- Email: [email protected]
Regulation of the toxinogenesis in Clostridium botulinum and Clostridium tetani
Biology of the Cell 92 (2000) 455−457 © 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S0248490000010972/SCO
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Regulation of the toxinogenesis in Clostridium botulinum and Clostridium tetani Jean-Christophe Marvaud, Stéphanie Raffestin, Maryse Gibert, Michel Robert Popoff* CNR anaérobies, Institut Pasteur, 28 rue du Dr-Roux, 75724 Paris cedex 15, France Received 26 September 2000; accepted 2 October 2000
1. INTRODUCTION Clostridium botulinum and Clostridium tetani produce highly potent neurotoxins, botulinum toxins (BoNTs) and tetanus toxin (TeTx) respectively. C. botulinum is responsible for botulism in human and animals. Food borne botulism is most often due to the ingestion of BoNT preformed in food. In some circumstances, ingested C. botulinum bacteria or spores can grow in the intestine and subsequently produce BoNT in situ. This is the mode of acquisition in neonatal botulism and in some cases of adult botulism. More rarely, botulism can be acquired after wound contamination by C. botulinum spores. This mode of contamination is usual in C. tetani infections. The toxin produced in food, intestine or wound is a prerequisite step for the onset of the disease. The environmental factors that control the toxin production are unknown. BoNTs are divided into seven toxinotypes according to their antigenic properties, whereas only one type of TeTx is known. BoNTs are associated to non-toxic proteins (ANTPs) to form botulinum complexes of various sizes. In contrast, TeTx does not associate to other proteins and does not form any complex. The genetic organisation of neurotoxin and ANTP genes have been determined in representative strains of C. botulinum and C. tetani. However, the regulation of toxin gene expression in these bacteria is largely unknown.
2. GENETIC ORGANISATION OF THE BOTULINUM AND TETANUS LOCI Neurotoxin genes have different genomic localisation in C. botulinum and C. tetani strains. The neurotoxin genes are localised in a large plasmid in C. tetani and C. argentinense, in chromosomal DNA in C. botulinum A, B, E, and F, and in phage DNA in C. botulinum C and D. In all the localisations, the genes encoding the neurotoxins and ANTPs are clustered in close vicinity and constitute the botulinum and tetanus loci. The organisation of the botulinum locus is conserved in the 3’ part, but differs in the 5’ part in the different toxinotypes of C. botulinum (Popoff and Marvaud, 1999). The bont genes lie in the 3’ part of the locus and are immediately preceded by the gene of the non-toxic and non-hemagglutinin component (NTNH). The ntnh and bont genes are transcribed in the same orientation. The genes encoding hemagglutinin (HA) components (HA33, HA17 and HA70 in C. botulinum A) are usptream of the ntnh–bont genes and are transcribed in the opposite direction. A gene (botR, previously called orf21 or orf22) encoding a 21–22 kDa protein which has the feature of a regulatory protein, is localised in the 5’ part of the botulinum locus in C. botulinum C and D, whereas it is localised between ntnh–bont and ha genes in C. botulinum A, B and G. In C. tetani, one gene (tetR), equivalent to botR, was found upstream of the tetx gene (Marvaud et al., 1998a; Marvaud et al., 1998b).
3. TRANSCRIPTIONAL ANALYSIS * Correspondence and reprints. E-mail address: [email protected] (M.R. Popoff).
Regulation of botulinum and tetanus neurotoxin production
Transcriptional analysis has been investigated in C. botulinum A and C (Hauser et al., 1995; Henderson et Marvaud et al.
456
al., 1996). In C. botulinum C, a transcription start site was mapped 113 nucleotides upstream of the ATG of ntnh/C, and another one 100 nucleotides upstream of the initial codon of bont/C1. Both genes are preceded by a consensus ribosome binding site (GGAGG). The analysis of mRNA by reverse transcriptase PCR (RT–PCR) showed that a mRNA overlapped bont/C1 and ntnh/C1 genes, possibly encoding both genes (Hauser et al., 1995). Similar results were found in C. botulinum A (Henderson et al., 1996). Two transcripts have been identified for the bont/A gene. The longer (7.5 kb) encompasses the ntnh–bont/A genes, and the shorter (4 kb) corresponds to bont/A alone. This indicates that bont/A and bont/C1 are transcribed as a mono or a bicistronic messenger in association with the corresponding ntnh gene. Using RT–PCR, it was found that at least one mRNA overlaps the three ha genes in C. botulinum C (Hauser et al., 1995). In Northern blots of C. botulinum A, a single 3.2 kb transcript encompasses ha35, ha17 and ha70 genes (Henderson et al., 1996). The ha genes seem to form a tricistronic operon. However, the HA35 or HA33 components in C. botulinum A and C respectively, were found to be produced in higher amounts than the other HA components (Inoue et al., 1996). This suggests that the relative levels of synthesis of the three HA components are controlled at the translational level as it was found for other toxins, such as the cholera toxin (Hirst, 1995). The genetic organisation in the other C. botulinum types shows that bont and antp genes are localised in two clusters which can be considered as polycistronic units by comparison with the findings reported for C. botulinum A and C.
Biology of the Cell 92 (2000) 455–457
BotRs from the different toxinotypes, and TetR have 51 to 97% identity and are related to other known regulatory proteins such as Uvia (25–28% identity) which regulates the bacteriocin production in C. perfringens, Msmr protein (21–26%) which regulates the sugar transport in Streptococcus mutans, and TxeR (20–24 %) in C. difficile. Interestingly, txeR has a similar localisation than that of botR and tetR, since it lies upstream of the locus of the Toxin A and Toxin B genes. It has been found that TxeR is a positive regulator by studying the expression of a reporter gene fused to toxA and toxB gene promoter in E. coli (Moncrief et al., 1997). In addition, BotR and TetR show some similarity, but to a lower extent, with the –35 binding domain of sigma factors.
5. FUNCTIONAL ANALYSIS OF botR AND tetR GENES
It was first reported that in C. botulinum C, a gene (botR/C, previously called orf22) encodes a 22 kDa protein having the features of a DNA binding protein: basic pI (10.4), and presence of a helix-turn-helix motif (Hauser et al., 1995). The botR/C gene is localised upstream of the ha genes. A homologous gene (botR/A, previously called orf21) was characterised in C. botulinum A (Henderson et al., 1996). The botR/A gene has a different localisation than that of botR/C, since it is inserted between ha and ntnh–bont genes. This gene is also conserved in proteolytic and non-proteolytic C. botulinum B, D, F and G, and in C. tetani (Bhandari et al., 1997; East et al., 1996; Henderson et al., 1996; Li et al., 1998). However, it has not yet been detected in C. botulinum E. The tetR gene is the only gene related to a gene from the botulinum locus, which is conserved in C. tetani. The tetR gene is localised immediately upstream of the Tetanus toxin gene.
We have analysed the function of botR/A by overexpressing this gene in C. botulinum A. The botR/A gene was cloned under the control of its own promoter, in a high copy number shuttle vector transferred in C. botulinum A by electroporation. A significant increase of the BoNT/A and ANTPs production, and of the corresponding mRNA levels, were detected in the recombinant strain. Inversely, partial inhibition of the botR/A expression by antisense mRNA resulted in a lower production of BoNT/A and ANTPs. It was concluded that BotR/A is a positive regulator at the transcriptional level of bont and antp genes. In the same way, we showed that TetR is a positive regulator of the tetx gene expression and that BotR/A, and to a lesser extent BotR/C, are functional in C. tetani. This indicates that the regulation of the bont and tetx genes is conserved in C. botulinum and C. tetani, and constitutes further evidence that bont and tetx gene loci derive from a common ancestor (Marvaud et al., 1998a). Many regulator factors act by directly interacting with the DNA promoter region of the target genes. Bacterial lysates from the recombinant C. botulinum strain overexpressing botR/A was used in gel retardation assays. Using DNA fragments corresponding to the promoter region upstream of the two operons of the botulinum locus, we observed a specific gel shift in the presence of the recombinant C. botulinum lysates. This indicates the formation of a protein–DNA complex migrating slower than the DNA fragment alone. By immunoprecipitation, it has been shown that BotR/A is part of the protein–DNA complex. However, recombinant BotR/A purified from E. coli in the presence of DNA did not cause gel retardation. BotR/A alone seems not to interact with the promoter region. BotR/A possibly requires a specific activation in C. botulinum to be active, or additional protein(s) to interact with DNA. Moreover, it was found that BotR/A
Regulation of botulinum and tetanus neurotoxin production
Marvaud et al.
4. THE botR AND tetR GENES
Biology of the Cell 92 (2000) 455–457
interacts directly with the promoter region upstream of the ntnh–bont and ha genes (Marvaud et al., 1998b). Interestingly, the –10 and –35 regions of the two operons are conserved in C. botulinum A and C (Henderson et al., 1996), C. botulinum A2, F, G, and also in C. tetani (Bhandari et al., 1997; Henderson et al., 1996). These regions could represent the target sites of BotR and TetR.
6. CONCLUSION The synthesis of many virulence factors is regulated. Environmental factors can stimulate a sensor protein which activates a cascade of protein kinases. Finally, the virulence factor genes are regulated at the transcriptional level. Findings on toxin gene regulation in C. botulinum and C. tetani have emerged recently. It is not known whether BotR and TetR are involved in a regulation cascade, and what environmental signals trigger the production of the neurotoxins and ANTPs. It was reported that the nitrogen source, such as small peptides, modulates the TeTx production (Porfirio et al., 1997). However, the involvement of a twocomponent signal transduction remains to be elucidated. In C. difficile, a catabolic repression of the expression of Toxin A and Toxin B genes by rapidly metabolisable sugars, such as glucose, has been shown (Dupuy and Sonenshein, 1998). No such regulation has been identified in C. botulinum and C. tetani.
REFERENCES Bhandari, M., Campbell, K.D., Collins, M.D., East, A.K., 1997. Molecular characterization of the clusters of genes encoding the botulinum neurotoxin complex in Clostridium botulinum (Clostridium argentinense) type G and nonproteolytic Clostridium botulinum type B. Curr. Microbiol. 35, 207–214.
Regulation of botulinum and tetanus neurotoxin production
457 Dupuy, B., Sonenshein, A.L., 1998. Regulated transcription of Clostridium difficile toxin genes. Mol. Microbiol. 27, 107–120. East, A.K., Bhandari, M., Stacey, J.M., Campbell, K.D., Collins, M.D., 1996. Organization and phylogenetic interrelationships of genes encoding components of the botulinum toxin complex in proteolytic Clostridium botulinum types A, B, and F: evidence of chimeric sequences in the gene encoding the nontoxic nonhemagglutinin component. Int. J. Syst. Bacteriol. 46, 1105–1112. Hauser, D., Gibert, M., Marvaud, J.C., Eklund, M.W., Popoff, M.R., 1995. Botulinal neurotoxin C1 complex, Clostridial neurotoxin homology and genetic transfer in Clostridium botulinum. Toxicon 33, 515–526. Henderson, I., Whelan, S.M., Davis, T.O., Minton, N.P., 1996. Genetic characterization of the botulinum toxin complex of Clostridium botulinum strain NCTC2916. FEMS Microbiol. Lett. 140, 151–158. Hirst, T.R., 1995. Biogenesis of cholera toxin and related oligomeric toxins. In: Moss, J., Iglewsski, B., Vaughan, M., Tu, A.T. (Eds.), Bacterial Toxins and Virulence Factors in Disease. Marcel Dekker, New York, pp. 123–184. Inoue, K., Fujinaga, Y., Watanabe, T., Ohyama, T., Takeshi, K., Moriishi, K., Nakajima, H., Inoue, K., Oguma, K., 1996. Molecular composition of Clostridium botulinum type A progenitor toxins. Infect. Immun. 64, 1589–1594. Li, B., Qian, X., Sarkar, H.K., Singh, B.R., 1998. Molecular characterization of type E Clostridium botulinum and comparison to other types of Clostridium botulinum. Biochim. Biophys. Acta. 1395, 21–27. Marvaud, J.C., Eisel, U., Binz, T., Niemann, H., Popoff,, M.R., 1998a. tetR is a positive regulator of the Tetanus toxin gene in Clostridium tetani and is homologous to botR. Infect. Immun. 66, 5698–5702. Marvaud, J.C., Gibert, M., Inoue, K., Fujinaga, V., Oguma, K., Popoff, M.R., 1998b. botR is a positive regulator of botulinum neurotoxin and associated non toxic protein genes in Clostridium botulinum A. Mol. Microbiol. 29, 1009–1018. Moncrief, J.S., Barroso, L.A., Wilkins, T.D., 1997. Positive regulation of Clostridium difficile toxins. Infect. Immun. 65, 1105–1108. Popoff, M.R., Marvaud, J.C., 1999. Structural and genomic features of clostridial neurotoxins. In: Alouf, J.E., Freer, J.H. (Eds.), The Comprehensive Sourcebook of Bacterial Portein Toxins, 2d Ed. Academic Press, London, pp. 174–201. Porfirio, Z., Prado, S.M., Vancetto, M.D.C., Fratelli, F., Alves, E.W., Raw, I., Fernandes, B.L., Camargo, A.C.M., Lebrun, I., 1997. Specific peptides of casein pancreatic digestion enhance the production of tetanus toxin. J. Appl. Microbiol. 83, 678–684.
Marvaud et al.
Minireview
Regulation of the toxinogenesis in Clostridium botulinum and Clostridium tetani Jean-Christophe Marvaud, Stéphanie Raffestin, Maryse Gibert, Michel Robert Popoff* CNR anaérobies, Institut Pasteur, 28 rue du Dr-Roux, 75724 Paris cedex 15, France Received 26 September 2000; accepted 2 October 2000
1. INTRODUCTION Clostridium botulinum and Clostridium tetani produce highly potent neurotoxins, botulinum toxins (BoNTs) and tetanus toxin (TeTx) respectively. C. botulinum is responsible for botulism in human and animals. Food borne botulism is most often due to the ingestion of BoNT preformed in food. In some circumstances, ingested C. botulinum bacteria or spores can grow in the intestine and subsequently produce BoNT in situ. This is the mode of acquisition in neonatal botulism and in some cases of adult botulism. More rarely, botulism can be acquired after wound contamination by C. botulinum spores. This mode of contamination is usual in C. tetani infections. The toxin produced in food, intestine or wound is a prerequisite step for the onset of the disease. The environmental factors that control the toxin production are unknown. BoNTs are divided into seven toxinotypes according to their antigenic properties, whereas only one type of TeTx is known. BoNTs are associated to non-toxic proteins (ANTPs) to form botulinum complexes of various sizes. In contrast, TeTx does not associate to other proteins and does not form any complex. The genetic organisation of neurotoxin and ANTP genes have been determined in representative strains of C. botulinum and C. tetani. However, the regulation of toxin gene expression in these bacteria is largely unknown.
2. GENETIC ORGANISATION OF THE BOTULINUM AND TETANUS LOCI Neurotoxin genes have different genomic localisation in C. botulinum and C. tetani strains. The neurotoxin genes are localised in a large plasmid in C. tetani and C. argentinense, in chromosomal DNA in C. botulinum A, B, E, and F, and in phage DNA in C. botulinum C and D. In all the localisations, the genes encoding the neurotoxins and ANTPs are clustered in close vicinity and constitute the botulinum and tetanus loci. The organisation of the botulinum locus is conserved in the 3’ part, but differs in the 5’ part in the different toxinotypes of C. botulinum (Popoff and Marvaud, 1999). The bont genes lie in the 3’ part of the locus and are immediately preceded by the gene of the non-toxic and non-hemagglutinin component (NTNH). The ntnh and bont genes are transcribed in the same orientation. The genes encoding hemagglutinin (HA) components (HA33, HA17 and HA70 in C. botulinum A) are usptream of the ntnh–bont genes and are transcribed in the opposite direction. A gene (botR, previously called orf21 or orf22) encoding a 21–22 kDa protein which has the feature of a regulatory protein, is localised in the 5’ part of the botulinum locus in C. botulinum C and D, whereas it is localised between ntnh–bont and ha genes in C. botulinum A, B and G. In C. tetani, one gene (tetR), equivalent to botR, was found upstream of the tetx gene (Marvaud et al., 1998a; Marvaud et al., 1998b).
3. TRANSCRIPTIONAL ANALYSIS * Correspondence and reprints. E-mail address: [email protected] (M.R. Popoff).
Regulation of botulinum and tetanus neurotoxin production
Transcriptional analysis has been investigated in C. botulinum A and C (Hauser et al., 1995; Henderson et Marvaud et al.
456
al., 1996). In C. botulinum C, a transcription start site was mapped 113 nucleotides upstream of the ATG of ntnh/C, and another one 100 nucleotides upstream of the initial codon of bont/C1. Both genes are preceded by a consensus ribosome binding site (GGAGG). The analysis of mRNA by reverse transcriptase PCR (RT–PCR) showed that a mRNA overlapped bont/C1 and ntnh/C1 genes, possibly encoding both genes (Hauser et al., 1995). Similar results were found in C. botulinum A (Henderson et al., 1996). Two transcripts have been identified for the bont/A gene. The longer (7.5 kb) encompasses the ntnh–bont/A genes, and the shorter (4 kb) corresponds to bont/A alone. This indicates that bont/A and bont/C1 are transcribed as a mono or a bicistronic messenger in association with the corresponding ntnh gene. Using RT–PCR, it was found that at least one mRNA overlaps the three ha genes in C. botulinum C (Hauser et al., 1995). In Northern blots of C. botulinum A, a single 3.2 kb transcript encompasses ha35, ha17 and ha70 genes (Henderson et al., 1996). The ha genes seem to form a tricistronic operon. However, the HA35 or HA33 components in C. botulinum A and C respectively, were found to be produced in higher amounts than the other HA components (Inoue et al., 1996). This suggests that the relative levels of synthesis of the three HA components are controlled at the translational level as it was found for other toxins, such as the cholera toxin (Hirst, 1995). The genetic organisation in the other C. botulinum types shows that bont and antp genes are localised in two clusters which can be considered as polycistronic units by comparison with the findings reported for C. botulinum A and C.
Biology of the Cell 92 (2000) 455–457
BotRs from the different toxinotypes, and TetR have 51 to 97% identity and are related to other known regulatory proteins such as Uvia (25–28% identity) which regulates the bacteriocin production in C. perfringens, Msmr protein (21–26%) which regulates the sugar transport in Streptococcus mutans, and TxeR (20–24 %) in C. difficile. Interestingly, txeR has a similar localisation than that of botR and tetR, since it lies upstream of the locus of the Toxin A and Toxin B genes. It has been found that TxeR is a positive regulator by studying the expression of a reporter gene fused to toxA and toxB gene promoter in E. coli (Moncrief et al., 1997). In addition, BotR and TetR show some similarity, but to a lower extent, with the –35 binding domain of sigma factors.
5. FUNCTIONAL ANALYSIS OF botR AND tetR GENES
It was first reported that in C. botulinum C, a gene (botR/C, previously called orf22) encodes a 22 kDa protein having the features of a DNA binding protein: basic pI (10.4), and presence of a helix-turn-helix motif (Hauser et al., 1995). The botR/C gene is localised upstream of the ha genes. A homologous gene (botR/A, previously called orf21) was characterised in C. botulinum A (Henderson et al., 1996). The botR/A gene has a different localisation than that of botR/C, since it is inserted between ha and ntnh–bont genes. This gene is also conserved in proteolytic and non-proteolytic C. botulinum B, D, F and G, and in C. tetani (Bhandari et al., 1997; East et al., 1996; Henderson et al., 1996; Li et al., 1998). However, it has not yet been detected in C. botulinum E. The tetR gene is the only gene related to a gene from the botulinum locus, which is conserved in C. tetani. The tetR gene is localised immediately upstream of the Tetanus toxin gene.
We have analysed the function of botR/A by overexpressing this gene in C. botulinum A. The botR/A gene was cloned under the control of its own promoter, in a high copy number shuttle vector transferred in C. botulinum A by electroporation. A significant increase of the BoNT/A and ANTPs production, and of the corresponding mRNA levels, were detected in the recombinant strain. Inversely, partial inhibition of the botR/A expression by antisense mRNA resulted in a lower production of BoNT/A and ANTPs. It was concluded that BotR/A is a positive regulator at the transcriptional level of bont and antp genes. In the same way, we showed that TetR is a positive regulator of the tetx gene expression and that BotR/A, and to a lesser extent BotR/C, are functional in C. tetani. This indicates that the regulation of the bont and tetx genes is conserved in C. botulinum and C. tetani, and constitutes further evidence that bont and tetx gene loci derive from a common ancestor (Marvaud et al., 1998a). Many regulator factors act by directly interacting with the DNA promoter region of the target genes. Bacterial lysates from the recombinant C. botulinum strain overexpressing botR/A was used in gel retardation assays. Using DNA fragments corresponding to the promoter region upstream of the two operons of the botulinum locus, we observed a specific gel shift in the presence of the recombinant C. botulinum lysates. This indicates the formation of a protein–DNA complex migrating slower than the DNA fragment alone. By immunoprecipitation, it has been shown that BotR/A is part of the protein–DNA complex. However, recombinant BotR/A purified from E. coli in the presence of DNA did not cause gel retardation. BotR/A alone seems not to interact with the promoter region. BotR/A possibly requires a specific activation in C. botulinum to be active, or additional protein(s) to interact with DNA. Moreover, it was found that BotR/A
Regulation of botulinum and tetanus neurotoxin production
Marvaud et al.
4. THE botR AND tetR GENES
Biology of the Cell 92 (2000) 455–457
interacts directly with the promoter region upstream of the ntnh–bont and ha genes (Marvaud et al., 1998b). Interestingly, the –10 and –35 regions of the two operons are conserved in C. botulinum A and C (Henderson et al., 1996), C. botulinum A2, F, G, and also in C. tetani (Bhandari et al., 1997; Henderson et al., 1996). These regions could represent the target sites of BotR and TetR.
6. CONCLUSION The synthesis of many virulence factors is regulated. Environmental factors can stimulate a sensor protein which activates a cascade of protein kinases. Finally, the virulence factor genes are regulated at the transcriptional level. Findings on toxin gene regulation in C. botulinum and C. tetani have emerged recently. It is not known whether BotR and TetR are involved in a regulation cascade, and what environmental signals trigger the production of the neurotoxins and ANTPs. It was reported that the nitrogen source, such as small peptides, modulates the TeTx production (Porfirio et al., 1997). However, the involvement of a twocomponent signal transduction remains to be elucidated. In C. difficile, a catabolic repression of the expression of Toxin A and Toxin B genes by rapidly metabolisable sugars, such as glucose, has been shown (Dupuy and Sonenshein, 1998). No such regulation has been identified in C. botulinum and C. tetani.
REFERENCES Bhandari, M., Campbell, K.D., Collins, M.D., East, A.K., 1997. Molecular characterization of the clusters of genes encoding the botulinum neurotoxin complex in Clostridium botulinum (Clostridium argentinense) type G and nonproteolytic Clostridium botulinum type B. Curr. Microbiol. 35, 207–214.
Regulation of botulinum and tetanus neurotoxin production
457 Dupuy, B., Sonenshein, A.L., 1998. Regulated transcription of Clostridium difficile toxin genes. Mol. Microbiol. 27, 107–120. East, A.K., Bhandari, M., Stacey, J.M., Campbell, K.D., Collins, M.D., 1996. Organization and phylogenetic interrelationships of genes encoding components of the botulinum toxin complex in proteolytic Clostridium botulinum types A, B, and F: evidence of chimeric sequences in the gene encoding the nontoxic nonhemagglutinin component. Int. J. Syst. Bacteriol. 46, 1105–1112. Hauser, D., Gibert, M., Marvaud, J.C., Eklund, M.W., Popoff, M.R., 1995. Botulinal neurotoxin C1 complex, Clostridial neurotoxin homology and genetic transfer in Clostridium botulinum. Toxicon 33, 515–526. Henderson, I., Whelan, S.M., Davis, T.O., Minton, N.P., 1996. Genetic characterization of the botulinum toxin complex of Clostridium botulinum strain NCTC2916. FEMS Microbiol. Lett. 140, 151–158. Hirst, T.R., 1995. Biogenesis of cholera toxin and related oligomeric toxins. In: Moss, J., Iglewsski, B., Vaughan, M., Tu, A.T. (Eds.), Bacterial Toxins and Virulence Factors in Disease. Marcel Dekker, New York, pp. 123–184. Inoue, K., Fujinaga, Y., Watanabe, T., Ohyama, T., Takeshi, K., Moriishi, K., Nakajima, H., Inoue, K., Oguma, K., 1996. Molecular composition of Clostridium botulinum type A progenitor toxins. Infect. Immun. 64, 1589–1594. Li, B., Qian, X., Sarkar, H.K., Singh, B.R., 1998. Molecular characterization of type E Clostridium botulinum and comparison to other types of Clostridium botulinum. Biochim. Biophys. Acta. 1395, 21–27. Marvaud, J.C., Eisel, U., Binz, T., Niemann, H., Popoff,, M.R., 1998a. tetR is a positive regulator of the Tetanus toxin gene in Clostridium tetani and is homologous to botR. Infect. Immun. 66, 5698–5702. Marvaud, J.C., Gibert, M., Inoue, K., Fujinaga, V., Oguma, K., Popoff, M.R., 1998b. botR is a positive regulator of botulinum neurotoxin and associated non toxic protein genes in Clostridium botulinum A. Mol. Microbiol. 29, 1009–1018. Moncrief, J.S., Barroso, L.A., Wilkins, T.D., 1997. Positive regulation of Clostridium difficile toxins. Infect. Immun. 65, 1105–1108. Popoff, M.R., Marvaud, J.C., 1999. Structural and genomic features of clostridial neurotoxins. In: Alouf, J.E., Freer, J.H. (Eds.), The Comprehensive Sourcebook of Bacterial Portein Toxins, 2d Ed. Academic Press, London, pp. 174–201. Porfirio, Z., Prado, S.M., Vancetto, M.D.C., Fratelli, F., Alves, E.W., Raw, I., Fernandes, B.L., Camargo, A.C.M., Lebrun, I., 1997. Specific peptides of casein pancreatic digestion enhance the production of tetanus toxin. J. Appl. Microbiol. 83, 678–684.
Marvaud et al.