A Novel Mechanism for Site-Directed Mutagenesis of Large Catabolic Plasmids Using Natural Transformation

Natural transformation is the process by which cells take up DNA from the surrounding medium under physiological conditions, altering the genotype in a heritable fashion. This occurs without chemical or physical treatment of the cells. Certain Acinetobacter strains exhibit a strong tendency to incorporate homologous DNA into their chromosomes by natural transformation. Transformation in Acinetobacter exhibits several unique properties that indicate this system's superiority as a model for transformation studies or studies which benefit from the use of transformation as an experimental method of gene manipulation. Pseudomonas putida is the natural host of TOL plasmids, ranging between 50 kbp and 300 kbp in size and encoding genes for the catabolism of toluene, meta-toluate, and xylene. These very large, single-copy plasmids are difficult to isolate, manipulate, or modify in vitro. In this study, the TOL plasmid pDKR1 was introduced into Acinetobacter calcoaceticus strains and genetically engineered utilizing natural transformation as part of the process. Following engineering by transformation, the recombinant DNA molecule was returned to the native genetic background of the original host P. putida strain. Specific parameters for the successful manipulation of large plasmids by natural transformation in Acinetobacter were identified and are outlined. The effects of growth phase, total transforming DNA concentration, transforming DNA conformation, and gene dosage on transformation efficiency are presented. Addition of Acinetobacter plasmid DNA sequences to the manipulated constructs did not have an effect on transformation rates. Results suggest that a broadly applicable and efficient method to carry out site-directed genetic manipulations of large plasmids has been identified. The ability to easily reintroduce the recombinant DNA molecules back into the original host organism was maintained.

Grantor dc:publisher

University of North Texas

Year dc:date

2001

Author dc:creator

• Williamson, Phillip C.

Contributors dc:contributor

• Benjamin, Robert C.
• O'Donovan, Gerard A.
• Shanley, Mark S.
• Kunz, Daniel A.
• McIntire, Sarah A.

• Genetic engineering.
• Plasmids -- Genetics.
• Mutagenesis.
• natural transformation
• gene manipulation
• Acintobactor calcoaceticas
• plasmid
• genetic engineering

Statement dc:rights

• Public
• Copyright
• Williamson, Phillip C.
• Copyright is held by the author, unless otherwise noted. All rights reserved.

Language dc:language

English

Identifier

oclc: 51106421
https://digital.library.unt.edu/ark:/67531/metadc2828/
ark: ark:/67531/metadc2828

OAI identifier oai:identifier

info:ark/67531/metadc2828