Metabolism of 4-chloro-2-methylphenoxyacetate by a soil pseudomonad. Preliminary evidence for the metabolic pathway

1. A pseudomonad capable of utilizing the herbicide 4-chloro-2-methylphenoxyacetate as a sole carbon source was isolated from soil and cultured in liquid medium. 2. Analysis of induction patterns of 4-chloro-2-methylphenoxyacetate-grown cells suggests that 5-chloro-o-cresol and 5-chloro-3-methylcatechol are early intermediates in the oxidation of 4-chloro-2-methylphenoxyacetate. Cells were not adapted to oxidize 4-chloro-6-hydroxy-2-methylphenoxyacetate. 3. In culture, 4-chloro-2-methylphenoxyacetate rapidly disappeared and the chlorine in the molecule was quantitatively released as Cl(-) ion. 4. A lactone (gamma-carboxymethylene-alpha-methyl-Delta(alphabeta)-butenolide) was isolated from cultures and established as an intermediate. 5. The following metabolic pathway is suggested: 4-chloro-2-methylphenoxyacetate --> 5-chloro-o-cresol --> 5-chloro-3-methylcatechol --> cis-cis-gamma-chloro-alpha-methylmuconate --> gamma-carboxymethylene-alpha-methyl-Delta(alphabeta)-butenolide --> gamma-hydroxy-alpha-methylmuconate. 6. The tentative identification of 5-chloro-o-cresol, a gamma-chloro-alpha-methylmuconate and gamma-hydroxy-alpha-methylmuconate in culture extracts supports this scheme. However, the catechol was never observed to accumulate in cultures. 7. The detection of 4-chloro-6-hydroxy-2-methylphenoxyacetate, 2-methyl-phenoxyacetate, a dehalogenated cresol and oxalate in culture extracts is discussed in relation to the proposed metabolic pathway.     

Metabolism of 3-chloro-, 4-chloro-, and 3,5-dichlorobenzoate by a pseudomonad.

Pseudomonas sp. WR912 was isolated by continuous enrichment in three steps with 3-chloro-, 4-chloro-, and finally 3,5-dichlorobenzoate as sole source of carbon and energy. The doubling times of the pure culture with these growth substrates were 2.6, 3.3, and 5.2 h, respectively. Stoichiometric amounts of chloride were eliminated during growth. Oxygen uptake rates with chlorinated benzoates revealed low stereospecificity of the initial benzoate 1,2-dioxygenation. Dihydrodi-hydroxybenzoate dehydrogenase, catechol 1,2-dixoygenase, and muconate cycloisomerase activities were found in cell-free extracts. The ortho cleavage activity for catechols appeared to involve induction of isoenzymes with different stereospecificity towards chlorocatechols. A catabolic pathway for chlorocatechols was proposed on the basis of similarity to chlorophenoxyacetate catabolism, and cometabolism of 3,5-dimethylbenzoate by chlorobenzoate-induced cells yielded 2,5-dihydro-2,4-dimethyl-5-oxo-furan-2-acetic acid.     

Metabolism of 3-chloro-, 4-chloro-, and 3,5-dichlorobenzoate by a pseudomonad.

Pseudomonas sp. WR912 was isolated by continuous enrichment in three steps with 3-chloro-, 4-chloro-, and finally 3,5-dichlorobenzoate as sole source of carbon and energy. The doubling times of the pure culture with these growth substrates were 2.6, 3.3, and 5.2 h, respectively. Stoichiometric amounts of chloride were eliminated during growth. Oxygen uptake rates with chlorinated benzoates revealed low stereospecificity of the initial benzoate 1,2-dioxygenation. Dihydrodi-hydroxybenzoate dehydrogenase, catechol 1,2-dixoygenase, and muconate cycloisomerase activities were found in cell-free extracts. The ortho cleavage activity for catechols appeared to involve induction of isoenzymes with different stereospecificity towards chlorocatechols. A catabolic pathway for chlorocatechols was proposed on the basis of similarity to chlorophenoxyacetate catabolism, and cometabolism of 3,5-dimethylbenzoate by chlorobenzoate-induced cells yielded 2,5-dihydro-2,4-dimethyl-5-oxo-furan-2-acetic acid.     

Metabolism of 2-chloro-4-methylphenoxyacetate by Alcaligenes eutrophus JMP 134

2-Chloro-4-methylphenoxyacetate is not a growth substrate for Alcaligenes eutrophus JMP 134 and JMP 1341. It is, however, being transformed by enzymes of 2,4-dichlorophenoxyacetic acid metabolism to 2-chloro-4-methyl-cis, cis-muconate, which is converted by enzymatic 1,4-cycloisomerization to 4-carboxymethyl-2-chloro-4-methylmuconolactone as a dead end metabolite. Chemically, only 3,6-cycloisomerization occurs, giving rise to both diastereomers of 4-carboxychloromethyl-3-methylbut-2-en-4-olide. Those lactones harbonring a chlorosubstituent on the 4-carboxymethyl side chain were surprisingly stable under physiological as well as acidic conditions.     

Bacterial metabolism of 4-chloro-2-methylphenoxyacetate. Formation of glyoxylate by side-chain cleavage

Crude extracts of Pseudomonas sp. grown on 4-chloro-2-methylphenoxyacetate as sole source of carbon were shown to oxidize 4-chloro-2-methylphenoxyacetate to 5-chloro-o-cresol and glyoxylate. A labelled 2,4-dinitrophenylhydrazone was isolated from an incubation mixture in which 4-chloro-2-methylphenoxy[carboxy-¹⁴C]acetate was used. The hydrazone was shown to behave identically on thin-layer chromatograms with the authentic 2,4-dinitrophenylhydrazone of glyoxylate. Radioactivity assay showed that 82% of the side chain of 4-chloro-2-methylphenoxyacetate was recovered as glyoxylate.     

Metabolism of 4-chloro-2-methylphenoxyacetic Acid by soil bacteria

A microorganism capable of degrading 4-chloro-2-methylphenoxyacetic acid (MCPA) was isolated from soil and identified as Flavobacterium peregrinum. All of the chlorine of MCPA was released as chloride, and the carboxyl-carbon was converted to volatile products by growing cultures of the bacterium, but a phenol accumulated in the medium. The phenol was identified as 4-chloro-2-methylphenol on the basis of its gas chromatographic and infrared characteristics. Extracts of cells of F. peregrinum and of a phenoxyacetate-metabolizing Arthrobacter sp. dehalogenated MCPA and several catechols but not 4-chloro-2-methylanisole. The Arthrobacter sp. cell extract was fractionated, and an enzyme preparation was obtained which catalyzed the conversion of MCPA to 4-chloro-2-methylphenol. The latter compound was not metabolized unless reduced nicotinamide adenine dinucleotide phosphate was added to the fractionated extract. The phenol in turn was apparently oxidized to a catechol by components of the enzyme preparation.     

Metabolism of beta-methylaspartate by a pseudomonad

A bacterium was isolated from soil which utilizes threo-beta-methyl-l-aspartate, certain other amino acids, and a variety of organic substances as single energy sources. It is, or closely resembles, Pseudomonas putida biotype B. The ability of this organism to rapidly decompose such amino acids is dependent on inducible enzyme systems. Dialyzed cell-free extracts of this bacterium metabolize beta-methylaspartate only when catalytic amounts of alpha-ketoglutarate, or pyruvate, and pyridoxal phosphate are also present. The main products formed from beta-methylaspartate under these conditions are alpha-aminobutyrate, carbon dioxide, and alpha-ketobutyrate. When l-aspartate is substituted for beta-methylaspartate in this system, it is converted mainly to alanine and carbon dioxide. beta-Methyloxalacetate is decarboxylated, and the resulting alpha-ketobutyrate is converted enzymatically in the presence of glutamate to alpha-aminobutyrate which accumulates. The added keto acids are converted, in part, to the corresponding amino acids probably by transamination. The data indicate that beta-methylaspartate is converted to alpha-aminobutyrate, and aspartate to alanine, by a circuitous transamination-beta-decarboxylation-transamination sequence rather than by a direct beta-decarboxylation.     

N-Alkane oxidation enzymes of a pseudomonad.

A nicotinamide adenine dinucleotide (NAD)-dependent n-alkane dehydrogenase and an NAD phosphate (reduced form)-dependent alkane hydroxylase have been purified from cell-free extracts of Pseudomonas sp. strain 196Aa grown anaerobically on n-alkane. The n-alkane dehydrogenase (fraction R-3), obtained as a single peak from Bio-Gel P-60, showed an overall 135-fold purification and was demonstrated by infrared spectroscopy and gas chromatography to convert n-decane to 1-decene. The alkene hydroxylase activity in the S-3 fraction, purified 167 times from diethylaminoethyl-cellulose, was shown by the same methodology to convert decene to decanol. Commercial ferredoxin has been shown to increase the alkane dehydrogenase activity. An NAD-, flavine adenine dinucleotide-, and iron-dependent alcohol dehydrogenase was demonstrated in the R-3 fraction. A mechanism for the anaerobic conversion of n-alkane to fatty acid has been proposed.     

Purification and properties of benzoate-4-hydroxylase from a soil pseudomonad.

Benzoate-4-hydroxylase from a soil pseudomonad was isolated and purified about 50-fold. Polyacrylamide gel electrophoresis of this enzyme preparation showed one major band and one minor band. The approximate molecular weight of the enzyme was found to be 120,000. Benzoate-4-hydroxylase was most active around pH 7.2. The enzyme showed requirements for tetrahydropteridine as the cofactor and molecular oxygen as the electron acceptor. NADPH, NADH, dithiothreitol, β-mercaptoethanol, and ascorbic acid when added alone to the reaction mixture did not support the hydroxylation reaction to any significant extent. However, when these compounds were added together with tetrahydropteridine, they stimulated the hydroxylation. This stimulation is probably due to the reduction of the oxidized pteridine back to the reduced form. This enzyme was activated by Fe²⁺ and benzoate. It was observed that benzoate-4-hydroxylase could catalyze the oxidation of NADPH in the presence of benzoate,p-aminobenzoate, p-nitrobenzoate, p-chlorobenzoate, and p-methylbenzoate, with only benzoate showing maximum hydroxylation. Inhibition studies with substrate analogs and their kinetic analysis revealed that the carboxyl group is involved in binding the substrate to the enzyme at the active center. The enzyme catalyzed the conversion of 1 mol of benzoate to 1 mol of p-hydroxybenzoate with the consumption of slightly more than 1 mol of NADPH and oxygen.     

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.     

Utilization of cutin by a pseudomonad isolated from soil

Summary A Pseudomonas sp., which has been isolated from orchard soil, is able to utilize cutin as a sole source of carbon. Products obtained from the culture filtrate corresponded to that obtained by alkaline hydrolysis of cutin.     

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     

Degradation of 2-fluorobenzoate by a pseudomonad

Pseudomonas (spp), isolated from a complex petrochemical sludge, was able to utilize 2-fluorobenzoate as its sole source of carbon and energy. At the end of the growth phase, about 42% of the organically bound fluoride was released. Catechol, 3-fluorocatechol, and 6-fluorodihydrodihydroxybenzoate were confirmed as intermediates by chromatographic and spectral analyses. During 2-fluorobenzoate metabolism, fluoride is eliminated before the aromaticity of the ring is lost. Twofold higher levels of catechol 1,2-oxygenase were detected in 2-fluorobenzoate-grown cells compared with cells grown on benzoate. When used as assay substrates, 3-chlorocatechol showed less catechol 1,2-oxygenase activity than catechol or 4-chlorocatechol. The ability to degrade 4-fluorobenzoate could be transferred toPseudomonas (spp) by the conjugal transfer of plasmid pWR1 fromPseudomonas sp. B13.     

Biotransformation of 4-chloro-2-nitrophenol into 5-chloro-2-methylbenzoxazole by a marine Bacillus sp. strain MW-1

Decolourization, detoxification and biotransformation of 4-chloro-2-nitrophenol (4C2NP) by Bacillus sp. strain MW-1 were studied. This strain decolorized 4C2NP only in the presence of an additional carbon source. On the basis of thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS), 4-chloro-2-aminophenol, 4-chloro-2-acetaminophenol and 5-chloro-2-methylbenzoxazole were identified as metabolites. Resting cells depleted 4C2NP with stoichiometric formation of 5-chloro-2-methyl benzoxazole. This is the first report of the formation of 5-chloro-2-methylbenzoxazole from 4C2NP by any bacterial strain.     

2, 4-Dichlorophenoxyacetate Metabolism by Arthrobacter sp.: Accumulation of a Chlorobutenolide

An enzyme preparation from 2,4-dichlorophenoxyacetate-grown Arthrobacter sp. converted cis,cis-2,4-dichloromuconate to chloromaleylacetate. The enzyme lactonizing the dichloromuconate to yield 2-chloro-4-carboxymethylene but-2-enolide was separated from the butenolide-delactonizing enzyme.     

The bacterial degradation of flavonoids. Hydroxylation of the A-ring of taxifolin by a soil pseudomonad

Cell-free extracts prepared from a Pseudomonas sp. grown on (+)-catechin oxidized taxifolin in the presence of NAD(P)H and molecular oxygen. The enzyme catalysing the reaction was partially purified and shown to be a flavoprotein. The product was identified as 3',4',5,7,8-pentahydroxyflavanonol (dihydrogossypetin).