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Applied and Environmental Microbiology, May 2004, p. 2928-2934, Vol. 70, No. 5
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.5.2928-2934.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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

Maria Lövenklev,^1, Ingrid Artin,² Oskar Hagberg,³ Elisabeth Borch,⁴ Elisabet Holst,² and Peter Rådström^1*

Applied Microbiology, Lund Institute of Technology,¹ Mathematical Statistics, Lund University, SE-221 00 Lund,³ Medical Microbiology, Dermatology and Infection, Lund University, SE-223 62 Lund,² SIK—The Swedish Institute for Food and Biotechnology, SE-223 70 Lund, Sweden⁴

Received 11 August 2003/ Accepted 28 January 2004

The effects of carbon dioxide, sodium chloride, and sodium nitrite on type B botulinum neurotoxin (BoNT/B) gene (cntB) expression in nonproteolytic Clostridium botulinum were investigated in a tryptone-peptone-yeast extract (TPY) medium. Various concentrations of these selected food preservatives were studied by using a complete factorial design 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 extracellular BoNT/B production. Multiple linear regression was used to set up six statistical models to quantify and predict these responses. All combinations of NaCl and NaNO₂ in the growth medium resulted in a prolonged lag phase duration and in a reduction in the specific growth rate. In contrast, the relative BoNT/B gene expression was unchanged, as determined by the cntB-specific quantitative reverse transcription-PCR method. This was confirmed when we measured the extracellular BoNT/B concentration by an enzyme-linked immunosorbent assay. CO₂ was found to have a major effect on gene expression when the cntB mRNA levels were monitored in the mid-exponential, late exponential, and late stationary growth phases. The expression of cntB relative to the expression of the 16S rRNA gene was stimulated by an elevated CO₂ concentration; the cntB mRNA level was fivefold greater in a 70% CO₂ atmosphere than in a 10% CO₂ atmosphere. These findings were also confirmed when we analyzed the extracellular BoNT/B concentration; we found that the concentrations were 27 ng · ml^–1 · unit of optical density^–1 in the 10% CO₂ atmosphere and 126 ng · ml^–1 · unit of optical density^–1 in the 70% CO₂ atmosphere.

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* Corresponding author. Mailing address: Applied Microbiology, Lund Institute of Technology, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden. Phone: 46 46-222 34 12. Fax: 46 46-222 42 03. E-mail: Peter.Radstrom{at}tmb.lth.se.

Maiden name, Maria Dahlenborg.

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Applied and Environmental Microbiology, May 2004, p. 2928-2934, Vol. 70, No. 5
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.5.2928-2934.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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