title: Quantitative interaction effects of carbon dioxide, sodium chloride, and sodium nitrite on neurotoxin gene expression in nonproteolytic Clostridium botulinum type B.
abstract: 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 NaNO(2) 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. CO(2) 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 CO(2) concentration; the cntB mRNA level was fivefold greater in a 70% CO(2) atmosphere than in a 10% CO(2) atmosphere. These findings were also confirmed when we analyzed the extracellular BoNT/B concentration; we found that the concentrations were 27 ng x ml(-1). unit of optical density(-1) in the 10% CO(2) atmosphere and 126 ng x ml(-1). unit of optical density(-1) in the 70% CO(2) atmosphere.
authors.affiliation: Applied Microbiology, Lund Institute of Technology, Mathematical Statistics, Lund University, SE-221 00 Lund, Sweden.
authors.forename: Maria
authors.initials: M
authors.lastname: Lövenklev
authors.forename: Ingrid
authors.initials: I
authors.lastname: Artin
authors.forename: Oskar
authors.initials: O
authors.lastname: Hagberg
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Borch
authors.forename: Elisabet
authors.initials: E
authors.lastname: Holst
authors.forename: Peter
authors.initials: P
authors.lastname: Rådström
mesh_terms: D001905:Botulinum Toxins / Q000235:genetics / Q000378:metabolism*; D019274:Botulinum Toxins, Type A; D002245:Carbon Dioxide / Q000378:metabolism*; D003014:Clostridium botulinum / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D003470:Culture Media; D004797:Enzyme-Linked Immunosorbent Assay; D005520:Food Preservatives / Q000378:metabolism*; D016014:Linear Models; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012965:Sodium Chloride / Q000378:metabolism*; D012977:Sodium Nitrite / Q000378:metabolism*
chemical_list: D003470:Culture Media; D005520:Food Preservatives; C096323:rimabotulinumtoxinB; D002245:Carbon Dioxide; D012965:Sodium Chloride; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; D012977:Sodium Nitrite