Efficient transformation and genome editing in a nondomesticated, biocontrol strain, Bacillus subtilis GLB191
Abstract Bacillus subtilis has been widely used as a biological control agent in agricultural production. Environmental strains of B. subtilis are an important source of biological control agents. However, due to its low genetic transformation efficiency, the genetic manipulation of the environmenta...
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2024-12-01
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| Online Access: | https://doi.org/10.1186/s42483-024-00287-0 |
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| author | Yu Zhao Zhenshuo Wang Qian Wang Bing Wang Xiaoning Bian Qingchao Zeng Daowan Lai Qi Wang Yan Li |
| author_facet | Yu Zhao Zhenshuo Wang Qian Wang Bing Wang Xiaoning Bian Qingchao Zeng Daowan Lai Qi Wang Yan Li |
| author_sort | Yu Zhao |
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| description | Abstract Bacillus subtilis has been widely used as a biological control agent in agricultural production. Environmental strains of B. subtilis are an important source of biological control agents. However, due to its low genetic transformation efficiency, the genetic manipulation of the environmental and nondomesticated strains is challenging. In this study, the impact of competent cell preparation, pulse electroporation, and recovery culture on the electroporation efficiency of B. subtilis GLB191 was assessed utilizing response surface methodology. Results indicated that the concentration of glycine, DL-threonine, and Tween 80 used in a cell wall weakening solution during competent cell preparation, and the voltage applied during pulse electroporation were the primary factors affecting electroporation efficiency. Optimization of these factors led to nearly a three-fold increase (reaching 74.00 ± 5.10 CFU/µg DNA) in electroporation efficiency. The elimination of dam and dcm modifications to mitigate the influence of host restriction-modification systems was integrated to further increase the electroporation efficacy. An electroporation efficiency for replicative plasmids of 1.96 ± 0.05 × 106 CFU/µg DNA was achieved using the optimized strategy. Utilizing this improved methodology, the temperature-sensitive plasmid pJOE8899 was efficiently transformed into B. subtilis GLB191, resulting in a markerless knockout of pdeH. The optimized transformation strategy significantly enhances the efficiency of markerless genome editing of nondomesticated B. subtilis, offering the potential for future interpretation of their modes of action, which is critical for the development of the nondomesticated B. subtilis strains. |
| format | Article |
| id | doaj-art-622f9ced3ca142f49fa6684eafaa0ae4 |
| institution | OA Journals |
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| language | English |
| publishDate | 2024-12-01 |
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| series | Phytopathology Research |
| spelling | doaj-art-622f9ced3ca142f49fa6684eafaa0ae42025-08-20T01:56:24ZengBMCPhytopathology Research2524-41672024-12-016111210.1186/s42483-024-00287-0Efficient transformation and genome editing in a nondomesticated, biocontrol strain, Bacillus subtilis GLB191Yu Zhao0Zhenshuo Wang1Qian Wang2Bing Wang3Xiaoning Bian4Qingchao Zeng5Daowan Lai6Qi Wang7Yan Li8Department of Plant Pathology, MOA Key Lab of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural UniversityDepartment of Plant Pathology, MOA Key Lab of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural UniversityDepartment of Plant Pathology, MOA Key Lab of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural UniversityDepartment of Plant Pathology, MOA Key Lab of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural UniversityDepartment of Plant Pathology, MOA Key Lab of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural UniversityDepartment of Plant Pathology, MOA Key Lab of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural UniversityDepartment of Plant Pathology, MOA Key Lab of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural UniversityDepartment of Plant Pathology, MOA Key Lab of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural UniversityDepartment of Plant Pathology, MOA Key Lab of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural UniversityAbstract Bacillus subtilis has been widely used as a biological control agent in agricultural production. Environmental strains of B. subtilis are an important source of biological control agents. However, due to its low genetic transformation efficiency, the genetic manipulation of the environmental and nondomesticated strains is challenging. In this study, the impact of competent cell preparation, pulse electroporation, and recovery culture on the electroporation efficiency of B. subtilis GLB191 was assessed utilizing response surface methodology. Results indicated that the concentration of glycine, DL-threonine, and Tween 80 used in a cell wall weakening solution during competent cell preparation, and the voltage applied during pulse electroporation were the primary factors affecting electroporation efficiency. Optimization of these factors led to nearly a three-fold increase (reaching 74.00 ± 5.10 CFU/µg DNA) in electroporation efficiency. The elimination of dam and dcm modifications to mitigate the influence of host restriction-modification systems was integrated to further increase the electroporation efficacy. An electroporation efficiency for replicative plasmids of 1.96 ± 0.05 × 106 CFU/µg DNA was achieved using the optimized strategy. Utilizing this improved methodology, the temperature-sensitive plasmid pJOE8899 was efficiently transformed into B. subtilis GLB191, resulting in a markerless knockout of pdeH. The optimized transformation strategy significantly enhances the efficiency of markerless genome editing of nondomesticated B. subtilis, offering the potential for future interpretation of their modes of action, which is critical for the development of the nondomesticated B. subtilis strains.https://doi.org/10.1186/s42483-024-00287-0Bacillus subtilisElectroporationResponse surface methodologyGenome editing |
| spellingShingle | Yu Zhao Zhenshuo Wang Qian Wang Bing Wang Xiaoning Bian Qingchao Zeng Daowan Lai Qi Wang Yan Li Efficient transformation and genome editing in a nondomesticated, biocontrol strain, Bacillus subtilis GLB191 Phytopathology Research Bacillus subtilis Electroporation Response surface methodology Genome editing |
| title | Efficient transformation and genome editing in a nondomesticated, biocontrol strain, Bacillus subtilis GLB191 |
| title_full | Efficient transformation and genome editing in a nondomesticated, biocontrol strain, Bacillus subtilis GLB191 |
| title_fullStr | Efficient transformation and genome editing in a nondomesticated, biocontrol strain, Bacillus subtilis GLB191 |
| title_full_unstemmed | Efficient transformation and genome editing in a nondomesticated, biocontrol strain, Bacillus subtilis GLB191 |
| title_short | Efficient transformation and genome editing in a nondomesticated, biocontrol strain, Bacillus subtilis GLB191 |
| title_sort | efficient transformation and genome editing in a nondomesticated biocontrol strain bacillus subtilis glb191 |
| topic | Bacillus subtilis Electroporation Response surface methodology Genome editing |
| url | https://doi.org/10.1186/s42483-024-00287-0 |
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