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

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 degrees C, whereas at 10 degrees 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.