Relative Neurotoxin Gene Expression inClostridium botulinumType B, Determined Using Quantitative Reverse Transcription-PCR

Abstract

A quantitative reverse transcription-PCR (qRT-PCR) method was developed to monitor the relative expression of the type B botulinum neurotoxin (BoNT/B) gene (cntB) in Clostridium botulinum. The levels of cntB mRNA in five type B strains were accurately monitored by using primers specific for cntB and for the reference gene encoding the 16S rRNA. The patterns and relative expression of cntB were different in the different strains. Except for one of the strains investigated, an increase in cntB expression was observed when the bacteria entered the early stationary growth phase. In the proteolytic strain C. botulinum ATCC 7949, the level of cntB mRNA was four-to fivefold higher than the corresponding levels in the other strains. This was confirmed when we quantified the production of extracellular BoNT/B by an enzyme-linked immunosorbent assay and measured the toxicity of BoNT/B by a mouse bioassay. When the effect of exposure to air on cntB expression was investigated, no decline in the relative expression was observed in spite of an 83% reduction in the viable count based on the initial cell number. Instead, the level of cntB mRNA remained the same. When there was an increase in the sodium nitrite concentration, the bacteria needed a longer adjustment time in the medium before exponential growth occurred. In addition, there was a reduction in the expression of cntB compared to the expression of the 16S rRNA gene at higher sodium nitrite concentrations. This was most obvious in the late exponential growth phase, but at the highest sodium nitrite concentration investigated, 45 ppm, a one-to threefold decline in the cntB mRNA level was observed in all growth phases.

Quantitative Interaction Effects of Carbon Dioxide, Sodium Chloride, and Sodium Nitrite on Neurotoxin Gene Expression in Nonproteolytic Clostridium botulinum Type B

“…Ten milliliters of the suspension was removed from each flask after shaking and chilled on ice, and the cells were harvested by centrifugation at 4°C and 10,400 ϫ g for 10 min and resuspended in ice-cold TES buffer (50 mM Tris [ICN Biochemicals Inc.], 5 mM EDTA [Sigma Chemical Co.], 50 mM NaCl [Sigma Chemical Co.]; pH 7.5). Total RNA was recovered by the method described in the accompanying paper (19). Before reverse transcription, contaminating DNA was degraded by treatment with DNase (Promega Co., Madison, Wis.).…”

“…The cntB-specific qRT-PCR method used in this study is described in the accompanying paper (19). To quantify the transcript levels of cntB, the same amount of total RNA (0.5 g) from each growth phase culture was used in the reverse transcription reaction.…”

“…A quantitative reverse transcription-PCR (qRT-PCR) method was recently developed for relative quantification of type B cnt (cntB) expression (19). The results obtained showed that the maximum cntB mRNA accumulation occurred as the bacteria entered the early stationary growth phase.…”

“…In the present study, the effects of selected food preservatives on cntB expression in a nonproteolytic C. botulinum Eklund type B strain were investigated by using the cntB-specific qRT-PCR method (19). Various concentrations of carbon di-oxide, sodium chloride, and sodium nitrite were studied in a factorial-design experiment in order to quantitatively study interaction effects, as well as main effects, on the following responses: lag phase duration (LPD), growth rate, relative cntB expression, and neurotoxin production.…”

Subtyping Botulinum Neurotoxins by Sequential Multiple Endoproteases In-Gel Digestion Coupled with Mass Spectrometry

“…Nucleotide sequencing of BoNT genes using polymerase chain reaction (PCR) or real-time PCR techniques can subtype BoNTs. [10][11][12][13][14][15] More recently, researchers have reported a multiplex PCR assay for the detection and identification of BoNT subtypes. [16][17] These PCR techniques require the presence of DNA, however.…”