Conjugal transfer of chromosomal DNA contributes to genetic variation in the oral pathogen Porphyromonas gingivalis

Abstract

Porphyromonas gingivalis is a major oral pathogen that contributes to the development of periodontal disease. There is a significant degree of genetic variation among strains of P. gingivalis, and the population structure has been predicted to be panmictic, indicating that horizontal DNA transfer and recombination between strains are likely. The molecular events underlying this genetic exchange are not understood, although a putative type IV secretion system is present in the genome sequence of strain W83, implying that DNA conjugation may be responsible for genetic transfer in these bacteria. In this study, we provide in vitro evidence for the horizontal transfer of DNA using plasmid- and chromosome-based assays. In the plasmid assays, Bacteroides-derived shuttle vectors were tested for transfer from P. gingivalis strains into Escherichia coli. Of the eight strains tested, five were able to transfer DNA into E. coli by a mechanism most consistent with conjugation. Additionally, strains W83 and 33277 tested positive for the transfer of chromosomally integrated antibiotic resistance markers. Ten chimeras resulting from the chromosomal transfer assay were further analyzed by Southern hybridization and were shown to have exchanged DNA fragments of between 1.1 and 5.6 kb, but the overall strain identity remained intact. Chimeras showed phenotypic changes in the ability to accrete into biofilms, implying that DNA transfer events are sufficient to generate measurable changes in complex behaviors. This ability to transfer chromosomal DNA between strains may be an adaptation mechanism in the complex environment of the host oral cavity.

FIG. 1.

(A) A putative Porphyromonas DNA transfer region. The genome region from P. gingivalis strain W83 that is similar to the Bacteroides DNA transfer genes from conjugative transposons is shown. PCR regions used as probes for Southern blots are indicated by thick horizontal bars. (B) Southern blots of P. gingivalis strains for tra genes. Results are shown for strains W83 (lanes 1), W50 (lanes 2), ATCC 49417 (lanes 3), 5083 (lanes 4), ATCC 33277 (lanes 5), 381 (lanes 6), A1A7-28 (lanes 7), and MP4-504 (lanes 8). Band sizes for strain W83 are indicated to the left of the blots.

FIG. 2.

Southern blot analysis of chimera strains. All blots are BamHI digests of chimera chromosomal DNA. (A) ISPG4 probe of chimeras to identify 33277- or W83-derived strains. (B) ISPG1 probe of chimera DNA. The arrow indicates the additional ISPG1 band acquired by the W83-derived chimera. (C) Location of tetQ and ermF markers in chimera genomes. The arrow indicates the ermF band in chimera 9, which shows a unique restriction profile compared to the donating parent, W83Em. (D) A PG0653 probe of the same blot as that in panel C. The PG0653 band in chimera 9 (indicated with an arrow) has the same BamHI restriction sites as the 33277Tc strain, with an additional 1.1 kb due to the presence of the ermF cassette from the donating parent, W83Em. (E) Homologous DNA recombination events that result in chimeras 2, 4, and 9. These chimeras result from the W83Em donation of ermF to 33277Tc. Bold lines represent the donated DNA, and thin lines represent the chromosomal DNA in the recipient. “B” indicates BamHI restriction sites. The bold X represents putative regions of homologous recombination.

FIG. 3.

Quantitative and qualitative biofilm assays. (A) Quantitative analysis of biofilm accretion by P. gingivalis strains and chimeras. Chimeric strains 2, 4, and 9 (dark bars) were compared to strain 33277TcEm for statistical analysis by t test. All other chimeras (light bars) were compared to W83TcEm. Results represent three independent experiments, each done in triplicate (n = 9). Symbols * and # represent P values of <0.05. (B) Fluorescent microscopy images of 24-h biofilm formation by nonchimeric strain 33277TcEm and chimera 4. Magnification, ×40.