Regulated expression systems for the development of whole-cell biocatalysts expressing oxidative enzymes in a sequential manner

This work reports the preparation of two recombinant strains each containing two enzymatic activities mutually expressed through regulated systems for production of functionalized epoxides in one-pot reactions. One strain was Pseudomonas putida PaW340, containing the gene coding for styrene monooxygenase (SMO) from Pseudomonas fluorescens ST under the auto-inducing Ptou promoter and the TouR regulator of Pseudomonas sp. OX1 and the gene coding for naphthalene dihydrodiol dehydrogenase (NDDH) from P. fluorescens N3 under the Ptac promoter inducible by IPTG. The second strain was Escherichia coli JM109, in which the expression of SMO was under the control of the Pnah promoter and the NahR regulator of P. fluorescens N3 inducible by salicylate, while the gene expressing NDDH was under the control of the Plac promoter inducible by IPTG. SMO and NDDH activities were tested in bioconversion experiments using cinnamyl alcohol as reference substrate. The application that we selected is one example of the sequential use of the two enzymatic activities which require a temporal control of the expression of both genes.     

Cloning of the <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. FG1 dehalogenase genes and construction of hybrid pathways in <Emphasis Type="Italic">Pseudomonas putida</Emphasis> strains

An Arthrobacter strain, able to utilize 4-chlorobenzoic acid as the sole carbon and energy source, was isolated and characterized. The first step of the catabolic pathway was found to proceed via a hydrolytic dehalogenation that leads to the formation of 4-hydroxybenzoic acid. The dehalogenase encoding genes (fcb) were sequenced and found highly homologous to and organized as those of other 4-chlorobenzoic acid degrading Arthrobacter strains. The fcb genes were cloned and successfully expressed in the heterologous host Pseudomonas putida PaW340 and P. putida KT2442 upper TOL, which acquired the ability to grow on 4-chlorobenzoic acid and 4-chlorotoluene, respectively. The cloned dehalogenase displayed a high specificity for para-substituted haloaromatics with affinity Cl > Br > I > F, in the order.     

Stable Coexistence of Five Bacterial Strains as a Cellulose-Degrading Community

A cellulose-degrading defined mixed culture (designated SF356) consisting of five bacterial strains (Clostridium straminisolvens CSK1, Clostridium sp. strain FG4, Pseudoxanthomonas sp. strain M1-3, Brevibacillus sp. strain M1-5, and Bordetella sp. strain M1-6) exhibited both functional and structural stability; namely, no change in cellulose-degrading efficiency was observed, and all members stably coexisted through 20 subcultures. In order to investigate the mechanisms responsible for the observed stability, “knockout communities” in which one of the members was eliminated from SF356 were constructed. The dynamics of the community structure and the cellulose degradation profiles of these mixed cultures were determined in order to evaluate the roles played by each eliminated member in situ and its impact on the other members of the community. Integration of each result gave the following estimates of the bacterial relationships. Synergistic relationships between an anaerobic cellulolytic bacterium (C. straminisolvens CSK1) and two strains of aerobic bacteria (Pseudoxanthomonas sp. strain M1-3 and Brevibacillus sp. strain M1-5) were observed; the aerobes introduced anaerobic conditions, and C. straminisolvens CSK1 supplied metabolites (acetate and glucose). In addition, there were negative relationships, such as the inhibition of cellulose degradation by producing excess amounts of acetic acid by Clostridium sp. strain FG4, and growth suppression of Bordetella sp. strain M1-6 by Brevibacillus sp. strain M1-5. The balance of the various types of relationships (both positive and negative) is thus considered to be essential for the stable coexistence of the members of this mixed culture.     

Biodegradation kinetics of 4-fluorocinnamic acid by a consortium of Arthrobacter and Ralstonia strains

Arthrobacter sp. strain G1 is able to grow on 4-fluorocinnamic acid (4-FCA) as sole carbon source. The organism converts 4-FCA into 4-fluorobenzoic acid (4-FBA) and utilizes the two-carbon side-chain for growth with some formation of 4-fluoroacetophenone as a dead-end side product. We also have isolated Ralstonia sp. strain H1, an organism that degrades 4-FBA. A consortium of strains G1 and H1 degraded 4-FCA with Monod kinetics during growth in batch and continuous cultures. Specific growth rates of strain G1 and specific degradation rates of 4-FCA were observed to follow substrate inhibition kinetics, which could be modeled using the kinetic models of Haldane–Andrew and Luong–Levenspiel. The mixed culture showed complete mineralization of 4-FCA with quantitative release of fluoride, both in batch and continuous cultures. Steady-state chemostat cultures that were exposed to shock loadings of substrate responded with rapid degradation and returned to steady-state in 10–15 h, indicating that the mixed culture provided a robust system for continuous 4-FCA degradation.     

Characterization of the 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE) degradation system in Janibacter sp. TYM3221

Bacterial degradation of 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE) has been previously reported, however, its degradation enzyme system has not been characterized. In this study, a DDE-degrading bacterium, Janibacter sp. TYM3221, was isolated and characterized. Transformation of DDE was demonstrated by TYM3211 resting cells grown in LB in the presence and absence of biphenyl. Gas chromatography–mass spectrometry analysis revealed five metabolites of DDE containing a meta-ring cleavage product and 4-chlorobenzoic acid, suggesting that TYM3221 degrades DDE to 4-chlorobenzoic acid via a meta-ring cleavage product. A gene cluster, bphAaAbAcAd, which codes for biphenyl dioxygenase subunits, was cloned from TYM3221. A mutant strain with a bphAa-gene inactivation did not grow on biphenyl, and showed no DDE degradation activity. These results indicate that in strain TYM3221, the bphAa-coded biphenyl dioxygenase is involved not only in the metabolism of biphenyl but also in the degradation of DDE.     

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.     

Toluene-Degrading Bacteria Are Chemotactic towards the Environmental Pollutants Benzene, Toluene, and Trichloroethylene

The bioremediation of polluted groundwater and toxic waste sites requires that bacteria come into close physical contact with pollutants. This can be accomplished by chemotaxis. Five motile strains of bacteria that use five different pathways to degrade toluene were tested for their ability to detect and swim towards this pollutant. Three of the five strains (Pseudomonas putida F1, Ralstonia pickettii PKO1, and Burkholderia cepacia G4) were attracted to toluene. In each case, the response was dependent on induction by growth with toluene. Pseudomonas mendocina KR1 and P. putida PaW15 did not show a convincing response. The chemotactic responses of P. putida F1 to a variety of toxic aromatic hydrocarbons and chlorinated aliphatic compounds were examined. Compounds that are growth substrates for P. putida F1, including benzene and ethylbenzene, were chemoattractants. P. putida F1 was also attracted to trichloroethylene (TCE), which is not a growth substrate but is dechlorinated and detoxified by P. putida F1. Mutant strains of P. putida F1 that do not oxidize toluene were attracted to toluene, indicating that toluene itself and not a metabolite was the compound detected. The two-component response regulator pair TodS and TodT, which control expression of the toluene degradation genes in P. putida F1, were required for the response. This demonstration that soil bacteria can sense and swim towards the toxic compounds toluene, benzene, TCE, and related chemicals suggests that the introduction of chemotactic bacteria into selected polluted sites may accelerate bioremediation processes.     

Study of Biochemical Pathways and Enzymes Involved in Pyrene Degradation by Mycobacterium sp. Strain KMS

Pyrene degradation is known in bacteria. In this study, Mycobacterium sp. strain KMS was used to study the metabolites produced during, and enzymes involved in, pyrene degradation. Several key metabolites, including pyrene-4,5-dione, cis-4,5-pyrene-dihydrodiol, phenanthrene-4,5-dicarboxylic acid, and 4-phenanthroic acid, were identified during pyrene degradation. Pyrene-4,5-dione, which accumulates as an end product in some gram-negative bacterial cultures, was further utilized and degraded by Mycobacterium sp. strain KMS. Enzymes involved in pyrene degradation by Mycobacterium sp. strain KMS were studied, using 2-D gel electrophoresis. The first protein in the catabolic pathway, aromatic-ring-hydroxylating dioxygenase, which oxidizes pyrene to cis-4,5-pyrene-dihydrodiol, was induced with the addition of pyrene and pyrene-4,5-dione to the cultures. The subcomponents of dioxygenase, including the alpha and beta subunits, 4Fe-4S ferredoxin, and the Rieske (2Fe-2S) region, were all induced. Other proteins responsible for further pyrene degradation, such as dihydrodiol dehydrogenase, oxidoreductase, and epoxide hydrolase, were also found to be significantly induced by the presence of pyrene and pyrene-4,5-dione. Several nonpathway-related proteins, including sterol-binding protein and cytochrome P450, were induced. A pyrene degradation pathway for Mycobacterium sp. strain KMS was proposed and confirmed by proteomic study by identifying almost all the enzymes required during the initial steps of pyrene degradation.     

mRNA Differential Display in a Microbial Enrichment Culture: Simultaneous Identification of Three Cyclohexanone Monooxygenases from Three Species

mRNA differential display has been used to identify cyclohexanone oxidation genes in a mixed microbial community derived from a wastewater bioreactor. Thirteen DNA fragments randomly amplified from the total RNA of an enrichment subculture exposed to cyclohexanone corresponded to genes predicted to be involved in the degradation of cyclohexanone. Nine of these DNA fragments are part of genes encoding three distinct Baeyer-Villiger cyclohexanone monooxygenases from three different bacterial species present in the enrichment culture. In Arthrobacter sp. strain BP2 and Rhodococcus sp. strain Phi2, the monooxygenase is part of a gene cluster that includes all the genes required for the degradation of cyclohexanone, while in Rhodococcus sp. strain Phi1 the genes surrounding the monooxygenase are not predicted to be involved in this degradation pathway but rather seem to belong to a biosynthetic pathway. Furthermore, in the case of Arthrobacter strain BP2, three other genes flanking the monooxygenase were identified by differential display, demonstrating that the repeated sampling of bacterial operons shown earlier for a pure culture (D. M. Walters, R. Russ, H. Knackmuss, and P. E. Rouvière, Gene 273:305-315, 2001) is also possible for microbial communities. The activity of the three cyclohexanone monooxygenases was confirmed and characterized following their expression in Escherichia coli.     

Bacterial Decarboxylation of o-Phthalic Acids

The decarboxylation of phthalic acids was studied with Bacillus sp. strain FO, a marine mixed culture ON-7, and Pseudomonas testosteroni. The mixed culture ON-7, when grown anaerobically on phthalate but incubated aerobically with chloramphenicol, quantitatively converted phthalic acid to benzoic acid. Substituted phthalic acids were also decarboxylated: 4,5-dihydroxyphthalic acid to protocatechuic acid; 4-hydroxyphthalic and 4-chlorophthalic acids to 3-hydroxybenzoic and 3-chlorobenzoic acids, respectively; and 3-fluorophthalic acid to 2-and 3-fluorobenzoic acids. Bacillus sp. strain FO gave similar results except that 4,5-dihydroxyphthalic acid was not metabolized, and both 3- and 4-hydroxybenzoic acids were produced from 4-hydroxyphthalic acid. P. testosteroni decarboxylated 4-hydroxyphthalate (to 3-hydroxybenzoate) and 4,5-dihydroxyphthalate but not phthalic acid and halogenated phthalates. Thus, P. testosteroni and the mixed culture ON-7 possessed 4,5-dihydroxyphthalic acid decarboxylase, previously described in P. testosteroni, that metabolized 4,5-dihydroxyphthalic acid and specifically decarboxylated 4-hydroxyphthalic acid to 3-hydroxybenzoic acid. The mixed culture ON-7 and Bacillus sp. strain FO also possessed a novel decarboxylase that metabolized phthalic acid and halogenated phthalates, but not 4,5-dihydroxyphthalate, and randomly decarboxylated 4-hydroxyphthalic acid. The decarboxylation of phthalic acid is suggested to involve an initial reduction to 1,2-dihydrophthalic acid followed by oxidative decarboxylation to benzoic acid.     

Characterization of biosurfactant produced by Pseudoxanthomonas sp. PNK-04 and its application in bioremediation

Biosurfactant producing bacterium was identified as Pseudoxanthomonas sp. PNK-04 based on morphological, physiological, biochemical tests and 16S rRNA gene sequencing. This strain was screened for biosurfactant production using different carbon sources by measuring the surface tension of the medium at different time intervals, and hemolytic activity. The produced biosurfactant was found to be a rhamnolipid based on the formation of dark blue haloes around the colonies in CTAB–methylene blue agar plates and the content of rhamnose sugar. The rhamnolipids produced by this bacterium were found to contain mono- and dirhamnose units linked to β-hydroxy alkonic acids containing 8–12 carbon atoms. This biosurfactant has high emulsifying activity when compared to chemical surfactants such as Tween-80 and Triton X-100 with respect to aliphatic and aromatic hydrocarbons. Further, the biosurfactant stimulates the degradation of 2-chlorobenzoic acid, 3-chlorobenzoic acid and 1-methyl naphthalene by Pseudoxanthomonas sp. PNK-04 probably by aiding in the uptake and increasing the solubility.     

Induction of the tod Operon by Trichloroethylene in Pseudomonas putida TVA8

Bioluminescence, mRNA levels, and toluene degradation rates in Pseudomonas putida TVA8 were measured as a function of various concentrations of toluene and trichloroethylene (TCE). TVA8 showed an increasing bioluminescence response to increasing TCE and toluene concentrations. Compared to uninduced TVA8 cultures, todC1 mRNA levels increased 11-fold for TCE-treated cultures and 13-fold for toluene-treated cultures. Compared to uninduced P. putida F1 cultures, todC1 mRNA levels increased 4.4-fold for TCE-induced cultures and 4.9-fold for toluene-induced cultures. Initial toluene degradation rates were linearly correlated with specific bioluminescence in TVA8 cultures.     

Phenanthrene degradation by bacteria of the genera <Emphasis Type="Italic">Pseudomonas</Emphasis> and <Emphasis Type="Italic">Burkholderia</Emphasis> in model soil systems

Degradation of phenanthrene by strains Pseudomona, Moscow, KMK, 2004simova, I.A. and Chernov, I.s putida BS3701 (pBS1141, pBS1142), Pseudomonas putida BS3745 (pBS216), and Burkholderia sp. BS3702 (pBS1143) were studied in model soil systems. The differences in accumulation and uptake rate of phenanthrene intermediates between the strains under study have been shown. Accumulation of 1-hydroxy-2-naphthoic acid in soil in the course of phenanthrene degradation by strain BS3702 (pBS1143) in a model system has been revealed. The efficiency of phenanthrene biodegradation was assessed using the mathematical model proposed previously for assessment of naphthalene degradation efficiency. The efficiency of degradation of both phenanthrene and the intermediate products of its degradation in phenanthrene-contaminated soil is expected to increase with the joint use of strains P. Putida BS3701 (pBS1141, pBS1142) and Burkholderia sp. BS3702 (pBS1143).     

Plasmid-coded degradation of salicylate using the catechol cleavage pathway of the host

Degradation rates of salicylate and phenol by Pseudomonas putida PpG1064 carrying the nahG gene on a multicopy plasmid were compared with those in NAH-carrying P. putida. Degradation rates of salicylate and phenol and the growth rate of the recombinant were higher than those in NAH-carrying P. putida in SP medium. The catechol 1,2 oxygenase activity of the recombinant in Sp medium was about twice that of the catechol 2,3 oxygenase and catechol 1,2 oxygenase activities of NAH-carrying P. putida. It was suggested that in simultaneous degradation of phenol and salicylate, the recombinant stimulated its ortho cleavage pathway and attained the higher degradation rates and growth rate.     

ACC deaminase producing Pseudomonas putida strain PSE3 and Rhizobium leguminosarum strain RP2 in synergism improves growth, nodulation and yield of pea grown in alluvial soils

Plant growth promoting rhizobacteria affects the overall performance of plants by one or combination of mechanisms. However, little information is available on how ACC deaminase secreting bacteria enhance crop production. The present study aimed at identifying ACC deaminase producing and phosphate solubilizing bacterial strains and to assess their plant growth promoting activities. Additionally, the effect of two ACC deaminase positive bacterial strains Pseudomonas putida and Rhizobium leguminosarum on pea plants was determined to find a novel and compatible bacterial pairing for developing efficient inoculants for enhancing legume production and reducing dependence on chemical fertilizers. The isolated bacterial cultures were characterized biochemically and by 16S rRNA sequence analysis. The plant growth promoting activities was determined using standard microbiological methods. The impact of P. putida and R. leguminosarum, on pea plants was determined both in pots and in field environments. Of the total 40 bacterial strains, strain PSE3 isolated from Mentha arvenss rhizosphere and RP2 strain from pea nodules produced ACC deaminase, solubilized insoluble phosphate, synthesized indole acetic acid, ammonia, cyanogenic compounds, exopolysaccharides and had antifungal activity. The dual inoculation of P. putida strain PSE3 and R. leguminosarum strain RP2 had largest positive effect and markedly increased the growth, symbiotic characteristics, nutrient pool and quantity and quality of pea seeds. The measured parameters were further augmented when inoculated pea plants were grown in soils treated with urea or DAP. A significant variation in the measured parameters of pea plants was observed under both pot and field trials following microbial inoculation but the bacterial cultures did not differ significantly in growth promoting activities. The results suggest that ACC deaminase positive bacterial cultures endowed with multiple potential can be targeted to develop mixed inoculants for enhancing pea production and hence, to reduce dependence on synthetic fertilizers.     

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.     

Cloning and sequencing of the sarcosine oxidase gene from Arthrobacter sp. TE1826

The gene encoding sarcosine oxidase from Arthrobacter sp. TE1826 (soxA) was cloned in Escherichia coli by a convenient plate assay. It was located within a 1.7-kbp PstI-EcoRI fragment of the recombinant plasmid pSAOEP3. The purified sarcosine oxidase from the recombinant strain was found to be the same as that from the parental strain. The DNA sequence of soxA was determined, and an open reading frame composed of 389 amino acid residues was found. By Edman degradation of the enzyme, it was revealed that the amino-terminal amino acid (methionine) was eliminated in the parental strain and E. coli. The molecular weight (43,249) of the enzyme was consistent with the result from SDS-polyacrylamide gel electrophoresis. The FAD-binding site was found in the amino-terminal region of sarcosine oxidase by a homology search. The soxA gene was subcloned on a shuttle vector, pHY300PLK, and was expressed in both E. coli and Bacillus subtillis in the absence of an inducer, although the enzyme was induced with sarcosine in the parental strain.     

Degradation of a Sodium Acrylate Oligomer by an Arthrobacter sp

Arthrobacter sp. strain NO-18 was first isolated from soil as a bacterium which could degrade the sodium acrylate oligomer and utilize it as the sole source of carbon. When 0.2% (wt/wt) oligomer was added to the culture medium, the acrylate oligomer was found to be degraded by 70 to 80% in 2 weeks, using gel permeation chromatography. To determine the maximum molecular weight for biodegradation, the degradation test was done with the hexamer, heptamer, and octamer, which were separated from the oligomer mixture by fractional gel permeation chromatography. The hexamer and heptamer were consumed to the extents of 58 and 36%, respectively, in 2 weeks, but the octamer was not degraded. Oligomers with three different terminal groups were synthesized to examine the effect of the different terminal groups on biodegradation, but few differences were found. Arthrobacter sp. NO-18 assimilated acrylic acid, propionic acid, glutaric acid, 2-methylglutaric acid, and 1,3,5-pentanetricarboxylic acid. Degradation of the acrylic unit structure by this strain is discussed.     

Model based evaluation of a contaminant plume development under aerobic and anaerobic conditions in 2D bench-scale tank experiments

The influence of transverse mixing on competitive aerobic and anaerobic biodegradation of a hydrocarbon plume was investigated using a two-dimensional, bench-scale flow-through laboratory tank experiment. In the first part of the experiment aerobic degradation of increasing toluene concentrations was carried out by the aerobic strain Pseudomonas putida F1. Successively, ethylbenzene (injected as a mixture of unlabeled and fully deuterium-labeled isotopologues) substituted toluene; nitrate was added as additional electron acceptor and the anaerobic denitrifying strain Aromatoleum aromaticum EbN1 was inoculated to study competitive degradation under aerobic / anaerobic conditions. The spatial distribution of anaerobic degradation was resolved by measurements of compound-specific stable isotope fractionation induced by the anaerobic strain as well as compound concentrations. A fully transient numerical reactive transport model was employed and calibrated using measurements of electron donors, acceptors and isotope fractionation. The aerobic phases of the experiment were successfully reproduced using a double Monod kinetic growth model and assuming an initial homogeneous distribution of P. putida F1. Investigation of the competitive degradation phase shows that the observed isotopic pattern cannot be explained by transverse mixing driven biodegradation only, but also depends on the inoculation process of the anaerobic strain. Transient concentrations of electron acceptors and donors are well reproduced by the model, showing its ability to simulate transient competitive biodegradation.     

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.