INTRODUCTION

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Tetanus toxin (TeTx) and botulinum neurotoxins (BoNTs) are the most potent protein toxins. They have similar structures and modes of action at the molecular level, and they are synthesized as single-chain proteins (approximately 150 kDa) which are proteolytically activated to dichain derivatives involving a light chain (L) (approximately 50 kDa) and a heavy chain (H) (approximately 100 kDa). Both chains remain linked by a single disulfide bridge. In the culture supernatants and contaminated food, BoNTs are associated with nontoxic proteins (ANTPs) to form complexes whose molecular sizes range from 230 to 900 kDa. In contrast, TeTx does not form such complexes (21). Certain ANTPs are hemagglutinins (HA). The TeTx and BoNT genes in various strains have been characterized. In Clostridium botulinum, the BoNT genes are localized in the 3' part of the C. botulinum locus and are preceded by the gene encoding the nontoxic, non-HA component (NTNH). The HA genes lie upstream of the NTNH-BoNT genes and are transcribed in the opposite orientation. A gene encoding a 21- to 22-kDa protein is localized in the 5' part of the C. botulinum locus in C. botulinum C and D (11, 17) or between the NTNH-BoNT and HA genes in C. botulinum A, B, F, and G (1, 5, 6, 12, 13). This protein called BotR shows the feature of a transcriptional regulator (basic pI [10.4] and the presence of helix-turn-helix motifs), and it is related (25 to 28% identity according to the Bestfit program) to other regulatory proteins such as UviA from Clostridium perfringens (10), a protein (TxeR) from Clostridium difficile (16), and, to a lesser extent, MsmR from Streptococcus mutans (20). The txeR gene is located directly upstream from the tcdB and tcdA genes encoding the C. difficile toxins B and A, respectively, which are responsible for the gastrointestinal disorders caused by this bacterium. It was shown to be a positive regulator of tcdB and tcdA gene promoters in Escherichia coli (16).

In Clostridium tetani, a gene homologous to botR was found in the flanking regions of the TeTx gene. Thus, it was reported that a DNA sequence upstream of the TeTx gene encodes 29 C-terminal amino acids homologous to BotR (1, 7, 8). No gene related to those encoding NTNH and HA components of botulinum complexes was detected. Here, we report the complete characterization of tetR from C. tetani, and we report that TetR and also BotR from C. botulinum A (BotR/A) and C. botulinum C (BotR/C) are positive regulators of TeTx gene expression in C. tetani.

	MATERIALS AND METHODS

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Bacterial strains and plasmids. C. tetani CN655 and recombinant strains were grown in broth containing trypticase (30 g/liter), yeast extract (20 g/liter), glucose (5 g/liter), and cysteine-HCl (0.5 g/liter) (pH 7.2) under anaerobic conditions. Clostridium DNA was extracted and purified as previously described (18).

DNA techniques. Ligation, transformation, sequencing, and preparation of plasmid DNA from E. coli were conducted by standard procedures (22).

Transformation of C. tetani by electroporation. Competent cells from C. tetani CN655 were prepared in an anaerobic chamber. The bacteria of a Trypticase-glucose-yeast extract (TGY) culture (100 ml) were recovered by centrifugation in the middle of the exponential growth phase, washed in distilled water, and suspended in 0.5 ml of 7 mM Na₂HPO₄ (pH 7.4) containing 1 mM MgCl₂ and 270 mM sucrose. Plasmid DNA (1 to 5 µg) produced in E. coli HB101 was added to 50 µl of cell suspension. Electroporation was performed outside the anaerobic chamber with a Bio-Rad gene pulser (2.5 kV, 200 , and 25 µF) and a hermetically sealed cuvette with an anaerobic atmosphere. The bacteria were diluted in TGY, incubated for 3 h at 37°C, and plated onto TGY agar containing 5 µg of erythromycin per ml in an anaerobic chamber.

Construction of plasmids for expression of tetR and botR/C gene expression plasmids. A DNA fragment containing the coding region of tetR was amplified by PCR from C. tetani CN655 with primers introducing a NcoI site at the translational start codon and a PstI site immediately downstream of the stop codon. The amplification product cut by NcoI and PstI was cloned into the high-copy-number vector pAT19 downstream of the C. perfringens iota toxin gene promoter and upstream of the 3' part of the iota toxin ibp gene as previously described (pMRP306) (15). The resulting plasmid, pMRP365, was transferred into C. tetani CN655 yielding the CN655-OE strain.

RNA isolation and RNA dot blots. Total RNA was extracted from C. tetani cultures in the middle of the exponential growth phase by using Trizol (GibcoBRL, Cergy Pontoise, France). The bacterial pellet from a 10-ml culture (optical density at 600 nm [OD₆₀₀], 1.6 to 1.8) was washed twice in distilled water and suspended in 200 µl of 10 mM Tris-HCl (pH 7)-10 mM EDTA-20% sucrose 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 the suspension was incubated for 5 min at room temperature. The subsequent steps were performed according to the manufacturer's recommendations.

PAGE and immunoblotting procedure. Proteins were precipitated from the supernatant of cultures (OD, 1.8) of wild-type and recombinant C. tetani strains with 10% trichloroacetic acid. The precipitate was collected by centrifugation and was washed with acetone. The solubilized proteins in 50 mM Tris-HCl (pH 8) and serial dilutions were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) in a 10% acrylamide gel.

Toxicity to mice. Serial twofold dilutions of samples (0.5 ml) in 50 mM sodium phosphate buffer (pH 6.3) containing 0.2% (wt/vol) gelatin were injected intraperitoneally into mice weighing 18 to 20 g. Four mice were used for each dilution. The mice were observed over 4 days, and the numbers that died were recorded.

Nucleotide sequence accession number. The nucleotide sequence reported in this paper has been submitted to the EMBL Data Library under accession no. AJ006534.

	RESULTS

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Characterization of the tetR gene. Previous reports of the TeTx nucleotide sequence indicated the presence of tetR immediately upstream of the TeTx gene (7, 8). To obtain the complete sequence of tetR, we cloned a 2.4-kbp HindIII/EcoRI fragment and established the sequence of an area of 480 bp located upstream of the previously reported sequence (8). The nucleotide sequence and the deduced amino acid sequence of tetR are presented in Fig. 1. TetR has a calculated molecular mass of 21,562 Da and consists of 178 amino acids. It displays 50 to 65% identity with the corresponding proteins of C. botulinum strains. A sequence alignment is shown in Fig. 2. TetR exhibits the characteristic features of a DNA binding protein, i.e., a calculated basic pI (9.53) and a helix-turn-helix motif, and shows significant homology to UviA from C. perfringens and TxeR from C. difficile, two other putative Clostridium regulatory proteins (Fig. 2).

View larger version (57K): [in this window] [in a new window]   FIG. 1.   Nucleotide and deduced amino acid sequences of TetR. The amino acid sequence is shown in single-letter code below the nucleotide sequence. The nontranslated regions upstream of the tetR and between the tetR and TeTx open reading frames are indicated by lowercase letters. Arrows identify the translational start codons of tetR and the TeTx gene.

View larger version (109K): [in this window] [in a new window]   FIG. 2.   Alignment of TetR; BotRs from C. botulinum A1, A2, B proteolytic (Bp), B nonproteolytic (Bnp), C, F proteolytic (Fp), and G; and the related regulatory proteins from C. difficile (TxeR) and C. perfringens (UviA).

Overexpression of the tetR gene in C. tetani. To analyze the function of TetR, the tetR gene was overexpressed in C. tetani. A truncated promoter region of tetR was present only in the 2.4-kbp HindIII-EcoRI fragment and was too short to permit expression of the tetR gene. Thus, the coding region of tetR was amplified by PCR and cloned into the vector containing the promoter of the C. perfringens iap gene, which was used for Clostridium gene expression (15). The resulting plasmid, pMRP365, was transferred by electroporation into C. tetani CN655, yielding CN655-OE.

View larger version (14K): [in this window] [in a new window]   FIG. 3.   Mouse lethal activity in culture supernatants of wild-type C. tetani CN655 () and recombinant strains overexpressing the tetR (), botR/A (), and botR/C () genes. The mouse lethal activity (LD50) is plotted against the OD600 for each culture. The means and standard deviations of the values from two experiments are indicated.

View larger version (41K): [in this window] [in a new window]   FIG. 4.   Production of TeTx assayed by Western blotting with anti-TeTx antibodies in wild-type C. tetani CN655 (A) and in recombinant strains overexpressing the tetR (CN655-OE) (B), botR/A (CN655-BotR/A) (C), and botR/C (CN655-BotR/C) (D) genes. Supernatants of each culture (OD600) were concentrated by trichloroacetic acid precipitation, 20 µg of protein was loaded on lane 1, and serial twofold dilutions were loaded in the subsequent lanes. In panels B and D, the upper bands correspond to the whole TeTx and the lower bands correspond to the H chain.

View larger version (38K): [in this window] [in a new window]   FIG. 5.   PAGE of extracellular proteins (50 µg) of recombinant strain overexpressing tetR (CN655-OE) (lane 1) and of C. tetani wild-type CN655 (lane 2). H and L, heavy and light chains of TeTx, respectively.

View larger version (58K): [in this window] [in a new window]   FIG. 6.   mRNA dot blots from the wild-type strain CN655 (wt) and from recombinant strains CN655-OE (OE), CN655-BotR/A (botR/A), and CN655-botR/C (botR/C) with probes specific for the TeTx gene. The mRNA was prepared from cultures at an OD600 of 1.4. The total amounts of mRNA loaded in each lane are indicated in micrograms.

Overexpression of botR/A and botR/C genes in C. tetani. Since TetR shows a high similarity to the equivalent regulatory genes (botRs) from C. botulinum, it was interesting to know if these genes of the same family can be functionally interchangeable. BotR/A shows an overall identity of 60% with TetR (Fig. 2). It was found that plasmid pMRP309 containing the botR/A gene under the control of its own promoter induced a significant increase in the production of BoNT/A and ANTPs and in the corresponding mRNAs in C. botulinum A (15). C. tetani CN655 was transformed by electroporation with pMRP309, yielding strain CN655-BotR/A. A comparable increase in the amount of TeTx assayed by the mouse test and immunoblotting was observed during the exponential growth phase of CN655-BotR/A and CN655-OE (Fig. 3 and 4). Mouse lethal activity in the culture supernatant of CN655-OE and CN655-botR/A was approximately eight times higher than that in the wild-type strain (Fig. 3). The overproduction of TeTx assayed by immunoblotting was increased by approximately 16 times in CN655-OE and 8 times in CN655-botR/A compared to the wild-type strain (Fig. 4). Moreover, the amounts of mRNAs of the TeTx gene were increased (approximately four times) in both strains (Fig. 6). This indicates that BotR/A was able to positively regulate the TeTx gene in C. tetani.

	DISCUSSION

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We report the complete sequence of the tetR gene from C. tetani, which is highly related to the botR genes from C. botulinum A, B, C, D, F, and G (1, 5, 6, 12, 13). TetR shows an overall level of identity of from 50% with BotR/C to 65% with BotR/F. This family of genes is related to other putative regulatory genes in Clostridium, such as uviA in C. perfringens and txeR in C. difficile (10, 16). TetR and the other related proteins possess the features of DNA binding proteins, i.e., high pI (pH 9.53) and the presence of a helix-turn-helix motif.

We succeeded in transforming C. tetani with pAT19, which is a shuttle vector between gram-positive and gram-negative bacteria and which contains a replication origin from Enterococcus fecalis (23). The electroporation conditions were similar to those used for the C. botulinum transformation (15). The present article is the first report of genetic transformation in C. tetani and construction of recombinant C. tetani strains to investigate a gene function in this microorganism.

Overexpression of tetR in trans position induces an increase in TeTx production, as monitored by mouse lethality in culture supernatant and by Western blotting. A corresponding increase in the specific mRNA of the TeTx gene indicates that tetR positively regulates the transcriptional level of the toxin gene in C. tetani. It can not be ruled out that tetR in cis position could be more efficient. tetR seems to regulate specifically the TeTx gene and to have no pleomorphic effect. We explored if the equivalent genes (botR) from C. botulinum are functional in C. tetani. The high-copy-number vector pAT19 containing botR/A or botR/C was transferred by electroporation into C. tetani CN655. BotR/A, which is more closely related to TetR than BotR/C, produced a higher increase in TeTx production and in the specific mRNA, compared with BotR/C. This shows that BotR/A and BotR/C are functional in C. tetani. The different levels of effect between BotR/A and BotR/C could be due to a lower level of expression of botR/C, since botR/C was under the control of the iap promoter (pMRP365) and botR/A was under the control of its own promoter (pMRP309). However, tetR was constructed under the control of the iap promoter and induced an equivalent activation of TeTx gene expression equivalent to that of botR/A. The more distant relatedness of BotR/C to TetR than to BotR/A could explain the reduced efficiency of BotR/C in C. tetani. These data suggest a common mechanism of regulation of the neurotoxin genes in C. tetani and C. botulinum.

We found that BotR/A stimulates expression of both the BoNT and ANTP genes (15). The 10 and 35 regions of the neurotoxin and ANTP gene promoters in C. botulinum A, B, C, D, F, and G and C. tetani contain conserved sequences (1, 12). Moreover, BotR/A seems to interact directly with the promoter region and the conserved motifs could represent binding sites for the regulatory proteins (15). TetR could be also a regulatory protein which binds the promoter region of the TeTx gene. Whether Tet/R and BotR are involved in a cascade of regulatory proteins is unknown. It has been found that short peptides from casein hydrolysates are important for toxinogenesis in C. tetani (19), but the environmental signals which trigger neurotoxin production remain to be determined.

The presence of highly conserved genes in the close vicinity of the clostridial neurotoxin genes, which are functionally interchangeable, constitutes additional evidence that the locus of clostridial neurotoxin genes derived from a common ancestor. However, the NTNH and HA genes, which lie upstream from the BoNT genes in the different C. botulinum toxinotypes, are missing in C. tetani. The tetR gene is the only ANTP gene which was found in C. tetani.

Vaccination against tetanus is extremely effective in preventing this disease, and widespread vaccination has almost eradicated tetanus from developed countries. Current tetanus vaccines are produced by formaldehyde treatment of TeTx produced by wild-type C. tetani to yield the immunogenic toxoid. A novel generation of tetanus vaccines involves production of the C-terminal part (fragment C) of TeTx, which is nontoxic and is able to induce neutralizing antibodies. The production of large quantities of recombinant fragment C in various organisms such as E. coli, Lactococcus lactis, Baculovirus, and Pichia pastoris (3, 4, 9, 14, 24) was attempted. Our findings on the genetic transformation of C. tetani and on the identification of TetR as a positive regulator open the possibility of using C. tetani as an engineering system for vaccine production. It may be possible to construct C. tetani strains which produce large amounts of TeTx or fragment C. C. tetani has the advantage of secreting a soluble form of TeTx, and this organism is already used in industrial fermentation.