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

Abstract

ABSTRACT 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.