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PubMed2026

Microbial aerobic degradation of 4-isopropylnitrobenzene by Sphingobium yanoikuyae strain SG1.

4-Isopropylnitrobenzene (4-IPNB) is a nitroaromatic compound commonly employed as an intermediate in pesticide synthesis and chemical manufacturing. Despite its potential environmental persistence and ecological risks, the microbial degradation pathway of 4-IPNB remains largely unknown. In this study, a Gram-negative bacterium, designated Sphingobium yanoikuyae strain SG1, was isolated from a pesticide manufacturing site in Brazil for its ability to utilize 4-IPNB as the sole added source of carbon, nitrogen, and energy. Aerobic degradation of 4-IPNB by strain SG1 was accompanied by nitrite release, and intermediate-trapping experiments revealed the transient accumulation of 4-isopropylcatechol (4-IPC). Together, these findings support the initial conversion of 4-IPNB to 4-IPC through oxidative denitration and dihydroxylation. Genomic and transcriptomic analyses further inferred several candidate nitroarene dioxygenases that may catalyze this initial reaction. Furthermore, the downstream metabolism of 4-IPC proceeded via both meta- and ortho-cleavage pathways, with cell-extract enzyme assays demonstrating predominant meta-cleavage activity under the tested conditions. Strain SG1 also degraded 4-IPNB in nonsterile soil slurry microcosms, extending its degradation capability beyond defined liquid culture. This study provides the first comprehensive insight into the microbial aerobic degradation of 4-IPNB, advances our understanding of the environmental fate of emerging nitroaromatic contaminants, and supports the potential of strain SG1 in 4-IPNB biodegradation and removal.

Sphingomonadaceae↗

PubMed1973

Benzoate metabolism in Pseudomonas putida(arvilla) mt-2: demonstration of two benzoate pathways.

Benzoate-grown cells of Pseudomonas putida(arvilla) mt-2 contain both metapyrocatechase and pyrocatechase activities, although the former activity is much higher than that of the latter. A spontaneous mutant deficient in metapyrocatechase and 2-hydroxymuconic semialdehyde hydrolyase, the first two enzymes in the meta-cleavage pathway of the ring of catechol, has been isolated from this strain. This mutant grows well on a minimal medium containing benzoate as a sole carbon source and has the high activity of pyrocatechase. These findings indicate that the strain mt-2 possesses the genetic capacity for enzymes of both the meta- and ortho-cleavage pathways of benzoate degradation, but its phenotypic expression is the meta pathway.

Benzoates↗

PubMed2001

Cloning and expression of the benzoate dioxygenase genes from Rhodococcus sp. strain 19070.

The bopXYZ genes from the gram-positive bacterium Rhodococcus sp. strain 19070 encode a broad-substrate-specific benzoate dioxygenase. Expression of the BopXY terminal oxygenase enabled Escherichia coli to convert benzoate or anthranilate (2-aminobenzoate) to a nonaromatic cis-diol or catechol, respectively. This expression system also rapidly transformed m-toluate (3-methylbenzoate) to an unidentified product. In contrast, 2-chlorobenzoate was not a good substrate. The BopXYZ dioxygenase was homologous to the chromosomally encoded benzoate dioxygenase (BenABC) and the plasmid-encoded toluate dioxygenase (XylXYZ) of gram-negative acinetobacters and pseudomonads. Pulsed-field gel electrophoresis failed to identify any plasmid in Rhodococcus sp. strain 19070. Catechol 1,2- and 2,3-dioxygenase activity indicated that strain 19070 possesses both meta- and ortho-cleavage degradative pathways, which are associated in pseudomonads with the xyl and ben genes, respectively. Open reading frames downstream of bopXYZ, designated bopL and bopK, resembled genes encoding cis-diol dehydrogenases and benzoate transporters, respectively. The bop genes were in the same order as the chromosomal ben genes of P. putida PRS2000. The deduced sequences of BopXY were 50 to 60% identical to the corresponding proteins of benzoate and toluate dioxygenases. The reductase components of these latter dioxygenases, BenC and XylZ, are 201 residues shorter than the deduced BopZ sequence. As predicted from the sequence, expression of BopZ in E. coli yielded an approximately 60-kDa protein whose presence corresponded to increased cytochrome c reductase activity. While the N-terminal region of BopZ was approximately 50% identical in sequence to the entire BenC or XylZ reductases, the C terminus was unlike other known protein sequences.

Benzoates↗

PubMedDate not supplied

[Degradation of phenanthrene by mutant strains--naphthalene degraders].

Five naphthalene- and salicylate-utilizing Pseudomonas putida strains cultivated for a long time on phenanthrene produced mutants capable of growing on this substrate and 1-hydroxy-2-naphthoate as the sole sources of carbon and energy. The mutants catabolize phenanthrene with the formation of 1-hydroxy-2-naphthoate, 2-hydroxy-1-naphthoate, salicylate, and catechol. The latter products are further metabolized by the meta- and ortho-cleavage pathways. In all five mutants, naphthalene and phenanthrene are utilized with the involvement of plasmid-borne genes. The acquired ability of naphthalene-degrading strains to grow on phenanthrene is explained by the fact that the inducible character of the synthesis of naphthalene dioxygenase, the key enzyme of naphthalene and phenanthrene degradation, becomes constitutive.

Biodegradation, Environmental↗

PubMed1990

Degradation of halogenated aromatic compounds.

Due to their persistence, haloaromatics are compounds of environmental concern. Aerobically, bacteria degrade these compounds by mono- or dioxygenation of the aromatic ring. The common intermediate of these reactions is (halo)catechol. Halocatechol is cleaved either intradiol (ortho-cleavage) or extradiol (meta-cleavage). In contrast to ortho-cleavage, meta-cleavage of halocatechols yields toxic metabolites. Dehalogenation may occur fortuitously during oxygenation. Specific dehalogenation of aromatic compounds is performed by hydroxylases, in which the halo-substituent is replaced by a hydroxyl group. During reductive dehalogenation, haloaromatic compounds may act as electron-acceptors. Herewith, the halosubstituent is replaced by a hydrogen atom.

Bacteria↗

PubMed1997

Microbial degradation of chloroaromatics: use of the meta-cleavage pathway for mineralization of chlorobenzene.

Pseudomonas putida GJ31 is able to simultaneously grow on toluene and chlorobenzene. When cultures of this strain were inhibited with 3-fluorocatechol while growing on toluene or chlorobenzene, 3-methylcatechol or 3-chlorocatechol, respectively, accumulated in the medium. To establish the catabolic routes for these catechols, activities of enzymes of the (modified) ortho- and meta-cleavage pathways were measured in crude extracts of cells of P. putida GJ31 grown on various aromatic substrates, including chlorobenzene. The enzymes of the modified ortho-cleavage pathway were never present, while the enzymes of the meta-cleavage pathway were detected in all cultures. This indicated that chloroaromatics and methylaromatics are both converted via the meta-cleavage pathway. Meta cleavage of 3-chlorocatechol usually leads to the formation of a reactive acylchloride, which inactivates the catechol 2,3-dioxygenase and blocks further degradation of catechols. However, partially purified catechol 2,3-dioxygenase of P. putida GJ31 converted 3-chlorocatechol to 2-hydroxy-cis,cis-muconic acid. Apparently, P. putida GJ31 has a meta-cleavage enzyme which is resistant to inactivation by the acylchloride, providing this strain with the exceptional ability to degrade both toluene and chlorobenzene via the meta-cleavage pathway.

Biodegradation, Environmental↗

PubMed1998

Key enzymes for the degradation of benzoate, m- and p-hydroxybenzoate by some members of the order Actinomycetales.

A preliminary screening of numerous species of the order Actinomycetales, especially of the genera Mycobacterium, Nocardia, Rhodococcus, Pseudonocardia, and Streptomyces, showed that many of them are able to metabolize benzoate (B) and p-hydroxybenzoate (pHB) as indicated by growth and change of color of the pH-indicator of an agar medium. Subsequent experiments with liquid cultures which allowed the analysis of substrate utilization by thin layer chromatography confirmed these results. The study of the degradative pathway proved that B was metabolized via catechol (C), pHB via protocatechuate (P) and m-hydroxybenzoate (mHB) via gentisate (G). The aromatic ring of C and P was subjected to an ortho-cleavage; only one strain of Noc. asteroides degraded C via a meta-cleavage, but P via an ortho-cleavage. Cell free extracts of four selected organisms exhibited activity of C-1,2-dioxygenase (C-1,2-O) and/or P-3,4-dioxygenase (P-3,4-O), depending on the growth substrate used for precultivation. In Streptomyces C-1,2-O was only found in cells grown on B, and P-3,4-O only in cells grown on pHB. On the contrary, in Rhodococcus rhodochrous B-cells oxidized C as well as P, while P-cells possessed only P-3,4-O-activity.

Actinomycetales↗

PubMed1994

Identification of a meta-cleavage pathway for metabolism of phenoxyacetic acid and phenol in Pseudomonas cepacia AC1100.

Pseudomonas cepacia strain AC1100 grows luxuriantly on 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) but does not utilize phenoxyacetic acid. After long-term selective pressure on phenoxyacetic acid, mutants designated as strain PAA, capable of utilizing phenoxyacetic acid as well as phenol, emerged spontaneously at a frequency of 1 x 10(-8). A deletion mutant strain PT88, which is devoid of a part of 2,4,5-T metabolic pathway, generated neither phenoxyacetic acid utilizing nor phenol-utilizing mutants. The wild type (Wt) strain AC1100 and all its mutants utilized benzoate via ortho-cleavage pathway. However, only mutant strain PAA harbored the whole set of enzymes required for utilization of phenol via meta-cleavage pathway. The results suggest that Wt strain AC1100 carries silent genes for meta-cleavage pathway which are expressed in strain PAA enabling it to utilize phenoxyacetic acid and phenol. Gene activation is presumed to be due to the translocation of insertion elements.

Biodegradation, Environmental↗

PubMed1982

Metabolism of dibutylphthalate and phthalate by Micrococcus sp. strain 12B.

Micrococcus sp. strain 12B was isolated by enriching for growth with dibutylphthalate as the sole carbon and energy source. A pathway for the metabolism of dibutylphthalate and phthalate by micrococcus sp. strain 12B is proposed: dibutylphthalate leads to monobutylphthalate leads to phthalate leads to 3,4-dihydro-3,4-dihydroxyphthalate leads to 3,4-dihydroxyphthalate leads to protocatechuate (3,4-dihdroxybenzoate). Protocatechuate is metabolized both by the meta-cleavage pathway through 4-carboxy-2-hydroxymuconic semialdehyde and 4-carboxy-2-hydroxymuconate to pyruvate and oxaloacetate and by the ortho-cleavage pathway to beta-ketoadipate. Dibutylphthalate- and phthalate-grown cells readily oxidized dibutylphthalate, phthalate, 3,4-dihydroxyphthalate, and protocatechuate. Extracts of cells grown with dibutylphthalate or phthalate contained the 3,4-dihydroxyphthalate decarboxylase and the enzymes of the protocatechuater 4,5-meta-cleavage pathway. Extracts of dibutylphthalate-grown cells also contained the protocatechuate ortho-cleavage pathway enzymes. The dibutylphthalate-hydrolyzing esterase and 3,4-dihydroxyphthalate decarboxylase were constitutively synthesized; phthalate-3,4-dioxygenase (and possibly the "dihydrodiol" dehydrogenase) was inducible by phthalate or a metabolite occurring before protocatechuate in the pathway; two protocatechuate oxygenases and subsequent enzymes were inducible by protocatechuate or a subsequent metabolic product. During growth at 37 degrees C, strain 12B gave clones at high frequency that had lost the ability to grow with phthalate esters. One of these nonrevertible mutants, strain 12B-Cl, lacked all of the enzymes required for the metabolism of dibutylphthalate through the protocatechuate meta-cleavage pathway. Enzymes for the metabolism of protocatechuate by the ortho-cleavage pathway were present in this strain grown with p-hydroxybenzoate or protocatechuate.

Dibutyl Phthalate↗

PubMed2000

Arrangement and regulation of the genes for meta-pathway enzymes required for degradation of phenol in Comamonas testosteroni TA441.

Comamonas testosteroni TA441 degrades phenol by a meta-cleavage pathway after the occurrence of a spontaneous mutation that derepresses the aphKLMNOPQB operon encoding phenol hydroxylase and catechol 2,3-dioxygenase, the enzymes for the initial two steps of the degradation pathway. A gene cluster, aphCEFGHJI, encoding the meta-pathway enzymes for degradation of 2-hydroxymuconic semialdehyde (HMS) to TCA cycle intermediates was found downstream of the aphK operon. The upstream operon and the downstream gene cluster were found to be separated by two open reading frames of unknown function and an oppositely oriented aphT gene, which is similar to regulatory genes for ortho-cleavage of catechol or chlorinated catechols. A promoter assay using an aphC::lacZ transcriptional fusion plasmid revealed that the aphC promoter activity is induced by both phenol and HMS. The phenol-dependent induction was mediated by AphR and the HMS-dependent induction was mediated by AphT. The aphC promoter in strain TA441 was not silenced, unlike the cases of the aphK and aphR promoters, and was highly induced by HMS.

Biodegradation, Environmental↗

PubMed1990

Loss of Tdn catabolic genes by deletion from and curing of plasmid pTDN1 in Pseudomonas putida: rate and mode of loss are substrate and pH dependent.

The ability to degrade aromatic amines and m-toluate (Tdn+ phenotype), encoded by plasmid pTDN1, was lost from Pseudomonas putida hosts after subculture in benzoate, succinate, acetate and glucose minimal medium, the fastest rate of loss occurring where benzoate was the substrate. Tdn- cells had either lost the entire pTDN1 plasmid or suffered a recombinational deletion of a specific 26 kbp region. Proportional increase of Tdn- cells resulted from their growth-rate advantage, and additionally, where benzoate was the substrate, from its metabolism via the chromosomal ortho-cleavage pathway incorporating a short lag phase. The ratio of whole plasmid loss to deletion was substrate and pH dependent. Deletion of catabolic genes was not required for loss of pTDN1 but by comparison was a prerequisite for loss of TOL plasmid pWW0. It appeared that m-toluate and benzoate were channelled via chromosomally encoded benzoate oxygenase and dihydroxycyclohexadiene carboxylate dehydrogenase prior to pTDN1 encoded meta-cleavage.

Aniline Compounds↗

PubMed1994

Loss of the TOL meta-cleavage pathway functions of Pseudomonas putida strain PaW1 (pWW0) during growth on toluene.

A derivative of Pseudomonas putida strain PaW1 bearing the TOL plasmid pWW0 was isolated from a culture which has grown unlimited on toluene. In contrast to the parent strain PaW1, the derivative, strain CG220, is unable to grow with xylenes and toluates, while toluene and benzoate served as substrates. Strain CG220 had a remarkable growth advantage against the wild type when grown with toluene. Biochemical analysis showed that in strain CG220 toluene was metabolised through the TOL plasmid upper pathway to benzoate and the latter to amphibolic intermediates by the chromosomal encoded ortho-cleavage pathway. No activities of the TOL plasmid encoded toluate dioxygenase and catechol 2,3-dioxygenase were detectable in strain CG220. No reversion of strain CG220 to growth with xylenes or toluates was observed. Hybridisation experiments with TOL plasmid-derived gene probes and oligonucleotides revealed that genes xylY to xylG were absent, while xylX and xylK were still present.

Base Sequence↗

PubMed1999

Utilization of phenoxyacetic acid, by strains using either the ortho or meta cleavage of catechol during phenol degradation, after conjugal transfer of tfdA, the gene encoding a 2,4-dichlorophenoxyacetic acid/2-oxoglutarate dioxygenase.

The degradation of recalcitrant pollutants in contaminated soils and waters could be facilitated by broadening the degradative capabilities of indigenous microbes by the conjugal transfer of catabolic genes. The feasibility of establishing bacterial populations that degrade phenoxyacetic acid by conjugal transfer of tfdA, the gene encoding 2,4-dichlorophenoxyacetic acid/2-oxoglutarate dioxygenase, to phenol-degrading strains of Pseudomonas and Ralstonia was examined. The mobilizable plasmid pKJS32 served as a vector for delivery of tfdA and the regulatory gene, tfdS. Transconjugant strains that degraded phenol by an ortho cleavage of catechol grew well on phenoxyacetic acid while those employing a meta cleavage could only grow on phenoxyacetic acid in the presence of benzoic acid or after a prolonged lag period and the appearance of mutants that had gained catechol 1,2-dioxygenase activities. Thus, an ortho cleavage of catechol was essential for degradation of phenoxyacetic acid, suggesting that a product of the ortho-cleavage pathway, probably cis, cis-muconic acid, is an inducer of tfdA gene expression. Establishment of phenoxyacetic-acid-degrading soil populations by conjugal transfer of tfdA would depend on the presence of phenol-degrading recipients employing an ortho cleavage of catechol.

2,4-Dichlorophenoxyacetic Acid↗

PubMed2002

The enhancement of 2-chlorophenol degradation by a mixed microbial community when augmented with Pseudomonas putida CP1.

The effect of the introduction of Pseudomonas putida CP1 to a commercial mixed microbial community for the degradation of 1.56mM 2-chlorophenol was investigated. Degradation of 2-chlorophenol by the commercial mixture was via a meta-cleavage pathway leading to incomplete degradation, while P. putida CPI was shown to be capable of the complete degradation of 2-chlorophenol via an ortho-cleavage pathway. Augmentation of the commercial mixed culture with P. putida CP1 resulted in complete degradation of 2-chlorophenol via an ortho-cleavage pathway. The augmented mixed culture displayed increased degradative capabilities, with times of degradation reduced when compared to those achieved by P. putida CP1 in isolation. The ability of P. putida CP1 to degrade 2-chlorophenol was increased with the addition of increasing concentrations of the mixed culture. Increasing the mixed culture inoculum size added to P. putida CP1 decreased lag periods and increased rates of degradation, resulting in decreased times of degradation.

Biodegradation, Environmental↗

PubMed1991

Degradation of 2-methylaniline and chlorinated isomers of 2-methylaniline by Rhodococcus rhodochrous strain CTM.

Rhodococcus rhodochrous strain CTM co-metabolized 2-methylaniline and some of its chlorinated isomers in the presence of ethanol as additional carbon source. Degradation of 2-methylaniline proceeded via 3-methylcatechol, which was metabolized mainly by meta-cleavage. In the case of 3-chloro-2-methylaniline, however, only a small proportion (about 10%) was subjected to meta-cleavage; the chlorinated meta-cleavage product was accumulated in the culture fluid as a dead-end metabolite. In contrast, 4-chloro-2-methylaniline was degraded via ortho-cleavage exclusively. Enzyme assays showed the presence of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase as inducible enzymes in strain CTM. Extended cultivation of strain CTM with 2-methylaniline and 3-chloro-2-methylaniline yielded mutants, including R. rhodochrous strain CTM2, that had lost catechol 2,3-dioxygenase activity; these mutants degraded the aromatic amines exclusively via the ortho-cleavage pathway. DNA hybridization experiments using a gene probe revealed the loss of the catechol 2,3-dioxygenase gene from strain CTM2.

Aniline Compounds↗

PubMed1983

Degradation of chlorophenols by a defined mixed microbial community.

Synthetic sewage containing phenol, acetone, and alkanols plus 4-chlorophenol or a mixture of isomeric chlorophenols is completely degraded by a defined mixed culture with Pseudomonas sp. strain B13 as a chlorocatechol-dissimilating member of the community. Total degradation of the organic carbon was indicated by release of stoichiometric amounts of chloride and low content of dissolved organic carbon in the cell-free effluents. During adaptation to high loads of chlorophenols the initial meta-cleavage activity was completely replaced by ortho-cleavage activity of type I and II. In the fully acclimated culture, hybrid strains such as Alcaligenes sp. strain A7-2 were detected, which are more competitive than Pseudomonas sp. strain B13 with respect to chlorophenol degradation.

Alcaligenes↗

PubMed2002

Monocyclic aromatic hydrocarbon degradation by Rhodococcus sp. strain DK17.

Rhodococcus sp. strain DK17 was isolated from soil and analyzed for the ability to grow on o-xylene as the sole carbon and energy source. Although DK17 cannot grow on m- and p-xylene, it is capable of growth on benzene, phenol, toluene, ethylbenzene, isopropylbenzene, and other alkylbenzene isomers. One UV-generated mutant strain, DK176, simultaneously lost the ability to grow on o-xylene, ethylbenzene, isopropylbenzene, toluene, and benzene, although it could still grow on phenol. The mutant strain was also unable to oxidize indole to indigo following growth in the presence of o-xylene. This observation suggests the loss of an oxygenase that is involved in the initial oxidation of the (alkyl)benzenes tested. Another mutant strain, DK180, isolated for the inability to grow on o-xylene, retained the ability to grow on benzene but was unable to grow on alkylbenzenes due to loss of a meta-cleavage dioxygenase needed for metabolism of methyl-substituted catechols. Further experiments showed that DK180 as well as the wild-type strain DK17 have an ortho-cleavage pathway which is specifically induced by benzene but not by o-xylene. These results indicate that DK17 possesses two different ring-cleavage pathways for the degradation of aromatic compounds, although the initial oxidation reactions may be catalyzed by a common oxygenase. Gas chromatography-mass spectrometry and 300-MHz proton nuclear magnetic resonance spectrometry clearly show that DK180 accumulates 3,4-dimethylcatechol from o-xylene and both 3- and 4-methylcatechol from toluene. This means that there are two initial routes of oxidation of toluene by the strain. Pulsed-field gel electrophoresis analysis demonstrated the presence of two large megaplasmids in the wild-type strain DK17, one of which (pDK2) was lost in the mutant strain DK176. Since several other independently derived mutant strains unable to grow on alkylbenzenes are also missing pDK2, the genes encoding the initial steps in alkylbenzene metabolism (but not phenol metabolism) appear to be present on this approximately 330-kb plasmid.

Catechol 1,2-Dioxygenase↗

PubMed1990

Complete nucleotide sequence and polypeptide analysis of multicomponent phenol hydroxylase from Pseudomonas sp. strain CF600.

Pseudomonas sp. strain CF600 metabolizes phenol and some of its methylated derivatives via a plasmid-encoded phenol hydroxylase and meta-cleavage pathway. The genes encoding the multicomponent phenol hydroxylase of this strain are located within a 5.5-kb SacI-NruI fragment. We report the nucleotide sequence and the polypeptide products of this 5.5-kb region. A combination of deletion analysis, expression of subfragments in tac expression vectors, and identification of polypeptide products in maxicells was used to demonstrate that the polypeptides observed are produced from the six open reading frames identified in the sequence. Expression of phenol hydroxylase activity in a laboratory Pseudomonas strain allows growth on phenol, owing to expression of this enzyme and the chromosomally encoded ortho-cleavage pathway. This system, in conjunction with six plasmids that each expressed all but one of the polypeptides, was used to demonstrate that all six polypeptides are required for growth on phenol.

Amino Acid Sequence↗