In Gram-positive bacteria, CodY (control of dciA (decoyinine induced operon) Y) is a master regulator of metabolism, sporulation, and virulence. In Bacillus subtilis, CodY controls more than 100 genes involved in the adaptation to nutrient restriction and transition from the exponential growth phase to the stationary growth phase. Typically, CodY acts by binding to the promoter of target genes in a GTP(guanosine triphosphate)- and branched-chain amino acid-dependent manner; these are indicators of the general metabolism status of the bacterium [51]. Thereby, in B. subtilis, CodY senses intracellular levels of GTP and branched amino acids such as isoleucine, whose levels are high during the exponential growth and decrease in mostly repressed gene transcription. In contrast, at low GTP or isoleucine levels, CodY induces the de-repression of genes which are involved in adaptive responses to nutrient limitation, such as those coding for extracellular degradative enzymes, transport systems, and catabolic pathways [51,52]. CodY is conserved in clostridia, including the toxigenic species C. botulinum, C. tetani, C. perfringens, and C. difficile. In C. botulinum A strain ATCC3502, CodY binds to the promoter of the ntnh-bont operon at high GTP levels, whereas isoleucine is ineffective, and stimulates toxin gene transcription and BoNT/A synthesis (as tested with the ClosTron system, and determining BoNT/A levels by ELISA and bont/A transcription by qPCR) [53]. The precise role of CodY in C. botulinum is still elusive: does CodY directly regulate ntnh-bont transcription or interfere with botR or a TCS gene such as by repressing the negative TCS regulator? CodY is also a positive regulator of TeNT synthesis in C. tetani (as tested by the antisense mRNA strategy, and determining TeNT levelsby ELISA, and tent transcription by qPCR) [32]. CodY binds to the tent promoter but not to that of tetR [32]. BoNT and TeNT synthesis is dependent on the availability of a carbon source such as glucose [54,55,56]. CodY controls carbon metabolism in B. subtilis. Notably, under glucose-rich conditions in culture medium, CodY and CcpA (catabolite control protein A), a regulator of carbon catabolism, facilitate the conversion of excess pyruvate resulting from glycolysis into excretable overflow compounds such as acetate, lactate, and acetoin [57]. A similar mechanism of CodY in glucose/pyruvate metabolism has been suggested in C. botulinum A [53]. CcpA is conserved in C. botulinum and C. tetani. However, the role of CcpA in these microorganisms remains to be elucidated. In contrast, in C. difficile, CodY and CcpA are negative regulators of toxin A (TcdA) and toxin B (TcdB). Glucose and rapidly metabolizable carbohydrates inhibit toxin synthesis in C. difficile. CodY and CcpA, which are activated by glucose and rapidly metabolizable carbohydrates, bind to the promoter of TcdR and repress its transcription and subsequently that of tcdA and tcdB [58,59,60]. The opposite regulatory pathways of toxin synthesis linked to carbohydrate metabolism controlled by CodY and CcpA between C. botulinum/C. tetani and C. difficile are intriguing. This indicates that toxin synthesis in C. botulinum and C. tertani requires energy from carbohydrate metabolism, mainly glucose, the main carbohydrate fermented by these bacteria, while C. difficile mainly uses amino acid metabolism as an energy source, notably through the Stickland reaction, for toxin production [60,61,62,63]. These divergent regulatory pathways might have evolved during bacterial adaptation to different environments: soil for C. botulinum/C. tetani and the intestine for C. difficile.

Spo0A is a master regulator of the initial steps of sporulation in Bacillus and clostridia. However, the mode of activation of Spo0A differs in the two classes of bacteria. Nutrient limitation, notably carbohydrate, nitrogen, and phosphorus limitation, is the major signal leading to Spo0A activation through a phosphorelay including five sensor kinases and subsequent positive transcriptional regulation of critical sporulation-essential genes [64]. The kinases that activate Spo0A in Bacillus are not conserved in clostridia. Orphan Spo0A-activating histidine kinases have been identified in clostridia, such as Clostridium acetobutylicum, C. perfringens, C. difficile, C. botulinum, and C. tetani. Clostridia sense different environmental stimuli to initiate sporulation, including external pH resulting from fermentation, with the subsequent release of acidic end-products (acetate, butyrate) and unknown factors [65,66,67,68,69]. In clostridia, Spo0A displays additional functions apart from sporulation initiation. In C. acetobutylicum, Spo0A is activated at the end of the exponential growth phase and controls the shift between acidogenesis that occurs during the exponential growth, and solventogenesis that is coupled to the onset of sporulation [70,71]. In C. perfringens, Spo0A controls the production of toxins (C. perfringens enterotoxin and TpeL), which are synthesized during the sporulation process [72,73]. The role of Spo0A in the regulation of TcdA and TcdB synthesis is variable according to the genetic background of C. difficile strains [62,74]. Spo0A coordinates the expression of a large number of C. difficile genes involved in multiple additional functions, such as nutrient transport, metabolic pathways including the production of butyrate, surface protein assembly, and flagellar biosynthesis [75].

Spo0A is highly conserved in all C. botulinum genomes: an orphan sensor histidine kinase that is able to phosphorylate Spo0A has been identified [69]. In C. botulinum ATCC3502, Spo0A is expressed during the exponential growth and its expression decreases during the entry into the stationary phase, while the subsequent transcription of sigma factors essential for sporulation increases [76,77]. It is not known whether Spo0A affects the expression of bont in group I C. botulinum. Adaptation to cultivation at high temperatures (45 °C) represses both bont/A and sporulation genes in strain ATCC3502, but no co-regulation of these genes has been evidenced [78]. No correlation between sporulation and the production of BoNT/A has been observed in two other C. botulinum A strains [33]. Moreover, the strain Hall A-hyper produces high levels of BoNT/A and is unable to sporulate [79]. Similarly, the highly TeNT-producing C. tetani strain used for vaccine production is a non-sporulating strain [80], and Spo0A has not been found to control TeNT synthesis (as tested by the antisense mRNA strategy, TeNT monitoring by ELISA, and tent transcription by qPCR) [32]. In contrast, in group II C. botulinum E, Spo0A is a positive regulator of BoNT/E synthesis and sporulation (as tested with the ClosTron system, toxin monitoring by ELISA, and gene transcription by qPCR). Spo0A binds to a conserved motif in the promoters of the ntnh-bont/A operon together with CodY, AbrB (putative repressor of bont/E), sigma K (belonging to the sigma factor cascade of sporulation), and an UviA-like regulator [43]. Thus, Spo0A might directly and indirectly regulate the transcription of bont/E. Thereby, group II C. botulinum E strains that have an UviA-like regulator instead of BotR likely use specific and common regulatory pathways of bont expression compared to C. botulinum A strains which belong to the distinct physiological and genetic group 1.

In group III C. botulinum C and D, the production of the C2 toxin, which is an ADP-ribosyltransferase targeting monomeric actin, is linked to sporulation [81]. However, the regulatory pathway of C2 toxin genes, and the possible involvement of spo0A and/or other sporulation genes, has not yet been elucidated.

C. botulinum and C. tetani produce high levels of toxins in complex media rich in peptones and other nutrients, whereas chemically defined media even containing almost all amino acids and vitamins as well as a carbon source usually yield 10- to 100-fold lower toxin titers [55,82,83,84]. Licona-Cassani et al. showed that, although C. tetani grew in a chemically defined medium, toxin production was obtained only when casein-derived peptides were added to the medium [82]. In addition to variations in toxin production according to different media, variations in BoNT or TeNT yields are often observed from batch to batch of the same culture medium, even using the same bacterial strain. The transcription of neurotoxin genes and toxin synthesis occur mainly within a short time interval between the late exponential growth and early stationary growth phase [33,85,86]. Thus, nutritional and environmental factors influence the regulation of toxin synthesis in C. botulinum and C. tetani, which takes place in a restricted phase of bacterial growth. Peptides and amino acids appear to be important regulatory factors at the transcriptional and posttranscriptional levels. Indeed, large amounts (0.8–0.9 g/L) of amino acids (aspartate, glutamate, serine, histidine, threonine) downregulate tetR and tent by a yet non-identified regulatory pathway [86]. Arginine is an essential amino acid for C. botulinum growth, but an excess of arginine represses BoNT production in proteolytic group I C. botulinum [87]. Arginine deiminase leads to arginine catabolites that increase the pH and induce subsequent BoNT degradation by not yet characterized proteases. BoNT and botulinum complexes are stable at acidic pH in media without excess of arginine [88]. Supplementation with a high amount of glucose (50 g/L) that induces acidification counteracts the effect of arginine. Interestingly, BoNT synthesis is coupled to protease production [89]. Likely, proteases that are active at alkaline pH induce BoNT degradation. Secreted proteases are required for protein substrate degradation, resulting in peptides and amino acids that are taken up into the bacteria through transport systems and used for protein synthesis, including neurotoxin synthesis. Indeed, the C. botulinum A and C. tetani genomes contain numerous protease/peptidase and transport system genes [37,80].

Peptides in culture media were found to be critical for TeNT synthesis by C. tetani. Since culture media containing casein pancreatic digests support high levels of TeNT production, peptides derived from casein tryptic digestion were investigated. Histidine-containing peptides as well as hydrophobic peptides containing the motif proline–aromatic acid–proline were the most effective in promoting TeNT production [84,90,91]. It is noteworthy that genome analysis of C. tetani shows the presence of numerous peptidases and amino acid degradation pathways [92]. The kinetics of C. tetani growth in a complex medium show rapid exponential growth (stage I, around 10–12 h), then a slower linear growth (stage II, around 30 h), followed by a stationary phase and subsequent autolysis. During stage I, the amino acids are consumed and the genes involved in amino acid degradation pathways are overexpressed, corroborating amino acid catabolism that provides energy used for the rapid biomass formation during this growth phase. The pH decreases due to organic acid production, tetR and tent are not expressed, and TeNT is not synthesized. Once free amino acids are depleted in the culture medium, C. tetani uses peptides, whose metabolism requires transporters that are more energy-costly, and enters the linear growth phase II. The transition from free amino acid to peptide consumption is associated with increased pH due to the reduction of organic acids to alcohols and solvents, and the production of ammonia from peptide metabolism. During phase II, tetR and tent are highly expressed, as well as codY, the TCS that positively regulates tent, and two additional sigma factors located on the large plasmid containing tent, resulting in TeNT synthesis [54,82,86,93]. In complex media, glucose is consumed during the first phase of growth, leading to rapid bacterial multiplication and pH decrease. Then, the nitrogen source is used and the pH increases. TeNT is synthesized only during this second phase, when glucose is no longer or weakly available and when peptides are used for energy production. Thus, as shown by Fratelli et al., the balance between the nitrogen and carbon sources, as well as the subsequent pH of culture media, are critical factors [54,93]. C. botulinum and C. tetani likely share common metabolic pathways and subsequent toxin gene regulatory networks. In both microorganisms, the transition from amino acid utilization to peptides that are more common substrates in the environment seems to elicit the production of proteases. BoNT and TeNT are metalloproteases, but which recognize specific substrates in host neuronal cells. BoNT and TeNT possibly evolve from ancestor metalloproteases with a broader substrate range that were used by the bacteria for nutrient acquisition and that were regulated as the other proteases. Thus, the regulation of toxin synthesis in C. botulinum and C. tetani might represent a reminiscent common regulatory circuit controlling protease synthesis.

In addition to nutrients required for growth and protein synthesis, some nutritional and environmental factors might influence, directly or indirectly, toxin synthesis.

CO₂—A high concentration of CO₂ in the gas phase increases bont expression and BoNT synthesis in non-proteolytic group II C. botulinum B and E, although the growth rate is decreased. Indeed, a 70% CO₂ atmosphere versus 10% stimulates 2- to 5-fold greater toxin gene expression and BoNT formation. In high and low CO₂ concentrations, toxin gene expression occurs in the same growth phase, mainly in the late exponential growth and early stationary phase [94,95]. The signaling pathways in the regulation by CO₂ are not known. CO₂ can dissolve in the liquid medium and generate bicarbonate, which influences protein synthesis through carboxylation reactions. CO₂ (35%) in the gas phase of C. tetani culture versus nitrogen atmosphere (unpublished) or the addition of sodium carbonate (100 mM) in the culture medium increases the production of TeNT approximately two-fold [32], despite reduced growth in the CO₂-rich atmosphere (approximately three-fold). In contrast, elevated CO₂ in the gas phase of proteolytic group I C. botulinum B and E has no effect on toxin gene expression [96], suggesting that CO₂ triggers a signaling pathway controlling toxin synthesis in non-proteolytic strains.

Inorganic phosphate—Inorganic phosphate has been found to control TeNT synthesis in C. tetani. Supplementation of culture medium with inorganic phosphate (optimum concentration 40 mM) stimulates tent expression and TeNT production approximately three-fold without impairing the growth rate [32]. Inorganic phosphate is involved in multiple biochemical reactions; its effect on toxin gene transcription might be mediated by TCSs. C. tetani genome contains two TCSs putatively involved in phosphate uptake, one of which has been found to negatively regulate TeNT synthesis [32]. Inorganic phosphate is apparently not involved in BoNT production in C. botulinum A, as tested by supplementation of the TGY (trypticase-glucose-yeast extract) culture medium with 20 to 150 mM Na₂HPO₄ and monitoring BoNT/A production (strain Hall) in the culture supernatant by titration of the mouse lethal activity (unpublished). Control of the virulence mechanism by inorganic phosphate and TCS from the PhoP/PhoR family has been found in several pathogens [97,98]. TCSs control the homeostasis of phosphate according to the availability in the environment. However, the precise subsequent phosphate-dependent signaling pathways controlling virulence remain largely unknown.

pH—C. botulinum and C. tetani grow and produce the neurotoxins in a wide range of pH (pH 4.5–5 to 9). The initial pH of the growth medium was found to influence the autolysis of C. tetani. An initial pH of 6.1 seems optimal for TeNT production [84]. In a complex medium, high pH (7.8) downregulates tent [99]. The mechanism of toxin gene regulation by pH is not yet identified.

The culture pH of proteolytic C. botulinum A, B grown in complex media typically drops (pH 6–6.3 with initial glucose concentration up to 1%, and until pH 5.5 with glucose 1.5%) during the exponential growth phase, and then stabilizes and slightly increases during the stationary phase [56,85,88,100]. Maintaining an acidic pH (pH 5.7–6) during the culture does not modify the BoNT yield in the culture supernatant, whereas an alkaline pH (pH 7.2 and above, manually adjusted or by supplementation of the culture medium with 2% arginine) decreases the BoNT level [56,88]. The pH does not influence BoNT synthesis at the transcriptional level, but affects BoNT stability by activating a BoNT degrading metalloprotease in alkaline conditions [88].

Temperature—In contrast to C. difficile, in which a high temperature (42 °C) prevents tcdR and toxin gene expression, temperatures of 37–44 °C have no influence on botR and bont transcription in group I C. botulinum A. However, a high temperature induces the production of protease(s), which inactivate BoNT/A [33]. TeNT production is usually obtained by C. tetani culture at 33–35 °C [82,84,99].

Group II C. botulinum has an optimum temperature of 25 °C for growth and toxin production, but the strains of this group can grow and form toxins at temperatures as low as 3.0–3.3 °C in 5 to 7 weeks [101]. Investigation with C. botulinum E showed that growth and toxin production are lower at 10 °C than at 30 °C. However, bontE transcription relative to growth was similar at 10 °C and 30 °C [102]. A TCS is involved in the cold adaptation of C. botulinum E [103,104]. Similarly, cold tolerance of growth at 15 °C in C. botulinum A strain ATCC3502 requires the contribution of a TCS [105]. This TCS is conserved in C. botulinum A strain Hall, but it has not been identified as a regulator of BoNT/A synthesis [50]. Thus, temperature is important for growth and toxin production, but temperature seems to have no direct role in the regulation of toxin synthesis in C. botulinum and C. tetani.