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Applied and Environmental Microbiology, November 2005, p. 7075-7082, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.7075-7082.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Survival of Genetically Modified and Self-Cloned Strains of Commercial Baker's Yeast in Simulated Natural Environments: Environmental Risk Assessment

Akira Ando, Chise Suzuki,{dagger} and Jun Shima*

National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan

Received 6 April 2005/ Accepted 16 June 2005


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ABSTRACT
 
Although genetic engineering techniques for baker's yeast might improve the yeast's fermentation characteristics, the lack of scientific data on the survival of such strains in natural environments as well as the effects on human health prevent their commercial use. Disruption of acid trehalase gene (ATH1) improves freeze tolerance, which is a crucial characteristic in frozen-dough baking. In this study, ATH1 disruptants constructed by genetic modification (GM) and self-cloning (SC) techniques were used as models to study such effects because these strains have higher freeze tolerance and are expected to be used commercially. Behavior of the strains in simulated natural environments, namely, in soil and water, was studied by measuring the change in the number of viable cells and in the concentration of DNA that contains ATH1 loci. Measurements were made using a real-time PCR method during 40 days of cultivation. Results showed that the number of viable cells of GM and SC strains decreased in a time-dependent manner and that the decrease rate was nearly equal to or higher than that for wild-type (WT) yeast. For all three strains (SC, GM, and WT) in the two simulated natural environments (water and soil), the DNA remained longer than did viable cells but the decrease patterns of either the DNA or the viable cells of SC and GM strains had tendencies similar to those of the WT strain. In conclusion, disruption of ATH1 by genetic engineering apparently does not promote the survival of viable cells and DNA in natural environments.


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INTRODUCTION
 
Molecular genetic engineering techniques for breeding of commercial baker's yeast are well established. Such techniques could improve the yeast's characteristics, such as fermentation ability and stress tolerance, and could decrease the cost for baker's yeast production and for bakery processes (16, 18, 22, 23, 25). Genetic engineering techniques produce two categories of yeasts: genetically modified (GM) yeast, which contains a heterologous DNA segment derived from organisms taxonomically different from their host cells, and self-cloning (SC) yeast, which does not contain any DNA derived from other organisms and does not produce any additional proteins except for proteins originally produced in the yeast (2, 10, 29). SCprocesses are considered the same as naturally occurring gene conversion, such as recombination, deletion, and transposition, and thus SC yeast is not considered a GM organism. For this reason, SC yeast might be more acceptable for consumers than GM yeast. However, genetically engineered baker's yeasts, not only GM yeasts but also SC yeasts, are currently not used commercially. One reason for the hesitation in commercial use of GM or SC strains of yeast is the lack of scientific data on the survival of such strains in natural environments as well as the effects on human health (5, 12, 14).

Assessment of the viability of yeasts constructed by GM and SC techniques in natural environments is important because such yeast might be inadvertently or intentionally released into natural environments, such as soil and water environments, during propagation processes of yeast products in factories or during baking processes in bakeries. It is important to provide the general public with accurate information about the behavior of genetically engineered yeast under natural conditions so that consumers can comfortably accept such techniques and the resultant products, resulting in a boost of the commercial use of GM or SC yeasts in the food industry. The aim of this study was to clarify the survival of viable cells and DNA of SC and GM yeast at the molecular level in natural environments.

In this study, gene disruptants of acid trehalase gene (ATH1) derived from commercial baker's yeast were constructed by using GM or SC techniques and then used as models of genetically engineered yeast. In ATH1 disruptants, trehalose is highly accumulated and functional as a cryoprotectant under freezing conditions (22). Because disruption of ATH1 improves the freeze tolerance of commercial baker's yeast, the commercial use of ATH1 disruptants is expected in frozen-dough baking (22).

Despite the increased studies on the genetic engineering techniques of microorganisms, only a few studies on the survival of GM and SC yeasts under natural environments have been reported previously (3, 8). For example, Fujimura et al. (8) showed that under simulated environmental conditions, Saccharomyces cerevisiae that overproduces human coagulation factor XIIIa showed the same survival rate as the strain that harbors an empty vector. Specific methods for detecting genetically engineered yeast, however, have not yet been established. In contrast to only a few studies on genetically engineered yeasts, many studies on genetically engineered bacteria have been reported, such as Pseudomonas strains used for bioremediation and lactic acid bacteria used for probiotics (1, 7, 19, 21). Specific methods for the detection of genetically engineered bacteria have been reported previously (11, 26, 27, 30).

The goal of this current study was to clarify the survival of cells and specific DNA fragments of GM and SC yeasts in natural environments. Soil and water were chosen as models of natural environments because deliberate or accidental releases to such natural environments might occur. Diploid strains derived from commercial strains were used to simulate industrial baker's yeast in this study. First, a system to detect GM and SC yeasts in natural environments was constructed using quantitative real-time PCR (RTm-PCR) technology recently used to rapidly quantify genes and microorganisms in complex environments (6, 9, 24, 30). Then, the changes in the number of viable cells and in the concentration of DNA during 40 days in the two simulated natural environments (soil and water) were measured and compared for these three strains, namely, GM type of ATH1 disruptants that harbor an antibiotic resistance marker gene derived from bacteria (28), SC type of ATH1 disruptant constructed using an auxotrophic marker gene that was originally cloned from yeast (20), and wild-type (WT) strain.


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MATERIALS AND METHODS
 
Construction of strains.
Table 1 summarizes the strains and oligonucleotide primers used in this study. Strains T7 and T18 are haploid strains that are derivatives of commercial baker's yeast (22). T118 is a diploid strain obtained by mating haploid strains T7 and T18. The strain T118 has high fermentation ability (22). To discriminate these strains from indigenous yeast strains, diploid strains that harbor cycloheximide resistance were constructed. The spontaneous cycloheximide-resistant mutants (13) were isolated from strains T7 and T18, yielding T7CR and T18CR. T118CR-WT was constructed by mating T7CR and T18CR. T118CR-WT was resistant to 10 µg/ml of cycloheximide. Because T118CR-WT had an intact ATH1 locus, T118CR-WT was defined as the WT strain in this study. The diploid strain of the SC type of ATH1 disruptant was constructed as follows. To allow for utilization of the URA3 gene as a selective marker, spontaneous ura3 mutants from T7CR and T18CR were obtained by 5-fluoroorotic acid selection (4), yielding T7CRu and T18CRu. Gene disruption of ATH1 with URA3 was carried out as described previously (22), yielding T7CR-SC and T18CR-SC. T118CR-SC was constructed by mating T7CR-SC and T18CR-SC, and T118CR-SC was defined as the SC strain in this study. The diploid strain of GM type of the ATH1 disruptant was constructed as follows. The ath1::kanMX4 fragment was obtained by PCR using primers P6 and P7 (Table 1 and Fig. 1A) and genomic DNA of BY4741{Delta}ath1 as a template. Gene disruption of the ATH1 locus in strains T7CR and T18CR was achieved using the PCR fragment of ath1::kanMX4, yielding T7CR-GM and T18CR-GM. T118CR-GM was constructed by mating strains T7CR-GM and T18CR-GM, and T118CR-GM was defined as the GM strain in this study.


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  		TABLE 1. Strains and oligonucleotide primers used in this study



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  		FIG. 1. (A) Schematic model of ATH1 loci and PCR primers. Coding region of the ATH1 gene is indicated by open boxes. Regions of URA3 and kanMX4 genes are indicated by shaded and filled boxes, respectively. Positions and directions of PCR primers are indicated by arrows. (B) ATH1 loci of WT, SC, and GM strains were confirmed by PCR amplification. Lane M shows the molecular size marker. Lanes 1 to 6 show PCR product amplified using primers P1 and P3 from genome of T118CR-WT (lane 1), P1 and P3 from that of T118CR-SC (lane 2), P1 and P3 from that of T118CR-GM (lane 3), P1 and P5 from that of T118CR-WT (lane 4), P1 and P5 from that of T118CR-SC (lane 5), and P1 and P5 from that of T118CR-GM (lane 6). (C) Schematic model of cyh2 locus and PCR primers. Coding region of the CYH2 gene is indicated by open boxes. Position and direction of PCR primers are indicated by arrows. Mutation position of cyh2 (CAA to AAA) is indicated by a large open triangle.

Cocultivation systems in soil and water environments.
Model soil and water environments were inoculated with WT, SC, and GM yeast cells grown at 30°C for 48 h in YPD medium that contained 10 g of yeast extract (Difco, Detroit, Mich.), 20 g of peptone (Difco), and 20 g of glucose (per liter). Nonsterile river sand (Matsuzaki, Japan) for horticulture was used as the model soil, where the water content was 7.2% (wt/wt) and pH was 6.5. Sterile distilled water was used as the model water.

Two cocultivation systems were used: a series I cocultivation system, which harbored WT and GM strains, and series II, which harbored SC and GM strains. For the soil environment, two strains (either WT and GM or SC and GM) were coinoculated into 70 g of soil in a 125-ml plastic bottle at a cell density of 106 cells (each strain) per 1 g of dry soil and then immediately mixed. For the water environment, the strains were coinoculated into 500 ml of sterile distilled water in a 1-liter flask at a cell density of 106 cells (each strain) per 1 ml of water and then immediately mixed. These soil and water cocultivation systems were then incubated at 25°C for 40 days and 42 days, respectively, under dark conditions without shaking. During incubation, samples were taken for measurement of the number of viable cells and the DNA concentration. At the time of sampling, the cocultivation systems were mixed to ensure homogenous samples.

Measurement of the number of viable cells remaining in soil and water environments.
The number of viable cells of inoculated yeast cells remaining in the soil and water environments (series I and II cocultivation systems) were measured using the cultivation method as follows. The soil environment samples (0.5 g) were suspended in distilled water, and total volume was adjusted to 5 ml. Serial 10-fold dilutions of the soil suspension and the water environment samples were prepared using distilled water. The dilutions (200 µl) were then individually plated onto CPS agar medium, which is YPD medium containing 5 mg of cycloheximide (Sigma-Aldrich, St. Louis, Mo.), 0.75 g of sodium propionate, 0.3 g of streptomycin (Sigma-Aldrich), 5 g of lactic acid, and 20 g of agar (perliter). The colonies that appeared after incubation for 3 days at 30°C on CPS medium were considered total viable cells (VCt) of the inoculated yeast cells. These total survivors appearing in CPS agar medium (20 to 200 colonies) were then replica plated onto YPD-G418 agar medium (i.e., YPD agar medium supplemented with an aminoglycoside antibiotic, G418 [Sigma], at a concentration of 0.3 g/liter) and incubated at 30°C for 1 day. The colonies that appeared on YPD-G418 agar were considered cells that harbored ath1::kanMX4 locus (VCg). The number of viable cells that harbored either wild-type ATH1 (VCw) or self-cloned ath1::URA3 locus (VCs) were calculated by subtraction of VCg from VCt. The number of viable cells was expressed as the mean from triplicate experiments.

To measure the number of indigenous bacteria, portions of each suspension of the soil and water environment samples were plated onto tryptic soy broth agar (Difco) and then incubated at 37°C for 3 days. The colonies that appeared on the agar were counted as the number of indigenous bacteria.

DNA extraction from soil and water environments and from yeast cells.
DNA contained in 0.5-g soil environment samples was extracted using a FastDNA spin kit for soil (Q-Biogene, Carlsbad, Calif.) according to the manufacturer's instructions. The extracted DNA was then dissolved in 50 µl of distilled water and used for RTm-PCR analysis.

DNA contained in 1-ml water environment samples was extracted according to a protocol described by Philippsen et al. (17). The extracted DNA was then dissolved in 60 µl of distilled water and used for RTm-PCR analysis.

Yeast DNA used as standard DNA in RTm-PCR analysis was extracted from yeast cells, which were grown in 2 ml of YPD medium at 30°C for 48 h, using a FastDNA spin kit for soil according to the manufacturer's instructions. The extracted DNA was dissolved in 50 µl of distilled water. The DNA concentration was measured by using a spectrophotometer (Ultraspec UV2100 pro; Amersham Biosciences, Piscataway, N.J.).

Measurement of yeast DNA concentration in soil and water environments.
The concentration of DNA from WT, SC, and GM strains in the soil or water environments was quantified using the RTm-PCR method (15, 24). RTm-PCR was done using a hot-start PCR kit (LightCycler FastStart DNA Master SYBR Green I; Roche, Mannheim, Germany) and LightCycler instrument (Roche), and the analysis was done using the LightCycler software version 3.5 (Roche). For RTm-PCR, 20 µl of the reaction mixture was used, consisting of 0.05% (wt/vol) of bovine serum albumin, 1 µM of each respective primer (Table 1) (also discussed in the next paragraph), 3 mM of MgCl2, 0.29 µl of LightCycler FastStart enzyme (included in the kit), 1.71 µl of LightCycler FastStart reaction mix (included in the kit), and 2 µl of template DNA solution.

Primers for the detection of ATH1 and cyh2 loci were as follows. ATH1 locus in the WT strain and ath1::URA3 locus in the SC strain were detected as the 105bp of PCR product using primers P1 and P2 (Table 1 and Fig. 1A). ATH1 locus replaced by kanMX4 in the GM strain was detected as the 101 bp of PCR product using primers P4 and P5 (Table 1 and Fig. 1A). cyh2 locus of the WT, SC, and GM strains was detected as the 119 bp of PCR product using primers P8 and P9 (Table 1 and Fig. 1C). Primer P8 was designed based on the mutation position in the CYH2 gene to enable the detection of the cyh2 locus.

Thermal cycling conditions for detection of ATH1 and cyh2 loci were as follows. The thermal cycling conditions for ATH1 and ath1::URA3 locus in the WT and SC strains consisted of initially heating samples to 95°C and storage at 95°C for 10 min, followed by 40 cycles of heating at 20°C/s to 95°C and storage at 95°C for 15 s, cooling at 20°C/s to 63°C and storage at 63°C for 4 s, and heating at 10°C/s to 72°C and storage at 72°C for 5 s. The thermal cycling conditions for detection of the kanMX4 locus in the GM strain consisted of initially heating samples to 95°C and storage at95°C for 10 min, followed by 40 cycles of heating at 20°C/s to 95°C and storage at 95°C for 15 s, cooling at 20°C/s to 61°C and storage at 61°C for 5 s, and heating at10°C/s to 72°C and storage at 72°C for 6 s. The thermal cycling conditions for detection of the cyh2 locus of the WT, SC, and GM strains consisted of initially heating samples to 95°C and storage there for 10min, followed by 40 cycles of heating at 20°C/s to 95°C and storage at 95°C for 15 s, cooling at 20°C/s to 65°C and storage at 65°C for 4 s, and heating at 20°C/s to 72°C and storage at 72°C for 5 s. Fluorescence of double-stranded DNA-SYBR Green I complex was measured at the end of each 72°C cycle (extension process).

Melting curve analysis was done to confirm that the correct amplification of ATH1 or cyh2 loci occurred. The condition for this analysis consisted of heating at 20°C/s to 95°C, cooling at 20°C/s to 65°C and storage at 65°C for 15 s, and heating at 0.1°C/s to 95°C with continuous monitoring of the fluorescence. Amelting curve was obtained by plotting the negative first derivative of fluorescence against temperature (i.e., –dF/dT). The melting temperature (Tm) of the double-stranded DNA products was represented by a peak in the melting curve.

Concentrations of the DNA contained in the soil and water environment samples were determined using the standard curves. The standard curves for measurement of ATH1 and cyh2 loci were generated by plotting the log of the number of copies in a 10-fold dilution series of the standard DNA extracted from the WT, SC, and GM strains against the CT value, in which CT was defined as the fractional cycle number (calculated using the LightCycler software) where the fluorescence increased above the detection threshold. The standard curve was then represented by a linear regression line of these CT values. The quality of the standard curve was confirmed by the correlation coefficient (r2) of CT and DNA quantity. The DNA concentration was normalized based on the copy number per cell (two copies of ATH1 loci and two copies of cyh2 locus were contained in a diploid cell). DNA concentration was expressed as an equivalent of cell number and was the mean from triplicate experiments.


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RESULTS
 
Construction of discriminative measurement systems of viable GM, SC, and WT strains.
Homozygous SC and GM strains of ATH1 disruptants were constructed in the genetic background of T118. The expected ATH1 loci of these strains are illustrated in Fig. 1A. Disruption of the ATH1 loci of SCand GM was confirmed by PCR using primers P1 and P3 designed for the amplification of both intact ATH1 and ath1::URA3 and using primers P1 and P5 designed for the amplification of ath1::kanMX4, respectively (Fig. 1A). Amplifications of expected lengths of DNA from the WT, SC, and GM strains were observed (Fig. 1B), thus confirming that ATH1 loci of the strains were correctly constructed.

Spontaneous mutations for cycloheximide resistance were introduced into the WT, SC, and GM strains to distinguish these strains from indigenous yeast cells under simulated soil environments. Sequence analysis of CYH2 locus of the WT strain revealed that mutation occurred at position 623 (C to A) in the CYH2 gene, suggesting the replacement of Gln37 by Lys (Fig. 1C). These strains grew on CPS agar plates, which contained 5 µg/ml of cycloheximide. Indigenous yeast included in the soil used in this study could not grow on CPS agar medium (data not shown). These results suggest that the three strains constructed here (WT, SC, and GM) are suitable for discriminative measurements from indigenous yeast using CPS agar medium.

The G418 resistance of strains constructed here was examined to determine suitable conditions for discriminative measurement between the GM strain that harbors kanMX4 and the WT and SC strains. The GM strain was resistant to 300 µg/ml of G418, whereas the WT and SC strains did not grow on agar medium containing G418 (data not shown). These data suggest that discriminative measurement between the GM strain and the SC and WT strains is possible by using agar medium containing 300 µg/ml of G418.

Molecular detection of ATH1 and cyh2 loci in soil and water environments.
To construct a quantification system using RTm-PCR for the DNA fragments of the ATH1 locus that survived in the soil and water samples, we designed specific PCR primers (P1 and P2) for amplification of the ATH1 locus in both the WT and SC strains and specific primers (P4 and P5) for that in the GM strain (Table 1). The positions of the primers are indicated in Fig. 1A. The cyh2 locus that was not modified by genetic engineering techniques was used for the neutral control to confirm the analysis results of the ATH1 locus. Specific primers (P8 and P9) for amplification of the cyh2 locus were designed based on the nucleotide sequence of cyh2 in these strains (Table 1 and Fig. 1C).

Figure 2 shows the RTm-PCR profiles of DNA fragments amplified from the WT (ATH1) strain and those from the GM (ath1::kanMX4) strain. To confirm the correct amplification of each target locus, Tm of the PCR products was measured using melting curve analysis (Fig. 2A, C, and E). Figure 2A shows the melting curve analysis results for the ATH1 locus amplified from the WT strain using primers P1 and P2. The Tm of the PCR fragment amplified from DNA directly extracted from the soil samples was identical with that from standard DNA of the WT strain (Fig. 2A). In our assay system, the ATH1 fragment amplified from the WT (ATH1) strain was expected to be the same as that from the SC (ath1::URA3) strain. Consistent with this expectation, both the Tm of the ATH1 fragment amplified from DNA directly extracted from the soil samples and that from standard DNA of the SC strain were identical to that from the WT strain (data not shown). The Tm of the DNA fragments of ath1::kanMX4 locus amplified from the soil samples was confirmed to be identical to the Tm from standard DNA of the GM strain using primers P4 and P5 (Fig. 2C). The Tm of the DNA fragments of cyh2 locus amplified from the soil samples was confirmed to be identical to the Tm from standard DNA of the WT strain using primers P8 and P9 (Fig. 2E). The Tm of the amplified fragments from standard DNA of the SCand GM strains was identical to that of the WT strain (data not shown). Melting curve analysis using the genomic DNA of strain T118 (CYH2) using primers P8 and P9 as a template revealed that specific amplification did not occur (data not shown), suggesting that the cyh2 locus is correctly detected by using primers P8 and P9. The molecular sizes of the PCR products from the ATH1, ath1::kanMX4, and cyh2 loci measured by agarose gel electrophoresis were consistent with predicted DNA sizes (105, 101, and 119 bp, respectively) (data not shown). These results of Tm and molecular size analyses confirm the accuracy and high specificity of the PCR amplification of ATH1 loci with cyh2 locus as the neutral control.



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  		FIG. 2. Melting curves and standard curves of RTm-PCR products. (A, C, and E) Melting curves generated by plotting the negative first derivative of fluorescence against temperature (–dF/dT), which is a parameter for the rate of change in fluorescence based on DNA melting. (B, D, and F) Standard curves and correlation coefficients (r2) by plotting CT versus DNA quantity. CT values are expressed as means ± standard deviations from triplicate experiments. Panel A is the melting curve of the ATH1 locus amplified from control DNA (solid line) and from DNA extracted from soil (dotted line) using primers P1 and P2. Panel B is the standard curve for the ATH1 locus. Panel C is the melting curve of the ath1::kanMX4 locus amplified from control DNA (solid line) and from DNA extracted from soil (dotted line) using primers P4 and P5. Panel D is the standard curve for the ath1::kanMX4 locus. Panel E is the melting curve of the cyh2 locus amplified from control DNA (solid line) and from DNA extracted from soil (dotted line) using primers P8 and P9. Panel F is the standard curve for the cyh2 locus.

To quantify the DNA fragments of ATH1, ath1::kanMX4, and cyh2 in the water and soil environment samples, standard curves were constructed by using standard DNA (Fig. 2B, D, and F, respectively). The standard curves for each DNA fragment had a high r2 (>0.99) and were not affected by other components, except for the concentration of DNA included in the samples directly extracted from the soil (data not shown). These curves shown in Fig. 2 confirm the reliability of the measurements of DNA containing the ATH1, ath1::kanMX4, and cyh2 loci contained in a soil environment. The same experiments were done using DNA extracted from water samples. The RTm-PCR profiles for these water samples were identical to those for the standard DNA (data not shown), confirming the reliability of quantification of DNA in a water environment.

Survival rates of viable yeast cells and DNA concentration in the soil environment.
To compare the survival rates of viable yeast cells and DNA concentrations of the three strains under strictly identical conditions, we constructed two series of cocultivation assay systems, namely, cocultivation of GM and WT strains (series I) and that of GM and SC strains (series II). In brief, two different strains (either GM and WT or GM and SC) were inoculated into soil and water samples and then cultured for 40 days and 42 days, respectively. During the culturing, the number of viable cells and the DNA concentration were measured using the discriminative methods described above (see Materials and Methods).

Figure 3 shows the measured number of viable cells and DNA concentration of the GM, SC, and WT strains in the soil environment. Figure 3A shows the results from the series Icocultivation system containing GM and WT strains. The number of viable cells of both strains logarithmically decreased in a similar time-dependent manner. The DNA fragment of the ATH1 and ath1::kanMX4 loci specified WT and GM strains, respectively, in the soil. The DNA concentration decreased more slowly than did the number of viable cells (Fig. 3C). The DNA concentration of cyh2, which was the neutral control for DNA quantification, decreased similarly to the concentrations of ATH1 and ath1::kanMX4 and was nearly equal to the sum of the concentrations of these two ATH1 loci (Fig. 3C). Figure 3B and D show data from the series II cocultivation system containing GM and SC strains. Although the decrease rate of DNA concentration was lower than that of the number of viable cells, the DNA of both strains contained in the soil decreased logarithmically with the decrease in the number of viable cells (Fig. 3D). The data shown in Fig. 3 indicate that viable cells of both the GM and SC strains decreased in the same manner as those of the WT strain.



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  		FIG. 3. Number of viable cells (A and B) and concentration of DNA containing the AHT1 loci and cyh2 locus (C and D) during 40days (d) of cultivation in the soil environment. Panel A shows the number of viable cells in series I cocultivation (WT+GM) and panel B in series II cocultivation (SC+GM). Panel C shows DNA concentration of the ATH1, ath1::kanMX4, and cyh2 loci in series I cocultivation (WT+GM) and panel D, that of the ath1::URA3, ath1::kanMX4, and cyh2 loci in series II cocultivation (SC+GM). Number of viable cells and DNA concentration are expressed as means ± standard deviations from triplicate experiments.

Based on these results, there was no significant difference in the survival of viable cells and DNA in the soil environment among the GM, SC, and WT strains.

Survival rates of viable yeast cells and DNA concentrations in the water environment.
Figure 4 shows the survival rates of viable yeast cells and the concentrations of DNA from the GMand WT strains (series I) in the water environment. Although the decrease rates of both strains in water were lower than those in soil, the number of viable cells and the DNA concentration of the GM strain decreased in a time-dependent manner but the decrease rate of the GM strain was significantly higher than that of the WT strain.



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  		FIG. 4. Number of viable cells (A) and concentration of DNA containing AHT1 loci and cyh2 locus (B) of series I cocultivation (WT+GM) during 42 days (d) of cultivation in the water environment. Number of viable cells and DNA concentration are expressed as means ± standard deviations from triplicate experiments.


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DISCUSSION
 
In summary, the number of viable cells and DNA concentration of the GM and SC yeast strains in simulated natural environments, namely, in soil and water, were compared with that of the WT strain. The survival rate of yeast cells was not related to the presence or absence of genetically engineered ATH1 loci in the soil environment because all three strains tested (GM, SC, and WT) showed similar kinetics of survivability. Decrease in the ATH1 loci and the neutral gene cyh2 were also investigated under the same conditions. The decrease in DNA that contained either ATH1 or cyh2 loci was correlated with the decrease in viable cells. For all three strains in both simulated environments, DNA remained longer than did viable cells, although the decrease rates of the SC and GM strains were the same as or higher than that of the WT strain.

The effectiveness of techniques using an RTm-PCR method to quantitatively measure a specific yeast DNA contained in soil was tested and demonstrated. Under the experimental soil conditions used here, the sensitivity of the RTm-PCR method was the same as that of the viable-cell-count method. After inoculation of yeast cells at a concentration of 106 cells/g of dry soil (Fig. 3C and D), the measured DNA concentration was approximately 105 cell equivalents/g of dry soil whereas the number of viable cells was approximately 105 cells/g of soil. The recovery rates of DNA and viable cells were approximately 10%. Although the behavior of the nonrecoverable cells and DNA remains unclear at present, the behavior of the recoverable cells and DNA is assumed to represent that of the nonrecoverable cells and DNA because we obtained similar data from three independent experiments with reproducibility. Under the experimental conditions for the water environment used in our study, the sensitivity of the RTm-PCR method was almost the same as that of the viable-cell-count method (Fig. 4). The RTm-PCR method therefore should be a useful tool for the detection of yeast cells in natural environments as well because the method can measure the number of specific yeast cells more rapidly and more easily than other detection methods, including the viable-cell-count method. We attempted to apply the RTm-PCR method to yeast detection in more complicated environments such as kitchen garbage, but we could not obtain specific amplification of the yeast DNA (data not shown). Although further research into the detection of DNA in such complicated environments is necessary, the RTm-PCR method should be a useful tool for detection in natural environments.

Bröker (3) and Fujimura et al. (8) reported that no differences could be detected in the survival rate of either recombinant or wild-type yeast cells under either sterile conditions (in water) or nonsterile conditions (in soil). Although in this study the presence of the genetically engineered loci of ATH1 might not directly affect the survival of GM and SC types of commercial baker's yeast in nonsterile soil conditions, our results suggested that the GM type of baker's yeast was less stable than the WT in the sterile water condition.

The RTm-PCR assay showed that the DNA fragment derived from yeast strains decreased at a slower rate than did the viable cells under soil and water environments. The rates of decrease in the concentration of DNA of the GM and SC strains were not significantly different from that of the WT strain. These results suggest that the disruption of ATH1 by genetic engineering does not promote the survival of viable cells and DNA in natural environments.

Although the survivability of baker's yeast constructed by GM and SC techniques was clarified here, the effects of any release of GM and SC yeasts on indigenous microflora remain unknown. Further research is planned to study these effects by determining the effect of yeast inoculation on the microflora of indigenous bacteria, fungi, and yeast.


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ACKNOWLEDGMENTS
 
This study was supported in part by a grant-in-aid (Japan Comprehensive Research Project on the Assurance of Safe Use of Genetically Modified Organisms) from the Ministry of Agriculture, Forestry, and Fisheries, Japan; in part by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN); and in part by the Cooperative System for Supporting Priority Research from the Japan Science and Technology Agency.

We thank Ryoichi Nakajima (Oriental Yeast Co. Ltd.) for supplying the baker's yeast strains and for critical comments on this study.


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FOOTNOTES
 
* Corresponding author. Mailing address: National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan. Phone: 81 (29) 838 8066. Fax: 81 (29) 838 7996. E-mail: shimaj{at}nfri.affrc.go.jp. Back

{dagger} Present address: National Institute of Livestock and Grassland Science, 2 Ikenodai, Tsukuba, Ibaraki 305-0901, Japan. Back


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Applied and Environmental Microbiology, November 2005, p. 7075-7082, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.7075-7082.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.




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