RepA and RepB exert plasmid incompatibility repressing the transcription of the repABC operon Ángeles Pérez-Oseguera, Miguel A. Cevallos ⇑ Programa de Genómica evolutiva, Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Apartado Postal 565-A, Cuernavaca, Morelos, Mexico article info Article history: Received 7 May 2013 Accepted 16 August 2013 Available online 7 September 2013 Communicated by Dr. Dhruba K. Chattoraj Keywords: Symbiotic plasmid Rhizobium RepA RepB parS abstract Rhizobium etli CFN42 has a multipartite genome composed of one chromosome and six large plasmids with low copy numbers, all belonging to the repABC plasmid family. All ele- ments essential for replication and segregation of these plasmids are encoded within the repABC operon. RepA and RepB direct plasmid segregation and are involved in the tran- scriptional regulation of the operon, and RepC is the initiator protein of the plasmid. Here we show that in addition to RepA (repressor) and RepB (corepressor), full transcriptional repression of the operon located in the symbiotic plasmid (pRetCFN42d) of this strain requires parS, the centromere-like sequence, and the operator sequence. However, the co-expression of RepA and RepB is sufficient to induce the displacement of the parental plasmid. RepA is a Walker-type ATPase that self associates in vivo and in vitro and binds specifi- cally to the operator region in its RepA-ADP form. In contrast, RepA-ATP is capable of bind- ing to non-specific DNA. RepA and RepB form high molecular weight DNA–protein complexes in the presence of ATP and ADP. RepA carrying ATP-pocket motif mutations induce full repression of the repABC operon without the participation of RepB and parS. These mutants specifically bind the operator sequence in their ATP or ADP bound forms. In addition, their expression in trans exerts plasmid incompatibility against the parental plasmid. RepA and RepB expressed in trans induce plasmid incompatibility because of their ability to repress the repABC operon and not only by their capacity to distort the plasmid segregation process. Ó 2013 Elsevier Inc. All rights reserved. 1. Introduction ‘‘Plasmid incompatibility, as defined by Richard Novick and coworkers (1976), is ‘‘the inability of two different plasmids to coexist stably in the same host cell in the ab- sence of continued selection pressure’’. Plasmid incompat- ibility is a consequence of functional interference between the replication systems and/or segregation machinery of the involved plasmids (Austin and Nordström, 1990; Ebersbach et al., 2005; Bouet et al., 2007). Incompatible plasmids frequently share genes and sites involved in replication or segregation, indicating that these plasmids share a common evolutionary history. Plasmid incompati- bility is a limiting factor in the acquisition of new plasmids and plays a central role in shaping bacterial evolution. Alphaproteobacteria frequently possess multipartite genomes composed of one chromosome and several large plasmids present in low copy numbers. This genome architecture is especially common within the Rhizobiales, Rhodospirillales and Rhodobacterales bacterial orders (Cevallos et al., 2008; Petersen et al., 2009). Genomic anal- yses of members of these bacterial orders indicate that these large plasmids usually belong to the repABC plasmid family, even when they come from the same strain, indicat- ing that this plasmid family encompasses several incom- patibility groups. 0147-619X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.plasmid.2013.08.001 ⇑ Corresponding author. Fax: +52 7773 17 55 81. E-mail address: mac@ccg.unam.mx (M.A. Cevallos). Plasmid 70 (2013) 362–376 Contents lists available at ScienceDirect Plasmid journal homepage: www.elsevier.com/locate/yplas