| Abstract: | Clostridium botulinum cultures are classified into seven types, types A to G, based on the antigenicity of the neurotoxins produced. Of these seven types, only types C and D produce C2 toxin in addition to the neurotoxin. The C2 toxin consists of two components designated C2I and C2II. The genes encoding the C2 toxin components have been cloned, and it has been stated that they might be on the cell chromosome. The present study confirmed by using pulsed-field gel electrophoresis and subsequent Southern hybridization that these genes are on a large plasmid. The complete nucleotide sequence of this plasmid was determined by using a combination of inverse PCR and primer walking. The sequence was 106,981 bp long and contained 123 potential open reading frames, including the c2I and c2II genes. The 57 products of these open reading frames had sequences similar to those of well-known proteins. It was speculated that 9 these 57 gene products were related to DNA replication, 2 were responsible for the two-component regulatory system, and 3 were σ factors. In addition, a total of 20 genes encoding proteins related to diverse processes in purine catabolism were found in two regions. In these regions, there were 9 and 11 genes rarely found in plasmids, indicating that this plasmid plays an important role in purine catabolism, as well as in C2 toxin production.Clostridium botulinum is a gram-positive, spore-forming, anaerobic bacterium. Cultures of this species produce poisonous botulinum neurotoxins (BoNTXs) that are lethal to humans and animals and are classified into seven types, types A to G, based on the antigenicity of the BoNTXs produced (actually, the cultures producing type G toxin were recently classified in a new species, Clostridium argentinens 40]).C. botulinum type C and D cultures produce a binary C2 toxin in addition to C (or C1) and D BoNTXs; this additional toxin consists of two nonlinked proteins, C2I and C2II (28), that occur independently in the culture supernatant and are not chemically joined to each other. The C. botulinum C2 toxin used here is a representative of the family of binary actin-ADP-ribosylating toxins, which includes, in addition to C2 toxin, the Clostridium perfringens iota toxin, Clostridium difficile toxin, Clostridium spiroforme toxin, and the vegetative insecticidal proteins from Bacillus cereus (4).The enzyme component of C2 toxin (C2I) ADP ribosylates G-actin at arginine 177 (1). This leads to depolymerization of actin filaments and finally to cell rounding. The proteolytically activated binding-translocation component (C2IIa) forms heptamers, which assemble with C2I and bind to the cellular receptor (5). Following receptor-mediated endocytosis, C2IIa forms pores in the membrane of acidic endosomes. Subsequently, C2I translocates across the membrane into the cytosol through these C2IIa pores.The production of C1 and D BoNTXs is governed by bacteriophages (12, 13, 18, 19, 26), and both toxin genes have been cloned from the corresponding phage DNAs (16, 21). Recently, we determined the whole-genome sequence of a type C toxin-converting phage (c-st) genome (31). Eklund et al. reported that C2 toxin toxigenicity (mouse lethality) became clear when strains were cultured in fortified egg-meat medium and the culture supernatants were treated with trypsin (12). They also reported that C2 toxin production was not related to the BoNTX-converting phages; some non-BoNTX-producing cells produced C2 toxin. Fujii et al. (15) and Kimura et al. (22) determined the whole nucleotide sequences of the c2I and c2II genes and speculated that these genes might be located on the bacterial chromosome (15, 22).In this study, we determined that these genes are present not on the cell chromosome but on a large plasmid; we first speculated that this was this was the case based on the results of both pulsed-field gel electrophoresis (PFGE) and Southern hybridization analysis and then confirmed it by determining the complete nucleotide sequence of the plasmid. Since the plasmid was extremely unstable, we could not purify the complete plasmid DNA; therefore, we determined the whole-genome sequence by using the inverse PCR method, which enables rapid determination of the flanking regions of unknown sequences and determination of unidentified sequences in the genome, and the primer-walking method. This is the first case in which an entire DNA sequence of a large plasmid was determined using only these two procedures. The process used to determine the whole-plasmid DNA sequence and several interesting features of the plasmid are described below. |
