Organization and sequence analysis of the 2,4-dichlorophenol hydroxylase and dichlorocatechol oxidative operons of plasmid pJP4

Abstract

Growth of Alcaligenes eutrophus JMP134 on 2,4-dichlorophenoxyacetate requires a 2,4-dichlorophenol hydroxylase encoded by gene tfdB. Catabolism of either 2,4-dichlorophenoxyacetate or 3-chlorobenzoate involves enzymes encoded by the chlorocatechol oxidative operon consisting of tfdCDEF, which converts 3-chloro-and 3,5-dichlorocatechol to maleylacetate and chloromaleylacetate, respectively. Transposon mutagenesis has localized tfdB and t.fdCDEF to EcoRI fragment B of plasmid pJP4 (R. H. Don, A. J. Wieghtman, H.-J. Knackmuss, and K. N. Timmis, J. Bacteriol. 161:85-90, 1985). We present the complete nucleotide sequence of tfdB and tfdCDEF contained within a 7,954-base-pair HindIII-SstI fragment from EcoRI fragment B. Sequence and expression analysis of tfdB in Escherichia coli suggested that 2,4-dichlorophenol hydroxylase consists of a single subunit of 65 kilodaltons. The amino acid sequences of proteins encoded by tfdD and tfdE were found to be 63 and 53% identical to those of functionally similar enzymes encoded by clcB and ckD, respectively, from plasmid pAC27 of Pseudomonas putida. P. putida(pAC27) can utilize 3-chlorocatechol but not dichlorinated catechols. A region of DNA adjacent to clcD in pAC27 was found to be 47% identical in amino acid sequence to tfdiF, a gene important in catabolizing dichlorocatechols. The region in pAC27 does not appear to encode a protein, suggesting that the absence of a functional trans-chlorodienelactone isomerase may prevent P. putida(pAC27) from utilizing 3,5-dichlorocatechol.Bacteria play a crucial role in the dissimilation of environmental pollutants. In general, the catabolism of aromatic compounds is channeled through catechol intermediates (33). Hence, the ability of an organism to form and catabolize catechols or their derivatives can play a major part in the utilization of an aromatic compound. Comparison of the degradative pathways of different aromatics may help in understanding the evolution of growth substrate specificity.Chlorinated aromatics, such as 2,4-dichlorophenoxyacetic acid (2,4D) and 3-chlorobenzoate (3CBA), are catabolized via a modified ortho pathway (11,12,20,28,34,35; for a review, see reference 33). Pseudomonas putida plasmid pAC27, a deletion derivative of pAC25, confers catabolism of 3CBA (5), whereas Alcaligenes eutrophus JMP134 plasmid pJP4 enables degradation of both 2,4D and 3CBA (10, 11). In both cases, 3CBA appears to be degraded to 3-chlorocatechol by enzymes encoded by chromosomal genes. 2,4D is catabolized by A. eutrophus JMP134 to 2,4-dichlorophenol (2,4DCP) by 2,4D monooxygenase. 2,4DCP is then hydroxylated by 2,4DCP hydroxylase to form 3,5-dichlorocatechol (11; Fig. 1).3-Chlorocatechol is oxidized to maleylacetic acid by enzymes encoded by the clcABD operon in pAC27 (13) and by the tfdCDEF operon of pJP4 (11). The maleylacetic acid is then catabolized by chromosomally encoded enzymes (21). Enzymes involved in this transformation are chlorocatechol 1,2-dioxygenase (catechol 1,2-dioxygenase II or pyrocatechase II; EC 1.13.11.1), encoded b...

Sequence analysis of the Pseudomonas sp. strain P51 tcb gene cluster, which encodes metabolism of chlorinated catechols: evidence for specialization of catechol 1,2-dioxygenases for chlorinated substrates

“…frameshift mutation in the ORF3 of the tcb operon did not verall patterns of relative activities were similar in cell affect the conversion of 3,4-DCC in E. coli cell extracts (38 experiment. P51 has a high relative activity on 3,4-dichlorocatechol In contrast to the clc operon (8,21), it appears that the tcb (Fig. 3B), we could not detect any trans-chlorodienelactone isomerase activity, which is ascribed to the tfdF gene product (5,21,32,38).…”

“…P51 has a high relative activity on 3,4-dichlorocatechol In contrast to the clc operon (8,21), it appears that the tcb (Fig. 3B), we could not detect any trans-chlorodienelactone isomerase activity, which is ascribed to the tfdF gene product (5,21,32,38). These suggestions, however, are not supported by biochemical studies of the tfd-encoded enzymes, which indicated that cis-2-chloro-4-carboxymethylenebut-2-en-4-olide is formed directly from 2,4-dichlorocis,cis-muconate by the activity of chloromuconate cycloisomerase (14,22,28).…”

Identification and characterization of genes encoding polycyclic aromatic hydrocarbon dioxygenase and polycyclic aromatic hydrocarbon dihydrodiol dehydrogenase in Pseudomonas putida OUS82

“…2). The pahB gene was thought to flank the pahA gene because genes that encode catabolic enzymes are often clustered (13,19,22). We sequenced the nucleotides of a 6-kb region between the EcoRV and Sacl sites (Fig.…”

Flow Injection Biosensor System for 2,4-Dichlorophenoxyacetate Based on a Microbial Reactor and Tyrosinase-modified Electrode

“…Most of the 2,4-D-degrading bacteria isolated from human-disturbed sites include copiotrophic and fast-growing genera in the and subdivisions of Proteobacteria, which has been classified as class I 2,4-D degraders. The degradation pathway of 2,4-D has been extensively characterized with Ralstonia eutrophus JMP134, one of the class I degraders (Don et al, 1985;Streber et al, 1987;Perkins et al, 1990). Degradation of 2,4-D in this class of degraders is considered to be initiated by cleavage of the ether linkage to yield 2,4-dichlorophenol (2,4-DCP), which is then hydroxylated to 3,5-dichlorocatechol (3,5-DCC), followed by ring cleavage (Fig.…”