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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 18h. 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 18h, 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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In order to survive a temperature downshift, bacteria have to sense the changing environment and adjust their metabolism and structure. Two-component signal transduction systems (TCSs) play a central role in sensing and responding to many different environmental stimuli. Although the nonproteolytic (group II) Clostridium botulinum represents a major hazard in chilled foods, the cold adaption mechanisms of group II C. botulinum organisms are not known. Here, we show that the CLO3403/CLO3404 TCS of C. botulinum E1 Beluga is involved in the cold shock response and growth at 12°C. Cold shock induced the expression of the genes encoding the histidine kinase (clo3403) and the response regulator (clo3404) by more than 100-fold after 5 h relative to their expression in a nonshocked culture at the corresponding time point. The involvement of CLO3403/CLO3404 in growth at low temperature was demonstrated by impaired growth of the insertional clo3403 and clo3404 knockout mutants at 12°C compared to the growth of the wild-type culture. Additionally, the inactivation of clo3403 had a negative effect on motility. The growth efficiency at 12°C of the TCS mutants and the motility of the kinase mutants were restored by introducing a plasmid harboring the operon of the CLO3403/CLO3404 TCS. The results suggest that the CLO3403/CLO3404 TCS is important for the cold tolerance of C. botulinum E1 Beluga.

A key survival mechanism of Clostridium botulinum, the notorious neurotoxic food pathogen, is the ability to form heat-resistant spores. While the genetic mechanisms of sporulation are well understood in the model organism Bacillus subtilis, nothing is known about these mechanisms in C. botulinum. Using the ClosTron gene-knockout tool, sigK, encoding late-stage (stage IV) sporulation sigma factor K in B. subtilis, was disrupted in C. botulinum ATCC 3502 to produce two different mutants with distinct insertion sites and orientations. Both mutants were unable to form spores, and their elongated cell morphology suggested that the sporulation pathway was blocked at an early stage. In contrast, sigK-complemented mutants sporulated successfully. Quantitative real-time PCR analysis of sigK in the parent strain revealed expression at the late log growth phase in the parent strain. Analysis of spo0A, encoding the sporulation master switch, in the sigK mutant and the parent showed significantly reduced relative levels of spo0A expression in the sigK mutant compared to the parent strain. Similarly, sigF showed significantly lower relative transcription levels in the sigK mutant than the parent strain, suggesting that the sporulation pathway was blocked in the sigK mutant at an early stage. We conclude that σ(K) is essential for early-stage sporulation in C. botulinum ATCC 3502, rather than being involved in late-stage sporulation, as reported for the sporulation model organism B. subtilis. Understanding the sporulation mechanism of C. botulinum provides keys to control the public health risks that the spores of this dangerous pathogen cause through foods.