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Construction of a hybrid plasmid capable of replication in Amycolatopsis mediterranei
1Lehrstuhl für Gentechnologie/Mikrobiologie, Fakultät für Biologie, Universität Bielefeld, Federal Republic of Germany.
Abstract:
A new plasmid, pA387, has been isolated from "Amycolatopsis sp." (DSM 43387). This plasmid could be isolated from liquid culture as well as mycelium from agar plates by a modified procedure. Plasmid pA387 is about 29.6 kb and can be cured at low frequency by protoplasting and ethidium bromide and heat treatment. Hybridization experiments showed that this plasmid is present in free form and does not integrate into the chromosome. A hybrid plasmid was constructed by cloning a 5.1-kb fragment of pA387 into the Escherichia coli vector pDM10. This hybrid plasmid, termed pRL1, could be transformed into Amycolatopsis mediterranei and A. orientalis by electroporation. A transformation frequency of 2.2 x 10(3) transformants per micrograms of DNA at 12.5 kV/cm and a pulse duration of 10.8 ms was obtained in A. mediterranei, whereas 1.1 x 10(5) transformants per microgram of DNA were obtained at a field strength of 7.5 kV/cm and a pulse duration of 7.6 ms in A. orientalis. Plasmid pRL1 is the first hybrid plasmid which could be used successfully for the transformation of A. mediterranei. The plasmid has a rather high copy number, is genetically stable, and can be easily reisolated from A. mediterranei. Plasmid pRL1 will be useful for further construction of a shuttle vector for E. coli and A. mediterranei and becomes the basis for the development of gene cloning techniques in Amycolatopsis spp.
Insights
A novel hybrid plasmid, pRL1, was developed for efficient gene transfer in Amycolatopsis species. This tool enables genetic manipulation and gene cloning in Amycolatopsis mediterranei and related bacteria.
Area of Science:
- Microbiology
- Molecular Biology
- Genetic Engineering
Background:
- Plasmids are essential tools for genetic manipulation in bacteria.
- Amycolatopsis species are important industrial microorganisms, but lack efficient genetic tools.
- Isolation and characterization of native plasmids are crucial for developing these tools.
Purpose of the Study:
- To isolate and characterize a native plasmid from Amycolatopsis sp. (DSM 43387).
- To construct a hybrid plasmid for transformation of Amycolatopsis mediterranei and A. orientalis.
- To establish a basis for gene cloning techniques in Amycolatopsis species.
Main Methods:
- Isolation of plasmid pA387 from Amycolatopsis sp. using a modified procedure.
- Construction of a hybrid plasmid (pRL1) by cloning a fragment of pA387 into an E. coli vector.
- Transformation of Amycolatopsis mediterranei and A. orientalis using electroporation.
Main Results:
- Plasmid pA387 (29.6 kb) was isolated and found to exist in a free form, not integrated into the chromosome.
- Hybrid plasmid pRL1 was successfully constructed and transformed into A. mediterranei and A. orientalis with high efficiency.
- pRL1 demonstrated high transformation frequencies, genetic stability, and a high copy number in A. mediterranei.
Conclusions:
- Plasmid pRL1 is the first hybrid plasmid enabling successful transformation of Amycolatopsis mediterranei.
- pRL1 serves as a valuable tool for developing gene cloning techniques in Amycolatopsis species.
- This work lays the foundation for creating shuttle vectors for E. coli and Amycolatopsis.