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Applied and Environmental Microbiology, August 2005, p. 4455-4460, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4455-4460.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Use of Saccharomyces cerevisiae BLYES Expressing Bacterial Bioluminescence for Rapid, Sensitive Detection of Estrogenic Compounds

John Sanseverino,^1,2 Rakesh K. Gupta,^1, Alice C. Layton,^1,2 Stacey S. Patterson,^1, Steven A. Ripp,^1,2 Leslie Saidak,¹ Michael L. Simpson,^1,3 T. Wayne Schultz,^1,4 and Gary S. Sayler^1,2*

The Center for Environmental Biotechnology, The University of Tennessee, Knoxville, Tennessee 37996,¹ The Department of Microbiology, The University of Tennessee, Knoxville, Tennessee 37996,² Molecular Scale Engineering and Nanoscale Technologies Research Group, Oak Ridge National Laboratory, P.O. Box 2008, MS 6006, Oak Ridge, Tennessee 37831,³ Department of Comparative Medicine, The University of Tennessee, Knoxville, Tennessee 37996⁴

Received 9 November 2004/ Accepted 11 March 2005

Top Abstract Introduction Materials and Methods Results and Discussion References

 
An estrogen-inducible bacterial lux-based bioluminescent reporter was developed in Saccharomyces cerevisiae for applications in chemical sensing and environmental assessment of estrogen disruptor activity. The strain, designated S. cerevisiae BLYES, was constructed by inserting tandem estrogen response elements between divergent yeast promoters GPD and ADH1 on pUTK401 (formerly pUA12B7) that constitutively express luxA and luxB to create pUTK407. Cotransformation of this plasmid with a second plasmid (pUTK404) containing the genes required for aldehyde synthesis (luxCDE) and FMN reduction (frp) yielded a bioluminescent bioreporter responsive to estrogen-disrupting compounds. For validation purposes, results with strain BLYES were compared to the colorimetric-based estrogenic assay that uses the yeast lacZ reporter strain (YES). Strains BLYES and YES were exposed to 17ß-estradiol over the concentration range of 1.2 x 10^–8 through 5.6 x 10^–12 M. Calculated 50% effective concentration values from the colorimetric and bioluminescence assays (n = 7) were similar at (4.4 ± 1.1) x 10^–10 and (2.4 ± 1.0) x 10^–10 M, respectively. The lower and upper limits of detection for each assay were also similar and were approximately 4.5 x 10^–11 to 2.8 x 10^–9 M. Bioluminescence was observed in as little as 1 h and reached its maximum in 6 h. In comparison, the YES assay required a minimum of 3 days for results. Strain BLYES fills the niche for rapid, high-throughput screening of estrogenic compounds and has the ability to be used for remote, near-real-time monitoring of estrogen-disrupting chemicals in the environment.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Evidence suggests a wide variety of xenobiotic compounds (e.g., pesticides, plasticizers, and synthetic hormones) and naturally occurring chemicals possess steroid-like activities that lead to the disruption of the endocrine system in vertebrates (5, 8, 9, 15, 17, 34). In response to public health concerns, the United States Congress directed the Environmental Protection Agency (EPA) to develop a screening program for evaluating the potential of pesticides and other substances to induce hormone-related health effects (Food Quality Protection Act [Public Law 104-170]). This screening approach is enormous in scope, with the EPA estimating that 87,000 existing and new chemicals require testing (7). Furthermore, the U.S. Geological Survey recently reported a low-level occurrence of steroid growth hormones in 80% of 139 water systems examined in the United States (16). Other developed countries, including the United Kingdom, Germany, The Netherlands, Italy, Canada, Brazil, and Japan, share the problem of endocrine-disrupting chemicals in the environment.

Several in vivo mammalian assays (reviewed in reference 23) and in vitro assays (reviewed in references 8 and 36) exist for measuring estrogenic effects. In vitro assays fall into the following broad categories: competitive ligand binding assays, cell proliferation assays, postconfluent cell accumulation, induction of protein expression/enzyme activities, and recombinant receptor/reporter gene assays (for a complete review, see reference 36). Recombinant receptor/reporter gene assays are designed to detect the induction or repression of a biological process via specific endocrine receptors. These assays usually have a high responsiveness and sensitivity and can be used to assess the relative potency of alleged receptor-mediated agonists and antagonists (4, 36).

A widely used receptor/reporter assay for detecting estrogenic compounds is the yeast estrogen screen (YES) (26). The Saccharomyces cerevisiae strain contains the human estrogen receptor (hER-) and a plasmid-based estrogen response element (ERE)-lacZ reporter fusion. When an estrogen-like compound binds to the estrogen receptor protein, it in turn binds to the ERE, inducing transcription of lacZ. ß-Galactosidase transforms the chromogenic substrate chlorophenol red-ß-D-galactopyranoside (CPRG) to a red product measured by absorbance at 540 nm. This assay has been used extensively to measure estrogenic responses to polychlorinated biphenyls and hydroxylated derivatives (18, 28, 30), polynuclear aromatic hydrocarbons (31), and other compounds (32), as well as detection of estrogens in wastewater treatment systems (18) and dairy manure (25). In addition, the YES assay has been used for screening contaminated water for antiestrogenic activity (24). Although proven effective for the in vitro determination of estrogenic activity, the standard colorimetric YES assay's incubation time of 3 to 5 days (29) is impractical when considering the 87,000 chemicals requiring tier I screening. To overcome this issue, a bioluminescent version of this reporter has been constructed.

Bioluminescent bioreporter technology based on activation of gene fusions using the firefly (luc) or bacterial (lux) luciferase is as well established as lacZ, cat, or gfp reporter systems (6). The luc and lux reporters offer unique capabilities for functional transcriptional profiling (22, 35), in vivo monitoring of transcriptional logic gates (33), whole-body imaging (10, 11, 13), and reagentless microluminometer-based hybrid bio/silico sensors (3). Reporter constructs based on the complete bacterial lux cassette (luxCDABE) offer the distinct advantage over the luc genetic system of autonomous light generation without the requirement for exogenous substrate addition or secondary excitation. Further, the bacterial bioluminescence reaction generates a visible light signal that can be detected easily and quantified within hours rather than days, making it more amenable to rapid, high-throughput screening protocols.

Recently, Gupta and coworkers functionally expressed the luxA, -B, -C, -D, and -E genes from Photorhabdus luminescens and the frp gene from Vibrio harveyi in Saccharomyces cerevisiae (12). This bioreporter was engineered using two pBEVY yeast expression vectors (20), which allowed bidirectional constitutive expression of the individual luxA, -B, -C, -D, and -E genes. The luxA and luxB genes were independently expressed from divergent yeast constitutive promoters GPD and ADH1 on pBEVY-U. The luxCD and luxE-frp genes were independently expressed from a second plasmid (pBEVY-L), also using the GPD and ADH1 promoters. An internal ribosome entry site (IRES) was inserted between the luxC and luxD genes and the luxE and frp genes. The IRES allows translation of multiple genes from a single promoter in eukaryotes (14). This present work extends the bioreporter of Gupta and coworkers (12) by developing an estrogen-responsive yeast-based bioluminescent bioreporter and demonstrating its usefulness against known estrogenic and nonestrogenic compounds.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Strains, plasmids, and growth conditions.
Strains and plasmids used in this study are listed in Table 1. Escherichia coli DH5, used as a host for plasmid construction and maintenance, was grown in Luria-Bertani (LB) broth at 37°C with or without 100 µg ampicillin/ml, depending on the requirement for plasmid maintenance.

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TABLE 1. Escherichia coli and Saccharomyces cerevisiae strains and plasmids used in this study

YPD liquid medium (1% yeast extract, 2% peptone, 2% glucose) was used for routine growth of plasmid-free S. cerevisiae strains. S. cerevisiae strains harboring plasmids with leucine and uracil selective markers were grown in modified minimal medium without leucine and uracil (YMM leu^–, ura^–) (26).

Chemicals.
17ß-Estradiol (98% purity), 17-estradiol, 17-ethynyl estradiol (98% purity), diethylstilbestrol (DES; 99% purity), estrone (99% purity), 4,4'-cyclohexlidene bisphenol (98% purity), 4-andostrenedione (98% purity), and ethanol were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO). para-Nonylphenol was purchased from Supelco (Bellefonte, PA). Stock solutions of each chemical were made in ethanol.

Molecular biology techniques.
DNA manipulations were performed according to standard protocols (27). Plasmids were transformed into E. coli and S. cerevisiae by electroporation using ECM600 (BTX Inc., Holliston, MA) as described by the manufacturer. For E. coli strains, electroporation conditions were as follows: charging voltage of 2.5 kV, resistance of 125 , capacitance of 50 µF, pulse length of 5 ms. Immediately following transformation, 960 µl of SOC or LB medium was added and cells were allowed to recover at 37°C with shaking at 200 rpm for at least 1 hour. For S. cerevisiae, cells were prepared according to the manufacturer's instructions (BTX protocols). Cells were transformed with 300 to 500 ng of each plasmid DNA. Electroporation conditions were the same as for E. coli. Immediately following transformation, 1 ml cold 1 M sorbitol was added to the transformed cells. After 10 min, cells were plated on YMM (leu^–, ura^–) noble agar plates.

Plasmid isolation was performed using Wizard mini- or midi-prep kits (Promega, San Luis Obispo, CA) or the RPM yeast plasmid isolation kit (Bio101 Inc., Carlsbad, CA). PCR was performed in 25-µl volumes using Ready-to-Go PCR beads (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) and the oligonucleotide primers listed in Table 2. DNA sequencing was performed with an ABI Big Dye Terminator cycle sequencing reaction kit on an ABI 3100 DNA sequencer (Perkin-Elmer, Inc., Foster City, CA).

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TABLE 2. Oligonucleotide primer sequences used for the construction of plasmid pUTK407

Construction of strain BLYES.
S. cerevisiae strain BLYES contains the plasmids pUTK404 and pUTK407 and the hER- gene in the chromosome (Fig. 1). The construction of pUTK404 has been described previously (12). Briefly, this plasmid contains the luxC, -D, and -E genes from Photorhabdus luminescens and the flavin oxidoreductase gene (frp) from Vibrio harveyi for provision of the FMNH₂ cofactor required for the bioluminescent reaction. The translation of luxD and frp genes is mediated by inclusion of a yeast IRES (14).

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FIG. 1. Schematic representation of S. cerevisiae BLYES. Estrogenic compounds cross the cell membrane and bind to the estrogen receptor. This complex interacts with the ERE, initiating transcription of luxA and luxB. The gene for the human estrogen receptor is located on the chromosome.

Sequential PCRs were used to insert tandem ERE between the bidirectional constitutive promoters of pUTK401 (formerly pUA12B7 [12]). In the first PCR, promoter GPD was amplified using primers GPDR and EREGPDF (Table 2; Fig. 2). Primer EREGPDF contains the entire sequence of the tandem ERE. Primer GPDR contains a unique BamHI site at the 5' end. This 25-µl PCR mixture included Ready-to-Go PCR beads (Amersham Biosciences Corp., Piscataway, NJ) with 250 nM EREGPDF primer, 250 nM GPDR primer, and 1 ng pUTK401 template. A stringent touchdown thermal cycler program was used to amplify this promoter: 95°C for 15 min of initial denaturation, followed by 15 cycles of denaturation at 95°C for 30 s, annealing at 72°C for 30 s, and elongation at 72°C for 30 s. The annealing temperature was reduced by 1°C in each of the first 15 cycles. Then followed 30 cycles of denaturation at 95°C for 30 s, annealing at 57°C for 30 s, and elongation at 72°C for 30 s. There was a final elongation step at 72°C for 2 min.

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FIG. 2. PCR and cloning strategy for generating pUTK407. Sequential PCR steps were used to generate GPDEREADH. This construct was cloned into the BamHI-SpeI site of pUTK401 to generate pUTK407. Restriction site abbreviations: A, AvrII; B, BamHI; E, EcoRI; K, KpnI; N, NotI; P, PstI; Sc, SacI; Sl, SalI; Sp, SpeI; X, XmaI. Figure is not drawn to scale.

In the second PCR, the promoter ADH1 was amplified using primers ADHR and EREADHF (Table 2; Fig. 2). Primer EREADHF contains the entire sequence of the tandem ERE. The 25-µl PCR mixture consisted of 1x buffer D (FailSafe PCR system; Epicentre, Madison, WI), 250 nM primer EREADHF, 250 nM primer ADHR, 1 ng pUTK401 template, and 2.5 U of FailSafe PCR enzyme mix. A stringent touchdown thermal cycler program was used: 95°C for 15 min of initial denaturation, followed by eight cycles of denaturation at 95°C for 2 min, annealing at 72°C for 30 s, and elongation at 72°C for 1 min. The annealing temperature was reduced by 1°C in each of the first eight cycles. Then followed 25 cycles of denaturation at 95°C for 1 min followed by annealing at 65°C for 30 s and elongation at 72°C for 1 min. There was a final elongation step at 72°C for 4 min.

Each PCR product was gel purified (QIAquick gel extraction kit; QIAGEN Inc., Valencia, CA), quantified, and combined in a third PCR to amplify the entire GPD-ERE-ADH region (Fig. 2). This 25-µl PCR mixture consisted of 1x buffer D, 250 nM primer GPDR, 250 nM primer ADHR, 18 ng purified EREGPD from the first PCR, 20 ng purified EREADH from the second PCR, and 2.5 U of FailSafe PCR enzyme mix. The touchdown program used for this reaction was 95°C for 15 min of initial denaturation, followed by eight cycles of denaturation at 95°C for 2 min, annealing at 72°C for 30 s, and elongation at 72°C for 1 min. The annealing temperature was reduced by 1°C in each of the first eight cycles. Then followed 30 cycles of denaturation at 95°C for 1 min followed by annealing at 65°C for 30 s and elongation at 72°C for 1 min. There was a final elongation step at 72°C for 2 min. This PCR product, named GPDEREADH, was gel purified and cloned into pCR2.1-TOPO (Invitrogen Corp., Carlsbad, CA), creating pUTK408. The identity of the insert was confirmed by DNA sequencing.

GPDEREADH was cloned into pUTK401 to create pUTK407. To accomplish this, the GPD/ADH1 promoter region was removed from pUTK401 by double digestion with BamHI and SpeI and subsequent gel purification of the vector. Plasmid pUTK408 was similarly digested to excise GPDEREADH. A ligation of 1:10 vector pUTK401-insert GPDEREADH was performed using standard methods (27). The resulting ligand was electroporated into TransforMax EC100 electrocompetent E. coli cells (Epicentre, Madison, WI) and spread on LB plates with 100 µg ampicillin/ml.

S. cerevisiae hER containing hER- in the chromosome was cotransformed with plasmids pUTK404 and pUTK407 to create strain BLYES (Fig. 1). Transformants were selected on YMM (leu^–, ura^–).

Bioluminescence estrogen assay.
Strain BLYES was grown in YMM (leu^–, ura^–) overnight at 30°C and 200 rpm shaking to an approximate optical density at 600 nm (OD₆₀₀) of 1.0. Cells were centrifuged and resuspended in fresh YMM (leu^–, ura^–) to an OD₆₀₀ of 1.0. Two hundred microliters was transferred to each well of a black 96-well Microfluor microtiter plate (Dynex Technologies, Chantilly, VA). Appropriate dilutions of test chemicals were added to each well. Bioluminescence was measured every 60 min for 12 h in a Perkin-Elmer Victor² multilabel counter with an integration time of 1 s/well.

Colorimetric estrogen assay.
The YES assay was performed as described previously (28). ß-Galactosidase activity was measured at 540 nm using the chromogenic substrate CPRG and normalized to growth rate via concurrent OD₆₀₀ measurements.

EC₅₀ calculations.
For each chemical, bioluminescence or ß-galactosidase activity (OD₅₄₀) versus the log of chemical concentration was plotted. A linear regression was determined using three or four points falling on the linear portion of the curve. Each 50% effective concentration (EC₅₀) (x axis) was calculated using the linear regression formula and the midpoint y value of each dose-response curve. For the 17ß-estradiol standards, the EC₅₀ was calculated individually for seven assays. The mean and standard deviation was calculated from the seven EC₅₀ values to determine the variability between assays.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
S. cerevisiae BLYES.
Using a genetic scheme similar to S. cerevisiae BLYEV (12), S. cerevisiae hER was cotransformed with plasmids pUTK404 and pUTK407. Tandem estrogen response elements were inserted between the two constitutive divergent promoters GPD and ADH1 on pUTK401 (12), regulating luxA and luxB, respectively (Fig. 1). The resulting estrogen reporter was designated S. cerevisiae BLYES.

One advantage of bioluminescence assays compared to colorimetric assays is speed. When the BLYES strain was exposed to 2.8 x 10^–9 M 17ß-estradiol, quantifiable bioluminescence was observed in 60 min (Fig. 3). By 6 h, the assay reached a maximum bioluminescence and a lower limit of detection of 4.9 x 10^–11 M (defined as twofold over background). In contrast, the colorimetric assay required 3 days before a response was measured and, for target compounds with low estrogenicity, 5 days of incubation were required for detection of the estrogenic response (18, 19, 25, 28). Although the incubation time needed to measure estrogenic responses differed between BLYES and YES, the measured responses to 1.2 x 10^–8 M through 5.6 x 10^–12 M 17ß-estradiol were similar for both strains (Fig. 4). This result was not unexpected, as the same host yeast strain containing the human estrogen receptor protein gene was used for both bioreporter constructs.

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FIG. 3. Three-dimensional plot of bioluminescence versus time for 17ß-estradiol. Initial bioluminescence was observed in as little as 60 min for 2.8 x 10–9 M and reached a maximum at approximately 360 min. CPS, counts per second.

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FIG. 4. EC50 dose-response profile of 17ß-estradiol using S. cerevisiae BLYES (open circles) and S. cerevisiae hER (closed circles) reporter strains. The lower and upper limits of detection for each assay with 17ß-estradiol were approximately 4.5 x 10–11 and 2.8 x 10–9 M, respectively.

Bioluminescent dose-response profiles for specific estrogenic chemicals.
Additional comparison of the two bioreporters was performed by challenging BLYES and YES with nine chemical compounds known to induce a range of estrogenic responses (Table 3). In these experiments, a series of six 1:10 dilutions of 17ß-estradiol (ranging from 1.2 x 10^–8 to 1.2 x 10^–13 M) were used to generate the standard curves. Data for the bioluminescence and colorimetric assays were collected at 6 h and 3 days, respectively. The reproducibility of the bioreporter assays was determined by calculating the EC₅₀ of 17ß-estradiol from seven independent experiments. The calculated EC₅₀ values from the colorimetric and bioluminescence assays were (4.4 ± 1.1) x 10^–10 and (2.4 ± 1.0) x 10^–10 M. These values are in agreement with previously published data (2, 9).

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TABLE 3. Comparison of EC50 values obtained by the colorimetric and bioluminescent yeast estrogenic assays using six-point 17ß-estradiol standard curves

Seven chemicals tested were moderately to strongly estrogenic (8, 32). The EC₅₀ values obtained by the two assays were very similar for 17ß-estradiol, 17-estradiol, DES, 4,4'-cyclohexlidene bisphenol, and bisphenol A, but they differed slightly for estrone and 17-ethinylestradiol (Table 3). The EC₅₀ values for these seven estrogenic compounds calculated from the bioluminescence assay were plotted against the EC₅₀ values calculated from the colorimetric assay (Fig. 5). A linear regression analysis resulted in a slope of 0.99 and a coefficient of determination (r²) of 0.99 and confirmed that strain BLYES provides data similar to strain YES for moderately to strongly estrogenic chemicals.

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FIG. 5. Linear regression analysis of EC50 values determined by the bioluminescence and colorimetric assays. EC50 values are listed in Table 3. A coefficient of determination of 0.99 was achieved.

Two other chemicals tested, p-nonylphenol and 4-andostrenedione, resulted in different estrogenic responses between the two assays (Table 3). In the BLYES assay, nonylphenol induced a weak estrogenic response in the range consistent with the literature (9). However, in the colorimetric YES assay, nonylphenol reduced cell growth by 90% in the estrogenic concentration ranges and thus resulted in no estrogenic response. The difference in response between the BLYES and YES assays to p-nonylphenol suggests that the BLYES assay, which is a resting cell assay, is more immune to toxic effects than the YES assay, which is a growing cell assay. 4-Andostrenedione was included as a negative control. The negative response of the BLYES assay to 4-andostrenedione contrasts to the very weak response (more than a million-fold less estrogenic than 17ß-estradiol) seen in the YES assay. Again, this difference may be due to the length of the incubation period for each assay. The 4-andostrenedione may be transformed over the 3-day incubation period of the YES assay, thus giving a false-positive result.

Conclusions.
The environmental deposition of natural, pharmaceutical, and synthetic chemicals with estrogenic activities is associated with numerous human and wildlife physiological disorders, prompting the development of various assays to screen for estrogenic potencies (1). As a model towards demonstrating the applicability and inherent advantages of self-bioluminescent yeast bioreporters, a lux-based assay for environmental estrogens was developed and functionally compared to the established lacZ-based YES assay (26). Although proven effective for the in vitro determination of estrogenic activity, the YES incubation time of 3 to 5 days is impractical when considering the thousands of chemicals requiring screening. In contrast, the BLYES assay demonstrated a response time of <6 h for each chemical tested. In addition, the BLYES assay had the same or better sensitivity to the test chemicals as the YES assay.

Although the rapidity of the BLYES assay has been significantly improved compared to the YES assay, other potential limitations still exist. As demonstrated with the YES assay, the yeast cell wall and transport system can selectively decrease a particular chemical's potency or remain fully impermeable to it (1). Yeast-based assays have been criticized for their inability to differentiate between estrogen agonists and antagonists (4). However, Beresford and coworkers (2) demonstrated that these issues can be overcome or diminished through careful experimental design. However, we agree with their overall conclusion that a single assay system coherently functional for the thousands of chemicals requiring screening is unrealistic. Rather, a suite of assays will likely be needed.

The incorporation of the bacterial lux cassette under the control of estrogen response elements into S. cerevisiae hER will allow high-throughput screening of chemicals as required by the Food Quality Protection Act (Public Law 104-170). Further, S. cerevisiae BLYES, when combined with appropriate photodetection technology, can be used for remote, near-real-time monitoring of our nation's waterways for endocrine-disrupting activity (3, 21).

 
We express appreciation for technical support provided by Victoria Garrett and James Easter. We express appreciation to E. A. Meighen for the P. luminescens luxCDABE operon and J. P. Sumpter for the Glaxo-Wellcome YES strain.

This research was supported in part by the U.S. EPA's STAR program through grant RD-831302, a National Institutes of Health grant from the National Institute for Diabetes and Digestive Disorders (5R21 RR14169-02), a U.S. Department of Energy Cooperative Research and Development Award (ORNL 98-0520), and a NASA Advanced Environmental Monitoring and Control grant (NAG5-8760).

Although the research described in this article has been funded wholly or in part by the United States Environmental Protection Agency through grant/cooperative agreement RD-831302, it has not been subjected to the Agency's required peer and policy review and therefore does not necessarily reflect the views of the Agency, and no official endorsement should be inferred.

 
* Corresponding author. Mailing address: The Center for Environmental Biotechnology, The University of Tennessee, 676 Dabney Hall, Knoxville, TN 37996-1605. Phone: (865) 974-8080. Fax: (865) 974-8086. E-mail: Sayler{at}utk.edu.

Present address: Department of Microbiology, RLA College, University of Delhi, New Delhi 110021, India.

Present address: College of Marine Science, University of South Florida, St. Petersburg, FL 33701.

Top Abstract Introduction Materials and Methods Results and Discussion References

 

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Applied and Environmental Microbiology, August 2005, p. 4455-4460, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4455-4460.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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