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Journal of Bacteriology, October 2006, p. 6793-6801, Vol. 188, No. 19
0021-9193/06/$08.00+0     doi:10.1128/JB.00869-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Molecular and Population Analyses of a Recombination Event in the Catabolic Plasmid pJP4

Juanita Larraín-Linton, Rodrigo De la Iglesia, Francisco Melo, and Bernardo González*

Departamento de Genética Molecular y Microbiología, Millennium Nucleus on Microbial Ecology and Environmental Microbiology and Biotechnology, Center for Advanced Studies on Ecology and Biodiversity, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile

Received 16 June 2006/ Accepted 24 July 2006

Cupriavidus necator JMP134(pJP4) harbors a catabolic plasmid, pJP4, which confers the ability to grow on chloroaromatic compounds. Repeated growth on 3-chlorobenzoate (3-CB) results in selection of a recombinant strain, which degrades 3-CB better but no longer grows on 2,4-dichlorophenoxyacetate (2,4-D). We have previously proposed that this phenotype is due to a double homologous recombination event between inverted repeats of the multicopies of this plasmid within the cell. One recombinant form of this plasmid (pJP4-F3) explains this phenotype, since it harbors two copies of the chlorocatechol degradation tfd gene clusters, which are essential to grow on 3-CB, but has lost the tfdA gene, encoding the first step in degradation of 2,4-D. The other recombinant plasmid (pJP4-FM) should harbor two copies of the tfdA gene but no copies of the tfd gene clusters. A molecular analysis using a multiplex PCR approach to distinguish the wild-type plasmid pJP4 from its two recombinant forms, was carried out. Expected PCR products confirming this recombination model were found and sequenced. Few recombinant plasmid forms in cultures grown in several carbon sources were detected. Kinetic studies indicated that cells containing the recombinant plasmid pJP4-FM were not selectable by sole carbon source growth pressure, whereas those cells harboring recombinant plasmid pJP4-F3 were selected upon growth on 3-CB. After 12 days of repeated growth on 3-CB, the complete plasmid population in C. necator JMP134 apparently corresponds to this form. However, wild-type plasmid forms could be recovered after growing this culture on 2,4-D, indicating that different plasmid forms can be found in C. necator JMP134 at the population level.

* Corresponding author. Mailing address: Laboratorio de Microbiología, Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Casilla 114-D, Santiago, Chile. Phone: 56-2-6862845. Fax: 56-2-6862185. E-mail: bgonzalez{at}bio.puc.cl.

Journal of Bacteriology, October 2006, p. 6793-6801, Vol. 188, No. 19
0021-9193/06/$08.00+0     doi:10.1128/JB.00869-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.





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