Degradation of glyphosate by Pseudomonas sp. PG2982 via a sarcosine intermediate

The bacterium Pseudomonas PG2982 metabolizes glyphosate (N-(phosphonomethyl)glycine) by converting it to glycine, a one-carbon unit, and phosphate. Here we show that this conversion involves the intermediate formation of sarcosine. When cells are incubated with [14C]glyphosate, the 14C can be entrapped in glycine or sarcosine. With added sarcosine, 14C from all three carbons of glyphosate is recovered solely in sarcosine. In experiments with glycine, radioactivity from the carboxymethyl moiety of glyphosate is trapped in glycine as well as serine, whereas radioactivity from the phosphonomethyl carbon is only incorporated into serine. These results are consistent with a pathway involving the conversion of glyphosate to sarcosine by cleavage of its carbon-phosphorus (C-P) bond, followed by the oxidation of sarcosine to glycine and formaldehyde.     

Degradation of the Phosphonate Herbicide Glyphosate by Arthrobacter atrocyaneus ATCC 13752

Of nine authentic Arthrobacter strains tested, only A. atrocyaneus ATCC 13752 was capable of using the herbicide glyphosate [N-(phosphonomethyl)glycine] as its sole source of phosphorus. Contrary to the previously isolated Arthrobacter sp. strain GLP-1, which degrades glyphosate via sarcosine, A. atrocyaneus metabolized glyphosate to aminomethylphosphonic acid. The carbon of aminomethylphosphonic acid was entirely converted to CO₂. This is the first report on glyphosate degradation by a bacterial strain without previous selection for glyphosate utilization as a source of phosphorus.     

Glyphosate catabolism by Pseudomonas sp. strain PG2982.

The pathway for the degradation of glyphosate (N-phosphonomethylglycine) by Pseudomonas sp. PG2982 has been determined by using metabolic radiolabeling experiments. Radiorespirometry experiments utilizing [3-14C]glyphosate revealed that approximately 50 to 59% of the C-3 carbon was oxidized to CO2. Fractionation of stationary-phase cells labeled with [3-14C]glyphosate revealed that from 45 to 47% of the assimilated label is distributed to proteins and that the amino acids methionine and serine are highly labeled. Adenine and guanine received 90% of the C-3 label found in the nucleic acid fraction, and the only pyrimidine base labeled was thymine. These results indicated that C-3 of glyphosate was at some point metabolized to a C-1 compound whose ultimate fate could be both oxidation to CO2 and distribution to amino acids and nucleic acid bases that receive a C-1 group from the C-1-donating coenzyme tetrahydrofolate. Pulse-labeling of PG2982 cells with [3-14C]glyphosate resulted in the isolation of [3-14C]sarcosine as an intermediate in glyphosate degradation. Examination of crude extracts prepared from PG2982 cells revealed the presence of a sarcosine-oxidizing enzyme that oxidizes sarcosine to glycine and formaldehyde. These results indicate that the first step in glyphosate degradation by PG2982 is cleavage of the carbon-phosphorus bond, resulting in the release of sarcosine and a phosphate group. The phosphate group is utilized as a source of phosphorus, and the sarcosine is degraded to glycine and formaldehyde. This pathway is supported by the results of [1,2-14C]glyphosate metabolism studies, which show that radioactivity in the proteins of labeled cells is found only in the glycine and serine residues.     

Isolation and Characterization of a Mutant of Arthrobacter sp. Strain GLP-1 Which Utilizes the Herbicide Glyphosate as Its Sole Source of Phosphorus and Nitrogen

Arthrobacter sp. strain GLP-1, grown on glucose as a carbon source, utilizes the herbicide glyphosate [N-(phosphonomethyl)glycine] as its sole source of phosphorus as well as its sole source of nitrogen. The mutant strain GLP-1/Nit-1 utilizes glyphosate as its sole source of nitrogen as well. In strain GLP-1, P_i was a potent competitive inhibitor of glyphosate uptake (K_i, 24 μM), while the affinity of P_i for the uptake system of strain GLP-1/Nit-1 was reduced by 2 orders of magnitude (K_i, 2.3 mM). It is concluded that the inability of strain GLP-1 to utilize glyphosate as a source of nitrogen is due to the stringent control of glyphosate uptake by excess phosphate released during the degradation of the herbicide.     

The Site of the Inhibition of the Shikimate Pathway by Glyphosate: I. INHIBITION BY GLYPHOSATE OF PHENYLPROPANOID SYNTHESIS IN BUCKWHEAT (FAGOPYRUM ESCULENTUM MOENCH)

The nonselective herbicide glyphosate (n-[phosphonomethyl]glycine) inhibited the light-induced accumulation of phenylpropanoid substances (chlorogenic acid, procyanidin, rutin, anthocyanin) in etiolated buckwheat hypocotyls 90% at 1 millimolar. Structurally related compounds, such as n,n-bis[phosphonomethyl]glycine, aminomethylphosphonate, methylglycine, and iminodiacetate, had little or no inhibiting effects. Of all amino acids tested, only l-phenylalanine reversed the inhibition, and partial reversal of anthocyanin synthesis was achieved with chorismate, phenylpyruvate, trans-cinnamate, p-coumarate, and naringenin. Phenylalanine concentrations were reduced in glyphosate-treated hypocotyls, and glyphosate effectively reduced the high level of phenylalanine that was caused by the phenylalanine ammonia-lyase inhibitor l-alpha-aminooxy-beta-phenylpropionate. Glyphosate had no significant effect on the time course of phenylalanine ammonia-lyase activity in hypocotyls incubated either in the dark or in the light. Under appropriate feeding conditions, glyphosate inhibited the incorporation of [(14)C]shikimate into all three aromatic amino acids, and radioactive shikimate accumulated in the tissue. The results lead to the conclusion that glyphosate interferes with the shikimate pathway at or prior to the formation of chorismate.     

Metabolism of glyphosate in Pseudomonas sp. strain LBr

Metabolism of glyphosate (N-phosphonomethylglycine) by Pseudomonas sp. strain LBr, a bacterium isolated from a glyphosate process waste stream, was examined by a combination of solid-state 13C nuclear magnetic resonance experiments and analysis of the phosphonate composition of the growth medium. Pseudomonas sp. strain LBr was capable of eliminating 20 mM glyphosate from the growth medium, an amount approximately 20-fold greater than that reported for any other microorganism to date. The bacterium degraded high levels of glyphosate, primarily by converting it to aminomethylphosphonate, followed by release into the growth medium. Only a small amount of aminomethylphosphonate (about 0.5 to 0.7 mM), which is needed to supply phosphorus for growth, could be metabolized by the microorganism. Solid-state 13C nuclear magnetic resonance analysis of strain LBr grown on 1 mM [2-13C,15N]glyphosate showed that about 5% of the glyphosate was degraded by a separate pathway involving breakdown of glyphosate to glycine, a pathway first observed in Pseudomonas sp. strain PG2982. Thus, Pseudomonas sp. strain LBr appears to possess two distinct routes for glyphosate detoxification.     

Metabolism of glyphosate in Pseudomonas sp. strain LBr.

Metabolism of glyphosate (N-phosphonomethylglycine) by Pseudomonas sp. strain LBr, a bacterium isolated from a glyphosate process waste stream, was examined by a combination of solid-state 13C nuclear magnetic resonance experiments and analysis of the phosphonate composition of the growth medium. Pseudomonas sp. strain LBr was capable of eliminating 20 mM glyphosate from the growth medium, an amount approximately 20-fold greater than that reported for any other microorganism to date. The bacterium degraded high levels of glyphosate, primarily by converting it to aminomethylphosphonate, followed by release into the growth medium. Only a small amount of aminomethylphosphonate (about 0.5 to 0.7 mM), which is needed to supply phosphorus for growth, could be metabolized by the microorganism. Solid-state 13C nuclear magnetic resonance analysis of strain LBr grown on 1 mM [2-13C,15N]glyphosate showed that about 5% of the glyphosate was degraded by a separate pathway involving breakdown of glyphosate to glycine, a pathway first observed in Pseudomonas sp. strain PG2982. Thus, Pseudomonas sp. strain LBr appears to possess two distinct routes for glyphosate detoxification.     

Physiological aspects of glyphosate degradation in Alcaligenes spec. strain GL

Alcaligenes spec. strain GL (IMET 11314) is able to grow on glyphosate (N-[phosphonomethyl]glycine) and other phosphonates as sole source of phosphorus. Degradation of glyphosate to inorganic phosphate and sarcosine by this strain is subject to several regulatory principles. While uptake and dephosphonation of glyphosate are regulated by P_i starvation, the intensity of glyphosate degradation is also controlled by the cellular ability to utilize the C-skeleton derived from glyphosate. Depending on the external concentration of glyphosate, the liberated sarcosine is differentially metabolised. Utilization of the sarcosine moiety and complete incorporation of 3-[¹⁴C]-label of glyphosate into cellular material occur only in cultures adapted to higher concentrations (5 mM) of the herbicide. At low concentrations of glyphosate (1 mM) only the P_i required by the growing cultures is utilized but not the sarcosine. Initially high rates of glyphosate uptake obtained after P_i-starvation decrease in the presence of low glyphosate concentrations. It is suggested that uptake and metabolism of glyphosate are connected with the expression of the sarcosine metabolizing capacity of the Alcaligenes cells.Abbreviation AMPA aminomethylphosphonic acid     

Uptake of Glyphosate by an Arthrobacter sp

The uptake of glyphosate (N-[phosphonomethyl]glycine) by an Arthrobacter sp. which can utilize this herbicide as its sole source of phosphorus was investigated. Orthophosphate suppressed the expression of the uptake system for glyphosate and also competed with glyphosate for uptake. The K_m for glyphosate uptake was 125 μM, and the K_i for orthophosphate was 24 μM. Organophosphonates as well as organophosphates inhibited glyphosate uptake, but only organophosphates and orthophosphate suppressed the uptake system. Glyphosate uptake was energy dependent, had a pH optimum of 6 to 7, and was differentially affected by divalent cations.     

Glyphosate utilization byPseudomonas sp. andAlcaligenes sp. isolated from environmental sources

A total of 21 bacterial cultures were isolated that could utilize glyphosate (N-phosphonomethyl glycine) as a sole source of phosphorus in a mineral salts medium. Sources of inocula for enrichment cultures included aerobic digester liquid, raw sewage, trickling filter effluent, pesticide disposal pit liquid, and soil. Eleven cultures were identified asPseudomonas sp., one asPseudomonas stutzeri, and nine asAlcaligenes sp. Aminomethylphosphonic acid, the major metabolic intermediate of glyphosate degradation in soil, could also serve as a sole phosphorus source for all 21 isolates. Neither glyphosate nor aminomethylphosphonic acid could serve as carbon sources in mineral salts media. Experiments withPseudomonas sp. SG-1 (isolated from aerobic digester liquid) suggested that enzymatic activity responsible for glyphosate degradation was intracellular, inducible, and required the cofactors pyruvate and pyridoxal phosphate. The degradation pathway for glyphosate in this culture may be similar to that previously reported for aminoethylphosphonic acid.     

Differential sensitivity of bacterial 5-enolpyruvylshikimate-3-phosphate synthases to the herbicide glyphosate

Abstract The potent inhibition of the shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase by the broad-spectrum herbicide glyphosate ( N -[phosphonomethyl]glycine) was confirmed for the enzymes extracted from various bacteria, a green alga and higher plants. However, 5 out of 6 species belonging to the genus Pseudomonas were found to have EPSP synthases with a 50- to 100-fold decreased sensitivity to the inhibitor. Correspondingly, growth of these 5 species was not inhibited by 5 mM glyphosate, and the organisms did not excrete shikimate-3-phosphate in the presence of the herbicide.     

Insensitivity of 5-enolpyruvylshikimic acid-3-phosphate synthase to glyphosate confers resistance to this herbicide in a strain of Aerobacter aerogenes

The broadspectrum herbicide glyphosate (N-[phosphonomethyl]glycine), an inhibitor of the shikimate pathway enzyme 5-enolpyruvyl-shikimic acid-3-phosphate (EPSP)-synthase, inhibits the growth of Aerobacter aerogenes and causes the excretion of shikimic acid-3-phosphate. A strain of A. aerogenes, resistant to inhibition of growth by glyphosate, was isolated and found to have a glyphosate-insensitive EPSP-synthase and to no longer excrete shikimic acid-3-phosphate in the presence of glyphosate. Partial identity of EPSP-synthases from the glyphosate-sensitive and-resistant A. aerogenes strains was demonstrated by immunological procedures.Abbreviation EPSP-synthase 5-enolpyruvylshikimic acid-3-phosphate synthase (EC 2.5.1.19; 3-phosphoshikimate 1-carboxyvinyltransferase)     

The Site of the Inhibition of the Shikimate Pathway by Glyphosate: II. INTERFERENCE OF GLYPHOSATE WITH CHORISMATE FORMATION IN VIVO AND IN VITRO

In the presence of the nonselective herbicide glyphosate (N-[phosphonomethyl]glycine), buckwheat (Fagopyrum esculentum Moench) hypocotyls and cultured cells of Galium mollugo L. accumulate an organic acid, which was identified as shikimate by mass-spectroscopy of its methyl ester. After growth in 0.5 millimolar glyphosate for 10 days, G. mollugo cells contained shikimate in amounts of up to 10% of their dry weight. Synthesis of chorismate-derived anthraquinones in G. mollugo was blocked by glyphosate. Chorismate and o-succinylbenzoate (an anthraquinone precursor) alleviated the inhibition. The conclusion drawn from these experiments, that glyphosate inhibits a step in the biosynthetic sequence from shikimate to chorismate, was substantiated by the finding that glyphosate is a powerful inhibitor of the conversion of shikimate to chorismate in cell-free extracts from Aerobacter aerogenes 62-1.     

Genomic heterogeneity within conservedmetabolic pathways of Arthrobacter species - a bioinformatic approach

A comparative genomic analysis of three species of the soil bacterium Arthrobacter was undertaken with specific emphasis on genes involved in important and core energy metabolism pathways like glycolysis and amino acid metabolism. During the course of this study, it was revealed that codon bias of a particular species, namely Arthrobacter aurescens TC1, is significantly lower than that of the other two species A. chlorophenolicus A6 and Arthrobacter sp. FB24. The codon bias was also found to be negatively correlated with gene expression level which is determined by computing codon adaptation index of the genes. Uniformity in codon usage pattern among three species is evident in terms of genes which has high codon bias and multifunctional nature. Further, it was observed that this trend is present amongst the genes of important metabolic pathways, such as glycolysis and amino acid metabolism. The evolutionary divergence of the pathway gene sequences was calculated and was found to be equivalent in nature in the case of Arthrobacter sp. FB24 and Arthrobacter chlorophenolicus A6, but turned out to be dissimilar in the case of Arthrobacter aurescens TC1. A strong correlation between synonymous substitution rate and effective codon number or Nc was also observed. These observations clearly point out that the genes having low bias, in Arthrobacter aurescens TC1, and even of those that are part of highly conserved metabolic pathways like glycolysis and amino acid ensemble pathways have undergone a different type of evolution and might be subjected to positive selection pressure in comparison with Arthrobacter sp. FB24 and Arthrobacter chlorophenolicus A6.     

The essential role of cobalt in the inhibition of the cytosolic isozyme of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Nicotiana silvestris by glyphosate

The prime molecular target of glyphosate (N-[phosphonomethyl]glycine), a potent herbicidal and antimicrobial agent, is known to be the shikimate-pathway enzyme, 5-enol-pyruvylshikimate-3-phosphate synthase. Inhibition by glyphosate of an earlier pathway enzyme that is located in the cytosol of higher plants, 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DS-Co), has raised the possibility of dual enzyme targets in vivo. With the recent appreciation that magnesium (and manganese) can replace cobalt as the divalent-metal activator of DS-Co, it has now been possible to show that sensitivity of DS-Co to inhibition by glyphosate is obligately dependent upon the presence of cobalt. Evidence for a cobalt(II):glyphosate complex with octahedral coordination was obtained through examination of the effect of glyphosate upon the visible electronic spectrum of aqueous solutions of cobalt(II) chloride. The presence of glyphosate increased the concentration of cobalt(II) chloride required for enzyme activity, and the concentration of cobalt(II) chloride markedly affected the concentration of glyphosate required for inhibition of DS-Co activity. The extent to which DS-Co is vulnerable to inhibition by glyphosate in vivo depends, therefore, upon the unknown extent to which DS-Co molecules in the cytosol might be associated with cobalt.     

Influence of glyphosate on flower morphogenesis and pigmentation in Petunia hybrida

Glyphosate showed a remarkable effect inducing the change of flower symmetry from the actinomorphic to the zygomorphic type in Petunia hybrida. Glyphosate [N-(phosphonomethyl)glycine] reduced the anthocyanin content and showed a weak inhibitory effect against phenylalanine ammonia-lyase (PAL) activity. L-2-Aminooxy-3-phenylpropionic acid (APA), an inhibitor of PAL activity, reduced the anthocyanin content but had no effect on flower shape. Additional phenylalanine or trans-cinnamic acid, the intermediates of glyphosate inhibition against PAL activity, could not recover the change of flower shape induced by glyphosate. These results suggested that the reduction of PAL activity alone could not account for the two characteristic changes of flower symmetry and pigmentation induced by glyphosate. On the other hand, the results of application of glyphosate-related compounds suggested that the structure of glyphosate contributed to induce the morphological change of Petunia flower. Glyphosate may thus be a very useful agent in the elucidation of unresolved questions of flower morphogenesis and the related metabolism.     

Evidence for a reactive gamma-carboxyl group (Glu-418) at the herbicide glyphosate binding site of 5-enolpyruvylshikimate-3-phosphate synthase from Escherichia coli

Incubation of 5-enolpyruvylshikimate-3-phosphate synthase, a target for the nonselective herbicide glyphosate (N-(phosphonomethyl)glycine), with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the presence of glycine ethyl ester resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo-first order kinetics, with a second order rate constant of 2.2 M-1 min-1 at pH 5.5 and 25 degrees C. The inactivation is prevented by preincubation of the enzyme with a combination of the substrate shikimate 3-phosphate plus glyphosate, but not by shikimate 3-phosphate, phosphoenolpyruvate, or glyphosate alone. Increasing the concentration of glyphosate during preincubation resulted in decreasing the rate of inactivation of the enzyme. Complete inactivation of the enzyme required the modification of 4 carboxyl groups per molecule of the enzyme. However, statistical analysis of the residual activity and the extent of modification showed that among the 4 modifiable carboxyl groups, only 1 is critical for activity. Tryptic mapping of the enzyme modified in the absence of shikimate 3-phosphate and glyphosate by reverse phase chromatography resulted in the isolation of a [14C]glycine ethyl ester-containing peptide that was absent in the enzyme modified in the presence of shikimate 3-phosphate and glyphosate. By amino acid sequencing of this labeled peptide, the modified critical carboxyl group was identified as Glu-418. The above results suggest that Glu-418 is the most accessible reactive carboxyl group under these conditions and is located at or close to the glyphosate binding site.     

Effect of glyphosate on indole-3-acetic acid metabolism in tolerant and susceptible plants

A comparison study was conducted on the effect of glyphosate (N-[phosphonomethyl]glycine) on indole-3-[2-¹⁴C]acetic acid (IAA) metabolism, ethylene production, and growth of 7-day-old seedlings of different plants. The plants tested were American germander (Teucrium canadense L.), soybean (Glycine max L. Merr.), pea (Pisum sativum L. cv. Alaska and Little marvel), mungbean (Vigna radiata L.), and buckwheat (Fagopyrum esculentum Moench). A spray with 2 mM glyphosate affected IAA metabolism to a varied degree. The induced increase of IAA metabolism was greater in buckwheat, Alaska pea, and mungbean than soybean, Little marvel pea, and American germander. The increased IAA metabolism was correlated with the inhibition of growth and with the decrease of ethylene production.The natural rate of IAA metabolism was markedly different among the plant species and cultivars tested and appeared to be related to the sensitivity of the plants to glyphosate. American germander and Little marvel pea with high rates of IAA metabolism were more tolerant to glyphosate than buckwheat and Alaska pea, which had low rates of IAA metabolism. Plants with a high natural rate of IAA metabolism were probably less dependent on IAA and thus less susceptible to glyphosate.     

Synthesis,cytotoxicity and clastogenicity of novel α-aminophosphonic acids

Summary. α-Ethyl-N-(phosphonomethyl) glycine is synthesized and characterized by NMR and FAB spectroscopy. The cytotoxicity, clastogenic and antiproliferative effect of 3-ethyl-2-hydroxyl-2-oxo-1,4,2-oxazaphosphorinane, sodium salt of 3-ethyl-2-hydroxyl-2-oxo-1,4,2-oxazaphosphorinane, α-ethyl-α-N-(hydroxyethylamino) methylphosphonic acid, α-ethyl-N-(phosphonomethyl) glycine, α-ethyl-N-(phosphonomethyl) glycine isopropylammonium salt, glyphosate isopropylammonium salt are tested.