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Journal of Bacteriology, August 2004, p. 5003-5016, Vol. 186, No. 15
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.15.5003-5016.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Replication of Enterococcus faecalis Pheromone-Responding Plasmid pAD1: Location of the Minimal Replicon and oriV Site and RepA Involvement in Initiation of Replication

Maria Victoria Francia,1,2 Shuhei Fujimoto,1,{dagger} Patricia Tille,3 Keith E. Weaver,3 and Don B. Clewell1,4*

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

Received 14 April 2004/ Accepted 23 April 2004

The hemolysin-determining plasmid pAD1 is a member of a widely disseminated family of highly conjugative elements commonly present in clinical isolates of Enterococcus faecalis. The determinants repA, repB, and repC, as well as adjacent iteron sequences, are believed to play important roles in pAD1 replication and maintenance. The repA gene encodes an initiator protein, whereas repB and repC encode proteins related to stability and copy number. The present study focuses specifically on repA and identifies a replication origin (oriV) within a central region of the repA determinant. A small segment of repA carrying oriV was able to support replication in cis of a plasmid vector otherwise unable to replicate, if an intact RepA was supplied in trans. We demonstrate that under conditions in which RepA is expressed from an artificial promoter, a segment of DNA carrying only repA is sufficient for stable replication in E. faecalis. We also show that RepA binds specifically to oriV DNA at several sites containing inverted repeat sequences (i.e., IR-1) and nonspecifically to single-stranded DNA, and related genetic analyses confirm that these sequences play an important role in replication. Finally, we reveal a relationship between the internal structure of RepA and its ability to recognize oriV. An in-frame deletion within repA resulting in loss of 105 nucleotides, including at least part of oriV, did not eliminate the ability of the altered RepA protein to initiate replication using an intact origin provided in trans. The relationship of RepA to other known initiator proteins is also discussed.

* Corresponding author. Mailing address: Biologic and Materials Sciences, School of Dentistry, University of Michigan, Ann Arbor, MI 48109-1078. Phone: (734) 763-0117. Fax: (734) 763-9905. E-mail: dclewell{at}umich.edu.

{dagger} Present address: Department of Microbiology, Gunma University School of Medicine, Maebashi, Gunma, Japan.

Journal of Bacteriology, August 2004, p. 5003-5016, Vol. 186, No. 15
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.15.5003-5016.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Kwong, S. M., Lim, R., LeBard, R. J., Skurray, R. A., Firth, N. (2008). Analysis of the pSK1 replicon, a prototype from the staphylococcal multiresistance plasmid family. Microbiology 154: 3084-3094 [Abstract] [Full Text]  
  • Francia, M. V., Weaver, K. E., Goicoechea, P., Tille, P., Clewell, D. B. (2007). Characterization of an Active Partition System for the Enterococcus faecalis Pheromone-Responding Plasmid pAD1. J. Bacteriol. 189: 8546-8555 [Abstract] [Full Text]  
  • Tanaka, T., Ishida, H., Maehara, T. (2005). Characterization of the Replication Region of Plasmid pLS32 from the Natto Strain of Bacillus subtilis. J. Bacteriol. 187: 4315-4326 [Abstract] [Full Text]  


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