COMPARATIVE-ANALYSIS OF C3 AND BOTULINAL NEUROTOXIN GENES AND THEIR ENVIRONMENT IN CLOSTRIDIUM-BOTULINUM TYPE-C AND TYPE-D

The C3 exoenzyme gene is located on a bacteriophage in Clostridium bot ulinum types C and D (M. R. Popoff, D. Hauser, P. Boquet, M. W. Eklund , and D. M. Gill, Infect. Immun. 59:3673-3679, 1991). A derivative CN phage from phage C of C. botulinum Stockholm (C-St) (K. Oguma, H. lida , and K. Inoue, Jpn. J. Microbiol. 19:167-172, 1975), isolated as neur otoxin negative, also does not produce exoenzyme C3. The botulinal neu rotoxin C1 gene is present on the CN phage but contains a stop mutatio n in the DNA region encoding the N-terminal part of the heavy chain (c odon 553). The putative truncated botulinal neurotoxin C1 protein was not recovered in a C. botulinum strain harboring the CN phage. We foun d that the C3 gene is localized on a 21.5-kbp DNA fragment flanked by the core motif 5'-AAGGAG-3' in DNAs of phage C of C. botulinum 468 (C- 468), C-St phage, and phage D of C. botulinum 1873 (D-1873). The 21.5- kbp DNA fragment is deleted in CN phage DNA, and the motif 5'-AAGGAG-3 ' is present only in one copy at the deletion junction, but the deleti on in the CN phage could be nonspecific, since this phage was obtained by nitrosoguanidine treatment. These findings could indicate that the C3 gene is localized on a 21.5-kbp mobile element. C. botulinum type C strain 003-9 produces a C3 exoenzyme (Y. Nemoto, T. Namba, S. Kozaki , and S. Narumiya, J. Biol. Chem. 266:19312-19319, 1991), and Staphylo coccus aureus El produces a related C3 enzyme which is named epidermal cell differentiation inhibitor (S. Inoue, M. Sugai, Y. Murooka, S. Y. Paik, Y. M. Hong, H. Oghai, and H. Suginaka, Biochem. Biophys. Res. C ommun. 174:459-464,1991) and which shares 80.6 and 56.6% similarity, r espectively with the C3 enzymes from C-468 or C-St and D-1873 phages a t the amino acid level. The features of the putative 21.5-kbp transpos on were not found in C. botulinum 003-9 and S. aureus E1, as determine d by analysis of the C3 and epidermal cell differentiation inhibitor g ene-flanking DNA regions. These data suggest a common ancestral origin and divergent evolution of the C3 genes in these three groups of bact erial strains and dissemination of a 21.5-kbp element carrying the C3 gene in C-468, C-St, and D-1873 phages.