Degradation of chlorosubstituted aromatic compounds by Pseudomonas sp. strain B13: fate of 3,5-dichlorocatechol

The degradation of 3,5-dichlorocatechol by enzymes of 3-chlorobenzoate-grown cells of Pseudomonas sp. strain B13 was studied. The following compounds were formed from 3,5-dichlorocatechol: trans-2-chloro-4-carboxymethylenebut-2-en-4-olide, cis-2-chloro-4-carboxymethylenebut-2-en-4-olide, and chloroacetylacrylate as the decarboxylation product of 2-chloromaleylacetate. They were identified by chromatographic and spectroscopic methods (UV, MS, PMR). An enzyme activity converting trans-2-chloro-4-carboxymethylenebut-2-en-4-olide into the cis-isomer was observed.Abbreviations 3CB 3-chlorobenzoate - 4CB 4-chlorobenzoate - 3,5DCB 3,5-dichlorobenzoate - 2,4D 2,4-dichlorophenoxyacetate - NOE Nuclear-Overhauser-Effect     

Degradation of 1,4-dichlorobenzene by enriched and constructed bacteria

Summary Three strains, RHO1, R3 and B1, tentatively identified as a Pseudomonas sp., an Alcaligenes sp. and a Pseudomonas sp. which were able to use 1,4-dichlorobenzene as the sole carbon and energy source were isolated from water of the Rhine river and from the sewage plant at Leverkusen-Bürrig. A hybrid strain, WR1313, which uses chlorobenzene as the growth substrate, was obtained by mating the benzene-growing Pseudomonas putida strain F1 with strain B13, a Pseudomonas sp. degrading chlorocatechols. Further selection of this strain for growth on 1,4-dichlorobenzene allowed the isolation of strain WR1323. During growth on 1,4-dichlorobenzene the strains released stoichiometric amounts of chloride. The affinity of the organisms to 1,4-dichlorobenzene was measured with strain R3 showing a K_s value of 1.2 mg/l. Respiration data and enzyme activities in cell extracts as well as the isolation of 3,6-dichlorocatechol from the culture fluid are consistent with the degradation of 1,4-dichlorobenzene via 3,6-dichlorocatechol, 2,5-dichloro-cis,cis-muconate, 2-chloro-4-carboxymethylenebut-2-en-4-olide.     

Metabolism of 2,4-dichlorophenoxyacetic acid, 4-chloro-2-methylphenoxyacetic acid and 2-methylphenoxyacetic acid by Alcaligenes eutrophus JMP 134

Of eleven substituted phenoxyacetic acids tested, only three (2,4-dichloro-, 4-chloro-2-methyl- and 2-methylphenoxyacetic acid) served as growth substrates for Alcaligenes eutrophus JMP 134. Whereas only one enzyme seems to be responsible for the initial cleavage of the ether bond, there was evidence for the presence of three different phenol hydroxylases in this strain. 3,5-Dichlorocatechol and 5-chloro-3-methylcatechol, metabolites of the degradation of 2,4-dichlorophenoxyacetic acid and 4-chloro-2-methylphenoxyacetic acid, respectively, were exclusively metabolized via the ortho-cleavage pathway. 2-Methylphenoxyacetic acid-grown cells showed simultaneous induction of meta- and ortho-cleavage enzymes. Two catechol 1,2-dioxygenases responsible for ortho-cleavage of the intermediate catechols were partially purified and characterized. One of these enzymes converted 3,5-dichlorocatechol considerably faster than catechol or 3-chlorocatechol. A new enzyme for the cycloisomerisation of muconates was found, which exhibited high activity against the ring-cleavage products of 3,5-dichlorocatechol and 4-chlorocatechol, but low activities against 2-chloromuconate and muconate.Non-standard abbreviations MCPA 4-chloro-2-methylphenoxyacetic acid - 2MPA 2-methylphenoxyacetic acid - PA phenoxyacetic acid     

Dependence of the conversion of chlorophenols by rhodococci on the number and position of chlorine atoms in the aromatic ring

Study of the conversion of chlorophenols byRhodococcus opacus 1G,R. rhodnii 135,R. rhodochrous 89, andR. opacus 1cp disclosed the dependence of the conversion rate and pathway on the number and position of chlorine atoms in the aromatic ring. The most active chlorophenol converter, strainR. opacus 1cp, grew on each of the three isomeric monochlorophenols and on 2,4-dichlorophenol; the rate of growth decreased from 4-chlorophenol to 3-chlorophenol and then to 2-chlorophenol. The parameters of growth on 2,4-dichlorophenol were the same as on 3-chlorophenol. None of the strains studied utilized trichlorophenols. A detailed study of the pathway of chlorophenol transformation showed that 3-chloro-, 4-chloro-, and 2,4-dichlorophenol were utilized by the strains via a modifiedortho-pathway. 2-Chlorophenol and 2,3-dichlorophenol were transformed by strainsR. opacus 1cp andR. rhodochrous 89 via corresponding 3-chloro- and 3,4-dichlorocatechols, which were then hydroxylated with the formation of 4-chloropyrogallol and 4,5-dichloropyrogallol; this route had not previously been described in bacteria. Phenol hydroxylase ofR. opacus 1G exhibited a previously undescribed catalytic pattern, catalyzing oxidative dehalogenation of 2,3,5-trichlorophenol with the formation of 3,5-dichlorocatechol but not hydroxylation of the nonsubstituted position 6.     

Regioselective hydroxylation of chlorobenzene and chlorophenols by a Pseudomonas putida

Summary Pseudomonas putida MST, previously isolated in the presence of -methylstyrene, has been shown to transform several substituted aromatic compounds. It was able to modify halogenated aromatic compounds by co-oxidation. It regiospecifically hydroxylates chlorobenzene and 2-chlorophenol to 3-chlorocatechol, and 4-chlorophenol to 4-chlorocatechol; both metabolites were identified in the cultures.     

Degradation of mixtures of chloroaromatic compounds in soil slurry by mixed cultures of specialized organisms

The degradation of a mixture of 13 chloroaromatics, 2-chloro-, 3-chloro-, 4-chloro- and 3,4-dichloroaniline,2-chloro-, 3-chloro-,4-chloro-, 3,4-dichloro-and 3,5-dichlorobenzoate, and chloro-,1,2-dichloro-, 1,4-dichloro- and 1,2,4-trichlorobenzene in soil slurries by a mixed culture of Pseudomonas acidovorans strain BN 3.1, Pseudomonas ruhlandii strain FRB2, Pseudomonas cepacia strain JH230 and Pseudomonas aeruginosa strain RHO1 was studied. About 70% of the organic bound chlorine was eliminated after 25 days from soil with a carbon content of 8% (soil 1) when 2–3 × 10⁵ cells/g soil of each of the strains were added to the slurries. The effect of the clean-up was demonstrated by a biological test using cress and wheat. Both plants showed good germination and growth on both non-contaminated soils and the contaminated soil 1 after the biotreatment with the strains. No growth was observed when the plants were incubated with the contaminated soil 1 and with the contaminated and biotreated soil 2 (carbon content 2.6%). This indicates that the remaining 30% of organic chlorine in soil 1 after biotreatment does not influence the germination and growth of the two plants tested. *** DIRECT SUPPORT *** AG903062 00010     

Transformation of chlorinated phenolic compounds in the genusRhodococcus

The ability of strains of the genusRhodococcus to transform chlorinated phenolic compounds was studied. Noninduced cells of several strains ofRhodococcus, covering at least eight species, were found to attack mono-, di-, and trichlorophenols by hydroxylation at theortho position to chlorocatechols. 3-chlorophenol and 4-chlorophenol were converted to 4-chlorocatechol, 2,3-dichlorophenol to 3,4-dichlorocatechol, and 3,4-di-chlorophenol to 4,5-dichlorocatechol. The chlorocatechols accumulated to nearly stoichiometric amounts. Other mono- and dichlorophenols were not transformed. The ability of the strains to hydroxylate chlorophenols correlated with the ability to grow on unsubstituted phenol as the sole source of carbon and energy. SeveralRhodococcus strains attacked chlorophenolic compounds by both hydroxylation and O-methylation. 2,3,4-, 2,3,5- and 3,4,5-trichlorophenol were hydroxylated to trichlorocatechol and then sequentially O-methylated to chloroguaiacol and chloroveratrole. Tetrachlo-rohydroquinone was O-methylated sequentially to tetrachloro-4-methoxy-phenol and tetrachloro-1,4-dimethoxybenzene. Several of the active strains had no known history of exposure to any chloroaromatic compound. Rhodococci are widely distributed in soil and sludge and these results suggest that this genus may play an important role in transformation of chlorinated phenolic compounds in the environment.     

Degradation of chloroanilines in soil slurry by specialized organisms

The microbial degradation of 2-chloro-, 3-chloro-, 4-chloro-, and 3,4-dichloroaniline was examined as single compounds as well as a mixture in soil slurries. At 30°C the degradation of chloroanilines by indigenous soil populations in soil slurries was observed when soil slurry was freshly contaminated or precontaminated to allow binding of chloroanilines to the soil matrix. Within 6 weeks, 3-chloro- and 3,4-dichloroaniline (each 2 mm) were degraded more rapidly (about 50% chloride elimination) than 4-chloro- and 2-chloroaniline, due to stronger adsorption of 4-chloroaniline and greater resistance of 2-chloroaniline. The addition of various supplements such as buffer, mineral salts and acetate only slightly influenced the degradation of chloroanilines by the indigenous soil populations. The mineralization was drastically enhanced when laboratory-selected chloroaniline-degraders (8·10⁶ cells/g) such as Pseudomonas acidovorans strain BN3.1 were supplemented to the soil slurries so that complete elimination of chloride from the chloroanilines occurred within 10 days. Correspondence to: F. R. Brunsbach     

Mineralization of low-chlorinated biphenyls by Burkholderia sp. strain LB400 and by a two-membered consortium upon directed interspecies transfer of chlorocatechol pathway genes

The biphenyl-mineralizing bacterium Burkholderia sp. strain LB400 also utilized 3-chloro-, 4-chloro-, 2,3′-dichloro- and 2,4′-dichlorobiphenyl for growth. By the attack of the initial enzyme a chlorine was eliminated dioxygenolytically from position 2 of one of the aromatic rings when hydrogens of both were substituted by chlorine. The strain mineralized 3-chloro- and 2,3′-dichlorobiphenyl via the central intermediate 3-chlorobenzoate through its chlorocatechol pathway enzymes, but excreted stoichiometric amounts of 4-chlorobenzoate from 4-chloro- and 2,4^′-dichlorobiphenyl. These two compounds were mineralized by a co-culture of strain LB400 and a derivative of the (methyl-) benzoate-degrading strain Pseudomonas putida mt-2 (TOL). The complete degradation was achieved upon transfer of a cluster of at least five genes, encoding the regulated chlorocatechol pathway operon, from strain LB400 to strain mt-2. This transfer was demonstrated by the polymerase chain reaction. Received: 15 April 1998 / Received revision: 12 June 1998 / Accepted: 19 June 1998     

Co-metabolism of methyl- and chloro-substituted catechols by an Achromobacter sp. possessing a new meta-cleaving oxygenase

Co-metabolism of 3-methylcatechol, 4-chlorocatechol and 3,5-dichlorocatechol by an Achromobacter sp. was shown to result in the accumulation of 2-hydroxy-3-methylmuconic semialdehyde, 4-chloro-2-hydroxymuconic semialdehyde and 3,5-dichloro-2-hydroxymuconic semialdehyde respectively. Formation of these products indicated that cleavage of the aromatic nucleus of the substituted catechols was accomplished by a new meta-cleaving enzyme, catechol 1,6-oxygenase. This enzyme was equally active on both chloro- and methyl-substituted catechols.     

A Nag-like dioxygenase initiates 3,4-dichloronitrobenzene degradation via 4,5-dichlorocatechol in Diaphorobacter sp. strain JS3050

The chemical synthesis intermediate 3,4-dichloronitrobenzene (3,4-DCNB) is an environmental pollutant. Diaphorobacter sp. strain JS3050 utilizes 3,4-DCNB as a sole source of carbon, nitrogen and energy. However, the molecular determinants of its catabolism are poorly understood. Here, the complete genome of strain JS3050 was sequenced and key genes were expressed heterologously to establish the details of its degradation pathway. A chromosome-encoded three-component nitroarene dioxygenase (DcnAaAbAcAd) converted 3,4-DCNB stoichiometrically to 4,5-dichlorocatechol, which was transformed to 3,4-dichloromuconate by a plasmid-borne ring-cleavage chlorocatechol 1,2-dioxygenase (DcnC). On the chromosome, there are also genes encoding enzymes (DcnDEF) responsible for the subsequent transformation of 3,4-dichloromuconate to β-ketoadipic acid. The fact that the genes responsible for the catabolic pathway are separately located on plasmid and chromosome indicates that recent assembly and ongoing evolution of the genes encoding the pathway is likely. The regiospecificity of 4,5-dichlorocatechol formation from 3,4-DCNB by DcnAaAbAcAd represents a sophisticated evolution of the nitroarene dioxygenase that avoids misrouting of toxic intermediates. The findings enhance the understanding of microbial catabolic diversity during adaptive evolution in response to xenobiotics released into the environment.     

Production of halogenated compounds byBjerkandera adusta

The white-rot fungusBjerkandera adusta produces volatile chlorinated phenyl compounds. The main compounds identified were 3-chloro-4-methoxybenzaldehyde (3-chloro-p-anisaldehyde), 3-chloro-4-methoxybenzyl alcohol (3-chloro-p-anisyl alcohol), 3,5-dichloro-4-methoxybenzaldehyde (3,5-dichloro-p-anisaldehyde), and 3,5-dichloro, 4-methoxybenzyl alcohol (3,5-dichloro-p-anisyl alcohol).p-Anisaldehyde, veratraldehyde and the corresponding alcohols,p-anisyl alcohol and veratryl alcohol were produced simultaneously. Even with a very low concentration of chloride in the medium (< 10^–5 m), chlorinated aromatic compounds were still observed. Addition of bromide to the culture medium led to the production of brominated compounds: 3-bromo-4-methoxybenzaldehyde, 3-bromo-4-methoxybenzyl alcohol, 3,5-dibromo-4-methoxybenzaldehyde and 3-bromo-5-chloro-4-methoxybenzaldehyde. These brominated compounds have not previously been reported as natural products. Although iodo-aromatic compounds were not produced by supplementation of the medium with iodide, isovanillin was found in the culture broth under these conditions. This compound may be formed by substitution of the iodine intermediate by a hydroxyl group on the third carbon of the ring. Diiodomethane or chloroiodomethane were also found. It is the first time that the production of halomethane has been related to the production of halogenated aromatic compounds. All the strains tested have these capabilities.     

Effect of Enhancers on the Pyridopyridazine–Peroxide–HRP Reaction

4-Substituted phenyl boronic acids (e.g., 4-iodo, 4-bromo, 4-phenyl) are effective enhancers of the horseradish peroxidase (Type VIA) catalysed chemiluminescent oxidation of various pyrido[3,4-d]pyridazine-1,4(2H,3H)dione derivatives. The most effective combination was 4-biphenylboronic acid and 8-amino-5-chloro-7- phenylpyrido[3,4-d]- pyridazine-1,4(2H,3H)dione. Generally, the intensity of light emission in the presence of peroxidase was higher with the pyridopyridazines than with sodium luminol. However, the blank light emission was much lower with sodium luminol than with the pyridopyridazines. A synergistic enhancement phenomenon was demonstrated for the combination of a 4-iodophenol and a 4-biphenylboronic acid enhancer with 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)dione. The combination of these two enhancers produced a light emission intensity in an assay for 5 fmol of peroxidase that was 25% higher than expected from the sum of the individual light intensities.     

Diphenylurea Derivatives Induce Somatic Embryogenesis in Citrus

The present research investigates the possibility that three diphenylurea (DPU) derivatives, N-phenyl-N′-benzothiazol-6-ylurea (PBU), N,N′-bis-(2,3-methilendioxyphenyl)urea (2,3-MDPU) and N,N′-bis-(3,4-methilendioxyphenyl)urea (3,4-MDPU), stimulate the induction of somatic embryogenesis in three Citrus species. The hypothetical embryogenic activity was assessed using stigma and styles of Citrus myrtifolia Raf., Citrus madurensis Lour. and Citrus limon (L.) Burm. The three compounds influenced the production of somatic embryos differently as regards the concentrations tested and the citrus species. PBU was able to induce somatic embryogenesis at all the concentrations tested and in all the three species with percentages that ranged from 44 (C. limon) to 85% (C. myrtifolia). 2,3-MDPU and 3,4-MDPU were completely unable to induce the production of somatic embryos in C. myrtifolia while both the compounds at the higher concentration (12 μM) acted positively in both C. madurensis and C. limon (68% of embryogenic explants). The phenylurea derivatives, used for the first time in this study to induce somatic embryogenesis in plant, showed a higher embriogenic performance when compared with 6-benzylaminopurine (BAP), a classical adenine-cytokinin, and with N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU), a classical DPU derivative.     

Studies on the Lipids of Scale Insects

Corynebacterium cultures, isolated from soil by the use of n-paraffin as the sole carbon source have been shown to bring about significant oxidation of a new compound, dl-cistetrahydro-2-oxo-4-n-pentyl-thieno-(3,4-d)-imidazoline (dl-TOPTI), in the presence of hydrocarbon.

The co-oxidation products were isolated from culture broth and identified as dl-biotinol and dl-biotin by IR, NMR and MS spectroscopies.     

Poly(3-hydroxyalkanoate) polymerase synthesis and in vitro activity in recombinant Escherichia coli and Pseudomonas putida

We tested the synthesis and in vitro activity of the poly(3-hydroxyalkanoate) (PHA) polymerase 1 from Pseudomonas putida GPo1 in both P. putida GPp104 and Escherichia coli JMU193. The polymerase encoding gene phaC1 was expressed using the inducible PalkB promoter. It was found that the production of polymerase could be modulated over a wide range of protein levels by varying inducer concentrations. The optimal inducer dicyclopropylketone concentrations for PHA production were at 0.03% (v/v) for P. putida and 0.005% (v/v) for E. coli. Under these concentrations the maximal polymerase level synthesized in the E. coli host (6% of total protein) was about three- to fourfold less than that in P. putida (20%), whereas the maximal level of PHA synthesized in the E. coli host (8% of total cell dry weight) was about fourfold less than that in P. putida (30%). In P. putida, the highest specific activity of polymerase was found in the mid-exponential growth phase with a maximum of 40 U/g polymerase, whereas in E. coli, the maximal specific polymerase activity was found in the early stationary growth phase (2 U/g polymerase). Our results suggest that optimal functioning of the PHA polymerase requires factors or a molecular environment that is available in P. putida but not in E. coli.     

Reduction of aryl acids by white-rot fungi for the biocatalytic production of aryl aldehydes and alcohols

Ligninolytic basidiomycetes were screened for their ability to reduce aryl acids to the corresponding aldehydes and alcohols. Seven fungal strains converted p-anisic acid in high molar yields to the reduced products. The white-rot fungus Bjerkandera sp. strain BOS55 was one of the best reducing strains and was highly tolerant towards high concentrations of different aromatic acids. It was tested for the reduction of p-anisic, veratric, 3-chloro-4-methoxybenzoic, 3,5-dichloro-4-methoxybenzoic, 3,4-dichlorobenzoic, 4-fluorobenzoic, and 3-nitrobenzoic acids. All of these compounds were reduced to their corresponding aldehydes and alcohols. Received: 22 March 1999 / Received revision: 12 July 1999 / Accepted: 1 August 1999     

Oxidation of 2-(2-bromoethyl)bromobenzene with toluene dioxygenase: Isolation and identification of new chiral synthons

2(2-Bromoethyl)bromobenzene was subjected to microbial oxidation by the whole cells of Pseudomonas putida 39/D and JM109(pDTG601) yielding (3R,4S)-2-(2-bromoethyl)-bromocyclohexa-1,5-diene-3,4-diol. © 1995 Wiley-Liss, Inc.     

Biosynthesis and cytoplasmic accumulation of a chlorinated catechol pigment during 3-chlorobenzoate aerobic co-metabolism in Pseudomonas fluorescens

A strain of Pseudomonas fluorescens was capable of co-metabolizing 3-chlorobenzoic acid with the production of a chlorinated catechol black pigment. A peroxidase and another enzymatic activity referred to as a polyphenol oxidase were found to be involved in the oxidation of 4-chlorocatechol to 4-chloro-1,2-benzoquinone, i.e. in the production of highly reactive substrates for pigment formation. Therefore, P. fluorescens cells were seen to take an active part not only in 3-chlorobenzoate mineralization but also in overall pigment production. pH was found to be a key parameter in the regulation of the activity of P. fluorescens oxidoreductive enzymes. Ultrastructural investigations showed that electron dense granules of pigment were distributed throughout the cytoplasm of Pseudomonas fluorescens cells grown in presence of 3-chlorobenzoate, as confirmed also by Thiéry cytochemical investigations.In these cells, an extensive contraction of the cytoplasm as well as a significant damage to the cell wall after two days of incubation, suggested that pigment production caused a premature death of the cells accompanied by the leakage of the cell content. Pigment production seemed to occur mostly in the cytoplasmic context where the electron dense material accumulates until it is released in the medium after the cell lysis.Abbreviations 3-CBA 3-chlorobenzoic acid - BA benzoic acid - 4-CC 4-chlorocatechol - 3-CC 3-chlorocatechol - MBTH 3-methyl-2-benzothiazolinone hydrazone - l-DOPA l-3,4-dihydroxyphenyl-alanine - SPB sodium phosphate buffer     

Improved <Emphasis Type="Italic">p</Emphasis>-hydroxybenzoate production by engineered <Emphasis Type="Italic">Pseudomonas putida</Emphasis> S12 by using a mixed-substrate feeding strategy

The key precursors for p-hydroxybenzoate production by engineered Pseudomonas putida S12 are phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P), for which the pentose phosphate (PP) pathway is an important source. Since PP pathway fluxes are typically low in pseudomonads, E4P and PEP availability is a likely bottleneck for aromatics production which may be alleviated by stimulating PP pathway fluxes via co-feeding of pentoses in addition to glucose or glycerol. As P. putida S12 lacks the natural ability to utilize xylose, the xylose isomerase pathway from E. coli was introduced into the p-hydroxybenzoate producing strain P. putida S12palB2. The initially inefficient xylose utilization was improved by evolutionary selection after which the p-hydroxybenzoate production was evaluated. Even without xylose-co-feeding, p-hydroxybenzoate production was improved in the evolved xylose-utilizing strain, which may indicate an intrinsically elevated PP pathway activity. Xylose co-feeding further improved the p-hydroxybenzoate yield when co-fed with either glucose or glycerol, up to 16.3 Cmol% (0.1 g p-hydroxybenzoate/g substrate). The yield improvements were most pronounced with glycerol, which probably related to the availability of the PEP precursor glyceraldehyde-3-phosphate (GAP). Thus, it was demonstrated that the production of aromatics such as p-hydroxybenzoate can be improved by co-feeding different carbon sources via different and partially artificial pathways. Moreover, this approach opens new perspectives for the efficient production of (fine) chemicals from renewable feedstocks such as lignocellulose that typically has a high content of both glucose and xylose and (crude) glycerol.