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Analysis of atrazine-degrading microbial communities in soils using most-probable-number enumeration, DNA hybridization, and inhibitors
Soil Biology & Biochemistry 34 (2002) 1449–1459 www.elsevier.com/locate/soilbio
Analysis of atrazine-degrading microbial communities in soils using most-probable-number enumeration, DNA hybridization, and inhibitors Ellen B. Ostrofskya,1, Jayne B. Robinsonb, Samuel J. Trainac, Olli H. Tuovinena,* a
Department of Microbiology, The Ohio State University, 484 West 12th Avenue, Columbus, OH 43210-1292, USA b Department of Biology, University of Dayton, Dayton, OH 45469-2320, USA c School of Natural Resources, The Ohio State University, Columbus, OH 43210-1085, USA Received 4 May 2001; received in revised form 8 May 2002; accepted 22 May 2002
Abstract The purpose of this study was to determine whether there was an association between kinetics of atrazine mineralization, the number and type of atrazine-degrading microorganisms, and the presence of three genes representing different steps in the degradative pathway of atrazine in three soils with different histories of atrazine application. Composite soil samples were collected from two agricultural fields and one riparian zone soil. The samples were amended with atrazine, and mineralization was measured in biometers as 14CO2 evolution in samples spiked with [ring-U-14C]-labeled atrazine. Atrazine-degrading microorganisms were enumerated by a most-probable-number (MPN) method, which was based on loss of atrazine (HPLC assay) in acetate media. Dot-blot hybridization assays were performed on total DNA extracted from the MPN samples. The DNA probes used in dot-blot assays were the genes atzA (atrazine chlorohydrolase from Pseudomonas ADP), atrA (cytochrome P-450 from Rhodococcus TE1), and trzD (cyanuric acid amidohydrolase from Pseudomonas NRRLB-12228). The herbicide amendment enhanced the subsequent rate of mineralization of atrazine in all three soil samples. The MPN numbers were in the range of 100 – 102 cells g21 dry wt. soil, indicating that atrazine-degrading microorganisms could not be quantitatively enumerated by this technique. Positive hybridization signals of DNA extracted from MPN samples were frequent with the atzA probe; the atrA dot blots had fewer positive signals. The trzD signals were negligible or undetectable, although the parallel mineralization studies showed fast and extensive 14CO2 evolution from [ring-U-14C]-atrazine. The results suggested that trzD, the only gene known to encode striazine ring-cleavage, is not dominant among the atrazine-degrading populations of these soils. Streptomycin and cycloheximide were added to soil samples in biometers to determine the relative contributions of bacteria and fungi in the soils to the mineralization of atrazine. The relative suppression of mineralization in the presence of the bacterial or fungal specific inhibitor was approximately the same, indicating that both groups contributed to the mineralization of atrazine. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Atrazine; Biodegradation of atrazine; Gene probing; Hybridization; Triazine
1. Introduction Atrazine (2-chloro-4-N-isopropyl-6-N-ethyl-s-triazine) has been in worldwide use for control of annual grasses and broadleaved weeds for over 40 years. Microbial degradation is the dominant mode of atrazine attenuation in soils. Several pure and mixed bacterial cultures have been described that can mineralize or partially degrade atrazine (Assaf and Turco, 1994; Yanze-Kontchou and Gschwind, 1994; Mandelbaum et al., 1995; Radosevich et al., 1995; Alvey and Crowley, 1996; Topp et al., 2000). The catabolic * Corresponding author. Tel.: þ 1-614-292-3379; fax: þ1-614-292-8120. E-mail address: [email protected] (O.H. Tuovinen). 1 Present address: Darrin Freshwater Institute, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY 12110-3590, USA.
pathways and genes have been only partially characterized (Wackett et al., 2002), and the molecular and biochemical basis of atrazine biodegradation, especially in soils, is poorly understood. The intermediates of atrazine biodegradation through to the formation of cyanuric acid are believed to be common, but the sequence of pathway steps varies among the known degraders. For example, Pseudomonas ADP dechlorinates atrazine before deamination of the ethylamino group, whereas Rhodococcus NI86/ 21 N-dealkylates atrazine as its initial degradative step (de Souza et al., 1995; Nagy et al., 1995; Van Zwieten and Kennedy, 1995). Genes encoding enzymes that are involved in atrazine biodegradation have been identified and cloned from Pseudomonas and Rhodococcus spp. (Eaton and Karns, 1991a,b; Mulbry, 1994; Shao and Behki, 1995; Shao et al.,
0038-0717/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 0 2 ) 0 0 0 8 9 - 5
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Fig. 1. The reactions of three enzymes involved in atrazine biodegradation: AtzA from Pseudomonas ADP, AtrA from Rhodococcus TE1, and TrzD from Pseudomonas NRRLB-12228.
1995; de Souza et al., 1996; Boundy-Mills et al., 1997). Some genes encode enzymes with similar functions, but nucleotide and amino acid homology studies have revealed little similarity among these genes and gene products from different organisms. One notable exception is the atzABC genes from Pseudomonas sp. ADP, which have been found in several other atrazine-degrading bacteria (de Souza et al., 1998). The purpose of this study was to characterize the mineralization of atrazine in soils with different histories of application of atrazine. Kinetics of the mineralization of atrazine were determined to establish whether the kinetic parameters were related to the number and type of atrazine-degrading microorganisms and the presence of atzA, atrA, and trzD genes in the test soils. Fig. 1 shows the reactions catalyzed by the enzymes encoded by these three genes associated with the degradation of atrazine. The gene atzA encodes a chlorohydrolase and was originally identified and cloned from Pseudomonas ADP that can mineralize atrazine (de Souza et al., 1996). The gene atrA encodes a cytochrome P-450 monooxygenase, which causes spontaneous N-dealkylation of either the ethyl or isopropyl group after enzymecatalyzed hydroxylation. This gene was identified and cloned from Rhodococcus TE1 and Rhodococcus NI86/ 21, both of which are capable of degrading S-ethyl dipropylthiocarbamate (EPTC) and atrazine (Nagy et al., 1995; Shao and Behki, 1995). The gene trzD was originally found in Pseudomonas strains NRRLB-12227 and NRRLB-12228 (Eaton and Karns, 1991a,b; Karns,
1999), which can utilize melamine, ammeline, ammelide, and cyanuric acid as N sources but they do not degrade atrazine. The gene trzD encodes cyanuric acid amidohydrolase, and its homologs have been found by PCR amplification in many atrazine-degrading soil bacteria (Rousseaux et al., 2001). Amendment of soils with cyanuric acid has a variable effect on the subsequent mineralization of atrazine by soil microorganisms, ranging from no response to great enhancement (Ostrofsky et al., 2001). In the present study, amendment with atrazine followed by incubation was used in an attempt to enhance subsequent mineralization and to enrich for atrazine-degrading microorganisms. A mostprobable-number (MPN) method was employed to enumerate atrazine-degrading microorganisms and to assess their changes in response to atrazine treatment. Genomic DNA samples from the MPN cultures were examined to establish whether incubation with atrazine selected for bacteria that contained DNA sequences homologous to the atzA, atrA, or trzD genes. Levanon (1993) used streptomycin and cycloheximide to differentiate between bacterial and fungal contributions, respectively, to the biodegradation of atrazine. Suppression of the mineralization of atrazine was evident with both antibiotics, indicating that bacteria and fungi are involved in the mineralization. In the present studies, soil samples were amended with streptomycin (an inhibitor of bacteria) and cycloheximide (an inhibitor of eukaryotes) to discriminate between eukaryotic and bacterial biodegradation.
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2. Materials and methods 2.1. Site description and sampling The surface soil samples were collected at various times between 1996 and 1998 from two field plots (CC, CR) and a reference site (RZ) at a research farm in Piketon, Ohio, designated as the Ohio Management Systems Evaluation Area (Ohio MSEA). A composite of four surface soil samples (0 –5 cm) was collected at each site. CC was a continuous cornfield with annual application of atrazine at the maximum allowable rate (2.5 kg ha21). CR was a corn – soybean– wheat crop rotation field with atrazine applied at a reduced rate (1.5 kg ha21) during corn years (corn planted and atrazine applied in April 1994 and again in April 1997). RZ was a riparian zone adjacent to the agricultural fields and had never received atrazine other than by aerial drift or by vernal flooding of a nearby river (Scioto River) that has an agricultural watershed. Descriptive details of these soils can be found in Ostrofsky et al. (1997, 2001). The field moisture content of the CC soil samples was 10 ^ 1.4% (w/w) in May 1996, 15 ^ 0.3% in June 1997, and 5.1 ^ 1.9% in October 1997. The field moisture content of the CR soil samples was 12 ^ 1.5% in May 1996 and 5.1 ^ 0.9% in October 1997. The field moisture content of the RZ soil samples was 20 ^ 2.0% in May 1996, 24 ^ 1.1% in June 1997, and 16 ^ 0.35% in October 1997. 2.2. Soil amendments Samples for soil amendment experiments were collected in May 1996 and June 1997. The composite soil samples (100 g wet weight) were placed into sterile plastic trays and amended with sterile double-distilled H2O or an aqueous solution of atrazine by drop-wise addition and mixing thoroughly with a sterile spatula. Atrazine was added to a final concentration of 1.0 mg kg21 soil to approximate a field application of 2.5 kg ha21. Because the moisture content of field samples was low, it was normalized to 25% (v/w) with the amendments. All incubations were at 22 ^ 2 8C. At various time intervals following amendment, subsamples (50 g) were removed for determining the mineralization of atrazine and for MPN enumeration. 2.3. Mineralization of atrazine The mineralization of atrazine was measured in biometers, reaction vessels that are designed to measure the evolution of 14CO2 from 14C-labeled substrates. These biometers consisted of 60 ml serum bottles equipped with suspended 2 ml vials to trap evolved CO2 (Ostrofsky et al., 1997). Each biometer received 5 g dry wt of soil and 1.0 ml of double-distilled water containing 0.1 mCi of [ringU-14C]-atrazine ($ 95% pure, Sigma Chemical Co., St Louis, MO) and unlabeled atrazine (99.5% pure, Chem Service, West Chester, PA) to yield a final concentration of
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2.5 mg atrazine kg soil21 for the mineralization experiments. The mineralization of atrazine was tested for all field samples. In the soil amendment experiments, the mineralization of atrazine was monitored as 14CO2 evolution over time in duplicate biometers for subsamples that had been pre-incubated with atrazine or H2O for various time intervals. For sterile controls, duplicate soil samples in biometers were autoclaved for 20 min at 121 8C. To compare rates of mineralization, the data were fitted to a first-order rate equation, which is normally used by regulatory agencies to assess half-lives and rate constants of attenuation of pesticidal molecules in the environment. It was not within the scope of this work to find the best-fitting rate expressions for each of the incubations. The first-order data fitting was used to determine the rate constants (k) and half-lives (t1/2) (Ostrofsky et al., 1997) P ¼ Pmax ð1 2 e2kt Þ P is cumulative %14CO2 evolved at time t (d), Pmax is the maximum cumulative %14CO2 evolved during the mineralization time-course. The coefficient of determination (r 2) was calculated to assess the goodness of the first-order fit. The level of residual atrazine was analyzed in amended samples: soil samples (10 g wet weight from the 50 g subsamples) were mixed with a 3:1 methanol/water solution in plastic screw-capped tubes, shaken by hand, and then incubated for 20 min in an ultrasonic bath. The samples were extracted overnight at 22 ^ 2 8C. Supernatants were filtered (0.45 mm GHP glass fiber, Gelman Sciences, Ann Arbor, MI) and analyzed with an ELISA specific for atrazine (Strategic Diagnostics, Inc., Newark, DE). According to the manufacturer, the ELISA has been developed for aqueous samples with a lower limit of detection of 0.046 mg atrazine l21 water. Other triazines, including three atrazine metabolites, cross-react with the assay. 2.4. MPN enumeration The medium for MPN enumeration was formulated on the basis of growth of pure cultures of atrazine-mineralizing bacteria. The medium consisted of (per liter) 21.5 mg atrazine, 0.5 g KH2PO4, 0.65 g Na acetate, 0.2 g MgSO4· 7H2O, and 10 ml of a trace metals stock solution containing (per liter stock solution) 2.0 g nitrilotriacetic acid, 1.0 g MnSO4·H2O, 0.9 g CaSO4, 0.8 g Fe(NH4)2(SO4)2·6H2O, 0.2 g CoSO4·7H2O, 0.2 g ZnSO4·7H2O, 0.03 g CuSO4·7H2O, 0.02 g Na2WO4, 0.02 g Na2SeO3, and 0.02 g NiCl2·6H2O. The medium was sterilized by autoclaving and aseptically dispensed in 5 ml aliquots into sterile glass test tubes (16 £ 100 mm2). For the initial dilution, 10 g samples of field moist soil were added to 90 ml of sterile phosphate buffered saline in sterile centrifuge bottles. The bottles were shaken horizontally for 30 min. Four additional 10-fold serial dilutions
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Table 1 List of cultures used as reference sample or plasmid DNA in dot-blot hybridization studies. Information on cultivation of these cultures can be found in the listed references Culture
Source
Reference
E. coli DH5a (pJK206) E. coli DH5a (pMD4) E. coli DH5a (pKL1) E. coli ATCC 10798 Rhodococcus TE1 Pseudomonas 12228 Pseudomonas ADP M91-3
J.S. Karns M. de Souza W.A. Dick Department of Microbiology, Ohio State University W.A. Dick J. S. Karns M. de Souza M. Radosevich
Eaton and Karns, 1991a de Souza et al., 1996 Shao and Behki, 1995 LBa Shao and Behki, 1995 Eaton and Karns, 1991b de Souza et al., 1996 Radosevich et al., 1995
a
Grown in Luria broth (LB) at 378C with shaking overnight.
were then made, and 1 ml aliquots of the dilutions were used to inoculate the MPN tubes. The number of replicate MPN tubes was either three or five depending on the experiment. For sterile controls, a 1021 dilution of each soil was autoclaved and 1 ml aliquots were used to inoculate a set of five MPN tubes. These sterile controls were used to account for abiotic losses of atrazine. MPN tubes were incubated without shaking at 22 ^ 2 8C for 30 d. At 30 d, water lost by evaporation was replaced (determined either by an initial marking of the solution level or gravimetrically) with sterile water, and the samples mixed thoroughly by vortexing. A 1 ml sample from each MPN tube was removed, centrifuged to remove particles, and analyzed by HPLC. The HPLC parameters were a flow rate of 0.75 ml min21, absorbance at 225 nm, degassed mobile phase of 70% acetonitrile (ACN) and 30% H2O, and a C18 column (Alphabond, Alltech Associates, Deerfield, IL). A linear equation derived from regression analysis of peak areas of standards of known concentration was used to calculate atrazine concentration in the MPN samples. Samples with an atrazine loss of $ 25% of the initial concentration, as determined by HPLC analysis of samples from five uninoculated MPN tubes, were scored as positive. The scoring was based on atrazine utilization rather than observance of growth, because the intention of the MPN assay was to enumerate specifically atrazine-degraders. The extent of the degradation of atrazine varied considerably in the MPN tubes. A cut-off of 25% degradation of atrazine was chosen to score tubes positive. Turbidity as an indication of growth was not used as a scoring basis because it was presumed that organisms could grow in the medium (using acetate as a C source and various N sources from the inoculum) without degrading the atrazine. Estimates of numbers of atrazine-degraders with 95% confidence intervals were determined using an MPN program written in BASIC (Hurley and Roscoe, 1983). 2.5. DNA extraction from MPN samples Genomic DNA was extracted from the three-dilution, three-replicate-tube series that were used to calculate the
MPN estimate. The remaining solutions in MPN tubes were centrifuged to pellet microorganisms. The pellets were subjected to the rapid genomic bacterial DNA extraction and purification procedure (Davis et al., 1994). Briefly, the pellets were treated with aqueous solutions of sodium dodecyl sulfate (SDS) and proteinase K and then extracted sequentially with phenol and chloroform/isoamyl alcohol. The resulting pellets of DNA were resuspended in 100 ml TE buffer (10 mM Tris– HCl, 0.1 mM EDTA, pH 8.0). For dot-blot hybridization analysis, 50 ml aliquots (1 – 3 mg) of the DNA were applied to the membranes (Hybond-Nþ nylon, Amersham Pharmacia Biotech, Amersham, UK). 2.6. Bacterial cultures and media Table 1 lists the sources of cultures used for genomic and plasmid DNA isolation, which were used as positive and negative controls in the hybridization experiments. Cultures were grown for 1– 3 d under conditions specified in the respective references in Table 1. Overnight cultures of strains of Escherichia coli DH5a were grown at 37 ^ 2 8C with shaking for purification of plasmid and probe DNA. Genomic DNA from bacterial cultures was extracted and purified by the modified protocol provided with the QIAamp Tissue kit (Qiagen, Chatsworth, CA). 2.7. Description and production of DNA gene probes The gene probes used in the hybridization experiments are described in Table 2. At the beginning of this study, sequence information for the genes atzA, atrA, and trzD was not available. Sequence information became available only after hybridization analyses had been completed. The size of the atzA gene is 1.4 kb and is contained entirely within the 1.9 kb AvaI/AvaI restriction fragment used as a probe (de Souza et al., 1996). The remainder of the 1.9 kb probe consists of remnant DNA from Pseudomonas ADP. The size of the trzD gene is 1.1 kb and is contained entirely within the 2.0 kb HindIII/PstI fragment; the remainder of this fragment consists of Pseudomonas 12228 DNA (Karns, 1999). The atrA probe used in this study was the 6.2 kb
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Table 2 Description of the gene probes used in the dot-blot hybridization studies Gene
Size (kb)
GenBank accession numbera
Restriction fragment used as probe
Plasmid vector
AtzA AtrA TrzD
1.4 1.0, 1.3b 1.1
U55933 U17130c AF086815
1.9 kb AvaI/AvaI 6.2 kb KpnI/KpnI 2.0 kb HindIII/PstI
pMD4 pKL1 pJK206
a
At the time of the present study, the sequences of these three DNA fragments used as probes had not been determined. The information included in this Table was made available only after this study had been completed. b Sizes of the thcR and thcB genes, respectively. c The sequences of the genes are from Rhodococcus NI86/21.
KpnI/KpnI fragment of pKL1. Shao and Behki (1996) reported that this 6.2 kb fragment was found in Rhodococcus NI86/21. Nagy et al. (1995) sequenced this fragment in Rhodococcus NI86/21 and found that it contained two genes, thcB and thcR, which are involved in encoding a cytochrome P-450 monooxygenase. For the production of probes and quantitative standards, plasmid DNA was extracted from cultures of E. coli by an alkaline lysis protocol (Sambrook and Russell, 2001). Plasmid DNA was digested with the appropriate restriction enzymes at 37 8C for 1– 2 h to generate probe DNA. Restriction digest fragments were separated by gel electrophoresis on 1% agarose and visualized by staining with ethidium bromide. HindIII-digested lambda DNA was used as a size standard. The appropriate restriction fragment was excised from the gel and purified using the protocol provided with the Qiaquick Gel Extraction kit (Qiagen). The resulting purified DNA was used in probe labeling reactions. Reference samples for dot-blot hybridization analysis included the plasmids pJK206 (contains the gene trzD), pMD4 (atzA), and pKL1 (atrA) as quantitative standards. Pseudomonas NRRLB-12228, Pseudomonas ADP, and Rhodococcus TE1 genomic DNA served as positive controls for trzD, atzA and atrA, respectively. Genomic DNA from an LB-grown culture of E. coli ATCC 10798 served as a negative control. 2.8. DNA hybridization Probe labeling, hybridization, and detection were performed as instructed by the protocol provided with the Gene Images Chemiluminescent Labeling and Detection kit (Amersham Pharmacia Biotech). Hybridizations and washes were performed at 50 8C. The blots were washed once for 15 min each with 2X SSC (20X: 3 M NaCl, 300 mM Na-citrate) þ 0.1% SDS and 0.1X SSC þ 0.1% SDS. DNA samples were loaded onto the hybridization membrane using a vacuum dot-blot manifold (Bio-Dotw Microfiltration Apparatus, Bio-Rad, Hercules, CA). The quantity of DNA in all samples was 1 mg, except for the quantitative plasmid standards, which are indicated in the figure legends. Chemiluminescence was detected by exposure of X-ray
film (Hyperfilme-MP, Amersham Life Science) to the blots. Film was developed in an automatic film developer. Developed films were scanned using Adobe Photoshop software (version 4, Adobe Systems, San Jose, CA), and intensity measurements were determined with Scion Image for Windows software (version 2, Scion Corp., Frederick, MD). Before re-hybridization with another gene, blots were stripped of probe with three 10 min washes of boiling 0.1% SDS. After stripping of the last gene probe, re-exposure of the blot to X-ray film confirmed the complete removal of the probe. 2.9. Effect of bacterial and fungal inhibitors on the mineralization of atrazine in soil To determine the contribution of bacteria and fungi to the mineralization of atrazine observed in CC soils, antibiotics were added to soil samples in the biometers singly or together. Streptomycin was selected as an inhibitor of bacteria and cycloheximide as an inhibitor of eukaryotes. Biometers were established in duplicate, as previously described. The inhibitors were added to the stock solutions of atrazine and 1 ml (0.1 mCi 14C-atrazine, 2.5 mg atrazine kg21 soil) was applied to 5 g (dry wt) of field moist CC soil (collected in January 1998). To distinguish the effects of inhibitors, the CC soil was used in these biometer experiments because it demonstrated relatively rapid and extensive mineralization of atrazine. The concentrations of streptomycin and cycloheximide were 1, 3, and 5 g kg21 soil for the single addition experiments, and it was 3 g kg21 soil for each inhibitor for the multiple addition experiment. For the single addition experiment, inhibitors were added at the beginning of the incubation, and biometers were incubated and monitored for 14CO2 evolution every other day for 10 d. For the multiple addition experiment, the inhibitors were added at the beginning and every other day for a total of five additions. In an additional inhibitor experiment, duplicate biometers containing 5 g of CC soil (collected in June 1998) were amended once with 5 g streptomycin kg21 soil, 3 g cycloheximide kg21, or both, and spiked with 14C-ring-atrazine (2.5 mg atrazine kg21 soil). Evolution of 14CO2 was monitored on days 5 and 10 d after the addition of inhibitors.
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Fig. 2. Time course data for the mineralization of atrazine in CC, CR, and RZ soil samples collected in May 1996. These samples were amended with 1 mg kg21 atrazine and incubated for 30, 60, 90, or 138 d. Subsequently, subsamples were collected, spiked with [U-14C-ring]-atrazine, and the time course of 14CO2 evolution was measured in biometers. Mineralization of atrazine was also measured in initial soil samples and corresponding sterile controls. The bars represent the standard deviation which, in most cases, are smaller than the size of the symbol.
3. Results 3.1. Mineralization of atrazine and microbial enumeration Three different soils were sampled in May 1996 and amended in the laboratory with 1.0 mg atrazine kg21 soil or with double-distilled water as a control. At 30, 60, 90, and 138 d of incubation after amendment, samples were transferred to biometers and spiked with [U-14C-ring]atrazine to monitor mineralization of atrazine as evolution of 14CO2. Amendment and incubation for 30 d enhanced the subsequent rate of mineralization of atrazine for the CR and RZ soils as evidenced by the production of 14CO2 from the
ring-labeled atrazine (Fig. 2). This enhancement persisted throughout the 138 d pre-incubation (Fig. 2). The CC soils already had a high level of atrazine-mineralization activity even without amendment with atrazine (Fig. 2(A)). Autoclaved sterile control soils showed less than 1% evolution of 14 CO2 in all three soils (Fig. 2). Water-amended samples of the three soils demonstrated rates of mineralization that were similar to the activities of the respective preamendment samples at all incubation times, indicating that native mineralization activity persisted throughout the 138 d pre-incubation period. The kinetic data for CC soils with all treatments yielded mineralization rate constants of 0.04 –0.14 d21 and halflives of 5 –19 d (Table 3). The kinetic data derived from fitting the mineralization data to a first-order rate expression showed increases in rate constants and decreases in halflives of the mineralization of atrazine in the CR and RZ soils after amendment with atrazine (Table 3). The kinetic data for the water-amended samples did not vary during the time course of this experiment. These kinetic data indicated that the mineralization of atrazine was fastest in the CC samples. The kinetics of the pre-amended CR samples were slower than in the CC samples but increased after amendment with atrazine to be comparable with CC. Amendment with atrazine accelerated mineralization also in RZ soils. Mineralization of atrazine was also compared with the residual levels of atrazine in these three soils. Before any amendments, ELISA results showed residual levels of 13, 7, and 4 mg atrazine kg soil21 in the CC, CR, and RZ soils, respectively. All water-amended samples had residual atrazine levels similar to those in the pre-amendment samples. The residual levels after pre-incubation with 1 mg atrazine kg21 soil were 14– 17 mg kg21 for the CC, 26– 36 mg kg21 for the CR, and 31– 40 mg kg21 for the RZ samples, respectively. These data are in keeping with the relative extent of mineralization of atrazine in the three soils. The CC and RZ soils were sampled in June 1997 for the mineralization of atrazine. The CC soil, which had a history of atrazine application, had a greater extent of mineralization than the RZ soil, which had never received direct atrazine application (Table 4). After amendment and incubation of the CC soil, a transient decrease in evolved 14 CO2 was observed, but the final extent of mineralization of atrazine was comparable with that in the unamended CC soil. The amendment of 1 mg atrazine kg21 soil followed with 60 d of pre-incubation resulted in an increase in the extent of the mineralization of atrazine from 10 to 60% in the RZ samples (Table 4). The mineralization of atrazine in sterile CC soils was less than 0.2% after 30 d. In samples collected in October 1997, the CC soil reached 85% mineralization in 30 d, consistent with the sample collected in June (Table 4). The 14CO2 evolution in the CR soil was about 35%, whereas the mineralization in the RZ soil was about 3% after 30 d of incubation. The CR soils receive atrazine treatment only once every three years, and
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Table 3 Kinetic data and residual atrazine concentration in the amendment and mineralization experiments Soila
CC
CR
RZ
a
Amendment
Length of pre-incubation (d)
Parameter Pmax (%)
k (d21)
r2
t1/2 (d)
Pre-amendment H2 O H2 O H2 O H2 O
0 30 60 90 138
79.5 68.7 95.1 74.5 84.8
0.09 0.08 0.06 0.04 0.06
7.9 8.9 12 19 12
0.82 0.85 0.83 0.59 0.88
Atrazine Atrazine Atrazine Atrazine
30 60 90 138
69.6 98.8 78.8 86.1
0.14 0.09 0.08 0.07
5.1 8.2 8.7 10
0.98 0.92 0.89 0.84
Pre-amendment H2 O H2 O H2 O H2 O
0 30 60 90 138
19.0 20.6 19.5 9.34 16.1
0.003 0.004 0.002 0.001 0.001
217 178 347 693 495
0.96 0.94 0.94 0.66 0.97
Atrazine Atrazine Atrazine Atrazine
30 60 90 138
36.9 57.0 63.8 75.5
0.008 0.008 0.02 0.04
90 84 35 17
0.89 0.90 0.88 0.93
Pre-amendment H2 O H2 O H2 O H2 O
0 30 60 90 138
15.8 8.1 13.4 9.33 16.87
0.003 0.001 0.001 0.001 0.002
277 495 496 693 462
0.95 0.97 0.96 0.80 0.96
Atrazine Atrazine Atrazine Atrazine
30 60 90 138
39.9 51.9 35.9 56.3
0.012 0.008 0.006 0.02
58 90 116 42
0.88 0.96 0.77 0.91
CC, continuous corn; CR, crop rotation; RZ, riparian zone.
mineralization in this soil was consistent with its history of intermittent atrazine application. The numbers of atrazine-degrading microorganisms estimated by MPN enumeration was 812 (95% confidence interval of 246– 2700) and 12 (4 – 40) cells g21 dry soil in the CC and RZ soils sampled in June 1997, respectively.
After the CC and RZ soils were amended with atrazine and incubated for 60 d, the MPN of atrazine-degraders diminished to less than 3 cells g21 dry soil. For the October 1997 samples, the MPN of atrazine-degrading organisms were in the range of 51– 75 (95% confidence interval of 21– 194) cells g21 dry soil for the CC, CR, and RZ soils.
Table 4 Mineralization of atrazine in soil samples collected in June and October, 1997 Sample
CC RZ Sterile CC CC RZ CC CR RZ a
Sampling date
June, 1997 June, 1997 June, 1997 June, 1997 June, 1997 October, 1997 October, 1997 October, 1997
Treatmenta
A A A B B A A A
% Cumulative mineralization ^ standard deviation 10 d
20 d
30 d
69 ^ 0.90 1.3 ^ 0.92 0.03 ^ 0.002 16 ^ 2.7 12 ^ 5.0 39 ^ 4.7 4.1 ^ 3.65 0.74 ^ 0.001
73 ^ 0.54 4.5 ^ 2.81 0.11 ^ 0.007 68 ^ 5.1 40 ^ 12.4 NDb ND ND
76 ^ 0.27 10 ^ 3.4 0.17 ^ 0.011 76 ^ 1.5 60 ^ 1.7 85 ^ 6.5 35 ^ 22.2 3.2 ^ 1.95
All soil samples were incubated at 22 ^ 2 8C for 60 d before measuring the mineralization. A, unamended soils; B, soils amended with 1 mg atrazine kg soil21. b ND, no data.
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Fig. 3. Dot-blot hybridizations with DNA extracted from MPN cultures. The MPN samples were from a three-dilution series with unamended CC, CR, and RZ soils from October 1997. Half of the DNA extraction mixture (50 ml) was added to the blot. Plasmid, genomic, and MPN DNA samples hybridized with atzA (A); with atrA (B); and with trzD (C); corresponding results of HPLC analyses used for MPN determination (% atrazine degradation) (D).
3.2. Gene probe analysis of DNA from MPN samples Plasmid DNA, genomic reference DNA samples, and DNA from MPN tubes were hybridized with genes atzA, atrA, and trzD (Fig. 3(A)–(C)). The HPLC results used to calculate the MPN and corresponding to the samples on the blot are shown in Fig. 3(D). Almost all the MPN samples showed a hybridization signal with atzA (Fig. 3(A)), but there did not seem to be a correlation between intensity of the signal and loss of atrazine. The number of signals did not decrease with dilution, as would be expected if organisms containing the gene were diluted. More positive hybridization signals were observed than there were samples that were positive for atrazine loss. Fewer hybridization signals with MPN samples probed with atrA were observed than with samples probed with atzA (Fig. 3(B)). Again, there did not seem to be a correlation between hybridization signals and loss of
atrazine or a decrease in frequency of signal detection with dilution (Fig. 3(B)). Hybridization signals were not observed when MPN samples were probed with trzD (Fig. 3(C)). 3.3. Effect of streptomycin and cycloheximide on the mineralization of atrazine There was no striking differential effect in mineralization of atrazine resulting from treatments of the soils with streptomycin, cycloheximide, and both. All treatments suppressed mineralization of atrazine until after 6 d of incubation. At 10 d, the extent of mineralization was nearly the same as the untreated CC control, except for the treatments with 5 g kg21 streptomycin (Fig. 4(A)) and the 3 g kg21 cycloheximide (Fig. 4(B)). In combination, the inhibitors at 3 and 5 g kg21 suppressed mineralization of atrazine as compared with the untreated control (Fig. 4(C)).
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mineralization of atrazine involved both eukaryotic and bacterial populations.
4. Discussion
Fig. 4. Time course data for the mineralization of atrazine in inhibitor experiments in CC soil. (A) Single amendment of 1, 3, or 5 g streptomycin kg21 dry soil. (B) Single amendment of 1, 3, or 5 g cycloheximide kg21 dry soil. (C) Single amendment of 1, 3, or 5 g of both streptomycin and cycloheximide kg21 dry soil. CC refers to untreated soil samples, and abiotic refers to autoclaved soil samples. (D) Multiple amendments of inhibitors at 3 g each kg21 dry soil every other day. The bars represent the standard deviation which, in most cases, are smaller than the size of the symbol.
All three inhibitor treatments suppressed mineralization of atrazine in the multiple addition experiment (Fig. 4(D)). In an additional experiment, the untreated CC soil showed an extent of mineralization of atrazine of about 50% after 10 d of incubation (Fig. 5). The individual streptomycin- and cycloheximide-treated samples showed lower extents (about 10% after 10 d) of mineralization as compared with the untreated CC soil (Fig. 5). The lowest extent of mineralization was seen in the sample treated with a combination of streptomycin and cycloheximide, which showed only about 5% mineralization activity after 10 d of incubation (Fig. 5). These results suggested that the
The two agricultural soils, CC and CR, which had a prior history of atrazine application contained resident microbial communities capable of mineralizing the herbicide without additional amendment, albeit at different rates and to different extents. The microbes in the RZ soil, however, mineralized atrazine only after additional amendment, and the rate increased almost to the same rate as in CC. These results suggest that atrazine-mineralizing microorganisms existed in the RZ soil but were inactive or less numerous when atrazine was not present. The MPN results suggest that the atrazine-degraders in these soils were relatively non-culturable in the atrazineacetate medium used. Although the quantitative relation between the kinetics of the mineralization of atrazine and the numbers of indigenous atrazine-degrading microorganisms has not been elucidated, the estimated numbers of ca. 800 atrazine-degraders g21 dry CC soil do not seem great enough to account for the high level (approx. 80%) of the mineralization of atrazine. Based on studies with atrazine where soils were inoculated with a mineralizing culture, cell numbers should be several orders of magnitude greater than those obtained in the present study to account for the relatively rapid mineralization (Yanze-Kontchou and Gschwind, 1995; Alvey and Crowley, 1996; Yassir et al., 1999). The cell numbers decreased in the amended and incubated CC and RZ soil samples. These decreases were within the statistical range of the MPN method, but they could also be the result of a loss of cells during the incubation or to the inherent variability and lack of culturability. At the initiation of the probe design and hybridization experiments, nucleotide sequence information was not available for any of the three genes. Sequence information is now available for genes atzA and trzD and for the two genes found on the atrA DNA fragment. The amino acid sequence derived from the nucleotide sequence of gene atzA is not related to any other amino acid sequence in either the SwissProt or PIR databases at a level greater than 20% homology (de Souza et al., 1996). Karns (1999) indicated that the gene sequence of trzD is not related to any other sequence in the GenBank database. The positive hybridization signal of Pseudomonas 12228 genomic DNA with atzA was an unexpected result, because this bacterium is a melamine-degrader and is not known to possess atrazine dechlorination activity. The atzA gene probe contained some genomic DNA from Pseudomonas ADP, and it is possible that Pseudomonas ADP may have complementary sequences to genomic DNA of Pseudomonas 12228 that are not related to atrazine biodegradation. Seffernick et al. (2001) reported that the amino acid
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Fig. 5. The mineralization of atrazine in a repeated inhibitor experiment with single addition of the inhibitors. The bars represent the standard deviation.
sequence of AtzA of Pseudomonas ADP differed by only nine residues from that of melamine deaminase (TriA) cloned from Pseudomonas 12227, a strain related to Pseudomonas 12228, but that the enzymes catalyze dissimilar reactions on different substrates. In addition, positive hybridization signals were observed for Pseudomonas ADP and Pseudomonas 12228 genomic DNA probed with gene atrA. According to Shao and Behki (1996), the DNA fragment called atrA contains a possible regulatory gene, thcR, and a structural gene, thcB, encoding a novel type of cytochrome P-450. Either of the two genes on the atrA DNA fragment could have complements in the Pseudomonas genomes and produce the hybridization signals. Levanon (1993) demonstrated that adding inhibitors of bacteria and fungi separately to soils reduced the total amount of atrazine mineralized from 29% in 30 d in untreated soils to less than 1% for both classes of inhibitors. In this study, inhibitors were added once at the initiation of the mineralization assays at concentrations of 2.5 and 5.85 g inhibitor kg21 soil for the fungal and bacterial inhibitors, respectively. These concentrations of inhibitors showed only a partial suppression of the mineralization of atrazine. Only when the inhibitors
were added together multiple times individually or together did the suppression of the mineralization activity persist. These results were similar to those seen by Levanon (1993) in that both bacteria and eukaryotes seemed to contribute to the mineralization of atrazine in these soils. Decomposition products of the antibiotics could include compounds that serve as C or N sources for the microorganisms involved in the mineralization of atrazine. The decomposition would diminish the antimicrobial effect of the antibiotics, and growth of the microorganisms could lead to enhanced mineralization, thereby also partially alleviating the inhibition. It is clear from this study that none of the gene probes gave consistent signals although the mineralization of atrazine was relatively fast, especially in the CC soil samples. It is conceivable that the genes involved in the biodegradation of atrazine are so diverse in soil microorganisms that they do not yield signals when probed with genes isolated from known pure cultures. Genes from other bacteria and also from fungi would expand the molecular tools and widen the search for signature sequences of degradative pathways of atrazine in the environment. Because of the apparent lack of culturability of atrazine-degraders in soils, the molecular approach is
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of key importance in furthering the understanding of environmental factors that modulate the degradation and mineralization of atrazine in soils.
Acknowledgments We are grateful to Dr J.S. Karns for the Pseudomonas strains and the E. coli construct containing the trzD gene, Dr W.A. Dick for Rhodococcus TE1 and the E. coli construct containing the atrA fragment, and Dr M. de Souza for Pseudomonas ADP and the E. coli construct containing the atzA gene. E.B.O. received partial support for this work from the US Dept. of Defense Environmental Fellowship Program. The research was partially funded by the Ohio MSEA project, which is a cooperative research and educational effort of the Ohio Agricultural Research and Development Center, The Ohio State University, OSU Extension, the USDA-ARS, USDA-CRRS, USDA-ES, USGS, USEPA, and other state and federal agencies. Salary and research support were provided to S.J.T. by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University.
References Alvey, S., Crowley, D.E., 1996. Survival and activity of an atrazinemineralizing bacterial consortium in rhizosphere soil. Environmental Science and Technology 30, 1596–1603. Assaf, N.A., Turco, R.F., 1994. Influence of carbon and nitrogen application on the mineralization of atrazine and its metabolites in soil. Pesticide Science 41, 41–47. Boundy-Mills, K.L., de Souza, M.L., Mandelbaum, R.T., Wackett, L.P., Sadowsky, M.J., 1997. The atzB gene of Pseudomonas sp strain ADP encodes the second enzyme of a novel atrazine degradation pathway. Applied and Environmental Microbiology 63, 916–923. Davis, L.B., Kuehl, W.M., Battey, J.F., 1994. Basic Methods in Molecular Biology, Second edition, Appleton and Lange, Norwalk, CT. Eaton, R.W., Karns, J.S., 1991a. Cloning and analysis of s-triazine catabolic genes from Pseudomonas sp. strain NRRLB-12227. Journal of Bacteriology 173, 1215–1222. Eaton, R.W., Karns, J.S., 1991b. Cloning and comparison of the DNA encoding ammelide aminohydrolase and cyanuric acid amidohydrolase from three s-triazine-degrading bacterial strains. Journal of Bacteriology 173, 1363–1366. Hurley, M.A., Roscoe, M.E., 1983. Automated statistical analysis of microbial enumeration by dilution series. Journal of Bacteriology 55, 159–164. Karns, J.S., 1999. Gene sequence and properties of an s-triazine ringcleavage enzyme from Pseudomonas sp. strain NRRLB-12227. Applied and Environmental Microbiology 65, 3512–3517. Levanon, D., 1993. Roles of fungi and bacteria in the mineralization of the pesticides atrazine, alachlor, malathion, and carbofuran in soil. Soil Biology and Biochemistry 25, 1097–1105. Mandelbaum, R.T., Allan, D.L., Wackett, L.P., 1995. Isolation and characterization of a Pseudomonas sp. that mineralizes the s-triazine herbicide atrazine. Applied and Environmental Microbiology 61, 1451– 1457. Mulbry, W.W., 1994. Purification and characterization of an inducible striazine hydrolase from Rhodococcus corallinus NRRL B-15444R. Applied and Environmental Microbiology 60, 613–618.
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Nagy, I., Compernolle, F., Ghys, K., Vanderleyden, J., De Mot, R., 1995. A single cytochrome P-450 system is involved in degradation of the herbicides EPTC (S-ethyl dipropylthiocarbamate) and atrazine by Rhodococcus sp. strain NI86/21. Applied and Environmental Microbiology 61, 2056– 2060. Ostrofsky, E.B., Traina, S.J., Tuovinen, O.H., 1997. Variation in atrazine mineralization rates in relation to agricultural management practice. Journal of Environmental Quality 26, 647–657. Ostrofsky, E.B., Robinson, J.B., Traina, S.J., Tuovinen, O.H., 2001. Effect of cyanuric acid amendment on atrazine mineralization in surface soils and detection of the s-triazine ring-cleavage gene trzD. Soil Biology and Biochemistry 33, 1539–1545. Radosevich, M., Traina, S.J., Hao, Y.-L., Tuovinen, O.H., 1995. Degradation and mineralization of atrazine by a soil bacterial isolate. Applied and Environmental Microbiology 61, 297–302. Rousseaux, S., Hartmann, A., Soulas, G., 2001. Isolation and characterisation of new Gram-negative and Gram-positive atrazine degrading bacteria from different French soils. FEMS Microbiology Ecology 36, 211–222. Sambrook, J., Russell, D.W., 2001. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Seffernick, J.L., de Souza, M.L., Sadowsky, M.J., Wackett, L.P., 2001. Melamine deaminase and atrazine chlorohydrolase: 98 percent identical but functionally different. Journal of Bacteriology 183, 2405–2410. Shao, Z.Q., Behki, R.M., 1995. Cloning of the genes for the degradation of the herbicides EPTC (S-ethyl dipropylthiocarbamate) and atrazine from Rhodococcus sp. strain TE1. Applied and Environmental Microbiology 61, 2061–2065. Shao, Z.Q., Behki, R.M., 1996. Characterization of the expression of the thcB gene, coding for a pesticide-degrading cytochrome P-450 in Rhodococcus strains. Applied and Environmental Microbiology 62, 403–407. Shao, Z.Q., Seffens, W., Mulbry, W., Behki, R.M., 1995. Cloning and expression of the s-triazine hydrolase gene (trzA) from Rhodococcus corallinus and development of Rhodococcus recombinant strains capable of dealkylating and dechlorinating the herbicide atrazine. Journal of Bacteriology 177, 5748–5755. de Souza, M.L., Newcombe, D., Alvey, S., Crowley, D.E., Hay, A., Sadowsky, M.J., Wackett, L.P., 1998. Molecular basis of a bacterial consortium—interspecies catabolism of atrazine. Applied and Environmental Microbiology 64, 178 –184. de Souza, M.L., Sadowsky, M.J., Wackett, L.P., 1996. Atrazine chlorohydrolase from Pseudomonas sp. strain ADP: gene sequence, enzyme purification, and protein characterization. Journal of Bacteriology 178, 4894–4900. de Souza, M.L., Wackett, L.P., Boundy-Mills, K.L., Mandelbaum, R.T., Sadowsky, M.J., 1995. Cloning, characterization, and expression of a gene region from Pseudomonas sp. strain ADP involved in the dechlorination of atrazine. Applied and Environmental Microbiology 61, 3373–3378. Topp, E., Zhu, H., Nour, S.M., Houot, S., Lewis, M., Cuppels, D., 2000. Characterization of an atrazine-degrading Pseudaminobacter sp. isolated from Canadian and French agricultural soils. Applied and Environmental Microbiology 66, 2773– 2782. Van Zwieten, L., Kennedy, I.R., 1995. Rapid degradation of atrazine by Rhodococcus sp. NI86/21 and by an atrazine-perfused soil. Journal of Agricultural and Food Chemistry 43, 1377–1382. Wackett, L.P., Sadowsky, M.J., Martinez, B., Shapir, N., 2002. Biodegradation of atrazine and related s-triazine compounds: from enzymes to field studies. Applied Microbiology and Biotechnology 58, 39–45. Yanze-Kontchou, C., Gschwind, N., 1994. Mineralization of the herbicide atrazine in soil inoculated with a Pseudomonas strain. Applied and Environmental Microbiology 60, 4297– 4302. Yanze-Kontchou, C., Gschwind, N., 1995. Mineralization of the herbicide atrazine in soil inoculated with a Pseudomonas strain. Journal of Agricultural and Food Chemistry 43, 2291–2294. Yassir, A., Lagacherie, B., Houot, S., Soulas, G., 1999. Microbial aspects of atrazine biodegradation in relation to history of soil treatment. Pesticide Science 55, 799–809.
Analysis of atrazine-degrading microbial communities in soils using most-probable-number enumeration, DNA hybridization, and inhibitors Ellen B. Ostrofskya,1, Jayne B. Robinsonb, Samuel J. Trainac, Olli H. Tuovinena,* a
Department of Microbiology, The Ohio State University, 484 West 12th Avenue, Columbus, OH 43210-1292, USA b Department of Biology, University of Dayton, Dayton, OH 45469-2320, USA c School of Natural Resources, The Ohio State University, Columbus, OH 43210-1085, USA Received 4 May 2001; received in revised form 8 May 2002; accepted 22 May 2002
Abstract The purpose of this study was to determine whether there was an association between kinetics of atrazine mineralization, the number and type of atrazine-degrading microorganisms, and the presence of three genes representing different steps in the degradative pathway of atrazine in three soils with different histories of atrazine application. Composite soil samples were collected from two agricultural fields and one riparian zone soil. The samples were amended with atrazine, and mineralization was measured in biometers as 14CO2 evolution in samples spiked with [ring-U-14C]-labeled atrazine. Atrazine-degrading microorganisms were enumerated by a most-probable-number (MPN) method, which was based on loss of atrazine (HPLC assay) in acetate media. Dot-blot hybridization assays were performed on total DNA extracted from the MPN samples. The DNA probes used in dot-blot assays were the genes atzA (atrazine chlorohydrolase from Pseudomonas ADP), atrA (cytochrome P-450 from Rhodococcus TE1), and trzD (cyanuric acid amidohydrolase from Pseudomonas NRRLB-12228). The herbicide amendment enhanced the subsequent rate of mineralization of atrazine in all three soil samples. The MPN numbers were in the range of 100 – 102 cells g21 dry wt. soil, indicating that atrazine-degrading microorganisms could not be quantitatively enumerated by this technique. Positive hybridization signals of DNA extracted from MPN samples were frequent with the atzA probe; the atrA dot blots had fewer positive signals. The trzD signals were negligible or undetectable, although the parallel mineralization studies showed fast and extensive 14CO2 evolution from [ring-U-14C]-atrazine. The results suggested that trzD, the only gene known to encode striazine ring-cleavage, is not dominant among the atrazine-degrading populations of these soils. Streptomycin and cycloheximide were added to soil samples in biometers to determine the relative contributions of bacteria and fungi in the soils to the mineralization of atrazine. The relative suppression of mineralization in the presence of the bacterial or fungal specific inhibitor was approximately the same, indicating that both groups contributed to the mineralization of atrazine. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Atrazine; Biodegradation of atrazine; Gene probing; Hybridization; Triazine
1. Introduction Atrazine (2-chloro-4-N-isopropyl-6-N-ethyl-s-triazine) has been in worldwide use for control of annual grasses and broadleaved weeds for over 40 years. Microbial degradation is the dominant mode of atrazine attenuation in soils. Several pure and mixed bacterial cultures have been described that can mineralize or partially degrade atrazine (Assaf and Turco, 1994; Yanze-Kontchou and Gschwind, 1994; Mandelbaum et al., 1995; Radosevich et al., 1995; Alvey and Crowley, 1996; Topp et al., 2000). The catabolic * Corresponding author. Tel.: þ 1-614-292-3379; fax: þ1-614-292-8120. E-mail address: [email protected] (O.H. Tuovinen). 1 Present address: Darrin Freshwater Institute, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY 12110-3590, USA.
pathways and genes have been only partially characterized (Wackett et al., 2002), and the molecular and biochemical basis of atrazine biodegradation, especially in soils, is poorly understood. The intermediates of atrazine biodegradation through to the formation of cyanuric acid are believed to be common, but the sequence of pathway steps varies among the known degraders. For example, Pseudomonas ADP dechlorinates atrazine before deamination of the ethylamino group, whereas Rhodococcus NI86/ 21 N-dealkylates atrazine as its initial degradative step (de Souza et al., 1995; Nagy et al., 1995; Van Zwieten and Kennedy, 1995). Genes encoding enzymes that are involved in atrazine biodegradation have been identified and cloned from Pseudomonas and Rhodococcus spp. (Eaton and Karns, 1991a,b; Mulbry, 1994; Shao and Behki, 1995; Shao et al.,
0038-0717/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 0 2 ) 0 0 0 8 9 - 5
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Fig. 1. The reactions of three enzymes involved in atrazine biodegradation: AtzA from Pseudomonas ADP, AtrA from Rhodococcus TE1, and TrzD from Pseudomonas NRRLB-12228.
1995; de Souza et al., 1996; Boundy-Mills et al., 1997). Some genes encode enzymes with similar functions, but nucleotide and amino acid homology studies have revealed little similarity among these genes and gene products from different organisms. One notable exception is the atzABC genes from Pseudomonas sp. ADP, which have been found in several other atrazine-degrading bacteria (de Souza et al., 1998). The purpose of this study was to characterize the mineralization of atrazine in soils with different histories of application of atrazine. Kinetics of the mineralization of atrazine were determined to establish whether the kinetic parameters were related to the number and type of atrazine-degrading microorganisms and the presence of atzA, atrA, and trzD genes in the test soils. Fig. 1 shows the reactions catalyzed by the enzymes encoded by these three genes associated with the degradation of atrazine. The gene atzA encodes a chlorohydrolase and was originally identified and cloned from Pseudomonas ADP that can mineralize atrazine (de Souza et al., 1996). The gene atrA encodes a cytochrome P-450 monooxygenase, which causes spontaneous N-dealkylation of either the ethyl or isopropyl group after enzymecatalyzed hydroxylation. This gene was identified and cloned from Rhodococcus TE1 and Rhodococcus NI86/ 21, both of which are capable of degrading S-ethyl dipropylthiocarbamate (EPTC) and atrazine (Nagy et al., 1995; Shao and Behki, 1995). The gene trzD was originally found in Pseudomonas strains NRRLB-12227 and NRRLB-12228 (Eaton and Karns, 1991a,b; Karns,
1999), which can utilize melamine, ammeline, ammelide, and cyanuric acid as N sources but they do not degrade atrazine. The gene trzD encodes cyanuric acid amidohydrolase, and its homologs have been found by PCR amplification in many atrazine-degrading soil bacteria (Rousseaux et al., 2001). Amendment of soils with cyanuric acid has a variable effect on the subsequent mineralization of atrazine by soil microorganisms, ranging from no response to great enhancement (Ostrofsky et al., 2001). In the present study, amendment with atrazine followed by incubation was used in an attempt to enhance subsequent mineralization and to enrich for atrazine-degrading microorganisms. A mostprobable-number (MPN) method was employed to enumerate atrazine-degrading microorganisms and to assess their changes in response to atrazine treatment. Genomic DNA samples from the MPN cultures were examined to establish whether incubation with atrazine selected for bacteria that contained DNA sequences homologous to the atzA, atrA, or trzD genes. Levanon (1993) used streptomycin and cycloheximide to differentiate between bacterial and fungal contributions, respectively, to the biodegradation of atrazine. Suppression of the mineralization of atrazine was evident with both antibiotics, indicating that bacteria and fungi are involved in the mineralization. In the present studies, soil samples were amended with streptomycin (an inhibitor of bacteria) and cycloheximide (an inhibitor of eukaryotes) to discriminate between eukaryotic and bacterial biodegradation.
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2. Materials and methods 2.1. Site description and sampling The surface soil samples were collected at various times between 1996 and 1998 from two field plots (CC, CR) and a reference site (RZ) at a research farm in Piketon, Ohio, designated as the Ohio Management Systems Evaluation Area (Ohio MSEA). A composite of four surface soil samples (0 –5 cm) was collected at each site. CC was a continuous cornfield with annual application of atrazine at the maximum allowable rate (2.5 kg ha21). CR was a corn – soybean– wheat crop rotation field with atrazine applied at a reduced rate (1.5 kg ha21) during corn years (corn planted and atrazine applied in April 1994 and again in April 1997). RZ was a riparian zone adjacent to the agricultural fields and had never received atrazine other than by aerial drift or by vernal flooding of a nearby river (Scioto River) that has an agricultural watershed. Descriptive details of these soils can be found in Ostrofsky et al. (1997, 2001). The field moisture content of the CC soil samples was 10 ^ 1.4% (w/w) in May 1996, 15 ^ 0.3% in June 1997, and 5.1 ^ 1.9% in October 1997. The field moisture content of the CR soil samples was 12 ^ 1.5% in May 1996 and 5.1 ^ 0.9% in October 1997. The field moisture content of the RZ soil samples was 20 ^ 2.0% in May 1996, 24 ^ 1.1% in June 1997, and 16 ^ 0.35% in October 1997. 2.2. Soil amendments Samples for soil amendment experiments were collected in May 1996 and June 1997. The composite soil samples (100 g wet weight) were placed into sterile plastic trays and amended with sterile double-distilled H2O or an aqueous solution of atrazine by drop-wise addition and mixing thoroughly with a sterile spatula. Atrazine was added to a final concentration of 1.0 mg kg21 soil to approximate a field application of 2.5 kg ha21. Because the moisture content of field samples was low, it was normalized to 25% (v/w) with the amendments. All incubations were at 22 ^ 2 8C. At various time intervals following amendment, subsamples (50 g) were removed for determining the mineralization of atrazine and for MPN enumeration. 2.3. Mineralization of atrazine The mineralization of atrazine was measured in biometers, reaction vessels that are designed to measure the evolution of 14CO2 from 14C-labeled substrates. These biometers consisted of 60 ml serum bottles equipped with suspended 2 ml vials to trap evolved CO2 (Ostrofsky et al., 1997). Each biometer received 5 g dry wt of soil and 1.0 ml of double-distilled water containing 0.1 mCi of [ringU-14C]-atrazine ($ 95% pure, Sigma Chemical Co., St Louis, MO) and unlabeled atrazine (99.5% pure, Chem Service, West Chester, PA) to yield a final concentration of
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2.5 mg atrazine kg soil21 for the mineralization experiments. The mineralization of atrazine was tested for all field samples. In the soil amendment experiments, the mineralization of atrazine was monitored as 14CO2 evolution over time in duplicate biometers for subsamples that had been pre-incubated with atrazine or H2O for various time intervals. For sterile controls, duplicate soil samples in biometers were autoclaved for 20 min at 121 8C. To compare rates of mineralization, the data were fitted to a first-order rate equation, which is normally used by regulatory agencies to assess half-lives and rate constants of attenuation of pesticidal molecules in the environment. It was not within the scope of this work to find the best-fitting rate expressions for each of the incubations. The first-order data fitting was used to determine the rate constants (k) and half-lives (t1/2) (Ostrofsky et al., 1997) P ¼ Pmax ð1 2 e2kt Þ P is cumulative %14CO2 evolved at time t (d), Pmax is the maximum cumulative %14CO2 evolved during the mineralization time-course. The coefficient of determination (r 2) was calculated to assess the goodness of the first-order fit. The level of residual atrazine was analyzed in amended samples: soil samples (10 g wet weight from the 50 g subsamples) were mixed with a 3:1 methanol/water solution in plastic screw-capped tubes, shaken by hand, and then incubated for 20 min in an ultrasonic bath. The samples were extracted overnight at 22 ^ 2 8C. Supernatants were filtered (0.45 mm GHP glass fiber, Gelman Sciences, Ann Arbor, MI) and analyzed with an ELISA specific for atrazine (Strategic Diagnostics, Inc., Newark, DE). According to the manufacturer, the ELISA has been developed for aqueous samples with a lower limit of detection of 0.046 mg atrazine l21 water. Other triazines, including three atrazine metabolites, cross-react with the assay. 2.4. MPN enumeration The medium for MPN enumeration was formulated on the basis of growth of pure cultures of atrazine-mineralizing bacteria. The medium consisted of (per liter) 21.5 mg atrazine, 0.5 g KH2PO4, 0.65 g Na acetate, 0.2 g MgSO4· 7H2O, and 10 ml of a trace metals stock solution containing (per liter stock solution) 2.0 g nitrilotriacetic acid, 1.0 g MnSO4·H2O, 0.9 g CaSO4, 0.8 g Fe(NH4)2(SO4)2·6H2O, 0.2 g CoSO4·7H2O, 0.2 g ZnSO4·7H2O, 0.03 g CuSO4·7H2O, 0.02 g Na2WO4, 0.02 g Na2SeO3, and 0.02 g NiCl2·6H2O. The medium was sterilized by autoclaving and aseptically dispensed in 5 ml aliquots into sterile glass test tubes (16 £ 100 mm2). For the initial dilution, 10 g samples of field moist soil were added to 90 ml of sterile phosphate buffered saline in sterile centrifuge bottles. The bottles were shaken horizontally for 30 min. Four additional 10-fold serial dilutions
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Table 1 List of cultures used as reference sample or plasmid DNA in dot-blot hybridization studies. Information on cultivation of these cultures can be found in the listed references Culture
Source
Reference
E. coli DH5a (pJK206) E. coli DH5a (pMD4) E. coli DH5a (pKL1) E. coli ATCC 10798 Rhodococcus TE1 Pseudomonas 12228 Pseudomonas ADP M91-3
J.S. Karns M. de Souza W.A. Dick Department of Microbiology, Ohio State University W.A. Dick J. S. Karns M. de Souza M. Radosevich
Eaton and Karns, 1991a de Souza et al., 1996 Shao and Behki, 1995 LBa Shao and Behki, 1995 Eaton and Karns, 1991b de Souza et al., 1996 Radosevich et al., 1995
a
Grown in Luria broth (LB) at 378C with shaking overnight.
were then made, and 1 ml aliquots of the dilutions were used to inoculate the MPN tubes. The number of replicate MPN tubes was either three or five depending on the experiment. For sterile controls, a 1021 dilution of each soil was autoclaved and 1 ml aliquots were used to inoculate a set of five MPN tubes. These sterile controls were used to account for abiotic losses of atrazine. MPN tubes were incubated without shaking at 22 ^ 2 8C for 30 d. At 30 d, water lost by evaporation was replaced (determined either by an initial marking of the solution level or gravimetrically) with sterile water, and the samples mixed thoroughly by vortexing. A 1 ml sample from each MPN tube was removed, centrifuged to remove particles, and analyzed by HPLC. The HPLC parameters were a flow rate of 0.75 ml min21, absorbance at 225 nm, degassed mobile phase of 70% acetonitrile (ACN) and 30% H2O, and a C18 column (Alphabond, Alltech Associates, Deerfield, IL). A linear equation derived from regression analysis of peak areas of standards of known concentration was used to calculate atrazine concentration in the MPN samples. Samples with an atrazine loss of $ 25% of the initial concentration, as determined by HPLC analysis of samples from five uninoculated MPN tubes, were scored as positive. The scoring was based on atrazine utilization rather than observance of growth, because the intention of the MPN assay was to enumerate specifically atrazine-degraders. The extent of the degradation of atrazine varied considerably in the MPN tubes. A cut-off of 25% degradation of atrazine was chosen to score tubes positive. Turbidity as an indication of growth was not used as a scoring basis because it was presumed that organisms could grow in the medium (using acetate as a C source and various N sources from the inoculum) without degrading the atrazine. Estimates of numbers of atrazine-degraders with 95% confidence intervals were determined using an MPN program written in BASIC (Hurley and Roscoe, 1983). 2.5. DNA extraction from MPN samples Genomic DNA was extracted from the three-dilution, three-replicate-tube series that were used to calculate the
MPN estimate. The remaining solutions in MPN tubes were centrifuged to pellet microorganisms. The pellets were subjected to the rapid genomic bacterial DNA extraction and purification procedure (Davis et al., 1994). Briefly, the pellets were treated with aqueous solutions of sodium dodecyl sulfate (SDS) and proteinase K and then extracted sequentially with phenol and chloroform/isoamyl alcohol. The resulting pellets of DNA were resuspended in 100 ml TE buffer (10 mM Tris– HCl, 0.1 mM EDTA, pH 8.0). For dot-blot hybridization analysis, 50 ml aliquots (1 – 3 mg) of the DNA were applied to the membranes (Hybond-Nþ nylon, Amersham Pharmacia Biotech, Amersham, UK). 2.6. Bacterial cultures and media Table 1 lists the sources of cultures used for genomic and plasmid DNA isolation, which were used as positive and negative controls in the hybridization experiments. Cultures were grown for 1– 3 d under conditions specified in the respective references in Table 1. Overnight cultures of strains of Escherichia coli DH5a were grown at 37 ^ 2 8C with shaking for purification of plasmid and probe DNA. Genomic DNA from bacterial cultures was extracted and purified by the modified protocol provided with the QIAamp Tissue kit (Qiagen, Chatsworth, CA). 2.7. Description and production of DNA gene probes The gene probes used in the hybridization experiments are described in Table 2. At the beginning of this study, sequence information for the genes atzA, atrA, and trzD was not available. Sequence information became available only after hybridization analyses had been completed. The size of the atzA gene is 1.4 kb and is contained entirely within the 1.9 kb AvaI/AvaI restriction fragment used as a probe (de Souza et al., 1996). The remainder of the 1.9 kb probe consists of remnant DNA from Pseudomonas ADP. The size of the trzD gene is 1.1 kb and is contained entirely within the 2.0 kb HindIII/PstI fragment; the remainder of this fragment consists of Pseudomonas 12228 DNA (Karns, 1999). The atrA probe used in this study was the 6.2 kb
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Table 2 Description of the gene probes used in the dot-blot hybridization studies Gene
Size (kb)
GenBank accession numbera
Restriction fragment used as probe
Plasmid vector
AtzA AtrA TrzD
1.4 1.0, 1.3b 1.1
U55933 U17130c AF086815
1.9 kb AvaI/AvaI 6.2 kb KpnI/KpnI 2.0 kb HindIII/PstI
pMD4 pKL1 pJK206
a
At the time of the present study, the sequences of these three DNA fragments used as probes had not been determined. The information included in this Table was made available only after this study had been completed. b Sizes of the thcR and thcB genes, respectively. c The sequences of the genes are from Rhodococcus NI86/21.
KpnI/KpnI fragment of pKL1. Shao and Behki (1996) reported that this 6.2 kb fragment was found in Rhodococcus NI86/21. Nagy et al. (1995) sequenced this fragment in Rhodococcus NI86/21 and found that it contained two genes, thcB and thcR, which are involved in encoding a cytochrome P-450 monooxygenase. For the production of probes and quantitative standards, plasmid DNA was extracted from cultures of E. coli by an alkaline lysis protocol (Sambrook and Russell, 2001). Plasmid DNA was digested with the appropriate restriction enzymes at 37 8C for 1– 2 h to generate probe DNA. Restriction digest fragments were separated by gel electrophoresis on 1% agarose and visualized by staining with ethidium bromide. HindIII-digested lambda DNA was used as a size standard. The appropriate restriction fragment was excised from the gel and purified using the protocol provided with the Qiaquick Gel Extraction kit (Qiagen). The resulting purified DNA was used in probe labeling reactions. Reference samples for dot-blot hybridization analysis included the plasmids pJK206 (contains the gene trzD), pMD4 (atzA), and pKL1 (atrA) as quantitative standards. Pseudomonas NRRLB-12228, Pseudomonas ADP, and Rhodococcus TE1 genomic DNA served as positive controls for trzD, atzA and atrA, respectively. Genomic DNA from an LB-grown culture of E. coli ATCC 10798 served as a negative control. 2.8. DNA hybridization Probe labeling, hybridization, and detection were performed as instructed by the protocol provided with the Gene Images Chemiluminescent Labeling and Detection kit (Amersham Pharmacia Biotech). Hybridizations and washes were performed at 50 8C. The blots were washed once for 15 min each with 2X SSC (20X: 3 M NaCl, 300 mM Na-citrate) þ 0.1% SDS and 0.1X SSC þ 0.1% SDS. DNA samples were loaded onto the hybridization membrane using a vacuum dot-blot manifold (Bio-Dotw Microfiltration Apparatus, Bio-Rad, Hercules, CA). The quantity of DNA in all samples was 1 mg, except for the quantitative plasmid standards, which are indicated in the figure legends. Chemiluminescence was detected by exposure of X-ray
film (Hyperfilme-MP, Amersham Life Science) to the blots. Film was developed in an automatic film developer. Developed films were scanned using Adobe Photoshop software (version 4, Adobe Systems, San Jose, CA), and intensity measurements were determined with Scion Image for Windows software (version 2, Scion Corp., Frederick, MD). Before re-hybridization with another gene, blots were stripped of probe with three 10 min washes of boiling 0.1% SDS. After stripping of the last gene probe, re-exposure of the blot to X-ray film confirmed the complete removal of the probe. 2.9. Effect of bacterial and fungal inhibitors on the mineralization of atrazine in soil To determine the contribution of bacteria and fungi to the mineralization of atrazine observed in CC soils, antibiotics were added to soil samples in the biometers singly or together. Streptomycin was selected as an inhibitor of bacteria and cycloheximide as an inhibitor of eukaryotes. Biometers were established in duplicate, as previously described. The inhibitors were added to the stock solutions of atrazine and 1 ml (0.1 mCi 14C-atrazine, 2.5 mg atrazine kg21 soil) was applied to 5 g (dry wt) of field moist CC soil (collected in January 1998). To distinguish the effects of inhibitors, the CC soil was used in these biometer experiments because it demonstrated relatively rapid and extensive mineralization of atrazine. The concentrations of streptomycin and cycloheximide were 1, 3, and 5 g kg21 soil for the single addition experiments, and it was 3 g kg21 soil for each inhibitor for the multiple addition experiment. For the single addition experiment, inhibitors were added at the beginning of the incubation, and biometers were incubated and monitored for 14CO2 evolution every other day for 10 d. For the multiple addition experiment, the inhibitors were added at the beginning and every other day for a total of five additions. In an additional inhibitor experiment, duplicate biometers containing 5 g of CC soil (collected in June 1998) were amended once with 5 g streptomycin kg21 soil, 3 g cycloheximide kg21, or both, and spiked with 14C-ring-atrazine (2.5 mg atrazine kg21 soil). Evolution of 14CO2 was monitored on days 5 and 10 d after the addition of inhibitors.
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Fig. 2. Time course data for the mineralization of atrazine in CC, CR, and RZ soil samples collected in May 1996. These samples were amended with 1 mg kg21 atrazine and incubated for 30, 60, 90, or 138 d. Subsequently, subsamples were collected, spiked with [U-14C-ring]-atrazine, and the time course of 14CO2 evolution was measured in biometers. Mineralization of atrazine was also measured in initial soil samples and corresponding sterile controls. The bars represent the standard deviation which, in most cases, are smaller than the size of the symbol.
3. Results 3.1. Mineralization of atrazine and microbial enumeration Three different soils were sampled in May 1996 and amended in the laboratory with 1.0 mg atrazine kg21 soil or with double-distilled water as a control. At 30, 60, 90, and 138 d of incubation after amendment, samples were transferred to biometers and spiked with [U-14C-ring]atrazine to monitor mineralization of atrazine as evolution of 14CO2. Amendment and incubation for 30 d enhanced the subsequent rate of mineralization of atrazine for the CR and RZ soils as evidenced by the production of 14CO2 from the
ring-labeled atrazine (Fig. 2). This enhancement persisted throughout the 138 d pre-incubation (Fig. 2). The CC soils already had a high level of atrazine-mineralization activity even without amendment with atrazine (Fig. 2(A)). Autoclaved sterile control soils showed less than 1% evolution of 14 CO2 in all three soils (Fig. 2). Water-amended samples of the three soils demonstrated rates of mineralization that were similar to the activities of the respective preamendment samples at all incubation times, indicating that native mineralization activity persisted throughout the 138 d pre-incubation period. The kinetic data for CC soils with all treatments yielded mineralization rate constants of 0.04 –0.14 d21 and halflives of 5 –19 d (Table 3). The kinetic data derived from fitting the mineralization data to a first-order rate expression showed increases in rate constants and decreases in halflives of the mineralization of atrazine in the CR and RZ soils after amendment with atrazine (Table 3). The kinetic data for the water-amended samples did not vary during the time course of this experiment. These kinetic data indicated that the mineralization of atrazine was fastest in the CC samples. The kinetics of the pre-amended CR samples were slower than in the CC samples but increased after amendment with atrazine to be comparable with CC. Amendment with atrazine accelerated mineralization also in RZ soils. Mineralization of atrazine was also compared with the residual levels of atrazine in these three soils. Before any amendments, ELISA results showed residual levels of 13, 7, and 4 mg atrazine kg soil21 in the CC, CR, and RZ soils, respectively. All water-amended samples had residual atrazine levels similar to those in the pre-amendment samples. The residual levels after pre-incubation with 1 mg atrazine kg21 soil were 14– 17 mg kg21 for the CC, 26– 36 mg kg21 for the CR, and 31– 40 mg kg21 for the RZ samples, respectively. These data are in keeping with the relative extent of mineralization of atrazine in the three soils. The CC and RZ soils were sampled in June 1997 for the mineralization of atrazine. The CC soil, which had a history of atrazine application, had a greater extent of mineralization than the RZ soil, which had never received direct atrazine application (Table 4). After amendment and incubation of the CC soil, a transient decrease in evolved 14 CO2 was observed, but the final extent of mineralization of atrazine was comparable with that in the unamended CC soil. The amendment of 1 mg atrazine kg21 soil followed with 60 d of pre-incubation resulted in an increase in the extent of the mineralization of atrazine from 10 to 60% in the RZ samples (Table 4). The mineralization of atrazine in sterile CC soils was less than 0.2% after 30 d. In samples collected in October 1997, the CC soil reached 85% mineralization in 30 d, consistent with the sample collected in June (Table 4). The 14CO2 evolution in the CR soil was about 35%, whereas the mineralization in the RZ soil was about 3% after 30 d of incubation. The CR soils receive atrazine treatment only once every three years, and
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Table 3 Kinetic data and residual atrazine concentration in the amendment and mineralization experiments Soila
CC
CR
RZ
a
Amendment
Length of pre-incubation (d)
Parameter Pmax (%)
k (d21)
r2
t1/2 (d)
Pre-amendment H2 O H2 O H2 O H2 O
0 30 60 90 138
79.5 68.7 95.1 74.5 84.8
0.09 0.08 0.06 0.04 0.06
7.9 8.9 12 19 12
0.82 0.85 0.83 0.59 0.88
Atrazine Atrazine Atrazine Atrazine
30 60 90 138
69.6 98.8 78.8 86.1
0.14 0.09 0.08 0.07
5.1 8.2 8.7 10
0.98 0.92 0.89 0.84
Pre-amendment H2 O H2 O H2 O H2 O
0 30 60 90 138
19.0 20.6 19.5 9.34 16.1
0.003 0.004 0.002 0.001 0.001
217 178 347 693 495
0.96 0.94 0.94 0.66 0.97
Atrazine Atrazine Atrazine Atrazine
30 60 90 138
36.9 57.0 63.8 75.5
0.008 0.008 0.02 0.04
90 84 35 17
0.89 0.90 0.88 0.93
Pre-amendment H2 O H2 O H2 O H2 O
0 30 60 90 138
15.8 8.1 13.4 9.33 16.87
0.003 0.001 0.001 0.001 0.002
277 495 496 693 462
0.95 0.97 0.96 0.80 0.96
Atrazine Atrazine Atrazine Atrazine
30 60 90 138
39.9 51.9 35.9 56.3
0.012 0.008 0.006 0.02
58 90 116 42
0.88 0.96 0.77 0.91
CC, continuous corn; CR, crop rotation; RZ, riparian zone.
mineralization in this soil was consistent with its history of intermittent atrazine application. The numbers of atrazine-degrading microorganisms estimated by MPN enumeration was 812 (95% confidence interval of 246– 2700) and 12 (4 – 40) cells g21 dry soil in the CC and RZ soils sampled in June 1997, respectively.
After the CC and RZ soils were amended with atrazine and incubated for 60 d, the MPN of atrazine-degraders diminished to less than 3 cells g21 dry soil. For the October 1997 samples, the MPN of atrazine-degrading organisms were in the range of 51– 75 (95% confidence interval of 21– 194) cells g21 dry soil for the CC, CR, and RZ soils.
Table 4 Mineralization of atrazine in soil samples collected in June and October, 1997 Sample
CC RZ Sterile CC CC RZ CC CR RZ a
Sampling date
June, 1997 June, 1997 June, 1997 June, 1997 June, 1997 October, 1997 October, 1997 October, 1997
Treatmenta
A A A B B A A A
% Cumulative mineralization ^ standard deviation 10 d
20 d
30 d
69 ^ 0.90 1.3 ^ 0.92 0.03 ^ 0.002 16 ^ 2.7 12 ^ 5.0 39 ^ 4.7 4.1 ^ 3.65 0.74 ^ 0.001
73 ^ 0.54 4.5 ^ 2.81 0.11 ^ 0.007 68 ^ 5.1 40 ^ 12.4 NDb ND ND
76 ^ 0.27 10 ^ 3.4 0.17 ^ 0.011 76 ^ 1.5 60 ^ 1.7 85 ^ 6.5 35 ^ 22.2 3.2 ^ 1.95
All soil samples were incubated at 22 ^ 2 8C for 60 d before measuring the mineralization. A, unamended soils; B, soils amended with 1 mg atrazine kg soil21. b ND, no data.
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Fig. 3. Dot-blot hybridizations with DNA extracted from MPN cultures. The MPN samples were from a three-dilution series with unamended CC, CR, and RZ soils from October 1997. Half of the DNA extraction mixture (50 ml) was added to the blot. Plasmid, genomic, and MPN DNA samples hybridized with atzA (A); with atrA (B); and with trzD (C); corresponding results of HPLC analyses used for MPN determination (% atrazine degradation) (D).
3.2. Gene probe analysis of DNA from MPN samples Plasmid DNA, genomic reference DNA samples, and DNA from MPN tubes were hybridized with genes atzA, atrA, and trzD (Fig. 3(A)–(C)). The HPLC results used to calculate the MPN and corresponding to the samples on the blot are shown in Fig. 3(D). Almost all the MPN samples showed a hybridization signal with atzA (Fig. 3(A)), but there did not seem to be a correlation between intensity of the signal and loss of atrazine. The number of signals did not decrease with dilution, as would be expected if organisms containing the gene were diluted. More positive hybridization signals were observed than there were samples that were positive for atrazine loss. Fewer hybridization signals with MPN samples probed with atrA were observed than with samples probed with atzA (Fig. 3(B)). Again, there did not seem to be a correlation between hybridization signals and loss of
atrazine or a decrease in frequency of signal detection with dilution (Fig. 3(B)). Hybridization signals were not observed when MPN samples were probed with trzD (Fig. 3(C)). 3.3. Effect of streptomycin and cycloheximide on the mineralization of atrazine There was no striking differential effect in mineralization of atrazine resulting from treatments of the soils with streptomycin, cycloheximide, and both. All treatments suppressed mineralization of atrazine until after 6 d of incubation. At 10 d, the extent of mineralization was nearly the same as the untreated CC control, except for the treatments with 5 g kg21 streptomycin (Fig. 4(A)) and the 3 g kg21 cycloheximide (Fig. 4(B)). In combination, the inhibitors at 3 and 5 g kg21 suppressed mineralization of atrazine as compared with the untreated control (Fig. 4(C)).
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mineralization of atrazine involved both eukaryotic and bacterial populations.
4. Discussion
Fig. 4. Time course data for the mineralization of atrazine in inhibitor experiments in CC soil. (A) Single amendment of 1, 3, or 5 g streptomycin kg21 dry soil. (B) Single amendment of 1, 3, or 5 g cycloheximide kg21 dry soil. (C) Single amendment of 1, 3, or 5 g of both streptomycin and cycloheximide kg21 dry soil. CC refers to untreated soil samples, and abiotic refers to autoclaved soil samples. (D) Multiple amendments of inhibitors at 3 g each kg21 dry soil every other day. The bars represent the standard deviation which, in most cases, are smaller than the size of the symbol.
All three inhibitor treatments suppressed mineralization of atrazine in the multiple addition experiment (Fig. 4(D)). In an additional experiment, the untreated CC soil showed an extent of mineralization of atrazine of about 50% after 10 d of incubation (Fig. 5). The individual streptomycin- and cycloheximide-treated samples showed lower extents (about 10% after 10 d) of mineralization as compared with the untreated CC soil (Fig. 5). The lowest extent of mineralization was seen in the sample treated with a combination of streptomycin and cycloheximide, which showed only about 5% mineralization activity after 10 d of incubation (Fig. 5). These results suggested that the
The two agricultural soils, CC and CR, which had a prior history of atrazine application contained resident microbial communities capable of mineralizing the herbicide without additional amendment, albeit at different rates and to different extents. The microbes in the RZ soil, however, mineralized atrazine only after additional amendment, and the rate increased almost to the same rate as in CC. These results suggest that atrazine-mineralizing microorganisms existed in the RZ soil but were inactive or less numerous when atrazine was not present. The MPN results suggest that the atrazine-degraders in these soils were relatively non-culturable in the atrazineacetate medium used. Although the quantitative relation between the kinetics of the mineralization of atrazine and the numbers of indigenous atrazine-degrading microorganisms has not been elucidated, the estimated numbers of ca. 800 atrazine-degraders g21 dry CC soil do not seem great enough to account for the high level (approx. 80%) of the mineralization of atrazine. Based on studies with atrazine where soils were inoculated with a mineralizing culture, cell numbers should be several orders of magnitude greater than those obtained in the present study to account for the relatively rapid mineralization (Yanze-Kontchou and Gschwind, 1995; Alvey and Crowley, 1996; Yassir et al., 1999). The cell numbers decreased in the amended and incubated CC and RZ soil samples. These decreases were within the statistical range of the MPN method, but they could also be the result of a loss of cells during the incubation or to the inherent variability and lack of culturability. At the initiation of the probe design and hybridization experiments, nucleotide sequence information was not available for any of the three genes. Sequence information is now available for genes atzA and trzD and for the two genes found on the atrA DNA fragment. The amino acid sequence derived from the nucleotide sequence of gene atzA is not related to any other amino acid sequence in either the SwissProt or PIR databases at a level greater than 20% homology (de Souza et al., 1996). Karns (1999) indicated that the gene sequence of trzD is not related to any other sequence in the GenBank database. The positive hybridization signal of Pseudomonas 12228 genomic DNA with atzA was an unexpected result, because this bacterium is a melamine-degrader and is not known to possess atrazine dechlorination activity. The atzA gene probe contained some genomic DNA from Pseudomonas ADP, and it is possible that Pseudomonas ADP may have complementary sequences to genomic DNA of Pseudomonas 12228 that are not related to atrazine biodegradation. Seffernick et al. (2001) reported that the amino acid
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Fig. 5. The mineralization of atrazine in a repeated inhibitor experiment with single addition of the inhibitors. The bars represent the standard deviation.
sequence of AtzA of Pseudomonas ADP differed by only nine residues from that of melamine deaminase (TriA) cloned from Pseudomonas 12227, a strain related to Pseudomonas 12228, but that the enzymes catalyze dissimilar reactions on different substrates. In addition, positive hybridization signals were observed for Pseudomonas ADP and Pseudomonas 12228 genomic DNA probed with gene atrA. According to Shao and Behki (1996), the DNA fragment called atrA contains a possible regulatory gene, thcR, and a structural gene, thcB, encoding a novel type of cytochrome P-450. Either of the two genes on the atrA DNA fragment could have complements in the Pseudomonas genomes and produce the hybridization signals. Levanon (1993) demonstrated that adding inhibitors of bacteria and fungi separately to soils reduced the total amount of atrazine mineralized from 29% in 30 d in untreated soils to less than 1% for both classes of inhibitors. In this study, inhibitors were added once at the initiation of the mineralization assays at concentrations of 2.5 and 5.85 g inhibitor kg21 soil for the fungal and bacterial inhibitors, respectively. These concentrations of inhibitors showed only a partial suppression of the mineralization of atrazine. Only when the inhibitors
were added together multiple times individually or together did the suppression of the mineralization activity persist. These results were similar to those seen by Levanon (1993) in that both bacteria and eukaryotes seemed to contribute to the mineralization of atrazine in these soils. Decomposition products of the antibiotics could include compounds that serve as C or N sources for the microorganisms involved in the mineralization of atrazine. The decomposition would diminish the antimicrobial effect of the antibiotics, and growth of the microorganisms could lead to enhanced mineralization, thereby also partially alleviating the inhibition. It is clear from this study that none of the gene probes gave consistent signals although the mineralization of atrazine was relatively fast, especially in the CC soil samples. It is conceivable that the genes involved in the biodegradation of atrazine are so diverse in soil microorganisms that they do not yield signals when probed with genes isolated from known pure cultures. Genes from other bacteria and also from fungi would expand the molecular tools and widen the search for signature sequences of degradative pathways of atrazine in the environment. Because of the apparent lack of culturability of atrazine-degraders in soils, the molecular approach is
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of key importance in furthering the understanding of environmental factors that modulate the degradation and mineralization of atrazine in soils.
Acknowledgments We are grateful to Dr J.S. Karns for the Pseudomonas strains and the E. coli construct containing the trzD gene, Dr W.A. Dick for Rhodococcus TE1 and the E. coli construct containing the atrA fragment, and Dr M. de Souza for Pseudomonas ADP and the E. coli construct containing the atzA gene. E.B.O. received partial support for this work from the US Dept. of Defense Environmental Fellowship Program. The research was partially funded by the Ohio MSEA project, which is a cooperative research and educational effort of the Ohio Agricultural Research and Development Center, The Ohio State University, OSU Extension, the USDA-ARS, USDA-CRRS, USDA-ES, USGS, USEPA, and other state and federal agencies. Salary and research support were provided to S.J.T. by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University.
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