Catabolism of 2,4,5-trimethyoxybenzoic acid and 3-methoxycrotonic acid.

4-Methoxygentisic acid was an intermediate formed when Arthrobacter degraded, 2,4,5-trimethoxybenzoic acid. Isolates of Pseudomonas and Arthrobacter from soil grew at the expense of 3-methoxycrotonic acid. Evidence is presented that enzymatic hydration, with elimination of methanol, accounted for replacement of the methoxyl group of 3-methoxycrotonic acid and also of one methoxyl group of 2,4,5-trimethoxybenzoic acid.     

Bacterial degradation of 3,4,5-trimethoxycinnamic acid with production of methanol.

When grown on 3,4,5-trimethoxycinnamic acid, a strain of Pseudomonas putida oxidized this compound and also 3,4,5-trimethoxybenzoic, 3,5-dimethoxy-4-hydroxybenzoic (syringic), and 3,4-dihydroxy-5-methoxybenzoic (3-O-methylgallic) acids, but 3,5-dimethoxy-4-hydroxycinnamic and other acids bearing structural resemblances to the growth substrate were oxidized only slowly. These results indicate that two carbon atoms of the side chain of 3,4,5-trimethoxycinnamate were released before oxidative demethylation occurred to give the ring-fission substrate, 3-O-methylgallate. Oxidation of 3,4,5-trimethoxycinnamate by intact cells gave equimolar amounts of methanol, which was derived from the methoxyl group of 3-O-methylgallate. The tricarboxylic acids, 4-carboxy-2-keto-3-hexenedioic and 4-carboxy-4-hydroxy-2-ketoadipic acids, were shown to be formed by the action of a cell extract upon 3-O-methylgallate; therefore, methanol was released either during or immediately after fission of the benzene nucleus. Cell extracts, prepared from several independent soil isolates after growth on 3,4,5-trimethoxy derivatives of benzoic, cinnamic, and beta-phenylpropionic acids, rapidly oxidized 3-O-methylgallate without added cofactors. It is concluded that the reactions investigated serve generally as a source of methanol in nature.     

Production of methanol from aromatic acids by Pseudomonas putida.

When grown at the expense of 3,4,5-trimethoxybenzoic acid, a strain of Pseudomonas putida oxidized this compound and also 3,5-dimethoxy-4-hydroxybenzoic (syringic) and 3,4-dihydroxy-5-methoxybenzoic (3-O-methylgallic) acids; but other hydroxy- or methoxy-benzoic acids were oxidized slowly or not at all. Radioactivity appeared exclusively in carbon dioxide when cells were incubated with [4-methoxyl-14C]trimethoxybenzoic acid, but was found mainly in methanol when[methoxyl-14C]3-O-methylgallic acid was metabolized. The identity of methanol was proved by analyzing the product from [methoxyl-13C]3-O-methylgallic acid by nuclear magnetic resonance spectroscopy and by isolating the labeled 3,5-dinitrobenzoic acid methyl ester, which was examined by mass spectrometry. These results, together with measurements of oxygen consumed in demethylations catalyzed by cell extracts, showed that two methoxyl groups of 3,4,5-trimethoxybenzoate and one of syringate were oxidized to give carbon dioxide and 3-O-methylgallate. This was then metabolized to pyruvate; the other product was presumed to be the 4-methyl ester of oxalacetic acid, for which cell extracts contained an inducible, specific esterase. P. putida did not metabolize the methanol released from this compound by hydrolysis. Support for the proposed reaction sequence was obtained by isolating mutants which, although able to convert 3,4,5-trimethoxybenzoic acid into 3-O-methylgallic acid, were unable to use either compound for growth.     

Degradation of 2,4,5-Trichlorophenoxyacetic acid by a Nocardioides simplex culture

A Nocardioides simplex strain 3E was isolated which totally dechlorinated 2,4,5-trichlorophenoxyacetic acid and was capable of its utilization as the sole source of carbon. The mechanism of 2,4,5-trichlorophenoxyacetic acid degradation by this strain was investigated. Chloroaromatic metabolites that occur in the lag, exponential and stationary growth phases of the strain Nocardioides simplex 3E were isolated and identified bases on a combination of TLC, GC-MS and HPLC data. Decomposition of 2,4,5-trichlorophenoxyacetic acid at the initial stage was shown to proceed by two pathways: via the splitting of the two-carbon fragment to yield 2,4,5-trichlorophenol and the reductive dechlorination to produce 2,4-dichlorophenoxyacetic acid. Hydrolytic dechlorination of 2,4,5-trichlorophenoxyacetic acid was found to yield dichlorohydroxyphenoxyacetic acid, thus pointing to the possible existence of a third branch at the initial stage of degradation of the xenobiotic. 2,4,5-Trichlorophenol and 2,4-dichlorophenoxyacetic acid produced during the metabolism of 2,4,5-trichlorophenoxyacetic acid and in experiments with resting cells are utilized by the strain Nocardioides simplex 3E as growth substrates.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - 2,4,5-TCP 2,4,5-trichlorophenol     

Demethylation of [14C]-labelled veratric acid and oxidation of methanol and formaldehyde by the white-rot fungus Phlebia radiata

Veratric acids 14C-labelled in carboxyl group, 3-OCH3, 4-OCH3, or aromatic ring together with unlabelled veratric acid were supplemented in the cultures of the white-rot fungus Phlebia radiata. The effect of various carbon sources on the release of 14CO2 was studied. Veratric acid was readily decarboxylated, maximally already on day 1 from the addition of [14COOH]-veratric acid. High amounts (4%) of glucose slightly repressed the decarboxylation. In medium supplemented with cellulose the methoxyl group in position 4 was much more readily mineralized to CO2 than the group in position 3. The maximum evolution was achieved on day 5, two days from the addition. Cellulose did not repress methanol oxidation but repression of methanol oxidation by glucose was detected in media supplemented with [O14CH3]-veratric acids and 14CH3OH. However, glucose did not repress oxidation of H14CHO. The apparent uptake of 14C by fungal mycelium, especially from methoxyl groups, but also from the aromatic ring, may partially be due to the strong slime formation observed in cellobiose medium. Also in cellobiose medium apparent uptake of 14C from 14C-labelled methoxyl groups was observed.     

Biodegradation of 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid by dichlorophenol-adapted microorganisms from freshwater,anaerobic sediments

Reductive dechlorination of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was investigated in anaerobic sediments by non-adapted microorganisms and by microorganisms adapted to either 2,4- or 3,4-dichlorophenol (DCP). The rate of dechlorination of 2,4-D was increased by adaptation of sediment microorganisms to 2,4-DCP while dechlorination by sediment microorganisms adapted to 3,4-DCP displayed a lag phase similar to non-adapted sediment slurries. Both 2,4- and 3,4-DCP-adapted microorganisms produced 4-chlorophenoxyacetic acid by ortho-chlorine removal. Lag phases prior to dechlorination of the initial addition of 2,4,5-T by DCP-adapted sediment microorganisms were comparable to those from non-adapted sediment slurries. However, the rates of dechlorination increased upon subsequent additions of 2,4,5-T. Biodegradation of 2,4,5-T by sediment microorganisms adapted to 2,4- and/ or 3,4-DCP produced 2,5-D as the initial intermediate followed by 3-chlorophenol and phenol indicating a para > ortho > meta order of dechlorination. Dechlorination of 2,4,5-T, by either adapted or non-adapted sediment microorganisms, progressed without detection of 2,4,5-trichlorophenol as an intermediate.     

Abscisic acid and gibberellic acid as factors in the translocation of auxin

The effects of abscisic acid (ABA) and gibberellic acid (GA)on the translocation of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)in beans (Phaseolus vulgaris L. cv. Stringless Greenpd) seedlingswere determined. ¹⁴C-labeled 2,4,5-T was injected in the stemtissue at the cotyledonary node in 1-µl amounts alongwith the AB or GA. ABA caused an enhancement of 2,4,5-T translocationto the lower hypocotyl and roots and promoted a decrease inthe accumulation of 2,4,5-T in the young expanding shoots andprimary leaves. GA promoted the accumulation of 2,4,5-T in youngshoots. The enhanced basipetal translocation of 2,4,5-T wasmeasurable after a few hours of treatment and was partiallyeffective even in the presence of the protein synthesis inhibitorcychloheximide. The GA effects on 2,4,5-T translocation werenullified by cycloheximide and were also noted after only afew hours of treatment. ¹Journal Article 2618 of the Agricultural Experiment Station,Oklahoma State University. (Received October 1, 1973; )     

Interaction of coumarin, gibberellic acid and abscisic acid in the translocation of auxin in bean seedlings

The effects of coumarin on the translocation of 2,4,5-trichlorophenoxyaceticacid (2,4,5-T) in bean (Phaseolus vulgaris L. cv. StringlessGreenpod) seedlings was determined. ¹⁴C-labeled 2,4,5-T wasinjected in the stem tissue at the cotyledonary node along withthe coumarin or the coumarin was added to the nutrient solutionprior to, or at the time of, 2,4,5-T treatment. The amount oftranslocation of radioactive 2,4,5-T to plant parts was thendetermined at various times after treatment. An immediate effectof coumarin was to enhance acropetal 2,4,5-T translocation tothe young shoots. This effect occurred at low 2,4,5-T treatmentlevels and appeared to be specific for 2,4,5-T since sucrosetranslocation was not affected. Prolonged treatment with coumarininhibited acropetal and basipetal 2,4,5-T translocation in amanner similar to prolonged treatment with abscisic acid (ABA).Gibberellin A₃ (GA) reversed the inhibitory effects of coumarinand ABA on 2,4,5-T acropetal translocation. ¹ Journal article 3244 of the Agricultural Experiment Station,Oklahoma State University. (Received December 6, 1976; )     

Raoultella planticola, a new strain degrading 2,4,5-trichlorophenoxyacetic acid

A new strain that degrades the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was isolated from soil, which was exposed to factors related to the petrochemical industry. According to its physiological, biochemical, cultural, and morphological traits, together with the sequence of the 16S rRNA gene, the strain was identified as Raoultella planticola 33-4ch. The strain could consume 2,4,5-T as a sole source of carbon and energy. The amount of 2,4,5-T in the culture medium decreased by 51% after five days of incubation. Raoultella planticola 33-4ch consumes 2,4,5-T to produce 4-chlorophenoxyacetic, phenoxyacetic, and 3-methyl-2,6-dioxo-4-hexenoic acids.     

Raoultella planticola, a new strain degrading 2,4,5-trichlorophenoxyacetic acid

A new strain that degrades the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was isolated from soil, which was exposed to factors related to the petrochemical industry. According to its physiological, biochemical, cultural, and morphological traits, together with the sequence of the 16S rRNA gene, the strain was identified as Raoultella planticola 33-4ch. The strain could consume 2,4,5-T as a sole source of carbon and energy. The amount of 2,4,5-T in the culture medium decreased by 51% after five days of incubation. Raoultella planticola 33-4ch consumes 2,4,5-T to produce 4-chlorophenoxyacetic, phenoxyacetic, and 3-methyl-2,6-dioxo-4-hexenoic acids.     

Positionsspezifische O-Demethylierung von Benzoesäuren in Weizenkeimpflanzen

Summary Various methoxybenzoic acids (anisic, veratric and 3,4,5-trimethoxybenzoic acid) labelled specifically in para and meta methoxyl groups as well as the corresponding 4-hydroxybenzoic acids were added to the nutrient solution of sterile cultures of wheat seedlings.The experiments show that the O-demethylation of benzoic acids is specific for para methoxy groups. meta-O-Methyl carbon atoms appeared only to a very low extent as CO₂ and no products formed by demethylation of these groups could be isolated.The products formed by O-demethylation of the para methoxyl groups could be identified as p-hydroxybenzoic acid from anisic acid, vanillic acid from veratric acid and syringic acid from trimethoxybenzoic acid. These 4-hydroxybenzoic acids are normally decarboxylated to a high extent after being fed to plants. When they are formed in the plants by O-demethylation they can be isolated partly as free acids but mainly as their glycosides and glucose esters. These observations and some other indications give evidence of a possible compartmentalisation of plant cells.

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Im Text verwendete Abkürzungen c COOH-¹⁴C - r Ring-¹⁴C - m O-Methyl-¹⁴C - As Anissäure - Hb p-Hydroxybenzoesäure - Vr Veratrumsäure - Vs Vanillinsäure - Sy Syringasäure - Tmb 3,4,5-Trimethoxybenzoesäure. Beispiel - mVr-3 Veratrumsäure-(3-O-Methyl-¹⁴C) - mVr-4 Veratrumsäure-(4-O-Methyl-¹⁴C) Herrn Prof. Dr. W. Flaig zum 60. Geburtstag gewidmet.     

Natural selection for 2,4,5-trichlorophenoxyacetic acid mineralizing bacteria in agent orange contaminated soil

Agent Orange contaminated soils were utilized in direct enrichment culture studies to isolate 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and 2,4-dichlorophenoxyacetic acid (2,4-D) mineralizing bacteria. Two bacterial cultures able to grow at the expense of 2,4,5-T and/or 2,4-D were isolated. The 2,4,5-T degrading culture was a mixed culture containing two bacteria, Burkholderia species strain JR7B2 and Burkholderia species strain JR7B3. JR7B3 was able to metabolize 2,4,5-T as the sole source of carbon and energy, and demonstrated the ability to affect metabolism of 2,4-D to a lesser degree. Strain JR7B3 was able to mineralize 2,4,5-T in pure culture and utilized 2,4,5-T in the presence of 0.01 yeast extract. Subsequent characterization of the 2,4-D degrading culture showed that one bacterium, Burkholderiaspecies strain JRB1, was able to utilize 2,4-D as a sole carbon and energy source in pure culture. Polymerase chain reaction (PCR) experiments utilizing known genetic sequences from other 2,4-D and 2,4,5-T degrading bacteria demonstrated that these organisms contain gene sequences similar to tfdA, B, C, E, and R (Strain JRB1) and the tftA, C, and E genes (Strain JR7B3). Expression analysis confirmed that tftA, C, and E and tfdA, B, and C were transcribed during 2,4,5-T and 2,4-D dependent growth, respectively. The results indicate a strong selective pressure for 2,4,5-T utilizing strains under field condition.     

Testing of 2,4,5-T-amino acid conjugates for mutagenic activity in Salmonella typhimurium strains

Since amino acid conjugates are plant metabolites of the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 5 amino acid conjugates (aspartic acid, glutamic acid, leucine, methionine and tryptophan) of 2,4,5-T were tested for possible mutagenic activity utilizing 5 strains of Salmonella typhimurium (TA97, TA98, TA100, TA1535 and TA1538) with and without rat-liver microsomal and cytosolic enzymes. These compounds did not cause any significant increase in reversions when compared with controls in the presence or absence of the activating system. Further, linear regression analysis showed no significant (p less than 0.05) dose-response relationships. Thus, it was concluded that the tested amino acid conjugates of 2,4,5-T are not mutagens or promutagens in these assays.     

Detoxification of 2,4,5-trichlorophenoxyacetic acid from contaminated soil by Pseudomonas cepacia

The strain of Pseudomonas cepacia, AC1100, capable of utilizing 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole source of carbon and energy can degrade 2,4,5-T in contaminated soil, removing more than 99% of 2,4,5-T present at 1 mg/g of soil within 1 week. Repeated application of AC1100 even allowed more than 90% removal of 2,4,5-T within 6 weeks from heavily contaminated soil containing as much as 20,000 ppm 2,4,5,-T (20 mg/g of soil). Microbial removal of 2,4,5-T allowed the soil to support growth of plants sensitive to low concentrations of 2,4,5-T. After 2,4,5-T removal, the titer of AC1100 in the soil rapidly fell to undetectable levels within a few weeks.     

Degradation of the chlorinated phenoxyacetate herbicides 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid by pure and mixed bacterial cultures

Combined cell suspensions of the 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)-metabolizing organism Pseudomonas cepacia AC1100, and the 2,4-dichlorophenoxyacetic acid (2,4-D)-metabolizing organism Alcaligenes eutrophus JMP134 were shown to effectively degrade either of these compounds provided as single substrates. These combined cell suspensions, however, poorly degraded mixtures of the two compounds provided at the same concentrations. Growth and viability studies revealed that such mixtures of 2,4-D and 2,4,5-T were toxic to AC1100 alone and to combinations of AC1100 and JMP134. High-pressure liquid chromatography analyses of culture supernatants of AC1100 incubated with 2,4-D and 2,4,5-T revealed the accumulation of chlorohydroquinone as an apparent dead-end catabolite of 2,4-D and the subsequent accumulation of both 2,4-dichlorophenol and 2,4,5-trichlorophenol. JMP134 cells incubated in the same medium did not catabolize 2,4,5-T and were also inhibited in initiating 2,4-D catabolism. A new derivative of strain AC1100 was constructed by the transfer into this organism of the 2,4-D-degradative plasmid pJP4 from strain JMP134. This new strain, designated RHJ1, was shown to efficiently degrade mixtures of 2,4-D and 2,4,5-T through the simultaneous metabolism of these compounds.     

Detoxification of 2,4,5-trichlorophenoxyacetic acid from contaminated soil by Pseudomonas cepacia.

The strain of Pseudomonas cepacia, AC1100, capable of utilizing 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole source of carbon and energy can degrade 2,4,5-T in contaminated soil, removing more than 99% of 2,4,5-T present at 1 mg/g of soil within 1 week. Repeated application of AC1100 even allowed more than 90% removal of 2,4,5-T within 6 weeks from heavily contaminated soil containing as much as 20,000 ppm 2,4,5,-T (20 mg/g of soil). Microbial removal of 2,4,5-T allowed the soil to support growth of plants sensitive to low concentrations of 2,4,5-T. After 2,4,5-T removal, the titer of AC1100 in the soil rapidly fell to undetectable levels within a few weeks.     

Sequence analysis of a gene cluster involved in metabolism of 2,4,5-trichlorophenoxyacetic acid by Burkholderia cepacia AC1100.

Burkholderia cepacia AC1100 utilizes 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole source of carbon and energy. PT88 is a chromosomal deletion mutant of B. cepacia AC1100 and is unable to grow on 2,4,5-T. The nucleotide sequence of a 5.5-kb chromosomal fragment from B. cepacia AC1100 which complemented PT88 for growth on 2,4,5-T was determined. The sequence revealed the presence of six open reading frames, designated ORF1 to ORF6. Five polypeptides were produced when this DNA region was under control of the T7 promoter in Escherichia coli; however, no polypeptide was produced from the fourth open reading frame, ORF4. Homology searches of protein sequence databases were performed to determine if the proteins involved in 2,4,5-T metabolism were similar to other biodegradative enzymes. In addition, complementation studies were used to determine which genes were essential for the metabolism of 2,4,5-T. The first gene of the cluster, ORF1, encoded a 37-kDa polypeptide which was essential for complementation of PT88 and showed significant homology to putative trans-chlorodienelactone isomerases. The next gene, ORF2, was necessary for complementation and encoded a 47-kDa protein which showed homology to glutathione reductases. ORF3 was not essential for complementation; however, both the 23-kDa protein encoded by ORF3 and the predicted amino acid sequence of ORF4 showed homology to glutathione S-transferases. ORF5, which encoded an 11-kDa polypeptide, was essential for growth on 2,4,5-T, but the amino acid sequence did not show homology to those of any known proteins. The last gene of the cluster, ORF6, was necessary for complementation of PT88, and the 32-kDa protein encoded by this gene showed homology to catechol and chlorocatechol-1,2-dioxygenases.     

Regulation of 2,4,5-trichlorophenoxyacetic acid and chlorophenol metabolism in Pseudomonas cepacia AC1100

The expression of the degradative genes encoding 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2,4,5-trichlorophenol (2,4,5-TCP), and pentachlorophenol (PCP) dechlorination in a 2,4,5-T-degrading strain of Pseudomonas cepacia was examined during growth on alternate carbon sources. The dechlorination mechanisms for all three compounds were expressed in 2,4,5-T- and 2,4,5-TCP-grown cells but were not expressed in cells grown on succinate, glucose, or lactate. The addition of 2,4,5-TCP or PCP to cells grown on succinate or lactate resulted in the expression of the 2,4,5-TCP dechlorination mechanism in resting cells after 1-h lag. This expression was prevented by the presence of chloramphenicol in the resting cell suspension. Succinate-plus-PCP-grown resting cells preincubated with 2,4,5-TCP fully induced the trichlorophenol dechlorination system and partially induced the PCP dechlorination system. Preincubation of succinate-plus-PCP-grown resting cells with PCP induced neither the 2,4,5-TCP nor the PCP dechlorinating system. Succinate-grown resting cells converted 2,4,5-T to 2,4,5-TCP even in the presence of chloramphenicol. Thus, the data indicate that the enzyme(s) which converts 2,4,5-T to 2,4,5-TCP is constitutively expressed, whereas those that convert 2,4,5-TCP to central intermediates are induced by 2,4,5-TCP but not by 2,4,5-T or PCP and are repressed in the presence of an alternate carbon source.     

Degradation of the chlorinated phenoxyacetate herbicides 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid by pure and mixed bacterial cultures.

Combined cell suspensions of the 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)-metabolizing organism Pseudomonas cepacia AC1100, and the 2,4-dichlorophenoxyacetic acid (2,4-D)-metabolizing organism Alcaligenes eutrophus JMP134 were shown to effectively degrade either of these compounds provided as single substrates. These combined cell suspensions, however, poorly degraded mixtures of the two compounds provided at the same concentrations. Growth and viability studies revealed that such mixtures of 2,4-D and 2,4,5-T were toxic to AC1100 alone and to combinations of AC1100 and JMP134. High-pressure liquid chromatography analyses of culture supernatants of AC1100 incubated with 2,4-D and 2,4,5-T revealed the accumulation of chlorohydroquinone as an apparent dead-end catabolite of 2,4-D and the subsequent accumulation of both 2,4-dichlorophenol and 2,4,5-trichlorophenol. JMP134 cells incubated in the same medium did not catabolize 2,4,5-T and were also inhibited in initiating 2,4-D catabolism. A new derivative of strain AC1100 was constructed by the transfer into this organism of the 2,4-D-degradative plasmid pJP4 from strain JMP134. This new strain, designated RHJ1, was shown to efficiently degrade mixtures of 2,4-D and 2,4,5-T through the simultaneous metabolism of these compounds.