Characterization of Clostridium perfringens iota-toxin genes and expression in Escherichia coli.
The iota toxin which is produced by Clostridium perfringens type E, is a binary toxin consisting of two independent polypeptides: Ia, which is an ADP-ribosyltransferase, and Ib, which is involved in the binding and internalization of the toxin into the cell. Two degenerate oligonucleotide probes deduced from partial amino acid sequence of each component of C. spiroforme toxin, which is closely related to the iota toxin, were used to clone three overlapping DNA fragments containing the iota-toxin genes from C. perfringens type E plasmid DNA. Two genes, in the same orientation, coding for Ia (387 amino acids) and Ib (875 amino acids) and separated by 243 noncoding nucleotides were identified. A predicted signal peptide was found for each component, and the secreted Ib displays two domains, the propeptide (172 amino acids) and the mature protein (664 amino acids). The Ia gene has been expressed in Escherichia coli and C. perfringens, under the control of its own promoter. The recombinant polypeptide obtained was recognized by Ia antibodies and ADP-ribosylated actin. The expression of the Ib gene was obtained in E. coli harboring a recombinant plasmid encompassing the putative promoter upstream of the Ia gene and the Ia and Ib genes. Two residues which have been found to be involved in the NAD+ binding site of diphtheria and pseudomonas toxins are conserved in the predicted Ia sequence (Glu-14 and Trp-19). The predicted amino acid Ib sequence shows 33.9% identity with and 54.4% similarity to the protective antigen of the anthrax toxin complex. In particular, the central region of Ib, which contains a predicted transmembrane segment (Leu-292 to Ser-308), presents 45% identity with the corresponding protective antigen sequence which is involved in the translocation of the toxin across the cell membrane.
Perelle
S
S
Laboratoire des Toxines Microbiennes, Institut Pasteur, Paris, France.
Gibert
M
M
Boquet
P
P
Popoff
M R
MR
D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D000942:Antigens, Bacterial; D001408:Bacillus anthracis / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D001427:Bacterial Toxins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D003016:Clostridium perfringens / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D017386:Sequence Homology, Amino Acid
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004269:DNA, Bacterial; C030325:anthrax toxin; C050737:iota toxin, Clostridium perfringens; D036002:ADP Ribose Transferases