Construction of an Escherichia coli-rhodococcus shuttle vector and plasmid transformation in Rhodococcus spp
A plasmid transformation system for Rhodococcus sp. strain H13-A was developed by using an Escherichia coli-Rhodococcus shuttle plasmid constructed in this study. Rhodococcus sp. strain H13-A contains three cryptic indigenous plasmids, designated pMVS100, pMVS200, and pMVS300, of 75, 19.5, and 13.4 kilobases (kb), respectively. A 3.8-kb restriction fragment of pMVS300 was cloned into pIJ30, a 6.3-kb pBR322 derivative, containing the E. coli origin of replication (ori) and ampicillin resistance determinant (bla), as well as a Streptomyces gene for thiostrepton resistance, tsr. The resulting 10.1-kb recombinant plasmid, designated pMVS301, was isolated from E. coli DH1 (pMVS301) and transformed into Rhodococccus sp. strain AS-50, a derivative of strain H13-A. The cloned 3.8-kb fragment of Rhodococcus DNA in pMVS301 contains a Rhodococcus origin of replication, since the hybrid plasmid was capable of replication in both genera. The plasmid was identical in E. coli and Rhodococcus transformants as determined by restriction analysis and was maintained as a stable, independent replicon in both organisms. A restriction map demonstrated 14 unique restriction sites in pMVS301, some of which are potentially useful for molecular cloning in Rhodococcus spp. and other actinomycetes. This is the first report of plasmid transformation and of heterologous gene expression in a Rhodococcus sp.
- Research Organization:
- Univ. of Georgia, Athens
- DOE Contract Number:
- FG09-86ER13588
- OSTI ID:
- 5285224
- Journal Information:
- J. Bacteriol.; (United States), Vol. 170:2
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
BACTERIA
GENETIC ENGINEERING
DNA-CLONING
ESCHERICHIA COLI
GENETIC MAPPING
PLASMIDS
RECOMBINANT DNA
REPLICONS
STREPTOMYCES
CELL CONSTITUENTS
CLONING
DNA
GENES
MAPPING
MICROORGANISMS
NUCLEIC ACIDS
ORGANIC COMPOUNDS
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