FR2778922A1 - Polynucleotide useful for regulating gene expression in bacteria, especially Clostridium, e.g. for vaccine production - Google Patents

Abstract

Polynucleotide comprising all or part of at least one regulatory sequence of a Clostridium toxin synthesis operon in a form isolated from the rest of the genes constituting the operon is new.- DETAILED DESCRIPTION - INDEPENDENT CLAIMS are also included for the following: - (1) a vector or plasmid containing at least part of the polynucleotide; - (2) a protein or peptide having at least 60% sequence identity with a protein or peptide comprising a substantial part of the 179 residue amino acid sequence fully defined in the specification; - (3) a protein or peptide having at least 60% sequence identity with a protein or peptide comprising a substantial part of the sequence consisting of amino acids 125-175 of the 179 residue sequence; - (4) a microorganism containing the polynucleotide or vector.- ACTIVITY - Antibacterial.- MECHANISM OF ACTION - Vaccine

Description

The subject of the present invention is the gene sequence for regulating

  synthesis of toxins produced by bacteria from

genus Clostridium.

  It also relates to polynucleotides comprising these sequences as well as to microorganisms carrying these vectors. Tetanus and botulinum toxins are neurotoxins secreted by bacteria of the genus Clostridium, respectively

C. tetani and C. botulinum.

  The structural genes of these toxins have been characterized in

0o various strains.

  In the case of botulinum toxin, the structural genes are associated with genes coding for non-toxic proteins, as well as regulatory genes within operons responsible for producing these toxins. The organization is substantially identical in the seven groups of known toxins (A, B, C1, D, E, F and G). Among other things, it has been described by HAUSER et al. (1994, Mol. Gen. Genet, 243, 631-640), EAST et al. (1994, System. Appl. Microbiol., 17, 306-312) or even

  EAST et al. (1996, Int. J. System. Bacteriol., 46, 1005-1012).

  A gene was identified in the C1 toxinotype. Then, this gene predicted to be a gene for regulating the expression of toxins was

  highlighted by BHANDARI et al. (1997, Curr. Microbiol., 35; 207-

  214) in strains of C. botulinum type B. This gene was called

  P-21, but is still known by the name Orf21 or BolR.

  The organization of genes expressing the toxin in C.tetani is

  much less complex than that demonstrated in C.botulinum.

  Thus, the operons or groups of genes controlling the synthesis of

  tetanus toxin include only one structural gene.

  BHANDARI et al. (1997, previously cited) point out that there is a homology between the regulatory gene called P-21 in Clostridium botulinum, and the C-terminal end (29 amino acids) of a protein encoded upstream of the structural gene toxin in Clostridium tetani. However, this is simply a hypothesis and there is no evidence to confirm that this sequence of 29

  amino acids to a regulatory protein.

  In any event, the complete sequence of this gene is not

not described.

  The study of the synthesis of these toxins and its genetic organization, however, is of great pharmaceutical interest, due to their nature. Indeed, the production of these toxins, or their fragments, has been attempted in various microorganisms such as Escherichia coi, Lactococcus lactis, Baculovirus and Pichia Pastoris, but these productions have proved to be insufficient for these microorganisms to be

  used on an industrial scale.

  1o The inventors have shown that there is a regulatory gene in Clostridium tetani, allowing positive regulation of the

  synthesis of the toxin of this bacterium.

  They also showed that this gene, as well as other genes for regulating bacteria of the genus Clostridium, could be used for the production of various proteins by bacteria of the genus Clostridium. The subject of the present invention is therefore a sequence having an identity of at least 60%, and preferably 75%, with at least a substantial part of the tetR regulatory gene having the sequence SEQ ID N 1 following, or hybridizing under conditions

  stringent, (or rigorous), with this part.

  The percentages of identity indicated in the present invention

  were calculated by the Best Phit program (GCG software).

SEQ ID N 1:

  AACTATATTA ATATACACAT CCTCGGTAAT ATATCAATAC AATACATGTA

  ATTAATAATG TTTTTTGTAA AAGMTTAAGC TTAGTGGTTT ACTTTCAAAA

  TATAATAAAA CGTTAATTAT AGAAAAGTGA AAGTAAGGTG ATG CTT ATG GAA

  AAT CTA TTT GTA AAA ATT AAG ACA TTA AAA AAT AAT AAT AAA GAA TTT

  GAA GTA ATT ATT ATA CAT TTC AAA AAT ACT GTA AAT ATA CTTATT CGA

  AAG TAT AAT TTA GAA AGTCAT TAT AAT GAT ATC TTA TAT CATTTA TGG

  AAT ATA CTT AAA AAA ATT GAT TTG AAT AAA TTT AAT ACA GAA AAT GAT

  TTA CAC AAA TAT ATT AGTAGA TCT TTA AAA AGA TAT TGC TTA GAT ATT

  TGT AAT AAA AAA AAT AGG GAT AAA AAA ATA ATA TAT AAC TCA GAA ATT

  ACA AAT ATA AAA TTA AAT TTA ATG GAA AAT AGT TGC TCA AAT TAT TTA

  AAC TTC GAA TTT AAT GAT TTA ATA TCC ATA TTA CCT GAA AAT CAA AGA

  AAA ATT TTA TAT ATG AAA TTT TTT GAA TAT ATG AAA GAA TGT GAT ATA

  GCT AAG CTT CAT ATG AGT CGT CAA GCT GTA TAT AAA AAT AAG GTT TTA

  GCA TTA AAA AAA TTA GAA CCT ATA GTA AAT AAA TTA ATT AAT ATA T

  AGTTTTTATA ATTTAATTAT GAATAATATT CTTAAGATAA AAAGTAAATT

  TTTAAAAAAT TTAAATTTTC AGTTTACAAA AAATAACCTG ATTATGTTAT

  ATGTAATTGT AAAAAACATA TAAAAAATCA GAAAAATTTA GGAGGTATAT

  TATTAATGGA TTAAATAATA ATTTTTTAAT TTACTTTTGA TTAATAAATA

  TTAAATGTTT ATTTTAATTA GGAGATGATA CGTATGCC

  Such a sequence preferably comprises at least

  minus part of the 3 'end of the sequence SEQ ID N 1.

  The term "part" or "fragment" of the nucleic acid designates any nucleic acid comprising at least a part of the sequence concerned (for example the sequence SEQ ID N 1) and retaining a regulatory activity. The part of the sequence advantageously comprises at least 50 bp, more preferably at least 100 bp. These "parts" can be easily generated by conventional techniques of molecular biology, either by enzymatic cleavage and digestion from the fragments described, or by synthesis

  using nucleic acid synthesizers.

  The term “hybridizer” designates, within the meaning of the invention, any hybridization under normal conditions, rigorous or not, as defined below. Rigorous or stringent hybridization conditions are for example: hybridization at 42 ° C., 50% of formamide, 5 × SSC, 1 × Denhardt; washing at 65 C in 0.1 x SSC, 0.1% SDS. Non-stringent conditions include: hybridization at 37 C, 40% formamide, 5 x SSC, 1 x Denhardt; washing at 50 C in 1 x SSC, 0.1% SDS. The rigorous conditions are particularly suitable when the nucleic acids are present in small quantities and / or in poorly purified form. Non-stringent conditions are more suitable when the nucleic acid is present in larger quantities and is

  found in the form significantly represented in the sample.

  Advantageously, the "hybridizing" sequences are sequences which hybridize under rigorous conditions, and which therefore have a high degree of structural homology with the sequence concerned (for example

  the sequence SEQ ID N 1) or its fragments.

  Proteins or peptides having an identity of at least 60% and preferably 75%, with a protein or peptide comprising a substantial part of the sequence SEQ ID N 2

  next, constitute another object of the present invention.

SEQ ID N 2:

  Met Leu Met Glu Asn Leu Phe Val Lys lie Lys Thr Leu Lys Asn Asn Asn Lys Glu Glu Val lie lie lie His Phe Lys Asn Thr Val Asn lile Leu lie Arg Lys Tyr Asn Leu Glu Ser His Tyr Asn Asp lie Leu Tyr His Leu Trp Asn lie Leu Lys Lys lie Asp Leu Asn Lys Phe Asn Thr Glu Asn Asp Leu His Lys Tyr lie Ser Arg Ser Leu Lys Arg Tyr Cys Leu Asp lie Cys Asn Lys Lys Asn Arg Asp Lys Lys lie lie Tyr Asn Ser Glu lie Thr Asn lie Lys Leu Asn Leu Met Glu Asn Ser Cys Ser Asn Tyr Leu Asn Phe Glu Phe Asn Asp Leu lie Ser lie Leu Pro Glu Asn GIn Arg Lys lie Leu Tyr Met Lys Phe Phe Glu Tyr Met Lys Glu Cys Asp lie Ala Lys Leu His Met Ser Arg Gln Ala Val Tyr Lys Asn Lys Val Leu Ala Leu Lys Lys Leu Glu Pro bind Val Asn Lys Leu bind Asn bind Advantageously, such a protein or such a peptide has a sequence identity of at least 60 % with a protein or peptide comprising a substantial part of the sequence constituted by the sequence of amino acids in positions 125 to 175

of the sequence SEQ ID N 2.

  The present invention further relates to polynucleotides comprising all or part of at least one regulatory sequence of a synthetic operon for toxins produced by a bacterium of the genus Clostridium, in a form isolated from the rest of the genes included in said operon . According to a preferred embodiment of the invention, said regulatory sequence is that of an operon, or group of genes, involved in the synthesis of toxins of the species C. botulinum and C.tetani, or a sequence having a identity of at least 60% with

0o this sequence.

  Advantageously, such a polynucleotide is present,

  under appropriate conditions, in the form of multiple copies.

  A polynucleotide is considered to be present in the form of multicopy when it is present in an amount of 200 to 300 copies per bacterium. Depending on the uses, an oligonucleotide according to the present invention may nevertheless be present at a lower level.

  number of copies, for example 20 to 30 copies per bacteria.

  Such a polynucleotide advantageously has a size between 4 and 15 kb, and even more preferably between 6 and 9

2o kb.

  It comprises part of a regulatory sequence, which can be the tet / R gene (SEQ ID N 1), or a sequence hybridizing under stringent conditions (stringent) with SEQ ID N 1, but which can be that of any other regulatory gene for a bacterium of the genus Clostridium, and in particular the botR / C or botR / A genes or parts of these genes, or sequences which hybridize under rigorous conditions

with these sequences.

  Such a polynucleotide can also include two or more

  two regulation sequences, up to 10 regulation sequences.

  It can also include at least one regulatory sequence of a toxin synthesis operon produced by a bacterium of the genus Clostridium, a suitable promoter sequence, and a structural gene coding for a protein homologous or heterologous with respect to the

  regulatory sequence, or part of one of these proteins.

  By "homologous protein" is meant a protein of

  structure from the same microorganism as the regulatory sequence.

  The term “heterologous protein” is understood to mean a structural protein derived from a microorganism different from the regulatory sequence. A "part of a protein" can be a fragment of a

  toxin lacking toxic activity.

  “Toxin” is understood to mean, within the meaning of the invention, any peptide, polypeptide or protein produced by a pathogenic bacteria, and involved in said pathogenic activity. It can be a factor directly

  responsible for the toxicity of the bacteria, or participating in this toxicity.

  A toxin "fragment" can consist of any part of a toxin, having retained certain immunogenic motifs of the toxin. In particular, it has been described that bacterial toxins often have different distinct functional domains, and in particular one domain involved in toxic activity (catalytic site) distinct from other domains involved in site recognition or in interactions with partners. . A toxin "fragment" according to the invention advantageously consists of a domain devoid of toxic activity, but retaining an immunogenic power. A toxin variant can consist, for example, of a derivative resulting from genetic modifications of the sequence coding for said toxin or fragment of toxin. Such genetic modifications are for example mutations, deletions, fusions, etc. Generally, mutations affect from 1 to 10 residues, preferably from 1 to 5 residues. These mutations are mutations modifying the encoded amino acid, and therefore the sequence of the protein. Deletions can be internal or terminal deletions. They can affect up to 40% of the entire sequence. Mergers consist of introducing additional regions 5 'and / or 3' of the sequence, or possibly inserting such regions within the sequence. These modifications can be carried out with the aim of either reducing or even eliminating the toxicity of these proteins, or improving their production or their stability for example. Such genetic modifications can be carried out according to the conventional conditions of molecular biology, and are

  illustrated in the state of the art.

  The expression of such genes can be governed by the original promoter, but this promoter can be replaced by any other promoter allowing expression of the gene. The polynucleotides according to the present invention must also contain sequences allowing their multiplication within the

  bacteria, as well as their functioning in general.

  Thus, such polynucleotides must include an origin 0o of replication, which according to a particular embodiment of the invention can make it possible to obtain a large number of copies of these

  vectors within the same bacteria.

  The promoter sequence may be that of the iota toxin gene or of the tetR regulatory gene or of a gene of a toxin of

  Clostridium as cited below.

  An oligonucleotide according to the invention can be contained in a

vector or plasmid.

  Such a vector can be pMRP309 (botR / A), pMRP365 (tet / R), pMRP319 (botR / C), which are plasmids obtained by cloning DNA fragments having the sequences SEQ ID N 16, SEQ ID N 12 and SEQ ID N 14 below, in the Smal site of the vector

pAT1 8.

SEQ ID N 12:

  AAGCTTATTT TACATTAAAT TACTTTTATT GATAAAAATA TATTTTTTGT

  ATTTATATAG AGATTTTCTT TGTCATATAC TGTATAATAT TTATATAATA

  TTAATACATA ATATTATAAC TAAATTCAAA ATAGAAAGGA

  GGTTATCTTATGTTAAAAAG AAAAATTTAA AATACTATTT TCATTATTTG

  AAGATTGAAT CTTAAATAAA CTTGAATTAG GAGGGAGTAC CC ATG

  GTT ATG GAAAAT CTA TTT GTA AAA ATT AAG ACA TTA AAA AAT AAT

  AAT AAA GAA TTT GAA GTA ATT ATT ATA CAT TTC AAA AAT ACT GTA

  AAT ATA CTT ATT CGA AAG TAT AAT TTA GAA AGT CAT TAT AAT GAT

  ATC TTA TAT CAT TTA TGG AAT ATA CTT AAA AAA ATT GAT TTG AAT

  AAA TTT AAT ACA GAA AAT GAT TTA CAC AAA TAT ATT AGT AGA TCT

  TTA AAA AGA TAT TGC TTA GAT ATT TGT AAT AAA AAA AAT AGG GAT

  AAA AAA ATA ATA TAT AAC TCA GAA ATT ACA AAT ATA AAA TTA AAT

  TTA ATG GAA AAT AGT TGC TCA AAT TAT TTA AAC TTC GAA TTT AAT

  GAT TTA ATA TCC ATA TTA CCT GAA AAT CAA AGA AAA ATT TTA TAT

  ATG AAA TTT TTT GAA TAT ATG AAA GAA TGT GAT ATA GCT AAG CTT

  CAT ATG AGT CGT CAA GCT GTA TAT AAA AAT AAG GTT TTA GCA TTA AAA AAA TTA GAA CCT ATA GTA AAT AAA TTA ATT AAT ATC TGCAGATAAA AATTATATTT AAAATTATTA ATAAATATCA AAATTAATAA 5 AGCTAGTTAT TCATACTGATATATATATATATATATATATATATATATATATATAT

  ATATTGAAAT TTGTTGAGGT TTAGATAATA TTTTATTTAA ACCTTAATTT TCTTTTTATG TAATTATAGT GAATATAAAT TGTACAAAAA CTAGAGATAT TTCTAGA10

SEQ ID N 14:

  AAGCTTATTT TACATTAAAT TACTTTTATT GATAAAAATA TATTTTTTGT ATTTATATAG AGATTTTCTT TGTCATATAC TGTATAATAT TTATATAATA

  TTAATACATA ATATTATAAC TAAATTCAAA ATAGAAAGGA GGTTATCTTA TGTTAAAAAG AAAAATTTAA AATATATTTT CATTATTTGA AGATTGAATC TTAAATAAAC TTGAATTAGG AGGGAGTACC20 ATGGGCAGCA GCCATCATCA TCATCAT TAG CGT AGT

  GAA AAT TTA AAG CAT GAT AAC CAG CAC TTT AAC TTT ATT GAA ATG TCT CTA AAA AAA TAT ATT GAA AAA ACT TCT AAA AAG TAT AAT TTA TAT TAT GAT TAT TAT AAT GAT ATA CTG TAT CAT TTA TGG AAA GAA25 CTT ATT GAA ATT A TTA AAG AAT TTT AAT TCT GAA TTA GAT TTA AGA AAA TAT ATT AGT ACA AGT ATA AAA AGA TAT TGT ATA AAT ATT

  TGT AAG AAA AAA AAT AGG GAT AAG AAA ATT ATA TAT AAT TCA GAA GTA ACA TAT AAA AAA TTG GAT GT GGTA AAT GTT TAT TCT TTA TAT TGT GAT AAT TTC GAG TTT TTG GAT TTG ATA TCC ATA TTA AAC30 TAC AAG GAA AAG CA ATA TAT ATG AAA TTT TTT GAA GGT AGA AAA GAT AAT GAA ATA GCT ATA AGG CTT CGT TTA AGT CGT CAA TCT

  ATA TAT AAG ATT AGA TCT TTA AAA AAG TTG TAT CCT ATA GTA ATG CAA TTA GTT AAT ATT T GACTGCAGAT AAAAATTATA TTTAAAATTA TTAATAAATA TCAAAATTAA TAAAGCTAGT TATTCATACTATATATATATATATATATATTA

  GGTTTAGATA ATATTTTATT TAAACCTTAA TTTTCTTTTT ATGTAATTAT AGTGAATATA AATTGTACAA AAACTAGAGA TATTTCTAGA

SEQ ID N 16:

  GGTAGAAAAG TTCTTTCAAC TGGACATATA ATTCACCTCT TTATAAATTA

  GATGATAGTA CTCATATTAA ATCTTTGTTG ATATTAATTT TATGGGTTAC GAATATTCCA TTTCTGATTA TCATCTCCAT GATAATTAAA TACTTGAATA

  GCAGTTCCGT TTGCTGTTTG GCTGTTATAT AAATCTAGAT CCATACTTTT TATTTTATAA GCAGCTTTAT TAGAATCATA TATAAGTCTC CATCTTTCAA GGTAATTTCT AGTTTGTTGA AATAAGCTAA CGTTACCGGC AACTTGATAA

  AAAAATAAAT TAGTATCGGC CTTACAGGAG ATGGTAACAA TTITGTCATT

  TAATGAATTT TGGATTACTG AATAGTGTTC CATTATGATT CCTCCTTTAT

  TTAAGAATTA ATCTTACATA TAACATATAA CATAATCAAG TTATTTTTTG

  TAAACCTAAA ATTTAAATAT ATCAAATIT TATTAGTATG TTTACATAAT

  TGATTATGGA TATTTCGTAA AAATGGCTTA TTAAAAATTT AAAGGCAATT

  AGTTTATTTA TAGTATAATA AAACAATAAT ATGTATATTA TGGAGGGATA

  GTGGTAAAT ATG AAT AAG TTG TTT TTA CAA ATT AAA ATG TTA AAA AAT

  GAC AAC GAG GAG TTT CAA GAA ATT mT AAG CAT T GAA AAA ACT ATA

  AAT ATA TTT ACT AGA AAA TAT AAT ATA TAT GAT AAT TAC AAT GAT ATT

  TTG TAC CAT TTA TGG TAT ACA CTT AAA AAA GTT GAT TTG AGC AAT TTC

  AAT ACA CAA AAT GAT TTA GAG AGA TAT ATT AGT AGG ACT TTA AAA AGA

  TAT TGC TTA GAT ATT TGC AAT AAA AGA AAG ATT GAT AAG AAA ATA ATA

  TAT AAT TCA GAA ATT GCA GAT AAG AAA TTA AGC TTA ATA GCA AAT AGT

  TAT TCA AGT TAT TCA GAA T GAA TTT AAT GAT TTA ATA TCC ATA TTA

  CCT GAC GAT CAA AAG AAA ATT ATA TAT ATG AAA T GTT GAA GAT ATT

  AAG GAG ATA GAT ATA GCT AAA AAA CTT AAT ATA AGT CGT CAA TCT GTA

  TAT AAA AAT AAA ATA ATG GCT TTA GAG AGA TTA GAA CCC ATA TTG AAA

  AAA TTA ATT AAT ATG T AGTTATATT TTTAAAAAAT TMAGGTMA

  CAAAAAATAG TGTGGCTATG TTATATATAA ATGATAAGAA TATACTGAAA

  AATGTATCCA AAATTAAGG GGGCGTGTAT AGTAAATAAT TAAAAGTATG

  TGCGTTGAAA TAAATTTAGG AGAGTGGTTA GATATGAATA TAAATGACAA

  CTTAAGTATA AATTCCCCGG TAGATAATAA AAATGTT

  PMRP309 is a recombinant plasmid allowing expression of the BotR / A regulatory gene for C. botulinum A under the control of its own promoter. The DNA fragment (nucleotides 2479 to 4425 of the sequence deposited at the EMBL under the access number L42537) was amplified by PCR from the strain of C. botulinum A 62 and cloned in the vector pAT18 at the site Smal. This DNA fragment contains the coding regions for the botR / A genes (also known as Orf21 or OrfX) and the 34 kDa component of hemaglutinin (HA34). The hemaglutinin gene was excised by digestion at the Xbal and EcoRV sites

  (deletion of nucleotides 2664 to 3253) and religation of the plasmid on it-

  even. The Xbal site of the vector had previously been eliminated by

  cleavage at BamHI-Sall sites and religation of the vector on itself.

  region 3450 - 3473 promoter region of the BotR / A gene region 3474 - 4210 coding region for BotR / A (179 amino acids) region 4211 -4425 3 'non-coding part of the BotR / A gene The construction is illustrated by FIG. 16 which represents the sequences of the three regions mentioned above. The part inserted into pAT18 corresponds to the sequence SEQ ID N 16. The plasmid pMRP365 contains the coding region for the regulatory gene tetR under the control of the promoter of the toxin gene iota de

  C. perfringens and the 3 'part of the iota toxin gene from C. perfringens.

  The iota toxin gene promoter region of C. perfringens io (nucleotides 1241 to 1464 of the sequence deposited at the EMBL under the access number X73562) was amplified by PCR from the strain of C. perfringens E NCIB10748 by adding a HindIII site at the 5 'end and the Ncol site at the 3' part. The promoter -10 and -35 regions have been

  determined by the primer extension technique.

  The coding region of the TetR gene (nucleotides 56 to 604) was amplified by PCR from the strain of C. tetani CN655, by adding an Ncol site in part 5 ′ at the level of the initial translation codon and a Pstl site in part 3 'just downstream of the termination codon. This part codes for 179 amino acids. The second amino acid Leu of the initial sequence was changed to Val due to the introduction of the Ncol site. The 3 'part of the iota toxin gene from C. perfringens was amplified by PCR (nucleotides 5504 to 5739 of the sequence X73562) by introducing a PstI site in part 5' and an Xbal site in part 3 '. This non-coding part is essential for the expression of Clostridium genes in the transformed pAT18 or pJIR750 shuttle vectors.

in Clostridium.

  DNA fragments were arranged in the shuttle vector

  pAT18 between the Hindlll and Xbal sites.

  Region 1241 to 1464 Promoter of the toxin iota of C. perfringens E Region 56 to 604 code for amino acids 1 to 179 of the

  tetR regulatory protein of C. tetani.

  111 Region 5504 to 5739 Part 3 'of the iota toxin gene from C. perfringens E. The construction is illustrated in FIG. 17 which represents the sequences of the three regions mentioned above. The part inserted into pAT18 corresponds to the sequence SEQ ID N 12. The plasmid pMRP319 is a recombinant plasmid allowing the expression of the regulatory gene BotR / C of C. botulinum C under control

  of the promoter of the iota toxin gene from C. perfringens.

  The iota toxin gene promoter region of C. perfringens 0o (nucleotides 1241 to 1464 of the sequence deposited at the EMBL under the access number X73562) was amplified by PCR from the strain of C. perfringens E NCIB10748 by adding a HindIII site at the 5 'end and the Ncol site at the 3' part. The promoter -10 and -35 regions have been

  determined by the primer extension technique.

  1 5 The coding region of the BotR / C gene (also known as Orf22) corresponding to nucleotides 302 to 841 (amino acids 1 to 179) of the sequence of the EMBL having the access number X72793 was amplified by generating at 5 'end the Ndel site and at the 3' end the Pstl site from the strain of C. botulinum C468. This DNA fragment was cloned 2o in the vector pET28b (Novagen) and the Ncol-Pstl fragment was included in the plasmid pMRP319. Therefore, the Ncol-Ndel fragment of the vector

  pET28b remains upstream of the BotR / C gene.

  The 3 'part of the iota toxin gene from C. perfringens was amplified by PCR (nucleotides 5504 to 5739 of the sequence X73562) by introducing a PstI site in part 5' and an Xbal site in part 3 '. This non-coding part is essential for the expression of Clostridium gene in the transformed pAT18 or pJIR750 shuttle vectors.

in Clostridium.

  DNA fragments were arranged in the shuttle vector

  pAT18 between the Hindlll and Xbal sites.

  Region 1241 to 1464 Promoter of the iota toxin of C. perfringens E Nde-Ncol region of pET28b Region 302 - 841 Coding region of BotR / C

(amino acids 1 to 179).

  Region 5504 to 5739 Part 3 'of the iota toxin gene from C.perfringens E The construction is illustrated in FIG. 18 which represents the sequences of the four regions mentioned above. The inserted part

  in pAT18 corresponds to the sequence SEQ ID N 14.

  These plasmids are included in the E. Coli bacteria deposited on May 12, 1998 with the National Collection of Cultures of Microorganisms of the Pasteur Institute (CNCM) respectively under

  n 1-2010 (pMRP309), 1-2012 (pMRP 365) and 1-2011 (pMRP319).

  They are also included in the bacteria C. tetani CN655 deposited on May 18, 1998 with the National Collection of Cultures of Microorganisms of the Pasteur Institute (CNCM) respectively under

  n 1-2020 (pMRP309), 1-2022 (pMRP365) and 1-2021 (pMRP319).

  Such a vector can also carry a structural gene coding for a protein heterologous with respect to the regulatory sequence, or for a part of a homologous protein, such as the C-terminal part.

tetanus toxin.

  The present invention further relates to a microorganism characterized in that it comprises at least one vector, a polynucleotide or a sequence as described above. Advantageously, such a microorganism carries a gene coding for a heterologous protein,

  a homologous protein or parts of these proteins.

  A heterologous protein can be any structural protein derived from a microorganism different from that of the regulatory sequence, and in particular butyl toxins, or fragments of these toxins, such as fragments corresponding functionally to fragment C of the tetanus toxin , or Clostridium toxins or variants

  immunogenic but non-toxic for vaccine use.

  A homologous protein can be for example a part devoid of toxic activity, of a toxin produced by a bacteria of the

genus Clostridium.

  As indicated above, the polynucleotide may comprise on the one hand the regulatory gene and on the other hand a structural gene coding for a homologous or heterologous protein or a part of these proteins. However, these two genes can be carried by independent vectors, both present within the microorganism. In a particularly advantageous manner, such a microorganism is a bacterium of the genus Clostridium chosen from C. absonum, C. baratii, C. bifermentans, C. chauvei, C. difficile, G. ghonii, C. lituseburense, C. novyi, C. perfringens, C. septicum, C. sordellii, C.

subterminale and C. tetani.

  Preferably, such a microorganism belongs to the species C. tetani, C. perfringens or E. coli. It can thus be in particular a strain of C. tetani, freed from the plasmid naturally encoding

for tetanus toxin.

  The use of the polynucleotides according to the present invention, carrying at least part of a regulatory gene, makes it possible to obtain a significant increase in the production yields of protein, the synthesis of which is put under the control of the adequate regulatory sequences of the genes. toxin synthesis, compared to

  bacteria not carrying these polynucleotides.

  As a result, it is possible to modify the strains of C. tetani in order to improve the production yields of tetanus toxin, or to produce other proteins such as fragments of tetanus toxin which are no longer toxic but which retain immunogenicity

whole toxin.

  Thus, the overexpression of the tetR gene using a multicopy vector (100 to 200 copies for pAT19) makes it possible to increase the

  minimum of a factor of 10 to 20 the production of tetanus toxin.

  As the tetanus toxin gene is carried on a large plasmid in wild strains, derived strains without plasmid and non-toxigenic can be easily obtained. It is then possible to introduce, using a shuttle vector such as pJIR750 described below (selection marker: chloramphenicol), recombinant genes coding for a non-toxic fragment of tetanus toxin io (fragment Hc) or for another protein, under the control of the promoter of the tetanus toxin gene. The TetR regulatory gene is overexpressed by transformation of these strains with the vector pAT19 described below (selection: erythromycin) carrying this gene. The two vectors pJIR750

and pAT19 can coexist.

  Such a property has a considerable advantage, in the case of industrial production of one of these proteins.

  In addition, the use of bacteria of the genus Clostridium makes it possible to recover the proteins in the culture supernatant, because these

  proteins are secreted by these microorganisms.

  The present invention thus relates to a process for the production of one or more heterologous or homologous proteins comprising the following steps: - culturing a microorganism as described above, and

  - recovery of said proteins.

  Advantageously, these proteins are recovered in

  the culture supernatant of microorganisms.

  Although the present invention is particularly intended for the production of proteins of pharmaceutical interest, this process can be used for any protein which can be expressed, and if possible

  secreted by bacteria of the genus Clostridium.

  The present invention is nevertheless particularly intended

  the production of proteins of pharmaceutical interest.

  More particularly, the invention is suitable for the production of the following toxins or variants: Beta 2 toxin from Clostridium peffringens or any immunogenic fragment. The hydrophilicity profile of the beta 2 toxin reveals several hydrophilic regions which define particular fragments within the meaning of the invention. These regions are located in particular at the amino acid residues 40-55, 105-120, 160-170, 175-188, 200-210 and 250-260. In addition, fragments of the beta 2 toxin lacking

  toxicity include the tryptic fragments of 24, 15 and 13 kDa.

  Clostridium perfringens beta 1 toxin or any immunogenic fragment. The sequence of this toxin has been described by HUNTER and

  Io al. (1993, Infect. Immun. 61, 3958-3965).

  Iota toxins from Clostridium perfringens or any immunogenic fragment. The sequence of the genes coding for the toxins iota1 (gene la) and iota2 (gene lb) has been described by PERELLE et al. (1993,

Infect. Immun. 61, 5147-5156).

  1 5 Alpha toxin from C. novyi or any immunogenic fragment. The gene sequence for this toxin (tcna gene) has been described by HOFMANN et al. (1995, Mol. Gen. Genet 247 670-679). Alpha toxin from C. septicum or any immunogenic fragment. The gene sequence for this toxin has been described by BALLARD et al.

(1995, Infect. Immun. 63, 340-344).

  Toxins a and B of C. difficile or any immunogenic fragment. The gene sequence of these toxins has been described in the literature, as well as different immunogenic regions by VON EICHEL-STREIBER et al. (1992, Mol. Gen. Genet 233, 260-268), VON EICHEL-STREIBER et al. (1989, J. Gen. Microbio. 135, 55-64); VON EICHEL-STREIBER et al.

  (1992, J. Bacteriol. 174, 6707-6710).

  C. perfringens epsilon toxin (WORTHINGTON et al. 1973,

  Onderstepoort J. Vet. Res. 40 (4) 145-152; HUNTER et al. 1992, Infect.

Immun. 60, 102-110).

  Enterotoxin from C. perfringens (McCLANE, Toxicon 34,1996,

1335-1343).

  C. chauvoei toxin (CRICHTON et al. 1986, Australian Vet.

J. 63, 68).

  Cytotoxin L from C. sordellii or any immunogenic fragment. The gene sequence for this toxin has been described by GREEN et al. (1995, Gene 161, 57-61), as well as various immunogenic regions. In particular, this toxin consists of a protein of approximately 270 kDa

  and different antigenic fragments have been described.

  Tetanus toxin, botulinum toxin, in particular the fragment C of these toxins which is the immunogenic fragment (MAKOFF et al. 1989, Bio / Technology 7, 1043; FIGUEIREDO et al. Infect. Immun. 63 1995, 3218-3221; WELLS and al., 1993, Molecular Microbiol. 8, 1155-1162; BOUCHER et al., 1994, Infect. Immun. 62, 449-456; CLARE et al. 1991,

  Bio / Technology 9, 455; CLAYTON et al. 1995, Infect. Immun. 63, 2738-

1o 2742).

  Bordetella pertussis toxin or any other bacterial toxin.

  The subject of the present invention is therefore a vaccine or a medicament, characterized in that it contains a polynucleotide, a sequence, a vector, a microorganism, or a protein obtained by culture of a microorganism according to the present invention, or of a

  protein obtained by culture of this microorganism.

  Such a vaccine is particularly suitable for infections by

C. tetani.

  It also relates to a pharmaceutical composition characterized in that it contains a pharmacologically effective amount of a polynucleotide, of a sequence, of a vector, of a microorganism or of a protein obtained by culture of this

  microorganism, and pharmaceutically acceptable excipients.

  Finally, it relates to the use of these polynucleotides, sequences, vectors, microorganisms or proteins obtained by culture of these microorganisms for immunization against diseases linked to bacteria of the genus Clostridium, or for the treatment

of these diseases.

  The present invention is illustrated without however being limited

by the following examples.

  FIG. 1 represents the nucleotide sequence and the

  amino acid sequence deduced from the tetR gene.

* Figure 2 illustrates the sequence identities between the different

  regulatory genes and the tetR gene.

  FIG. 3 illustrates the lethal activity of culture supernatants of the CN655 strains (wild strain, WT), CN655-OE overexpressing the

  tetR gene, CN655-botR / A overexpressing the BotR / A gene and CN655-

  BotR / C overexpressing the BotR / C gene in mice. The lethal activity (LD50) of these supernatants is measured as a function of the density

  optics at 600 nm from each culture.

  FIG. 4 illustrates the production of TeTx demonstrated by Western Blot using anti-TeTx antibodies, respectively in the wild-type CN655 source (FIG. 4A), the strain overexpressing the gene BotR / A (FIG. 4B), strain overexpressing the BotR / C gene (FIG. 4C), and the strain overexpressing the tetR gene (FIG. 4D). The supernatants of each of the cultures were concentrated by precipitation. 20 g of protein were loaded into well 1 and half dilutions made in series were loaded into the following wells (wells 2 to 4). In FIGS. 4B and 4D the upper band corresponds to the entire TeTx and the lower band to the heavy chain H. FIG. 5 represents an electrophoresis on a polyacrylamide gel of extracellular proteins of the wild strain CN655 from C.tetani (well 1) and of the recombinant strain overexpressing the tetR gene (well 2). The band identified by H corresponds to the string

  heavy with TeTx, while L corresponds to the light chain.

  FIG. 6 represents point hybridizations (dot blots) of messenger RNAs of the wild strain CN655 (WT), and recombinant strains overexpressing the tetR gene (OE), the botR / A gene, and the botR / C gene. Hybridization is carried out using specific probes

  of the TeTx gene. The amounts of messenger RNAs are indicated below.

  above each column (20 pg, 10pg, 5 pg and 2.5 pg).

  FIG. 7 is a schematic representation of the site coding for the botulinum neurotoxins of types A, B, C and D of C. botulinum

and type G of C. argentinense.

  Figure 8 illustrates the strategy used to build the gene

  encoding anti-sense messenger RNA for the botR / A gene.

  FIG. 9 illustrates the toxicity in mice of culture supernatants respectively of the wild strain 62, of the recombinant strain 62-OE overexpressing the botR / A gene and of the recombinant strain 62-AS producing antisense messenger RNAs for the botR / A gene. Toxicity (LD 50) is measured as a function of density

crop optics.

  FIGS. 10 to 10C respectively illustrate the production of BoNT / A, assayed by Western type hybridization, using anti-BoNT / A antibodies, respectively by the wild strain 62 (FIG. 10A), by the strain 62- OE (Figure 10B) and by the strain 62-AS (Figure 10C). The supernatant of each culture was loaded into wells N I1 and half dilutions were loaded into the following wells. The arrows indicate the positions corresponding to the whole neurotoxin (BoNT), the

  heavy chain (H) and light chain (L).

  Figure 11 illustrates the production of non-toxic proteins

  NTNH, assayed by Western type hybridization using anti-

  NTNH in the wild strain (Figure 11A), strain 62-OE (Figure

  11B) and strain 62-AS (Figure 11C).

  FIGS. 12A to 12C respectively illustrate the production of the HA34 and HA70 proteins partially cleaved by Western type hybridization, respectively in the wild strain (FIG. 12A) in

  strain 62-OE (Figure 12B) and in strain 62-AS (Figure 12C).

  FIG. 13 represents dot blot assays of messenger RNAs of wild strains 62 (wt), 62-OE (oe) and 62-AS (as) using probes specific for the bont / A, ntnh, ha70 genes , and ha34. The total amount of messenger RNA loaded in each well is indicated

(20 to 0.1 pg).

  Figure 14A shows a gel mobility test. A labeled DNA fragment corresponding to the ntnh promoter (well 2) was mixed with a cell extract of the strain 62-OE (well 3). The mobility observed with the cell extract is no longer observed when adding an excess of unlabeled DNA (well 1). Addition of polyclonal antibodies to BotR / C to cell extract and labeled DNA

  increases the gel delay (well 4).

  FIG. 14B illustrates the specificity of anti-polyclonal antibodies

  BotR / C by Western type hybridization. The cell extracts of wild strain 62 (well 1) and of recombinant strain 62-OE (well 2) were put to migrate on a PAGE-SDS gel and then transferred to

  nitrocellulose and hybridized with an anti-BotR / C antibody.

  FIG. 15 illustrates the immunoprecipitation of the botR / A protein linked to the promoter of the ntnh (FIG. 15A) and ha genes (FIG. 15B), but not to the coding regions of the botR / A gene (FIG. 15C). Cell extracts of strain 62-OE were incubated with labeled DNA fragments, immunopurified anti-BotR / C antibodies and were immunoprecipitated with agarose protein A beads (well 1). The labeled DNA bands were visualized by autoradiography of the dried SDS-PAGE 1o gel (FIGS. 15A to 15C) and the proteins were

  visualized by Western type hybridization with anti-

  BotR / C then by chemiluminescence (Figure 15D, Figure 15E and Figure F). Negative controls comprising labeled DNA fragments incubated with anti-BotR / C antibodies and devoid of cell extract (well 2) with cell extracts and without anti-BotR / C antibody (well 3) and with cell extracts and non-specific rabbit immunoglobulins (well 4) were performed. Positive controls consisting of labeled DNA mixed with the cell extract without

  immunoprecipitation with agarose-protein A were carried out (well 5).

  BotR / A was immunoprecipitated in all three assays (Figure 15D, Figure E and Figure 15F). The DNA fragments of the promoter regions of ntnh and of ha were co-immunoprecipitated with BotR / A, while the DNA fragment of an identical size of the coding region of bont / A

  (Figure 15C) was not immunoprecipitated.

  Figures 16, 17 and 18 illustrate the constructions

  plasmids pMRP309, pMRP365 and pMRP319 respectively.

  The materials and methods used are as follows.

Materials and methods

Batériennes strains and plasmids.

  The C. tetani CN655 strain and the recombinant strains were cultivated in medium containing trypticase (30 9 / l), yeast extract (20 9 / l), glucose (5 9 / l) and HCI cysteine (0.5 9 / l) (pH 7.2) under anaerobic conditions. Clostridium DNA was extracted and purified as previously described POPOFF et al. (1985, J.

1o Gen. Microbiol, 131: 1697-1703).

  Molecular biology techniques.

  The ligations, transformations, sequencing and preparations of plasmid DNA from Escherichia coli were carried out according to the procedures usually used and described by SAMBROOK et al., (1989 Molecular Cloning: a Laboratory Manual, 2nd edition. Cold Spring

  Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

  Transformation of C, tetani and C. botulinum by electroporation.

  Competent cells of C. tetani CN655 were prepared under anaerobic conditions. Bacteria from a culture in TGY (100 ml) were centrifuged in the middle of their exponential growth phase, washed in distilled water and suspended in 0.5 ml of Na2HPO4, pH 7.4, containing MgCI2 ( 1 mM) and sucrose (270 mM). The plasmid DNA (1 to 5 μg) produced in the E. coli HB101 strain was added to 50 μl of cell suspension. The electroporation was carried out under non-anaerobic conditions using the apparatus marketed under the name "gene pulser" by Bio-Rad (2.5 kV, 200 O and 25 mF) and using a hermetically sealed cuvette in an anaerobic atmosphere. . The bacteria were diluted in TGY, incubated for 3 hours at 37 ° C. and spread on TGY agar containing pg / ml of erythromycin under anaerobic conditions. The electroporation of the cells of the C.botulinum A NCTC 2916 and 62 strain was carried out as indicated previously for

strains of C. tetani.

  The transformation controls are pGK12 and pAT19 which can be transferred into various Gram-positive bacteria. The transformation efficiency of the NCTC2916 strain of C.botulinum by the plasmid pGK12 is from 102 to 103, and from 10 to 102 transforming per pg of DNA for the plasmid pAT19 isolated from the strain

of Escherichia coli HB101.

  Construction of the BotR / A gene expression plasmid A DNA fragment containing the BotR / A gene and its promoter region (between positions 3250 and 4426 numbered according to the sequence of the botulinum neurotoxin site of the strain NCTC2916 corresponding to the number L42537 from GenBank as described by HENDERSON et al. (1996, FEMS Microbiol. Lett. 140, 151-158)) was amplified by PCR using primers having the sequences SEQ ID N 7 and SEQ ID N 8 from the strain of C. botulinum NCTC2916 and was inserted into the plasmid pUC18 at the SmaI site. The fragment was removed by digestion with KpnI-Pstl and was then inserted between the Kpnlet Pstl sites of pAT19. The resulting plasmid (pMR309) was transformed into strain 62 of C.botulinum and gave rise to the

strain 62-OE.

  The plasmid pMRP309 was the subject of a deposit, in the SURe strain of Escherichia coli with the National Culture Collection of

  Microorganisms of the Pasteur Institute on May 12, 1998 under the N 1-2010.

  Construction of plasmids for the expression of the tetR and

botR / C.

  A DNA fragment containing the regions coding for the TetR gene was amplified by the PCR amplification technique from the CN655 strain of C tetani using primers having the sequences SEQ ID N 3 and SEQ ID N 4 following (P516 and P517):

SEQ ID N 3

NCOI

  (P516) CCATGG TTA TGG AAA-ATC TAT TTG TAA AAA

SEQID N 4

Pstl

  (P517) CTGCAG CTA TAT ATT AAT TAA TTT ATT TAC TAT AGG

  The amplification product cut by Ncol and Pst1 was cloned into a multicopy vector pAT19 downstream of the promoter of the iota toxin gene of C.perfringens and upstream of the 3 ′ part of the ibp gene of iota toxin as described below. above for expression of the BotR / A gene

(pMRP306).

  The resulting plasmid pMRP365 was transferred to the strain

  C.tetani CN655 giving rise to the CN655-OE strain.

  The Escherichia coli SURe strain containing pMRP365 has been deposited with the National Collection of Cultures of

  Microorganisms of the Pasteur Institute on May 12, 1998 under the number 1-2012.

  A similar construction was carried out with BotR / C.

  The BotR / C coding region was amplified by PCR from

  of C. botilinum 468 HAUSER et al. (1994, Mol. Gen.

  Genet, 243: 631-640), using the following primers of sequences SEQ ID N 5 and SEQ ID N 6:

SEQID N 5

  (P392) CATATG AATGATTTATTTTATGCTATAG

SEQ ID N06

  (P326) CTGCAGTCAAATATTAACTAATTGCATTAC

  The amplification product was then cloned and gave

birth at pMRP319.

  The resulting plasmid was transferred to the CN655 strain of

  C. tetani and the resulting strain was named CN655-BotR / C.

  The Escherichia coli SURe strain containing this plasmid was

  filed with the CNCM on May 12, 1998 under number 1-2011.

  Construction of a vector encoding anti-sense messenger RNAs

for the BotR / A gene.

  A 73 base pair nucleotide fragment containing the ribosome binding site and the first 18 5 'codons of the BotR / A gene was amplified by the PCR method and was inserted in a reverse orientation into the plasmid pAT19 downstream of the promoter of the iota toxin gene of C.perfringens, and upstream of the 3 'end of the lb gene

  the iota toxin described in the article by PERRELLE et al. (1993, Infect.

  Immun., 61, 5147-5156), as shown in Figure 8.

  The recombinant plasmid pMRP306 was introduced into strain 62 of C. botulinum by electroporation. The recombinant strain

was called 62-AS.

  RNA isolation and dot RNA blots.

  Total RNA was extracted from cultures of C. tetani in the middle of the growth phase using Trizol (GIBCO BRL, Cergy,

Pontoise, France).

  The bacterial pellet of a 10 ml culture was washed twice in distilled water and suspended in 200 ml of 10 mM Tris-HCl (pH

  7) -EDTA (10 mM) sucrose (20%), containing 1 mg of lysozyme.

  The mixture was incubated for 30 min at 37 ° C. and centrifuged. The pellet was suspended in 1 ml of Trizol and incubated for 5 min at room temperature. The following steps were carried out according to the

manufacturer's recommendations.

  Serial dilutions of total RNA in SSCx20 were transferred to Hybond N + membranes (Amersham, Paris, France), were incubated at 60 ° C for two hours in rapid hybridization buffer (Amersham) and the PCR amplified fragments corresponding to the tetx gene labeled with 32p using the Megaprime kit (Amersham). The membranes were washed in SSC x 0.1-SDS (0.1%) at 60 ° C and exposed to X-rays.

PAGE and immunoblots.

  1o The proteins were precipitated from culture supernatants (optical density 1.8) of the wild strain and the strains

  C. tetani recombinants using 10% trichloroacetic acid.

  The precipitates were collected by centrifugation and were

washed with acetone.

  The proteins solubilized in 50 mM Tris-HCl, pH 8, and serial dilutions were put to migrate on an acrylamide gel to

% SDS-PAGE.

  The immunoblot method is that described by BURNETTE et al. (1981, Anal. Biochem., 112, 115-203). The proteins, separated using the SDS-PAGE gel, were transferred onto nitrocellulose sheets (Hybond C, Amersham). The nitrocellulose sheets were incubated for 1 hour in a phosphate buffered saline containing 5% milk powder and were incubated overnight at room temperature with rabbit anti-Tetx antibodies at a dilution of 1: 400. Bound antibodies were detected using peroxidase-labeled protein A and the chemiluminescence kit

marketed by Amersham.

  Electrophoresis and immunoprecipitation.

  C.botulinum strain 62-OE was cultured in TGY until an optical density of 0.6 at 600 nm was obtained. The bacterial cells were centrifuged and resuspended in 10 mM HEPES, pH 7.8, containing KCI (10 mM), EDTA (100 mM), EGTA (100 mM), PMSF (0.5 mM) and DTT (1 mM ). The bacteria were treated with ultrasound for 4 minutes and centrifuged. The supernatant consists

cell extract.

  DNA fragments of 194 and 82 base pairs corresponding to the promoter regions of the ntnh and ha34 genes were amplified by PCR using the primers having the following sequences SEQ ID N 7 to 10:

SEQ ID N 7

io (P369) CAAGATTTTTATTATCTACCGGGG

SEQ ID N 8

(P419) GGTTTACAAAAAATAGTGTGGCTATG

SEQ ID N 9

(P259) CATTATGATTCCTCCTTTATTTAA

SEQ ID N 10

(P530) ATTTTAGGTTTACAAAAAATAAC

  The primers having the sequences SEQ ID N 7 and SEQ ID N 8 were used to amplify the promoter region of ntnh, while the primers having the sequences SEQ ID N 9 and SEQ ID N 10

  have been used to amplify the promoter sequence of the ha34 gene.

  The primers were labeled with [Y32P] ATP using o0 a polynucleotide kinase (Pharmacia), before amplification by the PCR method. The amplification products were extracted with a phenolchloroform mixture and precipitated with ethanol (HURME et al. 1996, J. Biol. Chem., 271, 12626-12631). DNA pellets have been

  dissolved in water and radioactivity was counted.

  Cell extracts (10 pg total protein) were added to the radioactively labeled DNA fragments (1-10 ng, 10,000 cpm) in TBE x 5 (Tris-HCl, 89 mM, pH 8.3, borate 89 mM , EDTA (2 mM); containing MgCI2 (1 mM) NaCI, (50 mM) glycerol (5%), 4 g of salmon sperm DNA and 2 g of acetylated bovine serum albumin (BSA)

in a total volume of 30 pl.

  The mixture was incubated for 20 minutes at 37 ° C. and then loaded onto a 5% polyacrylamide gel (29: 1 acrylamide-bisacrylamide) and subjected to electrophoresis in TBE x 5 at 10 V.cm'1 for 4 hours at 4 C. The gels were dried and autoradiographed. After a 20 min preincubation step in the presence of 1 μg of unlabeled DNA, the labeled probes were added. Anti BotR / C antibodies were added at a 1: 100 dilution in the reaction medium. Anti-BotR / C antibodies were obtained using the BotR / C protein fused to His-tag in the vector pET28b (Novagen), produced in the Escherichia coli BL21 strain and purified on an Ni column. For the immunopurification of anti-BotR / C antibodies, the cell extract of the C.botulinum 62-OE strain was subjected to a

  electrophoresis on PAGE 10% SDS 0.1%, and transferred to nitrocellulose.

  The 21 kD band was considered to be the cut off. The reaction was blocked by incubation in PBS containing 5% milk and then in 1:10 anti BotR / C serum. After washing with PBS, the specific antibodies were eluted with 0.1 M acetic acid and

  neutralized with 3M Tris-HCI at pH8.

  Immunoprecipitations were performed by incubating

  DNA fragments amplified by PCR and labeled with 32p as described above

  above (2x105 cpm), the cellular extract of C.botulinum 62-OE (10 μg of total proteins) in a buffer B Tris-HCl 20 mM, pH 7.5, containing MgCl 2 10 mM and 250 μg / ml of Salmon sperm DNA in a

  final volume of 60 μl, for 20 min at room temperature.

  As a negative control, DNA fragments of the coding region of the bont / A gene (positions 3638 to 3769 of the sequence M30196) were amplified by PCR. Immunopurified anti-botR / C antibodies (400 ng) were added to each sample and were incubated for 4 min at room temperature. Agarose-protein A (Sigma) (20 μg) was added and was incubated for 1 h at 4 C. The beads were isolated by centrifugation and then washed three times with buffer B. They were mixed with 15 μl of sample buffer, heated at 100 ° C. for two minutes and loaded onto a 10% SDS-0.1% PAGE gel. The experiments were performed in duplicate. One gel was dried and exposed to X-ray for 4 days in order to visualize the 32p DNA, and the other gel was transferred to nitrocellulose, hybridized with the immunopurified anti-BotR / C antibodies and the proteins were detected. using a kit

chemiluminescence.

Toxicity in mice.

  Serial dilutions of half the samples (0.5 ml), in 50 mM sodium phosphate buffer (pH 3) containing 0.2% (weight / volume) of gelatin, were injected through the peritoneum (i.p.)

in mice from 18 to 20 g.

  Four mice were used for each dilution. The mice were observed after four days and the death toll was

account.

EXAMPLE 1

  Characterization of the tetR gene and overexpression of this gene in

C. tetani.

  1) Characterization of the tetR gene The nucleotide sequence and the amino acid sequence deduced from this nucleotide sequence (respectively sequences SEQ ID N 1 and SEQ ID N 2) of the TetR gene are represented on the

figure 1.

  The deduced protein has a calculated molecular mass of 21,562 Da and is composed of -178 amino acids. It shows a sequence similarity of 50 to 65% with the corresponding proteins of the strains of C. botulinum, as can be seen from the alignment of

sequences of figure 2.

  This protein has the characteristics of proteins

binding to DNA.

  2) Overexpression of the tetR gene in C.tetani In order to analyze the function of the protein, the tetR gene was

overexpressed in C.tetani.

  The tetR gene of C. tetani was placed under the control of the promoter of the iota toxin gene of C. perfringens cloned into the shuttle vector pAT19 (selection marker: erythromycin). A 200 bp segment corresponding to the 3 'part of the iota toxin gene is inserted in part

  3 'of the tetR gene (plasmid pmRP365).

  The tetR gene could also be placed under the control of its own promoter by: - PCR amplification of the tetR promoter (143 nucleotides upstream of the tetR atg) by generating a HindIII site in part 5 'and Ncol in part 3' , and - replacement of the Hindll-Ncol fragment (promoter of the iota toxin gene) of pmRP365 with the amplified Hindll-Ncol fragment

(tetR promoter).

  The coding region of tetR was therefore amplified by PCR and cloned into the vector containing the promoter of the iap gene from C.perfringens. The resulting plasmid, pMRP365, was transferred by electroporation into C. tetani CN655, giving rise to the strain.

CN655-OE.

  The production of TeTx was monitored in the wild strain (CN655) and the recombinant strain (CN655-OE) by determination of the lethality in mice. As shown in FIG. 3, the activity in the culture supernatants during the exponential growth phase is 6 to 8 times higher in the CN655-OE strain, compared to the wild strain. The increase in the production of TeTx in the CN655-OE strain is confirmed by Western type hybridization (Western blot) using specific antibodies against TeTx, as shown in

figure 4.

  These results indicate that the tetR gene, present in large copy number and in trans position in C.tetani, induces an increase in the production of TeTx. No other extracellular 0o protein seems to have been overproduced as shown in FIG. 5. The messenger RNAs specific for the TeTx gene were sought by the dot blot technique in the

  wild strain and in the recombinant strain CN655-OE.

  The amount of TeTx specific messenger RNA is approximately four times higher in the recombinant strain compared to the wild strain, as shown in FIG. 5. This result is in agreement with the increase in the level of TeTx in the culture supernatant. measured by lethal activity in mice and

immunohybridization.

  These results clearly show that the tetR gene activates

  the expression of TeTx at the transcriptional level.

EXAMPLE 2

  Overexpression of the botR / A and botR / C genes in C.tetani Since the TetR and BotR genes have significant homology, it seemed interesting to determine whether these genes are

functionally identical.

  It had been shown that the plasmid pMRP309 containing the botR / A gene under the control of its own promoter induces a significant increase in the production of botNT / A and non-toxic proteins (ANTP), as well as corresponding messenger RNAs

in C.botulinum.

  The CN655 strain of C.tetani was transformed by electroporation with the plasmid pMRP309 giving rise to the CN655-BotR / A strain. A comparable increase in Tetx, measured by the mouse lethality test and by immunohybridization was observed during the exponential growth phase of CN655-BotR / A and CN655-OE, as shown in Figures 3 and 4. The activity lethal

  in mice culture supernatants of CN655-OE and CN655-

  BotR / A is approximately 8 times greater than the activity of

  supernatants from the wild strain, as shown in Figure 3.

  The overproduction of Tetx dosed by immunohybridization is 16

  times higher in CN655-OE and 8 times higher in CN655-

  BotR / A, compared to the wild strain, as illustrated in FIG. 4. In addition, the quantity of messenger RNAs of the Tetx gene is 4 times higher in the two strains, as illustrated in FIG. 6. This shows that BotR / A can up-regulate the Tetx gene in

C. tetani.

  The effect of BotR / C which is less related to TetR (50% identity) than BotR / A (60% identity) was analyzed in C.tetani. The plasmid pMRP319 corresponding to the vector pAT19 containing the coding region of botR / C, under the control of the promoter of the iap gene was transferred into the strain CN655 by electroporation, giving rise to the source CN655-botR / C. The production of Tetx, measured by lethal activity in mice is three times greater in the CN655-BotR / C strain than in the wild strain, and eight times

  higher if assayed by immunohybridization (Figures 3 and 4).

  No significant increase in the level of Tetx-specific messenger RNA is detected in the culture supernatant of the

  strain CN655-BotR / C, as shown in Figure 6.

  These results show that BotR / C stimulates the expression of

  Tetx gene, but less important than the BotR / A gene.

EXAMPLE 3

  Activity of the BotR / A regulatory gene 1) Overexpression of the botR / A gene in strain A 62 of

C. botulinum.

  The botR / A gene was overexpressed in C.botulinum by inserting it, under the control of its own promoter, into a vector with a large number of pAT19 copies. The recombinant plasmid was used to transform strain 62 by electroporation and gave rise to strain 62-OE. The level of transcription of the botR / A gene in strain 620E is higher than that obtained in wild strain 62, as shown by the assays of messenger RNAs carried out by dot

  blot with the botR / A gene labeled with 32p.

  The toxicity of the culture supernatant towards mice during the growth phase of the wild strain and of the 62-OE strain is illustrated in FIG. 9. The recombinant strain overexpressing botR / A in trans is more lethal towards with the mice (5 to 12 times) than the wild strain. The lethal activity in mice is due to the presence of BoNT / A in the culture supernatant of serotype A of C.botulinum. It has also been shown by Western type hybridization (FIG. 10) that there are 16 to 32 times more BoNT / A in the culture supernatant of the 62-OE strain than in that of the wild strain, after purification. partial

by acid precipitation.

  In order to determine whether botR / A also regulates the expression of the antp gene, Western type hybridizations were carried out on the culture supernatants partially purified by acid precipitation of the strain 62-OE and of the wild strain. An antiserum directed against the non-toxic proteins NTNH made it possible to detect two bands of respective sizes of 106 and 13 kDa, corresponding to the NTNH proteins which have undergone maturation. These bands are less intense for the 62-OE strain than for the wild strain, such as

shows figure 11.

  Similarly, Western-type hybridization with antibodies directed against hemagglutinin-like components shows that the proteins HA34 and HA70, which are partially cleaved in HA52 are

  overproduced in strain 62-OE, as illustrated in Figure 12.

  The level of overproduction of HA70 and .HA52 in the

  1o strain 62-OE is 8 to 16 times higher than that of the HA34 protein.

  A weak band corresponding to HA34 partially fuses with the band immediately above, and is observed in wells 1 and 2 of the Western-type hybridization of the wild strain (FIG. 12A) while the band HA34 is much more visible ( Figure 12B, wells 1 and 2)

with strain 62-OE.

  This shows that the overexpression of botR / A in trans is

  responsible for the overproduction of BoNT / A and ANTPs proteins.

  The levels of messenger RNAs for the proteins BoNT / A and ANTPs, measured by dot blot, are significantly greater 2o in the 62-OE strain, compared to the wild strain, as illustrated

Figure 13.

  The levels of messenger RNAs for the proteins boNT / A, NTNH and HA70 are 8 times higher in the strain 62-OE compared to the wild strain 62. This is consistent with the higher toxicity vis-à-vis mice, and higher levels of NTNH and HA70

  detected by immunohybridization for strain 62-OE.

  2) Partial inhibition of the expression of the botR / A gene and effect on the

  production of the proteins boNT / A and ANTP.

  Anti-sense messenger RNAs have been used to inhibit the expression of the botR / A gene. The DNA fragment used to produce antisense messenger RNAs (Figure 8) contains the ribosome binding site and the first 18 codons of the botR / A gene, and does not form any

secondary structure.

  The toxicity of the culture supernatant of strain 62-AS which 1o contains the plasmid pMRP306 is 4 to 10 times lower than that of the wild strain. This decrease in toxicity is more significant in the early culture stage (Figure 9). Inhibition of the boNT / A gene is also observed in strain 62-AS by immunohybridization

as shown in Figure 10.

  Lower levels of NTNH and HA35 were measured in strain 62-AS (Figures 11 and 12). However, the decrease in HA70 and HA52 is less marked. The dot blot detection tests for messenger RNAs show that the transcription of the boNT / A and antp genes is partially inhibited (by a factor of 4 to 16), as shown in

figure 13.

  3) Linkage of the BotR / A gene and the promoter region of the genes

ntnh and ha34.

  In the toxinotypes A of C.botulinum, the gene complexes coding for the botulinum toxin are organized into two polycistronic operons, as illustrated in FIG. 7. The promoter regions of the ntnh- bont / A and ha34 genes have sequences with

  -35 and -10 kept upstream of each operon.

  As BotR / A activates the expression of the two operons, the protein could directly interact with the promoter regions of

  ntnh-bont / A and ha34 genes, thereby regulating their transcription.

  In order to demonstrate this process, gel mobility retardation experiments were carried out with overproduced BotR / A in strain 62-OE and DNA fragments.

  corresponding to the promoter regions of the nt-nh and ha34 genes.

  Cell extract of strain 62-OE overexpressing the botR / A gene

  retards the DNA fragment from the promoter of ntnh (Figure 14A) and ha34.

  The specificity of this test was demonstrated by competition with

unlabeled DNA (Figure 14A).

  In addition, anti-BotR / C polyclonal antibodies, which recognize only BotR / A in the cell extract of toxinotype A of C. botulinum (Figure 14B) were used to confirm the specificity of the effects of BotR / A in cell extract of strain 62-OE. An increase in mobility was observed when the labeled DNA and the 62-OE extract were incubated in the presence of anti-BotR / C antibodies (FIG. 14A, well 4), while the antibodies directed to the against a Clostridium extract do not cause any delay in DNA mobility

2o marked.

  Additional experiments showing the binding of BotR / A with promoter DNA were performed by immunoprecipitation using immunopurified antibodies specific for BotR / C which cross-react with BotR / A. Co-immunoprecipitation of botR / A and DNA fragments corresponding to the promoter regions of ntnh and ha34 was observed, while a DNA fragment of size similar to that of the region coding for good / A n ' has not been immunoprecipitated under the same conditions, as illustrated by

figure 15.

EXAMPLE 4

  Construction of a plasmid for the expression of fragment C of the

tetanus toxin.

  The nucleotide sequence corresponding to the terminal fragment C of the tetanus toxin has already been cloned and used in bacteria Figueiredo et al., (1995, Infect. Immun., 63, 3218-3221; Clare

  et al., 1991, Biotechnology, 9, 455-460).

  o0 The construction of a plasmid allowing the expression of this fragment C in C.tetani can be carried out by the process comprising the following steps: - Amplification of the promoter region of the tetanus toxin gene (140 nucleotides upstream of the ATG codon of the tetanus toxin gene) by generating a Sphl site in part 5 ′ and the Ncol-Pstl sites in

  part 3 'including Ncol corresponding to the initial ATG.

  - Cloning of the Sphl-Pstl fragment into the corresponding sites of the polylinker of the vector pJIR750 (selection marker: chloramphenicol). Amplification by PCR of the 3 'part of the tetanus toxin gene coding for the C-terminal part (50 kDa) of the tetanus toxin and the 250 non-coding nucleotides downstream of the stop codon of this gene, by generating an Ncol site in part 5 'and in line with the

  coding sequence, and a PstI site in part 3 '.

  - Cloning of the Ncol-Pstl fragment in the corresponding sites

of the recombinant plasmid pMRP365.

* - Transformation by electroporation of a strain of C. tetani having lost its large plasmid carrying the tetanus toxin gene and therefore non-toxinogenic, with the plasmid pMRP365 or the

  equivalent plasmid with tetR under the control of its own promoter.

  Selection of transformants in an anaerobic chamber on erythromycin boxes (10 mg / ml). Transplanting a transforming clone and

  verification of its recombinant plasmid content.

  - Transformation of the clone obtained with the plasmid containing the gene for fragment C and selection in an anaerobic chamber on Petri dishes containing erythromycin (10 mg / ml) and chloramphenicol (20 mg / ml). Transplanting a transformant and checking its content in

recombinant plasmids.

  - Control of the production of fragment C by electrophoresis in polyacrylamide gel and Western blot with anti-toxin antibodies

  tetanus from the culture supernatant of the recombinant strain.

LIST OF SEQUENCES

(1) GENERAL INFORMATION:

(i) DEPOSITOR:

(A) NAME: INSTITUT PASTEUR

(B) STREET: 25 RUE DU DOCTEUR ROUX

(C) CITY: PARIS

(E) COUNTRY: FRANCE

(F) POSTAL CODE: 75015

  (ii) TITLE OF THE INVENTION: SEQUENCES (iii) NUMBER OF SEQUENCES: 17 (iv) FORM DETERMINABLE BY COMPUTER: (A) TYPE OF MEDIUM: Floppy disk (B) COMPUTER: IBM PC compatible

  (C) OPERATING SYSTEM: PC-DOS / MS-DOS

  (D) SOFTWARE: PatentIn Release # 1.0, Version # 1.30 (EPO)

  (2) INFORMATION FOR SEQ ID NO: 1:

  (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 916 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: double (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (gnomic) (iii ) HYPOTHETICAL: NO (ix) CHARACTERISTIC:

(A) NAME / KEY: CDS

(B) LOCATION: 141..678

  (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:

  AACTATATTA ATATACACAT CCTCGGTAAT ATATCAATAC AATACATGTA ATTAATAATG 60

  TTTTTTGTAA AAGTTTAAGC TTAGTGGTTT ACTTTCAAAA TATAATAAAA CGTTAATTAT 120

  AGAAAAGTGA AAGTAAGGTG ATG CTT ATG GAA AAT CTA TTT GTA AAA ATT 170

  Met Leu Met Glu Asn Leu Phe Val Lys Ile

1 5 10

  AAG ACA TTA AAA AAT AAT AAT AAA GAA TTT GAA GTA ATT ATT ATA CAT 218

  Lys Thr Leu Lys Asn Asn Asn Lys Glu Phe Glu Val Ile Ile Ile His

20. 25

  TTC AAA AAT ACT GTA AAT ATA CTT ATT CGA AAG TAT AAT TTA GAA AGT 266

  Phe Lys Asn Thr Val Asn Ile Leu Ile Arg-Lys Tyr Asn Leu Glu Ser

35 40

  CAT TAT AAT GAT ATC TTA TAT CAT TTA TGG AAT ATA CTT AAA AAA ATT 314

  His Tyr Asn Asp Ile Leu Tyr His Leu Trp Asn Ile Leu Lys Lys Ile

50 55

  GAT TTG AAT AAA TTT AAT ACA GAA AAT GAT TTA CAC AAA TAT ATT AGT 362

  Asp Leu Asn Lys Phe Asn Thr Glu Asn Asp Leu His Lys Tyr Ile Ser

65 70

  AGA TCT TTA AAA AGA TAT TGC TTA GAT ATT TGT AAT AAA AAA AAT AGG 410

  Arg Ser Leu Lys Arg Tyr Cys Leu Asp Ile Cys Asn Lys Lys Asn Arg

80 85 90

  GAT AAA AAA ATA ATA TAT AAC TCA GAA ATT ACA AAT ATA AAA TTA AAT 458

  Asp Lys Lys Ile Ile Tyr Asn Ser Glu Ile Thr Asn Ile Lys Leu Asn

100 105

  TTA ATG GAA AAT AGT TGC TCA AAT TAT TTA AAC TTC GAA TTT AAT GAT 506

  Leu Met Glu Asn Ser Cys Ser Asn Tyr Leu Asn Phe Glu Phe Asn Asp

115 120

  TTA ATA TCC ATA TTA CCT GAA AAT CAA AGA AAA ATT TTA TAT ATG AAA 554

  Leu Ile Ser Ile Leu Pro Glu Asn Gln Arg Lys Ile Leu Tyr Met Lys

130 135

  TTT TTT GAA TAT ATG AAA GAA TGT GAT ATA GCT AAG CTT CAT ATG AGT 602

  Phe Phe Glu Tyr Met Lys Glu Cys Asp Ile Ala Lys Leu His Met Ser

145 150

  CGT CAA GCT GTA TAT AAA AAT AAG GTT TTA GCA TTA AAA AAA TTA GAA 650

  Arg Gln Ala Val Tyr Lys Asn Lys Val Leu Ala Leu Lys Lys Leu Glu

160 165 170

  CCT ATA GTA AAT AAA TTA ATT AAT ATA T AGTTTTTATA ATTTAATTAT 698

  Pro Ile Val _Asn Lys Leu Ile Asn Ile

  GAATAATATT CTTAAGATAA AAAGTAAATT TTTAAAAAAT TTAAATTTTC AGTTTACAAA 758

  AAATAACCTG ATTATGTTAT ATGTAATTGT AAAAAACATA TAAAAAATCA GAAAAATTTA 818

  GGAGGTATAT TATTAATGGA TTAAATAATA ATTTTTTAAT TTACTTTTGA TTAATAAATA 878

  TTAAATGTTT ATTTTAATTA GGAGATGATA CGTATGCC 916

  (2) INFORMATION FOR SEQ ID NO: 2:

  (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 179 amino acids (B) TYPE: amino acid (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: protein

  (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:

  Met Leu Met Glu Asn Leu Phe Val Lys Ile Lys Thr Leu Lys Asn Asn

1 5 10 15

  Asn Lys Glu Phe Glu Val Ile Ile Ile His Phe Lys Asn Thr Val Asn

25 30

  Ile Leu Ile Arg Lys Tyr Asn Leu Glu Ser His Tyr Asn Asp Ile Leu

40 45

  Tyr His Leu Trp Asn Ile Leu Lys Lys Ile Asp Leu Asn Lys Phe Asn

55 60

  Thr Glu Asn Asp Leu His Lys Tyr Ile Ser Arg Ser Leu Lys Arg Tyr

70 75 80

  Cys Leu Asp Ile Cys Asn Lys Lys Asn Arg Asp Lys Lys Ile Tyr Island

90 95

  Asn Ser Glu Ile Thr Asn Ile Lys Leu Asn Leu Met Glu Asn Ser Cys

105 110

  Ser Asn Tyr Leu Asn Phe Glu Phe Asn Asp Leu Ile Ser Ile Leu Pro

120 125

  Glu Asn Gln Arg Lys Ile Leu Tyr Met Lys Phe Phe Glu Tyr Met Lys

135 140

  Glu Cys Asp Ile Ala Lys Leu His Met Ser Arg Gln Ala Val Tyr Lys

150 155 160

  Asn Lys Val Leu Ala Leu Lys Lys Leu Glu Pro Ile Val Asn Lys Leu

170 175

Ile Asn Ile

  (2) INFORMATION FOR SEQ ID NO: 3:

  (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii ) HYPOTHETICAL: NO

  (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3:

CCATGGTTAT GGAAAATCTA TTTGTAAAAA 30

  (2) INFORMATION FOR SEQ ID NO: 4:

  (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 36 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii ) HYPOTHETICAL: NO

  (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4:

  CTGCAGCTAT ATATTAATTA ATTTATTTAC TATAGG 36

  (2) INFORMATION FOR SEQ ID NO: 5:

  (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 28 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii ) HYPOTHETICAL: NO

  (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5:

CATATGAATG ATTTATTTTA TGCTATAG 28

  (2) INFORMATION FOR SEQ ID NO: 6:

  (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii ) HYPOTHETICAL: NO

  (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6:

CTGCAGTCAA ATATTAACTA ATTGCATTAC 30

  (2) INFORMATION FOR SEQ ID NO: 7:

  (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii ) HYPOTHETICAL: NO

  (xi) DESCRIPTION OF THE SEQUENCE: SEQ'ID NO: 7:

CAACATTTTT ATTATCTACC GGGG 24

  (2) INFORMATION FOR SEQ ID NO: 8:

  (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: RNA (genomic) (iii ) HYPOTHETICAL: NO

  (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8:

GGTAGAAAAG TTCTTTCAAC TGAC 24

  (2) INFORMATION FOR SEQ ID NO: 9:

  (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 26 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii ) HYPOTHETICAL: NO

  (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9:

GGTTTACAAA AAATAGTGTG GCTATG 26

  (2) INFORMATION FOR SEQ ID NO: 10:

  (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii ) HYPOTHETICAL: NO

  (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10:

CATTATGATT CCTCCTTTAT TTAA 24

  (2) INFORMATION FOR SEQ ID NO: 11:

  (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 23 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii ) HYPOTHETICAL: NO

  (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11:

ATTTTAGGTT TACAAAAAAT AAC 23

  (2) INFORMATION FOR SEQ ID NO: 12:

  (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1016 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: double (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii ) HYPOTHETICAL: NO (ix) CHARACTERISTIC:

(A) NAME / KEY: CDS

(B) LOCATION: 233..769

  (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12:

  AAGCTTATTT TACATTAAAT TACTTTTATT GATAAAAATA TATTTTTTGT ATTTATATAG 60

  AGATTTTCTT TGTCATATAC TGTATAATAT TTATATAATA TTAATACATA ATATTATAAC 120

  TAAATTCAAA ATAGAAAGGA GGTTATCTTA TGTTAAAAAG AAAAATTTAA AATACTATTT 180

  TCATTATTTG AAGATTGAAT CTTAAATAAA CTTGAATTAG GAGGGAGTAC CC ATG 235

Met

  GTT ATG GAA AAT CTA TTT GTA AAA ATT AAG ACA TTA AAA AAT AAT AAT 283

  Val Met Glu Asn Leu Phe Val Lys Ile Lys Thr Leu Lys Asn Asn Asn

190 195

  AAA GAA TTT GAA GTA ATT ATT ATA CAT TTC AAA AAT ACT GTA AAT ATA 331

  Lys Glu Phe Glu Val Ile Ile Ile His Phe Lys Asn Thr Val Asn Ile

205 210

  CTT ATT CGA AAG TAT AAT TTA GAA AGT CAT TAT AAT GAT ATC TTA TAT 379

  Leu Ile Arg Lys Tyr Asn Leu Glu Ser His Tyr Asn Asp Ile Leu Tyr

215 220 225

  CAT TTA TGG AAT ATA CTT AAA AAA ATT GAT TTG AAT AAA TTT AAT ACA 427

  His Leu Trp Asn Ile Leu Lys Lys Ile Asp Leu Asn Lys Phe Asn Thr

230 235 240

  GAA AAT GAT TTA CAC AAA TAT ATT AGT AGA TCT TTA AAA AGA TAT TGC 475

  Glu Asn Asp Leu His Lys Tyr Ile Ser Arg Ser Leu Lys Arg Tyr Cys

245 250 255 260

  TTA GAT ATT TGT AAT AAA AAA AAT AGG GAT AAA AAA ATA ATA TAT AAC 523

  Leu Asp Ile Cys Asn Lys Lys Asn Arg Asp Lys Lys Ile Tyr Island Asn

265,270,275

  TCA GAA ATT ACA AAT ATA AAA TTA AAT TTA ATG GAA AAT AGT TGC TCA 571

  Ser Glu Island Thr Asn Ile Lys Leu Asn Leu Met Glu Asn Ser Cys Ser

280 285 290

  AAT TAT TTA AAC TTC GAA TTT AAT GAT TTA ATA TCC ATA TTA CCT GAA 619

  Asn Tyr Leu Asn Phe Glu Phe Asn Asp Leu Ile Ser Ile Leu Pro Glu

295,300,305

  AAT CAA AGA AAA ATT TTA TAT ATG AAA TTT TTT GAA TAT ATG AAA GAA 667

  Asn Gln Arg Lys Ile Leu Tyr Met Lys Phe Phe Glu Tyr Met Lys Glu

310 315 320

  TGT GAT ATA GCT AAG CTT CAT ATG AGT CGT CAA GCT GTA TAT AAA AAT 715

  Cys Asp Ile Ala Lys Leu His Met Ser Arg Gln Ala Val Tyr Lys Asn

325 330 335 340

  AAG GTT TTA GCA TTA AAA AAA TTA GAA CCT ATA GTA AAT AAA TTA ATT 763

  Lys Val Leu Ala Leu Lys Lys Leu Glu Pro Ile Val Asn Lys Leu Ile

345 350 355

  AAT ATC TGCAGATAAA AATTATATTT AAAATTATTA ATAAATATCA AAATTAATAA 819

Asn Ile

  AGCTAGTTAT TCATACTGGC TACCTATATA AATATTTCTC GATTACTTAC AGAGGATGTC 879

  AATAATTTTG TGTAAACTAT CTAAAGAAAT ATATTGAAAT TTGTTGAGGT TTAGATAATA 939

  TTTTATTTAA ACCTTAATTT TCTTTTTATG TAATTATAGT GAATATAAAT TGTACAAAAA 999

CTAGAGATAT TTCTAGA 1016

  (2) INFORMATION FOR SEQ ID NO: 13:

  (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 179 amino acids (B) TYPE: amino acid

(D) CONFIGURATION: linear -

(ii) TYPE OF MOLECULE: protein

  (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13:

  Met Val Met Glu Asn Leu Phe Val Lys Ile Lys Thr Leu Lys Asn Asn

1 5 10 15

  Asn Lys Glu Phe Glu Val Ile le e Ile His Phe Lys Asn Thr Val Asn

25 30

  Ile Leu Ile Arg Lys Tyr Asn Leu Glu Ser His Tyr Asn Asp Ile Leu

40 45

  Tyr His Leu Trp Asn Ile Leu Lys Lys Ile Asp Leu Asn Lys Phe Asn

55 60

  Thr Glu Asn Asp Leu His Lys Tyr Ile Ser Arg Ser Leu Lys Arg Tyr

70 75 80

  Cys Leu Asp Ile Cys Asn Lys Lys Asn Arg Asp Lys Lys Ile Tyr Island

90 95

  Asn Ser Glu Ile Thr Asn Ile Lys Leu Asn Leu Met Glu Asn Ser Cys

105 110

  Ser Asn Tyr Leu Asn Phe Glu Phe Asn Asp Leu Ile Ser Ile Leu Pro

120 125

  Glu Asn Gln Arg Lys Ile Leu Tyr Met Lys Phe Phe Glu Tyr Met Lys

135 140

  Glu Cys Asp Ile Ala Lys Leu His Met Ser Arg Gln Ala Val Tyr Lys

150 155 160

  Asn Lys Val Leu Ala Leu Lys Lys Leu Glu Pro Ile Val Asn Lys Leu

170 175

Ile Asn Ile

  (2) INFORMATION FOR SEQ ID NO: 14:

  (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1078 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: double (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii ) HYPOTHETICAL: NO (ix) CHARACTERISTIC:

(A) NAME / KEY: CDS

(B) LOCATION: 291..828

  (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 14:

  AAGCTTATTT TACATTAAAT TACTTTTATT GATAAAAATA TATTTTTTGT ATTTATATAG 60

  AGATTTTCTT TGTCATATAC TGTATAATAT TTATATAATA TTAATACATA ATATTATAAC 120

  TAAATTCAAA ATAGAAAGGA GGTTATCTTA TGTTAAAAAG AAAAATTTAA AATATATTTT 180

  CATTATTTGA AGATTGAATC TTAAATAAAC TTGAATTAGG AGGGAGTACC ATGGGCAGCA 240

  GCCATCATCA TCATCATCAC AGCAGCGGCC TGGTGCCGCG CGGCAGCCAT ATG AAT 296

Met asn

  GAT TTA TTT TAT GCT ATA GAA AAT TTA AAG CAT GAT AAC CAG CAC TTT 344

  Asp Leu Phe Tyr Ala Ile Glu Asn Leu Lys His Asp Asn Gln His Phe

190 195

  AAC TTT ATT GAA ATG TCT CTA AAA AAA TAT ATT GAA AAA ACT TCT AAA 392

  Asn Phe Ile Glu Met Ser Leu Lys Lys Tyr Ile Glu Lys Thr Ser Lys

205 210

  AAG TAT AAT TTA TAT TAT GAT TAT TAT AAT GAT ATA CTG TAT CAT TTA 440

  Lys Tyr Asn Leu Tyr Tyr Asp Asp Tyr Tyr Asn Asp Ile Leu Tyr His.Leu

215 220 225

  TGG AAA GAA CTT ATT GAA ATT AAC TTA AAG AAT TTT AAT TCT GAA TTA 488

  Trp Lys Glu Leu Ile Glu Ile Asn Leu Lys Asn Phe Asn Ser Glu Leu

230 235 240 245

  GAT TTA AGA AAA TAT ATT AGT ACA AGT ATA AAA AGA TAT TGT ATA AAT 536

  Asp Leu Arg Lys Tyr Ile Ser Thr Ser Ile Lys Arg Tyr Cys Ile Asn

250 255 260

  ATT TGT AAG AAA AAA AAT AGG GAT AAG AAA ATT ATA TAT AAT TCA GAA 584

  Ile Cys Lys Lys Lys Asn Arg Asp Lys Lys Ile Ile Tyr Asn Ser Glu

265,270,275

  GTA ACA TAT AAA AAA TTG GAT GCT GTA AAT GTT TAT TCT TTA TAT TGT 632

  Val Thr Tyr Lys Lys Leu Asp Ala Val Asn Val Tyr Ser Leu Tyr Cys

280 285 290

  GAT AAT TTC GAG TTT TTG GAT TTG ATA TCC ATA TTA AAC TAC AAG GAA 680

  Asp Asn Phe Glu Phe Leu Asp Leu Ile Ser.Ile Leu Asn Tyr Lys Glu

295,300,305

  AAG CAA ATT ATA TAT ATG AAA TTT TTT GAA GGT AGA AAA GAT AAT GAA 728

  Lys Gln Ile Tyr Island Lys Phe Phe Glu Gly Arg Lys Asp Asn Glu

310 315 320 325

  ATA GCT ATA AGG CTT CGT TTA AGT CGT CAA TCT ATA TAT AAG ATT AGA 776

  Ile Ala Ile Arg Leu Arg Leu Ser Arg Gln Ser Tyr Lys Island Arg Island

330 335 340

  ATT ACG TCT TTA AAA AAG TTG TAT CCT ATA GTA ATG CAA TTA GTT AAT 824

* Ile Thr Ser Leu Lys Lys Leu Tyr Pro Ile Val Met Gln Leu Val Asn

345 350 355

  ATT T GACTGCAGAT AAAAATTATA TTTAAAATTA TTAATAAATA TCAAAATTAA 878

Isle

  TAAAGCTAGT TATTCATACT GGCTACCTAT ATAAATATTT CTCGATTACT TACAGAGGAT 938

  GTCAATAATT TTGTGTAAAC TATCTAAAGA AATATATTGA AATTTGTTGA GGTTTAGATA 998

  ATATTTTATT TAAACCTTAA TTTTCTTTTT ATGTAATTAT AGTGAATATA AATTGTACAA 1058

AAACTAGAGA TATTTCTAGA 1078

  (2) INFORMATION FOR SEQ ID NO: 15:

  (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 179 amino acids (B) TYPE: amino acid (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: protein

  (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 15:

  Met Asn Asp Leu Phe Tyr Ala Ile Glu Asn Leu Lys His Asp Asn Gln

1 5 10 15

  His Phe Asn Phe Ile Glu Met Ser Leu Lys Lys Tyr Ile Glu Lys Thr

25 30

  Ser Lys Lys Tyr Asn Leu Tyr Tyr Asp Asp Tyr Tyr Asn Asp Ile Leu Tyr

40 45

  His Leu Trp Lys Glu Leu Ile Glu Ile Asn Leu Lys Asn Phe Asn Ser

55 60

  Glu Leu Asp Leu Arg Lys Tyr Ile Ser Thr Ser Ile Lys Arg Tyr Cys

70 75 80

  Ile Asn Ile Cys Lys Lys Lys Asn Arg Asp Lys Lys Ile Tyr Island Asn

90 95

  Ser Glu Val Thr Tyr Lys Lys Leu Asp Ala Val Asn Val Tyr Ser Leu

105 110

  Tyr Cys Asp Asn Phe Glu Phe Leu Asp Leu Ile Ser Ile Leu Asn Tyr

120 125

  Lys Glu Lys Gln Ile Ile Tyr Met Lys Phe Phe Glu Gly Arg Lys Asp

135 140

  Asn Glu Ile Ala Ile Arg Leu Arg Leu Ser Arg Gln Ser Ile Tyr Lys

150 155 160

  Ile Arg Ile Thr Ser Leu Lys Lys Leu Tyr Pro Ile Val Met Gln Leu

170 175

Val Asn Ile

  (2) INFORMATION FOR SEQ ID NO: 16:

  (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1361 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: double (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (gnomic) (iii ) HYPOTHETICAL: NO (ix) CHARACTERISTIC:

(A) NAME / KEY: CDS

(B) LOCATION: 610..1144

  (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 16:

  GGTAGAAAAG TTCTTTCAAC TGGACATATA ATTCACCTCT TTATAAATTA GATGATAGTA 60

  CTCATATTAA ATCTTTGTTG ATATTAATTT TATGGGTTAC GAATATTCCA TTTCTGATTA 120

  TCATCTCCAT GATAATTAAA TACTTGAATA GCAGTTCCGT TTGCTGTTTG GCTGTTATAT 180

  AAATCTAGAT CCATACTTTT TATTTTATAA GCAGCTTTAT TAGAATCATA TATAAGTCTC 240

  CATCTTTCAA GGTAATTTCT AGTTTGTTGA AATAAGCTAA CGTTACCGGC AACTTGATAA 300

  AAAAATAAAT TAGTATCGGC CTTACAGGAG ATGGTAACAA TTTTGTCATT TAATGAATTT 360

  TGGATTACTG AATAGTGTTC CATTATGATT CCTCCTTTAT TTAAGAATTA ATCTTACATA 420

  TAACATATAA CATAATCAAG TTATTTTTTG TAAACCTAAA ATTTAAATAT ATCAAATTTT 480

  TATTAGTATG TTTACATAAT TGATTATGGA TATTTCGTAA AAATGGCTTA TTAAAAATTT 540

  AAAGGCAATT AGTTTATTTA TAGTATAATA AAACAATAAT ATGTATATTA TGGAGGGATA 600

  GTGGTAAAT ATG AAT AAG TTG TTT TTA CAA ATT AAA ATG TTA AAA AAT 648

  Met Asn Lys Leu Phe Leu Gln Ile Lys Met Leu Lys Asn

185 190

  GAC AAC GAG GAG TTT CAA GAA ATT TTT AAG CAT TTT GAA AAA ACT ATA 696

  Asp Asn Glu Glu Phe Gln Glu Ile Phe Lys His Phe Glu Lys Thr Ile

200 205

  AAT ATA TTT ACT AGA AAA TAT AAT ATA TAT GAT AAT TAC AAT GAT ATT 744

  Asn Ile Phe Thr Arg Lys Tyr Asn Ile Tyr Asp Asn Tyr Asn Asp Ile

210 215 220

  TTG TAC CAT TTA TGG TAT ACA CTT AAA AAA GTT GAT TTG AGC AAT TTC 792

  Leu Tyr His Leu Trp Tyr Thr Leu Lys Lys Val Asp Leu Ser Asn Phe

225 230 235 240

  AAT ACA CAA AAT GAT TTA GAG AGA TAT ATT AGT AGG ACT TTA AAA AGA 840

  Asn Thr Gln Asn Asp Leu Glu Arg Tyr Ile Ser Arg Thr Leu Lys Arg

245 250 255

  TAT TGC TTA GAT ATT TGC AAT AAA AGA AAG ATT GAT AAG AAA ATA ATA 888

  Tyr Cys Leu Asp Ile Cys Asn Lys Arg Lys Ile Asp Lys Lys Ile Ile

260 265 270

  TAT AAT TCA GAA ATT GCA GAT AAG AAA TTA AGC TTA ATA GCA AAT AGT 936

  Tyr Asn Ser Glu Ile Ala Asp Lys Lys Leu Ser Leu Ile Ala Asn Ser

275 280 285

  TAT TCA AGT TAT TCA GAA TTT GAA TTT AAT GAT TTA ATA TCC ATA TTA 984

  Tyr Ser Ser Tyr Ser Glu Phe Glu Phe Asn Asp Leu Ile Ser Ile Leu

290 295 300

  CCT GAC GAT CAA AAG AAA ATT ATA TAT ATG AAA TTT GTT GAA GAT ATT 1032

  Pro Asp Asp Gln Lys Lys Ile Ile Tyr Met Lys Phe Val Glu Asp Ile -50

305 310 315 320

  AAG GAG ATA GAT ATA GCT AAA AAA CTT AAT ATA AGT CGT CAA TCT GTA 1080

  Lys Glu Ile Asp Ile Ala Lys Lys Leu Asn Ile Ser Arg Gln Ser Val

325 330 335

  TAT AAA AAT AAA ATA ATG GCT TTA GAG AGA TTA GAA CCC ATA TTG AAA 1128

  Tyr Lys Asn Lys Ile Met Ala Leu Glu Arg Leu Glu Pro Ile Leu Lys

340 345 350

  -AAA TTA ATT AAT ATG T AGTTTATATT TTTAAAAAAT TTTAGGTTTA 1174

Lys Leu Ile Asn Met

  CAAAAAATAG TGTGGCTATG TTATATATAA ATGATAAGAA TATACTGAAA AATGTATCCA 1234

  AAATTTAAGG GGGCGTGTAT AGTAAATAAT TAAAAGTATG TGCGTTGAAA TAAATTTAGG 1294

  AGAGTGGTTA GATATGAATA TAAATGACAA CTTAAGTATA AATTCCCCGG TAGATAATAA 1354

AAATGTT 1361

  (2) INFORMATION FOR SEQ ID NO: 17:

  (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 178 amino acids (B) TYPE: amino acid (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: protein

  (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 17:

  Met Asn Lys Leu Phe Leu Gln Ile Lys Met Leu Lys Asn Asp Asn Glu

1 5 10 15

  Glu Phe Gln Glu Ile Phe Lys His Phe Glu Lys Thr Ile Asn Ile Phe

25 30

  Thr Arg Lys Tyr Asn Ile Tyr Asp Asn Tyr Asn Asp Ile Leu Tyr His

40 45

  Leu Trp Tyr Thr Leu Lys Lys Val Asp Leu Ser Asn Phe Asn Thr Gln

55 60

  Asn Asp Leu Glu Arg Tyr Ile Ser Arg Thr Leu Lys Arg Tyr Cys Leu

70 75 80

  Asp Ile Cys Asn Lys Arg Lys Ile Asp Lys Lys Ile Tyr Asn Ser

90 95

  Glu Ile Ala Asp Lys Lys Leu Ser Leu Ile Ala Asn Ser Tyr Ser Ser

105 110

  Tyr Ser Glu Phe Glu Phe Asn Asp Leu Ile Ser Ile Leu Pro Asp Asp

120 125

  Gln Lys Lys Ile Ile Tyr Met Lys Phe Val Glu Asp Ile Lys Glu Ile

135 140

  Asp Ile Ala Lys Lys Leu Asn Ile Ser Arg Gln Ser Val Tyr Lys Asn

150 155 160

  Lys Ile Met Ala Leu Glu Arg Leu Glu Pro Ile Leu Lys Lys Leu Ile

170 175

Asn Met

Claims (32)

  1. Polynucleotide comprising all or part of at least one regulatory sequence of a toxin synthesis operon produced by a bacterium of the genus Clostridium, in a form isolated from the rest of the genes included in said operon. 2. Polynucleotide according to claim 1 characterized in that said regulatory sequence is the sequence of a synthetic operon for toxins from C.botulinum or C. tetani, or a sequence having   at least 60% identity with this sequence.   3. Polynucleotide according to one of claims 1 to 2,   characterized in that it is present, in multicopy form in bacteria.   4. Polynucleotide according to one of claims 1 to 3,   characterized in that the regulatory sequence is the following sequence SEQ ID N 1, or a sequence which hybridizes under rigorous conditions,   (or stringent), with the sequence SEQ ID N 1.   AACTATATTA ATATACACAT CCTCGGTAAT ATATCAATAC AATACATGTA   ATTAATAATG TTTTTTGTAA AAGTTTAAGC TTAGTGGTTT ACTTTCAAAA   TATAATAAAA CGTTAATTAT AGAAAAGTGA AAGTAAGGTG ATG CTT ATG GAA   AAT CTA TTT GTA AAA ATT AAG ACA TTA AAA AAT AAT AAT AAA GAA TTT   GAA GTA ATTATTATA CAT TTC AAA AAT ACT GTA AAT ATA CTT ATT CGA   AAG TAT AATTTA GAA AGTCAT TAT AAT GAT ATC TTA TAT CATTTA TGG   AAT ATA CTTAAA AAA ATTGAT TTG AAT AAA M AAT ACA GAA AAT GAT   TTA CAC AAA TAT ATT AGTAGA TCT TTA AAA AGA TAT TGC TTA GAT ATT   TGT AATAAA AAA AAT AGG GAT AAA AAA ATA ATA TAT AACTCA GAA ATT   ACA AATATA AAA TTA AATTTA ATG GAA AAT AGTTGC TCA AAT TATTTA   AAC TTC GAA TTT AAT GATTTA ATA TCC ATA TTA CCT GAA AAT CAA AGA   AAA ATTTTA TAT ATG AAATTT iTTT GAA TAT ATG AAA GAA TGT GAT ATA   GCTAAG CTT CAT ATG AGTCGT CAA GCT GTA TAT AAA AATAAG GTT TTA   GCA TTA AAA AAA TTA GAA CCT ATA GTA AAT AAA TTA ATT AAT ATA T   AGTTTTTATA ATTTAATTAT GAATAATATT CTTAAGATAA AAAGTAAATT   TTTAAAAAAT TTAAATTTTC AGTTTACAAA AAATAACCTG ATTATGTTAT   ATGTAATTGT AAAAAACATA TAAAAAATCA GAAAAATTTA GGAGGTATAT   TATTAATGGA TTAAATAATA ATTTTTTAAT TTACTTTVGA TTAATAAATA   TTAAATGTTT ATTTTAATTA GGAGATGATA CGTATGCC   5. Polynucleotide according to one of claims 1 to 3,   characterized in that the regulatory sequence is that of the botR / C or botR / A gene or of a sequence hybridizing under conditions   rigorous with the sequence of one of these genes.   6. Polynucleotide according to one of claims 1 to 5   characterized in that it comprises at least two regulatory sequences, said sequences being the sequence SEQ ID N 1 or those of the BotR / C gene or of the BotR / A gene, parts of these sequences or sequences hybridizing with the sequences of these genes in   stringent (or stringent) conditions.   7. Polynucleotide according to one of claims 1 to 6,   characterized in that at least one regulatory sequence is associated with a structural gene coding for a protein heterologous to the regulatory sequence, a homologous protein, or part of a of these proteins.   8. Polynucleotide according to claim 7, characterized in that said structural gene codes for C.tetani toxin, fragment C of this toxin, a toxin of C. botulinum or a fragment of this   toxin, a toxin or a fragment of this Clostridium toxin.   9. Polynucleotide according to one of claims 1 to 8   characterized in that it comprises at least one regulatory sequence of a toxin synthesis operon produced by a bacterium of the genus Clostridium, a suitable promoter sequence, and a structural gene coding for a homologous or heterologous protein, or a part of one of these proteins.   10. Polynucleotide according to claim 9 characterized in that 3o that said promoter sequence is that of the iota toxin gene, or   tetR regulatory gene or a gene for a Clostridium toxin.   11. Vector or plasmid characterized in that it contains at   at least part of a polynucleotide according to one of claims 1 to   10. 12. Plasmid according to claim 11 called pMRP309, included in the bacterium E.Coli deposited on May 12, 1998 with the National Collection of Cultures of Microorganisms of the Pasteur Institute under the number 1-2010, or in the bacteria C tetani filed May 18, 1998   s under the number 1-2020 with this Collection.   13. Plasmid according to claim 11 called pMRP319 included in the bacterium E. Coli deposited on May 12, 1998 with the National Collection of Cultures of Microorganisms of the Pasteur Institute under the number 1-2011, or in the bacterium C. tetani deposited May 18, 1998   0o under the number 1-2021 with this Collection.   14. Plasmid according to claim 11 called pMRP365 included in the E.Coli bacterium deposited on May 12, 1998 with the National Collection of Cultures of Microorganisms of the Pasteur Institute under the number 1-2012, or in the bacterium C. tetani deposited May 18, 1998   s 5 under number 1-2022 with this Collection.   15. Sequence comprising at least a substantial part of the tetR regulatory gene having the following sequence SEQ ID N 1,   or hybridizing in rigorous conditions with this part. SEQ ID N I:   AACTATATTA ATATACACAT CCTCGGTAAT ATATCAATAC AATACATGTA   ATTAATAATG TTTTTTGTAA AAGTTTAAGC TTAGTGGTTT ACTTTCAAAA   TATAATAAAA CGTTAATTAT AGAAAAGTGA AAGTAAGGTG ATG CTT ATG GAA   AAT CTA TTT GTA AAA ATT AAG ACA TTA AAA AAT AAT AAT AAA GAA TTT   GAA GTA ATT ATTATA CAT TTC AAA AAT ACT GTA AAT ATA CTT ATT CGA   AAG TAT AAT TTA GAA AGTCAT TAT AAT GAT ATC TTA TAT CAT TTA TGG   AAT ATA CTT AAA AAA ATT GAT TTG AAT AAA TTT AAT ACA GAA AAT GAT   TTA CAC AAA TAT ATT AGTAGA TCT TTA AAA AGA TAT TGC TTA GAT ATT   TGT AAT AAA AAA AAT AGG GAT AAA AAA ATA ATA TAT AAC TCA GAA ATT   ACA AAT ATA AAA TTA AATTTA ATG GAA AAT AGTTGC TCA AAT TAT TTA   AAC TTC GAA TTT AAT GATTTA ATA TCC ATA TTA CCT GAA AAT CAA AGA   AAA ATT TTA TAT ATG AAAT TTT GAA TAT ATG AAA GAA TGT GAT ATA   GCTAAGCTT CAT ATG AGTCGT CAA GCT GTA TAT AAA AAT AAG GTT TTA   GCA TTA AAA AAA TTA GAA CCT ATA GTA AAT AAA TTA ATT AAT ATA T   AGTTTTTATA ATTTAATTAT GAATAATATT CTTAAGATAA AAAGTAAATT   TTTAAAAAAT TTAAATTTTC AGTTTACAAA AAATAACCTG ATTATGTTAT   ATGTAATTGT AAAAAACATA TAAAAAATCA GAAAAATMTA GGAGGTATAT   TATTAATGGA TTAAATAATA ATTTTTTAAT TTACTTTTGA TTAATAAATA   TTAAATGTTT ATTTTAATTA GGAGATGATA CGTATGCC   16. Sequence according to claim 15 characterized in that it comprises at least part of the 3 'end of the sequence SEQ ID N 1.   1o 17. Protein or peptide having a sequence identity of at least 60% with a protein or peptide comprising a substantial part of the following sequence SEQ ID N 2: Met Leu Met Glu Asn Leu Phe Val Lys lie Lys Thr Leu Lys Asn Asn Asn Lys Glu Phe Glu Val lie lie lie His Phe Lys Asn Thr Val Asn lie Leu lie Arg Lys Tyr Asn Leu Glu Ser His Tyr Asn Asp lie Leu Tyr His Leu Trp Asn lie Leu Lys Lys lie Asp Leu Asn Lys Phe Asn Thr Glu Asn Asp Leu His Lys Tyr lie Ser Arg Ser Leu Lys Arg Tyr Cys Leu Asp lie Cys Asn Lys Lys Asn Arg Asp Lys Lys lie lie Tyr Asn Ser Glu bind Thr Asn lie Lys Leu Asn Leu Met Glu Asn Ser Cys Ser Asn Tyr Leu Asn Phe Glu Phe Asn Asp Leu lie Ser lie Leu Pro Glu Asn Gin Arg Lys lie Leu Tyr Met Lys Phe Phe Glu Tyr Met Lys Glu Cys Asp lie Ala Lys Leu His Met Ser Arg Gin Ala Val Tyr Lys Asn Lys Val Leu Ala Leu Lys Lys Leu Glu Pro lie Val Asn Lys Leu lie Asn lie 18. Protein or peptide having a sequence identity of at least 60% with a protein e or a peptide comprising a substantial part of the sequence constituted by the chain of acids   amines in positions 125 to 175 of the sequence SEQ ID N 2.   19. Microorganism characterized in that it comprises at least one polynucleotide, a vector or a sequence according to one of claims 1 to 16.   20. Microorganism according to claim 19 characterized in that it carries a sequence coding for a homologous protein or   heterologous, or part of one of these proteins.   21. Microorganism according to claim 20 characterized in that the part of the heterologous protein, or of the homologous protein is   a part devoid of toxicity of a toxin produced by a bacterium.   22. Microorganism according to one of claims 20 and 21   characterized in that the heterologous protein, the homologous protein or their parts are coded by a gene carried by an independent vector   0o of the vector carrying the regulatory gene.   23. Microorganism according to one of claims 19 to 22,   characterized in that it is of the species C.tetani, C. perfringens, C. botulinum or E. coli.   24. Microorganism according to claim 19 characterized in that it is the strain 1-2010 deposited on May 12, 1998 with the   National Collection of Culture of Microorganisms of the Pasteur Institute.   25. Microorganism according to claim 19 characterized in that it is the strain 1-2011 deposited on May 12, 1998 with the National Collection of Cultures of Microorganisms of the institute Pastor.   26. Microorganism according to claim 19 characterized in that it is the strain 1-2012 deposited on May 12, 1998 with the National Collection of Cultures of Microorganisms of the Pasteur Institute.   27. Microorganism according to one of claims 19 and 23   characterized in that it is the strain 1-2020 of C. tetani deposited on May 18, 1998 with the National Collection of Cultures of   Microorganisms of the Pasteur Institute.   28. Microorganism according to one of claims 19 and 23   characterized in that it is the strain 1-2021 of C. tetani deposited on May 18, 1998 with the National Collection of Cultures of   Microorganisms of the Pasteur Institute.   29. Microorganism according to one of claims 19 and 23   characterized in that it is the strain 1-2022 of C. tetani deposited on May 18, 1998 with the National Collection of Cultures of   Microorganisms of the Institut Pasteur.   30. Process for the production of heterologous proteins or   homologs from a microorganism according to one of the claims   s 19 to 29 comprising the following stages: cultivation of said microorganism, and   - recovery of the proteins produced.   31. The method of claim 30 characterized in that said proteins are recovered in the culture supernatant of microorganisms.   32. Vaccine or drug characterized in that it contains a polynucleotide, a vector or a sequence according to one of   claims 1 to 16, or a microorganism according to one of   claims 19 to 29 or a protein obtained by culturing this 1 5 microorganism.   33. Vaccine or medicament according to claim 32 against C.tetani infections characterized in that it contains at least one   protein obtained by the process according to one of claims 30 and 31.   34. Pharmaceutical composition characterized in that it contains a polynucleotide, a vector or a sequence according to one of   claims 1 to 16, or a microorganism according to one of   claims 19 to 29, or a protein obtained by culturing this   microorganism, in pharmacologically effective amounts, as well as   pharmaceutically acceptable excipients.   35. Use of a vector or polynucleotide   sequence according to one of claims 1 to 16, or a microorganism   according to one of claims 19 to 29, or of a protein obtained by   culture of this microorganism, for the manufacture of a medicament or a vaccine for immunization against diseases linked to bacteria of the genus Clostridium, or for therapy against these diseases.