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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{triangledown}

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

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.

* 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

{triangledown} Published ahead of print on 15 August 2008.

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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