Nitrogen control of atrazine utilization in Pseudomonas sp. strain ADP

Pseudomonas sp. strain ADP uses the herbicide atrazine as the sole nitrogen source. We have devised a simple atrazine degradation assay to determine the effect of other nitrogen sources on the atrazine degradation pathway. The atrazine degradation rate was greatly decreased in cells grown on nitrogen sources that support rapid growth of Pseudomonas sp. strain ADP compared to cells cultivated on growth-limiting nitrogen sources. The presence of atrazine in addition to the nitrogen sources did not stimulate degradation. High degradation rates obtained in the presence of ammonium plus the glutamine synthetase inhibitor MSX and also with an Nas(-) mutant derivative grown on nitrate suggest that nitrogen regulation operates by sensing intracellular levels of some key nitrogen-containing metabolite. Nitrate amendment in soil microcosms resulted in decreased atrazine mineralization by the wild-type strain but not by the Nas(-) mutant. This suggests that, although nitrogen repression of the atrazine catabolic pathway may have a strong impact on atrazine biodegradation in nitrogen-fertilized soils, the use of selected mutant variants may contribute to overcoming this limitation.     

The atzABC Genes Encoding Atrazine Catabolism Are Located on a Self-Transmissible Plasmid in Pseudomonas sp. Strain ADP

Pseudomonas sp. strain ADP initiates atrazine catabolism via three enzymatic steps, encoded by atzA, -B, and -C, which yield cyanuric acid, a nitrogen source for many bacteria. In-well lysis, Southern hybridization, and plasmid transfer studies indicated that the atzA, -B, and -C genes are localized on a 96-kb self-transmissible plasmid, pADP-1, in Pseudomonas sp. strain ADP. High-performance liquid chromatography analyses showed that cyanuric acid degradation was not encoded by pADP-1. pADP-1 was transferred to Escherichia coli strains at a frequency of 4.7 × 10⁻². This suggests a potential molecular mechanism for the dispersion of the atzABC genes to other soil bacteria.     

Simultaneous Degradation of Atrazine and Phenol by Pseudomonas sp. Strain ADP: Effects of Toxicity and Adaptation

The strain Pseudomonas sp. strain ADP is able to degrade atrazine as a sole nitrogen source and therefore needs a single source for both carbon and energy for growth. In addition to the typical C source for Pseudomonas, Na₂-succinate, the strain can also grow with phenol as a carbon source. Phenol is oxidized to catechol by a multicomponent phenol hydroxylase. Catechol is degraded via the ortho pathway using catechol 1,2-dioxygenase. It was possible to stimulate the strain in order to degrade very high concentrations of phenol (1,000 mg/liter) and atrazine (150 mg/liter) simultaneously. With cyanuric acid, the major intermediate of atrazine degradation, as an N source, both the growth rate and the phenol degradation rate were similar to those measured with ammonia as an N source. With atrazine as an N source, the growth rate and the phenol degradation rate were reduced to ~35% of those obtained for cyanuric acid. This presents clear evidence that although the first three enzymes of the atrazine degradation pathway are constitutively present, either these enzymes or the uptake of atrazine is the bottleneck that diminishes the growth rate of Pseudomonas sp. strain ADP with atrazine as an N source. Whereas atrazine and cyanuric acid showed no significant toxic effect on the cells, phenol reduces growth and activates or induces typical membrane-adaptive responses known for the genus Pseudomonas. Therefore Pseudomonas sp. strain ADP is an ideal bacterium for the investigation of the regulatory interactions among several catabolic genes and stress response mechanisms during the simultaneous degradation of toxic phenolic compounds and a xenobiotic N source such as atrazine.     

Isolation and characterization of atrazine-degrading Arthrobacter sp. strains from Argentine agricultural soils

Three bacterial strains capable of degrading atrazine were isolated from Manfredi soils (Argentine) using enrichment culture techniques. These soils were used to grow corn and were treated with atrazine for weed control during 3 years. The strains were nonmotile Gram-positive bacilli which formed cleared zones on atrazine solid medium, and the 16S rDNA sequences indicated that they were Arthrobacter sp. strains. The atrazine-degrading activity of the isolates was characterized by the ability to grow with atrazine as the sole nitrogen source, the concomitant herbicide disappearance, and the chloride release. The atrazine-degrader strain Pseudomonas sp. ADP was used for comparative purposes. According to the results, all of the isolates used atrazine as sole source of nitrogen, and sucrose and sodium citrate as the carbon sources for growth. HPLC analyses confirmed herbicide clearance. PCR analysis revealed the presence of the atrazine catabolic genes trzN, atzB, and atzC. The results of this work lead to a better understanding of microbial degradation activity in order to consider the potential application of the isolated strains in bioremediation of atrazine-polluted agricultural soils in Argentina.     

Substrate Specificity of Atrazine Chlorohydrolase and Atrazine-Catabolizing Bacteria

Bacterial atrazine catabolism is initiated by the enzyme atrazine chlorohydrolase (AtzA) in Pseudomonas sp. strain ADP. Other triazine herbicides are metabolized by bacteria, but the enzymological basis of this is unclear. Here we begin to address this by investigating the catalytic activity of AtzA by using substrate analogs. Purified AtzA from Pseudomonas sp. strain ADP catalyzed the hydrolysis of an atrazine analog that was substituted at the chlorine substituent by fluorine. AtzA did not catalyze the hydrolysis of atrazine analogs containing the pseudohalide azido, methoxy, and cyano groups or thiomethyl and amino groups. Atrazine analogs with a chlorine substituent at carbon 2 and N-alkyl groups, ranging in size from methyl to t-butyl, all underwent dechlorination by AtzA. AtzA catalyzed hydrolytic dechlorination when one nitrogen substituent was alkylated and the other was a free amino group. However, when both amino groups were unalkylated, no reaction occurred. Cell extracts were prepared from five strains capable of atrazine dechlorination and known to contain atzA or closely homologous gene sequences: Pseudomonas sp. strain ADP, Rhizobium strain PATR, Alcaligenes strain SG1, Agrobacterium radiobacter J14a, and Ralstonia picketti D. All showed identical substrate specificity to purified AtzA from Pseudomonas sp. strain ADP. Cell extracts from Clavibacter michiganensis ATZ1, which also contains a gene homologous to atzA, were able to transform atrazine analogs containing pseudohalide and thiomethyl groups, in addition to the substrates used by AtzA from Pseudomonas sp. strain ADP. This suggests that either (i) another enzyme(s) is present which confers the broader substrate range or (ii) the AtzA itself has a broader substrate range.     

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-¹⁴C]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 H₂¹⁸O, 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 μM and a V_max of 31 μmol/min/mg of protein. The subunit molecular mass of the protein was 52 kDa. Atrazine hydrolysis was not inhibited by 500 μM EDTA but was inhibited by 100 μM 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.     

Biodegradation of Atrazine by Agrobacterium radiobacter J14a and Use of This Strain in Bioremediation of Contaminated Soil

We examined the ability of a soil bacterium, Agrobacterium radiobacter J14a, to degrade the herbicide atrazine under a variety of cultural conditions, and we used this bacterium to increase the biodegradation of atrazine in soils from agricultural chemical distribution sites. J14a cells grown in nitrogen-free medium with citrate and sucrose as carbon sources mineralized 94% of 50 μg of [¹⁴C-U-ring]atrazine ml⁻¹ in 72 h with a concurrent increase in the population size from 7.9 × 10⁵ to 5.0 × 10⁷ cells ml⁻¹. Under these conditions cells mineralized the [ethyl-¹⁴C]atrazine and incorporated approximately 30% of the ¹⁴C into the J14a biomass. Cells grown in medium without additional carbon and nitrogen sources degraded atrazine, but the cell numbers did not increase. Metabolites produced by J14a during atrazine degradation include hydroxyatrazine, deethylatrazine, and deethyl-hydroxyatrazine. The addition of 10⁵ J14a cells g⁻¹ into soil with a low indigenous population of atrazine degraders treated with 50 and 200 μg of atrazine g⁻¹ soil resulted in two to five times higher mineralization than in the noninoculated soil. Sucrose addition did not result in significantly faster mineralization rates or shorten degradation lag times. However, J14a introduction (10⁵ cells g⁻¹) into another soil with a larger indigenous atrazine-mineralizing population reduced the atrazine degradation lag times below those in noninoculated treatments but did not generally increase total atrazine mineralization.     

Atrazine degradation in saline wastewater by Pseudomonas sp strain ADP

Wastewater from atrazine manufacturing plants contains large amounts of residual atrazine and atrazine synthesis products, which must be removed before disposal. One of the obstacles to biological treatment of these wastewaters is their high salt content, eg, up to 4% NaCl (w/v). To enable biological treatment, bacteria capable of atrazine mineralization must be adapted to high-salinity conditions. A recently isolated atrazine-degrading bacterium, Pseudomonas sp strain ADP, originally isolated from contaminated soils was adapted to biodegradation of atrazine at salt concentrations relevant to atrazine manufacturing wastewater. The adaptation mechanism was based on the ability of the bacterium to produce trehalose as its main osmolyte. Trehalose accumulation was confirmed by natural-abundance ¹H NMR spectral analysis. The bacterium synthesized trehalose de novo in the cells, but could not utilize trehalose added to the growth medium. Interestingly, the bacterium could not produce glycine betaine (a common compatible solute), but addition of 1 mM of glycine betaine to the medium induced salt tolerance. Osmoregulated Pseudomonas sp strain ADP, feeding on citrate decreased the concentration of atrazine in non-sterile authentic wastewater from 25 ppm to below 1 ppm in less than 2 days. The results of our study suggest that salt-adapted Pseudomonas sp strain ADP can be used for atrazine degradation in salt-containing wastewater. Received 26 August 1997/ Accepted in revised form 06 December 1997     

Dual-Bioaugmentation Strategy To Enhance Remediation of Cocontaminated Soil

Although metals are thought to inhibit the ability of microorganisms to degrade organic pollutants, several microbial mechanisms of resistance to metal are known to exist. This study examined the potential of cadmium-resistant microorganisms to reduce soluble cadmium levels to enhance degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) under conditions of cocontamination. Four cadmium-resistant soil microorganisms were examined in this study. Resistant up to a cadmium concentration of 275 μg ml⁻¹, these isolates represented the common soil genera Arthrobacter, Bacillus, and Pseudomonas. Isolates Pseudomonas sp. strain H1 and Bacillus sp. strain H9 had a plasmid-dependent intracellular mechanism of cadmium detoxification, reducing soluble cadmium levels by 36%. Isolates Arthrobacter strain D9 and Pseudomonas strain I1a both produced an extracellular polymer layer that bound and reduced soluble cadmium levels by 22 and 11%, respectively. Although none of the cadmium-resistant isolates could degrade 2,4-D, results of dual-bioaugmentation studies conducted with both pure culture and laboratory soil microcosms showed that each of four cadmium-resistant isolates supported the degradation of 500-μg ml⁻¹ 2,4-D by the cadmium-sensitive 2,4-D degrader Ralstonia eutropha JMP134. Degradation occurred in the presence of up to 24 μg of cadmium ml⁻¹ in pure culture and up to 60 μg of cadmium g⁻¹ in amended soil microcosms. In a pilot field study conducted with 5-gallon soil bioreactors, the dual-bioaugmentation strategy was again evaluated. Here, the cadmium-resistant isolate Pseudomonas strain H1 enhanced degradation of 2,4-D in reactors inoculated with R. eutropha JMP134 in the presence of 60 μg of cadmium g⁻¹. Overall, dual bioaugmentation appears to be a viable approach in the remediation of cocontaminated soils.     

Molecular Basis of a Bacterial Consortium: Interspecies Catabolism of Atrazine

Pseudomonas sp. strain ADP contains the genes, atzA, -B, and -C, that encode three enzymes which metabolize atrazine to cyanuric acid. Atrazine-catabolizing pure cultures isolated from around the world contain genes homologous to atzA, -B, and -C. The present study was conducted to determine whether the same genes are present in an atrazine-catabolizing bacterial consortium and how the genes and metabolism are subdivided among member species. The consortium contained four or more bacterial species, but two members, Clavibacter michiganese ATZ1 and Pseudomonas sp. strain CN1, collectively mineralized atrazine. C. michiganese ATZ1 released chloride from atrazine, produced hydroxyatrazine, and contained a homolog to the atzA gene that encoded atrazine chlorohydrolase. C. michiganese ATZ1 stoichiometrically metabolized hydroxyatrazine to N-ethylammelide and contained genes homologous to atzB and atzC, suggesting that either a functional AtzB or -C catalyzed N-isopropylamine release from hydroxyatrazine. C. michiganese ATZ1 grew on isopropylamine as its sole carbon and nitrogen source, explaining the ability of the consortium to use atrazine as the sole carbon and nitrogen source. A second consortium member, Pseudomonas sp. strain CN1, metabolized the N-ethylammelide produced by C. michiganese ATZ1 to transiently form cyanuric acid, a reaction catalyzed by AtzC. A gene homologous to the atzC gene of Pseudomonas sp. strain ADP was present, as demonstrated by Southern hybridization and PCR. Pseudomonas sp. strain CN1, but not C. michiganese, metabolized cyanuric acid. The consortium metabolized atrazine faster than did C. michiganese individually. Additionally, the consortium metabolized a much broader set of triazine ring compounds than did previously described pure cultures in which the atzABC genes had been identified. These data begin to elucidate the genetic and metabolic bases of catabolism by multimember consortia.     

Bacterial Chemotaxis to Atrazine and Related s-Triazines

Pseudomonas sp. strain ADP utilizes the human-made s-triazine herbicide atrazine as the sole nitrogen source. The results reported here demonstrate that atrazine and the atrazine degradation intermediates N-isopropylammelide and cyanuric acid are chemoattractants for strain ADP. In addition, the nonmetabolized s-triazine ametryn was also an attractant. The chemotactic response to these s-triazines was not specifically induced during growth with atrazine, and atrazine metabolism was not required for the chemotactic response. A cured variant of strain ADP (ADP M13-2) was attracted to s-triazines, indicating that the atrazine catabolic plasmid pADP-1 is not necessary for the chemotactic response and that atrazine degradation and chemotaxis are not genetically linked. These results indicate that atrazine and related s-triazines are detected by one or more chromosomally encoded chemoreceptors in Pseudomonas sp. strain ADP. We demonstrated that Escherichia coli is attracted to the s-triazine compounds N-isopropylammelide and cyanuric acid, and an E. coli mutant lacking Tap (the pyrimidine chemoreceptor) was unable to respond to s-triazines. These data indicate that pyrimidines and triazines are detected by the same chemoreceptor (Tap) in E. coli. We showed that Pseudomonas sp. strain ADP is attracted to pyrimidines, which are the naturally occurring structures closest to triazines, and propose that chemotaxis toward s-triazines may be due to fortuitous recognition by a pyrimidine chemoreceptor in Pseudomonas sp. strain ADP. In competition assays, the presence of atrazine inhibited chemotaxis of Pseudomonas sp. strain ADP to cytosine, and cytosine inhibited chemotaxis to atrazine, suggesting that pyrimidines and s-triazines are detected by the same chemoreceptor.Atrazine [2-chloro-4-(N-ethylamino)-6-(N-isopropylamino)-1,3,5-s-triazine] is a human-made herbicide that is used worldwide to control broadleaf and grassy weeds. As one of the most heavily used herbicides in the United States, atrazine can be present in parts per million in agricultural runoffs (3), which exceeds the U.S. Environmental Protection Agency''s maximum allowable contaminant level of 3 ppb in ground and surface waters (13). Atrazine is persistent in soil (34) and was once considered nontoxic to animals. However, recent studies have shown that atrazine causes sexual abnormalities in frogs (21, 22, 50), reduced testosterone production in rats (53), and elevated levels of prostate cancer in workers at an atrazine-manufacturing factory (45). These studies suggest that there is cause for concern about atrazine residues in soil, groundwater, and surface waters.Several bacterial strains capable of mineralizing atrazine have been isolated (4, 27, 41, 49, 51, 52, 58). The best-studied atrazine-degrading strain, Pseudomonas sp. strain ADP (atrazine degrading pseudomonad), was isolated from an atrazine spill site in Minnesota (27). Strain ADP utilizes atrazine as a sole nitrogen source and mineralizes it in the process (27). The pathway of atrazine degradation in strain ADP has been characterized in detail (Fig. ​(Fig.1),1), and the genes encoding the six enzymes required for atrazine degradation have been cloned and sequenced (5, 7, 9, 10, 29, 42). The six genes are located in four distant locations on the atrazine catabolic plasmid (pADP-1) present in strain ADP (10, 29). atzA, atzB, and atzC, which encode the first three enzymes of the pathway, are constitutively expressed and highly conserved in atrazine-degrading bacteria isolated from geographically distinct locations (8, 11). Products of the atzDEF gene cluster catalyze the last three steps of atrazine degradation. This operon is divergently transcribed from atzR, the product of which has high homology to LysR-type regulatory proteins (29). AtzR and the inducer cyanuric acid are required for the expression of the atzDEF operon (14), and the operon is also subject to nitrogen control (15).Open in a separate windowFIG. 1.Pathway of atrazine degradation in Pseudomonas sp. strain ADP (reviewed in reference 55).In a study investigating the bioavailability of atrazine, Park et al. provided evidence that two atrazine-degrading strains, Pseudomonas sp. strain ADP and Agrobacterium radiobacter J14a, were chemotactically attracted to atrazine (38). Chemotaxis, the ability of motile bacteria to detect and respond to specific chemicals, can help bacteria find an optimal niche for their survival and growth and may play a role in the efficient degradation of pollutants in the environment (33, 37). Chemotaxis has been shown to enhance naphthalene biodegradation in both a heterogeneous aqueous system (30) and a non-aqueous-phase liquid system (24). In addition, a chemotactic naphthalene-degrading strain caused a higher rate of naphthalene desorption than was observed with nonchemotactic and nonmotile strains (24). Pseudomonas sp. strain ADP and recombinant strains expressing atz genes have been used to remove atrazine from soil in laboratory and field scale experiments (32, 48). If chemotaxis can enhance bioavailability in environments where the chemicals are sorbed to particles, the use of a motile chemotactic strain for bioremediation would be advantageous. Aside from the practical implications of atrazine chemotaxis, we are interested in understanding the evolution of a chemotactic response to a human-made chemical that was initially synthesized just 50 years ago (23). The results reported here indicate that Pseudomonas sp. strain ADP is chemotactically attracted to atrazine, atrazine metabolites, and the nonmetabolizable structural analog ametryn. The chemotactic response is not induced during growth with atrazine in strain ADP and does not require atrazine metabolism. We demonstrated that a single chemoreceptor (Tap) mediates chemotaxis to s-triazines and structurally similar pyrimidines in Escherichia coli. Additionally, we found that Pseudomonas sp. strain ADP is attracted to pyrimidines. In competition assays, cytosine inhibited atrazine chemotaxis, and vice versa. We therefore concluded that pyrimidines and s-triazines are detected by a single chemoreceptor in Pseudomonas sp. strain ADP.     

Adaptation of Pseudomonas sp. Strain 7-6 to Quaternary Ammonium Compounds and Their Degradation via Dual Pathways

Pseudomonas sp. strain 7-6, isolated from active sludge obtained from a wastewater facility, utilized a quaternary ammonium surfactant, n-dodecyltrimethylammonium chloride (DTAC), as its sole carbon, nitrogen, and energy source. When initially grown in the presence of 10 mM DTAC medium, the isolate was unable to degrade DTAC. The strain was cultivated in gradually increasing concentrations of the surfactant until continuous exposure led to high tolerance and biodegradation of the compound. Based on the identification of five metabolites by gas chromatography-mass spectrometry analysis, two possible pathways for DTAC metabolism were proposed. In pathway 1, DTAC is converted to lauric acid via n-dodecanal with the release of trimethylamine; in pathway 2, DTAC is converted to lauric acid via n-dodecyldimethylamine and then n-dodecanal with the release of dimethylamine. Among the identified metabolites, the strain precultivated on DTAC medium could utilize n-dodecanal and lauric acid as sole carbon sources and trimethylamine and dimethylamine as sole nitrogen sources, but it could not efficiently utilize n-dodecyldimethylamine. These results indicated pathway 1 is the main pathway for the degradation of DTAC.     

Biochemical and Genetic Analyses of Ferulic Acid Catabolism in Pseudomonas sp. Strain HR199

The gene loci fcs, encoding feruloyl coenzyme A (feruloyl-CoA) synthetase, ech, encoding enoyl-CoA hydratase/aldolase, and aat, encoding β-ketothiolase, which are involved in the catabolism of ferulic acid and eugenol in Pseudomonas sp. strain HR199 (DSM7063), were localized on a DNA region covered by two EcoRI fragments (E230 and E94), which were recently cloned from a Pseudomonas sp. strain HR199 genomic library in the cosmid pVK100. The nucleotide sequences of parts of fragments E230 and E94 were determined, revealing the arrangement of the aforementioned genes. To confirm the function of the structural genes fcs and ech, they were cloned and expressed in Escherichia coli. Recombinant strains harboring both genes were able to transform ferulic acid to vanillin. The feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase activities of the fcs and ech gene products, respectively, were confirmed by photometric assays and by high-pressure liquid chromatography analysis. To prove the essential involvement of the fcs, ech, and aat genes in the catabolism of ferulic acid and eugenol in Pseudomonas sp. strain HR199, these genes were inactivated separately by the insertion of omega elements. The corresponding mutants Pseudomonas sp. strain HRfcsΩGm and Pseudomonas sp. strain HRechΩKm were not able to grow on ferulic acid or on eugenol, whereas the mutant Pseudomonas sp. strain HRaatΩKm exhibited a ferulic acid- and eugenol-positive phenotype like the wild type. In conclusion, the degradation pathway of eugenol via ferulic acid and the necessity of the activation of ferulic acid to the corresponding CoA ester was confirmed. The aat gene product was shown not to be involved in this catabolism, thus excluding a β-oxidation analogous degradation pathway for ferulic acid. Moreover, the function of the ech gene product as an enoyl-CoA hydratase/aldolase suggests that ferulic acid degradation in Pseudomonas sp. strain HR199 proceeds via a similar pathway to that recently described for Pseudomonas fluorescens AN103.     

AtzC Is a New Member of the Amidohydrolase Protein Superfamily and Is Homologous to Other Atrazine-Metabolizing Enzymes

Pseudomonas sp. strain ADP metabolizes atrazine to cyanuric acid via three plasmid-encoded enzymes, AtzA, AtzB, and AtzC. The first enzyme, AtzA, catalyzes the hydrolytic dechlorination of atrazine, yielding hydroxyatrazine. The second enzyme, AtzB, catalyzes hydroxyatrazine deamidation, yielding N-isopropylammelide. In this study, the third gene in the atrazine catabolic pathway, atzC, was cloned from a Pseudomonas sp. strain ADP cosmid library as a 25-kb EcoRI DNA fragment in Escherichia coli. The atzC gene was further delimited by functional analysis following transposon Tn5 mutagenesis and subcloned as a 2.0-kb EcoRI-AvaI fragment. An E. coli strain containing this DNA fragment expressed N-isopropylammelide isopropylamino hydrolase activity, metabolizing N-isopropylammelide stoichiometrically to cyanuric acid and N-isopropylamine. The 2.0-kb DNA fragment was sequenced and found to contain a single open reading frame of 1,209 nucleotides, encoding a protein of 403 amino acids. AtzC showed modest sequence identity of 29 and 25%, respectively, to cytosine deaminase and dihydroorotase, both members of an amidohydrolase protein superfamily. The sequence of AtzC was compared to that of E. coli cytosine deaminase in the regions containing the five ligands to the catalytically important metal for the protein. Pairwise comparison of the 35 amino acids showed 61% sequence identity and 85% sequence similarity. AtzC is thus assigned to the amidohydrolase protein family that includes cytosine deaminase, urease, adenine deaminase, and phosphotriester hydrolase. Similar sequence comparisons of the most highly conserved regions indicated that the AtzA and AtzB proteins also belong to the same amidohydrolase family. Overall, the data suggest that AtzA, AtzB, and AtzC diverged from a common ancestor and, by random events, have been reconstituted onto an atrazine catabolic plasmid.     

Encapsulation of Pseudomonas sp. ADP cells in electrospun microtubes for atrazine bioremediation

Electrospun hollow polymeric microfibers (microtubes) were evaluated as an encapsulation method for the atrazine degrading bacterium Pseudomonas sp. ADP. Pseudomonas sp. ADP cells were successfully incorporated in a formulation containing a core solution of polyethylene oxide dissolved in water and spun with an outer shell solution made of polycaprolactone and polyethylene glycol dissolved in a chloroform and dimethylformamide. The resulting microtubes, collected as mats, were partially collapsed with a ribbon-like structure. Following encapsulation, the atrazine degradation rate was low (0.03?±?0.01?mg atrazine/h/g fiber) indicating that the electrospinning process negatively affected cell activity. Atrazine degradation was restored to 0.5?±?0.1?mg atrazine/h/g fiber by subjecting the microtubes to a period of growth. After 3 and 7?days growth periods, encapsulated cells were able to remove 20.6?±?3 and 47.6?±?5.9?mg atrazine/g mat, respectively, in successive batches under non-growth conditions (with no additional electron donor) until atrazine was detected in the medium. The loss of atrazine degrading capacity was regained following an additional cell-growth period.     

Metabolic ability and gene characteristics of Arthrobacter sp. strain DNS10, the sole atrazine-degrading strain in a consortium isolated from black soil

Strain DNS10 was the only member that could utilize atrazine as the sole nitrogen source for growth in an atrazine-degrading consortium which was isolated from black soil previously in our laboratory. It belongs to the genus Arthrobacter according to the sequence of 16S rRNA gene and is designated as Arthrobacter sp. DNS10. 16S rRNA gene phylogenetic analysis showed that strain DNS10 was located in a different evolutionary branch comparing with other Arthrobacter sp. atrazine-degrading strains. The degrading genes such as trzN, atzB and atzC harbored in strain DNS10 revealed high sequence similarity with those in Arthrobacter aurescens TC1 and Pseudomonas sp. ADP. These genes enabled the strain DNS10 to decompose atrazine to cyanuric acid. This was further proved by the results that the strain DNS10 (10⁸ CFU mL⁻¹) could degrade the whole atrazine (100 mg L⁻¹) in the medium within 24 h at 30 °C and there was 66.13 ± 2.11 mg L⁻¹ cyanuric acid accumulated at 24 h. These results imply that the strain DNS10 seems to be an excellent atrazine-degrading strain. Furthermore, this paper helps us in the better understanding of the strain evolution by comparing the metabolic ability and gene characteristics of strain DNS10 with other geographically distinct atrazine-degrading strains.     

Purification, Characterization, and Crystallization of the Components of the Nitrobenzene and 2-Nitrotoluene Dioxygenase Enzyme Systems

The protein components of the 2-nitrotoluene (2NT) and nitrobenzene dioxygenase enzyme systems from Acidovorax sp. strain JS42 and Comamonas sp. strain JS765, respectively, were purified and characterized. These enzymes catalyze the initial step in the degradation of 2-nitrotoluene and nitrobenzene. The identical shared reductase and ferredoxin components were monomers of 35 and 11.5 kDa, respectively. The reductase component contained 1.86 g-atoms iron, 2.01 g-atoms sulfur, and one molecule of flavin adenine dinucleotide per monomer. Spectral properties of the reductase indicated the presence of a plant-type [2Fe-2S] center and a flavin. The reductase catalyzed the reduction of cytochrome c, ferricyanide, and 2,6-dichlorophenol indophenol. The ferredoxin contained 2.20 g-atoms iron and 1.99 g-atoms sulfur per monomer and had spectral properties indicative of a Rieske [2Fe-2S] center. The ferredoxin component could be effectively replaced by the ferredoxin from the Pseudomonas sp. strain NCIB 9816-4 naphthalene dioxygenase system but not by that from the Burkholderia sp. strain LB400 biphenyl or Pseudomonas putida F1 toluene dioxygenase system. The oxygenases from the 2-nitrotoluene and nitrobenzene dioxygenase systems each had spectral properties indicating the presence of a Rieske [2Fe-2S] center, and the subunit composition of each oxygenase was an α₃β₃ hexamer. The apparent K_m of 2-nitrotoluene dioxygenase for 2NT was 20 μM, and that for naphthalene was 121 μM. The specificity constants were 7.0 μM⁻¹ min⁻¹ for 2NT and 1.2 μM⁻¹ min⁻¹ for naphthalene, indicating that the enzyme is more efficient with 2NT as a substrate. Diffraction-quality crystals of the two oxygenases were obtained.     

Metabolism of the Aliphatic Nitramine 4-Nitro-2,4-Diazabutanal by Methylobacterium sp. Strain JS178

The aliphatic nitramine 4-nitro-2,4-diazabutanal (NDAB; C₂H₅N₃O₃) is a ring cleavage metabolite that accumulates during the aerobic degradation of the energetic compound hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by various Rhodococcus spp. NDAB is also produced during the alkaline hydrolysis of either RDX or octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) and during the photolysis of RDX. Traces of NDAB were observed in a soil sampled from an ammunition-manufacturing facility contaminated with both HMX and RDX, suggesting natural attenuation. In this study, we report the isolation of a soil bacterium that is able to degrade NDAB under aerobic conditions. The isolate is a pink-pigmented facultative methylotroph affiliated with the genus Methylobacterium. The strain, named Methylobacterium sp. strain JS178, degrades NDAB as a sole nitrogen source, with concomitant growth and formation of 1 molar equivalent of nitrous oxide (N₂O). Comparison of the growth yield of strain JS178 grown on NDAB, nitrite (NO₂⁻), or ammonium (NH₄⁺) as a nitrogen source revealed that 1 N equivalent is assimilated from each mole of NDAB, which completes the nitrogen mass balance. In radiotracer experiments, strain JS178 mineralized 1 C of the [¹⁴C]NDAB produced in situ from [¹⁴C]RDX by Rhodococcus sp. strain DN22. Studies on the regulation of NDAB degradation indicated that allantoin, an intermediate in the purine catabolic pathway and a central molecule in the storage and transport of nitrogen in plants, up-regulated the enzyme(s) involved in the degradation of the nitramine. The results reveal the potential for the sequential participation of rhodococci and methylobacteria to effect the complete degradation of RDX.     

Survival in Sterile Soil and Atrazine Degradation of Pseudomonas sp. Strain ADP Immobilized on Zeolite

In laboratory settings, the ability of bacteria and fungi to degrade many environmental contaminants is well proven. However, the potential of microbial inoculants in soil remediation has not often been realized because catabolically competent strains rarely survive and proliferate in soil, and even if they do, they usually fail to express their desired catabolic potential. One method to address the survival problem is formulating the microorganisms with physical and chemical support systems. This study investigates the survival of Pseudomonas sp. strain ADP in sterile soil and its retention of atrazine-degrading functionality. Assessment was conducted with free and zeolite-immobilized bacteria incorporated into the soil. Pseudomonas sp. strain ADP remained viable for at least 10 weeks when stored at 15°C in sterile soil. Cell numbers increased for both free and zeolite-immobilized bacteria during this period, except for free cells when grown in Miller's Luria-Bertani medium, which exhibited constant cell numbers over the 10 weeks. Only the zeolite-immobilized cell retained full functionality to degrade atrazine after 10 weeks in sterile soil regardless of the medium used to culture Pseudomonas sp. strain ADP. Functionality was diminished in free-cell inoculations except when using an improved culture medium. Survival of zeolite-immobilized Pseudomonas sp. strain ADP separated from the soil matrix after 10 weeks’ incubation was significantly (p < .05) greater than in soil inoculated with free cells or in the soil fraction inoculated by release from zeolite-immobilized Pseudomonas sp. strain ADP.     

Simultaneous degradation of atrazine and phenol by Pseudomonas sp. strain ADP: effects of toxicity and adaptation

The strain Pseudomonas sp. strain ADP is able to degrade atrazine as a sole nitrogen source and therefore needs a single source for both carbon and energy for growth. In addition to the typical C source for Pseudomonas, Na(2)-succinate, the strain can also grow with phenol as a carbon source. Phenol is oxidized to catechol by a multicomponent phenol hydroxylase. Catechol is degraded via the ortho pathway using catechol 1,2-dioxygenase. It was possible to stimulate the strain in order to degrade very high concentrations of phenol (1,000 mg/liter) and atrazine (150 mg/liter) simultaneously. With cyanuric acid, the major intermediate of atrazine degradation, as an N source, both the growth rate and the phenol degradation rate were similar to those measured with ammonia as an N source. With atrazine as an N source, the growth rate and the phenol degradation rate were reduced to approximately 35% of those obtained for cyanuric acid. This presents clear evidence that although the first three enzymes of the atrazine degradation pathway are constitutively present, either these enzymes or the uptake of atrazine is the bottleneck that diminishes the growth rate of Pseudomonas sp. strain ADP with atrazine as an N source. Whereas atrazine and cyanuric acid showed no significant toxic effect on the cells, phenol reduces growth and activates or induces typical membrane-adaptive responses known for the genus Pseudomonas. Therefore Pseudomonas sp. strain ADP is an ideal bacterium for the investigation of the regulatory interactions among several catabolic genes and stress response mechanisms during the simultaneous degradation of toxic phenolic compounds and a xenobiotic N source such as atrazine.