Isolation and characterisation of Rhodococcus erythropolis TA57 able to degrade the triazine amine product from hydrolysis of sulfonylurea pesticides in soils

We isolated a bacterium capable of metabolising a methylated and methoxylated s-triazine ring as the only nitrogen source. On a weight basis, the s-triazine, commonly named triazine amine (TAM), constitutes approx. half of several sulfonylurea herbicides and is formed after hydrolysis of these herbicides. The isolate, strain TA57 was identified using multi-phasic taxonomy as a gram-positive Rhodococcus erythropolis. Strain TA57 mineralised over 50% 14C-labelled TAM within 4 days in growing cultures using all of the nitrogen for growth. The degradation capacity was found stable in cells grown on either tryptic soy broth agar plates or in minimal medium with NH4+. Among other s-triazines tested, only one other methylated, but de-methoxylated s-triazine amine supported growth. Inoculating 10(6) cells of TA57 per gram of soil (d.w.) resulted in 50% mineralisation of 14C labelled TAM (1 mg kg(-1)) within 25 days, in contrary to the indigenous population that mineralised only 6% in 50 days.     

Ametryne and Prometryne as Sulfur Sources for Bacteria

Bacteria were isolated that could utilize quantitatively the s-triazine herbicide prometryne [N,N′ -bis(1-methylethyl)-6-(methylthio)-1,3,5-triazine-2,4-diamine] or ametryne [N-ethyl-N′-(1-methylethyl)-6-(methylthio)-1,3,5-triazine- 2,4-diamine], or both, as a sole source of sulfur for growth. The success of enrichments depended on previous exposure of the soil inoculum to s-triazine herbicides. Deaminoethylametryne [4-(1-methylethyl)amino-6-(methylthio)-1,3,5-triazine-2-(1H)-one], methylsulfonic acid, and sodium sulfate could also be used as sulfur sources. Utilization of a compound was quantified as the growth yield per mole of sulfur supplied. Yields were about 6 kg of protein per mol of sulfur. The product of the desulfuration of an s-triazine was identified as the corresponding hydroxy-derivative. This is the first substantiated report of the utilization of these s-triazines as sulfur sources by bacteria.     

Gene sequence and properties of an s-triazine ring-cleavage enzyme from Pseudomonas sp. strain NRRLB-12227.

Pesticides based on the s-triazine ring structure are widely used in cultivation of food crops. Cleavage of the s-triazine ring is an important step in the mineralization of s-triazine compounds and hence in their complete removal from the environment. Cyanuric acid amidohydrolase cleaves cyanuric acid (2,4,6-trihydroxy-s-triazine), which yields carbon dioxide and biuret; the biuret is subject to further metabolism, which yields CO(2) and ammonia. The trzD gene encoding cyanuric acid amidohydrolase was cloned into pMMB277 from Pseudomonas sp. strain NRRLB-12227, a strain that is capable of utilizing s-triazines as nitrogen sources. Hydrolysis of cyanuric acid was detected in crude extracts of Escherichia coli containing the cloned gene by monitoring the disappearance of cyanuric acid and the appearance of biuret by high-performance liquid chromatography (HPLC). DEAE and hydrophobic interaction HPLC were used to purify cyanuric acid amidohydrolase to homogeneity, and a spectrophotometric assay for the purified enzyme was developed. The purified enzyme had an apparent K(m) of 0.05 mM for cyanuric acid at pH 8.0. The enzyme did not cleave any other s-triazine or hydroxypyrimidine compound, although barbituric acid (2,4, 6-trihydroxypyrimidine) was found to be a strong competitive inhibitor. Neither the nucleotide sequence of trzD nor the amino acid sequence of the gene product exhibited a significant level of similarity to any known gene or protein.     

Biotreatment of s-triazine-containing wastewater in a fluidized bed reactor

Mixed cultures of microorganisms immobilized on sand were used to degrade s-triazine-containing industrial wastewater in a fluidized bed reactor. Immobilized cell concentrations of up to 18 g/L volatile suspended solids could be achieved with the s-triazines as sole nitrogen source for growth and carbon sources added at a C--N ratio of about 12. Maximal removal efficiencies of 80% of the s-triazines could be maintained only if (a) the bio-film thickness was limited to avoid oxygen deficiency and (b) the carbon source and complete wastewater (/=20-25 h.     

Synthesis and antibacterial activity of 2,4,6-tri substituted s-triazines

Various 2,4,6-tri substituted s-triazines were synthesized and screened for antibacterial activity against Gram-positive and Gram-negative organisms. These s-triazine derivatives displayed high in vitro antibacterial activities comparable to penicillin and streptomycin against tested microorganisms.     

Arthrobacter aurescens TC1 metabolizes diverse s-triazine ring compounds

Arthrobacter aurescens strain TC1 was isolated without enrichment by plating atrazine-contaminated soil directly onto atrazine-clearing plates. A. aurescens TC1 grew in liquid medium with atrazine as the sole source of nitrogen, carbon, and energy, consuming up to 3,000 mg of atrazine per liter. A. aurescens TC1 is metabolically diverse and grew on a wider range of s-triazine compounds than any bacterium previously characterized. The 23 s-triazine substrates serving as the sole nitrogen source included the herbicides ametryn, atratone, cyanazine, prometryn, and simazine. Moreover, atrazine substrate analogs containing fluorine, mercaptan, and cyano groups in place of the chlorine substituent were also growth substrates. Analogs containing hydrogen, azido, and amino functionalities in place of chlorine were not growth substrates. A. aurescens TC1 also metabolized compounds containing chlorine plus N-ethyl, N-propyl, N-butyl, N-s-butyl, N-isobutyl, or N-t-butyl substituents on the s-triazine ring. Atrazine was metabolized to alkylamines and cyanuric acid, the latter accumulating stoichiometrically. Ethylamine and isopropylamine each served as the source of carbon and nitrogen for growth. PCR experiments identified genes with high sequence identity to atzB and atzC, but not to atzA, from Pseudomonas sp. strain ADP.     

Ralstonia basilensis M91-3, a denitrifying soil bacterium capable of using s-triazines as nitrogen sources

The purpose of this study was to characterize the phylogenetic and phenotypic traits of M91-3, a soil bacterium capable of mineralizing atrazine (2-chloro-4-N-isopropyl-6-N-ethyl-s-triazine). The isolate was identified as Ralstonia basilensis based on 99.5% homology of the 16S rRNA sequence and various chemotaxonomic data. The isolate used atrazine as the sole source of energy, carbon, and nitrogen. It could also use several other s-triazines as nitrogen sources. Ralstonia basilensis M91-3 was capable of denitrification, which was confirmed by gas chromatographic analysis of nitrous oxide under acetylene blockage conditions.     

Mineralization of the herbicide atrazine as a carbon source by a Pseudomonas strain.

Strain YAYA6 was isolated from a mixed microbial community that was growing on atrazine as a sole carbon source and formed quantitative amounts of chloride and nitrate. This strain was identified as a member of the true pseudomonad group (RNA group I) and was given the designation DMS 93-99. The growth yield when atrazine was the sole carbon and nitrogen source was 80 g (dry weight) of cells per mol of atrazine, and the cell doubling time was around 11 h. Approximately 20% of [U-ring 14C]atrazine was mineralized during primary degradation of atrazine. After atrazine disappeared from the culture supernatant, mineralization continued until the level of mineralization was more than 50%. Under different experimental conditions 10% of the atrazine supplied initially was converted to cyanuric acid and < 1% was converted to other s-triazines after prolonged incubation. Degradation proceeded via dechlorination and N-dealkylation. Atrazine was degraded until the concentration was circa 0.1 milligrams/liter. We obtained evidence showing that strain YAYA6 has specific uptake mechanisms for atrazine but less specific degradation mechanisms for s-triazines.     

Substrate specificity and colorimetric assay for recombinant TrzN derived from Arthrobacter aurescens TC1

The TrzN protein, which is involved in s-triazine herbicide catabolism by Arthrobacter aurescens TC1, was cloned and expressed in Escherichia coli as a His-tagged protein. The recombinant protein was purified via nickel column chromatography. The purified TrzN protein was tested with 31 s-triazine and pyrimidine ring compounds; 22 of the tested compounds were substrates. TrzN showed high activity with sulfur-substituted s-triazines and the highest activity with ametryn sulfoxide. Hydrolysis of ametryn sulfoxide by TrzN, both in vitro and in vivo, yielded a product(s) that reacted with 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) to generate a diagnostic blue product. Atrazine chlorohydrolase, AtzA, did not hydrolyze ametryn sulfoxide, and no color was formed by amending those enzyme incubations with NBD-Cl. TrzN and AtzA could also be distinguished by reaction with ametryn. TrzN, but not AtzA, hydrolyzed ametryn to methylmercaptan. Methylmercaptan reacted with NBD-Cl to produce a diagnostic yellow product having an absorption maximum at 420 nm. The yellow color with ametryn was shown to selectively demonstrate the presence of TrzN, but not AtzA or other enzymes, in whole microbial cells. The present study was the first to purify an active TrzN protein in recombinant form and develop a colorimetric test for determining TrzN activity, and it significantly extends the known substrate range for TrzN.     

Degradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Stenotrophomonas maltophilia PB1.

A mixed microbial culture capable of metabolizing the explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) was obtained from soil enrichments under aerobic and nitrogen-limiting conditions. A bacterium, Stenotrophomonas maltophilia PB1, isolated from the culture used RDX as a sole source of nitrogen for growth. Three moles of nitrogen was used per mole of RDX, yielding a metabolite identified by mass spectroscopy and 1H nuclear magnetic resonance analysis as methylene-N-(hydroxymethyl)-hydroxylamine-N'-(hydroxymethyl)nitroamin e. The bacterium also used s-triazine as a sole source of nitrogen but not the structurally similar compounds octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine, cyanuric acid, and melamine. An inducible RDX-degrading activity was present in crude cell extracts.     

Promotion of Sorghum Callus Growth by the s-Triazine Herbicides

Growth-promoting action of simazine and other s-triazine herbicides was detected by the use of sorghum (Sorghum bicolor [L]. Moench) callus tissue and the chlorophyll retention test. Soil application of simazine [2-chloro-4, 6-bis(ethylamino)-s-triazine] at sublethal levels nearly doubled the growth-promoting action of sorghum root exudates. Treated plants yielded up to 26% more total protein than untreated plants. This indicated that the level of callus growth-promoting action in the root exudate of the plant has a positive effect on its final total protein yield and confirms a positive effect of simazine on total protein content in certain instances. The results may provide a new understanding of the mode of action of s-triazines applied at sublethal levels in increasing protein content and certain enzymic activities of treated plants. It is speculated that the growth-promoting action of these herbicides is hormonal in nature and most likely kinetin-like.     

Mineralization of the s-triazine ring of atrazine by stable bacterial mixed cultures.

Enrichment cultures containing atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) at a concentration of 100 ppm (0.46 mM) as a sole nitrogen source were obtained from soils exposed to repeated spills of atrazine, alachlor, and metolachlor. Bacterial growth occurred concomitantly with formation of metabolites from atrazine and subsequent biosynthesis of protein. When ring-labeled [14C]atrazine was used, 80% or more of the s-triazine ring carbon atoms were liberated as 14CO2. Hydroxyatrazine may be an intermediate in the atrazine mineralization pathway. More than 200 pure cultures isolated from the enrichment cultures failed to utilize atrazine as a nitrogen source. Mixing pure cultures restored atrazine-mineralizing activity. Repeated transfer of the mixed cultures led to increased rates of atrazine metabolism. The rate of atrazine degradation, even at the elevated concentrations used, far exceeded the rates previously reported in soils, waters, and mixed and pure cultures of bacteria.     

Characterization of S-triazine herbicide metabolism by a Nocardioides sp. isolated from agricultural soils

Atrazine, a herbicide widely used in corn production, is a frequently detected groundwater contaminant. Nine gram-positive bacterial strains able to use this herbicide as a sole source of nitrogen were isolated from four farms in central Canada. The strains were divided into two groups based on repetitive extragenic palindromic (rep)-PCR genomic fingerprinting with ERIC and BOXA1R primers. Based on 16S ribosomal DNA sequence analysis, both groups were identified as Nocardioides sp. strains. None of the isolates mineralized [ring-U-(14)C]atrazine. There was no hybridization to genomic DNA from these strains using atzABC cloned from Pseudomonas sp. strain ADP or trzA cloned from Rhodococcus corallinus. S-Triazine degradation was studied in detail in Nocardioides sp. strain C190. Oxygen was not required for atrazine degradation by whole cells or cell extracts. Based on high-pressure liquid chromatography and mass spectrometric analyses of products formed from atrazine in incubations of whole cells with H(2)(18)O, sequential hydrolytic reactions converted atrazine to hydroxyatrazine and then to the end product N-ethylammelide. Isopropylamine, the putative product of the second hydrolytic reaction, supported growth as the sole carbon and nitrogen source. The triazine hydrolase from strain C190 was isolated and purified and found to have a K(m) for atrazine of 25 microM and a V(max) of 31 micromol/min/mg of protein. The subunit molecular mass of the protein was 52 kDa. Atrazine hydrolysis was not inhibited by 500 microM EDTA but was inhibited by 100 microM Mg, Cu, Co, or Zn. Whole cells and purified triazine hydrolase converted a range of chlorine or methylthio-substituted herbicides to the corresponding hydroxy derivatives. In summary, an atrazine-metabolizing Nocardioides sp. widely distributed in agricultural soils degrades a range of s-triazine herbicides by means of a novel s-triazine hydrolase.     

Cloning and analysis of s-triazine catabolic genes from Pseudomonas sp. strain NRRLB-12227.

Pseudomonas sp. strain NRRLB-12227 degrades the s-triazine melamine by a six-step pathway which allows it to use melamine and pathway intermediates as nitrogen sources. With the plasmid pLG221, mutants defective in five of the six steps of the pathway were generated. Tn5-containing-EcoRI fragments from these mutants were cloned and identified by selection for Tn5-encoded kanamycin resistance in transformants. A restriction fragment from ammelide-negative mutant RE411 was used as a probe in colony hybridization experiments to identify cloned wild-type s-triazine catabolic genes encoding ammeline aminohydrolase, ammelide aminohydrolase, and cyanuric acid amidohydrolase. These genes were cloned from total cellular DNA on several similar, but not identical, HindIII fragments, as well as on a PstI fragment and a BglII fragment. Restriction mapping and Southern hybridization analyses of these cloned DNA fragments suggested that these s-triazine catabolic genes may be located on a transposable element, the ends of which are identical 2.2-kb insertion sequences.     

Cloning and comparison of the DNA encoding ammelide aminohydrolase and cyanuric acid amidohydrolase from three s-triazine-degrading bacterial strains.

DNA encoding the catabolism of the s-triazines ammelide and cyanuric acid was cloned from Pseudomonas sp. strain NRRLB-12228 and Klebsiella pneumoniae 99 with, as a probe, a 4.6-kb PstI fragment from a third strain, Pseudomonas sp. strain NRRLB-12227, which also encodes these activities. In strains NRRLB-12228 and 99 the ammelide aminohydrolase (trzC) and cyanuric acid amidohydrolase (trzD) genes are located on identical 4.6-kb PstI fragments which are part of a 12.4-kb DNA segment present in both strains. Strain NRRLB-12227 also carries this 12.4-kb DNA segment, except that a DNA segment of 0.8 to 1.85 kb encoding a third enzyme, ammeline aminohydrolase (trzB), has been inserted next to the ammelide aminohydrolase gene with the accompanying deletion of 1.1 to 2.15 kb of DNA. In addition, the s-triazine catabolic genes are flanked in strain NRRLB-12227 by apparently identical 2.2-kb segments that are not present in the other two strains and that seem to cause rearrangements in adjacent DNA.     

Hydrogen Production by the Photosynthetic Bacterium Rhodospirillum rubrum

Continuous photosynthetic production of hydrogen by Rhodospirillum rubrum in batch cultures was observed up to 80 days with the hydrogen donor, pure lactate or lactic acid-containing wastes, supplied periodically. Hydrogen was produced at an average rate of 6 ml/h per g (dry weight) of cells with whey as a hydrogen donor. In continuous cultures with glutamate as a growth-limiting nitrogen source and lactate as a hydrogen donor, hydrogen was evolved at a rate of 20 ml/h per g (dry weight). The composition of the gas evolved remained practically constant (70 to 75% H₂, 25 to 30% CO₂). Photosynthetic bacteria processing specific organic wastes could be an advantage in large-scale production of hydrogen together with food protein of high value, compared to other biological systems.     

樟绒枝霉(Malbranchea cinnamomea)固体发酵产木聚糖酶的发酵条件优化

[目的] 研究樟绒枝霉(Malbranchea cinnamomea) CAU521利用农业废弃物固体发酵产木聚糖酶的发酵条件.[方法]采用单因素试验法优化影响菌株产酶的各个条件,包括碳源种类、氮源种类、初始pH、初始水分含量、培养温度及发酵时间共6个因素.[结果]获得的最佳产酶条件为:稻草为发酵碳源、2％(W/W)的酵母提取物为氮源、初始pH 7.0、初始水分含量80％和发酵温度45℃.在此条件下发酵6d后木聚糖酶的酶活力达到13 120 U/g干基碳源.[结论]樟绒枝霉固体发酵产木聚糖酶的产酶水平高,生产成本低,具有潜在的工业化应用前景.     

Synthesis of biologically active lipids by fungus Mucor lusitanicus 306D grown on media with various composition

Growth and lipogenesis of fungus Mucor lusitanicus 306 D producing gamma-linolenic acid was studied under various regimes of nitrogen and carbon nutrition. Media containing food industry wastes such as maize extract, molasses, and protein hydrolysate were used. Content of gamma-linolenic acid was higher when using carbohydrates such as glucose and molasses as carbon sources and urea as a nitrogen source. At high glucose concentration (100 g/l), fed batch cultivation provided high content of gamma-linolenic acid in lipids (1 g/l). After extraction of lipids, fungus biomass contained 42% proteins with all essential amino acids. Defatted biomass was shown to be effectively assimilated by minks.     

Cu(II) binding by substituted 1, 3, 5-triazine herbicides

s-Triazine herbicides form relatively stable complexes with cupric ions, complexes in which the major binding site was found to be a cyclic nitrogen N5. Polarographic data have shown that cuprous ions also form quite stable complexes with the studied ligands. The stability constants strongly suggest that s-triazines act in solutions as the monodentate ligands, a result which agrees well with earlier X-ray results.     

Bacterial degradation of N-cyclopropylmelamine. The steps to ring cleavage.

The s-triazine cyclopropylmelamine (N-cyclopropyl-1,3,5-triazine-2,4,6-triamine) was degraded to about 6 mol of NH4+/mol of substrate by a mixture of two bacteria (strains A and D, both Pseudomonas spp.) Only strain A grew with cyclopropylmelamine as sole and limiting source of nitrogen. The organism obtained 2 mol of nitrogen/mol of substrate and excreted a product that was identified as cyclopropylammelide [6-cyclopropylamino-1,3,5-triazine-2,4(1 H,3 H)-dione]. Proteins in extracts from strain A were separated on a Sephadex G-200 column. Cyclopropylmelamine was found to be deaminated in two separable steps to cyclopropylammelide via cyclopropylammeline [4-amino-6-cyclopropylamino-1,3,5-triazine-2(1 H)-one], which was identified. Strain D could not utilize cyclopropylmelamine or cyclopropylammeline, but could utilize cyclopropylammelide (or homologue) as sole and limiting source of nitrogen and obtain about 4 mol of nitrogen/mol of substrate. Proteins in cell extracts from strain D were separated on a DEAE-cellulose column. Alkylammelides were degraded quantitatively by one enzyme fraction to 1 mol of cyanuric acid plus 1 mol of alkylamine/mol of substrate. The specific activities of enzymes in extracts of the two strains were as high as the activities observed during growth. The three activities studied in the two strains were all active under aerobic and oxygen-free conditions. The reactions appear to be hydrolytic, yielding 2 mol of NH4+ plus 1 mol of cyclopropylamine and 1 mol of cyanuric acid/mol of substrate.