Degradation of 2,4,6-trichlorophenol via chlorohydroxyquinol inRalstonia eutropha JMP134 and JMP222

Padilla, Leandro; Matus, Valeria; Zenteno, Pamela; González, Bernardo

doi:10.1002/1521-4028(200008)40:4<243::aid-jobm243>3.0.co;2-d

Cited by 41 publications

(38 citation statements)

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“…The peaks at m/z 182 (M ϩ 4), 180 (M ϩ 2), and 178 (M ϩ ) and their relative intensities are characteristic of a molecule containing two chlorine atoms. The relative intensities are consistent with the natural abundance of 76% for 35 Cl and 24% for 37 Cl. The mass spectrum of the 10.65-min peak (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The reported cell yields for JMP134 and JMP222 growing on only 2,4,6-TCP are extremely low, and the culture's turbidity at 660 nm reaches a maximum of 0.043 after 5 days of incubation (10). The detection of 2,6-dichloro-p-hydroquinone (2,6-DiCH) during 2,4,6-TCP degradation by JMP134 whole cells and the identification of 6-chlorohydroxyquinol (6-CHQ) 1,2-dioxygenase activities in JMP134 cell extracts have led to a hypothesis that 2,4,6-TCP is degraded via 2,6-DiCH to 6-CHQ, which is subject to ring cleavage to 2-chloromaleylacetate (2-CMA) (37). However, the enzymes that convert 2,4,6-TCP to 2,6-DiCH and then to 6-CHQ are not detectable in the cell extracts (37).…”

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confidence: 99%

“…The detection of 2,6-dichloro-p-hydroquinone (2,6-DiCH) during 2,4,6-TCP degradation by JMP134 whole cells and the identification of 6-chlorohydroxyquinol (6-CHQ) 1,2-dioxygenase activities in JMP134 cell extracts have led to a hypothesis that 2,4,6-TCP is degraded via 2,6-DiCH to 6-CHQ, which is subject to ring cleavage to 2-chloromaleylacetate (2-CMA) (37). However, the enzymes that convert 2,4,6-TCP to 2,6-DiCH and then to 6-CHQ are not detectable in the cell extracts (37). Thus, the enzymes and genes responsible for the pathway have not been characterized.…”

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Genetic and Biochemical Characterization of a 2,4,6-Trichlorophenol Degradation Pathway in Ralstonia eutropha JMP134

2002

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Ralstonia eutropha JMP134 can grow on several chlorinated aromatic pollutants, including 2,4-dichlorophenoxyacetate and 2,4,6-trichlorophenol (2,4,6-TCP). Although a 2,4,6-TCP degradation pathway in JMP134 has been proposed, the enzymes and genes responsible for 2,4,6-TCP degradation have not been characterized. In this study, we found that 2,4,6-TCP degradation by JMP134 was inducible by 2,4,6-TCP and subject to catabolic repression by glutamate. We detected 2,4,6-TCP-degrading activities in JMP134 cell extracts. Our partial purification and initial characterization of the enzyme indicated that a reduced flavin adenine dinucleotide (FADH 2 )-utilizing monooxygenase converted 2,4,6-TCP to 6-chlorohydroxyquinol (6-CHQ). The finding directed us to PCR amplify a 3.2-kb fragment containing a gene cluster (tcpABC) from JMP134 by using primers designed from conserved regions of FADH 2 -utilizing monooxygenases and hydroxyquinol 1,2-dioxygenases. Sequence analysis indicated that tcpA, tcpB, and tcpC encoded an FADH 2 -utilizing monooxygenase, a probable flavin reductase, and a 6-CHQ 1,2-dioxygenase, respectively. The three genes were individually inactivated in JMP134. The tcpA mutant failed to degrade 2,4,6-TCP, while both tcpB and tcpC mutants degraded 2,4,6-TCP to an oxidized product of 6-CHQ. Insertional inactivation of tcpB may have led to a polar effect on downstream tcpC, and this probably resulted in the accumulation of the oxidized form of 6-CHQ. For further characterization, TcpA was produced, purified, and shown to transform 2,4,6-TCP to 6-CHQ when FADH 2 was supplied by an Escherichia coli flavin reductase. TcpC produced in E. coli oxidized 6-CHQ to 2-chloromaleylacetate. Thus, our data suggest that JMP134 transforms 2,4,6-TCP to 2-chloromaleylacetate by TcpA and TcpC. Sequence analysis suggests that tcpB may function as an FAD reductase, but experimental data did not support this hypothesis. The function of TcpB remains unknown.

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Genetic and Biochemical Characterization of a 2,4,6-Trichlorophenol Degradation Pathway in Ralstonia eutropha JMP134

2002

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“…They often are found in polluted sites where they contribute to degradation or containment of pollutants (Goris et al 2001;Hanada et al 1998). The possibility to use these bacteria in bioremediation of sites with organic or inorganic pollution or contamination is of current interest (Padilla et al 2000;Roux et al 2001). Survival in the presence of organic pesticides and copper would render oxstrain B advantageously compatible with agrochemicals used in chemical and biological control of plant diseases.…”

Section: Discussionmentioning

confidence: 99%

Oxalate-Degrading Bacteria Can ProtectArabidopsis thalianaand Crop Plants AgainstBotrytis cinerea

Schoonbeek¹,

Jacquat-Bovet²,

Mascher³

et al. 2007

MPMI

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Botrytis cinerea and Sclerotinia sclerotiorum secrete oxalic acid as a pathogenicity factor with a broad action. Consequently, it should be possible to interfere with the infection process by degrading oxalic acid during the interaction of these pathogens with their hosts. We have evaluated the potential of oxalate-degrading bacteria to protect plants against pathogenic fungi. Such bacteria were isolated from agricultural soil and selected on agar plates with Ca-oxalate as the sole carbon source. Four strains were retained with a medium-to-strong protective activity on Arabidopsis thaliana leaves against B. cinerea and S. sclerotiorum. They can provide 30 to 70% protection against fungal infection in different pathosystems, including B. cinerea on A. thaliana, cucumber, grapevine, and tomato. The oxalatedegrading bacteria induced only some marker genes for common plant signaling pathways for defenses, but protective effects were slightly reduced in A. thaliana mutants impaired in the ethylene and jasmonic acid signaling pathways. More detailed studies on the protective mechanism were performed in ox-strain B, identified as Cupriavidus campinensis, by analysis of transposon-tagged mutants that have a reduced ability to degrade oxalic acid.

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“…"Maleylacetate" is an intermediate not only in the degradation of chlorocatechols via the chlorocatechol ortho-cleavage pathway but also in the degradation of more highly substituted aromatics, such as 2,4,5-trichlorophenoxyacetate (1, 7), 2,4,6-trichlorophenol (16,20), pentachlorophenol (19,33), and lindane (18), even though different reaction sequences yielding maleylacetate have been proposed. Moreover, "maleylacetate" is assumed to be an intermediate in the degradation of resorcinolic compounds (5).…”

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Monitoring Key Reactions in Degradation of Chloroaromatics by In Situ ¹ H Nuclear Magnetic Resonance: Solution Structures of Metabolites Formed from cis -Dienelactone

et al. 2002

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A 1 H nuclear magnetic resonance ( 1 H NMR) assay was used to study the enzymatic transformation of cis-dienelactone, a central intermediate in the degradation of chloroaromatics. It was shown that the product of the cis-dienelactone hydrolase reaction is maleylacetate, in which there is no evidence for the formation of 3-hydroxymuconate. Under acidic conditions, the product structure was 4-carboxymethyl-4-hydroxybut-2-en-4-olide. Maleylacetate was transformed by maleylacetate reductase into 3-oxoadipate, a reaction competing with spontaneous decarboxylation into cis-acetylacrylate. One-dimensional 1 H NMR in 1 H 2 O could thus be shown to be an excellent noninvasive tool for monitoring enzyme activities and assessing the solution structure of substrates and products.A major route for mineralization of chloroaromatic compounds by microorganisms is their transformation into chlorocatechols and their further metabolism by enzymes of the chlorocatechol pathway (24). In this metabolic pathway, chlorocatechols are subject to intradiol cleavage to form the respective chloromuconates, which are converted by chloromuconate cycloisomerases into cis-or trans-dienelactone. The dienelactones undergo hydrolysis by dienelactone hydrolase. The hydrolysis product formed during the metabolism of 4-chlorocatechol was tentatively identified as "maleylacetate" based on its absorption characteristics ( max ϭ 243 nm in aqueous alkali) by Evans et al. (11). Similarly, Tiedje et al. (31) postulated maleylacetate and chloromaleylacetate as products formed during the metabolism of 4-chloro-and 3,5-dichlorocatechol, respectively. Those authors noted, however, that UV absorption was essentially quenched upon acidification, a behavior resembling keto-enol tautomerism, raising the question of the actual solution structure of maleylacetate. More recently, Seibert et al. claimed that the enol form (3-hydroxy-2,4-hexadienedioate, 3-hydroxymuconate) is thermodynamically favored under physiological conditions and exhibits an absorption maximum at 243 nm (28). The disappearance of this absorption under acidic conditions was believed to be due to the presence of the keto form, 3-oxo-cis-4-hexenedioate (maleylacetate in the strict sense), under these conditions. Despite the authors' assumption that 3-hydroxymuconate was the actual substrate of the purified reductase, the enzyme was termed "maleylacetate" reductase. Later, Prucha et al. (23) showed that the hydrolysis product of 3-methyldienelactone, supposedly 3-methylmaleylacetate or 3-hydroxy-4-methylmuconate, has a cyclic structure under acidic conditions (4-carboxymethylene-4-hydroxy-3-methylbut-2-en-4-olide, 4-hydroxy-3-methylmuconolactone), while no indication of the configuration under physiological conditions was given. Thus, although the structure of the decarboxylation product has been published (27), the actual solution structure of "maleylacetate" remained uncertain, presumably due to its reportedly high instability."Maleylacetate" is an intermediate not only in the degradation of chlo...

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Degradation of 2,4,6-trichlorophenol via chlorohydroxyquinol inRalstonia eutropha JMP134 and JMP222

Cited by 41 publications

References 25 publications

Genetic and Biochemical Characterization of a 2,4,6-Trichlorophenol Degradation Pathway in Ralstonia eutropha JMP134

Genetic and Biochemical Characterization of a 2,4,6-Trichlorophenol Degradation Pathway in Ralstonia eutropha JMP134

Oxalate-Degrading Bacteria Can ProtectArabidopsis thalianaand Crop Plants AgainstBotrytis cinerea

Monitoring Key Reactions in Degradation of Chloroaromatics by In Situ ¹ H Nuclear Magnetic Resonance: Solution Structures of Metabolites Formed from cis -Dienelactone

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Degradation of 2,4,6-trichlorophenol via chlorohydroxyquinol inRalstonia eutropha JMP134 and JMP222

Cited by 41 publications

References 25 publications

Genetic and Biochemical Characterization of a 2,4,6-Trichlorophenol Degradation Pathway in Ralstonia eutropha JMP134

Genetic and Biochemical Characterization of a 2,4,6-Trichlorophenol Degradation Pathway in Ralstonia eutropha JMP134

Oxalate-Degrading Bacteria Can ProtectArabidopsis thalianaand Crop Plants AgainstBotrytis cinerea

Monitoring Key Reactions in Degradation of Chloroaromatics by In Situ 1 H Nuclear Magnetic Resonance: Solution Structures of Metabolites Formed from cis -Dienelactone

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Monitoring Key Reactions in Degradation of Chloroaromatics by In Situ ¹ H Nuclear Magnetic Resonance: Solution Structures of Metabolites Formed from cis -Dienelactone