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Clostridium botulinum group III is the anaerobic Gram-positive bacteria producing the deadly neurotoxin responsible for animal botulism. Here, we used long-read sequencing to produce four complete genomes from Clostridium botulinum group III neurotoxin types C, D, C/D, and D/C. The protocol to obtain high-molecular-weight DNA from C. botulinum group III is described.

The activity of RNA helicase csdA ( cbo2802 ) after temperature downshift was compared to its activity at optimal growth temperature, and the effect of sense and antisense oriented insertional inactivation of cbo2802 on the growth of ATCC 3502 at suboptimal temperature was evaluated. The relative cbo2802 transcript level was significantly induced for 30 min to 5 h after cold shock. In contrast, a significant decrease in the relative transcript level of cbo2802 was observed within the same time frame at 37 °C. Inactivation of cbo2802 led to an extensive delay in initiation of exponential growth at 20 °C but not at 37 °C. In addition, the mean minimum growth temperatures of the mutant strains were higher than those of the wild-type strain. During a 24-hour incubation at 37 °C, all strains were motile, whereas at 20 °C the mutant strains showed severely impaired motility compared to the wild-type strain. This study shows that a functional csdA is needed for effective adaptation and initiation of growth and motility of Clostridium botulinum ATCC 3502 at suboptimal temperature.

Clostridium botulinum is a notable food pathogen and responsible for botulism due to production of botulinum neurotoxin. Strains of C. botulinum can adapt to and survive in stress conditions and food processing. The cold shock protein coding genes (csp) are involved in growth at low temperature, but they may also possess other functions. In this mutational analysis we show that cspB and cspC, but not cspA, are important for NaCl, pH and ethanol stress responses and for motility of C. botulinum ATCC 3502. In all NaCl concentrations tested, the cspB mutant had lower maximum growth rate and, together with the cspC mutant, a longer lag phase compared to the wild-type strain. At low pH, the cspB and cspC mutants showed either lower maximum growth rates or longer lag phases compared to the wild type. In all ethanol concentrations tested, the cspB mutant had lower maximum growth rates and the cspC mutant had a longer lag phase than the wild-type strain. Motility was reduced in cspA and cspC mutants, and flagella formation was affected. The results suggest that cspB plays a universal role in stress response and cspC aids C. botulinum in NaCl, pH and ethanol stress in C. botulinum ATCC 3502.

International audience; The goal of the AntiBotABE Program was the development of recombinant antibodies that neutralize botulinum neurotoxins (BoNT) A, B and E. These serotypes are lethal and responsible for most human botulinum cases. To improve therapeutic efficacy, the heavy and light chains (HC and LC) of the three BoNT serotypes were targeted to achieve a synergistic effect (oligoclonal antibodies). For antibody isolation, macaques were immunized with the recombinant and non-toxic BoNT/A, B or E, HC or LC, followed by the generation of immune phage-display libraries. Antibodies were selected from these libraries against the holotoxin and further analyzed in in vitro and ex vivo assays. For each library, the best ex vivo neutralizing antibody fragments were germline-humanized and expressed as immunoglobulin G (IgGs). The IgGs were tested in vivo, in a standardized model of protection, and challenged with toxins obtained from collections of Clostridium strains. Protective antibody combinations against BoNT/A and BoNT/B were evidenced and for BoNT/E, the anti-LC antibody alone was found highly protective. The combination of these five antibodies as an oligoclonal antibody cocktail can be clinically and regulatorily developed while their high "humanness" predicts a high tolerance in humans.

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.

Clostridium botulinum is a notorious foodborne pathogen. Its ability to adapt to and grow at low temperatures is of interest for food safety. Two-component systems (TCSs) have been reported to be involved in cold-shock and growth at low temperatures. Here we show the importance of TCS CBO2306/CBO2307 in the cold-shock response of C. botulinum ATCC 3502. The relative expression levels of the cbo2306 and cbo2307 were up to 4.4-fold induced in the cold-shocked cultures but negatively regulated in the late-log and stationary growth phase in relation to early logarithmic growth phase in non-shocked cultures. Importance of the CBO2306/CBO2307 in the cold stress was further demonstrated by impaired growth of insertional cbo2306 or cbo2307 knockout mutants in relation to the wild-type strain ATCC 3502. The results suggest that the TCS CBO2306/CBO2307 is important for cold-shock response and adaptation of C. botulinum ATCC 3502 to low temperature.

International audience; Botulinum neurotoxins are diverse proteins. They are currently represented by at least seven serotypes and more than 40 subtypes. New clostridial strains that produce novel neurotoxin variants are being identified with increasing frequency, which presents challenges when organizing the nomenclature surrounding these neurotoxins. Worldwide, researchers are faced with the possibility that toxins having identical sequences may be given different designations or novel toxins having unique sequences may be given the same designations on publication. In order to minimize these problems, an ad hoc committee consisting of over 20 researchers in the field of botulinum neurotoxin research was convened to discuss the clarification of the issues involved in botulinum neurotoxin nomenclature. This publication presents a historical overview of the issues and provides guidelines for botulinum neurotoxin subtype nomenclature in the future.