Characterization of a gene cluster involved in 4-chlorocatechol degradation by Pseudomonas reinekei MT1

Abstract

Pseudomonas reinekei MT1 has previously been reported to degrade 4- and 5-chlorosalicylate by a pathway with 4-chlorocatechol, 3-chloromuconate, 4-chloromuconolactone, and maleylacetate as intermediates, and a gene cluster channeling various salicylates into an intradiol cleavage route has been reported. We now report that during growth on 5-chlorosalicylate, besides a novel (chloro)catechol 1,2-dioxygenase, C12O(ccaA), a novel (chloro)muconate cycloisomerase, MCI(ccaB), which showed features not yet reported, was induced. This cycloisomerase, which was practically inactive with muconate, evolved for the turnover of 3-substituted muconates and transforms 3-chloromuconate into equal amounts of cis-dienelactone and protoanemonin, suggesting that it is a functional intermediate between chloromuconate cycloisomerases and muconate cycloisomerases. The corresponding genes, ccaA (C12O(ccaA)) and ccaB (MCI(ccaB)), were located in a 5.1-kb genomic region clustered with genes encoding trans-dienelactone hydrolase (ccaC) and maleylacetate reductase (ccaD) and a putative regulatory gene, ccaR, homologous to regulators of the IclR-type family. Thus, this region includes genes sufficient to enable MT1 to transform 4-chlorocatechol to 3-oxoadipate. Phylogenetic analysis showed that C12O(ccaA) and MCI(ccaB) are only distantly related to previously described catechol 1,2-dioxygenases and muconate cycloisomerases. Kinetic analysis indicated that MCI(ccaB) and the previously identified C12O(salD), rather than C12O(ccaA), are crucial for 5-chlorosalicylate degradation. Thus, MT1 uses enzymes encoded by a completely novel gene cluster for degradation of chlorosalicylates, which, together with a gene cluster encoding enzymes for channeling salicylates into the ortho-cleavage pathway, form an effective pathway for 4- and 5-chlorosalicylate mineralization.

FIG. 1.

Ratio of maleylacetate and protoanemonin formed from 3-chloromuconate by mixtures of MCI_ccaB (8.8 nM) with various amounts of trans-DLH (0 to 88 nM) of P. reinekei MT1. The reaction mixtures contained 50 mM Tris-HCl, 2 mM MnCl₂, pH 7.5, and 120 μM 3-chloromuconate. Substrate and product concentrations were analyzed by HPLC.

FIG. 2.

Gene organization of a 5,129-bp region from P. reinekei MT1 containing the cca gene cluster. The arrows indicate gene orientations: ccaA, C12O gene; ccaB, MCI gene; ccaC, trans-DLH gene; ccaD, putative MAR gene; and ccaR, putative transcriptional regulator gene. The encoded enzymes are given below the gene clusters.

FIG. 3.

Dendrograms showing the relatedness of intradiol dioxygenases (A) and MCIs (B). The evolutionary history was inferred with MEGA4 (59) using the neighbor-joining algorithm with p-distance correction and pairwise deletion of gaps and missing data. A total of 100 bootstrap replications were performed to test for branch robustness. The scale bars indicate amino acid differences per site.

FIG. 4.

Relative expression levels of catabolic genes in salicylate- and 5-chlorosalicylate-grown cells of P. reinekei MT1 as determined by quantitative RT-PCR. The values represent n-fold change (mean of triplicate samples) in the ratio of gene expression between the target gene and the reference gene (rpsL) compared to expression under noninducing conditions (for acetate-grown cells, this ratio was set at 1). The error bars indicate standard deviations.

FIG. 5.

Metabolism of 5-chlorosalicylate (A) or 4-methylsalicylate (B) by P. reinekei MT1. The kinetic constants of SalOH, C12O_salD, C12O_ccaA, MCI_salC, and MCI_ccaB are indicated. The specific activity (U/g protein) was determined in cell extracts, and the contribution of each of the (chloro)catechol 1,2-dioxygenases or (chloro)muconate cycloisomerases to the total activity against 0.1 mM 4-chlorocatechol or 0.1 mM 3-chloromuconate (A) or against 0.1 mM 4-methylcatechol or 0.1 mM 3-methylmuconate (B) in 5-chlorosalicylate-grown (gray) or 4-methylsalicylate-grown (boxed) cells was calculated after enzyme partial purification (given in percent and U/g protein). The enzyme concentrations (μmol/g protein) in the cell extracts were calculated based on the kinetic parameters of the enzyme of interest. The contributions of isoenzymes to the total metabolic flux of 0.1 mM 5-chlorosalicylate or 4-methylsalicylate by 5-chlorosalicylate-grown (gray) or 4-methylsalicylate-grown (boxed) cells were calculated by MATLAB and are given in percentages in the arrows.

FIG. 6.

Degradation of 5-chlorosalicylate by P. reinekei MT1. Designations of gene products are given below the reaction steps.