TetR is a positive regulator of the tetanus toxin gene in Clostridium tetani and is homologous to botR

Abstract

The TetR gene immediately upstream from the tetanus toxin (TeTx) gene was characterized. It encodes a 21,562-Da protein which is related (50 to 65% identity) to the equivalent genes (botR) in Clostridium botulinum. TetR has the feature of a DNA binding protein with a basic pI (9.53). It contains a helix-turn-helix motif and shows 29% identity with other putative regulatory genes in Clostridium, i.e., uviA from C. perfringens and txeR from C. difficile. We report for the first time the transformation of C. tetani by electroporation, which permitted us to investigate the function of tetR. Overexpression of tetR in C. tetani induced an increase in TeTx production and in the level of the corresponding mRNA. This indicates that TetR is a transcriptional activator of the TeTx gene. Overexpression of botR/A (60% identity with TetR at the amino acid level) in C. tetani induced an increase in TeTx production comparable to that for overexpression of tetR. However, botR/C (50% identity with TetR at the amino acid level) was less efficient. This supports that TetR positively regulates the TeTx gene in C. tetani and that a conserved mechanism of regulation of the neurotoxin genes is involved in C. tetani and C. botulinum.

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.

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).

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.

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.

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.

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.