Multilocus sequence analysis of Treponema denticola strains of diverse origin

Abstract

Background: The oral spirochete bacterium Treponema denticola is associated with both the incidence and severity of periodontal disease. Although the biological or phenotypic properties of a significant number of T. denticola isolates have been reported in the literature, their genetic diversity or phylogeny has never been systematically investigated. Here, we describe a multilocus sequence analysis (MLSA) of 20 of the most highly studied reference strains and clinical isolates of T. denticola; which were originally isolated from subgingival plaque samples taken from subjects from China, Japan, the Netherlands, Canada and the USA.

Results: The sequences of the 16S ribosomal RNA gene, and 7 conserved protein-encoding genes (flaA, recA, pyrH, ppnK, dnaN, era and radC) were successfully determined for each strain. Sequence data was analyzed using a variety of bioinformatic and phylogenetic software tools. We found no evidence of positive selection or DNA recombination within the protein-encoding genes, where levels of intraspecific sequence polymorphism varied from 18.8% (flaA) to 8.9% (dnaN). Phylogenetic analysis of the concatenated protein-encoding gene sequence data (ca. 6,513 nucleotides for each strain) using Bayesian and maximum likelihood approaches indicated that the T. denticola strains were monophyletic, and formed 6 well-defined clades. All analyzed T. denticola strains appeared to have a genetic origin distinct from that of 'Treponema vincentii' or Treponema pallidum. No specific geographical relationships could be established; but several strains isolated from different continents appear to be closely related at the genetic level.

Conclusions: Our analyses indicate that previous biological and biophysical investigations have predominantly focused on a subset of T. denticola strains with a relatively narrow range of genetic diversity. Our methodology and results establish a genetic framework for the discrimination and phylogenetic analysis of T. denticola isolates, which will greatly assist future biological and epidemiological investigations involving this putative 'periodontopathogen'.

Figure 1

Bayesian phylogenetic trees of Treponema denticola strains based on individual 16S rRNA, flaA, recA, pyrH, ppnK, dnaN, era and radC gene datasets. The Bayesian 50% majority-rule consensus tree of 9,000 trees, following the removal of 1,000 trees as burn-in, is shown for each gene. Numbers above branches are posterior probabilities. Corresponding gene homologoues from Treponema vincentii LA-1 (ATCC 33580) and Treponema pallidum subsp. pallidum SS14 were included in the phylogenetic analysis as outgroups. The radC gene is absent from the T. pallidum genome.

Figure 2

Taxonomic resolution based on the ranges of intraspecific sequence similarity (%) for the individual 16S rRNA, flaA, recA, pyrH, ppnK, dnaN, era and radC genes, within the 20 Treponema denticola strains analyzed. The y-axis indicates the levels of nucleotide identity (%) shared between the eight individual gene sequences analyzed from each strain, with the range represented as a bar.

Figure 3

Phylogenetic trees of Treponema denticola strains based on a concatenated 7-gene dataset (flaA, recA, pyrH, ppnK, dnaN, era and radC), using Maximum Likelihood and Bayesian methods.A: Maximum likelihood (ML) tree generated under the GTR + I + G substitution model, with bootstrap values shown above branches. The scale bar represents 0.015 nucleotide changes per site. Numbers beneath the breakpoints in the branches indicate the respective nucleotide changes per site that have been removed. B: Ultrametric Bayesian (BA) 50% majority-rule consensus tree of 9,000 trees following the removal of 1,000 trees as burn-in. Numbers above branches are posterior probabilities. The respective clades formed in each tree are indicated with a Roman numeral (I-VI). Corresponding gene homologoues from Treponema vincentii LA-1 (ATCC 33580) and Treponema pallidum subsp. pallidum SS14 were included in the phylogenetic analysis as outgroups.