Native plasmids of Fusobacterium nucleatum: characterization and use in development of genetic systems

Abstract

Three native plasmids of Fusobacterium nucleatum were characterized, including DNA sequence analysis of one plasmid, pFN1. A shuttle plasmid, pHS17, capable of transforming Escherichia coli and F. nucleatum ATCC 10953 was constructed with pFN1. pHS17 was stably maintained in the F. nucleatum transformants, and differences in the transformation efficiencies suggested the presence of a restriction-modification system in F. nucleatum.

FIG. 1

Restriction maps and Southern blots of the F. nucleatum plasmids. (A) Partial restriction map of F. nucleatum plasmids pFN1, pFN2 and pFN3 and shuttle plasmid pHS17. Selected restriction endonuclease sites in the native plasmids are presented. Restriction endonuclease sites indicated for pHS17 relate to the plasmid construction. The pFN1 portion of pHS17 is indicated by the thick solid bar, with the position of the repA homologue (ORF5) and putative ori indicated. (B and C) Plasmids from F. nucleatum strains 12230 (pFN1, lanes 1), 10113 (pFN2, lanes 2), and ATCC 10953 (pFN3, lanes 3) were probed with pFN1 DNA with EcoRI digests (B) or pFN3 DNA with EcoRV digests (C). The HincII-digested pFN1 and AseI-digested pFN3 probes were ³²P labeled (specific activity of 25 × 10⁸ and 4.5 × 10⁸ dpm/μg of DNA, respectively). The positions of molecular size markers are indicated on the left, and the linear forms of pFN1, pFN2, and pFN3 are indicated on the right.

FIG. 2

Physical characteristics of pFN1 based on DNA sequence analysis. (A) ORFs, the putative origin of replication (ori), and selected restriction endonuclease sites are indicated. (B) Structural elements of putative origin of replication found upstream of ORF5, the repA homologue. The putative origin contains an A-T-rich region (crosshatched bar), six perfect 22-bp direct repeats (▸) termed iterons, and several putative DnaA-binding sites (█).

FIG. 3

Plasmid DNA from F. nucleatum ATCC 10953 transformants consists of the shuttle plasmid, pHS17, and the native plasmid, pFN3. Plasmid preparations from E. coli (pHS17), F. nucleatum ATCC 10953 transformant strain KH21 (pHS17 and pFN3), and F. nucleatum ATCC 10953 (pFN3) were analyzed. The preparations were either not digested (lanes 1) or predigested with EcoRV (lanes 2) or EcoRI (lanes 3), separated on 0.8% agarose gels, stained with ethidium bromide, and visualized under UV illumination. The open circular (OC), linear (L), and covalently closed circular (CC) forms of pHS17 and pFN3 are indicated on the left and right, respectively.