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The tra Region of the Conjugative Plasmid pIP501 Is Organized in an Operon with the First Gene Encoding the Relaxase

Brigitta Kurenbach,¹ Dagmar Grothe,¹ María Eugenia Farías,^2, Ulrich Szewzyk,¹ and Elisabeth Grohmann^1*

Fachgebiet Ökologie der Mikroorganismen, Institut für Technischen Umweltschutz, Technische Universität Berlin, D-10587 Berlin, Germany,¹ Centro de Investigaciones Biológicas, CSIC, E-28006 Madrid, Spain²

Received 16 August 2001/ Accepted 10 December 2001

The tra genes orf1 to orf11 of pIP501 were shown to be transcribed as a single operon of 11.3 kb in Enterococcus faecalis by reverse transcription-PCR. The transcriptional start site of the tra mRNA was mapped at 110 bp upstream from the predicted TTG start codon of the first gene of the operon, the traA relaxase. The TraA protein (660 amino acids) and a C-terminally truncated version of the TraA protein (293 amino acids) were purified as fusions with glutathione S-transferase. oriT cleavage activity of both TraA proteins was demonstrated in vitro on supercoiled plasmid pVA2241 DNA containing oriT_pIP501. The activity of the DNA relaxase TraA is strictly dependent on the presence of Mg²⁺ or Mn²⁺ and is highest at temperatures of between 42 and 45°C.

Present address: PROIMI-Universidad Nacional de Tucumán, Tucumán, Argentina.

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