Regulation of neurotoxin and protease formation in Clostridium botulinum Okra B and Hall A by arginine

Abstract

Supplementation of a minimal medium with high levels of arginine (20 g/liter) markedly decreased neurotoxin titers and protease activities in cultures of Clostridium botulinum Okra B and Hall A. Nitrogenous nutrients that are known to be derived from arginine, including proline, glutamate, and ammonia, also decreased protease and toxin but less so than did arginine. Proteases synthesized during growth were rapidly inactivated after growth stopped in media containing high levels of arginine. Separation of extracellular proteins by electrophoresis and immunoblots with antibodies to toxin showed that the decrease in toxin titers in media containing high levels of arginine was caused by both reduced synthesis of protoxin and impaired proteolytic activation. In contrast, certain other nutritional conditions stimulated protease and toxin formation in C. botulinum and counteracted the repression by arginine. Supplementation of the minimal medium with casein or casein hydrolysates increased protease activities and toxin titers. Casein supplementation of a medium containing high levels of arginine prevented protease inactivation. High levels of glucose (50 g/liter) also delayed the inactivation of proteases in both the minimal medium and a medium containing high levels of arginine. These observations suggest that the availability of nitrogen and energy sources, particularly arginine, affects the production and proteolytic processing of toxins and proteases in C. botulinum.

Growth Conditions of and Emetic Toxin Production by Bacillus cereus in a Defined Medium with Amino Acids

“…We found that emetic toxin production was sharply decreased in the media supplemented with a high concentration of leucine, isoleucine or glutamic acid. This finding is similar to previous observations that the production of a botulinum neurotoxin was suppressed by high levels of certain amino acids (8,10), and that the biosynthesis of antibiotic penicillin was negatively regulated by several amino acids (3,7).…”

“…The production of neurotoxin by C. botulinum, was decreased by the presence of high levels of arginine and tryptophan (8,10). We also examined the effect of high levels of amino acids in CADM.…”

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

“…The only known negative regulator of BoNT synthesis, CBO0787/CBO0786, is a TCS whose regulator component CBO0786 binds specifically to the promoter of ntnh-botA and inhibits transcription of this operon by blocking the binding of the RNA polymerase-BotR complex to its target promoter DNA (Zhang et al, 2013b). The nature of the environmental signals activating the neurotoxinregulating TCSs is scarcely known, but availability of certain carbohydrates (Bonventre and Kempe, 1959) and amino acids (Patterson-Curtis and Johnson, 1989;Leyer and Johnson, 1990;Fredrick et al, 2017) and temperature (Selby et al, 2017), influence neurotoxin production and provide interesting hypotheses for further TCS studies. Not only the extracellular environment but also the intracellular state, such as the metabolic status of C. botulinum cells, affects BoNT production via the transition-state central regulator CodY .…”

Roles of arginine in growth of Clostridium botulinum Okra B

“…During the study of regulation of neurotoxin formation in C. botulinum by arginine (17, 18), we observed that the Okra B strain consumed quantities of arginine well in excess of the growth requirement. Degradation of high levels of arginine was of particular interest since arginine is probably the limiting organic nutrient for growth of group I C. botulinum in many foods and media (23) and because protease and neurotoxin production are repressed by high levels of arginine (17,18). Because basic end products and proline are formed during arginine fermentation, catabolism of high levels of arginine could provide protection against environmental stresses, including increased acidity (3,21) and osmolarity (6,14).…”

“…To study arginine metabolism, Okra B was grown in a chemically defined minimal medium (MI) as previously described (17,18,23). MI contains a basal level of arginine (3 g/liter) that supports growth measured atA660 to -1 (1.08 mg [dry cell weight] per ml) (23).…”