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.

FIG. 1.

Genetic elements of the F. nucleatum pKH9 plasmid. (A) pKH9 map. ORFs are indicated by gray arrows. R1, R2, and R3 are the repeated sequences flanking the unstable region (D). The location of the pKH9 Rep-independent origin of replication (oriI) is indicated by a black rectangle. (B) Three RCR motifs found in the pKH9 Rep protein in comparison to the consensus sequences of the φX174 RCR family. U denotes hydrophobic residues (I, L, V, M, F, Y, and W). The conserved Tyr residue is underlined. (C) Conserved amino acid sequence of the pKH9 FtsK/SpoIIIE protein. The lysine essential for the ATP-dependent DNA pump function is underlined. (D) GTAG-rich repeat sequences flanking pKH9 ORF 3, which is unstable in E. coli. Bold and capital letters in panels B, C, and D denote conserved amino acids and nucleotides.

FIG. 2.

pKH9 axf-txf toxin-antitoxin system. (A) Stability of a pKH9 derivative containing axf-txf (pKH90) (triangles) versus that of a derivative lacking axf-txf (pORI9) (squares) in F. nucleatum ATCC 23726. Open symbols represent results of the first experiment, and filled symbols represent those of a second independent experiment. (B) Region of overlapping sequence between the axf and txf genes.

FIG. 3.

Deletion analysis for the identification of the pKH9 minimal replication region. Hatched boxes represent pKH9 fragments present in each construct. pORI91 is a derivative of pORI9 lacking the ampicillin resistance gene. The EcoRI site in pORI92 was created during cloning.

FIG. 4.

Compatibility of pHS30 and pORI91. F. nucleatum ATCC 23726 harboring pHS30 was transformed with pORI91. Transformants were selected on media with clindamycin. Plasmid DNA isolated from representative transformants (T-1 and T-2) was examined by restriction enzyme digestion and compared with similarly digested pHS30 and pORI91 isolated from E. coli. DNA in lanes 1 was digested with KpnI to linearize pORI91. DNA in lanes 2 was digested with EcoRV to linearize pHS30. The open circular (OC), linear (L), and covalently closed circular (CCC) forms of pHS30 and pORI91 are indicated on the left and right, respectively.