Mobilization and prevalence of a Fusobacterial plasmid

Abstract

Fusobacterium nucleatum is a Gram-negative anaerobic rod found in dental plaque biofilms, and is an opportunistic pathogen implicated in periodontitis as well as a wide range of systemic abscesses and infections. Genomic analyses of F. nucleatum indicate considerable genetic diversity and a prominent role for horizontal gene transfer in the evolution of the species. Several plasmids isolated from F. nucleatum, including pFN1, harbor relaxase gene homologs that may function in plasmid mobilization. In this investigation we examined the RP4-mediated mobilization properties of pFN1 and the prevalence of pFN1-related sequences in a panel of F. nucleatum clinical isolates. The fusobacterial plasmid pFN1 was mobilized by RP4 at a high frequency. Deletion analyses were used to delineate the core mobilon of pFN1, which consisted of the relaxase gene (rlx), an upstream open reading frame ORF4 and a region of DNA upstream of ORF4 with potential nic sites. To examine the prevalence of pFN1 in a panel of clinical isolates, total DNA isolated from the strains was hybridized with pFN1 replication (repA) and rlx gene probes. DNA from strains harboring plasmids known to be homologous to pFN1 hybridized with both the repA and rlx probes. Five additional strains were rlx-positive but repA-negative, indicating a greater prevalence of rlx-related genes in comparison with repA-related genes. Plasmid or plasmid-related sequences were identified in 11.5% of the strains examined. These findings demonstrate mobilization properties of a fusobacterial plasmid that may be important in horizontal gene transfer.

Figure 1. Schematic Illustration of pFN1 Components Associated with Plasmid Mobilization

The diagram illustrates the portions of pFN1 cloned into the backbone vector pTL1 for each of the plasmid constructs evaluated, and indicates the DNA fragments used for the repA and rlx probes in hybridization studies (see Figure 3). MOB, mobilization above background levels by the E. coli S17-1 host strain possessing the chromosomally-integrated RP4 plasmid with transfer frequencies as follows: ++: > 10⁻², +: > 10⁻⁶ but less than 10⁻²; − < 10⁻⁶. The backbone vector pTL1 was either not mobilized or mobilized with a transfer frequency < 10⁻⁶. This level is used to define the background level in interpreting the contribution of pFN1 to mobilization properties. See Materials and Methods and Table 2 for additional details on plasmid construction and transfer frequency, respectively.

Figure 2. Comparison of pFN1 Mobilon with Staphylococcal Plasmid Mobilons

(A) Alignment of pFN1 genes and elements predicted to be involved in plasmid mobilization with those of the mobilizable S. aureus plasmid pC221 and pC223. Elements indicated include nic sites (▾) and potential pFN1 nic sites 1 and 2; c, MobC motif; c_p, putative pFN1 MobC motif; relaxase motifs, III, z, IIa and II. (B) Alignment of MobC protein motif of pC221 and pC223 (Varsaki et al., 2009) with predicted MobC motif of pFN1 ORF2. Colon (:) indicates identity with consensus; period (.) indicates conservative substitution. (C) Alignment of potential pFN1 nic sites with previously defined consensus sequences for MOBp family nic sites. The RP4 consensus sequence, representative of a Gram-negative nic site, indicates conserved nucleotides in capital letters and semi-conserved nucleotides in lower case letters (Lawley et al., 2004). The Gram-positive consensus is based on an analysis of staphylococcal plasmids (Smith and Thomas, 2004) and for pC221 and pC223 the nic site corresponding to that of the consensus has been demonstrated. The positions of confirmed nic sites (▾) are indicated. Colon (:) indicates identity with consensus.

Figure 3. Dot Blot Hybridization of pFN1 repA and rlx probes to total DNA from isolates of F. nucleatum

Total cellular DNA (1 µg) from clinical isolates of F. nucleatum were spotted on nylon membranes and hybridized to DIG-labelled pFN1 repA (panel A) or pFN1 rlx probes. Strain designations indicated on the figure are as follows: numbers only indicate RMA strains, W indicates WAL strains. The clinical isolate WAL12230, known to harbor pFN1, was included as a positive control. The laboratory strain ATCC 10953 (A10953), known to harbor pFN3, was included as a negative control based on prior hybridization studies (Haake et al., 2000). The repA and rlx probes hybridized to DNA from the positive control strain as well as strains known to harbor plasmids pFN2 (WAL10113), pFN4 (RMA11532) and pFN5 (RMA8409). The rlx probe hybridized to DNA from 5 additional strains from which plasmids have not been identified, including RMA8560, RMA8561, RMA10352, RMA10686 and RMA11695.