Cloning and characterization of overlapping DNA fragments of the toxin A gene of clostridium difficile

Journal of General Microbiology
C von Eichel-StreiberU Hadding

Abstract

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.

Citations

Jan 1, 1991·Toxicon : Official Journal of the International Society on Toxinology·C Fiorentini, M Thelestam
Dec 1, 1989·FEMS Microbiology Reviews·M YoungW L Staudenbauer
Mar 25, 1990·Nucleic Acids Research·M Sauerborn, C von Eichel-Streiber
Mar 15, 1997·European Journal of Biochemistry·T HundsbergerC von Eichel-Streiber
May 1, 1992·Molecular & General Genetics : MGG·C von Eichel-StreiberM Sauerborn
Mar 1, 1997·Microbial Pathogenesis·G A HammondJ L Johnson
Feb 10, 2019·Biological Chemistry·Xin MengRobert C Ford
Jun 3, 2021·Microbiology and Molecular Biology Reviews : MMBR·Kathleen E Orrell, Roman A Melnyk

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