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Properties of in vitro recombinant derivatives of pJV1, a multi-copy plasmid from Streptomyces phaeochromogenes
Abstract:
The 10.8 kb plasmid pJV1, isolated from Streptomyces phaeochromogenes, has a high copy number (about 150) and a broad host range among Streptomyces spp. Several pJV1 derivatives carrying the thiostrepton resistance gene (tsr) of S. azureus were made. One derivative, pWOR191, was shown to promote its own transfer and to mobilize chromosomal markers in S. lividans. Another derivative, pWOR109, was non-transmissible. Deletion in vitro of a segment of pWOR109 gave pWOR120 (5.6 kb), which has single BamHI and Bg/II sites shown to be capable of accepting 'foreign' DNA such as a previously cloned S. antibioticus DNA fragment encoding tyrosinase, giving vectors (pWOR125, pWOR126) with properties resembling the well-established multicopy vector pIJ702. Shuttle vectors capable of functioning in both S. lividans and Escherichia coli were also constructed. The region of pJV1 essential for replication and maintenance was localized to a 2.5 kb segment. Stable maintenance of pWOR109 and pWOR120 was observed in the presence of derivatives of pIJ101, the progenitor of pIJ702.
Insights
Researchers developed novel Streptomyces vectors derived from the pJV1 plasmid. These engineered plasmids, including shuttle vectors, offer broad host range and high copy numbers for genetic manipulation in Streptomyces species.
Area of Science:
- Molecular Biology
- Microbial Genetics
- Biotechnology
Background:
- The plasmid pJV1 from Streptomyces phaeochromogenes is a high-copy-number (approx. 150) plasmid with a broad host range within Streptomyces species.
- Development of versatile vectors is crucial for genetic studies and manipulation in Streptomyces.
Purpose of the Study:
- To engineer derivatives of the pJV1 plasmid for use as molecular vectors in Streptomyces.
- To characterize the properties of these new vectors, including transmissibility, host range, and capacity for foreign DNA insertion.
- To construct shuttle vectors functional in both Streptomyces and Escherichia coli.
Main Methods:
- Isolation and characterization of the pJV1 plasmid.
- Construction of pJV1 derivatives, including those with the thiostrepton resistance gene (tsr).
- In vitro manipulation (deletion) to create new vectors.
- Cloning of foreign DNA (tyrosinase gene) into engineered vectors.
- Assessment of vector maintenance and stability in Streptomyces hosts.
Main Results:
- Several pJV1 derivatives were created, including pWOR191 (self-transmissible, mobilizes chromosomal markers) and pWOR109 (non-transmissible).
- Deletion of a segment from pWOR109 yielded pWOR120 (5.6 kb) with single cloning sites, suitable for foreign DNA insertion (e.g., tyrosinase gene), creating vectors pWOR125 and pWOR126.
- Shuttle vectors functional in S. lividans and E. coli were constructed.
- The essential replication and maintenance region of pJV1 was localized to a 2.5 kb segment.
- Stable maintenance of pWOR109 and pWOR120 was observed with pIJ101 derivatives.
Conclusions:
- Engineered pJV1 derivatives provide new tools for Streptomyces molecular biology, offering high copy numbers and cloning capabilities.
- The developed vectors, including shuttle vectors, expand the options for genetic manipulation and research in Streptomyces species.
- The study successfully localized the minimal replication region of pJV1, aiding in future vector design.