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Curing of plasmid pBMB28 from Bacillus thuringiensis YBT-020 using an unstable replication region
Pengxia Wang1, Qian Zhu1, Hui Shang1
1State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, P. R. China.
Abstract:
Bacillus thuringiensis serovar finitimus strain YBT-020 is the well-studied spore-crystal association (SCA) phenotypic strain, whose parasporal crystals adhere to spore after lysis of the mother cell. Its endogenous plasmids pBMB26 and pBMB28 were proved essential for this SCA phenotype. In our previous study, using conventional methods, pBMB26 cured derivative and both pBMB26 and pBMB28 cured derivative of YBT-020 were obtained. However, YBT-020 solely cured of pBMB28 could not be obtained. In this study, an unstable replication region of pBMB28 was identified and was used to construct an incompatible plasmid pRep28B. This incompatible plasmid was successfully used to cure plasmid pBMB28 and was easily eliminated through segregational instability under the optimum growth temperature of YBT-020. Therefore, an endogenous plasmid was cured from the B. thuringiensis strain utilizing plasmid incompatibility. Moreover, using an unstable replication region instead of a temperature sensitive (Ts) replication region is better to cure the incompatible plasmid because it can avoid culturing at higher temperature. This method provides an efficient method for plasmid curing in B. thuringiensis and other bacteria.
Insights
Bacillus thuringiensis plasmid curing was achieved using a novel incompatible plasmid. This method efficiently removes endogenous plasmids, offering a new tool for bacterial genetic manipulation.
Area of Science:
- Microbiology
- Molecular Biology
- Bacterial Genetics
Background:
- Bacillus thuringiensis serovar finitimus strain YBT-020 exhibits a spore-crystal association (SCA) phenotype, crucial for its biological activity.
- Endogenous plasmids pBMB26 and pBMB28 are essential for the SCA phenotype in YBT-020.
- Previous attempts to cure only the pBMB28 plasmid from YBT-020 using conventional methods were unsuccessful.
Purpose of the Study:
- To develop an efficient method for curing the pBMB28 plasmid from Bacillus thuringiensis YBT-020.
- To investigate the use of plasmid incompatibility for endogenous plasmid elimination.
- To compare the efficacy of using an unstable replication region versus a temperature-sensitive region for plasmid curing.
Main Methods:
- Identification of an unstable replication region within the pBMB28 plasmid.
- Construction of an incompatible plasmid, pRep28B, utilizing the identified replication region.
- Application of pRep28B to cure the pBMB28 plasmid from YBT-020 through segregational instability.
- Culturing of the modified strain at the optimum growth temperature of YBT-020.
Main Results:
- The incompatible plasmid pRep28B was successfully constructed and utilized to eliminate the endogenous pBMB28 plasmid from YBT-020.
- Plasmid curing was achieved efficiently through segregational instability at the optimal growth temperature, avoiding the need for high-temperature culturing.
- The use of an unstable replication region proved advantageous over temperature-sensitive systems for plasmid curing.
Conclusions:
- Plasmid incompatibility is an effective strategy for endogenous plasmid curing in Bacillus thuringiensis.
- Utilizing an unstable replication region offers a more convenient and efficient method for plasmid elimination compared to temperature-sensitive approaches.
- This novel method provides a valuable tool for genetic manipulation of Bacillus thuringiensis and potentially other bacterial species.
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