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Journal of Bacteriology, December 2007, p. 8546-8555, Vol. 189, No. 23
0021-9193/07/$08.00+0     doi:10.1128/JB.00719-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Characterization of an Active Partition System for the Enterococcus faecalis Pheromone-Responding Plasmid pAD1{triangledown}

Maria Victoria Francia,1,2* Keith E. Weaver,3 Patricia Goicoechea,1 Patricia Tille,3,{dagger} and Don B. Clewell2,4

Servicio de Microbiología, Hospital Universitario Marqués de Valdecilla, Avda. de Valdecilla s/n, 39008 Santander, Cantabria, Spain,1 Department of Biologic and Materials Sciences, School of Dentistry,2 Department of Microbiology and Immunology, School of Medicine, University of Michigan, Ann Arbor, Michigan 48109,4 Department of Microbiology, University of South Dakota School of Medicine, Vermillion, South Dakota 570693

Received 7 May 2007/ Accepted 13 September 2007

Enterococcus faecalis plasmid pAD1 is a 60-kb conjugative, low-copy-number plasmid that encodes a mating response to the peptide sex pheromone cAD1 and a cytolytic exotoxin that contributes to virulence. Although aspects of conjugation have been studied extensively, relatively little is known about the control of pAD1 maintenance. Previous work on pAD1 identified a 5-kb region of DNA sufficient to support replication, copy control, and stable inheritance (K. E. Weaver, D. B. Clewell, and F. An, J. Bacteriol. 175:1900-1909, 1993), and recently, the pAD1 replication initiator (RepA) and the origin of vegetative replication (oriV) were characterized (M. V. Francia, S. Fujimoto, P. Tille, K. E. Weaver, and D. B. Clewell, J. Bacteriol. 186:5003-5016, 2004). The present study focuses on the adjacent determinants repB and repC, as well as a group of 25 8-bp direct repeats (iterons with the consensus sequence TAGTARRR) located between the divergently transcribed repA and repB. Through mutagenesis and trans-complementation experiments, RepB (a 33-kDa protein, a member of the ParA superfamily of ATPases) and RepC (a protein of 14.4 kDa) were shown to be required for maximal stabilization. Both were active in trans. The iteron region was shown to act as the pAD1 centromere-like site. Purified RepC was shown by DNA mobility shift and DNase I footprinting analyses to interact in a sequence-specific manner with the iteron repeats upstream of the repBC locus. The binding of RepC to the iteron region was shown to be modified by RepB in the presence of ATP via a possible interaction with the RepC-iteron complex. RepB did not bind to the iteron region in the absence of RepC.

* Corresponding author. Mailing address: Servicio de Microbiología, Hospital Universitario Marqués de Valdecilla, Avda. de Valdecilla s/n, 39008 Santander, Cantabria, Spain. Phone: 34-942-203357. Fax: 34-942-203462. E-mail: mvfrancia{at}humv.es

{triangledown} Published ahead of print on 28 September 2007.

{dagger} Present address: Department of Biology, University of Sioux Falls, Sioux Falls, SD 57105.

Journal of Bacteriology, December 2007, p. 8546-8555, Vol. 189, No. 23
0021-9193/07/$08.00+0     doi:10.1128/JB.00719-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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