Evaluation of normalization reference genes for RT-qPCR analysis of spo0A and four sporulation sigma factor genes in Clostridium botulinum Group I strain ATCC 3502

• Eight candidate reference genes were evaluated in Clostridium botulinum ATCC 3502.
• 16S rrn was selected as an appropriate normalization reference gene for RT-qPCR.
• Expression of sporulation regulators spo0A, sigF, sigE, sigG, and sigK was examined.
• Expression of sigF, sigE, and sigG was induced in late-exponential growth

Heat-resistant spores of Clostridium botulinum can withstand the pasteurization processes in modern food processing. This poses a risk to food safety as spores may germinate into botulinum neurotoxin-producing vegetative cells. Sporulation in Bacillus subtilis, the model organism for sporulation, is regulated by the transcription factor Spo0A and four alternative sigma factors, SigF, SigE, SigG, and SigK. While the corresponding regulators are found in available genomes of C. botulinum, little is known about their expression. To accurately measure the expression of these genes using quantitative reverse-transcriptase PCR (RT-qPCR) during the exponential and stationary growth phases, a suitable normalization reference gene is required. 16S rrn, adK, alaS, era, gluD, gyrA, rpoC, and rpsJ were selected as the candidate reference genes. The most stable candidate reference gene was 16S ribosomal RNA gene (rrn), based on its low coefficient of variation (1.81%) measured during the 18-h study time. Using 16S rrn as the normalization reference gene, the relative expression levels of spo0A, sigF, sigE, sigG, and sigK were measured over 18 h. The pattern of expression showed spo0A expression during the logarithmic growth phase, followed by a drop in expression upon entry to the stationary phase. Expression levels of sigF, sigE, and sigG peaked simultaneously at the end of the exponential growth phase. Peak expression of sigK occurred at 18 h, however low levels of expression were detected during the exponential phase. These findings suggest these sigma factors play a role in C. botulinum sporulation that is similar, but not equal, to their role in the B. subtilis model.