Plasmids in Clostridium botulinum type A and Clostridium sporogenes

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Virginia Polytechnic Institute and State University


A procedure to rapidly screen Clostridium botulinum type A and Clostridium sporogenes for plasmids was developed. Plasmid profiles of five C. botulinum type A strains and seven C. sporogenes strains were determined and a possible relationship of plasmids to toxin production was examined. The differentiation of these organisms by plasmid fingerprinting was also studied. The plasmid isolation procedure was a modified cleared lysate technique, including lysis under alkaline conditions. Samples were subject to agarose gel electrophoresis to detect plasmid DNA. Culture age affected plasmid detection due to changes in the cell density and lysing efficiency. Middle to late log cultures were used throughout the study because they provided optimum plasmid detection. Four out of five C. botulinum type A strains and three out of seven C. sporogenes strains contained extrachromosomal DNA. For those C. botulinum type A strains which contained plasmids, there were always two, one 15 to 15.6 Mdal and the other 6.2 Mdal. C. sporogenes showed less consistency in plasmid size and number and their plasmids were generally of a greater molecular weight than those in C. botulinum type A. One C. sporogenes strain contained two plasmids and two strains contained one plasmid. Toxin production may be plasmid-mediated in the plasmid containing strains, but there was no apparent general relationship, because one of the toxic strains did not show the presence of plasmids. Plasmid screening may be useful in the differentiation of these closely related organisms without toxin testing.