The relaxase of the Rhizobium etli symbiotic plasmid shows nic site cis-acting preference.
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.
Pérez-Mendoza
Daniel
D
Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Cientificas, CSIC, Profesor Albareda 1, 18008 Granada, Spain.
Lucas
María
M
Muñoz
Socorro
S
Herrera-Cervera
José A
JA
Olivares
José
J
de la Cruz
Fernando
F
Sanjuán
Juan
J
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016000:Cluster Analysis; D003227:Conjugation, Genetic; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D022761:Gene Transfer, Horizontal; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D010802:Phylogeny; D010957:Plasmids; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D042163:Rhizobium etli / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D009841:Oligonucleotides; D004265:DNA Helicases