Cloning and characterization of overlapping DNA fragments of the toxin A gene of clostridium difficile.
Clostridium difficile, a human pathogen, produces two very large protein toxins, A and B (250-600 kDa), which resist dissociation into subunits. To clone the toxin A gene, a genomic library of 3-8 kb chromosomal DNA fragments of C. difficile strain VPI 10463 established in pUC12 was screened with a rabbit polyclonal toxin A antiserum. Thirty-five clones were isolated which carried 2.5-7.0 kb inserts representing a 10 kb region of the C. difficile genome. All the inserts were oriented in the same direction, suggesting that toxin A gene expression was under control of the lac promoter of the pUC12 vector. Western blot experiments revealed the presence of low amounts of fusion proteins of variable size (30-170 kDa) in Escherichia coli strains harbouring recombinant plasmids. As deduced from subcloning experiments, the DNA sequences encoding toxin A comprised about 4 kb, corresponding to about 140 kDa of the 300-600 kDa protein. This was either due to incomplete cloning of the gene or it might indicate a subunit composition of toxin A. No additional gene(s) with homology to the cloned toxin A gene was detected.
von Eichel-Streiber
C
C
Institut für Medizinische Mikrobiologie, Johannes-Gutenberg-Universität, Mainz, FRG.
Suckau
D
D
Wachter
M
M
Hadding
U
U
D001427:Bacterial Toxins / Q000235:genetics*; D015153:Blotting, Western; D003001:Cloning, Molecular; D003013:Clostridium / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis*; D004768:Enterotoxins; D005798:Genes, Bacterial; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping
D001427:Bacterial Toxins; D004269:DNA, Bacterial; D004768:Enterotoxins; D011993:Recombinant Fusion Proteins; C041348:tcdA protein, Clostridium difficile