Comparison of the genome of the oral pathogen Treponema denticola with other spirochete genomes.
ABSTRACT We present the complete 2,843,201-bp genome sequence of Treponema denticola (ATCC 35405) an oral spirochete associated with periodontal disease. Analysis of the T. denticola genome reveals factors mediating coaggregation, cell signaling, stress protection, and other competitive and cooperative measures, consistent with its pathogenic nature and lifestyle within the mixed-species environment of subgingival dental plaque. Comparisons with previously sequenced spirochete genomes revealed specific factors contributing to differences and similarities in spirochete physiology as well as pathogenic potential. The T. denticola genome is considerably larger in size than the genome of the related syphilis-causing spirochete Treponema pallidum. The differences in gene content appear to be attributable to a combination of three phenomena: genome reduction, lineage-specific expansions, and horizontal gene transfer. Genes lost due to reductive evolution appear to be largely involved in metabolism and transport, whereas some of the genes that have arisen due to lineage-specific expansions are implicated in various pathogenic interactions, and genes acquired via horizontal gene transfer are largely phage-related or of unknown function.
- SourceAvailable from: Jiannong Xu[Show abstract] [Hide abstract]
ABSTRACT: Metagenomics is a relatively new but fast growing field within environmental biology and medical sciences. It enables researchers to understand the diversity of microbes, their functions, cooperation, and evolution in a particular ecosystem. Traditional methods in genomics and microbiology are not efficient in capturing the structure of the microbial community in an environment. Nowadays, high-throughput next-generation sequencing technologies are powerfully driving the metagenomic studies. However, there is an urgent need to develop efficient statistical methods and computational algorithms to rapidly analyze the massive metagenomic short sequencing data and to accurately detect the features/functions present in the microbial community. Although several issues about functions of metagenomes at pathways or subsystems level have been investigated, there is a lack of studies focusing on functional analysis at a low level of a hierarchical functional tree, such as SEED subsystem tree.PLoS ONE 09/2014; 9(9):e106588. · 3.53 Impact Factor
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ABSTRACT: The oral spirochete bacterium Treponema putidum inhabits human periodontal niches. The complete genome sequence of the OMZ 758(T) (ATCC 700334(T)) strain of this species was determined, revealing a 2,796,913-bp chromosome, with a G+C content of 37.30% and a single plasmid (pTPu1; 3,649 bp) identical to pTS1 from Treponema denticola.Genome Announcements 09/2014; 2(5).
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ABSTRACT: Porphyromonas gingivalis and Treponema denticola are strongly associated with chronic periodontitis. These bacteria have been co-localized in subgingival plaque and demonstrated to exhibit symbiosis in growth in vitro and synergistic virulence upon co-infection in animal models of disease. Here we show that during continuous co-culture a P. gingivalis:T. denticola cell ratio of 6∶1 was maintained with a respective increase of 54% and 30% in cell numbers when compared with mono-culture. Co-culture caused significant changes in global gene expression in both species with altered expression of 184 T. denticola and 134 P. gingivalis genes. P. gingivalis genes encoding a predicted thiamine biosynthesis pathway were up-regulated whilst genes involved in fatty acid biosynthesis were down-regulated. T. denticola genes encoding virulence factors including dentilisin and glycine catabolic pathways were significantly up-regulated during co-culture. Metabolic labeling using 13C-glycine showed that T. denticola rapidly metabolized this amino acid resulting in the production of acetate and lactate. P. gingivalis may be an important source of free glycine for T. denticola as mono-cultures of P. gingivalis and T. denticola were found to produce and consume free glycine, respectively; free glycine production by P. gingivalis was stimulated by T. denticola conditioned medium and glycine supplementation of T. denticola medium increased final cell density 1.7-fold. Collectively these data show P. gingivalis and T. denticola respond metabolically to the presence of each other with T. denticola displaying responses that help explain enhanced virulence of co-infections.PLoS Pathogens 03/2014; 10(3):e1003955. · 8.06 Impact Factor
Comparison of the genome of the oral pathogen
Treponema denticola with other spirochete genomes
Rekha Seshadri*, Garry S. A. Myers*, Herve ´ Tettelin*, Jonathan A. Eisen*†, John F. Heidelberg*‡, Robert J. Dodson*,
Tanja M. Davidsen*, Robert T. DeBoy*, Derrick E. Fouts*, Dan H. Haft*, Jeremy Selengut*, Qinghu Ren*,
Lauren M. Brinkac*, Ramana Madupu*, Jamie Kolonay*, Scott A. Durkin*, Sean C. Daugherty*, Jyoti Shetty*,
Alla Shvartsbeyn*, Elizabeth Gebregeorgis*, Keita Geer*, Getahun Tsegaye*, Joel Malek*, Bola Ayodeji*,
Sofiya Shatsman*, Michael P. McLeod§, David Sˇmajs§, Jerrilyn K. Howell¶, Sangita Pal§, Anita Amin§, Pankaj Vashisth¶,
Thomas Z. McNeill§, Qin Xiang§, Erica Sodergren§, Ernesto Baca§, George M. Weinstock§, Steven J. Norris¶,
Claire M. Fraser*?, and Ian T. Paulsen*†**
*The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD 20850;†Johns Hopkins University, Charles and 34th Streets, Baltimore, MD
21218;?Departments of Pharmacology and Microbiology and Tropical Medicine, The George Washington University School of Medicine, 2300 Eye Street
Northwest, Washington, DC 20037;‡Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, MD 21202;¶Department of
Pathology and Laboratory Medicine and Graduate School of Biomedical Sciences, University of Texas Health Science Center, 6431 Fannin Street, Houston,
TX 77230; and§Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030
Edited by Robert Haselkorn, University of Chicago, Chicago, IL, and approved February 9, 2004 (received for review November 20, 2003)
We present the complete 2,843,201-bp genome sequence of Trepo-
nema denticola (ATCC 35405) an oral spirochete associated with
periodontal disease. Analysis of the T. denticola genome reveals
factors mediating coaggregation, cell signaling, stress protection,
and other competitive and cooperative measures, consistent with
its pathogenic nature and lifestyle within the mixed-species envi-
ronment of subgingival dental plaque. Comparisons with previ-
ously sequenced spirochete genomes revealed specific factors
contributing to differences and similarities in spirochete physiol-
ogy as well as pathogenic potential. The T. denticola genome is
considerably larger in size than the genome of the related syphilis-
causing spirochete Treponema pallidum. The differences in gene
content appear to be attributable to a combination of three
phenomena: genome reduction, lineage-specific expansions, and
horizontal gene transfer. Genes lost due to reductive evolution
appear to be largely involved in metabolism and transport,
whereas some of the genes that have arisen due to lineage-specific
expansions are implicated in various pathogenic interactions, and
genes acquired via horizontal gene transfer are largely phage-
related or of unknown function.
human periodontal disease (1). This polymicrobial infection and
inflammation of the gingiva occurs in 80% of the adult popu-
lation at some time in their lives and can evolve to severe forms
including refractory periodontitis and acute necrotizing gingivi-
tis, resulting in bone resorption and tooth loss. Treatment
regimens to combat periodontitis are difficult and costly involv-
ing extensive antibiotic treatment and intricate surgery. T.
denticola dwells in a complex and diverse microbial community
within the oral cavity, and as such is highly specialized to survive
within this milieu. This aerotolerant anaerobe (2) is related to
the syphilis-causing obligate human pathogen, Treponema pal-
lidum subsp. pallidum. T. denticola is one of ?60 treponemal
species or uncharacterized phylotypes found in dental plaque
(3). Spirochetes comprise a monophyletic phylum that exhibits
overall structural similarity and rRNA relatedness but great
variability in habitat, physiologic properties, and genome size
and organization (Table 1).
Comparative analysis with previously sequenced spirochetes
(T. pallidum, Borrelia burgdorferi, and Leptospira interrogans)
(4–6) yielded insights into the basis for differences in their
lifestyle and disease manifestations. The genome of T. denticola
clearly reflects its adaptations for colonization and survival
within the biofilm environment of subgingival dental plaque.
Compared to other spirochetes, T. denticola is relatively easy to
he Gram-negative oral spirochete Treponema denticola is
predominantly associated with the incidence and severity of
cultivate and manipulate genetically, making this an excellent
model for spirochete research.
Bacteria. T. denticola strain 35405 was initially isolated and
designated as the type strain by Chan et al. (7). Bacteria used in
this study were obtained from the American Type Culture
Sequencing and Gene Identification. The complete genome of T.
denticola strain 35405 was sequenced by using the random
shotgun method described for genomes sequenced by The
Institute for Genomic Research (5). ORFs likely to encode
proteins (CDSs) were predicted by GLIMMER (8). All predicted
proteins ?30 aa were analyzed for sequence similarity against a
nonredundant protein database. Two sets of hidden Markov
models were used to determine CDS membership in families and
superfamilies: PFAM V5.5 (9) and TIGRFAMS (10). Domain-based
paralogous families were built by performing all-versus-all
searches on the remaining protein sequences. The 5? regions of
each CDS were inspected to define initiation codons by using
homologies, position of ribosomal binding sites, and transcrip-
tional terminators. Sequences containing frameshifts and point
mutations were reexamined and corrected where appropriate.
Protein membrane-spanning domains were identified by
TOPPRED (11). Putative signal peptides were identified with
Trinucleotide Composition. Distribution of all 64 trinucleotides
(3-mers) for each chromosome was determined, and the 3-mer
distribution in 2,000-bp windows that overlapped by half their
length (1,000 bp) across the genome was computed. For each
window, we computed the ?2statistic on the difference between
its 3-mer content and that of the whole chromosome. A large
value for ?2indicates the 3-mer composition in this window is
different from the rest of the chromosome. Probability values for
this analysis are based on assumptions that the DNA composi-
tion is relatively uniform throughout the genome, and that 3-mer
composition is independent. Because these assumptions may be
This paper was submitted directly (Track II) to the PNAS office.
Abbreviation: CDS, protein-coding sequences.
Data deposition: The sequence reported in this paper has been deposited in the GenBank
database (accession no. AE017226).
**To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
© 2004 by The National Academy of Sciences of the USA
April 13, 2004 ?
vol. 101 ?
incorrect, we prefer to interpret high ?2values as indicators of
regions on the chromosome that appear unusual and demand
Comparative Genomics. The T. pallidum and T. denticola genomes
were compared at the nucleotide level by suffix tree analysis
using MUMMER, and their ORF sets were compared by using
BLAST. Additionally, all T. denticola CDSs were compared by
BLASTP against the complete set of CDSs from T. pallidum, L.
interrogans, and B. burgdorferi using an E value cutoff of 10?5.
Results and Discussion
General Genome Features. The 2,843,201-bp chromosome of T.
denticola (ATCC 35405) is predicted to encode 2,786 CDSs, of
which 26.5% (734 CDSs) are unique. Although the T. denticola
and T. pallidum genome sizes are significantly different, the
number of stable RNAs present in each is near identical (Table
1 and Fig. 1). The organization of the T. denticola chromosomal
origin of replication is disparate from the organization (dnaA-
dnaN-recF-gyrB) typical for bacteria, including other spirochetes
(Fig. 4, which is published as supporting information on the
PNAS web site). Base pair 1 was assigned in an intergenic region
between dnaA and dnaE based on previous analyses (13).
However the transition in GC skew (G ? C?G ? C) typically
a distally located recF, with an adjacent dnaN, and a gene
encoding an ATPase domain protein that may serve as a gyrase.
The relative disruption in the organization of replication genes
and the GC skew analysis suggests that there may have been a
lineage-specific genome rearrangement in the region of the
replication origin. Annotated genome sequence and analysis for
T. denticola is available at www.tigr.org?tigr-scripts?CMR2?
Comparative Genomics. It has been proposed that the 1.14 Mb T.
pallidum genome was derived from the genome of T. denticola or
other host-associated treponemes through deletion and diver-
gence (15). Comparisons reveal that the T. pallidum genome
shares limited nucleotide identity with the T. denticola genome.
About one-fourth of T. denticola genes (728 CDSs) have their
best matches to CDSs in the T. pallidum genome (representing
68% of its genome), and on average, these share only 53% amino
acid identity (70.6% similarity). Essentially no synteny (conser-
vation of gene order) exists between the T. denticola and T.
pallidum genomes [with the exception of highly conserved
operons encoding ribosomal (TDE0766-TDE0792) and flagellar
proteins (TDE1198-TDE1219)]. This result, as well as differ-
ences in G ? C content (Table 1) and rRNA sequences, indicates
that divergence of T. denticola and T. pallidum from a common
ancestor was an ancient event relative to the recent divergence
of many bacterial groups (for which complete genome sequences
are available), such as the Brucella and Rickettsia genera and
some members of the Enterobacteriaceae.
Comparisons of the total set of T. denticola CDSs with that of
the other sequenced spirochetes by using BLASTP reveals a core
set of 618 CDSs found conserved in the other three spirochete
genomes (Fig. 2 and Table 2, which is published as supporting
information on the PNAS web site). Of these, ?90% have an
assigned role, most of which are housekeeping functions (DNA
replication, repair, cell division, transcription, translation, en-
ergy metabolism, etc.); others include ATP-binding cassette
(ABC) transporters (?120 CDS), and flagellum and chemotaxis
genes (?55 CDS). A total of 1,268 T. denticola CDSs have no
matches in any of the other spirochete genomes. Over half of
these are hypothetical genes, whereas the remainder have their
best matches in various sequenced Gram-positive species like
Clostridium spp., Streptococcus spp., as well as Fusobacterium
nucleatum, a primary colonizer during dental plaque formation,
found in conjunction with T. denticola and Porphyromonas
gingivalis, and thought to be required for their colonization (16).
These include various membrane proteins, ABC transporters,
transcriptional regulators, and enzymes involved in amino acid
metabolism and glycogen synthesis.
A small subset of the 618 predicted gene products conserved
in all spirochetes did not have significant matches in other phyla;
these may represent spirochete-specific proteins. These include
characterized flagellar components, FliL (TDE2764), FliE
(TDE1214) (17), putative outer layer protein FlaA (TDE1408)
(18) and its distant paralogs TDE1712 and TDE1409, that
Table 1. General genome features
T. denticola T. pallidum*
G?C content, %
No. with assigned function
No. of unknown function‡
No. of conserved hypotheticals§
No. with no database match¶
Average CDS size, bp
37.952.8 28.6 36.0
*The distribution of CDSs in the T. pallidum and B. burgdorferi chromosomes are derived from the original
different with updated blast searches and annotation.
†The genome information for L. interrogans represents combined data from both chromosomes.
‡Unknown function, significant sequence similarity to a named protein for which no function is currently
§Conserved hypothetical protein, sequence similarity to a translation of another ORF, however no experimental
evidence for protein expression exists.
¶Hypothetical protein, no significant similarity to any other sequenced protein.
?Twenty-five of the total number of CDSs in T. denticola possess one or more authentic frameshifts, point
mutations, or are truncated.
Seshadri et al.
April 13, 2004 ?
vol. 101 ?
no. 15 ?
perhaps account for the unique periplasmic flagellar location
and motility properties of spirochetes. The other spirochete-
associated genes (TDE2598, TDE2751, TDE1300, and
TDE1334) have no predicted functions, and two have regions of
similarity to fibronectin (TDE0446), and tetratricopeptide re-
peat (TPR)-domain proteins (TDE2696). Overall, this group
may be responsible for spirochete-specific properties, including
morphology and host–pathogen interactions.
A total of 1,700 (?60%) T. denticola CDSs have no matches
in T. pallidum (Table 3, which is published as supporting
information on the PNAS web site), and 162 T. pallidum CDSs
have no matches in T. denticola (Table 4, which is published as
possible reasons for this disparity in genome size and encoded
CDSs, including (i) elimination of genes in one genome (reduc-
tive evolution), (ii) acquisition of genes via lateral transfer, and
(iii) lineage-specific expansions, all subsequent to the divergence
of the two species. Our comparisons reveal that all of these
factors contribute in varying degrees to the differences in gene
content (Fig. 2). About one-sixth of these 1,700 CDSs have their
best matches to other CDSs within T. denticola and may repre-
sent recent duplication events or expansions within the T.
denticola lineage (Table 5, which is published as supporting
information on the PNAS web site). Over half of these putative
duplicated genes are situated adjacent to each other, suggesting
that they arose via tandem duplication events. Many of these
may be implicated in virulence functions, for example, YD-
Of the 1,700 T. denticola CDSs that lack T. pallidum homologs,
129 have homologs in B. burgdorferi and 313 have homologs in
L. interrogans, suggesting that these may have been lost by gene
with no significant match in T. denticola (Table 4), 106 are
hypothetical proteins. The remainder includes transporters,
components of a V-type ATPase, some proteases and enzymes
involved in various metabolic activities including proline and
asparagine synthesis, and the oxidative branch of the pentose
phosphate pathway. In the case of the 106 hypothetical CDSs,
one-third are ?100 aa in length and may not necessarily repre-
sent expressed genes.
The biofilm nature of dental plaque allows for population
diversity and coexistence of various aerobes, anaerobes, and
microaerophiles. Exchange of genetic information is probable in
groups, and carriers; light green, cell envelope; red, cellular processes; brown, central intermediary metabolism; yellow, DNA metabolism; light gray, energy
metabolism; magenta, fatty acid and phospholipid metabolism; pink, protein synthesis and fate; orange, purines, pyrimidines, nucleosides, and nucleotides;
olive, regulatory functions and signal transduction; dark green, transcription; teal, transport and binding proteins; gray, unknown function; salmon, other
categories; blue, hypothetical proteins. The second circle shows predicted coding regions on the minus strand color-coded by role categories. The third circle
The fifth circle shows putative phage regions and isolated phage genes. The sixth circle shows IS elements in black. The seventh circle shows rRNA genes in black
and tRNA genes in red. The eighth circle shows trinucleotide composition in black. The ninth circle shows percentage G ? C in relation to the mean G ? C in a
2,000-bp window. The 10th circle shows GC-skew curve in red (positive residues) and blue (negative residues).
Circular representation of the T. denticola (ATCC 35405) overall genome structure. The outer scale designates coordinates in base pairs. The first circle
www.pnas.org?cgi?doi?10.1073?pnas.0307639101 Seshadri et al.
such a milieu; for example, plasmid transfer from T. denticola to
oral bacteria such as Streptococcus gordonii has been demon-
strated (19). A number of genomic regions with unusual trinu-
cleotide composition (see Methods) were identified that may
signify laterally acquired DNA. For example, a 65-kb region
(coordinates ?1,807,000–1,872,000, TDE1756–TDE1843) en-
codes 66 hypothetical proteins and 15 conserved hypothetical
proteins (all with the same transcriptional orientation). Genes
for a single tRNA and a site-specific recombinase (TDE1844)
flank this region, suggesting a phage-mediated integration event.
A second 13-kb intergenic region (coordinates 367,000–370,000)
possesses 54 copies of a 36-bp ‘‘clustered regularly interspaced
short palindromic repeat’’ (CRISPR) (20) with four adjacently
located CRISPR-associated cas genes (TDE0327–TDE0330).
These direct repeats are interspaced by a nonrepetitive sequence
of 30 bp. Although no function is assigned to the CRISPR locus,
it has been hypothesized to be a mobile element. A third region,
a single cryptic phage, is seen at coordinates 1,164,840–1,202,400
encoding a cluster including eight phage-related genes, 28
hypothetical genes, and a ParB nuclease domain protein. In
addition, miscellaneous phage remnants and seven assorted
insertion sequence transposases are also found scattered in the
genome. At least three operons encoding type -I and -II restric-
tion-modification (RM) systems are present. These are specu-
lated to serve as a barrier against interspecies gene transfer (21)
and may be important for survival in the biofilm. Homologs of
these RM components are absent in the other spirochetes, with
the exception of a single type I RM system seen in L. interrogans.
Three predicted type IV RM systems have also been described
in B. burgdorferi (22). In contrast, T. pallidum, which survives
predominantly in the relatively secluded environment of human
tissue, has no recognized RM systems, insertion sequence ele-
ments, or bacteriophage.
Overall, genes lost due to reductive evolution appear to be
largely involved in basic metabolic functions and transport,
whereas some of the genes that have arisen due to lineage-
specific expansions are implicated in pathogenesis, and genes
acquired via horizontal gene transfer are phage-related or of
unknown function. These analyses emphasize that differences in
genome sizes of T. pallidum and T. denticola are not solely due
to reductive evolution of T. pallidum. Like many other obligate
parasites, T. pallidum had undergone some reduction in meta-
bolic capabilities indicating an increased dependence on host
(and oral microbial community) for nutritional purposes. Lin-
eage-specific expansions and lateral gene transfer, on the other
hand, may reflect niche-specific adaptations and differences in
their pathogenic potential.
Host–Pathogen Interactions. T. denticola has been shown to adhere
to various cell types and basement membranes via binding to
fibronectin, collagen, laminin, fibrinogen, and other substrates
(23, 24). Additionally, because T. denticola is a late colonizer
during plaque biofilm formation, adhesion to other oral bacteria
is critical; binding to Fusobacterium, B. forsythus, and P. gingivalis
has been demonstrated (25, 26). A whole-genome survey for
potential surface-exposed proteins revealed an array of adhesins
and other factors that may play a role in binding to host-cell
components and?or coaggregation (Table 6, which is published
as supporting information on the PNAS web site). These genes
include a four-member family of YD repeat proteins (carbohy-
drate-binding), 11 tetratricopeptide (TPR) repeat-containing
proteins (protein–protein interactions), peptidases, and pro-
teases among others. Tracts of iterative DNA located either in
the promoter region or the N terminus of some of these genes
may suggest a potential for undergoing antigenic or phase
The T. pallidum genome encodes a 12-member family
(TprA-L) of putative membrane proteins (4). This T. pallidum
gene family exhibits heterogeneity among different subspecies
and strains, indicative of recombination events among the dif-
ferent tpr alleles (27). In addition, protective immune responses
and generation of opsonic activity against Tpr proteins has been
reported (28) but disputed (29). T. denticola possesses only ‘a
single member (TDE0405, major outer sheath protein) related
to this gene family, suggesting that this family has been specif-
ically expanded in the T. pallidum lineage. The outer sheath of
spirochetes is reported to be unusual in its composition and
properties compared to the outer membrane of typical Gram-
negative bacteria. Previous studies with T. denticola lipopoly-
saccharide (LPS) are contradictory, ranging from the detection
of LPS-like material (30) to the absence of any LPS structural
components (31). TDE1419–TDE1441 encode various glycosyl
hydrolases and RfbAB that may be involved in synthesis of
surface polysaccharides; however, other enzymes for synthesis of
Lipid A, KDO, or other LPS components are absent. However,
two forms of LPS have been isolated from Treponema pectino-
vorum, indicating that at least some of the oral treponemes are
capable of synthesizing LPS (32).
Proteinases and peptidases may aid in degradation of host
tissue and bacterial components to fulfill nutritional require-
ments, and may contribute to cytotoxicity of periodontal infec-
tions (33–35). Over 25 assorted peptidases, hydrolases, and other
putative degradative enzymes were identified in the genome.
Because T. denticola is known to disrupt epithelial barriers and
perturb actin and actin-regulating pathways in host cells (36), a
have signal peptides) may facilitate this process (Table 6).
Stress Responses. The dental biofilm environment transitions
continually between feast and famine, aerobic and anaerobic
conditions, and high and low pH. T. denticola possesses various
mechanisms to counteract oxidative, osmotic, and other stresses.
It has been reported to have NADH oxidase, NADH peroxidase,
and superoxide dismutase (SOD)?reductase activities (37).
These activities may be fulfilled by Nox (TDE0096), alkyl
hydroperoxide reductase?peroxiredoxin (AhpC, TDE0011) in
combination with Nox, and desulfoferrodoxin?neelaredoxin
(TDE1754) (38), respectively. In addition, a single rubrerythrin
with significant homology (E ? 10?5) with the predicted products of the
pathogenic spirochetes T. pallidum, B. burgdorferi, and L. interrogans. The
number outside the circles (1,268) represents the number of T. denticola
Seshadri et al.
April 13, 2004 ?
vol. 101 ?
no. 15 ?
(TDE0434), implicated in oxidative stress protection via cata-
lytic reduction of intracellular H2O2, may account for the
observed SOD activity (39). Homologs of Bacteroides fragilis
aerotolerance-associated genes, batAB (TDE1250, TDE1252)
(40), may be implicated in aerotolerance of T. denticola as well.
Prokaryotic selenoproteins catalyze redox reactions and for-
mation of selenoethers in (stress-induced) metabolism and en-
ergy production. Previous assessment of the nutritional require-
ments of T. denticola revealed a strict growth dependence on
selenium (41). Genome sequence reveals the complete set of
genes for selenoprotein synthesis: selA, (TDE2477), selD
(TDE2461), and selB (TDE1963). In E. coli, the selenium is
incorporated into formate dehydrogenase (fdh) subunits; al-
though no fdh is identified in the T. denticola genome, a
thioredoxin (TDE0238), glycine reductase complex selenopro-
teins (grdA, TDE0745, grdB, TDE2119, TDE0078), and gluta-
thione peroxidase (TDE1729), all contain predicted selenocys-
teine sites. Given the potential role of these selenoproteins in
antioxidant functions, deficiency of selenium may have detri-
mental effects on T. denticola’s ability to withstand oxidative
stress. Other stress–response mechanisms include an arginine
deiminase (TDE0451) to counter acidification by producing
ammonia from arginine, and betaine aldehyde dehydrogenase
(TDE0080) and a possible transporter (TDE1261) for synthesis
or uptake of betaine, an osmoprotectant. T. denticola possesses
enzymes for glycogen synthesis (unlike the other spirochetes),
which may be a specific adaptation for survival during starvation
conditions. T. denticola has a proportionately greater number of
regulatory factors (relative to genome size) compared to T.
pallidum, including seven sigma factor homologs (TDE0070,
TDE0091, TDE0937, TDE1346, TDE2320, TDE2404, and
TDE2683), nine CDSs encoding two-component signal trans-
Transport and Metabolism. Consistent with the known proteolytic
abilities of T. denticola, analysis of the transporter content of the
genome reveals an array of peptide and amino acid uptake
systems, including eight ABC-type peptide uptake systems. By
contrast, there are very limited capabilities for uptake of other
organic nutrients, including a glucose?galactose ABC trans-
porter (TDE2215–TDE2217), and probable transporters for
lactose, gluconate, and carboxylates. Like T. pallidum, T. denti-
cola possesses HPr, enzyme I and enzyme IIA of the phospho-
transferase system (PTS), but no PTS transporter complex,
suggesting that these proteins play a purely regulatory role. Iron
acquisition appears to be an important priority for T. denticola
with the identification of eight probable ABC-type uptake
systems for iron chelates or siderophores.
Unexpectedly, T. denticola encodes a very large complement
of predicted ABC-type drug efflux systems. Eighty-three pre-
dicted ABC family drug efflux proteins were identified, com-
prising at least 47 different ABC efflux systems, significantly
more than any sequenced spirochete or other prokaryotes (Fig.
3). Three of these systems (TDE0719–TDE0720; TDE0425–
TDE0426; TDE2431–TDE2430) are probable bacteriocin secre-
tion systems, based on the presence of three proximally encoded
bacteriocin-type signal domain-containing CDSs (TDE0416,
TDE0422, TDE0424). The secreted bacteriocins may be bacte-
riostatic for competition against other organisms, or may be for
signaling within the oral biofilm. Homologs of the lux genes are
not present in the T. denticola genome in contrast to other dental
microbes that produce autoinducer (AI-2) signaling peptides.
Speculatively, the remainder of the ABC efflux systems may play
a role in secretion of effectors mediating host interactions or in
providing resistance against antagonistic factors (toxins, second-
ary metabolites, antibiotics) produced by other microbes in the
dental biofilm. T. denticola also possesses nine probable sodium-
ion driven MATE family drug efflux systems as well as other
predicted drug efflux systems.
Comparative analysis of metabolic profiles across all four
sequenced spirochete genomes reveals some distinctions of T.
denticola metabolism. T. pallidum and B. burgdorferi have limited
biosynthetic abilities in accordance with their reduced genome
sizes, whereas L. interrogans and T. denticola have somewhat
greater capabilities. Genes encoding enzymes for glycolysis,
gluconeogenesis, and a pentose phosphate pathway are present
(Table 7, which is published as supporting information on the
PNAS web site). Unlike T. pallidum and B. burgdorferi, the
pentose phosphate pathway in T. denticola (and L. interrogans)
lacks the oxidative branch. The existence of glycolysis and
absence of a tricarboxylic acid (TCA) cycle in T. denticola
suggests that ATP is generated by sugar fermentation as in the
case of B. burgdorferi and T. pallidum, whereas L. interrogans
possesses both a TCA cycle and an electron transport chain
(ETC). The lack of cytochromes and quinone biosynthesis genes
in T. denticola indicates that it does not possess an ETC for
In T. pallidum and B. burgdorferi, the membrane potential
essential for transport, motility and other cellular functions is
apparently established by V1V0 ATPases; these V-type com-
plexes typically consume ATP to translocate H?or Na?ions
across the membrane. T. pallidum has two V1V0-type ATPase
operons, with different subunit compositions, each speculated to
possess different specificities for Na?or H?. The single V-type
ATPase in T. denticola shares similarity with H?-specific AT-
Pase systems; however, the operon has undergone considerable
rearrangement (genes encoding the subunits are ordered K-I-
D-B-A, with a subunit E gene located 300 kb upstream), and the
subunit I gene contains a frameshift, which might render the
ATPase nonfunctional. Another component that may also con-
tribute to the proton gradient is a proton-translocating pyro-
phosphatase (TDE0651), although this gene also has a frame-
shift. TDE0834–TDE0838 have sequence similarity to the Na?-
translocating NADH?quinone reductase complex (nqrABCDE)
of other organisms. It has been speculated that this complex in
T. denticola and other organisms maintains a Na?gradient used
by several transport systems (42). However, T. denticola and
many other host-associated organisms (including Chlamydia,
Porphyromonas, and Clostridium species) that encode NQR
complexes do not have recognizable biosynthetic pathways for
ubiquinone, a required cofactor for the NQR systems charac-
terized to date. An interesting possibility is that these bacteria
may scavenge quinone compounds from the surrounding micro-
Number of predicted ABC efflux transporter genes present in various
www.pnas.org?cgi?doi?10.1073?pnas.0307639101Seshadri et al.
environment. This concept is not unprecedented, in that Esch-
erichia coli and Salmonella typhimurium are not capable of
synthesizing the o-quinone pyrroloquinolone quinone and must
import it as a cofactor to express glucose dehydrogenase activity
(43). However, the T. denticola genome does not encode a
recognizable quinone oxidase. Therefore, it is likely that the T.
denticola system utilizes another electron donor?receptor, such
as ferridoxin or rubredoxin, to fulfill the electron exchange part
of this reaction. Finally, Na??H?antiporter family proteins
TDE2203 and TDE2204 may permit exchange between the Na?
The T. denticola genome also contains all five required genes
of a glycine reductase complex (grdA–grdE), including two
divergent copies each of grdB and grdE. This complex acts in
consort with thioredoxin and thioredoxin reductase to deami-
nate and phosphorylate the substrate, thus contributing to the
pool of phosphorylated compounds (44). Alternate substrates in
Clostridium and Eubacterium species include betaine, sarcosine,
and N-methyl derivatives of glycine; the polypeptide subunits
derived from the multiple grdB and grdE genes in T. denticola
may affect substrate specificity and permit the utilization of
amino acids and other compounds for substrate phosphorylation
and ATP production. This possibility is consistent with the
growth of T. denticola in peptone?yeast extract?serum medium
in the absence of glucose with conversion of amino acids to
ammonia and acetic, lactic, succinic, and formic acids (45).
acids, cofactors, and nucleotides is possible. T. denticola may also
use a number of sugars like glucose, galactose, glycerol, melibi-
ose, fucose, and sorbitol. In contrast to the other spirochetes,
enzymes for synthesis of glycogen (TDE2035 and TDE1582) as
well as degradation to maltodextrin (TDE2411 and additional
?-amylases) are apparent. Although amino acid biosynthesis is
deficient, numerous genes for uptake, interconversion, and
catabolism are present.
The analysis of the T. denticola genome and comparisons with
other spirochetes has revealed insights into the evolution and
adaptive responses within the spirochete phylum. T. denticola is
primarily restricted to the subgingival plaque and does not cause
the systemic infections and manifestations characteristic of T.
pallidum, Borrelia, and Leptospira. However, a number of factors
that may contribute to periodontal disease were identified. The
genome sequence together with recently developed genetic
techniques (46, 47) will permit analysis of genes involved in the
colonization, survival, growth, and pathobiology of T. denticola
in this unique polymicrobial environment.
We thank S. Salzberg, O. White, M. Heaney, S. Lo, M. Holmes,
M. Covarrubias, J. Sitz, A. Resnick, J. Zhao, M. Zhurkin, R. Deal,
R. Karamchedu, and V. Sapiro for informatics, database, and software
support. This work was supported by the National Institute of Dental and
Craniofacial Research Grant RO1-DE12488.
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