3-nitrophenol. The insert shows activity of the tcpR-tcpX′-lacZ fusion at different concentrations of 2,4,6-TCP. The values are averages for three independent tests conducted in duplicate.