Progress 12/30/14 to 12/31/18
Outputs
Target Audience:The target audience was primarily scientists in the food industry, academic laboratories, and regulatory agencies whose field is food safety, specifically botulism. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided strategies and methods to understand the impact of Clostridium botulinum and its neurotoxins on food safety. These methods are being used for training of scientists in university settings. This work has also provided valuable information for the awareness of botulism by industry and regulatory agencies. How have the results been disseminated to communities of interest?These findings have been disseminated to the scientiic investigators and regulatory agencies. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Impact: An enhanced understanding of Clostridium botulinum and Its Impact on Food Safety What was accomplished under these goals? Clostridium botulinumis a gram-positive, anaerobic, spore-forming bacterium that produces botulinum neurotoxin (BoNT), which is the most poisonous toxin known to humankind. BoNTs are the agents of botulism, a potentially lethal paralytic disease; they act by cleaving neuronal proteins, thereby preventing the release of neurotransmitters. While many BoNTs are encoded on bacterial chromosomes, some are present on plasmids. Our laboratory was the first to demonstrate that BoNT-encoding plasmids could be experimentally transferred betweenC. botulinumstrains. This project was designed to further explore the mechanisms of transfer of BoNT-encoding plasmids to clostridia. The movement of BoNT-encoding genes and expression of BoNTs among clostridial strains is of particular interest to human health and food safety. For example,C. botulinumstrains expressing multiple serotypes of BoNT have been isolated from cases of infant botulism, suggesting that strains have the opportunity to acquire new toxins in the intestinal microbiome. Furthermore, BoNTs have been isolated from normally nontoxigenic clostridial species such asC. sporogenes,C. baratiiandC. butyricum. Transfer of BoNTs to other species could jeopardize the efficacy of treatments designed to eliminateC. botulinumin food products and in the intestinal environment.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Nawrocki EM, Bradshaw M, Johnson EA. 2018. Botulinum neurotoxin⿿encoding plasmids can be conjugatively transferred to diverse clostridial strains. Scientific Reports 8(1):3100.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2018
Citation:
Nawrocki, E.M. 2018. Mechanisms underlying the transfer of plasmid-borne BoNTs in Group I C. botulinum. Ph.D. Thesis, Department of Bacteriology, University of Wisconsin - Madison.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Bradshaw M, Fredrick CM, Nawrocki EM, Johnson EA. Construction and evaluation of catP-RAM for generation of Group II intron mutants in Clostridium botulinum. Presented as a poster at the Interagency Botulism Research Coordinating Committee Meeting in Chicago, IL, October 2018.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Nawrocki EM, Fredrick CM, Bradshaw M, Johnson EA. Group I C. botulinum plasmids are capable of conjugation. Presented as a poster at the Molecular Genetics of Bacteria and Phages Meeting in Madison, WI and at the Interagency Botulism Research Coordinating Committee Meeting in San Francisco, CA, Fall 2017.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2019
Citation:
An overview of plasmids in C. botulinum and related species
- Type:
Journal Articles
Status:
Submitted
Year Published:
2019
Citation:
Nawrocki EM, Fredrick CM, Bradshaw M, Johnson EA. 2018. Molecular mechanism of plasmid transfer in Clostridium botulinum. Submitted to Journal of Bacteriology
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:Ongoing progress has been communicated to laboratory colleagues and to student thesis committees as necessary. Poster presentations of the data were given at the Molecular Genetics of Bacteria and Phages Meeting in Madison, WI, the Interagency Botulism Research Coordinating Committee (IBRCC) in San Francisco, CA, Food Research Institute Annual Meeting, UW-Madison, in the last calendar year. A manuscript regarding pCLJ transfer is under revision in Scientific Reports. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided technical training in maintenance of Clostridium strain, anaerobic culture, molecular biology, and experimental design for Ph.D. and M.S. students. Professional development training occurred by outreach and communication of the methods and results as described in the body of the report. How have the results been disseminated to communities of interest?Ongoing progress has been communicated to laboratory colleagues and to student thesis committees as necessary. Poster presentations of the data were given at the Molecular Genetics of Bacteria and Phages Meeting in Madison, WI, and the Interagency Botulism Research Coordinating Committee (IBRCC) in San Francisco, CA, and Food Research Institute Annual Meeting, UW-Madison in the this calendar year. A manuscript regarding pCLJ transfer and undergoes revision in Scientific Reports. What do you plan to do during the next reporting period to accomplish the goals?In the next year, we will continue analyzing putative conjugation genes to identify their relevance to plasmid transfer. We will also co-culture clostridial strains with other common intestinal and/or soil microbes and determine whether their presence promotes pCLJ conjugation. These experiments will emphasize the relevance of BoNT movement to public health and disease. Identifying the origin of transfer and the genes necessary for conjugation is becoming increasingly feasible as clostridial genetic tools improve; as described above, we have recently developed and implemented a second antibiotic resistance cassette for this purpose. Now that double mutant strains are more easily achievable, we may more accurately define the host range of BoNT-encoding plasmids and illuminate the ways in which BoNT-encoding plasmids naturally spread in the environment. We also will seek to develop additional genetic tools in C. botulinum. New tools such as recombination-mediated genetic engineering and CRISPR-Cas9 genome editing have been accomplished in several clostridial species. We will attempt to modify this system for generation of mutations and gene deletions in C. botulinum. Availability of such genetic tools will enable us to make mutations in C. botulinum plasmid genes or even delete genes and assess their impact on plasmid transfer in the clostridia.
Impacts
What was accomplished under these goals?
Accomplishments are described in the following aims: (1) Tagging of BoNT-encoding plasmids in donor strains Donor strains are tagged with the insertion of a group II intron into the BoNT gene using a ClosTron system. The intron's insertion inactivates the expression of the corresponding BoNT and confers erythromycin resistance on the cell. Tagging plasmids is crucial to this research, as it allows for selection of transconjugants on the basis of their antibiotic resistance. Furthermore, when BoNT genes are inactivated in such a way, their toxins cannot be produced in recipient strains, thus providing a safe experimental approach for the study of plasmid transfer. Three BoNT-encoding plasmids have thus far been tagged with this system and are being used in the plasmid transfer experiments. (2) Generation of additional counterselectable traits in clostridial strains for plasmid transfer experiments To facilitate BoNT-encoding plasmid transfer to a recipient clostridial strain, the presence of a counterselectable marker in the recipient strain is desirable. In our previous experiments, we used recipient strains that carried a conjugative Tn916 transposon that contains a tetracycline resistance gene. However, Tn916 may potentially facilitate plasmid transfer, and therefore clostridial strains that did not carry this conjugative transposon were needed as recipients. The ClosTron system generates strains that are erythromycin (Erm) resistant, and the same marker could not be used in both plasmid donor and recipient strains. To tag recipient strains a new ClosTron vector was needed that would utilize a different antibiotic resistance marker. Using the same strategy that was utilized in construction of the Erm-cassette, a thiamphenicol (Th) gene was modified and tested for functionality in C. botulinum strains. Then a new ClosTron vector was constructed containing Th marker that allowed us to tag clostridial strains conferring a resistance to thiamphenicol. Thus BoNT-encoding plasmid transfer experiments could be performed using previously Erm-tagged C. botulinum donor strains and Th-tagged recipient strains. As an alternative strategy, Erm-tagged plasmid donor strain 657Ba was chemically mutagenized with nitrosoguanidine and screened for auxotrophs. Putative auxotrophs were restreaked multiple times to verify their purity and stability. Several clones that were Erm-resistant and unable to grow on minimal media were selected for plasmid transfer experiments. Such Erm-resistant auxotrophic plasmid strains can be used in mating experiments with any prototrophic clostridial strain. This method will aid in the recovery of transconjugants by allowing their selection on minimal media containing erythromycin: auxotrophic donor strains will not grow on minimal medium and recipient strains will not grow in the presence of erythromycin unless they receive the tagged plasmid. (3) Matings between C. botulinum plasmid-bearing donor strains and other BoNT producing clostridia, as well as nontoxigenic clostridial species present in human gut such as C. sporogenes and C. butyricum Matings have been conducted between Erm-tagged auxotrophic donor strain (strain 657Ba/pCLJ) and several prototrophic clostridial recipient strains without the aid of a conjugative transposon. Transfer frequencies to these strains were on par with those involving Tn916, suggesting that the transposon plays little to no role in mobilizing pCLJ. Analysis of transconjugants showed that pCLJ is transferred in full and remains stable in the absence of antibiotic selective pressure. The transfer of pCLJ was then expanded to a wider array of clostridial strains. We were able to show that pCLJ plasmid can be transferred under laboratory conditions into various C. botulinum isolates and into C. sporogenes and C. butyricum recipients. Experimental data suggest that the BoNT-encoding plasmids themselves are capable of transfer, in which case they are conjugative and not simply mobilizable. These experiments suggest a means for distribution of BoNTs throughout the clostridia. The Erm-tagged BoNT-encoding plasmids were also transferred to a representative clostridial recipient tagged with thiamphenicol resistant marker. The plasmid transfer frequency was similar to that observed in the Tn916 and auxotrophic mating schemes. (4) Optimization of mating experiments on solid and liquid media Mating experiments are still being optimized. We have evaluated different mating times and concentrations of agar in solid media during mating. None of these conditions improved frequency of plasmid transfer. Recently, we determined that conjugation efficiency of plasmids from E. coli donor strains to C. difficile can be greatly increased by heat shock of recipient strains. We also found this true for plasmid transfer from E. coli donor strains to C. botulinum strains (transfer of ClosTron vectors from E. coli donor stain into C. botulinum strains). Next we will investigate if heat shock of clostridial donor and/or recipient strains increases plasmid transfer efficiency. In liquid media, mating experiments are being adapted for a higher-throughput assay using 96-well microtiter plates, to allow for more robust screen of genes that contribute to conjugation. (5) Mating in the presence of gut microbiota that likely increase transfer frequency No gut microbiota have been included in mating experiments to this point. In line with the grant objectives, this will be further examined in the forthcoming year. (6) Analysis of transconjugants by PCR and PFGE Putative transconjugant strains were restreaked on selective plates for purity. The colonies initially were analyzed by PCR using primer sets specific for the recipient strains. Selected transconjugants were then confirmed by PFGE followed by hybridization with plasmid-specific probes. Valid transconjugants showed the banding pattern of the recipient strain and hybridized with a probe corresponding to the group II intron and/or the tagged plasmid originally present in the donor. C. sporogenes transconjugants were additionally shown to express active BoNT/A4, as detected by immunoblot and mouse bioassay. (7) Identification of BoNT-encoding plasmid genes involved in plasmid transfer Analysis of genome annotations of the BoNT-encoding plasmids revealed several genes with putative function in plasmid transfer. We have selected seven putative conjugation genes for further evaluation of their involvement in plasmid transfer. Three of these genes have already been inactivated by ClosTron (ErmR) in the wild type strain 657Ba/pCLJ. The ClosTron vectors have been designed for the other four genes, and the mutant clones will be generated. The pCLJ plasmids with mutated putative conjugation genes were analyzed in mating experiments with a C. botulinum recipient strain to evaluate whether inactivation of these genes prevents plasmid transfer. In one of the three mutants tested thus far, inactivation of a plasmid-borne gene completely abolished pCLJ conjugation between strains. This gene's essentiality will be a major focus of study in the next reporting period, in hopes of determining its contribution to the conjugative process.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2018
Citation:
Nawrocki, E,M., Bradshaw, M., Johnson, E.A. Botulinum neurotoxin-encoding plasmids can be conjugatively transferred to diverse clostridial strains.
Scientific Reports
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Attended International Botulism Meeting (IBRCC - Interagency Botulism Research Botulism Coordinating Committee) and presented a poster scientific report to regulatory, academic and industry personnel. Changes/Problems:Additional goal of "Identification of BoNT-encoding plasmid genes involved in plasmid transfer" was added to the project. Our laboratory was the first to demonstrate that BoNT-encoding plasmids could be experimentally transferred between C. botulinum strains. The mechanism governing this transfer remains to be determined and is a priority of this project. What opportunities for training and professional development has the project provided?The graduate student attended an international conference on botulism and interacted with regulatory, industry and academic expers. How have the results been disseminated to communities of interest?Results have been disseminated through an international conference, laboratory meetings with colleagues, a meeting that was required for thesis committee and a pending manuscript that is in preparation for publication. What do you plan to do during the next reporting period to accomplish the goals?In the next year, we will analyze the selected putative plasmid conjugation genes to identify which genes are involved in plasmid transfer. Depending on these results, we may select additional genes for analyses. We will also coculture clostridial strains with other common intestinal and/or soil microbes and determine whether their presence promotes pCLJ conjugation. These experiments will emphasize the relevance of BoNT movement to public health and disease. Identifying the origin of transfer and the genes necessary for conjugation is becoming increasingly feasible as clostridial genetic tools improve, as described above we have recently developed and implemented a second antibiotic resistance cassette for this purpose. Now that the double mutants strains are more easily achievable, this will allow us to more accurately define the host range of BoNT-encoding plasmids and illuminate the ways in which BoNT-encoding plasmids naturally spread in the environment. We also will develop additional genetic tools in C. botulinum. New tools such as recombination-mediated genetic engineering and CRISPR-Cas9 genome editing has been accomplished in several clostridial species. We will attempt to modify this system for generation of mutations and gene deletions in C. botulinum. Availability of such genetic tools will enable us to make mutations in C. botulinum plasmid genes or even delete genes and assess their impact on plasmid transfer in the clostridia.
Impacts
What was accomplished under these goals?
This project was designed to further explore the mechanisms of transfer of BoNT-encoding plasmids to clostridia. The movement of BoNT encoding genes and expression of BoNTs among clostridial strains is of particular interest to human health and food safety. For example, C. botulinum strains expressing multiple serotypes of BoNT have been isolated from cases of infant botulism, suggesting that strains have the opportunity to acquire new toxins in the intestinal microbiome. Furthermore, BoNTs have been isolated from normally nontoxigenic clostridial species such as C. sporogenes, C. baratii and C. butyricum. Transfer of BoNTs to other species could jeopardize the efficacy of treatments designed to eliminate C. botulinum in food products and in the intestinal environment. 1) Tagging the BoNT-encoding plasmids in donor strains Donor strains are tagged with the insertion of a group II intron into the bont gene using a ClosTron system. The intron's insertion inactivates the expression of the corresponding BoNT and confers erythromycin resistance on the cell. Tagging plasmids is crucial to this research, as it allows for selection of transconjugants on the basis of their antibiotic resistance. Furthermore, when BoNT genes are inactivated in such a way, their toxins cannot be produced in recipient strains, thus providing a safe experimental approach for the study of plasmid transfer. Three BoNT-encoding plasmids have thus far been tagged with this system and are being used in the plasmid transfer experiments. Generation of additional counterselectable traits in clostridial strains for plasmid transfer experiments To facilitate BoNT-encoding plasmid transfer to a recipient clostridial strain, a presence of a counterselectable marker in the recipient strain is desirable. In our previous experiments we used recipient strains that carried a conjugative Tn916 transposon that contains a tetracycline resistance gene. However, Tn916 may potentially facilitate plasmid transfer, thus clostridial strains that did not carry this conjugative transposon were needed as recipients. The ClosTron system generates strains that are erythromycin (Erm) resistant, and the same marker could not be used in both plasmid donor and recipient strains. To tag recipient strains a new ClosTron vector was needed that would utilize a different antibiotic resistance marker. Using the same strategy that was utilized in construction of the Erm-cassette, a thiamphenicol (Th) gene was modified and tested for functionality in C. botulinum strains. Then a new ClosTron vector was constructed containing Th marker that allowed us to tag clostridal strains conferring a resistance to thiamphenicol. Thus BoNT-encoding plasmid transfer experiments could be performed using previously Erm-tagded C. botulinum donor strains and Th-tagged recipient strains. In addition, Erm-tagged plasmid donor strain 657Ba was chemically mutagenized with nitrosoguanidine and screened for auxotrops. . Putative auxotrophs were restreaked multiple times to verify their purity and stability. Several clones that were Erm-resistant and unable to grow on minimal media were selected for plasmid transfer experiments. Such Erm-resistant auxotropic plasmid strains can be used in mating experiments with any clostridial strain. This method will aid in the recovery of transconjugants by allowing them to be selected on minimal media containing erythromycin: auxotrophic donor strains will not grow on minimal medium and recipient strains will not grow in the presence of erythromycin unless they receive the tagged plasmid. (2) Conduct matings between C. botulinum plasmid-bearing donor strains and other BoNT producing clostridia, as well as nontoxigenic clostridial species present in human gut such as C. sporogenes and C. butyricum Matings have been conducted between Erm-tagged auxotropic donor strain (Strain 657Ba/pCLJ) and several protothropic clostridial recipient strains without the aid of conjugative transposon. Transfer frequencies to these strains were on par with those involving Tn916, suggesting that the transposon plays little to no role in mobilizing pCLJ. Analysis of transconjugants showed that pCLJ is transferred in full and remained stable in the absence of antibiotic selective pressure. The transfer of pCLJ was expanded to a wider array of clostridial strains. We were able to show that pCLJ plasmid can be transferred in laboratory conditions into various C. botulinum isolates as well as C. sporogenes and C. butyricum recipients. Experimental data show that the BoNT-encoding plasmids themselves are capable of transfer, making them conjugative and not simply mobilizable. These experiments suggest a means for distribution of BoNTs throughout the clostridia. The Erm-tagged BoNT-encoding plasmids were also transferred to other clostridial recipients tagged with thiamphenicol resistant marker. The plasmid transfer frequency was similar to that observed earlier. (3) Optimizing mating experiments on solid and liquid media Mating experiments are still being optimized. We have evaluated different mating times, as well as concentration of agar in solid media plates during mating. None of these conditions improved frequency of plasmid transfer. Recently, it has been determined that conjugation efficiency of plasmids from E. coli donor strains to C. difficile can be greatly increased by heat shock of the recipient strains. We have also found that this is true for plasmid transfer from E. coli donor strains to C. botulinum strains (transfer of ClosTron vectors from E. coli donor stain into C. botulinum strains). Next we will investigate if the heat shock of clostridial donor and/or recipient strains can increase plasmid transfer efficiency. (4) Mating in the presence of gut microbiota that likely increase transfer frequency No gut microbiota have been included in mating experiments to this point. In line with the grant objectives, this will be further examined in the forth year. (5) Analysis of transconjugants by PCR and PFGE Putative transconjugant strains were restreaked on selective plates for purity. The colonies initially were analyzed by PCR using primer sets specific for the plasmid and the recipient strains. Then selected number of transconjugants were confirmed by PFGE followed by hybridization with plasmid and recipient probes. Valid transconjugants showed the banding pattern of the recipient strain and hybridized with a probe corresponding to the group II intron and/or the tagged plasmid originally present in the donor. (6) Identification of BoNT-encoding plasmid genes involved in plasmid transfer Analysis of putative functions of the BoNT-encoding plasmids revealed several genes with putative function in plasmid transfer. We have selected seven putative conjugation genes for further evaluation of their involvement in plasmid transfer. Three of these genes have already been inactivated by ClosTron (ErmR) in the wild type strain 657Ba/pCLJ. The Clostron vectors have been designed for the other four genes, and the mutant clones will be generated. The pJCL plasmids with mutated putative conjugation genes will be analyzed in mating experiments with a C. botulinum recipient strain to evaluate if inactivation of any of these genes will still support plasmid transfer.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Large BoNT-Encoding Plasmids are Transferred by Conjugation. 2016. International Botulism Meeting.
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Progress 12/30/14 to 09/30/15
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Participation inMolecular Genetics of Bacteria and Phages Meeting in August 2015 by Erin Nawrocki, graduate student. How have the results been disseminated to communities of interest?Ongoing progress has been communicated to colleagues when appropriate, as is required for thesis committee meetings and doctoral seminar presentations. The body of results are accumulating and we anticipate of presenting an oral paper in a national meeting. What do you plan to do during the next reporting period to accomplish the goals?In the next year, we will continue engineering counterselectable plasmid donor strains in an effort to expand the set of possible mating pairs. Once this is completed, we will evaluate the transfer of BoNT-encoding plasmids within and between the taxonomic groups of C. botulinum. This will allow us to more accurately define the host range of BoNT-encoding plasmids and illuminate the ways in which BoNT-encoding plasmids naturally spread in the environment. We also will develop additional genetic tools in C. botulinum. The mechanism of plasmid transfer is unknown, but there are certain candidate genes on the plasmids that appear to be related to conjugation. New tools such as recombination-mediated genetic engineering and CRISPR-Cas9 genome editing, are being developed in C. botulinum and will enable us to make mutations in these genes and assess their impact on plasmid transfer in the clostridia.
Impacts
What was accomplished under these goals?
Clostridium botulinum is a gram-positive, anaerobic, spore-forming bacterium that produces botulinum neurotoxin (BoNT), which is the most poisonous toxin known to humankind. BoNTs are the agents of botulism, a potentially lethal paralytic disease; they act by cleaving neuronal proteins, thereby preventing the release of neurotransmitters. While many BoNTs are encoded on bacterial chromosomes, some are present on plasmids. Our laboratory was the first to demonstrate that BoNT-encoding plasmids could be experimentally transferred between C. botulinum strains. The mechanism governing this transfer remains to be determined and is a priority of this project. This project was designed to further explore the mechanisms of transfer of BoNT-encoding plasmids to clostridia. The movement of BoNT encoding genes and expression of BoNTs among clostridial strains is of particular interest to human health and food safety. For example, C. botulinum strains expressing multiple serotypes of BoNT have been isolated from cases of infant botulism, suggesting that strains have the opportunity to acquire new toxins in the intestinal microbiome. Furthermore, BoNTs have been isolated from normally nontoxigenic clostridial species such as C. baratii and C. butyricum. Transfer of BoNTs to other species could jeopardize the efficacy of treatments designed to eliminate C. botulinum in food products and in the intestinal environment. 1)Tagging the BoNT-encoding plasmids in donor strains: Donor strains are tagged with the insertion of a group II intron into the bont gene. The intron's insertion inactivates the expression of the corresponding BoNT and confers erythromycin resistance on the cell. Tagging plasmids is crucial to this research, as it allows for selection of transconjugants on the basis of their antibiotic resistance. Furthermore, when bont genes are inactivated in such a way, their toxins cannot be produced in recipient strains, thus providing a safe experimental approach for the study of plasmid transfer. Three BoNT-encoding plasmids have thus far been tagged with this system and are being used in the plasmid transfer experiments. 2) Conduct matings between C. botulinum plasmid-bearing donor strains and other botulinum neurotoxin producing clostridia: Matings have been conducted between tagged plasmid-bearing donor strains and select recipients. BoNT-encoding plasmids were successfully transferred to two C. botulinum strains containing the Tn916 transposon, which confers tetracycline resistance. It is possible, however, that this transposon aids in mobilizing plasmids. To evaluate whether Tn916-facilitates plasmid mobilization, mating experiments were performed in strains that did not carry Tn916. Experimental data show that the plasmids themselves are capable of transfer, making them conjugative and not simply mobilizable. 3) Optimizing mating experiments on solidand liquid media: Mating experiments are still being optimized. Current efforts area focus on engineering the tagged donor strains to be counterselectable by auxotrophies. Strains are subjected to chemical mutagenesis with nitrosoguanidine and screened for growth on minimal medium with and without desired supplements. Putative auxotrophs are restreaked multiple times to verify their purity and stability. This method will aid in the recovery of transconjugants by allowing them to be selected on minimal media containing erythromycin: auxotrophic donor strains will not grow on minimal medium and recipient strains will not grow in the presence of erythromycin unless they receive the tagged plasmid. Some mutants have been isolated by this procedure and will be used in mating experiments. Counterselectable donor strains will enable mating experiments to be conducted between a larger set of pairs, broadening our scope that was formerly restricted to recipient strains containing a conjugative transposon. Liquid mating has not yet been investigated, and will be evaluated using this same selection strategy. 4)Mating in the presence of gut microbiota that likely increase transfer frequency: No gut microbiota have been included in mating experiments to this point. In line with the project objectives, this will be further examined in the second year. 5) Analysis of transconjugants by PFGE: A number of transconjugants have been confirmed by PFGE. Valid transconjugants have the banding pattern of the recipient strain and hybridize with a probe corresponding to the group II intron and/or the tagged plasmid originally present in the donor.
Publications
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