Transfer Of Botulinum Neurotoxin-Encoding Plasmids From Clostridium Botulinum To Nontoxigenic Clostridia

Experimental procedures. Tagging of the BoNT-encoding plasmids (donor strains). The same approach of using a group II intron (ClosTron) as described above in the Preliminary section will be used to tag the BoNT-encoding plasmids selected for mobilization studies. The intron will be inserted into a BoNT gene residing on the plasmid. Already tagged plasmids pCLK from proteolytic C. botulinum subtype A3 strain Loch Maree and pCLL from nonproteolytic C. botulinum serotype B strain 17B will be used as models to study plasmid transfer between proteolytic and nonproteolytic strains of C. botulinum and other nontoxigenic clostridia. These plasmids have been chosen since we have already shown their transfer to other C. botulinum strains. Recipient strains. In our preliminary experiments we used several C. botulinum strains that carried a Tn916 transposon, and thus were tetracycline resistant. This trait was used for the selection of transconjugants. However, C. botulinum strains normally do not carry Tn916, therefore it is possible that the transposon could also facilitate the BoNT-encoding plasmid transfer. To test this hypothesis it would be beneficial to use clostridial strains as donors that are Tn916 free. Therefore a different approach will be used to prepare the recipient strains that carry a specific marker for transconjugant isolation. One of such approaches is adapting the strains to grow in the presence of certain antibiotics. We will use tetracycline, because this antibiotic has provided a good selection for transposon Tn916 mutants. Selected C. botulinum strains will be grown in liquid TPGY media supplemented with low concentrations of tetracycline, then the concentration of the antibiotic will be increased gradually with every subsequent passage of the strains, until bacteria are able to grow efficiently in media containing 5-10 mg/ml tetracycline. Alternatively, another antibiotic, thiamphenicol that has been approved for use in genetic studies of toxigenic C. botulinum strains will be used. Similar strategy as described above will be used to generate donor strains resistant to thiamphenicol. Other species of clostridial plasmid transfer recipient strains will also be prepared in a similar way. However, these strains will first be tested for the resistance/sensitivity to erythromycin, tetracycline and thiamphenicol. Then upon the test results a proper antibiotic will be selected to generate a strains suitable as plasmid recipients. If the strains will be resistant to all three antibiotics then rifampicin and nalidix acid treatment will be used. This approach has been utilized to create selection traits in C. perfringens and other clostridial species (33).Mating experiments. Mating experiments initially will be conducted using a mixture of donor and recipient cells grown to mid to late log-phase. The mating protocols will be optimized under various conditions such as mating at different cell densities, using both donor and recipient strains at different growth phases, at temperatures optimal for both donor and recipient strains, varying the mating time interval, performing filter matings and/or increasing the concentration of agar in the solid medium. Usually mating on solid media (agar plates with or without nitrocellulose filters) has been used in clostridia. Plasmid transconjugant analyses. To confirm that the plasmid transfer has occurred, several random transconjugants from each mating experiment will be selected, tested for purity, and then PFGE plugs will be prepared and analyzed. Both digested and undigested samples of the transconjugants, along with the donor and recipient strains as controls for comparison will analyzed by PFGE followed by hybridization with appropriate probes to prove that the plasmid transfer has taken place.Plasmid mobilization experiments. A. Mobilization experiments of BoNT-encoding plasmids from proteolytic C. botulinum strains (Group I).Initially C. botulinum strain Loch Maree that carries and group II intron tagged pCLK-Erm will be used in mating experiments with C. sporogenes, C. baratii,C. butyricum. Mating experiments will be performed as described above. In case if no transconjugants were obtained, optimization of the mating conditions will be performed. The matings will be performed at different cell densities, using both donor and recipient strains at different growth phases, at temperatures optimal for both donor and recipient strains, varying the mating time interval, performing filter matings and/or increasing the concentration of agar in the solid medium. Transconjugants will be analyzed by PFGE and Southern blots as described above. Additional mating experiments will be performed between clostridial donor and recipient strains by including common gut bacteria such as Enterococcus and Lactobacillus in the mating mix. These gut microbiota species are of particular interest since they contain various mobile genetic elements (IS elements, transposases, transposons) that can be transferred to C. botulinum such as Tn916. These experiments will help define if the BoNT-encoding plasmids are self-mobilizable or if they require the presence of other common gut microorganisms to enhance and/or facilite BoNT-encoding plasmid transfer. These experiments will be performed simultaneously, with or without the Enterococcus and Lactobacillus included in the mating mix, then the plasmid transfer frequencies will be compared to evaluate if the latter microbes have affected plasmid transfer. Appropriate selective markers and growth conditions will be employed to ensure that the non-clostridial bacteria in the matings do not acquire the BoNT-encoding plasmids.B. Mobilization experiments of BoNT-encoding plasmids from nonproteolytic C. botulinum strains (Group II).The mating experiments and the analyses of the potential transconjugants will be performed as described above for Group I plasmid studies.Additional notes per Dr. Wells email:I have clarified that the scope of work in this proposal ONLY will involve transfer of BoNT-containing plasmids to clostridial recipients that are already known to produce botulinum neurotoxin (C. botulinum, C. baratii, C. butyricum, and C. sporogenes). We have further clarified that BoNT-producing C. sporogenes strain has been isolated and this is documented in the references (refs 10, 11 and 24). Following on this, I have tried to make it very clear that no matings will be performed that will allow transfer of the BoNT-plasmids to non-clostridial organisms including Lactobacillus, Enterococcus, and other common gut bacteria. The use of these organisms will ONLY be used to address the hypothesis that they provide factors that promote conjugal transfer of the plasmids to the clostridium recipients. These experiments are necessary as the conjugal transfer of BoNT genes appears to occur primarily in the infant gut where these organisms are present. The origin of BoNT/B producing C. sporogenes strain is not clear and this strain has not been studied in detail. This warrants further studies of BoNT-producing C. sporogenes strains.