Abstract
Background Clostridium botulinum produces seven distinct serotypes of botulinum neurotoxins (BoNTs). The genes encoding different subtype neurotoxins of serotypes A, B, F and several dual neurotoxin-producing strains have been shown to reside on plasmids, suggesting that intra- and interspecies transfer of BoNT-encoding plasmids may occur. The objective of the present study was to determine whether these C. botulinum BoNT-encoding plasmids are conjugative.Methodology/Principal Findings C. botulinum BoNT-encoding plasmids pBotCDC-A3 (strain CDC-A3), pCLJ (strain 657Ba) and pCLL (strain Eklund 17B) were tagged with the erythromycin resistance marker (Erm) using the ClosTron mutagenesis system by inserting a group II intron into the neurotoxin genes carried on these plasmids. Transfer of the tagged plasmids from the donor strains CDC-A3, 657Ba and Eklund 17B to tetracycline-resistant recipient C. botulinum strains was evaluated in mating experiments. Erythromycin and tetracycline resistant transconjugants were isolated from donor∶recipient mating pairs tested. Transfer of the plasmids to the transconjugants was confirmed by pulsed-field gel electrophoresis (PFGE) and Southern hybridizations. Transfer required cell-to-cell contact and was DNase resistant. This indicates that transfer of these plasmids occurs via a conjugation mechanism.Conclusions/SignificanceThis is the first evidence supporting conjugal transfer of native botulinum neurotoxin-encoding plasmids in C. botulinum, and provides a probable mechanism for the lateral distribution of BoNT-encoding plasmids to other C. botulinum strains. The potential transfer of C. botulinum BoNT-encoding plasmids to other bacterial hosts in the environment or within the human intestine is of great concern for human pathogenicity and necessitates further characterization of these plasmids.
Highlights
Clostridium botulinum is classified as an anaerobic, gram-positive, sporeforming organism that produces an extraordinarily lethal toxin designated botulinum neurotoxin (BoNT)
Plasmids carrying neurotoxin genes have been identified in some proteolytic and nonproteolytic C. botulinum strains of serotypes A and B, and in bivalent subtypes Ba, Bf and Ab [6,8,17,18,24]
We hypothesize that BoNT-encoding plasmids are capable of intra- and interspecies transfer, since they are present in a variety of proteolytic and nonproteolytic C. botulinum strains
Summary
Clostridium botulinum is classified as an anaerobic, gram-positive, sporeforming organism that produces an extraordinarily lethal toxin designated botulinum neurotoxin (BoNT). BoNTs are categorized into seven different serotypes listed A–G, and multiple subtype BoNTs have been discovered and continue to be identified, especially among serotypes A, B, E and F [1,2,3,4,5,6]. This highly heterogeneous species of C. botulinum is divided into four groups based on metabolic, physiological and genetic properties [7]. The objective of the present study was to determine whether these C. botulinum BoNT-encoding plasmids are conjugative
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