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Applied and Environmental Microbiology, October 2008, p. 6132-6137, Vol. 74, No. 19
0099-2240/08/$08.00+0     doi:10.1128/AEM.00469-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Quantitative Real-Time Reverse Transcription-PCR Analysis Reveals Stable and Prolonged Neurotoxin Cluster Gene Activity in a Clostridium botulinum Type E Strain at Refrigeration Temperature

Ying Chen, Hannu Korkeala, Jere Lindén, and Miia Lindström^*

Centre of Excellence in Microbial Food Safety Research, Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, University of Helsinki, Finland

Received 27 February 2008/ Accepted 2 August 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The relative expression levels of six botulinum neurotoxin cluster genes in a group II Clostridium botulinum type E strain grown at 10 or 30°C were investigated using quantitative real-time reverse transcription-PCR. An enzyme-linked immunosorbent assay was used to confirm neurotoxin expression. Distinct mRNA and toxin production patterns were observed at the two temperatures. The average relative mRNA levels at 10°C were higher than (ntnh and p47), similar to (botE), or lower than (orfx1, orfx2, orfx3) those at 30°C. The maximum botE expression levels and average neurotoxin levels at 10°C were 45 to 65% of those at 30°C. The relative mRNA levels at 10°C declined generally slowly within 8 days, as opposed to the rapid decline observed at 30°C within 24 h. Distinct expression patterns of the six genes at the two temperatures suggest that the type E neurotoxin cluster genes are transcribed as two tricistronic operons at 30°C, whereas at 10°C monocistronic (botE or orfx1 alone) and bicistronic (ntnh-p47 and orfx2-orfx3) transcription may dominate. Thus, type E botulinum neurotoxin production may be involved with various temperature-dependent regulatory events. In light of group II C. botulinum type E being a dangerous food-borne pathogen, these findings may be important in terms of the safety of refrigerated packaged foods of extended durability.

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Botulinum neurotoxins ingested with food and drink can cause a potentially lethal paralysis to humans and animals. The neurotoxins are mainly produced by Clostridium botulinum and appear in seven distinct serotypes (A through G) (7, 20, 21). Upon secretion, the neurotoxins are associated with nontoxic proteins forming progenitor toxin complexes of differing structures and molecular sizes, depending on serotype and strain (7, 8, 21).

The genes encoding the nontoxic-neurotoxin-associated proteins are located immediately upstream of the neurotoxin gene and vary in structure, composition, and arrangement between different serotypes and different strains (7). All types of the neurotoxin gene clusters share the same organization of bot (also called bont, cnt, and ntx) and ntnh at the 3' end but differ at their 5' ends (20). Type A1, B, C, D, and G neurotoxin gene clusters are associated with three hemagglutinin-encoding genes (ha70, ha17, and ha33), whereas types A2 to A4 and E and F toxin clusters are associated with p47 and orfx1, orfx2, and orfx3, encoding proteins with unknown function (2, 4, 6, 11, 20). In addition, all other types of toxin gene clusters, with the exception of type E, carry botR that encodes a sigma factor responsible for positive regulation of the neurotoxin gene (16).

The role of the nontoxic-neurotoxin-associated proteins has not been fully identified. It has been proposed that they play a role in protecting the neurotoxin against the acidity of the gastrointestinal tract (19), and the hemagglutinin components seem to assist in the absorption of ingested neurotoxin from the gastrointestinal tract (17). Nothing is known about the activities of orfx1, orfx2, and orfx3 and p47 or the role of their corresponding protein products.

Group II C. botulinum type E is frequently present in marine foods, and being a psychrotrophic organism, it can grow and produce neurotoxin at refrigeration temperatures (5). Thus, this bacterium possesses a significant safety risk for the modern food industry, where the chill chain is the most important bacterial-growth-inhibiting factor (13). Understanding the activity of bacterial virulence genes at low temperatures is a key to establishing novel strategies to ensure the safety of refrigerated foods. To date, all published studies on type E neurotoxin gene expression have been conducted at 30°C or higher (3, 18, 22, 23). However, to the best of our knowledge, no report is available on the type E neurotoxin gene expression at low temperatures or on the relative expression of the six type E neurotoxin cluster genes.

In this paper, relative mRNA levels of all six type E neurotoxin cluster genes were monitored at 10°C and 30°C using quantitative real-time reverse transcription-PCR (qRT-PCR). An indirect enzyme-linked immunosorbent assay (ELISA) was performed to detect neurotoxin production levels. Different expression patterns as a function of time were observed at the two temperatures. The relative mRNA levels declined slowly at 10°C, as opposed to a sharp peak and rapid decline at 30°C. Evidence of different transcriptional mechanisms at 10°C and 30°C was obtained. To the best of our knowledge, this is the first report on botulinum neurotoxin gene expression at low temperatures and the first quantitative analysis of relative expression of all six botulinum neurotoxin cluster genes. The results may have an important impact on the safety of chilled foods, in which group II C. botulinum type E poses a considerable safety hazard.

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C. botulinum strain, culture, and sampling.
C. botulinum type E strain CB11/1-1 was isolated from a case of food-borne botulism in Finland (14) and stored as a spore suspension in sterile distilled water at 4°C. Spores were inoculated into 10 ml of anaerobic tryptone-peptone-glucose-yeast extract (Difco, Detroit, MI) broth and incubated anaerobically overnight at 30°C. Ten-milliliter volumes of fresh anaerobic tryptone-peptone-glucose-yeast broth adjusted to 10°C or 30°C were inoculated with 100 µl of the overnight cultures and incubated under anaerobic conditions at either 10°C or 30°C. Replicate tubes for qRT-PCR assays and ELISAs were withdrawn from both temperatures as explained below. One milliliter of culture from each withdrawn tube was used for optical density at 600 nm (OD₆₀₀) measurements to conduct growth curves. In addition, three replicate tubes incubated at 30°C for 2 h were withdrawn for OD₆₀₀ measurement alone to complement the growth curve.

DNA and RNA extraction and purification.
External standard curves were used in the qRT-PCR analysis to enable comparison of the relative expression levels between the six different genes. Genomic DNA was extracted and purified from the CB11/1-1 overnight culture as described by Keto-Timonen et al. (9).

For RNA extraction, three replicate tubes incubated at either 10°C or 30°C were withdrawn at time intervals of 4 (mid-exponential), 5 (late exponential), 6 (early stationary), and 8 (mid-stationary) days or 4 (mid-exponential), 6 (late exponential), 8 (early stationary), 12 (mid-stationary), 16, and 24 h, respectively. Total RNA was extracted using the RNeasy midi kit (Qiagen GmbH, Hilden, Germany) according to the kit instructions. In brief, 5 to 9 ml of bacterial culture (maximum, 5 x 10⁹ bacteria) was mixed with ice-cold phenol-ethanol mixture (1:9) and kept in ice for 30 min. A bacterial pellet was obtained by centrifugation at 5,000 x g and 4°C for 5 min and then stored at –70°C until RNA extraction. For cell lysis, the pellet was incubated in 1 ml of Tris-EDTA buffer (pH 8.0; Fluka Biochemica, Steinheim, Germany) containing lysozyme (25 mg/ml; Sigma-Aldrich, St. Louis, MO) and mutanolysin (250 IU/ml; Sigma-Aldrich) under agitation at 37°C for 30 min. A conventional on-column DNase digestion during the RNA purification protocol was performed using an RNase-free DNase set (Qiagen). To ensure elimination of all DNA contamination for the qRT-PCR assays, a second DNase treatment with the DNA-free kit (Ambion, Austin, TX) was conducted after RNA isolation according to the manufacturer's instructions.

DNA and RNA concentrations were determined by measuring the absorbance at 260 nm (A₂₆₀) with the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE). The A₂₆₀/A₂₈₀ ratio of 1.8 to 2.2 was used as a purity criterion for all samples. All DNA and RNA were stored at –70°C until use.

RT.
First-strand cDNA was synthesized using the DyNAmo cDNA synthesis kit (Finnzymes, Espoo, Finland) according to the manufacturer's instructions in a 20-µl final volume containing 300 ng random hexamers and 0.5 to 1 µg RNA. The cDNA synthesis reaction mixtures were incubated at 37°C for 60 min, and the reactions were terminated by heating at 85°C for 5 min. Negative RT controls were produced by identical reaction conditions without reverse transcriptase. The cDNA solutions diluted 100- and 1,000-fold were stored at –20°C before PCR amplification.

Quantitative real-time PCR.
The real-time PCR amplification was conducted using the DyNAmo Flash Sybr green quantitative PCR (qPCR) kit (Finnzymes) according to the manufacturer's instructions. Specific primer pairs were designed based on the neurotoxin cluster genes of strain CB11/1-1 (GenBank accession number AM941719) (Fig. 1A) and 16S rrn (GenBank accession number L37592) and are listed in Table 1.

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FIG. 1. (A) Schematic representation of neurotoxin gene cluster in Clostridium botulinum type E. The gene orientation is indicated by the arrows. The black bars within the arrows indicate the regions amplified in the qPCR. (B) Putative transcriptional models of type E neurotoxin cluster genes at 30°C (black arrows) and at 10°C (gray arrows).

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TABLE 1. Primers used for qRT-PCR

One microliter of each dilution (1:100 or 1:1,000) of the cDNA of each sample was used as a template for real-time PCR with a final volume of 20 µl and a concentration of 0.5 µM of each primer. One run for each primer pair encompassing all samples with two replicates of each dilution was conducted in a Rotor-Gene 3000 real-time thermal cycler (Corbett Life Science, Sydney, Australia). Each run comprised initial denaturation and polymerase activation at 95°C for 7 min, followed by 40 cycles of denaturation at 95°C for 20 s and combined annealing and extension at 60°C for 30 s, with fluorescence measurement at the end of the extension, and finally a cooling step at 60°C for 1 min. At the end of each run a melting curve was produced by ramping the temperature from 60 to 98°C while monitoring the fluorescence. All samples exhibited only one sharp melting peak with no signs of unspecific products and conversely there was no specific product in the negative RT controls. The specificity of the qPCR was further confirmed by conventional PCR and gel electrophoresis of selected samples.

Standard curves for each target gene and 16S rrn (reference) were constructed using purified genomic DNA from CB11/1-1 and specific primers (Table 1). The DNA was serially diluted 10-fold to achieve a standard curve spanning 5 log units.

For each cDNA sample, the cycle threshold (C_T) values of the target gene and 16S rrn were converted to absolute amounts of cDNA by using external standard curves and Rotor-Gene real-time analysis software 6.0.31. Then the absolute cDNA amounts of each target gene were normalized against those of 16S rrn to yield the relative expression of each gene, according to the following equation: relative expression = (absolute amount of transcript of target gene)/(absolute amount of transcript of 16S rrn).

The 16S rrn transcript was used to correct differences in the amount of reverse-transcribed RNA and RT reaction efficiency. The C_T values of all monitored transcripts in the 100- and 1,000-fold-diluted cDNA were between the endpoints of the external standard curves. Furthermore, there was a linear relationship between the two dilutions and the corresponding absolute amounts of transcripts, attesting comparable reaction efficiencies in the qPCR between genomic DNA standards and cDNA samples. The 16S rrn was stably expressed at both temperatures throughout the experiment.

Type E neurotoxin ELISA.
To confirm botE expression, type E neurotoxin production was monitored by using a commercial ELISA kit (Tetracore Inc., Rockville, MD) at time points corresponding to mid-exponential, late exponential, early stationary, and mid-stationary growth phases at 10°C and 30°C. The indirect capture ELISA was performed according to the manufacturer's instructions. In brief, two replicate tubes were withdrawn at each time point and temperature, and 1 ml of culture from each tube was centrifuged at 15,000 x g at room temperature for 5 min and diluted 30- to 100-fold with ELISA dilution buffer to fall into the linear OD₄₀₅ measurement range. Negative capture antibody and ELISA dilution buffer were used as negative control. One hundred microliters of each diluted sample was used in ELISA. The OD₄₀₅ of each sample was determined by a microplate photometer (Multiskan Ascent; Thermo Fisher Scientific Inc., Waltham, MA). The amounts of type E neurotoxin production were corrected according to dilution rate and expressed as OD₄₀₅ units.

Statistical analysis.
One-way analysis of variance with Tukey's honestly significant difference test was used to test the hypotheses that two or more of the six genes show similar expression patterns as a function of time and that two or more groups of similarly behaving genes have different expression levels. Student's t test was used to test differences between the average relative expression levels of individual genes at late exponential to mid-stationary growth phases. The SPSS 15.0.1 software (SPSS Inc., Chicago, IL) was used to conduct the statistical analysis.

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Growth curve of CB11/1-1 at 10°C and 30°C.
At 10°C, the maximum OD₆₀₀ of 0.5 was reached within 5 days, followed by a stable stationary phase without any decline in OD within the 8-day experiment period (Fig. 2). At 30°C, the exponential growth phase was observed to occur between 2 and 6 h of the start of incubation, and the maximum OD₆₀₀ value of 1.2 was reached after 8 h (Fig. 2). This OD remained until the end of the 24-hour experiment.

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FIG. 2. Relative-expression curves of type E neurotoxin cluster genes normalized against 16S rrn during the growth of group II C. botulinum type E strain CB11/1-1 at 30°C (solid lines) and at 10°C (dashed lines). The growth curves were determined by measuring OD600. , botE; , ntnh; , p47; x, orfx1; +, orfx2; , orfx3. The expression values are means of three replicate cultures.

Relative expression patterns of type E botulinum neurotoxin cluster genes at 10°C and 30°C.
Different expression curves as a function of time were observed at 10°C and 30°C among the six neurotoxin cluster genes (Fig. 2). At 10°C, maximum relative mRNA levels of all six genes were mainly observed at the late exponential phase, whereas at 30°C, maximum levels were observed after entry into the stationary phase. In relation to the growth curves, the expression of all the toxin cluster genes initiated earlier at 10°C than at 30°C. While a clear expression peak and a rapid decline were observed for all the six genes at 30°C, the mRNA profiles at 10°C were smooth and without a peak but substantially more long-term than at 30°C. A slow decline in mRNA levels was observed at 10°C within the 8-day experiment period (Fig. 2).

At 10°C, no clear clustering of genes based on their relative expression curves was observed (Fig. 2). The botE, p47, and orfx3 had their maximal relative expression levels in the late exponential phase (5 days) whereas ntnh, orfx1, and orfx2 reached their maximum levels in early stationary phase (6 days) (Fig. 2 and 3). At 10°C, orfx1 had the lowest relative expression level throughout the experiment.

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FIG. 3. Comparison between relative expression of neurotoxin cluster genes at mid-exponential (10°C, 4 days; 30°C, 4 h) (black bars), late exponential (10°C, 5 days; 30°C, 6 h) (dark-gray bars), early stationary (10°C, 6 days; 30°C, 8 h) (light-gray bars), and mid-stationary (10°C, 8 days; 30°C, 12 h) (white bars) growth phases and at the two incubation temperatures. (Data for all other time points have been omitted.) The gene expression levels of the target genes were normalized against those of 16S rrn. The values represent a mean of three replicate cultures, and the I-bars indicate standard deviations.

In the culture grown at 30°C, two clusters of genes with distinct expression curves were observed. The mRNA levels of botE, ntnh, and p47 followed a similar pattern, and their average level was one-fourth of the average level of the similarly expressed orfx1, orfx2, and orfx3 genes (Fig. 2 and 3). The expression patterns of the two gene groups at 30°C were significantly different (P < 0.01), while no difference was observed between the three genes within each group.

Comparison of relative expression levels of the six type E botulinum neurotoxin cluster genes at 10°C and 30°C.
Although the OD₆₀₀ values measured at 10°C were less than half (maximum, 0.5) of those at 30°C (maximum, 1.2) (Fig. 2), the average relative mRNA levels of botE, ntnh, and p47 at 10°C were similar (botE) or 1.5-fold higher (ntnh and p47) than those at 30°C. The average relative expression levels of orfx2 and orfx3 at 10°C were half (P < 0.05) of those at 30°C, while the levels of orfx1 at 10°C were as low as 1/10 (P < 0.01) of those at 30°C (Fig. 3). At 10°C, the average relative expression levels of orfx1 were one-fourth (P < 0.001) of those of orfx2 and orfx3.

The maximum relative mRNA level of botE at 10°C was 60% of that at 30°C. Having reached a maximum at the late exponential growth phase on day 5 at 10°C, relative botE mRNA levels remained stable until day 6 and declined to half of the maximum level by the end of the 8-day experiment (Fig. 2 and 3). In accordance with the relative botE expression levels measured at and after the late exponential phase, the type E neurotoxin levels at 10°C were 45 to 65% of those measured at 30°C (Fig. 4).

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FIG. 4. Values (OD405 units) for type E neurotoxin in culture supernatants at 30°C (solid line) and at 10°C (dashed line) at different growth phases. Each value represents the mean of two replicate tubes, and the I-bars indicate the standard deviations.

At 30°C, the maximum relative mRNA levels of each gene were about 6-fold higher than those at the first measurement in the mid-exponential growth phase (Fig. 3). Accordingly, the maximum relative mRNA levels at 10°C, measured in the late exponential or early stationary growth phase, seemed to be on average 3- to 4-fold higher than those measured at the mid-exponential growth phase. At 30°C, the relative expression levels of botE, ntnh, and p47 showed similar peak levels, with a ratio of 1:1.4:1, respectively (Fig. 3). Similarly, the corresponding ratio of orfx1, orfx2, and orfx3 was 1.3:1:1.1, respectively, at 30°C (Fig. 3).

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Temperature is the main factor controlling the growth and neurotoxin production of group II C. botulinum in modern food production systems (reviewed in reference 13). In this study, the expression of type E neurotoxin cluster genes at 10°C showed trends that may have serious consequences for food safety. First, the mRNA levels of the type E neurotoxin cluster genes relative to growth were not repressed by the low temperature of 10°C. Notwithstanding the lower biomass, as estimated by OD₆₀₀ measurements, the relative botE mRNA levels at 10°C were similar to those measured at the generally considered optimal growth temperature of 30°C. The type E neurotoxin levels measured in culture supernatants were in accordance with the measured mRNA levels. Our results are in line with other stress factors, such as low concentration of oxygen (15) or high temperature (3), having no or only little effect on bot activity. These results together with the finding that carbon dioxide induced bot expression in a group II type E strain ATCC 9564 (1) may suggest that the efficacy of the inhibitory hurdles applied in the food industry may require reevaluation. Second, the relative mRNA levels showed only little or no decline after 8 days of incubation at 10°C in contrast to those at 30°C. Furthermore, the OD remained at the maximum level until the end of experiment, indicating that substantial cell lysis was unlikely. Also previous studies confirm group II C. botulinum cultures to be more stable at the stationary phase than group I cultures (3, 4, 15). These phenomena show that the type E botulinum neurotoxin gene cluster is actively expressed and maintained stably for a rather long time at 10°C.

At 30°C, the relative expression of botE, ntnh, and p47 on one hand and orfx1, orfx2, and orfx3 on the other had similar patterns and levels throughout the experiment. The maximal levels of orfx1, orfx2, and orfx3 were about 4-fold higher than those of botE, ntnh, and p47. Based on these transcriptional patterns it could be inferred that, at 30°C, two separate tricistronic operons are transcribed, namely botE, ntnh, and p47 and orfx1, orfx2, and orfx3 (Fig. 1B). In another study, a similar conclusion was drawn on the type A2 strain Kyoto grown at 37°C (4). The authors of that study further suggested that, as the orfx operon was transcribed from a conserved neurotoxin-associated promoter, the orfx genes most probably were related to the neurotoxin production. Although we could not identify a definite promoter in the type E neurotoxin gene cluster for orfx1, orfx2, and orfx3 (2), the dynamic variations observed in the relative expression of the type E neurotoxin cluster genes further support the hypothesis that the orfx gene products are relative to type E neurotoxin production.

To the best of our knowledge, there are no previous reports on quantitative analysis of orfx1, orfx2, and orfx3 expression in C. botulinum. Interestingly, similar to the 4-fold difference between the expression levels of the botE and orfx operons observed at 30°C in our study, another study reported that the ha cluster genes were expressed at a 3- to 6-fold higher level than were botD and ntnh in the group III C. botulinum type D strain D-4947 at an early stationary phase (10). Further studies are naturally warranted to reveal whether the proteins encoded by the orfx cluster share functional analogy with the hemagglutinin complex.

The low temperature of 10°C seemed to cause more variation among the expression of the six type E neurotoxin cluster genes than the optimum temperature of 30°C. Two distinct phenomena in the relative expression levels of the different genes were observed at 10°C. First, the average relative mRNA levels of ntnh and p47 were generally higher at 10°C than at 30°C, whereas the other genes showed no difference or were less expressed at 10°C than at 30°C. This may suggest that, at a low temperature, ntnh and p47 were being transcribed separately from botE, as opposed to at 30°C where all the three genes seemed to be cotranscribed as discussed above (Fig. 1B). The existence of transcriptional promoters upstream of both p47 and botE supports the notion of both mono- (24) and tricistronic (12) transcriptional models for botE. In line with our observations at 30°C, botE and p47 were concomitantly expressed at 37°C (3), indicating that the tricistronic transcription model is characteristic for optimal and high temperatures.

Second, at 10°C, the relative mRNA levels of orfx1 were significantly lower than those of orfx2 and orfx3. This may suggest orfx1 to be transcribed alone at a very low level and orfx2 to be cotranscribed with orfx3 at a higher level than orfx1 at a low temperature (Fig. 1B). These apparent temperature-dependent modes of type E botulinum neurotoxin gene cluster transcription may further suggest alternative regulatory events at high and low temperatures and structural alteration of the type E toxin complex by temperature change.

The roles of the P47, Orfx1, Orfx2 and Orfx3 proteins are not known, nor is it known how botE is regulated. We found that the expression levels of orfx1, orfx2, and orfx3, located immediately upstream of the neurotoxin gene (2), are significantly higher than botE. As many regulators, such as botR in the type A strains Hall and NCTC 2916 (3), have been shown to express at levels 1/100 of their target genes, we suggest that none of the orfx1, orfx2, and orfx3 genes encode a transcriptional regulator for botE. Furthermore, the role of P47 as a transcriptional regulator was deconstructed by showing that inactivation of p47 by using antisense RNA did not attenuate neurotoxin production (3). Further research is thus required to identify the regulatory mechanism of botE.

In conclusion, distinct type E neurotoxin cluster gene expression patterns were observed at 10°C and 30°C. In relation to growth, the relative mRNA levels were not repressed by the low temperature. Neurotoxin gene expression was further confirmed by detecting the toxin in culture supernatants. Only slow decline in the relative mRNA levels of neurotoxin cluster genes was observed within 8 days at 10°C, whereas at 30°C, decline was rapid after peak expression at the early stationary growth phase. At 30°C, botE, ntnh, and p47 and orfx1, orfx2, and orfx3 seemed to be expressed as two tricistronic operons. At 10°C, however, it seemed evident that botE and orfx1 are monocistronically transcribed, suggesting that the low temperature may induce alternative regulatory events behind the neurotoxin production. This study shows that the type E neurotoxin gene cluster is active at the slightly abused food storage temperature frequently measured in retailers' and consumers' refrigerators. Understanding the activity of the neurotoxin genes at low temperatures is a key to establishing novel strategies to ensure the safety of chilled foods.

 
The work was supported by the Academy of Finland (grants 1115133, 1118602, 1120180, and 206319) and the Walter Ehrström Foundation.

We thank Hanna Korpunen for technical assistance.

 
* Corresponding author. Mailing address: Department of Food and Environmental Hygiene, P.O. Box 66, FIN-00014 University of Helsinki, Finland. Phone: 358-9-19157107. Fax: 358-9-19157101. E-mail: miia.lindstrom{at}helsinki.fi

Published ahead of print on 15 August 2008.

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Applied and Environmental Microbiology, October 2008, p. 6132-6137, Vol. 74, No. 19
0099-2240/08/$08.00+0     doi:10.1128/AEM.00469-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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