Efficient degradation of 2,4,6-Trichlorophenol requires a set of catabolic genes related to tcp genes from Ralstonia eutropha JMP134(pJP4)

Abstract

2,4,6-Trichlorophenol (2,4,6-TCP) is a hazardous pollutant. Several aerobic bacteria are known to degrade this compound. One of these, Ralstonia eutropha JMP134(pJP4), a well-known, versatile chloroaromatic compound degrader, is able to grow in 2,4,6-TCP by converting it to 2,6-dichlorohydroquinone, 6-chlorohydroxyquinol, 2-chloromaleylacetate, maleylacetate, and beta-ketoadipate. Three enzyme activities encoded by tcp genes, 2,4,6-TCP monooxygenase (tcpA), 6-chlorohydroxyquinol 1,2-dioxygenase (tcpC), and maleylacetate reductase (tcpD), are involved in this catabolic pathway. Here we provide evidence that all these tcp genes are clustered in the R. eutropha JMP134(pJP4) chromosome, forming the putative catabolic operon tcpRXABCYD. We studied the presence of tcp-like gene sequences in several other 2,4,6-TCP-degrading bacterial strains and found two types of strains. One type includes strains belonging to the Ralstonia genus and possessing a set of tcp-like genes, which efficiently degrade 2,4,6-TCP and therefore grow in liquid cultures containing this chlorophenol as a sole carbon source. The other type includes strains belonging to the genera Pseudomonas, Sphingomonas, or Sphingopixis, which do not have tcp-like gene sequences and degrade this pollutant less efficiently and which therefore grow only as small colonies on plates with 2,4,6-TCP. Other than strain JMP134, none of the bacterial strains whose genomes have been sequenced possesses a full set of tcp-like gene sequences.

FIG. 1.

Enzymes, intermediates, and genes involved in the degradation of 2,4,6-TCP in R. eutropha JMP134(pJP4). (a) Catabolic pathway for 2,4,6-TCP. (b) Genetic organization of tcp genes in R. eutropha JMP134(pJP4). MA, maleylacetate. tcpA (1,551 bp), tcpC (828 bp), and tcpD (1,065 bp): genes encoding TCP-MO, HQDO, and MAR, respectively. tcpR (969 bp); a putative LysR-type transcriptional activator regulatory gene. tcpB (594 bp), tcpX (465 bp), and tcpY (1,026 bp), ORFs of unknown function (see the text). Bar, 1 kb.

FIG. 2.

Comparison of the degradation of 2,4,6-TCP by different types of 2,4,6-TCP-degrading bacteria. Resting cells (OD₆₆₀ = 1; pyruvate grown and 2,4,6-TCP-induced) of R. eutropha MS1 (a and c), and P. tolaasii MS6 (b and d), chosen as representatives of each bacterial type, were incubated in the presence of 0.5 mM 2,4,6-TCP. Samples were taken at intervals, and the UV spectra of the cell-free fluid were obtained (a and b). Representative profiles from several independent determinations are shown. In addition, the concentration of 2,4,6-TCP in the culture fluid was determined by HPLC (c and d). Each point is the average of two replicates; standard deviations were too low to be depicted. Time points (hours) are indicated.

FIG. 3.

Detection of gene sequences involved in 2,4,6-TCP metabolism by PCR and Southern analysis. (a, c, and e) Gel images showing PCR products obtained with primer pairs for (a) TCP-MO (tcpA). (c) HQDO (tcpC), and (e) MAR (tcpD). (b, d, and f) Southern blot using the strain JMP222 tcpA probe (b), the strain MS1 tcpC probe (d), and the strain JMP222 tcpD probe (f). Total DNA from R. eutropha JMP134(pJP4) (lanes 1) R. eutropha JMP222 (lanes 2), R. eutropha MS1 (lanes 3), Ralstonia sp. strain PZK (lanes 4), S. chilensis S37 (lanes 5), S. paucimobilis MS2 (lanes 6), P. tolaasii MS6 (lanes 7), and P. putida MS7 (lanes 8) was digested with Clal. The left lane in panels a, c, and e shows 1-kb (a) or 100-bp (c and e) DNA standards. Arrows indicate fragment sizes for orientation.