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Applied and Environmental Microbiology, December 2004, p. 6957-6962, Vol. 70, No. 12
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.12.6957-6962.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Characterization of the Novel Fusobacterium nucleatum Plasmid pKH9 and Evidence of an Addiction System

Institute of Dental Sciences, Hadassah School of Dental Medicine, Hebrew University, Jerusalem, Israel,¹ Sections of Periodontics and Oral Biology and Medicine, Dental Research Institute, UCLA School of Dentistry, Los Angeles, California,² Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland³

Received 31 March 2004/ Accepted 14 July 2004

Fusobacterium nucleatum is an important oral anaerobic pathogen involved in periodontal and systemic infections. Studies of the molecular mechanisms involved in fusobacterial virulence and adhesion have been limited by lack of systems for efficient genetic manipulation. Plasmids were isolated from eight strains of F. nucleatum. The smallest plasmid, pKH9 (4,975 bp), was characterized and used to create new vectors for fusobacterial genetic manipulation. DNA sequence analysis of pKH9 revealed an open reading frame (ORF) encoding a putative autonomous rolling circle replication protein (Rep), an ORF predicted to encode a protein homologous to members of the FtsK/SpoIIIE cell division-DNA segregation protein family, and an operon encoding a putative toxin-antitoxin plasmid addiction system (txf-axf). Deletion analysis localized the pKH9 replication region in a 0.96-kbp fragment. The pKH9 rep gene is not present in this fragment, suggesting that pKH9 can replicate in fusobacteria independently of the Rep protein. A pKH9-based, compact Escherichia coli-F. nucleatum shuttle plasmid was constructed and found to be compatible with a previously described pFN1-based fusobacterial shuttle plasmid. Deletion of the pKH9 putative addiction system (txf-axf) reduced plasmid stability in fusobacteria, indicating its addiction properties and suggesting it to be the first plasmid addiction system described for fusobacteria. pKH9, its genetic elements, and its shuttle plasmid derivatives can serve as useful tools for investigating fusobacterial properties important in biofilm ecology and pathogenesis.