Studies on phage nucleic acid relating to toxin production of Clostridium botulinum types C and D.

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Toxigenicity of Clostridium botulinum types C and D is converted from toxigenic to nontoxigenic strains by the infection with specific bacteriophages. The nucleic acids were extracted from five converting phages, c-st, c-468, c-203, c-d6f, d-1873, and one nonconverting phage, c-n71, and observed their susceptibility to nucleases. All of the nucleic acids isolated were not digested by RNase A, but digested by DNase <I> and exonuclease <III> , indicating that they were double stranded DNA. From the digestion patterns on 0.8% agarose gel electrophoresis by restriction endonucleases of Hind <III> and Eco RI, the length of c-st, c-n71, c-468, and c-d6f phage DNAs was estimated to be about 110 Kbp, and that of c-203 and d-1873 was about 150 Kbp. All of the phage DNAs were also digested by Hpa <I> , but not by Xho <I> . The susceptibility to Pst <I> and Sau 3A was different depending on the phages. The DNAs from c-st, c-n71, and c-468 were susceptible to Pst <I> , but not to Sau 3A. The digestion patterns of c-st and c-n71 phage DNAs by Pst <I> were exactely the same, and that of c-468 phage DNA was only one band less from them. The Hind <III> digestion patterns of c-st and c-n71 phages were also very similar. In another cases of phages and nucleases, however, such a good similarity was not observed though a few bands common to c-203 and c-d6f phages, and also several bands common to six phage DNAs were demonstrated. In dot hybridization test with these phage DNAs, the existence of several base sequences (regions) common to all of six phage DNAs was demonstrated. A large amount of homology, however, existed only between c-st, c-n71, and c-468 DNAs. These results indicate that DNAs of c-st, c-n71, and c-468 are similar to each other, but different from those of another phages, and that loss of converting ability of c-n71 phage may be caused not by the deletion of "tox" gene but the base mutation in c-st phage DNA.