Degradation of Fluorobenzene by Rhizobiales Strain F11 via ortho Cleavage of 4-Fluorocatechol and Catechol

The aerobic metabolism of fluorobenzene by Rhizobiales sp. strain F11 was investigated. Liquid chromatography-mass spectrometry analysis showed that 4-fluorocatechol and catechol were formed as intermediates during fluorobenzene degradation by cell suspensions. Both these compounds, unlike 3-fluorocatechol, supported growth and oxygen uptake. Cells grown on fluorobenzene contained enzymes for the ortho pathway but not for meta ring cleavage of catechols. The results suggest that fluorobenzene is predominantly degraded via 4-fluorocatechol with subsequent ortho cleavage and also partially via catechol.     

Degradation of fluorobenzene and its central metabolites 3-fluorocatechol and 2-fluoromuconate by Burkholderia fungorum FLU100

A halobenzene-degrading bacterium, Burkholderia fungorum FLU100 (DSM 23736), was isolated due to its outstanding trait to degrade fluorobenzene. Besides fluorobenzene, it utilizes, even in random mixtures, chlorobenzene, bromobenzene, iodobenzene, benzene, and toluene as sole sources of carbon and energy. FLU100 mineralizes mono-halogenated benzenes almost stoichiometrically (according to halide balance); after a lag phase, it also degrades 3-fluorophenol and 3-chlorophenol completely. The FLU100-derived transposon Tn5-mutant FLU100-P14R22 revealed 3-halocatechol to be a central metabolite of this new halobenzene degradation pathway. In FLU100, halocatechols are—as expected—strictly subject to ortho-cleavage of the catechol ring, with meta-cleavage never been observed. The strain is able to completely mineralize 3-fluorocatechol, the principal catecholic metabolite being nearly exclusively formed from fluorobenzene. The temporarily excreted 2-fluoromuconate formed thereof in turn is subsequently metabolized completely. This important finding falsifies the customary opinion of the persistence of 2-fluoromuconate valid up to now. The degradation of 4-fluorocatechol, however, being a very minor intermediate in FLU100, is substantially slower and incomplete and leads to the accumulation of uncharacterized derivatives of muconic acid and muconolactone in the medium. This branch therefore does not seem to be productive. To our knowledge, this represents the first example of the complete metabolism of 3-fluorocatechol via 2-fluoromuconate, a metabolite hitherto described as a dead-end metabolite in fluoroaromatic degradation.     

Phenol and Benzoate Metabolism by Pseudomonas putida: Regulation of Tangential Pathways

Catechol occurs as an intermediate in the metabolism of both benzoate and phenol by strains of Pseudomonas putida. During growth at the expense of benzoate, catechol is cleaved ortho (1,2-oxygenase) and metabolized via the beta-ketoadipate pathway; during growth at the expense of phenol or cresols, the catechol or substituted catechols formed are metabolized by a separate pathway following meta (2,3-oxygenase) cleavage of the aromatic ring of catechol. It is possible to explain the mutually exclusive occurrence of the meta and ortho pathway enzymes in phenol- and benzoate-grown cells of P. putida on the basis of differences in the mode of regulation of these two pathways. By use of both nonmetabolizable inducers and blocked mutants, gratuitous synthesis of some of the meta pathway enzymes was obtained. All four enzymes of the meta pathway are induced by the primary substrate, cresol or phenol, or its analogue. Three enzymes of the ortho pathway that catalyze the conversion of catechol to beta-ketoadipate enol-lactone are induced by cis,cis-muconate, produced from catechol by 1,2-oxygenase-mediated cleavage. Observations on the differences in specificity of induction and function of the two pathways suggest that they are not really either tangential or redundant. The meta pathway serves as a general mechanism for catabolism of various alkyl derivatives of catechol derived from substituted phenolic compounds. The ortho pathway is more specific and serves primarily in the catabolism of precursors of catechol and catechol itself.     

3-Fluorobenzoate enriched bacterial strain FLB 300 degrades benzoate and all three isomeric monofluorobenzoates

The bacterial strain FLB300 was enriched with 3-fluorobenzoate as sole carbon source. Besides benzoate all isomeric monofluorobenzoates were utilized. Regioselective 1,2-dioxygenation rather than 1,6-dioxygenation yielded 4-fluorocatechol and minimized the production of toxic 3-fluorocatechol. Degradation of 4-fluorocatechol was mediated by reactions of ortho cleavage pathway activities. Chemotaxonomic and r-RNA data excluded strain FLB300 from a phylogenetically defined genus Pseudomonas and suggested its allocation to the alpha-2 subclass of Proteobacteria in a new genus of the Agrobacterium-Rhizobium branch.Abbreviations PYES peptone yeast extract soy medium - TLC thin layer chromatography - NTA nitrilotriacetate - SDS-PAGE sodium dodecylsulphate-polyacrylsulphate gel electrophoresis - FB fluorobenzoate - DHB 1,2-dihydro-1,2-dihydroxybenzoate - NB nutrient broth     

Metabolism of monofluorobenzoates by Acinetobacter calcoaceticus N.C.I.B. 8250 formation of monofluorocatechols

None of the monofluorobenzoates serves as sole source of carbon and energy for growth of Acinetobacter calcoaceticus but all can contribute to growth on other substrates. The monofluorobenzoates are oxidised by bacteria pre-induced for benzoate oxidation and can themselves induce the appropriate enzymes. The initial products of oxidation have been separated and identified by gas-liquid chromatography. 2-Fluorobenzoate is oxidised to catechol, fluoride and 3-fluorocatechol; 3-fluorobenzoate gives 3- and 4-fluorocatechol; 4-fluorobenzoate gives 4-fluorocatechol. The fluorocatechols appear to be partially oxidised beyond the stage of 3-oxoadipate by suitably pre-induced bacteria.     

Metabolism of monofluorobenzoates by Acinetobacter calcoaceticus N.C.I.B. 8250. Formation of monofluorocatechols.

None of the monofluorobenzoates serves as sole source of carbon and energy for growth of Acinetobacter calcoaceticus but all can contribute to growth on other substrates. The monofluorobenzoates are oxidised by bacteria pre-induced for benzoate oxidation and can themselves induce the appropriate enzymes. The initial products of oxidation have been separated and identified by gas-liquid chromatography. 2-Flurobenzoate is oxidised to catechol, fluoride and 3-fluorocatechol; 3-fluorobenzoate gives 3- and 4-fluorocatechol; 4-fluorobenzoate gives 4-fluorocatechol. The fluorocatechols appear to be partially oxidised beyond the stage of 3-oxoadipate by suitably pre-induced bacteria.     

邻苯二酚2,3-双加氧酶的结构和功能研究进展

邻苯二酚是所有芳香族化合物降解过程中的重要的中间产物,其降解有邻位和间位裂解两条裂解途径,分别由邻苯二酚1,2-双加氧酶(C12O)和邻苯二酚2,3-双加氧酶(C23O)催化裂解。本综述简要介绍了邻苯二酚2,3-双加氧酶的结构和功能的研究进展。     

Influence of Side-Chain Substituents on the Position of Cleavage of the Benzene Ring by Pseudomonas fluorescens

Pseudomonas fluorescens was grown on mineral salts media with phenol, p-hydroxybenzoic acid, p-hydroxy-phenylacetic acid, or p-hydroxy-trans-cinnamic acid as sole carbon and energy source. Each compound was first hydroxylated, ortho to the hydroxyl group on the benzene ring, to give catechol, protocatechuic acid (3,4-dihydroxy-benzoic acid), homoprotocatechuic acid (3,4-dihydroxy-phenylacetic acid), and caffeic acid (3,4-dihydroxy-trans-cinnamic acid), respectively, as the ultimate aromatic products before cleavage of the benzene nucleus. Protocatechuic acid and caffeic acid were shown to be cleaved by ortho fission, via a 3,4-oxygenase mechanism, to give beta-substituted cis, cis-muconic acids as the initial aliphatic products. However, catechol and homoprotocatechuic acid were cleaved by meta fission, by 2,3-and 4,5-oxygenases, respectively, to give alpha-hydroxy-muconic semialdehyde and alpha-hydroxy-gamma-carboxymethyl muconic semialdehyde as initial aliphatic intermediates. Caffeic acid: 3,4-oxygenase, a new oxygenase, consumes 1 mole of O(2) per mole of substrate and has an optimal pH of 7.0. The mechanism of cleavage of enzymes derepressed for substituted catechols by P. fluorescens apparently changes from ortho to meta with the increasing nephelauxetic (electron donor) effect of the side-chain substituent.     

Mineralization and transformation of monofluorophenols by Pseudonocardia benzenivorans

The aerobic metabolism of monofluorophenols (mono-FPs) by the actinomycete, Pseudonocardia benzenivorans, was studied. This strain was able to grow on 4-fluorophenol (4-FP) and readily transform 2- and 3-fluorophenol to the corresponding metabolites. The detailed mechanism of mono-FPs degradation by P. benzenivorans was elucidated from enzymatic assays and the identification of reaction intermediates by high-performance liquid chromatography (HPLC) and gas chromatography–mass spectrometry. Two types of fluorocatechols (i.e., 3- and 4-fluorocatechol) were identified as the key transformation products. During 4-FP degradation, only 4-fluorocatechol was detected, and a stoichiometric level of fluoride was released. Both fluorocatechols were observed together in cultures containing 3-fluorophenol (3-FP), while only 3-fluorocatechol was found to accumulate in 2-fluorophenol (2-FP)-containing cultures. Whole-cell extracts of P. benzenivorans expressed catechol 1,2-dioxygenase activity, indicating that the transformation of the three tested mono-FPs proceeded via ortho-cleavage pathway. The results presented in this paper provide comprehensive information regarding the metabolism of mono-FPs by a single bacterium.     

Degradation of 2-bromo-, 2-chloro- and 2-fluorobenzoate by Pseudomonas putida CLB 250

Abstract Pseudomonas putida strain CLB 250 (DSM 5232) utilized 2-bromo-, 2-chloro- and 2-fluorobenzoate as sole source of carbon and energy. Degradation is suggested to be initiated by a dioxygenase liberating halide in the first catabolic step. After decarboxylation and rearomatization catechol is produced as a central metabolite which is degraded via the ortho-pathway. After inhibition of ring cleavage activities with 3-chlorocatechol, 2-chlorobenzoate was transformed to catechol in nearly stoichiometric amounts. Other ortho -substituted benzoates like anthranilate and 2-methoxybenzoate seem to be metabolized via the same route.     

Three types of phenol and p-cresol catabolism in phenol- and p-cresol-degrading bacteria isolated from river water continuously polluted with phenolic compounds

A total of 39 phenol- and p-cresol-degraders isolated from the river water continuously polluted with phenolic compounds of oil shale leachate were studied. Species identification by BIOLOG GN analysis revealed 21 strains of Pseudomonas fluorescens (4, 8 and 9 of biotypes A, C and G, respectively), 12 of Pseudomonas mendocina, four of Pseudomonas putida biotype A1, one of Pseudomonas corrugata and one of Acinetobacter genospecies 15. Computer-assisted analysis of rep-PCR fingerprints clustered the strains into groups with good concordance with the BIOLOG GN data. Three main catabolic types of degradation of phenol and p-cresol were revealed. Type I, or meta-meta type (15 strains), was characterized by meta cleavage of catechol by catechol 2,3-dioxygenase (C23O) during the growth on phenol and p-cresol. These strains carried C23O genes which gave PCR products with specific xylE-gene primers. Type II, or ortho-ortho type (13 strains), was characterized by the degradation of phenol through ortho fission of catechol by catechol 1,2-dioxygenase (C12O) and p-cresol via ortho cleavage of protocatechuic acid by protocatechuate 3,4-dioxygenase (PC34O). These strains carried phenol monooxygenase gene which gave PCR products with pheA-gene primers. Type III, or meta-ortho type (11 strains), was characterized by the degradation of phenol by C23O and p-cresol via the protocatechuate ortho pathway by the induction of PC34O and this carried C23O genes which gave PCR products with C23O-gene primers, but not with specific xylE-gene primers. In type III strains phenol also induced the p-cresol protocatechuate pathway, as revealed by the induction of p-cresol methylhydroxylase. These results demonstrate multiplicity of catabolic types of degradation of phenol and p-cresol and the existence of characteristic assemblages of species and specific genotypes among the strains isolated from the polluted river water.     

Suicide Inactivation of Catechol 2,3-Dioxygenase from Pseudomonas putida mt-2 by 3-Halocatechols

The inactivation of catechol 2,3-dioxygenase from Pseudomonas putida mt-2 by 3-chloro- and 3-fluorocatechol and the iron-chelating agent Tiron (catechol-3,5-disulfonate) was studied. Whereas inactivation by Tiron is an oxygen-independent and mostly reversible process, inactivation by the 3-halocatechols was only observed in the presence of oxygen and was largely irreversible. The rate constants for inactivation (K₂) were 1.62 × 10⁻³ sec⁻¹ for 3-chlorocatechol and 2.38 × 10⁻³ sec⁻¹ for 3-fluorocatechol. The inhibitor constants (K_i) were 23 μM for 3-chlorocatechol and 17 μM for 3-fluorocatechol. The kinetic data for 3-fluorocatechol could only be obtained in the presence of 2-mercaptoethanol. Besides inactivated enzyme, some 2-hydroxyhexa-2,4-diendioic acid was formed from 3-chlorocatechol, suggesting 5-chloroformyl-2-hydroxypenta-2,4-dienoic acid as the actual suicide product of meta-cleavage. A side product of 3-fluorocatechol cleavage is a yellow compound with the spectral characteristics of a 2-hydroxy-6-oxohexa-2,4-dienoic acid indicating 1,6-cleavage. Rates of inactivation by 3-fluorocatechol were reduced in the presence of superoxide dismutase, catalase, formate, and mannitol, which implies that superoxide anion, hydrogen peroxide, and hydroxyl radical exhibit additional inactivation.     

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.     

TOL plasmid pWW0 in constructed halobenzoate-degrading Pseudomonas strains: prevention of meta pathway.

The hybrid pathway for chlorobenzoate metabolism was studied in WR211 and WR216, which were derived from Pseudomonas sp. B13 by acquisition of TOL plasmid pWW0 from Pseudomonas putida mt-2. Chlorobenzoates are utilized readily by these strains when meta cleavage of chlorocatechols is suppressed. When WR211 utilizes 3-chlorobenzoate (3CB), the expression of catechol 2,3-dioxygenase (C23O) and the catabolic activities for chloroaromatics via the ortho pathway coexist as a consequence of inactivation of the meta cleavage activity by 3-chlorocatechol. Utilization of 4-chlorobenzoate (4CB) by WR216 presupposes the suppression of C23O by a spontaneous mutation in the structural gene, so that 4-chlorocatechol is not misrouted into the meta pathway. Such C23O- mutants were also selected when WR211 was grown continuously on 3CB. Our data explain why the phenotypic characters 3CB+ and Mtol+ (m-toluate) are compatible, whereas 4CB+ and Mtol+ are incompatible.     

Simultaneous biodegradation of chlorobenzene and toluene by a Pseudomonas strain

Pseudomonas sp. strain JS6 grows on a wide range of chloro- and methylaromatic substrates. The simultaneous degradation of these compounds is prevented in most previously studied isolates because the catabolic pathways are incompatible. The purpose of this study was to determine whether strain JS6 could degrade mixtures of chloro- and methyl-substituted aromatic compounds. Strain JS6 was maintained in a chemostat on a minimal medium with toluene or chlorobenzene as the sole carbon source, supplied via a syringe pump. Strain JS6 contained an active catechol 2,3-dioxygenase when grown in the presence of chloroaromatic compounds; however, in cell extracts, this enzyme was strongly inhibited by 3-chlorocatechol. When cells grown to steady state on toluene were exposed to 50% toluene-50% chlorobenzene, 3-chlorocatechol and 3-methylcatechol accumulated in the medium and the cell density decreased. After 3 h, the enzyme activities of the modified ortho ring fission pathway were induced, the metabolites disappeared, and the cell density returned to previous levels. In cell extracts, 3-methylcatechol was degraded by both catechol 1,2- and catechol 2,3-dioxygenase. Strain JS62, a catechol 2,3-dioxygenase mutant of JS6, grew on toluene, and ring cleavage of 3-methylcatechol was catalyzed by catechol 1,2-dioxygenase. The transient metabolite 2-methyllactone was identified in chlorobenzene-grown JS6 cultures exposed to toluene. These results indicate that strain JS6 can degrade mixtures of chloro- and methylaromatic compounds by means of a modified ortho ring fission pathway.     

Simultaneous biodegradation of chlorobenzene and toluene by a Pseudomonas strain.

Pseudomonas sp. strain JS6 grows on a wide range of chloro- and methylaromatic substrates. The simultaneous degradation of these compounds is prevented in most previously studied isolates because the catabolic pathways are incompatible. The purpose of this study was to determine whether strain JS6 could degrade mixtures of chloro- and methyl-substituted aromatic compounds. Strain JS6 was maintained in a chemostat on a minimal medium with toluene or chlorobenzene as the sole carbon source, supplied via a syringe pump. Strain JS6 contained an active catechol 2,3-dioxygenase when grown in the presence of chloroaromatic compounds; however, in cell extracts, this enzyme was strongly inhibited by 3-chlorocatechol. When cells grown to steady state on toluene were exposed to 50% toluene-50% chlorobenzene, 3-chlorocatechol and 3-methylcatechol accumulated in the medium and the cell density decreased. After 3 h, the enzyme activities of the modified ortho ring fission pathway were induced, the metabolites disappeared, and the cell density returned to previous levels. In cell extracts, 3-methylcatechol was degraded by both catechol 1,2- and catechol 2,3-dioxygenase. Strain JS62, a catechol 2,3-dioxygenase mutant of JS6, grew on toluene, and ring cleavage of 3-methylcatechol was catalyzed by catechol 1,2-dioxygenase. The transient metabolite 2-methyllactone was identified in chlorobenzene-grown JS6 cultures exposed to toluene. These results indicate that strain JS6 can degrade mixtures of chloro- and methylaromatic compounds by means of a modified ortho ring fission pathway.     

Role of Catechol and the Methylcatechols as Inducers of Aromatic Metabolism in Pseudomonas putida

Pseudomonas putida NCIB 10015 metabolizes phenol and the cresols (methylphenols) by the meta pathway and metabolizes benzoate by the ortho pathway. Growth on catechol, an intermediate in the metabolism of both phenol and benzoate, induces both ortho and meta pathways; growth on 3- or 4-methylcatechols, intermediates in the metabolism of the cresols, induces only the meta pathway to a very limited degree. Addition of catechol at a growth-limiting rate induces virtually no meta pathway enzymes, but high levels of ortho pathway enzymes. The role of catechol and the methylcatechols as inducers is discussed. A method is described for assaying low levels of catechol 1,2-oxygenase in the presence of high levels of catechol 2,3-oxygenase and vice versa.     

丛毛睾丸酮单胞菌ZD4-1和铜绿假单胞菌ZD4-3降解芳香烃化合物的机理

从某农药厂二沉池污泥中筛选分离得到两株革兰氏阴性的芳香烃降解菌ZD41和ZD43。经鉴定,它们分别属于Comamonas testosteroni和Pseudomonas aeruginosa。基于16S rDNA 序列的系统分类分析,结果表明,在分类地位上菌株ZD41和ZD43 分别属于两个不同的分类亚组。苯酚降解产物紫外光谱扫描和双加氧酶检测证明,菌株ZD41利用邻裂途径降解苯酚,而ZD43则通过间裂途径降解苯酚,邻裂途径的1,2双加氧酶和间裂途径的2,3双加氧酶都是可诱导的双加氧酶,其活性强烈的依赖于降解底物的出现。芳香烃降解试验结果表明,邻裂和间裂两种途径的降解性能不一样,虽然ZD43降解苯酚的效率要高于菌株ZD41,但是ZD41降解苯酚的pH值范围以及芳烃利用基质谱宽于后者。     

The metabolism of benzoate by Moraxella species through anaerobic nitrate respiration. Evidence for a reductive pathway.

Moraxella sp. isolated from soil grows anaerobically on benzoate by nitrate respiration; nitrate or nitrite are obligatory electron acceptors, being reduced to molecular N2 during the catabolism of the substrate. This bacterium also grows aerobically on benzoate. 2. Aerobically, benzoate is metabolized by ortho cleavage of catechol followed by the beta-oxoadipate pathway. 3. Cells of Moraxella grown anaerobically on benzoate are devoid of ortho and meta cleavage enzymes; cyclohexanecarboxylate and 2-hydroxycyclohexanecarboxylate were detected in the anaerobic culture fluid. 4. [ring-U-14C]Benzoate, incubated anaerobically with cells in nitrate-phosphate buffer, gave rise to labelled 2-hydroxycyclohexanecarboxylate and adipate. When [carboxy-14C]benzoate was used, 2-hydroxycyclohexanecarboxylate was radioactive but the adipate was not labelled. A decarboxylation reaction intervenes at some stage between these two metabolites. 5. The anaerobic metabolism of benzoate by Moraxella sp. through nitrate respiration takes place by the reductive pathway (Dutton & Evans, 1969). Hydrogenation of the aromatic ring probably occurs via cyclohexa-2,5-dienecarboxylate and cyclohex-1-enecarboxylate to give cyclohexanecarboxylate. The biochemistry of this reductive process remains unclear. 6. CoA thiol esterification of cyclohexanecarboxylate followed by beta-oxidation via the unsaturated and hydroxy esters, would afford 2-oxocyclohexanecarboxylate. Subsequent events in the Moraxella culture differ from those occurring with Rhodopseudomonas palustris; decarboxylation precedes hydrolytic cleavage of the alicyclic ring to produce adipate in the former, whereas in the latter the keto ester undergoes direct hydrolytic fission to pimelate.     

Inhibition of catechol 2,3-dioxygenase from Pseudomonas putida by 3-chlorocatechol.

Partially purified preparations of catechol 2,3-dioxygenase from toluene-grown cells of Pseudomonas putida catalyzed the stoichiometric oxidation of 3-methylcatechol to 2-hydroxy-6-oxohepta-2,4-dienoate. Other substrates oxidized by the enzyme preparation were catechol, 4-methylcatechol, and 4-fluorocatechol. The apparent Michaelis constants for 3-methylcatechol and catechol were 10.6 and 22.0 muM, respectively. Substitution at the 4-position decreases the affinity and activity of the enzyme for the substrate. Catechol 2,3-dioxygenase preparations did not oxidize 3-chlorocatechol. In addition, incubation of the enzyme with 3-chlorocatechol led to inactivation of the enzyme. Kinetic analyses revealed that both 3-chlorocatechol and 4-chlorocatechol were noncompetitive or mixed-type inhibitors of the enzyme. 3-Chlorocatechol (Ki = 0.14 muM) was a more potent inhibitor than 4-chlorocatechol (Ki = 50 muM). The effect of the ion-chelating agents Tiron and o-phenanthrolene were compared with that of 3-chlorocatechol on the inactivation of the enzyme. Each inhibitor appeared to remove iron from the enzyme, since inactive enzyme preparations could be fully reactivated by treatment with ferrous iron and a reducing agent.