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.
The tensile bond strength of Epoxylite 9075 and Enamelite to enamel treated with NaF and SnF2, at two concentrations, in the etching acid (H3PO4) was determined. The average fracture stress of those samples in which fracture occurred in the resin only, may be considered as the tensile strength of the resin, 69.8 +/- 5.6 kg/cm2. The mean fracture stresses of the Epoxy 9075 systems etched with pure phosphoric acid or with acid solutions containing 0.5% F salts were significantly higher than those treated with acid solutions containing 2% F salts. Incorporation of NaF or SnF2 in the etching solutions highly increased the fluoride concentration of enamel. The fluoride increase dependend on the fluoride concentration of the etching solution and was greater for NaF than SnF2.
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