title: Characterization of the novel Fusobacterium nucleatum plasmid pKH9 and evidence of an addiction system.
abstract: 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.
authors.affiliation: Institute of Dental Sciences, Hadassah School of Dental Medicine, Hebrew University, Jerusalem, Israel. bgilad@md2.huji.ac.il
authors.forename: Gilad
authors.initials: G
authors.lastname: Bachrach
authors.forename: Susan Kinder
authors.initials: SK
authors.lastname: Haake
authors.forename: Alon
authors.initials: A
authors.lastname: Glick
authors.forename: Ronen
authors.initials: R
authors.lastname: Hazan
authors.forename: Ronit
authors.initials: R
authors.lastname: Naor
authors.forename: Roxanna N
authors.initials: RN
authors.lastname: Andersen
authors.forename: Paul E
authors.initials: PE
authors.lastname: Kolenbrander
mesh_terms: D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors* / Q000235:genetics; D001483:Base Sequence; D004261:DNA Replication; D029968:Escherichia coli Proteins; D016967:Fusobacterium nucleatum / Q000235:genetics*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010957:Plasmids; D017422:Sequence Analysis, DNA
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D029968:Escherichia coli Proteins; C096452:FtsK protein, E coli; D008565:Membrane Proteins