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.
G roup II (nonproteolytic)Clostridium botulinum type E is a notorious food-borne pathogen which is mainly found in aquatic environments in cold regions of the world (e.g., Northern Europe, Japan, Canada, Alaska, and Greenland) (1, 2), with a high prevalence of spores in the Baltic Sea (3). Type E botulism is usually caused through consumption of contaminated fish or seafood products such as vacuum-packed smoked fish, salted fish, and fermented marine mammals (4-11). Concerning modern food processing, C. botulinum type E is the principal food safety hazard in refrigerated, minimally processed packaged foods of aquatic origin (12). The mild heat treatments used in the production of these foods are not sufficient to eliminate all spores, and the limited use of salt and preservatives, as well as vacuum packaging, may support growth and toxin production by C. botulinum. Most importantly, thermal control is often not sufficient to prevent food poisoning, as some strains of group II C. botulinum are able to grow and produce toxin at temperatures as low as 3°C (13-15).The mechanisms of cold shock response and growth at low temperature have been thoroughly studied in the model organisms Escherichia coli and Bacillus subtilis (16-18) but are still unknown in the psychrotrophic C. botulinum type E. The cold shock proteins (Csps) are a universal group of cold-induced proteins occurring in a wide range of bacteria and other organisms (17,19). While group I C. botulinum strains carry Csp genes in their genome (20), none were found in the genomes of C. botulinum type E strains (21), which raises the question of alternative strategies for coping with...