Oral microbiology and genomics

MARGARET J. DUNCAN
This review presents a broad picture of the process of
microbial genome sequencing, and illustrates, with
specic examples, how genome sequences have
increased our knowledge base for oral microbes. In
addition, the scope of future genome projects will be
discussed, and the reader will be introduced to some
of the tools and databases that can help decipher how
oral bacteria function.
While publication of the whole genome sequence
of yet another microorganism, plant or animal is no
longer major news, for biomedical researchers it is
a landmark event that opens the door to a greater
understanding of the biological systems they study.
Whole genome sequencing began with the Human
Genome Project, and model bacterial genomes were
chosen for proof of principle studies of random
shotgun DNA sequencing and assembly strategies.
The rst microbial genome sequence to be pub-
lished was that of Haemophilus inuenzae (14) and
to date (December, 2004) 20 archeal and 188 bac-
terial genome sequences have been completed and
published, and genome projects are in progress for
534 additional prokaryotic genomes (Gold Genomes
Online Database; URL: http://www.genomesonline.
org/). Among these medically and environmen-
tally important microorganisms are at least 15
oral bacteria that include known and putative
oral pathogens, e.g. Streptococcus mutans and
Porphyromonas gingivalis, and so-called benecial
organisms such as Streptococcus mitis, Actinomyces
naeslundii, and Streptococcus gordonii (listed in
Table 1).
The bottom-up, random shot-gun strategy used
to sequence microbial genomes consists of four
phases. First is the preparation of a genomic
library of small fragments (ca. 2-kb) obtained by
mechanically shearing chromosomal DNA iso-
lated from the organism of interest. Mechanical
fragmentation minimizes biases that are often
found in libraries made from enzyme-digested DNA
because some restriction sites are more susceptible
to cutting than others. The resulting small DNA
fragments are ligated to a high copy number bac-
terial plasmid, and transformed into Escherichia
coli. In addition, larger insert libraries are prepared
in bacteriophage lambda or pBR322-based vectors;
these will be used later in the genome assembly
phase as backup DNA templates for closing
sequence and physical gaps. In the second phase,
the ends of all the small fragments are sequenced
to obtain approximately 8-fold coverage of the
genome with overlapping small random sequences.
In the third phase, the random sequences are
assembled, usually beginning with 30-bp overlaps,
building up increasingly larger fragments that ulti-
mately can be joined to yield one contiguous
sequence. During the nal phase, open reading
frames of at least 100 base pairs are identied, rst
by automated annotation based on the BLASTP
algorithm (2), and nally by visual inspection.
The completion of a genome sequence is the rst
step toward determining the function of all genes in
an organism. DNA sequence reveals protein coding
sequences or open reading frames (ORF) and inter-
genic regions that contain promoter elements; all
the information necessary to study the regulation of
gene expression. The expanding number of genome
sequences allows the comparison of genes across
a wide spectrum of bacterial phyla, so that the
determination of gene function through amino acid
similarity and conserved domain searches becomes
signicantly more reliable. By means of genome
comparisons it is possible to trace the spread of
transposons, antibiotic resistance genes, and extra-
chromosomal elements between species, and to
analyze the evolution of metabolic pathways as well
as the evolution of virulence. Such comparisons have
demonstrated the major role played by horizontal
gene transfer in bacterial evolution, raising the larger
question of what constitutes a bacterial species. For
biomedical research and clinical practice, genome
sequences will yield better probes for the detection of
pathogens and even the identication of specic
strains.
63
Periodontology 2000, Vol. 38, 2005, 6371
Printed in the UK. All rights reserved
Copyright Blackwell Munksgaard 2005
PERIODONTOLOGY 2000
Treponema denticola
The recent publication of the genome sequence of
strain 35405 included a comparison with the
genomes of spirochetes Treponema pallidum, Borre-
lia burgdorferi, and Leptospira interrogans (45). A
core set of 618 ORFs was found in all the species, and
as expected they encoded functions such as DNA
replication and repair, transcription, translation,
energy metabolism, and cell division. The genome of
Treponema denticola was signicantly larger than
that of T. pallidum, encoding 2786 and 1040 putative
ORFs, respectively. The nucleotide compositions
also differ signicantly (37.9% G + C content for
T. denticola vs. 52.8% for T. pallidum) and not sur-
prisingly there is little DNA sequence homology,
supporting the notion that strain divergence was an
ancient event. While approximately 25% of T. denti-
cola ORFs have best matches in T. pallidum, over
Table 1. Genome projects for oral bacteria
Genome Strain
Genome
size (Mb)
Collaborating
institutions Funding
Web sites for
searches annotation
Actinobacillus
actinomycetemcomitans
HK1651 2.105 University
of Oklahoma
NIDCR
a
http://www.genome.ou.edu
http://www.oralgen.lanl.gov
Actinomyces naeslundii MG1 3.0 TIGR
b
NIDCR http://www.tigr.org
Fusobacterium
nucleatum
ATCC 25586 2.17 Integrated
Genomics Inc.
Integrated
Genomics
Inc.
http://www.genome.org/cgi/doi
Fusobacterium
nucleatum
ATCC10953 2.4 BCM-HGSC
c
UCLA
d
NIDCR http://www.hgsc.tmc.edu/
Fusobacterium
nucleatum
subspecies vincentii
ATCC 49256 2.118 Integrated
Genomics Inc.
Integrated
Genomics
Inc.
http://www.genome.org/cgi/doi
Porphyromonas
gingivalis
W83 2.343 TIGR
The Forsyth
Institute
NIDCR http://www.tigr.org
http://www.oralgen.lanl.gov
Prevotella intermedia 17 2.8 TIGR
LANL
e
NIDCR http://www.tigr.org
Streptococcus gordonii NTCT7868 4.351 TIGR NIDCR http://www.tigr.org
Streptococcus mitis University of
Wuerzburg
Streptococcus mitis NTCT12261 2.2 TIGR NIDCR http://www.tigr.org
Streptococcus mutans UA159 2.03 University of
Oklahoma, Ohio
State University
NIDCR http://www.genome.ou.edu
http://www.oralgen.lanl.gov
Streptococcus sanguinis SK36 Commonwealth
Biotechnologies
Inc., Virginia
Commonwealth
University
NIDCR http://www.sanguis.mic.vcu.edu
Streptococcus sobrinus 6715 2.2 LANL TIGR NIDCR http://www.tigr.org
Tannerella forsythia ATCC 43037 TIGR NIDCR http://www.tigr.org
Treponema denticola ATCC 35405 2.843 TIGR
BCM-HGSC
NIDCR http://www.tigr.org
http://www.oralgen.lanl.gov
a
National Institute of Dental and Craniofacial Research.
b
The Institute for Genomic Research.
c
Baylor College of Medicine-Human Genome Sequencing Center.
d
University of California Los Angeles.
e
Los Alamos National Laboratory.
64
Duncan
1000 ORFs did not have homologs in other spiro-
chetes. Half of these are hypothetical proteins, the
rest share homology with ORFs from gram-positive
organisms such Streptococcus (oral and nonoral) and
Clostridium species, and Fusobacterium nucleatum,
also a component of dental plaque. As noted by
Seshadri et al. (45) the larger genomic content of
T. denticola may have occurred through mechanisms
involving gene duplications, as evidenced by tan-
demly duplicated genes in the chromosome, and
horizontal gene transfer exemplied by a 65-kb
region that may have originated from phage-medi-
ated transfer. Of particular note was the nding that
T. denticola possesses an unusually large number of
genes encoding ABC-type drug efux functions, 83
proteins representing 47 systems, more than any other
sequenced prokaryote. It was proposed that they were
involved with secretion of bacteriocin and host-dam-
aging effectors as well as drug efux systems.
T. denticola possesses genes that encode enzymes
for glycolysis, gluconeogenesis, and the pentose
phosphate pathway; and the lack of a tricarboxylic
acid cycle suggests that adenosine triphosphate (ATP)
is generated by fermentation (45). Although there are
no systematic experimental studies to conrm all
these pathways, in earlier work based on measure-
ments of glycolytic enzyme activities it was concluded
that the organism metabolized glucose to pyruvate via
the Emden-Meyerhof pathway (22). T. denticola
preferentially fermented amino acids producing
acetate and carbon dioxide from cysteine, serine,
alanine, glycine, and arginine (22). These ndings,
and a requirement of selenium for growth, suggested
that T. denticola could metabolize amino acids via
glycine reductase, the essential enzyme of the Stick-
land reaction that contains selenocysteine (43). Bio-
chemical evidence and examination of the genome
sequence conrmed the existence of this pathway.
The genome sequence also revealed several new
surface proteins that could potentially mediate
binding to host cells and tissues. Furthermore, it was
reported that some of their coding genes contained
DNA sequences that may afford the potential for
phase variation mechanisms (45). However, there
have been no reports so far of antigenic heterogeneity
of T. denticola genes encoding surface proteins, as
observed with the T. pallidum tprK gene (8).
Previously, it was hypothesized that a 65-kb region
of the T. denticola 35405 genome may have been
acquired by lateral gene transfer (45), and in a recent
analysis the presence of a large integron cassette was
discovered within the same region (11). Within gram-
negative bacteria, integrons are important players in
lateral gene transfer because of their ability to
capture, rearrange, express, and spread antibiotic
resistance genes. The T. denticola integron is the rst
identied outside the Proteobacteria, and is signi-
cant because of its large size (58 kb), its similar
orientation to the integron integrase gene, and the
relatively large size of cassette sequences that are the
recombination sites for gene capture. The region
possesses all the components for integron function-
ality, and raises the question of whether it confers on
T. denticola the capacity to act as a reservoir and
disseminator of antibiotic resistance genes (11).
Streptococcus mutans
The genome sequence, and hence gene content, of
S. mutans revealed its metabolic versatility (1). Spe-
cically, the ability to utilize a wide variety of car-
bohydrates illustrated how well the organism is
adapted to its ecologic niche on tooth surfaces facing
exposure to a wide variety of food carbohydrates. In
addition to pathways for di- and monosaccharides,
S. mutans also has pathways for sugar-alcohol util-
ization. While some sugars are transported from the
environment by ATP-binding cassette (ABC) trans-
port systems, most enter cells by the phosphoenol-
pyruvate sugar phosphotransferase systems, of which
there are 14 copies in the genome, presumably with
specicities for different substrates.
The S. mutans genome sequence has beenexploited
in a number of recent studies on acidogenic and
aciduric properties of the organism. Protein expres-
sion was compared in S. mutans during continuous
culture at pH 7.0 andpH 5.0 (33, 34) andproteins were
analyzed by two-dimensional gel electrophoresis and
matrix-assisted laser desorption ionization time-of-
ight mass spectrometry. The obtained peptide
sequences were used to search the S. mutans genome
to identify coding genes. The study dened the stress-
responsive expression of 25 proteins, among which
were those involved in DNA replication, transcription
and translation, ribosomal subunit proteins, and the
molecular chaperones DnaK and trigger factor (33).
Proteomic changes in response to pH were primarily
limited to metabolic pathways for glycolysis, acid
production, and branched-chain amino acid synthesis
(34). Results of this study were also consistent with
ndings that acid tolerance was due to the ability to
pumphydrogenions out of the cytosol via amembrane
bound, acid stable, F
0
F
1
ATPase (reviewed in 39).
The transfer of genes between species, i.e. lateral
gene transfer, has played a major role in bacterial
65
Oral microbiology and genomics
evolution (13). Transfer is mediated by mobile
genetic elements such as transposons, plasmids, and
bacteriophage, and evidence of transposon footprints
was found in the S. mutans genome (1). Interestingly,
20 years ago the transfer of tetracycline resistance
between streptococcal strains was detected in the
absence of plasmid DNA (20). Further analyses
showed that transfer was resistant to DNase and was
dependent on cell-to-cell contact. The authors of this
study concluded that the tet gene was located in the
chromosome, and its sequence and mode of transfer
were similar to that of the tet gene associated with the
conjugative transposon Tn916. These conclusions
were validated with the identication of a conjugative
transposon in the genome sequence that was sim-
ilar to, but distinct from, Tn916 from Enterococcus
faecalis.
Fusobacterium nucleatum
Dening a role for F. nucleatum in periodontitis is
complicated by the fact that the organism is a key
structural component of normal and disease-associ-
ated dental plaque (27, 29, 30, 47). Fusobacteria are
notable for their specic interactions with several
species of oral bacteria, as illustrated by the associ-
ation with gram-positive cocci to generate charac-
teristic corn-cob formations (12, 31), and are
identied as coaggregation bridge organisms (28).
The central importance of F. nucleatum in plaque
architecture has heightened interest in the organisms
role as an opportunistic pathogen, and in its surface
proteins that might be involved in adherence and
coaggregation with other bacteria (reviewed in 7).
The genome sequence of F. nucleatum strain ATCC
25586 (FN) was published in 2002 (24), and was fol-
lowed a year later by the draft sequence of a second
strain, F. nucleatum subspecies vincentii (FNV; 25).
In general the strains were very similar with 85%
overall gene synteny; however, some interesting and
signicant differences between the two were noted.
For example, FNV possesses several DNA restriction-
modication systems that are not present in FN. The
sequence also showed that FNV had been under
attack by foreign mobile DNA elements since it
contains genes encoding over 100 phage-associated
proteins in clusters of up to 66 ORFs. Interestingly,
the 28%G + C content of these genes and their codon
usage patterns are very similar to that of the host
genome, suggesting that phage infection of FNV
occurred long ago. Other genome differences indicate
an increased capacity for peptide utilization, and it
was suggested that ribonucleotide degradation
products from the DNA restriction-modication sys-
tem may be used as carbon and nitrogen sources.
While outer surface proteins in FN ATCC 25586 and
FNV may be similar, their O-antigenic polysaccharide
composition was reported to be very different (25) as
FNV has the biosynthetic potential to incorporate
galactopyranose, galacturonate, and sialic acid into
O-antigen. These differences, however, have yet to be
conrmed experimentally.
The importance of F. nucleatum in subgingival
plaque architecture and metabolic interactivity has
been long recognized, but the dissection of these
interactions has been hampered by the lack of a gene
transfer system. A recent development on this front
focused on the construction of a shuttle vector based
on pFN1, a small indigenous plasmid that could be
isolated in good yield (18). Furthermore, methods
were developed for electroporation of F. nucleatum,
with plasmid transformation frequencies falling
within the workable range. These advances, together
with the genome sequences, should make this
organism more tractable to mechanistic studies.
Genome comparisons
Multiple genome sequence comparison is the gold
standard for determining the extent of strain diversity
within a species. The genomes of different strains of
several important bacterial pathogens have been
sequenced to determine whether different virulence
phenotypes are associated with specic genes. Group
A Streptococcus cause a range of infections from
pharyngitis to necrotizing fasciitis. Although group A
Streptococcus are classied according to the serotype
of the M surface protein, this does not predict the
severity of the disease phenotype (6). Comparisons of
the genome sequence of an aggressively invasive
serotype M3 strain with those of less invasive sero-
type M1 and M18 strains established that approxi-
mately 90% of the genome was shared between the
strains, with phage-related sequences accounting for
the remaining 10% diversity such as unique phage-
encoded genes for pyrogenic toxins, a superantigen,
and phospholipase activity in the invasive M3 strain
(6). A comparison of the E. coli K12 laboratory strain
and virulent strain 0157:H7 showed that lateral gene
transfer, potentially mediated by prophages, played
an important role in the acquisition of at least 130
new virulence-associated genes (21, 38).
To date, F. nucleatum is the only oral organism
with multiple complete genome sequences. In addi-
66
Duncan
tion to the already sequenced strain ATCC 25586 and
F. nucleatum subspecies vincentii, the sequence of
F. nucleatum strain ATCC 10953 is nearing comple-
tion. Lack of multiple sequences has not prevented
comparisons between P. gingivalis strains, since
in lieu of sequencing multiple strains, comparative
studies were carried out with P. gingivalis gene
microarrays (9). The microarrays were used to com-
pare the total gene content of the virulent strain W83
and the avirulent type strain, ATCC 33277. The data
indicated that the chromosomes were very similar,
with approximately 93% of the predicted genes in
common. The remaining 7% showed signicant
diversity or were absent in ATCC 33277, and among
the divergent features were previously reported
insertion and ragB sequences, as well as function-
ally assigned genes such as those encoding enzymes
involved in capsular polysaccharide synthesis
that were organized in a putative operon in
strain W83. Should multiple strains of key organisms
be sequenced? A case could be made for draft
sequences, i.e. 3- to 6-fold coverage, which may be
sufcient to identify regions of difference between
virulent and avirulent strains or recent clinical iso-
lates and laboratory passaged strains, and may not be
too costly to support.
Lateral gene transfer and
pathogenicity islands
From the comparison of multiple bacterial genome
sequences, and illustrated in the small number of
genomes discussed here, it is clear that the transfer of
genes between species, i.e. lateral gene transfer, has
occurred more frequently than previously appreci-
ated. Although the spread of antibiotic resistance
genes between bacterial species is the most well
known indication of lateral gene transfer, clusters of
genes such as those encoding a metabolic pathway
may also be transferred and maintained if they give
the new host a growth advantage (32).
Signs that a gene may have been acquired by lateral
gene transfer include a different GC content and/or
different codon usage from host genes, antibiotic
resistance functions, activities associated with viru-
lence, and genetic linkage with known moveable
DNA elements. Many of these criteria are fullled by
pathogenicity islands, so called because they often
contain genes for virulence factors in microorgan-
isms that cause disease (reviewed in 19). Ranging in
size from 10 to 200 kb, pathogenicity islands often
carry genes encoding integrases and transposases
that are involved in DNA mobility. They may also be
associated with transfer RNA genes, favored sites for
the integration of foreign DNA.
In the P. gingivalis genome there are two paralo-
gous regions that have all the hallmarks of patho-
genicity islands, one of approximately 28 kb, and a
deleted version of approximately 18 kb (9). It is
probable that after the initial putative transfer the
paralogs were generated by duplication and intra-
chromosomal recombination. With an average G + C
composition of 41%, compared to a 48% average for
the whole genome, the regions are bound on one side
by homologs of Bacteroides transposon Tn5520, and
on the other by either a serine or aspartate tRNA. Half
the ORFs encode homologs of transcription regula-
tors, mobilization and transfer functions of Bactero-
ides conjugative transposons, excisases and integ-
rases, ISPg1, and an efux pump family protein. The
other ORFs encode either conserved hypothetical or
species-specic hypothetical ORFs, and identica-
tion of the function of unknown ORFs within these
regions will determine whether they are true patho-
genicity islands.
The conditions prevailing in dental plaque, i.e.
constant cell-to-cell contact, are favorable for the
transfer of conjugative transposons (41, 53). Indeed,
genes associated with conjugative transposons were
found in the genomes of P. gingivalis (36), Prevotella
intermedia (Oral Pathogen Sequence databases,
URL: http://www.oralgen.lanl.gov/) and S. mutans
(1). These ndings raise the question of whether
antibiotic resistance and other genes can be trans-
ferred between plaque bacteria, and to other com-
mensals that colonize humans. The ermF and tetQ
resistance genes were detected in both gram-posit-
ive and gram-negative bacteria found in the colon,
as well as oral Porphyromonas species (44). The
transfer of Bacteroides conjugative transposons of
the CTnDOTERL family that carry these resistance
genes is induced by the presence of low concen-
trations of tetracycline (54), raising the alarming
possibility that antibiotic treatment may actually
increase the spread of resistance genes to other
dental plaque bacteria.
Another mechanism of gene transfer that may be
operational in plaque relates to increased genetic
competence of biolm-grown bacteria, and the sub-
sequent uptake of DNA (35). In the study by Li et al.
(35), two interesting features of biolm life were
established. First, transformation frequencies in bio-
lm-grown cells of S. mutans were 10- to 600-fold
higher than planktonic cells. Second, working on the
premise that DNA in the environment originates from
67
Oral microbiology and genomics
dead bacteria, it was demonstrated that biolm-
grown cells were not only able to take up puried
chromosomal DNA, but also DNA released from
heat-killed biolms. While these results were
obtained with autologous DNA, Wang et al. (51)
carried the concept a step further and were able
to show the transfer of a shuttle plasmid from
T. denticola to S. gordonii during growth in a mixed
biolm. So far there is no direct evidence that gene
transfer can actually occur in dental plaque, but these
recent reports underscore this possibility.
New challenges: uncultivable
bacteria and microbial
communities
It is estimated that more than 99% of microorgan-
isms observed in nature cannot be cultivated using
established techniques (3), and in the case of the oral
microbiome, only about 50% of the approximately
700 species present in the oral cavity are cultivable
(37). In nature most bacteria live in communities and
the paradigm of sequencing the chromosome of a
single organism was recently extended to the
metagenomes of dened environmental niches, thus
including the genomes of both cultivable and non-
cultivable species. Comprising bacterial, archeal,
viral, and eukaryotic species, the oral microbiome is
the ideal community to apply this new paradigm.
The methods used for metagenome sequencing
depend to some extent on the goals of the specic
project. The pioneering study of the metagenome of
soil used gentle methods to isolate large DNA frag-
ments that were cloned into bacterial articial chro-
mosome (BAC) vectors, sopreserving large stretches of
genetic information that may encode novel enzyme
activities (42). More recently, shotgun sequencing the
metagenome of the Sargassosea useda plasmid-based
library of short DNA fragments (26 kb) for end-
sequencing. The project generated over 1 million
GenBank entries of partial genomic sequences that
revealed a comprehensive picture of microbial diver-
sity in the environment, and the identication of at
least 1800 genomic species (50). On the other hand, a
similar small insert plasmid library of the microbiome
from a more restricted environment, acid mine
drainage biolm, yielded up to 10-fold coverage of the
genomes of two predominant species (48). Non-cul-
ture-basedtargetedapproaches toclone andsequence
16S rRNA DNA fragments from subgingival plaque
associated with health and disease identied more
than 700 bacterial species or phylotypes (37). Studies
are now underway to characterize the healthy human
oral microbiome, i.e. gene and genome content, using
high throughput methods to sequence random and
targeted plasmid and fosmid libraries (16). Additional
goals of this work are to characterize the microbiome
associated with chronic periodontitis; develop a gene
database for the scientic community; and construct a
microarray that could be used for disease diagnosis
and prognosis.
An alternative approach, based on fosmid librar-
ies, is being used to obtain sequence information
from uncultivable oral bacteria (5). Genomic DNA
was extracted from enriched cultures of specic
organisms puried from subgingival plaque, and
large fragments were cloned into fosmid vectors.
Fosmid inserts containing DNA from the unculti-
vable organism can be identied from signature
16S rRNA sequences, and these will be seed frag-
ments and used to probe libraries for clones that
contain overlapping DNA. By this iterative process
as much of the genome as possible will be
retrieved.
Postgenomics
In the postgenomic era, attention has turned to
protein function rather than gene identication.
Proteomics is the large-scale characterization of the
full complement of proteins expressed in a cell, i.e.
their molecular and cellular functions. This eld is
growing rapidly in terms of methodology, analysis,
data management, and bioinformatics; for more
detailed information the reader is referred to a recent
comprehensive review by Graves & Haystead (17).
Oral bacteria have not escaped proteomic attack, and
so far the acid tolerant proteome of S. mutans has
been analyzed using two-dimensional gel electro-
phoresis, followed by mass spectrometry to identify
proteins of interest (33, 34, 55). Two studies of
P. gingivalis using similar approaches identied
proteins secreted during incubation in epithelial cell-
conditioned medium (10) and the protein comple-
ment during normal growth (52).
Another important facet of proteomics is protein
protein interaction, which can potentially yield useful
information for researchers working with less well-
studied andor genetically intractable organisms (e.g.
oral bacteria) and for those attempting to dene the
function of the large percentage of functionally
unknown ORFs present in every genome. Combina-
tions of genetic, biochemical, and bioinformatic
68
Duncan
approaches have been successfully applied to
genome-wide analyses of proteinprotein interac-
tions in several microorganisms. Starting with a small
genome encoding 55 proteins, that of the bacterio-
phage T7, 25 interactions were identied using the
yeast two-hydrid system (4) with, in some cases,
corroborating experimental evidence found in the
literature. Two subsequent studies also used the two-
hybrid approach to dissect the interaction map of
Saccharomyces cerevisiae (23, 49). Together these
investigations identied over 5000 interactions
involving approximately 4200 proteins. The protein
protein interactive network of Helicobacter pylori,
also revealed through yeast two-hybrid analysis,
yielded enough information to assign functionally
unknown proteins to biological pathways (40). A
strictly in silico approach was used to identify func-
tionally linked genes and proteins in Mycobacterium
tuberculosis (46). Using four methods, Rosetta Stone,
Phylogenetic prole, Operon, and Conserved Gene
Neighbor, genome- wide protein pairs could be
detected as well as more complex protein networks.
Previously uncharacterized genes were predicted to
be associated with several functions including cell
wall metabolism and chaperone activities, and the
authors indicate that the methods can be applied to
any sequenced bacterial genome.
Databases and tools
As the number of genome sequences has grown, so
has the number of databases and tools to house
and analyze genomes, their component genes, and
protein products. The principal databases with their
URLS are shown in Table 2. Most of the genomes
of oral bacteria were sequenced at The Institute for
Genomic Research (TIGR), and sequences and
analysis tools can be found in the Comprehen-
sive Microbial Resource database. A specialized
Oral Pathogen Sequence Database (Oralgen) was
established by the Los Alamos National Laboratory
Bioscience Division. To date, this database contains
the genomes of six organisms, and will expand with
the addition of other bacterial and viral genomes as
they become available. The TIGR and LANL data-
bases have both similar and complementary ana-
lysis tools, and Oralgen has also tailored some
analyses to specic organisms, e.g. a comparison of
the proteomes of T. denticola and T. pallidum. The
Bioinformatics Resource for Oral Pathogens (BROP)
at The Forsyth Institute also houses genome
sequences, complementary analysis tools, and
P. gingivalis microarray data. A comprehensive
site for the analysis of microbial proteins and ORFs is
the Munich Information Center for Protein Sequences
(MIPS) and the Protein Extraction, Description, and
Analysis Tool (PEDANT). This database provides
analysis of proteins from 240 complete, and over 100
incomplete, eukaryote and prokaryote genomes. For
more general applications, the January issue of
Nucleic Acids Research publishes articles on, and a
listing of, currently available databases, with a des-
cription and URL (15); the July issue is devoted to
web-based genomics tools (26).
Concluding remarks
Oral microbiology has been revitalized with the
advent of genome sequences for its constituent
organisms. In time this new information will provide
insights on metabolic pathways and lead to the
development of better growth conditions, and evi-
dence of past genetic exchanges will stimulate the
development of new methods for gene transfer. New
projects to sequence the oral microbiome will expand
our realm of interest beyond present horizons, and
hopefully encourage and draw new investigators to
this challenging eld.
Table 2. Databases for genomic tools
Database Description Website
BROP Bioinformatics Resource for Oral Pathogens http://www.brop.org
MIPSPEDANT Munich Information Center for Protein Sequences;
Protein Extraction, Description, and Analysis Tool
mips.gsf.de/
STRING Search Tool for the Retrieval of Interacting GenesProteins string.embl.de/
BIND Biomolecular Interaction Network Database http://www.bind.ca/
DIP Database of Interacting Proteins dip.doe-mbi.ucla.edu/
69
Oral microbiology and genomics
References
1. Ajdic D, McShan WM, McLaughlin RE, Savic G, Chang J,
Carson MB, Primeaux C, Tian R, Kenton S, Jia H, Lin S,
Qian Y, Li S, Zhu H, Najar F, Lai H, White J, Roe BA, Ferretti
JJ. Genome sequence of Streptococcus mutans UA159, a
cariogenic dental pathogen. Proc Natl Acad Sci U S A 2002:
99: 1443414499.
2. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z,
Miller W, Lipman DJ. Gapped BLAST, PSI-BLAST: a new
generation of protein database search programs. Nucleic
Acids Res 1997: 25: 33893402.
3. Amann RI, Ludwig W, Schleifer KH. Phylogenetic
identication and in situ detection of individual micro-
bial cells without cultivation. Microbiol Rev 1995: 59:
143169.
4. Bartel PL, Roecklein JA, SenGupta D, Fields S. A pro-
tein linkage map of Escherichia coli bacteriophage T7.
Nat Genet 1996: 12: 7277.
5. Beheshti A, Duncan MJ, Paster BJ. Enrichment of phylo-
types of the TM7 division from human subgingival plaque.
ASM 104th General Meeting 2004: D013243.
6. Beres SB, Sylva GL, Barbian KD, Lei B, Hoff JS, Mammarella
ND, Liu MY, Smoot JC, Porcella SF, Parkins LD, Campbell
DS, Smith TM, McCormick JK, Leung DY, Schlievert PM,
Musser JM. Genome sequence of a serotype M3 strain of
group A Streptococcus: phage-encoded toxins, the high-
virulence phenotype, and clone emergence. Proc Natl Acad
Sci USA 2002: 99: 1007810083.
7. Bolstad AI, Jensen HB, Bakken V. Taxonomy, biology,
and periodontal aspects of Fusobacterium nucleatum.
Clin Microbiol Rev 1996: 9: 5571.
8. Centurion-Lara A, LaFond RE, Hevner K, Godornes C, Mo-
lini BJ, Van Voorhis WC, Lukehart SA. Gene conversion: a
mechanism for generation of heterogeneity in the tprK
gene of Treponema pallidum during infection. Mol Micro-
biol 2004: 52: 15791596.
9. Chen T, Hosogi Y, Nishikawa K, Abbey K, Fleischmann RD,
Walling J, Duncan MJ. Comparative whole-genome analy-
sis of virulent and avirulent strains of Porphyromonas
gingivalis. J Bacteriol 2004: 186: 54735479.
10. Chen W, Laidig KE, Park Y, Park K, Yates JR 3rd, Lamont RJ,
Hackett M. Searching the Porphyromonas gingivalis gen-
ome with peptide fragmentation mass spectra. Analyst
2001: 126: 5257.
11. Coleman N, Tetu S, Wilson N, Holmes A. An unusual
integron in Treponema denticola. Microbiology 2004: 150:
35243526.
12. DiRienzo JM, Porter-Kaufman J, Haller J, Rosan B. Corn-
cob formation: a morphological model for molecular
studies of bacterial interactions. In: Mergenhagen SE,
Rosan B, editors. Molecular Basis of Oral Microbial
Adhesion. Washington DC: American Society for Micro-
biology, 1985: 172176.
13. Doolittle WF. Lateral genomics. Trends Cell Biol 1999: 9:
M5M8.
14. Fleischmann RD, Adams MD, White O, Clayton RA, Kirk-
ness EF, Kerlavage AR, Bult CJ, Tomb JF, Dougherty BA,
Merrick JM, et al. Whole-genome random sequencing and
assembly of Haemophilus inuenzae Rd. Science 1995: 269:
496512.
15. Galperin MY. The Molecular Biology Database Collection,
2004 update. Nucleic Acids Res 2004: 32 (Database issue):
D3D22.
16. Gill SR. Community genomics of the human oral microbi-
ome, 2004 [WWW document]. http://crisp.cit.nih.gov.
17. Graves PR, Haystead TA. Molecular biologists guide to
proteomics. Microbiol Mol Biol Rev 2002: 66: 3963.
18. Haake SK, Yoder SC, Attarian G, Podkaminer K. Native
plasmids of Fusobacterium nucleatum: characterization
and use in development of genetic systems. J Bacteriol
2000: 182: 11761180.
19. Hacker J, Kaper JB. Pathogenicity islands and the evolution
of microbes. Annu Rev Microbiol 2000: 54: 641679.
20. Hartley DL, Jones KR, Tobian JA, LeBlanc DJ, Macrina FL.
Disseminated tetracycline resistance in oral streptococci:
implication of a conjugative transposon. Infect Immun
1984: 45: 1317.
21. Hayashi T, Makino K, Ohnishi M, Kurokawa K, Ishii K,
Yokoyama K, Han CG, Ohtsubo E, Nakayama K, Murata T,
Tanaka M, Tobe T, Iida T, Takami H, Honda T, Sasakawa C,
Ogasawara N, Yasunaga T, Kuhara S, Shiba T, Hattori M,
Shinagawa H. Complete genome sequence of enter-
ohemorrhagic Escherichia coli O157:H7 and genomic
comparison with a laboratory strain K)12. DNA Res 2001: 8:
1122.
22. Hespell RB, Canale-Parola E. Amino acid and glucose fer-
mentation by Treponema denticola. Arch Mikrobiol 1971:
78: 234251.
23. Ito T, Chiba T, Ozawa R, Yoshida M, Hattori M, Sakaki Y. A
comprehensive two-hybrid analysis to explore the yeast
protein interactome. Proceedings of the Natl Acad Sci U S A
2001: 98: 45694574.
24. Kapatral V, Anderson I, Ivanova N, Reznik G, Los T, Lykidis
A, Bhattacharyya A, Bartman A, Gardner W, Grechkin G,
Zhu L, Vasieva O, Chu L, Kogan Y, Chaga O, Goltsman E,
Bernal A, Larsen N, DSouza M, Walunas T, Pusch G,
Haselkorn R, Fonstein M, Kyrpides N, Overbeek R. Genome
sequence and analysis of the oral bacterium Fusobacterium
nucleatum strain ATCC 25586. J Bacteriol 2002: 184: 2005
2018.
25. Kapatral V, Ivanova N, Anderson I, Reznik G, Bhattacharyya
A, Gardner WL, Mikhailova N, Lapidus A, Larsen N,
DSouza M, Walunas T, Haselkorn R, Overbeek R, Kyrpides
N. Genome analysis of F. nucleatum sub spp vincentii and
its comparison with the genome of F. nucleatum ATCC
25586. Genome Res 2003: 13: 11801189.
26. Kelley BP, Yuan B, Lewitter F, Sharan R, Stockwell BR,
Ideker T. PathBLAST: a tool for alignment of protein
interaction networks. Nucleic Acids Res 2004: 32 (Web
Server issue): W83W88.
27. Kolenbrander PE. Intergeneric coaggregation among
human oral bacteria and ecology of dental plaque. Annu
Rev Microbiol 1988: 42: 627656.
28. Kolenbrander PE. Oral microbial communities: biolms,
interactions, and genetic systems. Annu Rev Microbiol
2000: 4: 413437.
29. Kolenbrander PE, Andersen RN. Multigeneric aggregations
among oral bacteria: a network of independent celltocell
interactions. J Bacteriol 1986: 168: 851859.
30. Kolenbrander PE, London J. Adhere today, here tomor-
row: oral bacterial adherence. J Bacteriol 1993: 175: 3247
3252.
70
Duncan
31. Lancy PJr, Dirienzo JM, Appelbaum B, Rosan B, Holt SC.
Corncob formation between Fusobacterium nucleatum and
Streptococcus sanguis. Infect Immun 1983: 40: 303309.
32. Lawrence JG, Roth JR. Selsh operons: horizontal transfer
may drive the evolution of gene clusters. Genetics 1996:
143: 18431860.
33. Len AC, Harty DW, Jacques NA. Stress-responsive proteins
are upregulated in Streptococcus mutans during acid tol-
erance. Microbiology 2004: 150: 13391351.
34. Len AC, Harty DW, Jacques NA. Proteome analysis of
Streptococcus mutans metabolic phenotype during acid
tolerance. Microbiology 2004: 150: 13531366.
35. Li YH, Lau PC, Lee JH, Ellen RP, Cvitkovitch DG. Natural
genetic transformation of Streptococcus mutans growing in
biolms. J Bacteriol 2001: 83: 897908.
36. Nelson KE, Fleischmann RD, DeBoy RT, Paulsen IT, Fouts
DE, Eisen JA, Daugherty SC, Dodson RJ, Durkin AS, Gwinn
M, Haft DH, Kolonay JF, Nelson WC, Mason T, Tallon L,
Gray J, Granger D, Tettelin H, Dong H, Galvin JL, Duncan
MJ, Dewhirst FE, Fraser CM. Complete genome sequence
of the oral pathogenic bacterium Porphyromonas gingivalis
strain W83. J Bacteriol 2003: 185: 55915601.
37. Paster BJ, Boches SK, Galvin JL, Ericson RE, Lau CN, Lev-
anos VA, Sahasrabudhe A, Dewhirst FE. Bacterial diversity
in human subgingival plaque. J Bacteriol 2001: 183: 3770
3783.
38. Perna NT, Plunkett G 3rd, Burland V, Mau B, Glasner JD,
Rose DJ, Mayhew GF, Evans PS, Gregor J, Kirkpatrick HA,
Posfai G, Hackett J, Klink S, Boutin A, Shao Y, Miller L,
Grotbeck EJ, Davis NW, Lim A, Dimalanta ET, Potamousis
KD, Apodaca J, Anantharaman TS, Lin J, Yen G, Schwartz
DC, Welch RA, Blattner FR. Genome sequence of enter-
ohaemorrhagic Escherichia coli O157: H7. Nature 2001:
409: 529533.
39. Quivey RG, Kuhnert WL, Hahn K. Genetics of acid adapta-
tion in oral streptococci. Crit Rev Oral Biol Med 2001: 12:
301314.
40. Rain JC, Selig L, De Reuse H, Battaglia V, Reverdy C, Simon
S, Lenzen G, Petel F, Wojcik J, Schachter V, Chemama Y,
Labigne A, Legrain P. The proteinprotein interaction map
of Helicobacter pylori. Nature 2001: 409: 211215.
41. Roberts AP, Pratten J, Wilson M, Mullany P. Transfer of a
conjugative transposon, Tn5397 in a model oral biolm.
FEMS Microbiol Lett 1999: 177: 6366.
42. Rondon MR, August PR, Bettermann AD, Brady SF, Gross-
man TH, Liles MR, Loiacono KA, Lynch BA, MacNeil IA,
Minor C, Tiong CL, Gilman M, Osburne MS, Clardy J,
Handelsman J, Goodman RM. Cloning the soil metage-
nome: a strategy for accessing the genetic and functional
diversity of uncultured microorganisms. Appl Environ
Microbiol 2000: 66: 25412547.
43. Rother M, Bock A, Wyss C. Selenium-dependent growth of
Treponema denticola: evidence for a clostridial-type gly-
cine reductase. Arch Microbiol 2001: 177: 113116.
44. Salyers AA, Gupta A, Wang Y. Human intestinal bacteria as
reservoirs for antibiotic resistance genes. Trends Microbiol
2004: 12: 412416.
45. Seshadri R, Myers GS, Tettelin H, Eisen JA, Heidelberg JF,
Dodson RJ, Davidsen TM, DeBoy RT, Fouts DE, Haft DH,
Selengut J, Ren Q, Brinkac LM, Madupu R, Kolonay J,
Durkin SA, Daugherty SC, Shetty J, Shvartsbeyn A, Gebre-
georgis E, Geer K, Tsegaye G, Malek J, Ayodeji B, Shatsman
S, McLeod MP, Smajs D, Howell JK, Pal S, Amin A, Vashisth
P, McNeill TZ, Xiang Q, Sodergren E, Baca E, Weinstock
GM, Norris SJ, Fraser CM, Paulsen IT. Comparison of the
genome of the oral pathogen Treponema denticola with
other spirochete genomes. Proc Natl Acad Sci U S A 2004:
101: 56465651.
46. Strong M, Graeber TG, Beeby M, Pellegrini M, Thompson
MJ, Yeates TO, Eisenberg D. Visualization and interpret-
ation of protein networks in Mycobacterium tuberculosis
based on hierarchical clustering of genome-wide func-
tional linkage maps. Nucleic Acids Res 2003: 31: 70997109.
47. Suchett-Kaye G, Decoret D, Barsotti O. Intra-familial dis-
tribution of Fusobacterium nucleatum strains in healthy
families with optimal plaque control. J Clin Periodontol
1999: 26: 401404.
48. Tyson GW, Chapman J, Hugenholtz P, Allen EE, Ram RJ,
Richardson PM, Solovyev VV, Rubin EM, Rokhsar DS,
Baneld JF. Community structure and metabolism through
reconstruction of microbial genomes from the environ-
ment. Nature 2004: 428: 3743.
49. Uetz P, Giot L, Cagney G, Manseld TA, Judson RS, Knight
JR, Lockshon D, Narayan V, Srinivasan M, Pochart P,
Qureshi-Emili A, Li Y, Godwin B, Conover D, Kalbeisch T,
Vijayadamodar G, Yang M, Johnston M, Fields S, Rothberg
JM. A comprehensive analysis of proteinprotein interac-
tions in Saccharomyces cerevisiae. Nature 2000: 403: 623
627.
50. Venter JC, Remington K, Heidelberg JF, Halpern AL, Rusch
D, Eisen JA, Wu D, Paulsen I, Nelson KE, Nelson W, Fouts
DE, Levy S, Knap AH, Lomas MW, Nealson K, White O,
Peterson J, Hoffman J, Parsons R, Baden-Tillson H,
Pfannkoch C, Rogers YH, Smith HO. Environmental gen-
ome shotgun sequencing of the Sargasso Sea. Science 2004:
304: 6674.
51. Wang BY, Chi B, Kuramitsu HK. Genetic exchange between
Treponema denticola and Streptococcus gordonii in bio-
lms. Oral Microbiol Immunol 2002: 17: 08112.
52. Wang T, Zhang Y, Chen W, Park Y, Lamont RJ, Hackett M.
Reconstructed protein arrays from 3D HPLC/tandem mass
spectrometry and 2D gels: complementary approaches to
Porphyromonas gingivalis protein expression. Analyst 2002:
127: 14501456.
53. Waters VL. Conjugative transfer in the dissemination of
beta-lactam and aminoglycoside resistance. Front Biosci
1999: 4: D433D456.
54. Whittle G, Hund BD, Shoemaker NB, Salyers AA. Charac-
terization of the 13-kilobase ermF region of the Bacteroides
conjugative transposon CTnDOT. Appl Environ Microbiol
2001: 67: 34883495.
55. Wilkins JC, Homer KA, Beighton D. Analysis of Streptococ-
cus mutans proteins modulated by culture under acidic
conditions. Appl Environ Microbiol 2002: 68: 23822390.
71
Oral microbiology and genomics