Conjugative botulinum neurotoxin-encoding plasmids in Clostridium botulinum.
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.


RESULTS
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
This 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.
Marshall
Kristin M
KM
Department of Bacteriology, College of Agriculture and Life Sciences, University of Wisconsin, Madison, Wisconsin, USA.
Bradshaw
Marite
M
Johnson
Eric A
EA
D001483:Base Sequence; D015139:Blotting, Southern; D001905:Botulinum Toxins / Q000235:genetics*; D003014:Clostridium botulinum / Q000235:genetics*; D017931:DNA Primers; D016521:Electrophoresis, Gel, Pulsed-Field; D007438:Introns; D010957:Plasmids
D017931:DNA Primers; D001905:Botulinum Toxins