The CLO3403/CLO3404 Two-Component System of Clostridium botulinum E1 Beluga Is Important for Cold Shock Response and Growth at Low Temperatures

Abstract

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...

Neurotoxin synthesis is positively regulated by the sporulation transcription factor Spo0A in Clostridium botulinum type E

“…Loss of the plasmids was verified by sensitivity to thiamphenicol (15 µg ml −1 ). Insertion in correct site and correct orientation was confirmed by PCR and southern blot analysis confirmed single intron insertion (Mascher et al ., ).…”

“…The spo0A genes were knocked-out by inserting a mobile group II intron from Lactococcus lactis (Ll.ltrB) using the Clos-Tron mutagenesis tool (Heap et al, 2007;Heap et al, 2010). The spo0A mutants with intron insertion in sense (spo0As) and antisense (spo0Aa) orientation were obtained and confirmed as described earlier (Mascher et al, 2014). Briefly, retargeted pMTL007C-E2 plasmids were ordered from DNA2.0 (Menlo Park, CA) and conjugated into C. botulinum.…”

Construction of Nontoxigenic Mutants of Nonproteolytic Clostridium botulinum NCTC 11219 by Insertional Mutagenesis and Gene Replacement

“…Although the inactivation of specific genes in clostridial species has proven to be a rather difficult, slow, and inefficient task for a long time, the genetic toolbox for knockout mutagenesis has been expanding in the last few years. The ClosTron system, which makes use of a mobile intron that can be retargeted to a sequence of interest, has proven to be particularly efficient for insertional mutagenesis in a range of clostridial species (20), but only a few studies have used ClosTron mutagenesis in gIICb thus far (21,22). Interestingly, ClosTron has been used to knock out the bont gene in both gICb and gIICb (21,23), but the properties of the gIICb knockout strain in view of its possible usefulness for challenge or process validation studies in foods were not further investigated.…”

Characterization of a temperature-responsive two component regulatory system from the Antarctic archaeon, Methanococcoides burtonii

“…A role for TCSs in functioning as thermosensors and regulating gene expression was originally described for Escherichia coli 20 . Subsequent global gene expression and/or gene inactivation studies have linked the role of TCSs to low temperature gene regulation in: animal pathogens including Bacillus cereus 21 , Clostridium botulinum 22 23 , Edwardsiella tarda 24 , Flavobacterium psychrophilum 25 , Haemophilus influenzae 26 , Listeria monocytogenes 27 28 , Yersinia pseudotuberculosis 29 ; plant pathogens Agrobacterium tumefaciens 10 30 and Pseudomonas syringae 31 32 33 34 ; environmental bacteria Bacillus subtilis 11 35 , Sphingobacterium antarcticus 31 and Synechocystis sp. 12 ; and environmental archaea Methanococcoides burtonii 13 and Methanolobus psychrophilus 18 .…”