Lucile Garrigues

Thuy Duong Do

Carine Bideaux

Stéphane E Guillouet

Isabelle Meynial-Salles

Insights into Clostridium tetani: from genome to bioreactors

Keywords: Clostridium tetani culture, tetanus toxin production, central metabolism map, nutritional requirements, chemically defined medium

Tetanus vaccination is of major importance for public health in most countries in the world.

The World Health Organization indicated that 15,000 tetanus cases were reported in 2018 (World Health Organization, 2019). Currently, vaccine manufacturers use tetanus toxin produced by Clostridium tetani fermentation in complex media. The complex components, commonly derived from animal sources, introduce potential variability in cultures. To achieve replicable fermentation and to avoid toxic or allergic reactions from animal-source compounds, several studies have tried to switch from complex to chemically defined media without affecting toxin titers. The present review introduces the current knowledge on i) C. tetani strain diversity, whole-genome sequences and metabolic networks; ii) toxin regulation and synthesis; and iii) culture media, cultivation processes and growth requirements. We critically reviewed the reported data on metabolism in C. tetani and completed comparative genomic and proteomic analyses with other Clostridia species. We integrated genomic data based on whole-genome sequence annotation, supplemented with cofactor specificities determined by protein sequence identity, in a new map of C. tetani central metabolism. This is the first data review that integrates insights from omics experiments on C. tetani. The overview of C. tetani physiology described here could provide support for the design of new chemically defined media devoid of complex sources for toxin production.

Introduction

Clostridium tetani is the pathogen responsible for tetanus. The disease is caused by a toxin produced by the bacterium and is not contagious. People suffering from tetanus do not develop any immunization, as the toxin level needed to induce an immune reaction is lethal.

Tetanus remains a public health issue, especially in developing countries. Vaccines that are effective against tetanus are produced from inactivated C. tetani toxin.

C. tetani is a strictly anaerobic bacillus. Its spores are naturally present in soils, especially in warm and humid materials (ground, dejections). When these spores penetrate a wound, their germination is possible under favorable conditions (nonirrigated damaged tissues). The incubation time lasts between three and twenty-one days (World Health [START_REF] Organization | Tetanus vaccines: WHO position paper-February 2017[END_REF].

Bacteria stay in the necrotic wound and release the tetanus neurotoxin. This neurotoxin enters the tissues, reaches the lymphatic system and is then transported by nerves or blood to the central nervous system. The toxin blocks inhibitory neurotransmitters, causing the wellknown tetanus-associated muscular rigidity and spasms [START_REF] Evans | Bacterial infections of humans: epidemiology and control[END_REF].

The tetanus-related death rate reaches 100 % in the absence of treatment. Most of the cases are related to neonatal tetanus, occurring in newborns or their mothers when hygienic conditions are not maintained; approximately 34,000 newborns died from tetanus in 2015, mainly in low-income countries (World Health [START_REF] Organization | Tetanus vaccines: WHO position paper-February 2017[END_REF]. Wound-related tetanus usually occurs in elderly individuals who are not up to date on their vaccination. Thus, because of this high pathogenicity, massive preventive vaccination has to be implemented to protect the population.

The tetanus vaccine is produced from C. tetani culture. The produced native tetanus toxin is then inactivated with formaldehyde to produce a toxoid. Because of this inactivation, the addition of an adjuvant is required to induce an immune response without lethality [START_REF] Smith | Vaccine production, distribution, access, and uptake[END_REF].

For vaccine production, C. tetani strains are industrially grown in complex media derived from Mueller and Miller medium (World Health Organization, 1994). This medium is mainly composed of casein hydrolysate, glucose, beef heart infusion and vitamins. Since pancreatic digests of casein and beef heart infusion are undefined complex materials, they cause important batch-to-batch variability. Mueller reported halved toxin titers when using a new batch of casein digests. After analysis of samples from various stages of casein digest production, the batch-to-batch variability remained unexplained [START_REF] Mueller | Variable factors influencing the production of tetanus toxin[END_REF].

Because of this variability and to avoid toxic or allergic reactions from incomplete digestion of mammalian proteins in complex media, the World Health Organization encourages all vaccine manufacturers to cultivate C. tetani in chemically defined media (World Health Organization, 1994).

A better understanding of C. tetani metabolism and physiology is required in order to simplify media components or to accomplish fermentation with high titers in chemically defined medium. This knowledge could be used to develop a chemically defined medium suitable for tetanus toxin production. This review provides a functional genomics analysis focused on relevant metabolic pathways for toxin production at an industrial scale. The regulation of toxin synthesis is not discussed in depth, as it was already reviewed by the Institute of Microbiology and Genetics (Bruggemann and Gottschalk, 2004) and the Pasteur Institute [START_REF] Connan | Regulation of toxin synthesis in Clostridium botulinum and Clostridium tetani[END_REF].

Clostridium tetani genome and central metabolism

Clostridium tetani strain diversity and genome sequences

To date, 43 C. tetani strains have been isolated, mainly from human wounds, in different geographic locations worldwide (North America, China, France) [START_REF] Chapeton-Montes | The population structure of Clostridium tetani deduced from its pan-genome[END_REF][START_REF] Cohen | Comparative pathogenomics of Clostridium tetani[END_REF]; U.S. National Library of Medicine). Strains are classified into two main groups: the Harvard strains derived from the ancestral Harvard strain (Clade 1A) and the wild-type strains isolated from clinical cases (Clades 1B-H and 2). The ancestral Harvard strain was collected by the Hygienic Laboratory of the US Public Health Service by the end of the First World War. That strain was then distributed to laboratories and vaccine manufacturers from the 1920s to the 1950s. This led to the development of the Harvard family strains, which are characterized by low sporulation and high toxin production and are suitable for vaccine production. The strains E88 and CN655, isolated from the Harvard family, are commonly used in European laboratories.

The 2.8 Mb genome of the C. tetani reference strain was sequenced in 2003 by Brüggemann [START_REF] Bruggemann | The genome sequence of Clostridium tetani, the causative agent of tetanus disease[END_REF]. The genome of C. tetani strain Harvard E88 encodes 2,372 ORFs (28.6 % G + C content) and a unique 74-kbp plasmid (24.5 % G + C content) with 61 ORFs.

That plasmid includes the gene encoding the tetanus toxin (tent), its transcriptional regulator tetR, and genes encoding virulence factors (surface layer and adhesion proteins, collagenase).

On the plasmid, ABC transporter-encoding genes (CTP24-25) were found directly downstream of a two-component system (TCS) composed of a response regulator (CTP21) and a histidine kinase (CTP22). Brüggemann et al. (2004) suggested that this two-component system, located 25 kb before the tetanus toxin gene, might regulate the transcription of these ABC transporter genes. Virulence factors were also found on the chromosome, such as tetanolysin O (CTC1888), hemolysin (CTC586, CTC1574) and fibronectin-binding protein (CTC164, CTC471, CTC1606). These proteins help C. tetani infect damaged tissues.

Among the 20 genes annotated as surface layer proteins in the E88 genome, a surface layer protein encoded by the chromosomal slpA gene (CTC462) was characterized by [START_REF] Qazi | Identification and characterization of the surface-layer protein of Clostridium tetani[END_REF]. The results showed a variability in its molecular weight depending on the strain (160 kDa in CN655, 180 kDa in the clinical isolate NC06336-07, 160 to 180 kDa in the three clinical isolates CTHCM 19, 22 and 25) compared to a 118 kDa predicted protein. No glycosylation was found to explain this variability [START_REF] Qazi | Identification and characterization of the surface-layer protein of Clostridium tetani[END_REF]. Such observations were already described by [START_REF] Takumi | S layer protein of Clostridium tetani: purification and properties[END_REF] with the characterization of the protein from the AO174 strain with a molecular weight of 140 kDa. [START_REF] Takumi | S layer protein of Clostridium tetani: purification and properties[END_REF] found that SlpA had an unusually low proline content and showed various isoelectric forms from pH 4.0 to 4.5. This surface layer protein presented an antigen, making immunological detection possible [START_REF] Takumi | S layer protein of Clostridium tetani: purification and properties[END_REF]). Brüggemann et al. (2004) identified a heme oxygenase (CTC2478) in C. tetani that could confer oxygen tolerance to bacteria during wound infection by creating a local anaerobic environment. This enzyme was also found in another Clostridium wound colonizer (Clostridium perfringens) but not in the other pathogenic or nonpathogenic Clostridia. Thus, C. tetani is an anaerobic microorganism but is reported as an aerotolerant species, similar to C. perfringens (Bruggemann et al., 2004). [START_REF] Dedic | Aerobic cultivation of Clostridium tetani in the presence of cobalt[END_REF] succeeded in growing C. tetani under aerobic conditions by the addition of cobalt (as Co(NO3)2). Under anaerobic conditions, cobalt supplementation did not influence growth. The authors suggested that cobalt addition reduced the oxygen partial pressure of the cultivation medium [START_REF] Dedic | Aerobic cultivation of Clostridium tetani in the presence of cobalt[END_REF]; however, this observation could also be related to the presence of heme oxygenase.

A particularly high number of sodium ion-dependent genes (35) were found in the C. tetani chromosome, and these genes might be related to the wound colonization capacity of this organism. A gene cluster (CTC1337-1352) homologous to the Mrp system was found on the chromosome. This gene cluster, absent in other Clostridia, provides resistance to high levels of sodium and potassium ions and regulates the intracellular pH. The sodium-motive force can also be driven by a Rnf-like complex (RnfC, RnfD, RnfG, RnfE, RnfA and RnfB encoded by CTC1019-1024) that transports the sodium ions out and regenerates NADH and oxidized ferredoxin. This sodium flow could then potentially be used to conserve energy through a Vtype sodium ATP synthase (CTC2326-2332), producing ATP (Bruggemann and Gottschalk, 2004). E88 also harbors the gene encoding the CodY protein (CTC1260), which is a global regulator. This protein is known to indirectly downregulate toxin production in Clostridium difficile [START_REF] Dineen | Repression of Clostridium difficile toxin gene expression by CodY[END_REF] but positively regulate toxin synthesis in C. perfringens [START_REF] Li | CodY Is a Global Regulator of Virulence-Associated Properties for Clostridium perfringens Type D Strain CN3718[END_REF] and Clostridium botulinum [START_REF] Zhang | Positive regulation of botulinum neurotoxin gene expression by CodY in Clostridium botulinum ATCC 3502[END_REF].

There are 28 transposase genes in the E88 genome, but most of them seem to be nonfunctional because of damaged ORFs. Only a few regions have a high G + C content (approximately 50 %): six rRNA gene clusters and ribosomal protein-encoding genes [START_REF] Bruggemann | The genome sequence of Clostridium tetani, the causative agent of tetanus disease[END_REF].

To date, the whole-genome sequences of the 43 isolated strains have been reported (U.S.

National Library of Medicine). All the genome sequences were compared to the genome of the model strain E88. Analysis showed that genetic identity among variants was high. The toxin-encoding genes have similar sequences with 99.3-99.4 % identity among all Harvard strains harboring the tetanus plasmid (two strains lost their plasmid and thus their pathogenicity). These Harvard and wild-type strains also harbor an identical toxin transcriptional regulator gene sequence with 100 % identity [START_REF] Cohen | Comparative pathogenomics of Clostridium tetani[END_REF]. Strainspecific proteins are encoded in their prophage regions. Other differences among these strains are found in their CRISPR/Cas loci and spacer regions. They mainly encode a set of fitness functions that protect these strains from environmental stress or new prophage infections [START_REF] Bruggemann | Genomics of Clostridium tetani[END_REF]. Among the 43 C. tetani genome sequences reported, [START_REF] Chapeton-Montes | The population structure of Clostridium tetani deduced from its pan-genome[END_REF] analyzed 38 genome sequences and showed that the core genome represents 77 % of the E88 genome. The 38 strains share 1,266 coding sequences (CDS; 32 % of the total CDS). The Harvard strains only differ by 292 single-nucleotide polymorphisms (SNPs) in their whole genomes and share a 100 % identical tetanus toxin at the protein level. They harbor strain-specific genes, encoding "a plasmid-like element carrying a toxin-antitoxin system, a gene cluster encoding surface-layer proteins, an iron transport system and a putative cell wall/spore coat/envelope/membrane modification system" [START_REF] Chapeton-Montes | The population structure of Clostridium tetani deduced from its pan-genome[END_REF]. Phylogenetic analysis showed that the C. tetani plasmid evolved together with the chromosome and is not a recent genomic element. This reflects the genomic stability of C. tetani [START_REF] Chapeton-Montes | The population structure of Clostridium tetani deduced from its pan-genome[END_REF][START_REF] Cohen | Comparative pathogenomics of Clostridium tetani[END_REF]. The C. tetani genome is considered to be stable in comparison with the genomes of other species, such as C. botulinum [START_REF] Chapeton-Montes | The population structure of Clostridium tetani deduced from its pan-genome[END_REF]. The strains used for C. tetani experiments in the laboratory belong to the same genomic subgroup (the Harvard family, clade 1A) and have identical toxin-and toxin transcriptional regulator-encoding genes.

Clostridium tetani central metabolism

The substrates most commonly used by C. tetani are amino acids, as many genes for their transport and degradation were identified in its genome (Table 1-A). The amino acid transport seems to be mainly sodium dependent, occurring through symporters (Bruggemann and Gottschalk, 2004). C. tetani transports and catabolizes numerous amino acids. The regulation of this gene expression was, up to now, poorly understood. Recently, [START_REF] Orellana | Time-course transcriptomics reveals that amino acids catabolism plays a key role in toxinogenesis and morphology in Clostridium tetani[END_REF] used a time-course comparative transcriptomics approach to better understand how the amino acid degradation pathways were expressed when the E88 strain was grown in complex medium with or without supplementation with five amino acids. The results showed that histidine and aspartate were mainly degraded to glutamate, which was degraded to acetate, butyrate, pyruvate and ammonium via the methylaspartate pathway. Serine was degraded mainly to pyruvate via the serine ammonia-lyase pathway (CTC1981-1982), threonine to glycine and acetaldehyde, and tyrosine to pyruvate and phenol via the tyrosine phenol lyase pathway (CTC818). Methionine was found to be mainly converted to 2-oxobutanoate and propionyl-CoA. However, other degradation pathways of these amino acids were found to be induced at the transcriptional level but to a lesser extent, and this induction was dependent on the cultivation phase (Table 1-B) As C. tetani assimilates many amino acids, its genome lacks biosynthetic pathways for at least phenylalanine, histidine, isoleucine, lysine, leucine, methionine, tryptophan and valine, causing amino acid auxotrophy. This is typical for pathogenic bacteria with small genomes: they do not develop pathways for amino acid biosynthesis because they live in a host [START_REF] Yu | Amino acid biosynthesis deficiency in bacteria associated with human and animal hosts[END_REF]. In addition to amino acid transport and degradation genes, C. tetani harbors numerous extracellular and intracellular peptidase-and (phospho)lipase-encoding genes. This set of peptidases, which are characteristic of pathogenic Clostridia, provides free amino acids to the bacteria. Genome sequence analysis data indicates that C. tetani, similar to Clostridium butyricum, could ferment glycerol to produce 1,3-propanediol [START_REF] Gonzalez-Pajuelo | Microbial conversion of glycerol to 1,3-propanediol: physiological comparison of a natural producer, Clostridium butyricum VPI 3266, and an engineered strain, Clostridium acetobutylicum DG1(pSPD5)[END_REF] (glycerol kinases CTC1758, CTC2462; glycerol-3-phosphate dehydrogenases CTC596, CTC1139, CTC1808, CTC2436; glycerol dehydratases CTC936, CTC1449), ethanolamine (gene cluster CTC2163-2181) and inositol (gene cluster CTC508-514) (Bruggemann and Gottschalk, 2004). (Bruggemann and Gottschalk, 2004). It was experimentally demonstrated in Clostridium acetobutylicum [START_REF] Yoo | A Quantitative System-Scale Characterization of the Metabolism of Clostridium acetobutylicum[END_REF] and Clostridium kluyveri (F. [START_REF] Li | Coupled ferredoxin and crotonyl coenzyme A (CoA) reduction with NADH catalyzed by the butyryl-CoA dehydrogenase/Etf complex from Clostridium kluyveri[END_REF] that butyryl-CoA dehydrogenase (Bcd) is an NADH-and ferredoxin-dependent enzyme. The Bcd enzyme in C. tetani (CTC2426) showed 82.6 % and 74.9 % identity with the Bcd enzyme from C. acetobutylicum and C. kluyveri, respectively, strongly suggesting its NADH and ferredoxin dependence in C. tetani. The Hbd enzyme was shown to be NADH dependent in C. acetobutylicum and NADPH dependent in C. kluyveri [START_REF] Yoo | Trends in Systems Biology for the Analysis and Engineering of Clostridium acetobutylicum Metabolism[END_REF]. The Hbd CTC2423 from C. tetani has a higher sequence identity with C. acetobutylicum (80.5 %) than with C. kluyveri (68.9 %), suggesting that it is an NADH-dependent enzyme. Both the AdhE1 and AdhE2 aldehyde-alcohol dehydrogenases from C. acetobutylicum were biochemically characterized in vitro and shown to be strictly NADH dependent [START_REF] Yoo | A Quantitative System-Scale Characterization of the Metabolism of Clostridium acetobutylicum[END_REF]. Both enzymes have dual functionality, participating in the conversion of i) butyryl-CoA to butyraldehyde and ii) acetyl-CoA to acetaldehyde. A similar enzyme, CTC1366, sharing 68.2 % amino acid identity with AdhE1 and 67.2 % amino acid identity with AdhE2, was identified in C. tetani. The three enzymes showed a conserved GCGXWG domain, which is commonly involved in coenzyme binding [START_REF] Fontaine | Molecular characterization and transcriptional analysis of adhE2, the gene encoding the NADHdependent aldehyde/alcohol dehydrogenase responsible for butanol production in alcohologenic cultures of Clostridium acetobutylicum ATCC824[END_REF]. Thus, on the basis of amino acid identity, aldehyde-alcohol dehydrogenase in C. tetani is likely NADH dependent.

Finally, a butanol dehydrogenase, CTC408, catalyzing the conversion of butyraldehyde to butanol, was also identified in C. tetani. This enzyme shares 66.8 % amino acid identity with BdhA and 63.8 % amino acid identity with BdhB from C. acetobutylicum, which were biochemically demonstrated in vitro to be NADPH dependent [START_REF] Yoo | A Quantitative System-Scale Characterization of the Metabolism of Clostridium acetobutylicum[END_REF]. Thus, butanol dehydrogenase in C. tetani could be considered to be NADPH dependent.

Extrapolation to C. tetani based on the above % amino acid identity suggests that butanol production could be dependent on NADH and NADPH in C. tetani as well. The general metabolic network in C. tetani is outlined in Figure 1. The enzymes involved in this metabolism are detailed with the number for the encoding gene. Their in vivo functionality has not been demonstrated. This metabolic map is not exhaustive. However, some uncertainties remain. Several amino acid transporters have not been identified (histidine, aspartate, arginine and lysine transporters). The glutamate/glutamine degradation pathway that functions through methylaspartate was first shown to be functional in C. tetani NCTC 5404 using C-labeled glutamate [START_REF] Buckel | Two pathways of glutamate fermentation by anaerobic bacteria[END_REF]. In 2016, Licona-Cassani et al., using in-depth transcriptional analysis, observed high expression of the methyl aspartate mutase cluster genes during the exponential phase, suggesting that glutamate is probably also metabolized via the mesaconate pathway in the E88 strain.

Clostridium tetani bioenergetics

Twenty-one iron-sulfur proteins with a [4Fe4S] core pattern and twenty-six homologous flavoproteins were found in C. tetani, suggesting a significant role of these electron transport proteins in metabolism. Among these iron-sulfur proteins, ferredoxins take part in electron transfer in C. tetani. Ferredoxin is reduced during the conversion of pyruvate to acetyl-CoA.

Reoxidation of reduced ferredoxin generates an H + flow, which can be converted to dihydrogen by hydrogenases or used by ferredoxin NAD + oxidoreductases such as the Rnf complex. In addition, FeS clusters are found, in particular, in a membrane-bound system (CTC1019-1024) homologous to the Rnf complex (Rhodobacter-specific nitrogen fixation), described in acetogenic bacteria as a respiratory enzyme complex catalyzing the oxidation of reduced ferredoxin and the reduction of NAD + , generating a transmembrane ion gradient. In Acetobacterium woodii, the Rnf complex was proposed to be coupled to the translocation of sodium ions across the cytoplasmic membrane [START_REF] Schuchmann | Autotrophy at the thermodynamic limit of life: a model for energy conservation in acetogenic bacteria[END_REF]. Thus, the Rnf-like system in C. tetani is suspected to maintain the sodium-motive force in the cell, which is essential for amino acid transport and for transport of electron flow from ferredoxin activity to regenerate the NADH pool. The (V)-type ATPases are the main ATPases in C. tetani (CTC993-1001, CTC2326-2332). The ATPase CTC993-1001 also participates in the transport of Na + ions out of the cell, reinforcing the sodium-motive force of the Rnf-like system. This sodium transport is then combined with energy dissipation. No F0-F1-type ATPase was found in C. tetani, which is unusual. Nonetheless, in contrast to other Clostridia, C. tetani harbors a gene cluster (CTC2326-2332) encoding a V-type sodium ATP synthase homologous to the archaeal-type ATP synthase (Bruggemann and Gottschalk, 2004). This complex could use the extracted sodium from the Rnf system to conserve energy with ATP synthesis. [START_REF] Licona-Cassani | Tetanus toxin production is triggered by the transition from amino acid consumption to peptides[END_REF] showed, by generating a transcriptional molecular map of an E88 culture on complex medium, the activation of genes encoding Rnf complex proteins, a flagellum-specific ATP synthase, calcium-transporter ATPases and genes associated with V-type ATPase synthase.

An H + pump (V-Type pyrophosphatase CTC383), coupled with the cleavage of inorganic pyrophosphate, was also found in C. tetani, which is uncommon in bacteria. In addition, a specific sodium ABC transporter (CTC1485) and several H + /Na + antiporters (CTC567, CTC901, CTC1183, CTC1423, CTC1853, CTC2161, CTC2520, CTC2529) were identified in the genome. Brüggemann et al. (2004) suggested that this predominant sodium ion bioenergetic profile could be linked to main fermentation pathways in C. tetani: the amino acid utilization pathways.

Tetanus toxin regulation and synthesis

Tetanus toxin regulation

Toxin regulation by an alternative sigma factor

The genes encoding the tetanus toxin and its regulator are located on the mega-plasmid of C. tetani [START_REF] Raffestin | BotR/A and TetR are alternative RNA polymerase sigma factors controlling the expression of the neurotoxin and associated protein genes in Clostridium botulinum type A and Clostridium tetani[END_REF]: i) the tetX gene (also written tent) encodes the toxin, and ii) the tetR gene, which is located immediately upstream of tetX, encodes the regulator of the tetanus toxin. TetR is an alternative sigma factor (from group 5 of the σ 70 family) that positively regulates the expression of tetX. It binds to an enzyme, the RNA polymerase core enzyme, to initiate the transcription of the tetX gene. The core enzyme and its sigma factor, combined with the tetX promoter, are necessary for tetanus toxin production. The tetanus toxin locus is described in Figure 2 [START_REF] Raffestin | BotR/A and TetR are alternative RNA polymerase sigma factors controlling the expression of the neurotoxin and associated protein genes in Clostridium botulinum type A and Clostridium tetani[END_REF]. The promoter of the tetanus toxin gene (PtetX), transcribed by TetR, is expanded with conserved sequences -35/-10 (gray boxes). The solid bar represents a putative promoter that is not transcribed by TetR, located upstream of the tetR gene (ptetR) [START_REF] Raffestin | BotR/A and TetR are alternative RNA polymerase sigma factors controlling the expression of the neurotoxin and associated protein genes in Clostridium botulinum type A and Clostridium tetani[END_REF].

The genetic organization of the tetanus toxin operon is similar to that of the botulinum toxin operon. Indeed, the botulinum sigma factor BotR/A showed 60 % amino acid identity with TetR. Moreover, in vivo overexpression of the exogenous BotR/A in C. tetani cells positively regulated TetX synthesis, illustrating the functional similarity between TetR and BotR/A [START_REF] Marvaud | TetR is a positive regulator of the tetanus toxin gene in Clostridium tetani and is homologous to BotR[END_REF].

Toxin regulation by Two-Component Systems

Tetanus toxin synthesis is regulated by a complex network of TCSs. For toxin synthesis, at

Toxin regulation related to virulence factors and cell division

Chapeton-Montes et al. ( 2020) showed that toxin synthesis was also regulated by CodY. This protein, often involved in toxin and virulence regulation in other Gram-positive pathogenic bacteria, exhibited binding to the tetX promoter. This increased tetX transcription, which positively regulated toxin synthesis. As CodY is known to respond to starvation conditions, the authors suggested that this toxin regulation could be related to nutritional stress [START_REF] Chapeton-Montes | Tetanus Toxin Synthesis is Under the Control of A Complex Network of Regulatory Genes in Clostridium tetani[END_REF]. Comparative transcriptional analysis of E88 grown in complex medium supplemented or not with an amino acid mixture suggested also that toxin production and virulence factors were related [START_REF] Orellana | Time-course transcriptomics reveals that amino acids catabolism plays a key role in toxinogenesis and morphology in Clostridium tetani[END_REF]. Specifically, the decrease in expression of virulence-related surface/cell-adhesion genes (CTC769-770, CTC772, CTC776-777) and flagellar genes considered as virulence-related genes in other pathogens (CTC1653-1679) affected toxin production. However, the genes encoding the homologous TCS of the virulence genes VirS/VirR in C. perfringens showed no difference in expression, regardless of the tetanus toxin production level.

C. tetani phenotypes seemed to depend on toxin synthesis, with the long-chained filaments appearing in the culture with the highest toxin production [START_REF] Orellana | Time-course transcriptomics reveals that amino acids catabolism plays a key role in toxinogenesis and morphology in Clostridium tetani[END_REF]). Yet, the expression of cell division and cell wall elongation genes was not linked to toxin synthesis when the E88 strain was grown in complex medium supplemented or not with a mixture of five amino acids. However, the transcription of the genes (CTC280, CTC316, CTC595, CTC2066) encoding autolysin proteins, which are involved in cell separation and cell shape, was inhibited under high toxin production, explaining these different cell morphologies.

Toxin production and maturation

The tetanus toxin is synthesized in the bacterial cytosol as a 151-kDa chain protein (1315 amino acids). Its maturation process is illustrated in Figure 3.

After translation of the RNA to protein, the first methionine residue is removed, and two cysteine disulfide bridges are formed. The tetanus toxin is released in the culture medium.

The toxin peptide chain is then hydrolyzed by a 27-kDa protease in the culture broth, resulting in a toxin composed of a 52-kDa light chain and a 98-kDa heavy chain (T. B. [START_REF] Helting | Structure of tetanus toxin. Demonstration and separation of a specific enzyme converting intracellular tetanus toxin to the extracellular form[END_REF]. One disulfide bond links the heavy chain to the light chain, and another one forms a loop on the heavy chain. The single-chain toxin is cleaved between Glu449 and Asn450.

However, toxins were also found in the culture broth as cleaved from Glu449 to Ser457, missing the junction residue. This protein nicking considerably increases its toxicity [START_REF] Krieglstein | Limited proteolysis of tetanus toxin. Relation to activity and identification of cleavage sites[END_REF]. both on light and heavy chains) and fragment C (47-kDa, on heavy chain). Fragment C is the toxin part that triggers the strongest antigenic reaction, although it is not toxic [START_REF] Helting | Structure of tetanus toxin. I. Breakdown of the toxin molecule and discrimination between polypeptide fragments[END_REF]. [START_REF] Schiavo | Tetanus Toxin Is a Zinc Protein and Its Inhibition of Neurotransmitter Release and Protease Activity Depend on Zinc[END_REF] identified one zinc atom linked to the light chain playing a catalytic role.

This atom is linked to a two-histidine motif, characteristic of metalloendopeptidases. Without zinc, the tetanus toxin cannot inhibit neurotransmitters and therefore cannot cause disease [START_REF] Schiavo | Tetanus Toxin Is a Zinc Protein and Its Inhibition of Neurotransmitter Release and Protease Activity Depend on Zinc[END_REF].

The tetanus toxin is very stable among the different strains because of its preserved amino acid sequence. Only a few changes in its amino acid composition were identified in clinical isolate strains. Its histidine pattern (HExxH) and its binding residues are always conserved [START_REF] Chapeton-Montes | The population structure of Clostridium tetani deduced from its pan-genome[END_REF].

Toxin tridimensional structure

The crystal structure of the full-length tetanus toxin was obtained for the first time by With solution scattering analysis, [START_REF] Masuyer | The structure of the tetanus toxin reveals pHmediated domain dynamics[END_REF] showed that the tetanus toxin structure is pH dependent. At acidic pH (under 5.5), the toxin is in a compact form (Figure 4).

Its structure adopts an extended conformation at pH values greater than 6.3. Between these two values, the toxin is in a semiopen conformation [START_REF] Masuyer | The structure of the tetanus toxin reveals pHmediated domain dynamics[END_REF].

Analytical methods for toxin detection

Flocculation method

Historically, the amount of tetanus toxin has been visually evaluated using the Ramon flocculation method [START_REF] Ramon | La floculation dans les mélanges de toxine et de sérum antidiphtérique[END_REF]. This method was first designed for diphtheria toxin titration and was subsequently adopted for tetanus toxin. This titration consists of an antigenantibody reaction. The antigen is located on the toxin, and the antibody is an antitetanus serum. This method requires a range of standard test tubes prepared with a serum in which the concentration of antitetanus antibody is well known. Then, after sample addition, white flocs appear in the tube with the best balance between toxin and serum. It is the first test tube in which flocculation occurs that indicates the toxin titer of the sample. For faster flocculation, tubes should be incubated in a water bath at up to 45°C. Titers determined with the Ramon method are expressed in Lf/mL of supernatant (where Lf stands for limit of flocculation). As it was for decades the only titration method for tetanus toxin, it is the only method currently approved by health authorities.

Minimal lethal dose

Few researchers use the minimal lethal dose value (MLD/mL of supernatant) to evaluate the toxin content and activity. This test, often performed on mice, consists of injecting diluted supernatant into animals. Then, knowing the greatest dilution associated with animal death, it is possible to calculate the MLD units contained in one sample. For reference, the MLD for humans is 2.5 ng/kg [START_REF] Gill | Bacterial toxins: a table of lethal amounts[END_REF]. The MLD method yielded more accurate results. However, as it requires animal resources and is labor intensive, this method is not often used.

Enzyme-linked immunosorbent assay

A routine test for tetanus antibody detection has been described by the World Health Organization (World Health Organization, 2013). This test, based on a capture enzyme-linked immunosorbent assay (ELISA), determines the tetanus toxoid content in the vaccine. The procedure consists of coating a monoclonal antibody on microplates. This antibody links the heavy chain of the toxoid. Then, wells are filled with standards and samples. Then, a polyclonal antibody is dropped in the wells. This antibody is recognized by a labeled secondary antibody, which is visualized by a colorimetric substrate. The optical density of the sample is correlated in log scale with the amount of toxoid in the sample. The detection limit of this method is 0.002 Lf/mL.

As the toxin and toxoid have the same antibody recognition sites, this test can be performed for toxin quantification. Monoclonal and polyclonal antibodies can be switched using the appropriate secondary antibody. This method is more accurate for toxin determination than the flocculation test or MLD measurement. However, it is not routinely used because it is not approved by the World Health Organization.

Clostridium tetani fermentation processes

Clostridium tetani growth phases in batch mode

C. tetani is mainly cultivated under batch mode in closed tubes or bottles. Only the end biomass and final toxin titer were reported, with no information on growth or toxin kinetics.

Some bioreactor cultures were also described (Table 3). [START_REF] Jagicza | Large-scale production of tetanus toxin in fermentor[END_REF] reported an industrial culture of the Harvard strain in Mueller and Miller medium. The biomass grew for three days from 10 5 to 10 8 cells/mL, with a pH decrease from 7.3 to 6.6. The toxin was intracellularly produced after one day of culture until the end of growth. The toxin was then released in the medium at a final toxin titer of 55 Lf/mL. A similar growth profile was also observed in a closed flask by [START_REF] Fratelli | Effect of medium composition on the production of tetanus toxin by Clostridium tetani[END_REF] with a maximal biomass concentration of 4.4 g/L and a final toxin titer of 40 Lf/mL using the Harvard-Caracas strain grown in Massachusetts medium [START_REF] Fratelli | Effect of medium composition on the production of tetanus toxin by Clostridium tetani[END_REF]. In 2016, Licona-Cassani et al. reported batch cultivation of the E88 strain on Massachusetts medium supplemented with cystine, uracil, vitamins and traces amounts of FeCl3 (Table 3). During cultivation (in five independent experiments), regular sampling was applied to rigorously follow the growth of C. tetani in the bioreactor. Two distinct growth phases were observed: i) an exponential growth (µ = 0.24 h -1 ) phase leading to a decrease in pH (from 6.8 to 6.6), lasting 10 h and exhibiting preferential consumption of free amino acids and ii) a slower growth (µ = 0.083 h -1 ) phase with an increase in pH (from 6.6 to 7.0), lasting 30 h, and associated with the consumption of peptides and glucose. The main virulence factor genes were activated, including tetX. The growth was then followed by a cell autolysis phase, lasting 30 h, with a decrease in optical density and an increase in pH (from 7.0 to 8.0). This autolysis appeared to be related to the physiological cell state. These five cultivations resulted in the production of 30.2 ± 2.86 Lf/mL toxin [START_REF] Licona-Cassani | Tetanus toxin production is triggered by the transition from amino acid consumption to peptides[END_REF], which is lower than the values for all other batch cultures reported in the literature (Table 3).

Continuous culture of Clostridium tetani

A continuous culture of a C. tetani Harvard derivative strain (strain 107) was reported in the literature. In 1968, Zacharias and Björklund studied the effects of different i) dilution rates (0.030 h -1 to 0.169 h -1 ), ii) temperatures (32°C to 39°C), iii) pH values (5.9 to 9.5) and iv) salts (0.1 g/L KCl, 0.15 g/L CaCl2) in a modified Massachusetts medium (all concentrations reduced by half). For each culture, one of these parameters was varied. The best toxin titer (120 Lf/mL toxin) was reached at a dilution rate of 0.125 h -1 , a temperature of 34°C and a pH of 7.4 in the modified Massachusetts medium supplemented with 0.1 g/L of potassium chloride. Growth rates from 0.03 h -1 to 0.125 h -1 resulted in similar biomass and toxin production, with a biomass concentration of 0.7 g/L and a toxin titer of 70-75 Lf/mL.

Potassium was used to increase membrane permeability and thus toxin release in the extracellular cultivation medium. The element controlling the growth at steady state was not described [START_REF] Zacharias | Continuous production of Clostridium tetani toxin[END_REF].

Fed-batch culture of Clostridium tetani

In 2010, Fratelli et al. cultured the C. tetani Harvard-Caracas strain in fed-batch conditions.

The authors evaluated the impact of different initial concentrations of casein and glucose in static bottles with pulsed glucose addition. The best culture conditions were obtained with an initial casein peptone concentration between 50 and 62.5 g/L and an initial glucose concentration from 0.75 to 1.25 g/L. The glucose concentration was then adjusted to 3 g/L by pulsed glucose addition at 16, 56 and 88 h of culture. These fed-batch cultures produced 60

Lf/mL tetanus toxin (compared to 15 Lf/mL in the reference batch cultures) [START_REF] Fratelli | Fed-batch production of tetanus toxin by Clostridium tetani[END_REF].

Based on the previously performed cultures (Table 3), the process that produced the highest toxin concentration seemed to be the continuous cultivation mode, with the concentration reaching 120 Lf/mL in modified Massachusetts medium. The advantage of the continuous culture process is that the cells are maintained in the exponential growth phase.

Optimal pH for Clostridium tetani cultivation

In continuous culture, the optimal pH for toxin production was 7.8, with the ability to produce toxin from pH values of 6.2 to 8.8 [START_REF] Zacharias | Continuous production of Clostridium tetani toxin[END_REF]). However, it was recommended that cultivation be performed at pH 7.4 since a higher pH leads to iron precipitation, producing a black color that interferes with biomass determination. To better understand the effect of pH, toxin stability was evaluated at lower and higher pH values, but no toxin degradation was found at these extreme pH values. Therefore, the authors suggested that the pH value affected either some enzymes involved in toxin production or the release capacity of the membrane.

In batch cultures, pH is usually not regulated. The initial pH is commonly in the range 6.8-7.2. Demain et al. (2003) showed that the initial pH had an influence on the autolysis phase.

The cell lysis percentage decreased with increasing initial pH values. The authors obtained the best toxin titer (67.5 Lf/mL) at an initial pH of 6.1, corresponding to a final pH of 7.75 (Demain and Fang, 2003).

6. Nutritional needs of Clostridium tetani for bacterial growth and toxin production

Histidine peptide consumption from complex amino acid sources

To determine the role of the consumption of amino acids released upon hydrolysis of peptides, [START_REF] Mueller | Participation of peptides in tetanus toxin production[END_REF] studied the effect of pancreatic digest of casein used for C.

tetani Harvard strain cultures. The pancreatic digest of casein was fractionated into three parts (acidic, neutral and basic). They found that the omission of any one of these parts eliminated toxin production. Toxin production was also inhibited when one of the fractions was hydrolyzed by acid. They also learned that free histidine did not satisfy the histidine requirements. Histidine had to be present in peptide form, such as glycyl-histidine or acetylhistidine, to start toxin production. Thus, Mueller's team suggested that this requirement for the peptide-bound form could also exist for serine, glutamic acid and aspartic acid [START_REF] Mueller | Participation of peptides in tetanus toxin production[END_REF]. [START_REF] Mueller | Essential role of histidine peptides in tetanus toxin production[END_REF] ran further experiments to study the effect of histidine peptides on toxin production by substituting the histidine part of the basic fraction of the pancreatic digest of casein with synthetic peptides. Eight different histidine peptides were evaluated: i) glycylhistidine was found to be the most effective histidine peptide, since its use produced as much toxin as the use of the whole pancreatic digest of casein (130 Lf/mL and 135-140 Lf/mL, respectively), followed by L-α-amino-n-butyryl-L-histidine; ii) β-L-aspartyl-L-histidine, acetyl-histidine and L-carnosine were efficient only at very high concentrations; and iii) anserine, 1-methyl-histidine, and 3-methyl-histidine resulted in no toxin production. They concluded that the structural specificity of the linkage of histidine was the stimulatory element. Moreover, free histidine still had to be part of the medium, but it contributed only to bacterial growth [START_REF] Mueller | Essential role of histidine peptides in tetanus toxin production[END_REF].

High histidine peptidase activity was measured from the beginning of growth, whereas intracellular toxin production started only after 10 h of cultivation [START_REF] Miller | Formation and action of a peptidase which hydrolyzes histidine peptides required in tetanus toxin synthesis[END_REF]. The enzyme was only synthesized in media containing histidine peptides, leading to enhanced toxin production. Moreover, Fe 2+ stimulated peptidase activity. The histidine peptidase was able to hydrolyze glycyl-L-histidine and L-α-amino-n-butyryl-L-histidine. The hydrolysis of acetyl-histidine was weaker and needed a high concentration of this peptide, corresponding to the observation already made by [START_REF] Mueller | Essential role of histidine peptides in tetanus toxin production[END_REF]. The peptidase activity was the same in cell-free assays. Therefore, the superiority of glycylhistidine as a peptidase substrate or toxin synthesis effector was not dependent on its membrane transport. The authors demonstrated that toxin production was directly proportional to histidine peptide hydrolysis. They also checked that the toxin and the histidine peptidase were not the same protein [START_REF] Miller | Formation and action of a peptidase which hydrolyzes histidine peptides required in tetanus toxin synthesis[END_REF]. Later, Helting et al. found this enzyme in the supernatant and confirmed that it was active toward glycyl-histidine and not responsible for the proteolysis to a mature toxin [START_REF] Helting | Structure of tetanus toxin. Demonstration and separation of a specific enzyme converting intracellular tetanus toxin to the extracellular form[END_REF].

Casein peptide consumption from complex amino acid sources

To further evaluate the impact of the casein pancreatic digest (NZ-Case) and identify the casein peptides with an effect on toxin synthesis, cultivation was performed for NZ-Case fractions with a Harvard strain in modified Massachusetts medium (supplemented with 1.01 g/L KH2PO4, 0.72 g/L CaCl2 and 0.36 g/L active charcoal). [START_REF] Porfirio | Specific peptides of casein pancreatic digestion enhance the production of tetanus toxin[END_REF] found that only the omission of the neutral fraction of the casein digest decreased toxin production, by approximately 30 %, suggesting that this fraction contains one or several elements promoting toxin synthesis. The omission of the acidic or the basic fraction of the casein digest did not affect the toxin titer. Hence, they purified peptides from the neutral part and individually added them to the Massachusetts medium. They showed that the following peptides were the most active in toxin production, with a final toxin titer reaching 200 % to 265 % of the toxin production in Massachusetts medium:

-Ile -Pro -Ile -Gln -Tyr -Val -Val -Leu -Gly -Pro -Val -Ala -Val -Pro -Tyr -Pro -Gln -Asp -Met -Pro -Ile -Val -Ala -Pro -Phe -Pro -Glu -Val -Phe -Glu -Met -Pro -Phe -Pro -Lys
They also pointed out that the glycyl-L-histidine peptide from the basic part of the casein digest favored toxin synthesis.

Zinc proteases are found in casein the tryptic digest, which is a component of Massachusetts medium (Table 2). The pattern Pro-hydrophobic-Pro is characteristic of several zinc peptidase inhibitors, and the tetanus toxin is a zinc protein. Thus, the authors suggested that the presence of these Pro-hydrophobic-Pro peptides could increase toxin production by protecting the toxin from proteolytic degradation. They also proposed that these peptide patterns could act as peptide-hormone signals in bacteria for toxin synthesis [START_REF] Porfirio | Specific peptides of casein pancreatic digestion enhance the production of tetanus toxin[END_REF].

Licona-Cassani et al. ( 2016) identified specific peptides from casein digestion (NZ-Case) that were specifically consumed by C tetani E88. The authors pointed out that peptides were consumed after exhaustion of free amino acids. They suggested that this peptide consumption could explain the decrease in the specific growth rate, as peptide transport requires more energy than free amino acid transport. The consumed peptides presented the following specific shared patterns:

-Val -Pro -Gln -Leu -Glu -Ile -Val -Val -Tyr -Pro -Phe -Pro -Gly -Pro -Ile

The last peptide harbors the Pro-Phe-Pro pattern, which was described by Porfirio et al.

(1997) as a zinc protease inhibitor.

Licona-Cassani et al. ( 2016) also noticed that peptide consumption was related to tetX expression in a transcriptional analysis, concluding that these specific peptides were essential for activation of the production of tetanus toxin [START_REF] Licona-Cassani | Tetanus toxin production is triggered by the transition from amino acid consumption to peptides[END_REF].

Furthermore, toxin synthesis has been shown to depend on the content of Maillard reaction products. When the medium was sterilized by filtration or autoclaved for too long, no toxin was detected. The production of toxin required an adequate amount of Maillard reaction products, produced from the peptide-sugar reaction under heat [START_REF] Chung | Tetanus toxin production from Clostridium tetani, using a casein-based medium in a single-use bioreactor[END_REF].

Considering the studies on the role of peptides in toxin production, it seems that glycylhistidine and the Pro-Phe-Pro peptide pattern have a significant effect on toxin production.

They could be used to design a chemically defined medium promoting toxin synthesis.

Free amino acid consumption for C. tetani growth and toxin production

Despite the influence of peptides in toxin production, studies were conducted to design a chemically defined medium that could support C. tetani growth and toxin production. Two different media were evaluated (Table 4-A) (Feeney et al., 1943b;[START_REF] Licona-Cassani | Tetanus toxin production is triggered by the transition from amino acid consumption to peptides[END_REF]). Glucose was present at the same concentration in both media, but the initial µ = 0.24 h -1 during exponential growth, then µ = 0.083 h -1 during slower growth µ = 0.69 h -1 1 Strain not specified; probably E88, as that was the only one sequenced at that time 2 Massachusetts medium supplemented with cystine, uracil and vitamins and with trace amounts of FeCl3 29 3 Tryptophan was not tested in this chemically defined medium but was identified as being essential in a previous experiment in complex medium (Mueller & Miller, 1942).

Essential: no growth when the amino acid was omitted. Stimulated: little growth after 48 h of culture when the amino acid was omitted. Less effective: Good growth after 48 h of culture when the amino acid was omitted.

Xmax: maximum biomass concentration; µ: specific growth rate Among the eight putative auxotrophic amino acids [START_REF] Yu | Amino acid biosynthesis deficiency in bacteria associated with human and animal hosts[END_REF], only five (His, Ile, Leu, Trp, Val) were considered essential by . The authors also identified arginine, isoleucine, tryptophan and valine as being essential for C. tetani growth. [START_REF] Mueller | Glutamine in the production of tetanus toxin[END_REF] pointed out that addition of glutamine could partially replace casein digestion. Using the Harvard strain in test tubes, a lowered casein content in the medium resulted in 3-fold lower toxin production. The addition of 0.25 g/L glutamine recovered half of the usual toxin titer (50 Lf/mL with glutamine to 90 Lf/mL for the control tube) [START_REF] Mueller | Glutamine in the production of tetanus toxin[END_REF]. This amino acid was totally consumed in the chemically defined medium described by [START_REF] Licona-Cassani | Tetanus toxin production is triggered by the transition from amino acid consumption to peptides[END_REF].

Among the amino acids potentially identified as auxotrophic by [START_REF] Yu | Amino acid biosynthesis deficiency in bacteria associated with human and animal hosts[END_REF], only histidine and leucine were significantly consumed in the culture grown in Licona-Cassani chemically defined medium (Table 4-B). Arginine, isoleucine, tryptophan and valine (identified as essential by Feeney et al.) were slightly or not consumed in the Licona-Cassani experiment in both complex and chemically defined media. Therefore, one can wonder if these amino acids are required for C. tetani growth. Growth stopped after the depletion of five amino acids (Glu, Ser, Gln, His, Thr), suggesting possible new auxotrophies for glutamate, serine, glutamine and threonine. By the end of the stationary phase, three other amino acids were completely consumed (Asp, Asn, Met). Overall, C. tetani assimilated thirteen amino acids to perform growth until a maximal optical density (530 nm) of 1.3 at a specific growth rate of µ = 0.69 h -1 (Figure 5). From the observations in these two experiments in chemically defined medium and in combination with casein peptide studies, histidine seems to be of major importance for C. tetani growth. 2016) observed in a modified Massachusetts medium that the depletion of free amino acids was correlated with the beginning of the slower growth phase when peptides started to be consumed. On the chemically defined medium, no toxin production was observed. As tetX expression is triggered when metabolism changes from amino acid to peptide consumption, tetanus toxin production might be caused by this metabolic transition.

In the culture performed in a modified Massachusetts medium, the genes encoding the degradation of glutamate, histidine, serine, threonine and aspartate were found to be highly expressed during the exponential and the slower growth phases, showing the strong consumption of these amino acids [START_REF] Licona-Cassani | Tetanus toxin production is triggered by the transition from amino acid consumption to peptides[END_REF]. In further experiments, these five amino acids were supplemented in the modified Massachusetts medium (+ 0.8 g/L of each amino acid) [START_REF] Orellana | Time-course transcriptomics reveals that amino acids catabolism plays a key role in toxinogenesis and morphology in Clostridium tetani[END_REF]. This resulted in a 2.5-fold increase in cell density, with a longer exponential growth phase (8.5 h instead of 5 h, with the same growth rate (0.46 h -1 ) as that in the control experiment). The slower growth phase showed a twofold increase in growth rate with the addition of amino acids (0.145 h -1 instead of 0.06 h -1 for the control culture); however, the final toxin titer was twofold lower. Moreover, the genes involved in the aspartate degradation pathway for pyrimidine metabolism (CTC2383-2384) were expressed during the exponential growth phase, rather than during the slower growth phase, as observed in the control culture. Therefore, aspartate utilization in the pyrimidine pathway could be related to toxin regulation. More generally, the expression levels of the genes encoding glutamate, histidine, threonine, methionine and aspartate degradation differed in the two cultures, with stronger gene expression observed in the first hours of growth in the amino acid-supplemented culture. The expression of the genes related to serine and tyrosine metabolism was similar in the two conditions. The authors suggested that amino acid supplementation could result in amino acid overflow, leading to the accumulation of metabolic intermediates or by-products, which could inhibit tetR transcription [START_REF] Orellana | Time-course transcriptomics reveals that amino acids catabolism plays a key role in toxinogenesis and morphology in Clostridium tetani[END_REF]. The studies presented in Table 4-B do not provide information on the effect of cysteine on the C. tetani growth requirements. In complex medium, addition of cystine (Cys-Cys peptide) after autoclaving instead of before autoclaving decreased toxin production, probably because of the effect of heat on cystine. Moreover, cystine inhibited toxin synthesis when added at a concentration of more than 125 mg/L [START_REF] Latham | Tetanus toxin production in the absence of protein[END_REF].

Glucose consumption during C. tetani cultivation

In 1943, Mueller et al. reported that growth and toxin production occurred on complex medium with the omission of either glucose or cystine, but growth failed if both were omitted.

Then, they suggested that both components might be involved in the maintenance of anaerobic conditions [START_REF] Mueller | Production of Tetanus Toxin on Peptone-Free Media[END_REF].

On casamino acid semisynthetic medium, [START_REF] Kaufman | Studies of the nutritional requirements of Clostridium tetani. I. A chemically defined medium[END_REF] showed on four strains from the University of Kentucky laboratories that glucose was nonessential and nonstimulatory for C. tetani growth. However, on a chemically defined medium, glucose was nonessential for half of the strains but stimulatory for the other strains: with glucose omission, two strains exhibited normal growth (51 % light transmission), whereas the other two showed poorer growth (87 % light transmission). The authors suggested that glucose degradation compounds might be involved in some amino acid utilization processes. No toxin production was observed in this study [START_REF] Kaufman | Studies of the nutritional requirements of Clostridium tetani. I. A chemically defined medium[END_REF]. [START_REF] Martinez | Glucose dissimilation by Clostridium tetani[END_REF] showed that glucose was consumed only after 24 to 30 h of growth on a complex medium containing casein, yeast extract and glucose. From this time point, growth increased proportionally to glucose addition in the medium. By observing glucose phosphorylation in cell-free extract, the authors then confirmed the presence of a glucokinase in C. tetani Harvard and 45e strains. This phenomenon was specific to glucose, as other hexoses were not affected. They suggested that this enzyme was inducible because it showed no activity in cells grown in a medium without glucose [START_REF] Martinez | Glucose dissimilation by Clostridium tetani[END_REF].

In continuous culture on complex medium (Harvard derivative strain), regardless of whether the dilution rate or the initial glucose concentration was changed, no toxin was found in the supernatant until glucose was completely consumed. The toxin release was then related to glucose limitation [START_REF] Zacharias | Continuous production of Clostridium tetani toxin[END_REF]. [START_REF] Fratelli | Effect of medium composition on the production of tetanus toxin by Clostridium tetani[END_REF] studied glucose requirements when a Harvard variant strain was cultured with an NZ-Case TT nitrogen source. They showed that the optimal glucose concentration was 9.7 g/L for 43.5 g/L NZ. With a low glucose concentration (2.3 g/L), growth occurred but did not lead to toxin production in the supernatant. In addition to optimizing the glucose concentration, they revealed that the ratio between carbon and nitrogen sources was a key factor. They established a relationship between glucose and nitrogen source described by the following formula, where G0 (g/L) is the initial glucose concentration, and NZ0 (g/L) is the initial casein digest concentration [START_REF] Fratelli | Effect of medium composition on the production of tetanus toxin by Clostridium tetani[END_REF]:

Toxin

Effects of inorganic compounds on C. tetani growth and toxin production

Iron

The role of some growth factors can depend on other medium components. [START_REF] Lerner | The role of glutamine in the glucose metabolism of Clostridium tetani[END_REF] related iron concentration to glucose consumption in Mueller and Miller medium.

Cells in iron-deficient medium were unable to ferment glucose. Glucose consumption was semilogarithmically proportional to iron content and was estimated through carbon dioxide production. They suggested that an enzyme or coenzyme of the glucose pathway was iron dependent. Later, this enzyme was identified as pyruvate:ferredoxin oxidoreductase, containing Fe-S clusters, which catalyzes pyruvate degradation to acetyl-CoA (Bruggemann and Gottschalk, 2004). This enzyme needs oxidized ferredoxin as a substrate and releases it in its reduced form. [START_REF] Lerner | The role of glutamine in the glucose metabolism of Clostridium tetani[END_REF] also experimented with glutamine addition in irondeficient complex medium. This resulted in stimulated carbon dioxide production, approaching the production levels observed in high-iron medium. Thus, addition of glutamine could overcome iron deficiency [START_REF] Lerner | The role of glutamine in the glucose metabolism of Clostridium tetani[END_REF].

According to Feeney et al. (1943), on casein hydrolysate medium, the optimum iron concentration was 50 mg/L reduced iron (powder) (Feeney et al., 1943a) or 0.3 mg/L FeSO4 - .7H2O [START_REF] Mueller | Variable factors influencing the production of tetanus toxin[END_REF]. In both studies, at concentrations above these values, growth was better, but toxin production decreased. Both growth and toxin production therefore were dependent on iron content.

In an initial study, Demain et al. (2003) observed that reduced powdered iron content affected the toxin titer only if the iron was autoclaved with the other compounds in Mueller and Miller medium. When the iron was autoclaved separately, its concentration had no influence on toxin production (Demain and Fang, 2003).

In powder. Charcoal provides an insoluble surface of growth for bacteria. When the insoluble parts of the medium were removed by filtration, toxin production was almost halved. It seems that solid iron could provide a surface for growth or could absorb some inhibitory compounds.

Iron in its solid form is also known to absorb oxygen, which is propitious for anaerobic bacteria [START_REF] Demain | The role of reduced iron powder in the fermentative production of tetanus toxin[END_REF]. [START_REF] Licona-Cassani | Tetanus toxin production is triggered by the transition from amino acid consumption to peptides[END_REF] observed no change in total soluble iron content (Fe 2+ and Fe 3+ ) in a modified Massachusetts medium during fermentation, but this conclusion could be biased because iron seemed to be present in excess in the medium (0.03 mg/L of FeCl3), even though it was diluted 1000x compared to the level in the initial Massachusetts medium (32 mg/L of FeCl3.6 H2O). They determined that the total intracellular iron (Fe 2+ and Fe 3+ ) content decreased slightly during glucose metabolism, which was in accordance with the observation of [START_REF] Lerner | The role of glutamine in the glucose metabolism of Clostridium tetani[END_REF] that related iron concentration and glucose consumption. Moreover, the transcriptomic analysis of E88 culture showed that Fe 2+ transport genes (CTC451-452, CTC534) were expressed in both growth phases, whereas Fe 3+ di-citrate transport genes (CTC784, CTC956, CTC961, CTC1371) were expressed during the slow growth phase, when toxin was produced [START_REF] Licona-Cassani | Tetanus toxin production is triggered by the transition from amino acid consumption to peptides[END_REF]. [START_REF] Mueller | Production of Tetanus Toxin on Peptone-Free Media[END_REF] reported that potassium, magnesium and phosphate inorganic salts were required for C. tetani growth [START_REF] Mueller | Production of Tetanus Toxin on Peptone-Free Media[END_REF]. In continuous culture, Zacharias and Björklund (1968) showed that the addition of 0.1 g/L potassium chloride in modified Massachusetts medium doubled the production of toxin (70 Lf/mL to 130 Lf/mL) in a

Mineral salts

Harvard strain derivative (strain 107). They pointed out that potassium has the property of increasing the permeability of the cell membrane. They observed the opposite phenomenon (70 Lf/mL to 40 LF/mL) with the addition of 0.15 g/L calcium chloride, which is known to reduce cell membrane permeability [START_REF] Zacharias | Continuous production of Clostridium tetani toxin[END_REF]. In TGY flask cultures of strain CN655, the optimal inorganic phosphate concentration for toxin production was found to be 40 mM (two-fold the extracellular toxin concentration in comparison to TGY without phosphate addition). Phosphate supplementation increased the transcription of tetX but not that of its transcriptional regulator tetR. Thus, the authors suggested that toxin regulation by phosphate is not related to TetR (Chapeton-Montes et al., 2020).

Vitamins

Vitamins and trace elements used to be supplied by beef heart infusion into Mueller and Miller medium for C. tetani growth. [START_REF] Latham | Tetanus toxin production in the absence of protein[END_REF] showed that beef heart infusion was not essential for growth and toxin production. Beef heart infusion can successfully be substituted with nicotinic acid, vitamin B12 and iron [START_REF] Latham | Tetanus toxin production in the absence of protein[END_REF]. When Hy-Soy® peptone was used, vitamin addition was not required. Hy-Soy® peptone may contain enough uracil, calcium pantothenate, thiamine, riboflavin, pyridoxal and biotin for C. tetani growth and toxin production. This complex material also satisfied the tyrosine and cystine requirements, as supplementation of these materials was no longer required [START_REF] Demain | Tetanus toxin production in soy-based medium: nutritional studies and scale-up into small fermentors[END_REF].

In chemically defined medium, Feeney et al. (1943) showed that ten vitamins and assimilated compounds were essential for Harvard C. tetani growth: biotin, calcium pantothenate, folic acid, nicotinic acid, oleic acid, riboflavin, pyridoxine, thiamin, adenine and uracil (Feeney et al., 1943b).

The chemically defined medium used by [START_REF] Licona-Cassani | Tetanus toxin production is triggered by the transition from amino acid consumption to peptides[END_REF] contained all of Feeney's vitamins and assimilated compounds except for oleic acid, which supported growth.

On complex media, they observed that calcium pantothenate and riboflavin were consumed during amino acid metabolism, whereas the concentrations of pyridoxine and nicotinic acid increased during fermentation. They also discovered that uracil was both taken up (amino acid metabolism) and released (autolysis) during cultivation [START_REF] Licona-Cassani | Tetanus toxin production is triggered by the transition from amino acid consumption to peptides[END_REF].

Conclusion

The C. tetani genome is highly conserved among all the sequenced strains, especially among strains used for toxin production (Harvard strains, clade 1A). Notably, the toxin-encoding genes show 99.3-99.4 % sequence identity. This identity reached 100 % for the transcriptional regulator gene sequence. Whole-genome sequencing and determination of C.

tetani metabolic pathways showed a metabolism oriented toward amino acid assimilation, with many enzymes dedicated to the transport and degradation of amino acids, which is common for pathogenic bacteria. Even though this microorganism has been used for many years, the regulators that induce the regulatory system for toxin synthesis have not been fully identified. Several experimental strategies have led to the development of different chemically defined media that are able to sustain C. tetani growth, but none of these strategies have led to toxin production. For now, peptides appear to be essential for induction of toxin production.

In particular, histidine seems to be of major importance in C. tetani growth and toxin production, either in its free or peptide form. The role of glucose in C. tetani toxin production is still unclear. It might contribute to toxin synthesis, for example, by generating metabolites of interest or by an unknown interaction. Iron appeared to enhance toxin production depending on form in which it was supplied.

This overview of the metabolism combined with the previous nutritional studies for C. tetani

Figure 1 :

 1 Figure 1: Clostridium tetani central metabolismYellow boxed compounds are fermentation products that are released in the medium

13Figure 2 :

 2 Figure 2: Genetic organization of the tetanus toxin locus in Clostridium tetani from Raffestin et al. (Raffestin et al., 2005)

  least two positive regulator TCSs and one negative regulator TCS were identified by[START_REF] Chapeton-Montes | Tetanus Toxin Synthesis is Under the Control of A Complex Network of Regulatory Genes in Clostridium tetani[END_REF]. The first positive regulator TCS is located 25 kb upstream of tetX on the plasmid, close to ATP-binding protein-encoding genes, but does not show any homology with other bacteria. The other positive regulator TCS is located on the chromosome and is putatively related to autolysis. These two TCSs are suspected to indirectly regulate toxin synthesis since their proteins do not bind to TetR or TetX promoters. In contrast, the negative regulator TCS directly represses toxin synthesis by binding to the TetR and TetX promoters. On the basis of homology, this TCS, located on the chromosome, is involved in cell division. Some other TCSs were suspected to act indirectly on toxin concentration by altering the cell membrane or controlling toxin secretion[START_REF] Chapeton-Montes | Tetanus Toxin Synthesis is Under the Control of A Complex Network of Regulatory Genes in Clostridium tetani[END_REF].

Figure 3 :

 3 Figure 3: Maturation process of Clostridium tetani toxin

  [START_REF] Masuyer | The structure of the tetanus toxin reveals pHmediated domain dynamics[END_REF] using X-ray crystallography (the subunit crystal structures were previously described separately). The tridimensional structure of the tetanus toxin is presented in Figure4. As previously described, the light chain (on fragment B) is the catalytic domain.The translocation domain (which is also part of fragment B) plays a role in transporting the toxin toward neurotransmitters. The binding domain (which is part of fragment C) binds to the neurons. Therefore, antibodies directed against fragment C are the most potent: they block the binding of the toxin to neurons[START_REF] Masuyer | The structure of the tetanus toxin reveals pHmediated domain dynamics[END_REF].

Figure 4 :

 4 Figure 4: Overall structure of tetanus toxin from Masuyer et al. (Masuyer et al., 2017)

  ): L enantiomer; (D-): D enantiomer; (DL-): racemic mixture

Figure 5 :

 5 Figure 5: Amino acid consumption in chemically defined medium, adapted from Licona-Cassani et al. (2016) Licona-Cassani et al. (2016) observed in a modified Massachusetts medium that the depletion

  growth and toxin synthesis gathered in this review could provide support for the design of new experiments on growth and toxin production in chemically defined media. For instance, cultivation with regular sampling and an in-depth analytical strategy (including substrate consumption and metabolite determination) could provide new knowledge on specific features of C. tetani. Combining transcriptomic and proteomic analyses within controlled fermentation systems should provide a better understanding of global metabolism and could help determine the key compounds involved in C. tetani growth and toxin synthesis. These experiments could help to provide a new chemically defined medium that would support both growth and tetanus toxin synthesis and would thus satisfy the World Health Organization recommendations for tetanus vaccine production.

Table 1 -A: Annotation of the amino acid transporter and degradation genes Amino acid Amino acid transporter gene annotation Amino acid degradation gene annotation

 1

	Alanine	CTC564, CTC1172, CTC1975	CTC695
	Arginine	Not annotated	CTC1763
			CTC561-562, CTC824-825,
	Aspartic acid	Not annotated	CTC1294, CTC1309, CTC1876,
			CTC2383-2384
	Cystine / Cysteine	CTC559	CTC1050
	Glutamic acid	CTC822, CTC2306, CTC2324	CTC1295, CTC2563, CTC2565, CTC2567-2568
	Glutamine	CTC559	CTC171
	Glycine	CTC564, CTC1172, CTC1975	Not annotated
	Histidine	Not annotated	CTC2318, CTC2321
	Leucine	CTC787, CTC1868, CTC2088	CTC1738
	Lysine	Not annotated	CTC890
	Methionine	CTC1355	CTC2530
	Proline	CTC1190	Not annotated
	Serine	CTC1514, CTC2307	CTC1981-1982
	Threonine	CTC1514, CTC2307	CTC2624
	Tryptophan	CTC1190	CTC1509
	Tyrosine	CTC819	CTC818

Table 1 -B: End products of the major and minor degradation pathways Amino acid Major degradation pathways Minor degradation pathways

 1

	Aspartate	Glutamate + Oxaloacetate	Ammonium + Fumarate
	Glutamate	Acetate + Butyrate + Pyruvate + Ammonium	2-oxoglutarate + Ammonium n-Formimino-L-Glutamate
	Histidine	Glutamate	Histamine
	Methionine	2-Oxobutanoate + Propionyl-CoA Propionate + ATP + Ammonium	S-Adenosyl-L-Methionine
	Serine	Pyruvate + Ammonium	L-Cysteine
	Threonine	Glycine + Acetaldehyde	Propionyl-CoA Propionate + ATP + Ammonium
	Tyrosine	Pyruvate + Phenol + Ammonia	-
	C. tetani ferments only glucose as a sugar source. The Harvard strains did not take up
	galactose, fructose, mannose, maltose, sucrose and lactose in complex (casein, yeast extract)
	medium (Martinez and Rittenberg, 1959). C. tetani harbors the genes encoding enzymes for
	glycolysis (CTC341, CTC378-382, CTC507, CTC2404, CTC2489-2490, CTC2515,
	CTC2637), glucose PTS transport (CTC278, CTC1771), or sodium symporter activity
	(CTC1237) but none for polysaccharide degradation. Unlike other Clostridium genomes, the
	C. tetani genome also contains genes from the pentose phosphate pathway (CTC228,
	CTC307, CTC1227, CTC1332, CTC1864-1865 and many putative genes encoding
	hydrolases). The degradation of glucose and many amino acids results mainly in pyruvate
	formation. No genes encoding citrate cycle enzymes were found in the C. tetani genome.
	Pyruvate is converted to acetyl-CoA through pyruvate ferredoxin oxidoreductase. This
	enzyme reduces ferredoxin, which is then reoxidized by a hydrogenase, releasing hydrogen.
	Acetyl-CoA can then be converted to acetate or butyrate through complete acetate and
	butyrate biosynthetic pathways. The C. tetani genome harbors one 3-hydroxybutyryl-CoA
	dehydrogenase (Hbd), two crotonases (Crt) and five homologous butyryl-CoA
	dehydrogenases (Bcd). Acetyl-CoA catabolism can also result in ethanol and butanol
	production

Table 2 : Comparison of the compositions of Mueller and Miller, Massachusetts, soy and TGY media

 2

	Constituents per	Final Mueller	Massachusetts	Soy medium	TGY medium
	liter			and Miller medium	medium (Latham et al.,	(Demain et al., 2005)	(Chapeton-Montes et al.,
				(Mueller and	1962)		2020)
				Miller, 1954)
	Digest of casein	22.5 g	25 g	-	30 g
	Beef heart infusion	50 mL	-	-	-
	Yeast extract		-	-	-	20 g
	Soy peptone		-	-	34 g	-
	Glucose		11 g	8.0 g	7.5 g	5 g
	NaCl			2.5 g	2.5 g	5.0 g	-
	Na2HPO4		2 g	-	0.5 g	-
	KH2PO4		0.15 g	-	0.175 g	-
	MgSO4			0.15 g	0.1 g	0.024 g	-
	Cystine			0.25 g	0.125 g	-	-
	Cysteine		-	-	0.125 g	-
	Cysteine HCl					0.5
	Tyrosine		0.5 g	-	0.125 g	-
	Calcium		1.0 mg	1.0 mg	-	-
	pantothenate
	Uracil			2.5 mg	1.25 mg	-	-
	Nicotinic acid		-	0.25 mg	-	-
	Thiamine		0.25 mg	0.25 mg	-	-
	Riboflavin		0.25 mg	0.25 mg	-	-
	Pyridoxine		0.25 mg	0.25 mg	-	-
	Biotin			2.5 µg	2.5 µg	-	-
	Vitamin B12		-	0.05 µg	-	-
	Reduced	iron	0.5 g	-	0.5 g	-
	(powder)
	FeCl3. 6 H2O		-	32 mg	-	-
	pH	adjustment	7.0 -7.2	7.0 ± 0.2	6.8	7.5
	(before autoclave)

5.1. Culture media for

Clostridium tetani A C. tetani growth medium was first designed in 1942 in the Mueller and Miller laboratory using a Harvard strain. Until the 1960s, their work focused on improving the final toxin titer of C. tetani cultures in this medium. The final Mueller and Miller medium was mainly composed of casein hydrolysate (usually NZ-Case), glucose and vitamins. Table

2

details the composition differences between the final Mueller and Miller medium and the modified medium made by

[START_REF] Latham | Tetanus toxin production in the absence of protein[END_REF][START_REF] Latham | Tetanus toxin production in the absence of protein[END_REF]

). Latham's medium was also called Massachusetts medium.

More than thirty years later, Demain et al. developed a plant medium free of animal-source compounds without milk hydrolysate. The raw materials were replaced by Quest Hy-Soy®

Table 3 : Performance comparison in different culture modes 455

 3

	Source	Strain	Cultivation	Cultivation	Gas	Growth	Toxin
			mode and	medium	stripping
			volume
	(Zacharias	Strain 107	Bioreactor	Modified	N2 gassing	0.125 h -1	120
	and Bjorklund, 1968)	(Harvard strain derivative)	mode 1 L Continuous	Massachusetts medium		dilution rate	Lf/mL ± 10 Lf/mL
	(Jagicza et	Harvard strain Bioreactor	Mueller and	N2 surface	Xmax = 10 8	55 Lf/mL
	al., 1981)		400 L	Miller	flushing for	cells/mL
			Batch mode	medium	16 h, then air flow
	(Gutiérrez	Massachusetts	Bioreactor	Modified	N2 bubbling	µ = 0.46	73 Lf/mL
	et al.,	strain	5 L	Massachusetts	during expo.	h -1
	2005)	(Harvard strain derivative)	Batch mode	medium	growth, then surface aeration	Xmax = 0.78 g/L
	(Demain et	Wyeth-Lederle	Bioreactor	Modified soy	N2 bubbling	Not	48 Lf/ml
	al., 2007)	Vaccine and	0.8 L	medium		reported
		Pediatrics strain	Batch mode
	(Fratelli et	Harvard-	Bottles 4 L	Modified	None	Xmax =	70 Lf/mL
	al., 2010)	Caracas (Harvard strain	mode Fed-batch	medium Massachusetts		4.34 g/L
		derivative)
	(Muniandi	Harvard strain	Bioreactor	Modified	Air surface	ODmax =	70 Lf/mL
	et al.,	derivative	400 L	Mueller	flow	2.3
	2013)		Batch mode	medium
	(Licona-	E88	Bioreactors	Modified	N2 surface	Expo: µ =	30.2
	Cassani et		1 L and 5 L	Massachusetts	flushing for 4	0.24 h -1	Lf/mL ±
	al., 2016)		Batch mode	medium	h, then air flow	Slow: µ = 0.08 h -1	2.9 Lf/mL
	(Chung et	Harvard strain	Bioreactor	Modified	Punctual N2	Xmax = 3	80 Lf/ml
	al., 2016)	derivative	50 L	Massachusetts	surface flow,	g/L
			Batch mode	medium	then air flow
	(Chawla et	Harvard strain	Bioreactor	Modified	Not reported Not	97.9 ± 3.3
	al., 2016)	derivative	1 L	Massachusetts		reported	Lf/mL
			Batch mode	medium with soy

Table 4 -B: Free amino acids involved in Clostridium tetani growth

 4

	Amino acids	Feeney et al., 1943	Yu et al., 2009	Licona-Cassani et al., 2016
	used for bacterial	Harvard strain	E88 strain 1	E88 strain
	growth	On chemically	Deficient in amino	Approximate consumption
		defined medium (Table 4-A)	acid biosynthetic pathways	On modified Massachusetts	On chemically defined medium
				medium 2	(Table 4-A)
	Arginine	essential	-	0 %	20 %
	Asparagine	not experimented	-	90 %	100 %
	Aspartate	less effective	-	100 %	100 %
	Cysteine	not experimented	-	90 %	not analyzed
	Glutamate	stimulated	-	> 95 %	100 %
	Glutamine	not experimented	-	90 %	100 %
	Histidine	essential	auxotrophic	> 95 %	100 %
	Isoleucine	essential	auxotrophic	0 %	15 %
	Leucine	essential	auxotrophic	0 %	60 %
	Lysine	less effective	auxotrophic	0 %	25 %
	Methionine	less effective	auxotrophic	75 %	100 %
	Phenylalanine	less effective	auxotrophic	0 %	20 %
	Serine	stimulated	-	> 95 %	100 %
	Threonine	stimulated	-	> 95 %	100 %
	Tryptophan	essential 3	auxotrophic	0 %	0 %
	Tyrosine	essential	-	100 %	60 %
	Valine	essential	auxotrophic	0 %	0 %
		Xmax = 80-85 % of
	Growth	the Xmax obtained in Mueller and
		Miller medium

  2006, when a Harvard derivative strain (from Wyeth-Lederle Vaccine and Pediatrics) was cultured in soy-based medium, Demain et al. showed that iron was essential for toxin production. However, only reduced iron powder yielded satisfactory toxin titers (56-73Lf/mL) at an optimal concentration of 0.5 g/L. When the insoluble iron was replaced by a soluble iron source such as ferric citrate, ferric gluconate or ferrous ammonium sulfate, moderate toxin production (36-43 Lf/mL) was observed. Ferrous sulfate, ferric chloride and

ferric nitrate failed to support toxin production. However, supplementation of activated charcoal with soluble iron sources (ferrous sulfate, ferric citrate, ferrous gluconate) increased toxin production (53-68 Lf/mL), which was consistent with the result obtained with iron

Acknowledgment

This work was supported by Sanofi-Pasteur and Toulouse Biotechnology Institute via an INRA-TWB competitive project. The authors thank AJE for English language editing of the document (certification code 7F3A-18CA-A42B-AF16-8A4P).

peptone (Table 2) (A. L. [START_REF] Demain | Effective levels of tetanus toxin can be made in a production medium totally lacking both animal (e.g., brain heart infusion) and dairy proteins or digests (e.g., casein hydrolysates)[END_REF]. However, due to its high toxin production performance (70 to 120 Lf/mL) and because it does not contain beef heart infusion, Massachusetts medium remains the most commonly used medium for C. tetani cultures in laboratories and for industrial toxin production ( Feeney et al. (1943) analyzed the effects of the amino acids in chemically defined medium on growth by one-at-a-time omission (Feeney et al., 1943b). [START_REF] Yu | Amino acid biosynthesis deficiency in bacteria associated with human and animal hosts[END_REF] identified possible auxotrophy resulting from the lack of an amino acid biosynthesis pathway in C. tetani by bioinformatics analysis of its genome [START_REF] Yu | Amino acid biosynthesis deficiency in bacteria associated with human and animal hosts[END_REF]. Licona-Cassani et al. (2016) followed the amino acid consumption kinetics in both complex and chemically defined media [START_REF] Licona-Cassani | Tetanus toxin production is triggered by the transition from amino acid consumption to peptides[END_REF]. No toxin production was observed on either chemically defined medium, but the analysis of the amino acids sustaining growth was convenient, as the media did not contain any complex materials.

Table 41-A: Initial glucose and amino acid compositions of chemically defined media

Conflicts of interest

The authors declare no conflicts of interest.