Cloning of genes encoding for C-P lyase fromPseudomonas isolates PG2982 and GLC11: Identification of a cryptic allele on the chromosome ofP. aeruginosa

Two isolates ofPseudomonas sp., GLC11 and PG2982, can use glyphosate as a sole source of phosphorus. This ability is indicative of enzymatic cleavage of a carbon-phosphorus bond, and the enzyme has been named C-P lyase. We have cloned, inEscherichia coli, gene/s coding for C-P lyase on a broad host range cosmid pLA2917. Restriction fragment arrangement of cloned fragments of PG2982 and GLC11 has been established. Analysis by Southern hybridization between two clones revealed a strong homology between threePstI fragments of pPG-CP-14 (derived from PG2982) and pGC-CP-4 (derived from GLC11). With the construct pGC-CP-4 as a probe, the presence of a cryptic allele for C-P lyase has been demonstrated on the chromosome of the parent isolate,pseudomonas aeruginosa PAO1. It is suggested that genetic rearrangement such as frame shift or a point mutation activated the cryptic C-P lyase gene. Metabolism of glyphosate byE. coli carrying pPG-CP-14 or pGC-CP-4 has been demonstrated by radiometric experiments.     

Phosphonate Utilization by the Glyphosate-Degrading Pseudomonas sp. Strain PG2982

The glyphosate-degrading Pseudomonas sp. strain PG2982 was found to utilize each of 10 organophosphonate compounds as a sole phosphorus source. Representative compounds tested included alkylphosphonates, 1-amino-substituted alkylphosphonates, amino-terminal phosphonates, and an arylphosphonate. This report demonstrates that PG2982 is capable of utilizing a wider range of structurally different organophosphonate compounds than any organism described to date.     

Mineralization of Paraoxon and Its Use as a Sole C and P Source by a Rationally Designed Catabolic Pathway in Pseudomonas putida

Organophosphate compounds, which are widely used as pesticides and chemical warfare agents, are cholinesterase inhibitors. These synthetic compounds are resistant to natural degradation and threaten the environment. We constructed a strain of Pseudomonas putida that can efficiently degrade a model organophosphate, paraoxon, and use it as a carbon, energy, and phosphorus source. This strain was engineered with the pnp operon from Pseudomonas sp. strain ENV2030, which encodes enzymes that transform p-nitrophenol into β-ketoadipate, and with a synthetic operon encoding an organophosphate hydrolase (encoded by opd) from Flavobacterium sp. strain ATCC 27551, a phosphodiesterase (encoded by pde) from Delftia acidovorans, and an alkaline phosphatase (encoded by phoA) from Pseudomonas aeruginosa HN854 under control of a constitutive promoter. The engineered strain can efficiently mineralize up to 1 mM (275 mg/liter) paraoxon within 48 h, using paraoxon as the sole carbon and phosphorus source and an inoculum optical density at 600 nm of 0.03. Because the organism can utilize paraoxon as a sole carbon, energy, and phosphorus source and because one of the intermediates in the pathway (p-nitrophenol) is toxic at high concentrations, there is no need for selection pressure to maintain the heterologous pathway.     

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.     

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.     

Mineralization of 4-fluorocinnamic acid by a Rhodococcus strain

A bacterial strain capable of aerobic degradation of 4-fluorocinnamic acid (4-FCA) as the sole source of carbon and energy was isolated from a biofilm reactor operating for the treatment of 2-fluorophenol. The organism, designated as strain S2, was identified by 16S rRNA gene analysis as a member of the genus Rhodococcus. Strain S2 was able to mineralize 4-FCA as sole carbon and energy source. In the presence of a conventional carbon source (sodium acetate [SA]), growth rate of strain S2 was enhanced from 0.04 to 0.14 h^?1 when the culture medium was fed with 0.5 mM of 4-FCA, and the time for complete removal of 4-FCA decreased from 216 to 50 h. When grown in SA-supplemented medium, 4-FCA concentrations up to 1 mM did not affect the length of the lag phase, and for 4-FCA concentrations up to 3 mM, strain S2 was able to completely remove the target fluorinated compound. 4-Fluorobenzoate (4-FBA) was transiently formed in the culture medium, reaching concentrations up to 1.7 mM when the cultures were supplemented with 3.5 mM of 4-FCA. Trans,trans-muconate was also transiently formed as a metabolic intermediate. Compounds with molecular mass compatible with 3-carboxymuconate and 3-oxoadipate were also detected in the culture medium. Strain S2 was able to mineralize a range of other haloorganic compounds, including 2-fluorophenol, to which the biofilm reactor had been exposed. To our knowledge, this is the first time that mineralization of 4-FCA as the sole carbon source by a single bacterial culture is reported.     

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.     

抗草甘膦酵母菌ZM-1的分离鉴定及其生长降解特性

以福州市郊区的耕作土壤为研究材料, 利用草甘膦为选择压力, 通过富集、驯化培养, 分离出一株对草甘膦具有高耐受和降解作用的酵母菌菌株ZM-1, 结合生理生化特征及26S rDNA D1/D2区序列分析将其初步鉴定为胶红酵母菌(Rhodotorula mucilaginosa)。菌株ZM-1能以草甘膦为唯一碳、氮源生长, 对草甘膦的最高耐受浓度为50 g/L。在草甘膦初始浓度为1 g/L的无机盐培养基中, 30°C、150 r/min 摇床振荡培养7 d, 草甘膦降解率为85.38%。适合菌株ZM-1生长及降解草甘膦的最佳条件为: 草甘膦初始浓度1 g/L, 接种量4%, 温度30°C, pH 值5.5-6.0, 装料量50 mL/250 mL。菌株ZM-1是一株良好的草甘膦耐受菌, 可用于草甘膦污染环境的生物修复, 也可能成为转基因抗草甘膦作物的一个很好资源。     

Phosphate starvation induces uptake of glyphosate by Pseudomonas sp. strain PG2982.

Pseudomonas sp. strain PG2982 has the ability to use the phosphonate herbicide, glyphosate, as a sole phosphorus source (J. K. Moore, H. D. Braymer, and A. D. Larson, Appl. Environ. Microbiol. 46:316-320, 1983). Glyphosate uptake is maximal in the late log phase of growth and is induced by phosphate starvation. Uptake is inhibited by phosphate and arsenate, but not by the amino acids glycine and sarcosine. The Km and Vmax for glyphosate uptake were calculated to be 23 microM and 0.97 nmol/mg (dry weight) per min, respectively. A phosphate transport system with a broad substrate specificity may be responsible for glyphosate uptake.     

Phosphate starvation induces uptake of glyphosate by Pseudomonas sp. strain PG2982

Pseudomonas sp. strain PG2982 has the ability to use the phosphonate herbicide, glyphosate, as a sole phosphorus source (J. K. Moore, H. D. Braymer, and A. D. Larson, Appl. Environ. Microbiol. 46:316-320, 1983). Glyphosate uptake is maximal in the late log phase of growth and is induced by phosphate starvation. Uptake is inhibited by phosphate and arsenate, but not by the amino acids glycine and sarcosine. The Km and Vmax for glyphosate uptake were calculated to be 23 microM and 0.97 nmol/mg (dry weight) per min, respectively. A phosphate transport system with a broad substrate specificity may be responsible for glyphosate uptake.     

Organophosphonate Utilization by the Thermophile Geobacillus caldoxylosilyticus T20

A strain of Geobacillus caldoxylosilyticus from central heating system water could utilize a number of organophosphonates as the sole phosphorus source for growth at 60°C. During growth on glyphosate, aminomethylphosphonate release to the medium was observed, and in cell extracts, a glyphosate oxidoreductase-type activity, producing stoichiometric amounts of aminomethylphosphonate and glyoxylate from glyphosate, was detectable.     

Glyphosate-Degrading Microorganisms from Industrial Activated Sludge

A plating medium was developed to isolate N-phosphonomethylglycine (glyphosate)-degrading microorganisms, with glyphosate as the sole phosphorus source. Two industrial biosystems treating glyphosate wastes contained elevated microbial counts on the medium. One purified isolate metabolized glyphosate to aminomethylphosphonic acid, mineralizing this accumulating intermediate during log growth. This microorganism has been identified as a Flavobacterium species.     

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.     

Prospects of in vivo 31P NMR method in glyphosate degradation studies in whole cell system

The degradation of the phosphonate herbicide glyphosate (N-phosphonomethylglycine) by four taxonomically distinct microorganisms was studied in vivo in whole cell system using phosphorus nuclear magnetic spectroscopy (³¹P NMR). The time-course of glyphosate metabolization in dense cell cultures was followed by means of ³¹P NMR up to 21 days after the addition. The results obtained by this non-invasive way confirmed that the cells of Spirulina platensis and Streptomyces lusitanus biodegrade herbicide. Moreover, phosphorus starvation influenced the rate of glyphosate degradation by S. platensis. On the other hand, the results of similar measurements in the cultures of green algae Chlorella vulgaris showed that this aquatic plant, however growing in the medium containing 1 mM of N-phosphonomethylglycine, did not seem to posses the ability of its biodegradation. Additionally, the use of this method allowed us to find the new fungal strain Fusarium dimerum, which is able to biodegrade and utilize the glyphosate as the sole source of phosphorus. The results of our studies on usefulness of in vivo ³¹P NMR for tracing glyphosate degradation in whole cell systems revealed that this non-invasive, one-step method, might be considered as a valuable tool in environmental biotechnology of organophosphonate xenobiotics.     

Biodegradation of Nitriles in Shale Oil

Enrichment cultures were obtained, after prolonged incubation on a shale oil as the sole source of nitrogen, that selectively degraded nitriles. Capillary gas chromatographic analyses showed that the mixed microbial populations in the enrichments degraded the homologous series of aliphatic nitriles but not the aliphatic hydrocarbons, aromatic hydrocarbons, or heterocyclic-nitrogen compounds found in this oil. Time course studies showed that lighter nitriles were removed more rapidly than higher-molecular-weight nitriles. A Pseudomonas fluorescens strain isolated from an enrichment, which was able to completely utilize the individual nitriles undecyl cyanide and undecanenitrile as sole sources of carbon and nitrogen, was unable to attack stearonitrile when provided alone as the growth substrate. A P. aeruginosa strain, also isolated from one of the enrichments, used nitriles but not aliphatic or aromatic hydrocarbons when the oil was used as a sole nitrogen source. However, when the shale oil was used as the sole source of carbon, aliphatic hydrocarbons in addition to nitriles were degraded but aromatic hydrocarbons were still not attacked by this P. aeruginosa strain.     

Cloning of a gene from Pseudomonas sp. strain PG2982 conferring increased glyphosate resistance.

A plasmid carrying a 2.4-kilobase-pair fragment of DNA from Pseudomonas sp. strain PG2982 has been isolated which was able to increase the glyphosate resistance of Escherichia coli cells. The increase in resistance was dependent on the presence of a plasmid-encoded protein with a molecular weight of approximately 33,000, the product of a translational fusion between a gene on the vector, pACYC184, and the insert DNA. An overlapping region of the PG2982 chromosome carrying the entire gene (designated igrA) was cloned, and a plasmid (pPG18) carrying the gene was also able to increase glyphosate resistance in E. coli. A protein with a molecular weight of approximately 40,000 was encoded by the PG2982 DNA contained in pPG18. This plasmid was not able to complement a mutation in the gene for 5-enolpyruvylshikimate-3-phosphate synthase (aroA) in E. coli, and modification of glyphosate by E. coli cells containing the plasmid could not be demonstrated. The nucleotide sequence of the PG2982 DNA contained an open reading frame able to encode a protein with a calculated molecular weight of 39,396.     

Cloning of a gene from Pseudomonas sp. strain PG2982 conferring increased glyphosate resistance

A plasmid carrying a 2.4-kilobase-pair fragment of DNA from Pseudomonas sp. strain PG2982 has been isolated which was able to increase the glyphosate resistance of Escherichia coli cells. The increase in resistance was dependent on the presence of a plasmid-encoded protein with a molecular weight of approximately 33,000, the product of a translational fusion between a gene on the vector, pACYC184, and the insert DNA. An overlapping region of the PG2982 chromosome carrying the entire gene (designated igrA) was cloned, and a plasmid (pPG18) carrying the gene was also able to increase glyphosate resistance in E. coli. A protein with a molecular weight of approximately 40,000 was encoded by the PG2982 DNA contained in pPG18. This plasmid was not able to complement a mutation in the gene for 5-enolpyruvylshikimate-3-phosphate synthase (aroA) in E. coli, and modification of glyphosate by E. coli cells containing the plasmid could not be demonstrated. The nucleotide sequence of the PG2982 DNA contained an open reading frame able to encode a protein with a calculated molecular weight of 39,396.     

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 New Clostridium sp. That Performs Effective Cellulosic Waste Digestion in a Thermophilic Methanogenic Bioreactor

A methanogenic bioreactor that utilized wastepaper was developed and operated at 55°C. Microbial community structure analysis showed the presence of a group of clostridia that specifically occurred during the period of high fermentation efficiency. To isolate the effective cellulose digester, the sludge that exhibited high fermentation efficiency was inoculated into a synthetic medium that contained cellulose powder as the sole carbon source and was successively cultivated. A comprehensive 16S rRNA gene sequencing study revealed that the enriched culture contained various clostridia that had diverse phylogenetic positions. The microorganisms were further enriched by successive cultivation with filter paper as the substrate, as well as the bait carrier. A resultant isolate, strain EBR45 (= Clostridium sp. strain NBRC101661), was a new member of the order Clostridiales phylogenetically and physiologically related to Clostridium thermocellum and Clostridium straminisolvens. Specific PCR-based monitoring demonstrated that strain EBR45 specifically occurred during the high fermentation efficiency period in the original methanogenic sludge. Strain EBR45 effectively digested office paper in its pure cultivation system with a synthetic medium.