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 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.     

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.     

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.     

Critical Reactions in Fluorobenzoic Acid Degradation by Pseudomonas sp. B13

3-Chlorobenzoate-grown cells of Pseudomonas sp. B13 readily cometabolized monofluorobenzoates. A catabolic pathway for the isomeric fluorobenzoates is proposed on the basis of key metabolites isolated. Only 4-fluorobenzoate was utilized and totally degraded after a short period of adaptation. The isoenzymes for total degradation of chlorocatechols, being found during growth with 3-chlorobenzoate or 4-chlorophenol, were not induced in the presence of fluorobenzoates. Correspondingly, only the ordinary enzymes of the benzoate pathway were detected in 4-fluorobenzoate-grown cells. Ring cleavage of 3-fluorocatechol was recognized as a critical step in 3-fluorobenzoate degradation. 2-Fluoro-cis,cis-muconic acid was identified as a dead-end metabolite from 2- and 3-fluorobenzoate catabolism. During 2-fluorobenzoate cometabolism, fluoride is eliminated by the initial dioxygenation.     

Effect of the metals iron, copper and silver on fluorobenzene biodegradation by Labrys portucalensis

Organic and metallic pollutants are ubiquitous in the environment. Many metals are reported to be toxic to microorganisms and to inhibit biodegradation. The effect of the metals iron, copper and silver on the metabolism of Labrys portucalensis F11 and on fluorobenzene (FB) biodegradation was examined. The results indicate that the addition of 1 mM of Fe²⁺ to the culture medium has a positive effect on bacterial growth and has no impact in the biodegradation of 1 and 2 mM of FB. The presence of 1 mM of Cu²⁺ was found to strongly inhibit the growth of F11 cultures and to reduce the biodegradation of 1 and 2 mM of FB to ca. 50 %, with 80 % of stoichiometrically expected fluoride released. In the experiments with resting cells, the FB degraded (from 2 mM supplied) was reduced ca. 20 % whereas the fluoride released was reduced to 45 % of that stoichiometrically expected. Ag⁺ was the most potent inhibitor of FB degradation. In experiments with growing cells, the addition of 1 mM of Ag⁺ to the culture medium containing 1 and 2 mM of FB resulted in no fluoride release, whereas FB degradation was only one third of that observed in control cultures. In the experiments with resting cells, the addition of Ag⁺ resulted in 25 % reduction in substrate degradation and fluoride release was only 20 % of that stoichiometrically expected. The accumulation of catechol and 4-fluorocatechol in cultures supplemented with Cu²⁺ or Ag⁺ suggest inhibition of the key enzyme of FB metabolism—catechol 1,2-dioxygenase.     

Catabolic degradation of 4-chlorobiphenyl byPseudomonas sp. DJ-12 via consecutive reaction ofmeta-cleavage and hydrolytic dechlorination

Pseudomonas sp. strain DJ-12 is a bacterial isolate capable of degrading 4-chlorobiphenyl (4CBP) as a carbon and energy source. The catabolic degradation of 4CBP by the strain DJ-12 was studied along with the genetic organization of the genes responsible for the crucial steps of the catabolic degradation. The catabolic pathway was characterized as being conducted by consecutive reactions of themeta-cleavage of 4CBP, hydrolytic dechlorination of 4-chlorobenzoate (4CBA), hydroxylation of 4-hydroxybenzoate, andmeta-cleavage of protocatechuate. ThepcbC gene responsible for themeta-cleavage of 4CBP only showed a 30 to 40% homology in its deduced amino acid sequence compared to those of the corresponding genes from other strains. The amino acid sequence of 4CBA-CoA dechlorinase showed an 86% homology with that ofPseudomonas sp. CBS3, yet only a 50% homology with that ofArthrobacter spp. However, thefcb genes for the hydrolytic dechlorination of 4CBA inPseudomonas sp. DJ-12 showed an uniquely different organization from those of CBS3 and other reported strains. Accordingly, these results indicate that strain DJ-12 can degrade 4CBP completely viameta-cleavage and hydrolytic dechlorination using enzymes that are uniquely different in their amino acid sequences from those of other bacterial strains with the same degradation activities.     

Degradation of toluene by ortho cleavage enzymes in Burkholderia fungorum FLU100

Burkholderia fungorum FLU100 simultaneously oxidized any mixture of toluene, benzene and mono‐halogen benzenes to (3‐substituted) catechols with a selectivity of nearly 100%. Further metabolism occurred via enzymes of ortho cleavage pathways with complete mineralization. During the transformation of 3‐methylcatechol, 4‐carboxymethyl‐2‐methylbut‐2‐en‐4‐olide (2‐methyl‐2‐enelactone, 2‐ML) accumulated transiently, being further mineralized only after a lag phase of 2 h in case of cells pre‐grown on benzene or mono‐halogen benzenes. No lag phase, however, occurred after growth on toluene. Cultures inhibited by chloramphenicol after growth on benzene or mono‐halogen benzenes were unable to metabolize 2‐ML supplied externally, even after prolonged incubation. A control culture grown with toluene did not show any lag phase and used 2‐ML as a substrate. This means that 2‐ML is an intermediate of toluene degradation and converted by specific enzymes. The conversion of 4‐methylcatechol as a very minor by‐product of toluene degradation in strain FLU100 resulted in the accumulation of 4‐carboxymethyl‐4‐methylbut‐2‐en‐4‐olide (4‐methyl‐2‐enelactone, 4‐ML) as a dead‐end product, excluding its nature as a possible intermediate. Thus, 3‐methylcyclohexa‐3,5‐diene‐1,2‐diol, 3‐methylcatechol, 2‐methyl muconate and 2‐ML were identified as central intermediates of productive ortho cleavage pathways for toluene metabolism in B. fungorum FLU100.     

Conversion of 2-Fluoromuconate to cis-Dienelactone by Purified Enzymes of Rhodococcus opacus 1cp

The present study describes the ¹⁹F nuclear magnetic resonance analysis of the conversion of 3-halocatechols to lactones by purified chlorocatechol 1,2-dioxygenase (ClcA2), chloromuconate cycloisomerase (ClcB2), and chloromuconolactone dehalogenase (ClcF) from Rhodococcus opacus 1cp grown on 2-chlorophenol. The 3-halocatechol substrates were produced from the corresponding 2-halophenols by either phenol hydroxylase from Trichosporon cutaneum or 2-hydroxybiphenyl 3-mono-oxygenase from Pseudomonas azelaica. Several fluoromuconates resulting from intradiol ring cleavage by ClcA2 were identified. ClcB2 converted 2-fluoromuconate to 5-fluoromuconolactone and 2-chloro-4-fluoromuconate to 2-chloro-4-fluoromuconolactone. Especially the cycloisomerization of 2-fluoromuconate is a new observation. ClcF catalyzed the dehalogenation of 5-fluoromuconolactone to cis-dienelactone. The ClcB2 and ClcF-mediated reactions are in line with the recent finding of a second cluster of chlorocatechol catabolic genes in R. opacus 1cp which provides a new route for the microbial dehalogenation of 3-chlorocatechol.     

Metabolism of 2-aminophenol by Pseudomonas sp. AP-3: modified meta-cleavage pathway

A novel pathway for 2-aminophenol metabolism by Pseudomonas sp. AP-3 is proposed. The proposed pathway is similar to that known for meta-cleavage of catechol except that one of the hydroxyl groups on the metabolites is replaced by an amino group. During the degradation of 2-aminophenol, 2-amino-2,4-pentadienoic acid is the last metabolite containing an amino group. We, therefore, propose a modified meta-cleavage pathway for the 2-aminophenol metabolism. Received: 27 November 1997 / Accepted: 14 May 1998     

Biodegradation of 3-nitrotoluene by Rhodococcus sp. strain ZWL3NT

A pure bacterial culture was isolated by its ability to utilize 3-nitrotoluene (3NT) as the sole source of carbon, nitrogen, and energy for growth. Analysis of its 16S rRNA gene showed that the organism (strain ZWL3NT) belongs to the genus Rhodococcus. A rapid disappearance of 3NT with concomitant release of nitrite was observed when strain ZWL3NT was grown on 3NT. The isolate also grew on 2-nitrotoluene, 3-methylcatechol and catechol. Two metabolites, 3-methylcatechol and 2-methyl-cis,cis-muconate, in the reaction mixture were detected after incubation of cells of strain ZWL3NT with 3NT. Enzyme assays showed the presence of both catechol 1,2-dioxygenase and catechol 2,3-dioxygenase in strain ZWL3NT. In addition, a catechol degradation gene cluster (catRABC cluster) for catechol ortho-cleavage pathway was cloned from this strain and cell extracts of Escherichia coli expressing CatA and CatB exhibited catechol 1,2-dioxygenase activity and cis,cis-muconate cycloisomerase activity, respectively. These experimental evidences suggest a novel pathway for 3NT degradation with 3-methylcatechol as a key metabolite by Rhodococcus sp. strain ZWL3NT.     

Formation of Catechols via Removal of Acid Side Chains from Ibuprofen and Related Aromatic Acids

Although ibuprofen [2-(4-isobutylphenyl)-propionic acid] is one of the most widely consumed drugs in the world, little is known regarding its degradation by environmental bacteria. Sphingomonas sp. strain Ibu-2 was isolated from a wastewater treatment plant based on its ability to use ibuprofen as a sole carbon and energy source. A slight preference toward the R enantiomer was observed, though both ibuprofen enantiomers were metabolized. A yellow color, indicative of meta-cleavage, accumulated transiently in the culture supernatant when Ibu-2 was grown on ibuprofen. When and only when 3-flurocatechol was used to poison the meta-cleavage system, isobutylcatechol was identified in the culture supernatant via gas chromatography-mass spectrometry analysis. Ibuprofen-induced washed-cell suspensions also metabolized phenylacetic acid and 2-phenylpropionic acid to catechol, while 3- and 4-tolylacetic acids and 2-(4-tolyl)-propionic acid were metabolized to the corresponding methyl catechols before ring cleavage. These data suggest that, in contrast to the widely distributed coenzyme A ligase, homogentisate, or homoprotocatechuate pathway for metabolism of phenylacetic acid and similar compounds, Ibu-2 removes the acidic side chain of ibuprofen and related compounds prior to ring cleavage.     

Identification of the degradation products of aspergillic acid by Trichoderma koningii M 102

Isolation and identification of the degradation products of aspergillic acid, a secondary metabolite of fungi, by Trichoderma koningii M 102 were undertaken. ¹⁴C-labeling experiments indicated that aspergillic acid was broken-down to a water-soluble degradation product and a chloroform-soluble one. Consequently, leucine and a new microbial metabolite, 2-hydroxyimino-3-methyl-1-pentanol, were isolated and identified as the degradation products formed by cleavage of the pyrazine ring of aspergillic acid by T. koningii M 102.     

Evidence that the neuroleptic fluphenazine replaces Ca2+ and adjusts the noradrenaline induced cyclic adenosine monophosphate synthesis in the rat retina

Intact rat retinae were incubated in Krebs-Ringer media with noradrenaline (NA) in the presence (0.75 mM) or absence of extracellular Ca²⁺ and at relatively high (10 mM) or low (1 mM) theophylline concentrations. Depending on the incubation conditions we found that the neuroleptic fluphenazine (FLU) affected cAMP-synthesis separately from cAMP-degradation of the NA-cAMP system in the retina. The main results were: At a relatively high theophylline concentration of 10 mM, where cAMP synthesis alone is operative, and at 0.75 Ca²⁺ we measured with 50 μM NA a NA-response of 110 pmol cAMP/mg prot. At a low theophylline concentration of 1 mM and again at 0.75 mM Ca²⁺ both cAMP-synthesis and -breakdown are operative. In this condition we found the NA-response of 26 pmol cAMP/mg prot. to be raised by 10 μM FLU to 130 pmol cAMP/mg prot. This enhancing effect might be due to inhibition of degradation of NA-induced cAMP by FLU. In the absence of extracellular calcium and again at 10 mM theophylline, 10 μM FLU raised the NA response nearly 4-fold from 42 pmol cAMP/mg prot. to 153 pmol cAMP/mg prot. The lowest effective concentration for obtaining this enhancing effect was 10 μM FLU and the effect is characterized by an apparent K_m of 0.5 μM. The use of 10 mM theophylline in this condition suggests that this FLU-Ca²⁺ effect is confined to the synthesis part of the NA-cAMP system. The effect points to a replacement of an intramembraneous Ca²⁺ function by FLU. In conclusion: our results suggest that FLU inhibits degradation of NA-induced synthesis of cAMP and that the neuroleptic renders the NA-response less dependent on extracellular Ca²⁺.     

Assimilation of aromatic compounds by Comamonas testosteroni: characterization and spreadability of protocatechuate 4,5-cleavage pathway in bacteria

Comamonas testosteroni strain CNB-1 was isolated from activated sludge and has been investigated for its ability to degrade 4-chloronitrobenzene. Results from this study showed that strain CNB-1 grew on phenol, gentisate, vanillate, 3-hydroxybenzoate (3HB), and 4-hydroxybenzoate (4HB) as carbon and energy sources. Proteomic data and enzyme activity assays suggested that vanillate, 3HB, and 4HB were degraded in strain CNB-1 via protocatechuate (PCA) 4,5-cleavage pathway. The genetics and biochemistry of the PCA 4,5-cleavage pathway were investigated. Results showed that the 4-oxalomesaconate (OMA) hydratase from C. testosteroni takes only enol-OMA as substrate. A previously functionally unknown gene pmdU encodes an OMA tautomerase and catalyzes conversion of OMA_keto into OMA_enol. The 4-carboxy-4-hydroxy-2-oxoadipate (CHA) aldolase is encoded by pmdF and catalyzes the last step of the PCA 4,5-cleavage pathway. We explored the 1,183 microbial genomes at GenBank for potential PCA 4,5-cleavage pathways, and 33 putative pmd clusters were found. Results suggest that PCA 4,5-cleavage pathways are mainly distributed in α- and β-Proteobacteria.     

Gene Encoding the Hydrolase for the Product of the meta-Cleavage Reaction in Testosterone Degradation by Comamonas testosteroni

In a previous study we isolated the meta-cleavage enzyme gene, tesB, that encodes an enzyme that carries out a meta-cleavage reaction in the breakdown of testosterone by Comamonas testeroni TA441 (M. Horinouchi et al., Microbiology 147:3367-3375, 2001). Here we report the isolation of a gene, tesD, that encodes a hydrolase which acts on the product of the meta-cleavage reaction. We isolated tesD by using a Tn5 mutant of TA441 that showed limited growth on testosterone. TesD exhibited ca. 40% identity in amino acid sequence with BphDs, known hydrolases of biphenyl degradation in Pseudomonas spp. The TesD-disrupted mutant showed limited growth on testosterone, and the culture shows an intense yellow color. High-pressure liquid chromatography analysis of the culture of TesD-disrupted mutant incubated with testosterone detected five major intermediate compounds, one of which, showing yellow color under neutral conditions, was considered to be the product of the meta-cleavage reaction. The methylation product was analyzed and identified as methyl-4,5-9,10-diseco-3-methoxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oate, indicating that the substrate of TesD in testosterone degradation is 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid. 4,5-9,10-Diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid was transformed by Escherichia coli-expressed TesD. Downstream of tesD, we identified tesE, F, and G, which encode for enzymes that degrade one of the products of 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid converted by TesD.     

Biosynthesis of 1α,2α,3β-trihydroxy-p-menthane by Fusicoccum amygdali

Measurement of isotope ratios in 1α,2α,3β-trihydroxy-p-menthane, which has been biosynthesized in Fusicoccum amygdali from ³H- and ¹⁴C-labelled mevalonate and in its degradation product diosphenol indicates that: (a) four tritium atoms arising from [5-³H₂, 2-¹⁴C]MVA are retained, one more than suggested from the hydroxylation pattern, (b) menth-2-ene-1-ol is generated from an α-terpinyl cation through a 1,3-hydride shift and (c) trans-cleavage of an α-epoxide by hydrolysis gives 1α,2α,3β-trihydroxy-p-menthane.     

Extracellular potassium controls responsiveness of the noradrenergic cAMP system in the rat retina to fluphenazine

The effects of fluphenazine (FLU) on the noradrenaline (NA) induced cAMP-synthesis in intact rat retinae were studied as a function of extracellular K⁺- and Ca²⁺-ions. Thus NA-induced cAMP levels were measured after incubating intact rat retinae with 50 μM NA in the presence or absence of FLU and in the presence of 1 or 10 mM theophylline. Results were: (1) Experimental condition a: standard NA-responses were measured after incubating retinae at 0.75 mM Ca²⁺, at 10 mM theophylline, at 10 μM FLU and at 2 and 0 mM K⁺. FLU does not affect the NA-response at 2 mM K⁺ significantly; however, it inhibits the NA-response at 0 mM K⁺ in this condition. (2) Experimental condition b: NA-responses were measured after incubating retinae at 0.125 mM Ca²⁺, 10 mM theophylline, 10 μM FLU and at 2 and 0 mM K⁺. At 2 mM K⁺ FLU replaces a Ca²⁺ function probably connected with the synthesis part of the NA-cAMP system and NA-responses in this low Ca²⁺ condition are consequently enhanced by FLU; however, FLU inhibits the NA-response at 0 mM K⁺ in this condition. (3) Experimental condition c: NA-responses were measured after incubating retinae at 0.75 mM Ca²⁺, 1 mM theophylline, 10 μM FLU and at 2 and 0 mM K⁺. At 2 mM K⁺ FLU enhances the NA-response by further inhibition of the degradation part of the NA-cAMP system; FLU inhibits the NA-response at 0 mM K⁺ in this condition. (4) The inhibitions of the NA-responses by FLU at 0 mM K⁺ in all three conditions a, b and c showed an apparent K_m of 1 μM. (5) Low concentrations of K⁺ (0.4–0.8 mM) maintain the property of FLU to enhance the NA-responses at condition b (0.125 mM Ca²⁺) and at condition c (1 mM theophylline). Results suggest that the activation of NA-receptor coupled adenylate cyclases (NA-AC-ases) by NA, resulting in activation of phosphodiesterase activity by the NA-elevated cAMP-levels, is sustained by (a) membraneous factor(s) connected to the NA-receptor. This (these) factor(s) is (are) switched off in the absence of K⁺. Evidence has been presented, that Ca²⁺ and FLU do not have access to this intramembraneous factor-enzyme activating moiety of the NA-cAMP system at 0 mM K⁺. Between 0.4 and 0.8 mM K⁺ the factor-enzyme-NA-receptor complex is still intact.