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

Abstract

Pseudomonas sp. strain P51 contains two gene clusters located on catabolic plasmid pP51 that encode the degradation of chlorinated benzenes. The nucleotide sequence of a 5,499-bp region containing the chlorocatechol-oxidative gene cluster tcbCDEF was determined. The sequence contained five large open reading frames, which were all colinear. The functionality of these open reading frames was studied with various Escherichia coli expression systems and by analysis of enzyme activities. The first gene, tcbC, encodes a 27.5-kDa protein with chlorocatechol 1,2-dioxygenase activity. The tcbC gene is followed by tcbD, which encodes cycloisomerase II (39.5 kDa); a large open reading frame (ORF3) with an unknown function; tcbE, which encodes hydrolase II (25.8 kDa); and tcbF, which encodes a putative trans-dienelactone isomerase (37.5 kDa). The tcbCDEF gene cluster showed strong DNA homology (between 57.6 and 72.1% identity) and an organization similar to that of other known plasmid-encoded operons for chlorocatechol metabolism, e.g., clkABD of Pseudomonas putida and tfdCDEF of Alcaligenes eutrophus JMP134. The identity between amino acid sequences of functionally related enzymes of the three operons varied between 50.6 and 75.7%, with the tcbCDEF and tfdCDEF pair being the least similar of the three. Measurements of the specific activities of chlorocatechol 1,2-dioxygenases encoded by tcbC, cicA, and tfdC suggested that a specialization among type II enzymes has taken place. TcbC preferentially converts 3,4-dichlorocatechol relative to other chlorinated catechols, whereas TfdC has a higher activity toward 3,5-dichlorocatechol. CIcA takes an intermediate position, with the highest activity level for 3-chlorocatechol and the second-highest level for 3,5-dichlorocatechol.Bacterial degradation of xenobiotic organic pollutants involves in many cases the use of altered metabolic functions (23) and can be regarded as a system of genetic adaptation to new substrates. As such, it presents a good model for studying evolution of enzymes and catabolic pathways. Of the vast array of synthetic compounds that have been introduced into the environment, chlorinated aromatic compounds are particularly refractory to bacterial degradation (11). In the past decade, various reports have described the degradation of chlorinated aromatic compounds (reviewed in reference 24). In many of these pathways chlorinated catechols are the central metabolites, whereas the only productive pathway for conversion of the chlorinated catechols was found to be the ortho-cleavage route (24). The ortho-cleavage pathway of chlorinated catechols was first described for Pseudomonas sp. strain B13 (7, 40), Pseudomonas putida (pAC27) (3), and Alcaligenes eutrophus JMP134(pJP4) (5). These bacterial strains were able to grow on 3-chlorobenzoate and, in the case of A. eutrophus, also on 2,4-dichlorophenoxyacetic acid as the sole carbon and energy source.

Characterization of a gene cluster encoding the maleylacetate reductase from Ralstonia eutropha 335T, an enzyme recruited for growth with 4-fluorobenzoate

“…Although the maleylacetate reductase gene, tfdF, of Ralstonia eutropha JMP134(pJP4) was initially misinterpreted as encoding a chlorodienelactone isomerase (Don et al, 1985), the chlorocatechol gene clusters of pJP4, pAC27, Pseudomonas sp. B13, and pP51 by DNA sequencing, by correspondence of experimentally determined N-terminal sequences to sequences predicted from DNA and by expression of cloned genes, were shown to contain maleylacetate reductase genes in addition to genes for chlorocatechol 1,2-dioxygenases, chloromuconate cycloisomerases and dienelactone hydrolases (Frantz & Chakrabarty, 1987;Perkins et al, 1990;Laemmli et al, 2000;van der Meer et al, 1991;Schell et al, 1994;Kasberg et al, 1995Kasberg et al, , 1997Seibert et al, 1993;Plumeier et al, 2002). Sequence similarities suggest that this is true also for the chlorocatechol gene clusters of various other plasmids or strains.…”

“…B13, and pP51 by DNA sequencing, by correspondence of experimentally determined N-terminal sequences to sequences predicted from DNA and by expression of cloned genes, were shown to contain maleylacetate reductase genes in addition to genes for chlorocatechol 1,2-dioxygenases, chloromuconate cycloisomerases and dienelactone hydrolases (Frantz & Chakrabarty, 1987;Perkins et al, 1990;Laemmli et al, 2000;van der Meer et al, 1991;Schell et al, 1994;Kasberg et al, 1995Kasberg et al, , 1997Seibert et al, 1993;Plumeier et al, 2002 During growth with 4-fluorobenzoate, R. eutropha 335 T and R. eutropha JMP222, a pJP4-free derivative of the 2,4-dichlorophenoxyacetate-utilizing strain JMP134, induce a maleylacetate reductase, but not the other enzymes typical for chlorocatechol catabolism (Schlömann et al, 1990a). Thus, these strains contain a presumably chromosomal maleylacetate reductase gene of still unknown metabolic affiliation.…”

Chlorocatechol 1,2‐Dioxygenase from Rhodococcus Erythropolis 1CP

“…Identities for painvise alignments of the Rhodococcus chlorocatechol 1,2-dioxygenase with the three other chlorocatechol 1,2-dioxygenases (Frantz and Chakrabarty, 1987;Perkins et al, 1990;van der Meer et al, 1991) and with three catechol 1,2-dioxygenases (Neidle et al, 1988;Kivisaar et al, 1991 ;Eck and Belter, 1993) ranged over 15 -22%. None of the compared intradiol dioxygenases appeared to be related to the enzyme from R. erythropolis 1 CP.…”

“…These enzymes differ in relative activity with various chlorinated catechols. DNA sequences of genes encoding chlorocatechol 1,2-dioxygenases from several Gram-negative strains are similar in spite of differences in their substrate specificity (Frantz and Chakrabarty, 1987;You, 1988, 1989;Perkitis et al, 1990;van der Meer et al, 1991;Miguez et al, 1993).…”

A linear megaplasmid, p1CP, carrying the genes for chlorocatechol catabolism of Rhodococcus opacus 1CP

“…In proteobacteria the genes of chlorocatechol pathways usually reside on plasmids (Frantz & Chakrabarty, 1987;Köiv et al, 1996;Perkins et al, 1990;van der Meer et al, 1991a) or other transferable genetic elements (van der Meer et al, 2001). In contrast, the genes for the degradation of numerous non-chlorinated aromatic compounds via the catechol and protocatechuate pathways in most strains investigated appear to be located on the chromosome (Doten et al, 1987;Holloway et al, 1994;Sauret-Ignazi et al, 1996;Zylstra et al, 1989).…”