Expression of botulinum neurotoxins A and E, and associated non-toxin genes, during the transition phase and stability at high temperature: analysis by quantitative reverse transcription-PCR.
Production of botulinum neurotoxin A (BoNT/A) and associated non-toxic proteins (ANTPs), which include a non-toxic non-haemagglutinin (NTNH/A) as well as haemagglutinins (HAs), was found previously to be dependent upon an RNA polymerase alternative sigma factor (BotR/A). Expression of the botR/A, bont/A and antp genes, monitored by reverse transcription and real-time PCR analysis, occurred concomitantly at the transition between the exponential and stationary growth phases of Clostridium botulinum A. The botR/A expression level was about 100-fold less than those of the bont/A and antp genes. Therefore, BotR/A is an alternative sigma factor controlling the botulinum A locus genes during the transition phase. The highest toxin concentration was released into the culture supernatant 12 h after maximum expression of the botR/A, bont/A and antp genes, without any apparent bacterial lysis. Toxin levels were then stable over 5 days in cultures at 37 degrees C, whereas a dramatic decrease in lethal activity was observed between 24 and 48 h in cultures at 44 degrees C. High temperature did inhibit transcription, since expression levels of the botR/A, bont/A and antp genes were similar in cultures at 37 and 44 degrees C. However, incubation at 44 degrees C triggered a calcium-dependent protease that degraded BoNT/A and NTNH/A, but not HAs. In C. botulinum E, which contains no gene related to botR, the bont/E and p47 genes were also expressed during the transition phase, and no protease activation at 44 degrees C was evident.
Couesnon
Aurélie
A
Unité des Bactéries anaérobies et Toxines, Institut Pasteur, Paris, France.
Raffestin
Stéphanie
S
Unité des Bactéries anaérobies et Toxines, Institut Pasteur, Paris, France.
Popoff
Michel R
MR
Unité des Bactéries anaérobies et Toxines, Institut Pasteur, Paris, France.
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D019274:Botulinum Toxins, Type A / Q000235:genetics / Q000378:metabolism*; D047013:Clostridium botulinum type A / Q000254:growth & development* / Q000378:metabolism; D047970:Clostridium botulinum type E / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D003470:Culture Media; D015964:Gene Expression Regulation, Bacterial; D006388:Hemagglutinins / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; C116103:BotR protein, Clostridium botulinum; D003470:Culture Media; D006388:Hemagglutinins; D015534:Trans-Activators; D019274:Botulinum Toxins, Type A