Transformation of Rhodococcus fascians by high-voltage electroporation and development of R. fascians cloning vectors

Journal Article · Sat Sep 01 04:00:00 EDT 1990 · Applied and Environmental Microbiology; (USA)

OSTI ID:6314855

Desomer, J; Dhaese, P; Montagu, M V ^[1]

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1. Laboratorium voor Genetica, Gent (Belgium)

The analysis of the virulence determinants of phytopathogenic Rhodococcus fascians has been hampered by the lack of a system for introducing exogenous DNA. We investigated the possibility of genetic transformation of R. fascians by high-voltage electroporation of intact bacterial cells in the presence of plasmid DNA. Electrotransformation in R. fascians D188 resulted in transformation frequencies ranging from 10{sup 5}/{mu}g of DNA to 10{sup 7}/{mu}g of DNA, depending on the DNA concentration. The effects of different electrical parameters and composition of electroporation medium on transformation efficiency are present. By this transformation method, a cloning vector (pRF28) for R. fascians based on an indigenous 160-kilobase (chloramphenicol and cadmium resistance-encoding) plasmid pRF2 from strain NCPPB 1675 was developed. The origin of replication and the chloramphenicol resistance gene on pRF28 were used to construct cloning vectors that are capable of replication in R. fascians and Escherichia coli. The electroporation method presented was efficient enough to allow detection of the rare integration of replication-deficient pRF28 derivatives in the R.fascians D188 genome via either homologous or illegitimate recombination.

• OSTI ID: 6314855
• Journal Information: Applied and Environmental Microbiology; (USA), Journal Name: Applied and Environmental Microbiology; (USA) Vol. 56:9; ISSN 0099-2240; ISSN AEMID
• Country of Publication: United States
• Language: English

Related Subjects

550400 -- Genetics
560400* -- Other Environmental Pollutant Effects
59 BASIC BIOLOGICAL SCIENCES
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
CELL CONSTITUENTS
CLONING
CURRENTS
DNA
DNA HYBRIDIZATION
DNA-CLONING
ELECTRIC CURRENTS
GENETIC ENGINEERING
HYBRIDIZATION
NUCLEIC ACIDS
ORGANIC COMPOUNDS
PATHOGENS
PLASMIDS
RECOMBINANT DNA
USES
VIRULENCE