Quantitative detection of gene expression and toxin complex produced by Clostridium botulinum serotype D strain 4947.
Botulinum toxin is produced by Clostridium botulinum as a large toxin complex (L-TC) non-covalently assembled with a neurotoxin (NT), a non-toxic non-hemagglutinin (NTNHA) and hemagglutinin subcomponents (HA-70, HA-33, and HA-17). In this study, the gene expressions of five individual L-TC components were examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in C. botulinum serotype D strain 4947 (D-4947) during cell growth. Transcripts for the five component genes were successfully detected in the mid-exponential growth phase (6.5 h), reaching a maximum at the early stationary growth phase (12 h). The ratio of the mRNA transcripts of nt and ntnha was approximately 1:1, suggesting that nt and ntnha are bicistronically transcribed. On the other hand, the transcript levels of the ha genes were several-fold higher than those of nt and ntnha, although the mRNA transcript level of ha-33 was less than the other two ha subcomponent genes. The results based on qRT-PCR indicate that a shortage of HA-33 among the proteins associated with botulinum TC could explain the production by D-4947 of other smaller-sized L-TCs (610, 540 and 410 kDa) with fewer HA-33 molecules than the mature 650 kDa L-TC. Western blot analysis demonstrated that TC species in cell lysate were initially observed in the mid-exponential phase, while extracellular TCs were detected subsequently in the early stationary phase.
Kouguchi
Hirokazu
H
Hokkaido Institute of Public Health, N19, W12, Kita-Ku, Sapporo 060-0819, Japan.
Suzuki
Tomonori
T
Hasegawa
Kimiko
K
Mutoh
Shingo
S
Watanabe
Toshihiro
T
Niwa
Koichi
K
Yoneyama
Tohru
T
Katoh
Yoshinobu
Y
Ohyama
Tohru
T
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D015153:Blotting, Western; D001905:Botulinum Toxins / Q000096:biosynthesis* / Q000235:genetics; D003014:Clostridium botulinum / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D020869:Gene Expression Profiling; D006388:Hemagglutinins / Q000096:biosynthesis* / Q000235:genetics; D012329:RNA, Bacterial / Q000032:analysis / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D001426:Bacterial Proteins; D006388:Hemagglutinins; D012329:RNA, Bacterial; D012333:RNA, Messenger; C096325:botulinum toxin type D; D001905:Botulinum Toxins