skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

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

Abstract

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.

Authors:
; ;  [1]
  1. Laboratorium voor Genetica, Gent (Belgium)
Publication Date:
OSTI Identifier:
6314855
Resource Type:
Journal Article
Journal Name:
Applied and Environmental Microbiology; (USA)
Additional Journal Information:
Journal Volume: 56:9; Journal ID: ISSN 0099-2240
Country of Publication:
United States
Language:
English
Subject:
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.; 59 BASIC BIOLOGICAL SCIENCES; ELECTRIC CURRENTS; USES; PATHOGENS; GENETIC ENGINEERING; DNA; DNA-CLONING; PLASMIDS; RECOMBINANT DNA; VIRULENCE; CELL CONSTITUENTS; CLONING; CURRENTS; DNA HYBRIDIZATION; HYBRIDIZATION; NUCLEIC ACIDS; ORGANIC COMPOUNDS; 560400* - Other Environmental Pollutant Effects; 550400 - Genetics

Citation Formats

Desomer, J, Dhaese, P, and Montagu, M V. Transformation of Rhodococcus fascians by high-voltage electroporation and development of R. fascians cloning vectors. United States: N. p., 1990. Web.
Desomer, J, Dhaese, P, & Montagu, M V. Transformation of Rhodococcus fascians by high-voltage electroporation and development of R. fascians cloning vectors. United States.
Desomer, J, Dhaese, P, and Montagu, M V. 1990. "Transformation of Rhodococcus fascians by high-voltage electroporation and development of R. fascians cloning vectors". United States.
@article{osti_6314855,
title = {Transformation of Rhodococcus fascians by high-voltage electroporation and development of R. fascians cloning vectors},
author = {Desomer, J and Dhaese, P and Montagu, M V},
abstractNote = {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.},
doi = {},
url = {https://www.osti.gov/biblio/6314855}, journal = {Applied and Environmental Microbiology; (USA)},
issn = {0099-2240},
number = ,
volume = 56:9,
place = {United States},
year = {Sat Sep 01 00:00:00 EDT 1990},
month = {Sat Sep 01 00:00:00 EDT 1990}
}