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The Relaxase of the Rhizobium etli Symbiotic Plasmid Shows nic Site cis-Acting Preference

Daniel Pérez-Mendoza,^1,2 María Lucas,² Socorro Muñoz,¹ José A. Herrera-Cervera,^1, José Olivares, Fernando de la Cruz,² and Juan Sanjuán^1*

Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas (CSIC), Granada, Spain,¹ Departamento de Biología Molecular (Unidad asociada al CIB, CSIC), Universidad de Cantabria, C/Herrera Oria s/n, 39011 Santander, Spain²

Received 17 May 2006/ Accepted 7 August 2006

Genetic and biochemical characterization of TraA, the relaxase of symbiotic plasmid pRetCFN42d from Rhizobium etli, is described. After purifying the relaxase domain (N265TraA), we demonstrated nic binding and cleavage activity in vitro and thus characterized for the first time the nick site (nic) of a plasmid in the family Rhizobiaceae. We studied the range of N265TraA relaxase specificity in vitro by testing different oligonucleotides in binding and nicking assays. In addition, the ability of pRetCFN42d to mobilize different Rhizobiaceae plasmid origins of transfer (oriT) was examined. Data obtained with these approaches allowed us to establish functional and phylogenetic relationships between different plasmids of this family. Our results suggest novel characteristics of the R. etli pSym relaxase for previously described conjugative systems, with emphasis on the oriT cis-acting preference of this enzyme and its possible biological relevance.

Present address: Dpto. Fisiología Vegetal, Facultad de Ciencias, Universidad de Granada, Campus Fuentenueva s/n, 18071 Granada, Spain.

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