Mutation analysis of the different tfd genes for degradation of chloroaromatic compounds in Ralstonia eutropha JMP134

Laemmli, Caroline; Werlen, Christoph; Meer, Jan Roelof van der

doi:10.1007/s00203-003-0634-4

Cited by 16 publications

(20 citation statements)

References 23 publications

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“…Except for chloromuconate cycloisomerases, no great differences in substrate specificity between the enzymes encoded by each tfd module have been found, although it has been shown that the maintenance of both modules in pJP4 results in a higher expression of key catabolic activities that prevents the accumulation of toxic chlorocatechols (28,30). An inactivation analysis of tfd functions was recently reported (19), suggesting that the tfdD II C II E II F II B II genes are not essential for growth on 2,4-D but are relevant for growth on MCPA. Since no further information was given on intermediate accumulation during the degradation of chlorophenoxyacetates by these mutants, and since gene complementation was not reported, it is unclear if some of these phenotypes could be derived from polar effects on the chlorophenol hydroxylase genes.…”

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confidence: 99%

Chlorophenol Hydroxylases Encoded by Plasmid pJP4 Differentially Contribute to Chlorophenoxyacetic Acid Degradation

Ledger

Pieper²,

González

2006

Appl Environ Microbiol

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Phenoxyalkanoic compounds are used worldwide as herbicides. Cupriavidus necator JMP134(pJP4) catabolizes 2,4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2-methylphenoxyacetate (MCPA), using tfd functions carried on plasmid pJP4. TfdA cleaves the ether bonds of these herbicides to produce 2,4-dichlorophenol (2,4-DCP) and 4-chloro-2-methylphenol (MCP), respectively. These intermediates can be degraded by two chlorophenol hydroxylases encoded by the tfdB I and tfdB II genes to produce the respective chlorocatechols. We studied the specific contribution of each of the TfdB enzymes to the 2,4-D/MCPA degradation pathway. To accomplish this, the tfdB I and tfdB II genes were independently inactivated, and growth on each chlorophenoxyacetate and total chlorophenol hydroxylase activity were measured for the mutant strains. The phenotype of these mutants shows that both TfdB enzymes are used for growth on 2,4-D or MCPA but that TfdB I contributes to a significantly higher extent than TfdB II . Both enzymes showed similar specificity profiles, with 2,4-DCP, MCP, and 4-chlorophenol being the best substrates. An accumulation of chlorophenol was found to inhibit chlorophenoxyacetate degradation, and inactivation of the tfdB genes enhanced the toxic effect of 2,4-DCP on C. necator cells. Furthermore, increased chlorophenol production by overexpression of TfdA also had a negative effect on 2,4-D degradation by C. necator JMP134 and by a different host, Burkholderia xenovorans LB400, harboring plasmid pJP4. The results of this work indicate that codification and expression of the two tfdB genes in pJP4 are important to avoid toxic accumulations of chlorophenols during phenoxyacetic acid degradation and that a balance between chlorophenol-producing and chlorophenol-consuming reactions is necessary for growth on these compounds.

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Chlorophenol Hydroxylases Encoded by Plasmid pJP4 Differentially Contribute to Chlorophenoxyacetic Acid Degradation

Ledger

Pieper²,

González

2006

Appl Environ Microbiol

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“…The best-studied 2,4-D degradation genes (located in a chromosome or a plasmid) are tfd-like (pJP4-like). The very recently sequenced 87,688-bp plasmid pJP4 (48) from Wautersia eutropha JMP134 (formerly Ralstonia eutropha) was originally isolated in Australia (8), and its tfd genes and the corresponding enzymes responsible for converting 2,4-D to 3-oxoadipate are well characterized (22,23,25,26,35,58). Besides pJP4, there are only two cases in which the DNA regions containing tfd genes for the whole 2,4-D degradation pathway have been sequenced, a chromosomal transposon-like structure (about 30 kb) from Delftia acidovorans P4a (15) and Tn5530 (41 kb) located in plasmid pIJB1 from Burkholderia cepacia 2a (36,56).…”

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confidence: 99%

The Completely Sequenced Plasmid pEST4011 Contains a Novel IncP1 Backbone and a Catabolic Transposon Harboring tfd Genes for 2,4-Dichlorophenoxyacetic Acid Degradation

2004

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The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium Achromobacter xylosoxidans subsp. denitrificans strain EST4002 contains plasmid pEST4011. This plasmid ensures its host a stable 2,4-D ؉ phenotype. We determined the complete 76,958-bp nucleotide sequence of pEST4011. This plasmid is a deletion and duplication derivative of pD2M4, the 95-kb highly unstable laboratory ancestor of pEST4011, and was self-generated during different laboratory manipulations performed to increase the stability of the 2,4-D ؉ phenotype of the original strain, strain D2M4(pD2M4). The 47,935-bp catabolic region of pEST4011 forms a transposon-like structure with identical copies of the hybrid insertion element IS1071::IS1471 at the two ends. The catabolic regions of pEST4011 and pJP4, the best-studied 2,4-D-degradative plasmid, both contain homologous, tfd-like genes for complete 2,4-D degradation, but they have little sequence similarity other than that. The backbone genes of pEST4011 are most similar to the corresponding genes of broad-host-range self-transmissible IncP1 plasmids. The backbones of the other three IncP1 catabolic plasmids that have been sequenced (the 2,4-D-degradative plasmid pJP4, the haloacetate-catabolic plasmid pUO1, and the atrazinecatabolic plasmid pADP-1) are nearly identical to the backbone of R751, the archetype plasmid of the IncP1 ␤ subgroup. We show that despite the overall similarity in plasmid organization, the pEST4011 backbone is sufficiently different (51 to 86% amino acid sequence identity between individual backbone genes) from the backbones of members of the three IncP1 subgroups (the ␣, ␤, and ␥ subgroups) that it belongs to a new IncP1subgroup, the ␦ subgroup. This conclusion was also supported by a phylogenetic analysis of the trfA2, korA, and traG gene products of different IncP1 plasmids.Microbial degradation of 2,4-dichlorophenoxyacetic acid (2,4-D), a xenobiotic herbicide used worldwide for almost 60 years, is a well-studied process. Various soil bacteria can use 2,4-D as a carbon and energy source. Therefore, this compound has become a model for studying the evolution and distribution of genes for the degradation of chloroaromatic compounds. A number of bacterial strains belonging to different phylogenetic groups able to mineralize this compound have been found to possess genetically and enzymatically different 2,4-D-catabolic pathways (9,10,(16)(17)(18). The best-studied 2,4-D degradation genes (located in a chromosome or a plasmid) are tfd-like (pJP4-like). The very recently sequenced 87,688-bp plasmid pJP4 (48) from Wautersia eutropha JMP134 (formerly Ralstonia eutropha) was originally isolated in Australia (8), and its tfd genes and the corresponding enzymes responsible for converting 2,4-D to 3-oxoadipate are well characterized (22,23,25,26,35,58). Besides pJP4, there are only two cases in which the DNA regions containing tfd genes for the whole 2,4-D degradation pathway have been sequenced, a chromosomal transposon-like structure (about 30 kb) from Delftia acidovor...

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“…The analysis of the three benzoate pathways in LB400 is a case study of functional redundancy, a recurring theme in studies of LB400 (30) and other large-genome environmental isolates (22,24,34). Where previous studies (12,13) suggested the involvement of the ben-cat, box C , and box M pathways in benzoate catabolism in LB400, the currently reported data provide definitive evidence that each pathway assimilates benzoate.…”

Section: Discussionmentioning

confidence: 78%

“…strain RHA1 (ϳ9.7 Mbp) have shown the presence of multiple pathways that catabolize phthalate, terephthalate, and ethylbenzene (22,34). In Ralstonia eutropha JMP134 (ϳ7.3 Mbp), redundant tfd operons involved in 2,4-dichlorophenoxyacetic acid degradation provide more-efficient degradation of 2-methyl-4-chlorophenoxyacetic acid (24), suggesting that the pathway redundancy confers a selective metabolic advantage.…”

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confidence: 99%

Genetic and Genomic Insights into the Role of Benzoate-Catabolic Pathway Redundancy in Burkholderia xenovorans LB400

Denef

Klappenbach

Patrauchan

et al. 2006

Appl Environ Microbiol

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Transcriptomic and proteomic analyses of Burkholderia xenovorans LB400, a potent polychlorinated biphenyl (PCB) degrader, have implicated growth substrate-and phase-dependent expression of three benzoate-catabolizing pathways: a catechol ortho cleavage (ben-cat) pathway and two benzoyl-coenzyme A pathways, encoded by gene clusters on the large chromosome (box C ) and the megaplasmid (box M ). To elucidate the significance of this apparent redundancy, we constructed mutants with deletions of the ben-cat pathway (the ⌬benABCD::kan mutant), the box C pathway (the ⌬boxAB C ::kan mutant), and both pathways (the ⌬benABCD⌬ boxAB C ::kan mutant). All three mutants oxidized benzoate in resting-cell assays. However, the ⌬benABCD::kan and ⌬ben-ABCD ⌬boxAB C ::kan mutants grew at reduced rates on benzoate and displayed increased lag phases. By contrast, growth on succinate, on 4-hydroxybenzoate, and on biphenyl was unaffected. Microarray and proteomic analyses revealed that cells of the ⌬benABCD::kan mutant growing on benzoate expressed both box pathways. Overall, these results indicate that all three pathways catabolize benzoate. Deletion of benABCD abolished the ability of LB400 to grow using 3-chlorobenzoate. None of the benzoate pathways could degrade 2-or 4-chlorobenzoate, indicating that the pathway redundancy does not directly contribute to LB400's PCB-degrading capacities. Finally, an extensive sigmaE-regulated oxidative stress response not present in wild-type LB400 grown on benzoate was detected in these deletion mutants, supporting our earlier suggestion that the box pathways are preferentially active under reduced oxygen tension. Our data further substantiate the expansive network of tightly interconnected and complexly regulated aromatic degradation pathways in LB400.

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Mutation analysis of the different tfd genes for degradation of chloroaromatic compounds in Ralstonia eutropha JMP134

Cited by 16 publications

References 23 publications

Chlorophenol Hydroxylases Encoded by Plasmid pJP4 Differentially Contribute to Chlorophenoxyacetic Acid Degradation

Chlorophenol Hydroxylases Encoded by Plasmid pJP4 Differentially Contribute to Chlorophenoxyacetic Acid Degradation

The Completely Sequenced Plasmid pEST4011 Contains a Novel IncP1 Backbone and a Catabolic Transposon Harboring tfd Genes for 2,4-Dichlorophenoxyacetic Acid Degradation

Genetic and Genomic Insights into the Role of Benzoate-Catabolic Pathway Redundancy in Burkholderia xenovorans LB400

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