Branching of o-nitrobenzoate degradation pathway in Arthrobacter protophormiae RKJ100: identification of new intermediates

We have earlier reported a novel reductive pathway for o-nitrobenzoate (ONB) degradation (at 0.5 mM) in Arthrobacter protophormiae RKJ100, which proceeds via the formation of o-hydroxylaminobenzoate (HABA) and anthranilate (AA). During growth of this organism at 40 times higher concentration (20 mM) of ONB, 3-hydroxyanthranilate (HAA) was identified as an intermediate by thin layer chromatography, gas chromatography and high performance liquid chromatography studies. Crude cell extracts of ONB-grown cells showed HAA 3,4-dioxygenase activity suggesting HAA as a terminal aromatic intermediate of the catabolic energy-yielding pathway as shown before in Pseudomonas fluorescens strain KU-7. HAA is further cleaved to 2-amino-3-carboxymuconic-6-semialdehyde by the action of HAA 3,4-dioxygenase. In this report we propose that ONB degradation occurs via the formation of HABA and the pathway branches at this point to form the two different aromatic intermediates AA and HAA by the action of a reductase and a mutase, respectively.     

Biodegradation of carbazole by Ralstonia sp. RJGII.123 isolated from a hydrocarbon contaminated soil

The use of microorganisms for bioremediation of contaminated soils may be enhanced with an understanding of the pathways involved in their degradation of hazardous compounds. Ralstonia sp. strain RJGII.123 was isolated from soil located at a former coal gasification plant, based on its ability to mineralize carbazole, a three-ring N-heterocyclic pollutant. Experiments were carried out with strain RJGHII.123 and 14C-carbazole (2 mg/L and 500 mg/L) as the sole organic carbon source. At 15 days, 80% of the 2 mg/L carbazole was recovered as CO2, and <1% remained as undegraded carbazole, while 24% of the 500 mg/L carbazole was recovered as CO2 and approximately 70% remained as undegraded carbazole. Several stable intermediates were formed during this time. These intermediates were separated by high performance liquid chromatography (HPLC) and were characterized using high resolution mass spectroscopy (HR-MS) and gas chromatography - mass spectroscopy (GC-MS). At least 10 ring cleavage products of carbazole degradation were identified; four of these were confirmed as anthranilic acid, indole-2-carboxylic acid, indole-3-carboxylic acid, and (1H)-4-quinolinone by comparison with standards. These data indicate that strain RJGII.123 shares aspects of carbazole degradation with previously described Pseudomonas spp., and may be useful in facilitating the bioremediation of NHA from contaminated soils.     

Oxygen-dependent catabolism of indole-3-acetic acid in Bradyrhizobium japonicum.

Some strains of Bradyrhizobium japonicum have the ability to catabolize indole-3-acetic acid (IAA). Examination of this catabolism in strain 110 by in vivo experiments has revealed an enzymatic activity catalyzing the degradation of IAA and 5-hydroxy-indole-3-acetic acid. The activity requires addition of the substrates for induction and is oxygen dependent. The highest activity is obtained when the concentration of inducer is 0.2 mM. Spectrophotometric data are consistent with the suggestion that the indole ring is broken during degradation of IAA. We hypothesize that the enzyme catalyzes an oxygen-consuming opening of the indole ring analogous to the one catalyzed by tryptophan 2,3-dioxygenase. The pattern of metabolite usage by known tryptophan-auxotrophic mutants and studies of metabolites by high-performance liquid chromatography indicate that anthranilic acid is a terminal degradation product in the proposed pathway.     

Degradation of 4-nitrocatechol by Burkholderia cepacia: a plasmid-encoded novel pathway

Pseudomonas cepacia RKJ200 (now described as Burkholderia cepacia) has been shown to utilize p-nitrophenol (PNP) as sole carbon and energy source. The present work demonstrates that RKJ200 utilizes 4-nitrocatechol (NC) as the sole source of carbon, nitrogen and energy, and is degraded with concomitant release of nitrite ions. Several lines of evidence, including thin layer chromatography, gas chromatography, 1H-nuclear magnetic resonance, gas chromatography-mass spectrometry, spectral analyses and quantification of intermediates by high performance liquid chromatography, have shown that NC is degraded via 1,2, 4-benzenetriol (BT) and hydroquinone (HQ) formation. Studies carried out on a PNP- derivative and a PNP+ transconjugant also demonstrate that the genes for the NC degradative pathway reside on the plasmid present in RKJ200; the same plasmid had earlier been shown to encode genes for PNP degradation, which is also degraded via HQ formation. It is likely, therefore, that the same sets of genes encode the further metabolism of HQ in NC and PNP degradation.     

Microbial metabolism of quinoline and related compounds. V. Degradation of 1H-4-oxoquinoline by Pseudomonas putida 33/1

A bacterial strain was isolated with the ability to use 1H-4-oxoquinoline as the sole source of carbon, nitrogen and energy. On the basis of its physiological properties, this isolate was classified as Pseudomonas putida. 1H-3-Hydroxy-4-oxoquinoline, N-formylanthranilic acid, anthranilic acid and catechol were identified as intermediates in the degradation pathway. The latter was further degraded by ortho-cleavage. The enzymatic conversion of 1H-4-oxoquinoline into 1H-3-hydroxy-4-oxoquinoline requires oxygen and NADH. Experiments with 18O2 showed that the oxygen consumed in this enzymatic reaction is derived from the atmosphere.     

Accumulation of anthranilic acid and N-glucosylanthranilic acid by a Corynebacterium glutamicum mutant resistant to DL-serine hydroxamate

During a study on the effect of DL-serine hydroxamate on Corynebacterium glutamicum (JCM1318, a wild strain), a mutant resistant to the drug, strain TO3002, was isolated. This mutant accumulated five Ehrlich's reagent positive fluorescent substances in the culture medium. Two major and one minor fluorescent products were isolated by preparative high-performance liquid chromatography following charcoal column chromatography from the culture supernatant. One major product was identified as anthranilic acid whose molecular ion was confirmed to be 137 by a measurement of liquid chromatography-mass spectrometry (LC-MS), and NMR spectrum coincided with that of anthranilic acid. LC-MS spectra of another major and the minor product showed that they had the same molecular weight of 299. This major product was supported to be N-glucosylanthranilic acid (N-o-carboxyphenyl-1-beta-glucosylamine) by two-dimensional (1)H and (13)C NMR analyses. The minor product was speculated to be an Amadori compound derived from N-glucosylanthranilic acid. N-Glucosylanthranilic acid accumulated in the early phase, then decreased in the late phase of the culture. In contrast, the accumulation of anthranilic acid increased remarkably in the late phase of the fermentation. Based on this phenomenon, it was assumed that N-glucosylanthranilic acid once accumulated was decomposed to form anthranilic acid, at least in large part, with the progress of fermentation. The strain TO3002 showed a leaky requirement for L-tryptophan or indole (but did not for anthranilic acid) and resistance to DL-serine hydroxamate.     

Metabolic pathway of homophthalic acid in Pseudomonas alcaligenes

Abstract A microorganism capable of degrading homophthalic acid as a sole source was isolated from garden soil. The strain was identified as Pseudomonas alcaligenes . The organism degraded homphthalate by a pathway which involved phenylactate and p -hydroxyphenylacetate as intermediates. The intermediates have been identified by physico-chemical methods. A tentative pathway for the degradation of homophthalate is proposed based on isolation of intermediates, oxygen uptake studies and presence of enzymes involved in the degradation.     

Degradation of terephthalic acid by a Bacillus species

Abstract A Gram-positive bacterium with the ability to utilize terephthalic acid as sole carbon source was isolated from soil. The strain was identified as a Bacillus sp. Protocatechuate was shown to be a key intermediate in the degradation of terephthalate. Oxygen uptake studies were carried out with the probable intermediates. The presence of different enzymes was tested for. A mechanism is proposed for the degradation of terephthalate.     

Isolation and characterization of an anaerobic dehydrodivanillin-degrading bacterium

A novel, strictly anaerobic, gram-negative, non-spore-forming, fusiform, rod-shaped bacterium having high dehydrodivanillin (DDV)-degrading activity was isolated from cow ruminal fluid. This strain degraded a range of six main lignin-related compounds such as DDV, ferulic acid, dehydrodiisoeugenol, guaiacoxyacetic acid, vanillin, and veratrylglycerol-beta-guaiacyl ether to the extent of 14 to 83% within 2 days under strictly anaerobic conditions. As DDV degradation intermediates, three aromatic compounds (dehydrodivanillic acid, vanillic acid, and 5-carboxyvanillic acid) and two alicyclic compounds (cyclohexanecarboxylic acid and cyclohexanol) were detected by thin-layer, high-performance liquid, and gas chromatography and mass spectrometry. The addition of 1% glucose and peptone in a synthetic medium stimulated growth of the strain but slowed down DDV degradation. The presence of 0.1% yeast extract increased both cell growth and DDV degradation. The growth yield in defined medium was 151.5 g (dry weight) of cells per mol of DDV utilized. Characterization of the strain indicated that it was distinct from known Fusobacterium and Clostridium species. The bacterium was easily induced to form protoplasts after treatment with either penicillin or lysozyme. The frequencies of protoplast formation and regeneration in the strain were 94 and 18%, respectively.     

Isolation and characterization of an anaerobic dehydrodivanillin-degrading bacterium.

A novel, strictly anaerobic, gram-negative, non-spore-forming, fusiform, rod-shaped bacterium having high dehydrodivanillin (DDV)-degrading activity was isolated from cow ruminal fluid. This strain degraded a range of six main lignin-related compounds such as DDV, ferulic acid, dehydrodiisoeugenol, guaiacoxyacetic acid, vanillin, and veratrylglycerol-beta-guaiacyl ether to the extent of 14 to 83% within 2 days under strictly anaerobic conditions. As DDV degradation intermediates, three aromatic compounds (dehydrodivanillic acid, vanillic acid, and 5-carboxyvanillic acid) and two alicyclic compounds (cyclohexanecarboxylic acid and cyclohexanol) were detected by thin-layer, high-performance liquid, and gas chromatography and mass spectrometry. The addition of 1% glucose and peptone in a synthetic medium stimulated growth of the strain but slowed down DDV degradation. The presence of 0.1% yeast extract increased both cell growth and DDV degradation. The growth yield in defined medium was 151.5 g (dry weight) of cells per mol of DDV utilized. Characterization of the strain indicated that it was distinct from known Fusobacterium and Clostridium species. The bacterium was easily induced to form protoplasts after treatment with either penicillin or lysozyme. The frequencies of protoplast formation and regeneration in the strain were 94 and 18%, respectively.     

Iron requirement of Rhizobium leguminosarum and secretion of anthranilic acid during growth on an iron-deficient medium

Rhizobium leguminosarum GF160 required iron for growth under aerobic conditions in a chemically defined medium. Maximal growth of bacteria previously depleted in iron was obtained with approximately 50 microM unchelated ferric iron and with glucose as the only carbon source. Growth under iron deficiency did not result in the production of detectable levels of siderophores of either the catechol or hydroxamate types. Growing cells released a Fe3+-reducing agent that was identified as anthranilic acid by paper and thin-layer chromatography, ultraviolet and nuclear magnetic resonance spectroscopy, and mass spectrometry. The amount of anthranilic acid secreted per unit of cell growth was inversely related to the iron concentration in the culture medium and reached concentrations up to 1 mM. Ferric but not ferrous ions were solubilized in the growth medium by anthranilic acid.     

Metabolism of 2-chloro-4-nitrophenol in a gram negative bacterium, Burkholderia sp. RKJ 800

A 2-chloro-4-nitrophenol (2C4NP) degrading bacterial strain designated as RKJ 800 was isolated from a pesticide contaminated site of India by enrichment method and utilized 2C4NP as sole source of carbon and energy. The stoichiometric amounts of nitrite and chloride ions were detected during the degradation of 2C4NP. On the basis of thin layer chromatography, high performance liquid chromatography and gas chromatography-mass spectrometry, chlorohydroquinone (CHQ) and hydroquinone (HQ) were identified as major metabolites of the degradation pathway of 2C4NP. Manganese dependent HQ dioxygenase activity was observed in the crude extract of 2C4NP induced cells of the strain RKJ 800 that suggested the cleavage of the HQ to γ-hydroxymuconic semialdehyde. On the basis of the 16S rRNA gene sequencing, strain RKJ 800 was identified as a member of genus Burkholderia. Our studies clearly showed that Burkholderia sp. RKJ 800 degraded 2-chloro-4-nitrophenol via hydroquinone pathway. The pathway identified in a gram negative bacterium, Burkholderia sp. strain RKJ 800 was differed from previously reported 2C4NP degradation pathway in another gram-negative Burkholderia sp. SJ98. This is the first report of the formation of CHQ and HQ in the degradation of 2C4NP by any gram-negative bacteria. Laboratory-scale soil microcosm studies showed that strain RKJ 800 is a suitable candidate for bioremediation of 2C4NP contaminated sites.     

Biodegradation of the nitroaromatic herbicide dinoseb (2-sec-butyl-4,6-dinitrophenol) under reducing conditions

The degradation pathway for dinoseb (2-sec-butyl-4,6-dinitrophenol) under reducing conditions was investigated. Cultures were inoculated with a dinoseb-degrading anaerobic enrichment culture used in field studies. Biotransformation intermediates were extracted with ethyl acetate and analyzed by high pressure liquid chromatography, gas chromatography, and mass spectrometry. Dinoseb degradation involves reduction of the nitro groups to amino groups followed by replacement with hydroxyl groups. Depending on the pH and redox potential in the culture, these intermediates may exist as quinones or hydroquinones.Publication No. 94506 of the Idaho Agricultural Experiment Station     

Tryptophan catabolism in Bacillus megaterium.

Bacillus megaterium grows in a medium containing L-tryptophan as the sole carbon, nitrogen, and energy source. Kynurenine, anthranilic acid, and catechol are metabolic intermediates, suggesting that this organism used the anthranilic acid pathway for tryptophan degradation. Cells that grow on L-tryptophan oxidize kynurenine, alanine, and anthranilic acid and the presence of tryptophan oxygenase (EC 1.13.1.12), kynureninase (EC 3.7.1.3), and catechol oxygenase (EC 1.13.1.1) in cell extracts provide additional evidence for the degradative pathway in B. megaterium. Tryptophan oxygenase is inhibited by sodium azide, potassium cyanide, and hydroxylamine, indicating that the enzyme has a functional heme group. D-Tryptophan is not a substrate for tryptophan oxygenase, and the D-isomer does not inhibit this enzyme. Formamidase (EC 3.5.1.9) and anthranilate hydroxylase are not detectable in extracts. Tryptophan catabolism is inducible in B megaterium and is subject to catabolite repression by glucose and glutamate. Arginine does not cause repression, and kynurenine induces both tryptophan oxygenase and kynureninase.     

Enzymology of oxidation of tropic acid to phenylacetic acid in metabolism of atropine by Pseudomonas sp. strain AT3.

Pseudomonas sp. strain AT3 grew with dl-tropic acid, the aromatic component of the alkaloid atropine, as the sole source of carbon and energy. Tropic acid-grown cells rapidly oxidized the growth substrate, phenylacetaldehyde, and phenylacetic acid. Crude cell extracts, prepared from dl-tropic acid-grown cells, contained two NAD+-linked dehydrogenases which were separated by ion-exchange chromatography and shown to be specific for their respective substrates, dl-tropic acid and phenylacetaldehyde. Phenylacetaldehyde dehydrogenase was relatively unstable. The stable tropic acid dehydrogenase was purified to homogeneity by a combination of ion-exchange, molecular-sieve, and affinity chromatography. It had a pH optimum of 9.5 and was equally active with both enantiomers of tropic acid, and at this pH, phenylacetaldehyde was the only detectable product of tropic acid oxidation. The formation of phenylacetaldehyde from tropic acid requires, in addition to dehydrogenation, a decarboxylation step. By analogy with NAD+-specific isocitrate and malate dehydrogenases, phenylmalonic semialdehyde, a 3-oxoacid, would be expected to be the precursor of phenylacetaldehyde. Other workers have established that isocitrate and malate dehydrogenases catalyze the decarboxylation of enzyme-bound or added 3-oxoacid intermediates, a reaction that requires Mn2+ or Mg2+ ions. Studies with tropic acid dehydrogenase were hampered by lack of availability of phenylmalonic semialdehyde, but in the absence of added divalent metal ions, both enantiomers of tropic acid were completely oxidized and we have not, by a number of approaches, found any evidence for the transient accumulation of phenylmalonic semialdehyde.     

Microbial metabolism of quinoline and related compounds. VI. Degradation of quinaldine by Arthrobacter sp

Quinaldine catabolism was investigated with the bacterial strain Arthrobacter sp., which is able to grow aerobically in a mineral salt medium with quinaldine as sole source of carbon, nitrogen and energy. The following degradation products of quinaldine were isolated from the culture fluid and identified: 1H-4-oxoquinaldine, N-acetylisatic acid, N-acetylanthranilic acid, anthranilic acid, 3-hydroxy-N-acetylanthranilic acid and catechol. 3-Hydroxy-N-acetylanthranilic acid was not further metabolized by this organism. A degradation pathway is proposed.     

Metabolism of diethylphthalate by a soil bacterium

A microorganism capable of degrading diethylphthalate as a sole carbon source was isolated from garden soil and tentatively identified asMicrococcus sp. Monoethylphthalate and phthalic acid were shown to be the intermediates by thin-layer chromatography and spectrophotometric and mass spectral analysis. The strain degraded diethylphthalate mainly through monoethylphthalate and phthalic acid as was evidenced by oxygen uptake and enzymatic studies. Ethanol also supported the growth of this organism. It appeared that the entire molecule was metabolized byMicrococcus sp.     

Enzymatic synthesis of cyclopeptine intermediates in Penicillium cyclopium

Cyclopeptine, a benzodiazepine alkaloid of Penicillium cyclopium, is formed from anthranilic acid, L-phenylalanine and the methyl group of L-methionine by cyclopeptine synthetase. The following partial activities of this enzyme system were determined in vitro: anthranilic acid and L-phenylalanine adenylyltransferase activity, binding of anthranilic acid and L-phenylalanine as thioesters to proteins, formation of thioester-bound N-methyl-L-phenylalanine and N-methyl-L-phenylalanylanthranilic acid. The obtained results indicate that cyclopeptine is formed via enzyme-bound intermediates by the thiotemplate mechanism of peptide biosynthesis.     

Finding new pathway-specific regulators by clustering method using threshold standard deviation based on DNA chip data of <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis>

A Gram-positive bacterial strain capable of aerobic biodegradation of 4-fluorophenol (4-FP) as the sole source of carbon and energy was isolated by selective enrichment from soil samples collected near an industrial site. The organism, designated strain IF1, was identified as a member of the genus Arthrobacter on the basis of 16S ribosomal RNA gene sequence analysis. Arthrobacter strain IF1 was able to mineralize 4-FP up to concentrations of 5 mM in batch culture. Stoichiometric release of fluoride ions was observed, suggesting that there is no formation of halogenated dead-end products during 4-FP metabolism. The degradative pathway of 4-FP was investigated using enzyme assays and identification of intermediates by gas chromatography (GC), GC–mass spectrometry (MS), high-performance liquid chromatography, and liquid chromatography–MS. Cell-free extracts of 4-FP-grown cells contained no activity for catechol 1,2-dioxygenase or catechol 2,3-dioxygenase, which indicates that the pathway does not proceed through a catechol intermediate. Cells grown on 4-FP oxidized 4-FP, hydroquinone, and hydroxyquinol but not 4-fluorocatechol. During 4-FP metabolism, hydroquinone accumulated as a product. Hydroquinone could be converted to hydroxyquinol, which was further transformed into maleylacetic acid and β-ketoadipic acid. These results indicate that the biodegradation of 4-FP starts with a 4-FP monooxygenase reaction that yields benzoquinone, which is reduced to hydroquinone and further metabolized via the β-ketoadipic acid pathway.     

Isolation and characterization of an atrazine-degrading Rhodococcus sp. strain MB-P1 from contaminated soil

Aims:  The aim of this study is to isolate and characterize organisms capable of utilizing high concentration atrazine from the contaminated sites.
Methods and Results:  A selective enrichment was used for isolating atrazine-degrading organisms from the contaminated sites resulting in isolation of an efficient atrazine-degrading organism designated as strain MB-P1. On the basis of 16S rRNA gene sequencing, total cellular fatty acid analysis and physiological and biochemical tests, strain MB-P1 was identified as a member of genus Rhodococcus . High performance liquid chromatography was performed to identify the atrazine degradation intermediates demonstrating that the degradation proceeds via formation of 'de-ethylatrazine' and 'de-isopropylatrazine'. Further, plasmid curing by SDS method showed atrazine-degrading gene(s) to be plasmid-encoded.
Conclusions:  We have successfully isolated a Rhodococcus sp. strain MB-P1 which is capable of utilizing atrazine as sole source of carbon and energy at very high concentrations of 1000 ppm. The pathway for degradation of atrazine has also been determined. The metabolic gene(s) responsible for atrazine degradation was found to be plasmid-encoded.
Significance and Impact of the Study:  Rhodococcus sp. strain MB-P1 could be used as an ideal model system for in-situ degradation and restoration of ecological niches which are heavily contaminated with atrazine.