Endodontic Topics 2004, 9, 37–51 Copyright r Blackwell Munksgaard

All rights reserved ENDODONTIC TOPICS 2004

1601-1538

Bacterial plasmids in the oral and

endodontic microflora
CHRISTINE SEDGLEY & DON B. CLEWELL

Bacterial plasmids typically represent covalently closed circular, double-stranded, supercoiled DNA molecules that
replicate independently of chromosomal DNA. Plasmids are frequently not essential to the bacterial host, but can
confer traits facilitating survival under atypical conditions e.g. resistance to antibiotics and heavy metals. Plasmids
have been identified in clinical and environmental bacteria from all over the world, and range in size from  1 to
4200 kb. Their copy number in the cell can range from one or two copies per chromosome, to well over 30; the
number is usually characteristic for the particular plasmid. Clinical strains of Enterococcus faecalis, a species
commonly associated with root canal infections, can carry as many as six co-resident plasmids with different sizes
and copy numbers. In part because of their ability to replicate autonomously, plasmids are used as tools or shuttle
vectors in basic molecular biological research. Many plasmids can transfer copies of themselves from one bacterial
cell to another by a process called conjugation. In some cases, small peptides called pheromones act as mating
signals in this process. Conjugative plasmids that make use of pheromones have been identified previously in
E. faecalis and the related plasmids tend to exhibit a narrow host range. Conjugative plasmids in E. faecalis that do
not make use of pheromones usually have a much broader host range. Pheromone-responding plasmids in E.
faecalis can carry genes relating to antibiotic resistance as well as virulence traits. In general, plasmids are capable of
adaptability and stable maintenance in a wide spectrum of bacterial hosts. The potential role of plasmids in oral and
endodontic microbiology is discussed.

Chromosomal DNA in bacteria usually corresponds to
a single large circular molecule and carries genes
necessary for cell survival and replication. Bacteria also
carry accessory genetic elements, collectively described
as the ‘horizontal gene pool’ (1). These include
plasmids (Fig. 1), transposons, and insertion sequences
(IS sequences), which, while not necessarily essential to
the cell’s survival, have the potential to profoundly
influence genome plasticity and evolution, playing an
integral role in the movement of genetic information
between different bacteria.
Plasmids represent extrachromosomal, autono-
mously replicating elements important for bacterial
adaptability and survival because they frequently
provide functions that might not be encoded by the
chromosome. For example, plasmids can carry genes
that code for antibiotic resistance as well as genes that
can enable the transfer of such genes from one bacterial
cell to another. In certain species, for example,
Enterococcus faecalis, the transfer of plasmids can be
greatly enhanced by small peptides called sex phero-
mones, which are secreted by a potential ‘recipient’ cell
and that ‘activate’ the transfer system of a potential
‘donor’ cell.
Transposons, or ‘jumping genes’, which are seg-
ments of DNA able to move from one location to
another, can move (‘jump’) from one DNA molecule
to another – for example, from the chromosome to a
resident plasmid. Transposons encode functions ne-
cessary for transposition and other functions (e.g.
antibiotic resistance), and frequently accumulate on
plasmids. IS sequences usually encode the ability to
transpose but do not carry accessory genes like
transposons do; however, IS sequences sometimes
represent components of a transposon (for reviews, see
(2, 3)).

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Sedgley & Clewell

Fig. 1. DNA in a bacterial cell. While chromosomal DNA predominates, more than one type of plasmid can occur stably
in a given cell provided they are compatible. Larger plasmids (e.g. 425 kb) are usually present as one to two copies per
chromosome, but the smaller plasmids (e.g. o10 kb) can be 430 copies.

Table 1. Glossary of terms

Horizontal gene pool: DNA with potential to transfer between bacteria by conjugation, transformation or transduction. Includes
plasmids, transposons, insertion sequences, bacteriophage genomes, as well as chromosomal DNA

Plasmids: DNA molecules distinct from chromosomal DNA that can replicate autonomously. Not necessarily essential for their
bacterial host survival except perhaps under atypical conditions (e.g. when carrying antibiotic resistance genes). Usually named by
using a designation preceded by the letter ‘p’, e.g. pAD1

Conjugation: Unidirectional transfer of DNA from a donor to potential recipient cells that are in physical contact. A recipient that
has received DNA from a donor is called a transconjugant

Conjugative plasmid: Self-transmissible plasmid that encodes all the functions needed for its own intercellular transmission by
conjugation

Bacterial sex pheromones: Small peptides secreted by potential recipient cells which activate the transfer system of a potential donor
cell

This review will first briefly provide an overview of
how DNA is exchanged between bacteria in nature, and
then discuss bacterial plasmids with some emphasis on
the role played by sex pheromones in the transfer of
plasmids between bacterial cells. The potential role of
plasmids in oral and endodontic microbiology, an area
of research not widely studied, will then be considered.
Table 1 lists important definitions.
Gene transfer systems in bacteria
Bacteria can transfer DNA by three basic methods:
transformation, transduction, and conjugation; ‘do-
nor’ cells are the source of the DNA while ‘recipient’
cells receive the DNA.
Natural genetic transformation of bacteria involves
the active uptake by a cell of free (extracellular) DNA
followed by its incorporation into the recipient genome
giving rise to ‘transformants’ (i.e. the recipient cell
becomes ‘transformed’). At a particular stage of
growth, the bacteria become ‘competent’, i.e. more
receptive in taking up donor DNA and thereby
becoming ‘transformed’. Uptake by the recipient
usually involves only ‘fragments’ of genomic DNA,
released from donor cells that may have lysed. The
process depends on the function of several genes

38
 Bacterial plasmids

located on the recipient’s chromosome (for a review of
natural transformation, see (4)). Natural transforma-
tion has been observed in the oral Gram-positive
bacteria Streptococcus mutans in biofilms (5) and
Streptococcus gordonii Challis (6) and has been studied
in great detail in Streptococcus pneumoniae (7).
Acquired DNA may correspond to intact plasmids,
able to replicate autonomously in the recipient, or as
fragments of DNA that recombine with homologous
regions of the recipient genome. Not all bacteria are
capable of natural transformation. Many that do not
achieve natural competence (i.e. a physiological state in
which DNA is taken up) can still be transformed in the
laboratory using artificial techniques. Essentially, donor
cells are broken open (‘lysed’) and the DNA isolated
and purified. Recipient cells are specially treated to
make them more receptive (‘competent’) in taking up
the donor DNA. Electroporation is a commonly used
in vitro transformation technique. It uses an electrical
pulse that facilitates the uptake of free, extracellular
DNA (Fig. 2). Successfully transformed cells will grow
on media selective for the phenotype expressed by a
gene marker on the newly acquired DNA (e.g. an
antibiotic resistance marker on a plasmid being used as
a vector in genetic manipulations).
Transduction involves bacterial viruses (called ‘bac-
teriophages’ or ‘phages’) as mediators of gene transfer
between bacteria. During the lytic phase of a viral
infection, some phage particles are packaged with
segments of bacterial DNA, which in turn may be
delivered to a potential recipient cell giving rise to
‘transductants’. Bacteriophages have been isolated
from dental plaque specific for oral bacteria such as
Actinobacillus actinomycetemcomitans (8) and Actino-
myces spp. (9), and from saliva samples specific for E.
faecalis (10) (for more information on bacteriophages,
see (11, 12)).
The third and most efficient genetic transfer system in
bacteria is conjugation, whereby DNA is transferred
between cells that are in physical (‘cell-to-cell’) contact
so that there is unidirectional transfer of genetic
information from donor to recipient. The requirement
for cell-to-cell contact distinguishes conjugation from
transduction and transformation. As such, it can
involve plasmids, chromosomal DNA, and conjugative
transposons (transposons that include genes enabling
their inter-cellular transfer). Conjugation can involve
the crossing of species barriers and can even occur
between bacteria and eukaryotic cells (13), to be
discussed later. Cells that have acquired DNA from a

Fig. 2. Transformation by electroporation. Donor cells are broken open and the plasmid DNA isolated and purified and
combined with specially treated recipient cells (‘competent’ cells). An electrical pulse is used to facilitate the uptake of
free, extracellular DNA by the recipient cells. Successfully transformed cells will grow on a medium selective for the
phenotype expressed by a gene marker on the newly acquired DNA (e.g. an antibiotic resistance marker). Analysis of
extracted plasmid from the purified transformed cells confirms that plasmid transfer occurred.

39
Sedgley & Clewell

donor by conjugation are called ‘transconjugants’.
Conjugative plasmids will be discussed further below.
Nature and properties of plasmids
In 1952, Joshua Lederberg introduced ‘plasmid’ as a
generic term for any extrachromosomal genetic parti-
cle. The word was constructed as a hybrid of
‘cytoplasm’ or ‘plasmagene’ and ‘-id’, as in plastid,
chromatid (14). A plasmid is usually named using a
designation (often reflecting an associated laboratory)
preceded by the letter ‘p’ (e.g. for the plasmid pAMb1,
‘A’ refers to Ann Arbor, ‘M’ refers to Michigan).
In the 1950s, in Japan, it was observed that multiple
antibiotic resistance was associated with Shigella
isolates from dysentry patients. In some cases, co-
isolated strains of Escherichia coli were also resistant and
the ability to transfer these traits between E. coli and
Shigella illustrated their characteristic genetic mobility
(15). Following cell contact, multiple drug resistance
(streptomycin, chloramphenicol, tetracycline, and sul-
fonamide) was transferred via what was then termed ‘R’
(resistance) factors from naturally occurring multiply
drug-resistant Shigella to E. coli, and Salmonella spp.
(16). Significantly, it was found that the R-factors
(plasmids) had both replication and transfer determi-
nants (17), indicating that part of the element coded
for self-maintenance. This was crucial to the realization
that R-factors were ‘extrachromosomal’. Meanwhile, in
Europe, observations showing that the production of
antibiotic substances called ‘bacteriocins’ by Entero-
bacteriaceae that killed other bacteria were frequently
encoded by plasmids (18).
It is now well established that plasmids are found in
Gram-negative and Gram-positive bacteria as well as in
archaea (19) and some yeasts, e.g. Saccharomyces
cerevisiae and other fungi (20, 21). Plasmids can also
exist ‘naturally’ in mammalian cells, for example the
double-stranded circular plasmids encoding the Ep-
stein–Barr virus, Karposi’s sarcoma-associated herpes-
virus, and papillomavirus (22). Plasmids are ubiquitous
in the bacterial world and may encode a variety of
different traits. It is not always clear which genes are
encoded on wild-type plasmids. Plasmids gain signifi-
cant notoriety from the fact that many of them carry a
series of genes that specifically enable them to transfer
copies of themselves to recipient bacteria. Such
plasmids are widespread in nature, and, as noted above,
are involved in the dissemination of antibiotic resis-
tance. Many plasmids have now had their DNA
sequenced and specific genes have been identified.
Figure 3 shows a simplified ‘map’ of pAD1, the
conjugative plasmid originally isolated from a clinical
isolate of E. faecalis. It should be noted that, while

Fig. 3. Simplified map of the conjugative plasmid pAD1 originally isolated from Enterococcus faecalis DS16. Segments are
described according to the functions encoded by genes contained within: (1) replication and maintenance; (2) regulation
of pheromone response; (3) structural genes relating to conjugation; (4) unknown; (5) cytolysin biosynthesis; (6)
unknown; (7) resistance to UV light. oriV, origin of replication; oriT, origin of transfer. Based on a figure from (108).
Reproduced with permission from Elsevier.

40

plasmids are commonly thought of as circular in nature,
linear plasmids have been isolated from actinomycetes
and streptomyces (23–25).
Nosocomial, or hospital-acquired, bacterial infec-
tions are often associated with the rapid spread of
antibiotic resistance, traits that are encoded on
plasmids. Plasmid research has been driven by the
profoundly negative impact of antibiotic resistance on
health care management. Concomitantly, the study of
plasmids was greatly enhanced by advances in mole-
cular biology and technology. A vital step in the early
stages of plasmid research was the development of a
method that used ethidium bromide–cesium chloride
buoyant density gradients (26) that facilitated their
separation from chromosomal DNA based on their
supercoiled (covalently closed) circular structure. Iso-
lation became greatly simplified by a subsequently
developed ‘cleared lysate’ technique (27). The size of
plasmids can vary, but they are significantly smaller than
chromosomal DNA. For example, plasmids recovered
from enterococci can range in size from  1 to
4200 kb, in contrast to the chromosomal DNA size
of 3218 kb, recently reported for E. faecalis V583 (28).
The number of copies of a given plasmid is generally a
characteristic property of the particular element. The
larger plasmids (e.g. 425 kb) usually are present as one
or two copies per chromosome; but the smaller
plasmids (e.g. o10 kb) can be 430 copies.
Although plasmids encode important traits related to
their autonomy, they rely on their host cell to provide
essential ingredients for DNA replication. Replication
of plasmids is not necessarily coupled to any specific
stage of the cell cycle but genes important to a balanced
distribution to daughter cells upon cell division
(‘partitioning’) are often present on the plasmid,
particularly on those existing at low copy number.
There are different replication mechanisms, including
the so-called rolling circle replication ubiquitous
among relatively small plasmids (29). Larger plasmids
usually replicate by a ‘theta’ mechanism (30); under
electron microscopy, the replication intermediates
typically are seen as theta-shaped molecules. Some
plasmids exhibit a broad host range able to propogate
in many different species of bacteria.
From the above, it is apparent that plasmids are a
diverse collection of genetic elements. In an effort to
provide some degree of organization, classification
systems have been developed. One commonly adopted
system classifies plasmids according to their ‘incompat-
Bacterial plasmids
ibility’ characteristics, i.e. whether the plasmid in
question can co-exist with other plasmids within the
same host. More than one type of plasmid can occur
stably in a given cell provided they are compatible (i.e.
are in different incompatibility, or ‘inc’, groups).
Incompatibility relates to a close relationship between
plasmids whereby there is competition for replication
machinery. Incompatibility relates to a necessary
consequence of the need for a plasmid to maintain a
certain copy number in the cell. Whichever plasmid is
able to replicate faster, or has some other advantage,
will eventually ‘kick out’ the competing element.
Bacterial conjugative plasmids
Conjugative plasmids can range in size from approxi-
mately 10 kb found in Streptomyces to well over 100 kb
(31, 32). Bacterial conjugation, in which DNA is
transferred from one bacterium to another, is an
energy-driven process. The array of genes transported
as a result of conjugation is extremely broad. Further,
conjugative plasmids can mobilize otherwise non-
transferable elements such as non-conjugative co-
resident plasmids and even chromosomal genes. Some
chromosome-borne transposons actually carry conju-
gative genes and although they cannot replicate
autonomously, they can excise from the chromosome,
generating a circular intermediate that can then
conjugate much like a plasmid. Such elements, known
as ‘conjugative transposons’, are widespread and like
plasmids, have played a major role in the dissemination
of antibiotic resistance (31, 33). One family of such
elements (the Tn916–Tn1545 family) is known to
transfer into well over 50 different species of bacteria
(34). Enteric Bacteroides species are known to have
conjugative transposons (35, 36). Once taken up by a
recipient, the element is believed to circularize prior to
inserting itself into the chromosomal DNA.
Bacterial conjugation was first reported in 1946 (37).
In experiments in the early 1950s, the ‘F’ (fertility)
factor was identified as controlling the transfer of genes
between strains of E. coli (38, 39). The ability to act as a
donor (in bacterial mating experiments) was a property
controlled by the ‘F-factor’. Transfer of the F-plasmid
involved establishment of cell-to-cell contact facilitated
by a plasmid-encoded ‘pilus’ structure that acts as a
‘grappling hook’ in binding and ‘pulling in’ the
recipient cell. A single strand of DNA is then
41
Sedgley & Clewell
Fig. 4. Conjugative transfer of plasmids in Gram-negative bacteria. Cell-to-cell contact is facilitated by a plasmid-
encoded pilus. A single strand of DNA is then transferred from the donor to the recipient. A complementary strand is
synthesized in the recipient and the resulting double-stranded DNA is circularized.
transferred to the recipient. A complementary strand is
synthesized in the recipient and the resulting double-
stranded DNA is circularized (Fig. 4). The F-factor is
also capable of integrating itself into the bacterial
chromosome, giving rise to a situation where chromo-
somal DNA can be transferred into a recipient
bacterium as well. Because of the large size of the
chromosome, usually only portions of it make it into
the recipient prior to breakage of the donor–recipient
union. That which is transferred may subsequently
recombine with homologous regions of recipient
DNA. Donors able to do this are known as high-
frequency recombination strains. Most information
available on conjugative plasmids or transposons in the
Gram-negative bacteria comes from work on members
of the Enterobacteriaceae family, particularly E. coli (32).
Because all or most of the events that take place
during conjugation are driven by the donor bacterium
and are typically plasmid encoded, other types of cells
can serve as the recipient. For example, conjugative
plasmid transfer from the prokaryote E. coli to a
eukaryote S. cerevisiae has been observed (40), as well
as to higher eukaryotes such as Chinese hamster ovary
cells (13). The Ti plasmids found in soil bacteria of the
Agrobacterium group are able to transfer genes into
plants that induce tumor formation (41).
Conjugative systems differ somewhat between Gram-
negative and Gram-positive bacteria perhaps due in
42
part to the inherent differences in structures of the cell
envelope. For example, Gram-positive hosts do not
make use of sophisticated structures, such as pili, to
achieve a close cell-to-cell contact (42). Earlier reports
where conjugative plasmids were found to be ubiqui-
tous in Gram-negative bacteria have been followed by
studies showing their presence in the Gram-positive
Enterococcus, Streptococcus, Staphylococcus, Lactococcus,
Listeria, Bacillus, Clostridium, Streptomyces, and Rho-
dococcus (31).
There are two significant groups of conjugative
plasmids in Gram-positive bacteria that have been
studied in some detail. Those such as pAMb1 and
pIP501 have a broad host range that includes many
Gram-positive species of bacteria. In the laboratory,
transfer of these plasmids is much more efficient when
matings are conducted on solid (agar) surfaces, in
contrast to broth. Members of the second group,
identified predominantly in E. faecalis, have a narrow
host range (e.g. pAD1, pCF10, pPD1, pAM373) and
encode recognition of recipient-produced peptide sex
pheromones (cAD1, cCF10, cPD1, cAM373, respec-
tively). In the laboratory, they transfer efficiently in
broth matings. (NB. A pheromone is usually named by
‘relating’ it to the plasmid whose transfer it initiates
preceded by the letter ‘c’. For example, cAD1 is the
pheromone to which pAD1 encodes a mating re-
sponse.)

Pheromone-initiated transfer of
plasmids
Pheromone-mediated conjugation was first observed in
E. faecalis (43, 44) and involved an oral strain
associated with marginal periodontitis. One E. faecalis
strain may secrete multiple pheromones, each specific
for inducing transfer of different families of plasmids.
Signals provided by pheromones are crucial to the
conjugation process resulting in transfer of the plasmid
DNA to a plasmid-free cell (Fig. 5). The pheromone
initiates a process that activates expression of conjuga-
tion functions, including a dramatic ‘clumping re-
sponse’ mediated by the appearance of a surface adhesin
called ‘aggregation substance’ (AS). This facilitates the
attachment of the donor cells to enterococcal binding
substance (EBS), which is present on the surface of
recipients as well as donors (44). ‘Aggregates’ formed
by AS-EBS then give rise to conjugal channels through
which the plasmid is transferred from the donor to the
recipient cell. Once the recipient cell has acquired the
plasmid, it assumes a ‘mating phenotype’ resembling
that of the original donor and shuts down the
production of endogenous pheromone; the transcon-
jugants, however, continue to produce pheromones
specific for donors harboring different classes of
plasmids (for recent reviews, see (31, 45)). As part of
the pheromone shut-down mechanism, a plasmid-
encoded ‘inhibitor peptide’ is secreted, which acts as a
specific competitor of any remaining endogenous
pheromone; this prevents self-induction of the donors.
Fig. 5. Pheromone-initiated conjugative plasmid transfer. The pheromone induces the appearance of a surface adhesin
(aggregation substance) that facilitates the attachment of the donor and recipient cells. Aggregates give rise to conjugal
channels through which the plasmid is transferred from the donor to the recipient cell.
Bacterial plasmids
Not all bacteria produce pheromones. For example,
the clinically important E. faecium is not known to
produce pheromone activity (31). Interestingly, a
peptide similar to the E. faecalis pheromone cAM373
has been detected in culture supernatants of Staphylo-
coccus aureus and S. gordonii; however, the use of
pheromone-initiated transfer of conjugative plasmids
within species other than E. faecalis has thus far not
been observed.
Antibiotic resistance and virulence
associated with plasmids
Plasmids may provide important survival properties to
their host; for example, by conferring resistance to an
antibiotic. Many plasmids encode other products such
as bacteriocins, toxins, adhesins, and special metabolic
enzymes (46). In hospitals where patients are taking
many antibiotics, the selective pressure on bacteria
containing plasmids carrying resistance genes is in-
tense; as a consequence, current antibiotics are losing
their effectiveness. Non-clinical practices such as the
feeding of antibiotics to farm animals to increase their
weight, or the rinsing of chickens in antibiotics to
increase their shelf-life in stores also contribute to the
selection and subsequent spread of antibiotic resistance
plasmids (47).
A particular concern relates to the emergence of
resistance to vancomycin, considered a ‘last resort’
antibiotic. Already prevalent in E. faecalis, there is fear
43
Sedgley & Clewell
that it will spread to S. aureus, with the potential for
catastrophic nosocomial infections. The scenario be-
came closer to reality in 2002 in Michigan, USA, with
the isolation of a high-level vancomycin-resistant S.
aureus strain from a patient also colonized by a resistant
E. faecalis (48, 49). In this case, it was fortunate that
the S. aureus strain was susceptible to another
antibiotic.
Plasmids can also carry genes for protecting a cell
against deleterious substances like mercury, copper, or
silver (50, 51), or they may carry genes that make it
possible for a cell to degrade or modify an unusual
substrate, such as a toxic hydrocarbon (52), allowing it
to be used as a nutrient or energy source, thereby
providing a survival advantage for their host. Some
plasmids provide genes that ultimately allow their host
to break down herbicides and industrial chemicals; this
property can be utilized in the management of
environmental pollution (53–55).
In other cases, plasmids carry genes that enhance the
bacterial host cell’s ability to cause a disease. One
recurring plasmid-based disease is associated with the
strain E. coli O157:H7, which has a 92 kb plasmid
(pO157) encoding a toxin that produces a severe
gastro-intestinal infection (afebrile hemorrhagic colitis)
with potentially life-threatening complications (56).
Virulence plasmids in S. aureus can encode a large
number of extracellular toxins that have been linked to
systemic shock – for example, enterotoxins associated
with food poisoning (57) and toxic shock syndrome
toxin (58).
Cytolysins produced by E. faecalis are capable of
lysing erythrocytes and other eukaryotic cells as well as
having the capacity to kill many species of Gram-
positive bacteria (59). E. faecalis cytolysins are more
likely to be associated with strains recovered from
clinical infections (60, 61) and have been shown to
enhance pathogenicity in animal models (62–65). In
addition, E. faecalis AS promotes resistance to killing
by human neutrophils in spite of phagocytosis and
neutrophil activation (66).
Some plasmid-encoded transfer functions may them-
selves play a role in bacterial virulence. For example, the
TraT protein is a cell-surface-exposed, outer membrane
lipoprotein specified by large, usually conjugative, F-
like plasmids in Gram-negative bacteria that resists the
bactericidal activities of serum (67).
The role of pheromones and pheromone inhibitors in
the activation of genes in mammalian cells has been less
44
well studied. Pheromones from E. faecalis, including
cAD1, were chemotactic for human neutrophils and
triggered superoxide production (68), while the
pheromone cAM373 was a potent chemotactic agent
for rat neutrophils and induced lysosomal granule
enzyme secretion (69).
Plasmids in the oral microflora
The very limited research involving plasmids associated
with oral microorganisms falls into two broad cate-
gories: (1) identification and transferability of plasmids
in oral species, especially oral streptococci, and (2)
epidemiological studies.
The first report of isolation and characterization of
plasmid DNA in oral streptococci related to S. mutans
LM-7, originally isolated from a human carious lesion
(70). Soon after, it was shown that a low copy number
plasmid from a Streptococcus pyogenes isolate from an
inflamed throat conferred resistance to erythromycin
(71). Similarly, a small plasmid, also with genes
determining resistance to erythromycin, was isolated
from Streptococcus sanguis from dental plaque; further,
this plasmid could be transferred into the plasmid-free
S. sanguis strain Challis (now designated S. gordonii) by
transformation (72). Later it was shown that oral
streptococci exchanged genetic information, including
plasmids bearing antibiotic markers, between them-
selves during mixed growth (73). A conjugative
transposon (Tn919) was also identified in a strain of
S. sanguis (74). More recently, in vitro transformation
of the naturally transformable S. gordonii occurred
both in broth and in biofilms with pKMR4PE DNA or
Treponema denticola harboring pKMR4PE as donor
sources, providing direct experimental evidence that
gene transfer can occur from T. denticola to S. gordonii
(6).
In an epidemiological study of S. mutans strains from
100 pedodontic patients, 13% contained a similar small
plasmid (75). The preservation of S. mutans strains
within a mother–child cohort by using a genotypic
marker, plasmid DNA showed that plasmid-containing
strains were significantly clustered in mother–child
pairs compared with non-related individuals (76). In
another study, a search for extrachromosomal elements
in 300 mutans streptococcal strains, isolated from
dental plaque, yielded only one 5.7 kb plasmid
(pJEB110) (77). Conceivably, differences in sampling

and plasmid extraction methods could account for
these disparities.
Analysis of 85 strains of bacterial species selected from
the predominant culturable dental plaque flora of
patients with different periodontal pathologies showed
plasmids in three Bacteroides melaninogenicus isolates
but none in Actinomyces species (78). Another study
proposed that the presence of the same plasmid in
different species of treponemes isolated from the same
patient suggested a naturally occurring genetic transfer
system within the oral spirochetes (79). Plasmids were
only found in a few Prevotella nigrescens strains from the
periodontium. No association was found between the
disease status of sampling sites and the presence of a
plasmid (80). Of 45 Fusobacterium nucleatum strains
from periodontal patients and 38 from healthy subjects,
plasmids were found in 26.7% of strains from patients
but not at all in healthy subjects; the plasmid profile was
described as similar (81).
The presence of a plasmid from E. faecalis ND539
from a carious lesion conferred sensitivity to a
bacteriocin produced by another clinical isolate (82).
Recently, plasmids were detected in seven of 11 E.
faecalis strains in oral rinse samples from 100 endo-
dontic patients (83).
Plasmids in the endodontic
microflora
There are even fewer data published relating to
plasmids associated with endodontic microbiology,
and these appear to be limited to E. faecalis. E. faecalis
has been recovered from root canals of failed endo-
dontically treated teeth in culture studies (84–86) and
using PCR-based methods of detection (87). The
mechanisms by which E. faecalis survives in or enters
the root canal despite endodontic treatment are not
fully understood (for a review, see reference (88)), but
as discussed above, plasmids known to contribute to
the dissemination of anti-microbial resistance are
commonly detected in these organisms.
Investigations of a 60 kb non-conjugative plasmid
from E. faecalis JG2, a multiple antibiotic resistant
isolate recovered from the periapical exudate of a failed
endodontically treated maxillary central incisor under-
going retreatment and periapical surgery (89), were
described in a preliminary report (90). In broth
dilution assays, E. faecalis JG2 was resistant to
Bacterial plasmids
tetracycline (Tc; MIC 250 mg/mL) and streptomycin
(Sm; MIC41 mg/mL). It was found that the plasmid
isolated from JG2 encoded these traits and could be
transferred by electroporation into the plasmid-free E.
faecalis strain JH2-2.
A more detailed study was carried out of 33
endodontic enterococcal isolates (comprising 31 E.
faecalis and two E. faecium) recovered from patients in
Gothenburg, Sweden (91). Plasmid DNA was detected
in 25 strains, with up to four plasmids per strain and a
similar (5.1 kb) plasmid occurring in 16 E. faecalis
isolates. Interestingly, several strains that appeared to
be clones based on pulsed field gel electrophoresis
analyses of total DNA were shown to have distinct
plasmid types (Fig. 6). Further, 16 of the plasmid-
positive strains exhibited a ‘clumping response’ (char-
acteristic of a response to pheromone) when exposed to
a culture filtrate of a plasmid-free strain (E. faecalis
JH2-2), suggesting the potential for conjugative
transfer of genetic elements.
From the above, it could be speculated that, if
endodontic strains contain conjugative plasmids that
enhance survival during or after endodontic treatment
‘procedures’, such properties might be transferred to
other enterococcal strains.
The exploitation of plasmids for
research purposes
This final section provides a brief overview of how
plasmids can be used in research, a topic not commonly
addressed in the dental literature. For more details, see
(92, 93).
In the early 1970s, it was realized that plasmids could
serve as valuable tools in molecular genetic research and
biotechnology, through their use in identifying restric-
tion enzymes and serving as cloning vectors in the new
recombinant DNA technology. Small naturally occur-
ring plasmids were modified for use as vectors with
convenient ‘markers’ and restriction sites into which
fragments containing desired portions of a particular
genome could be inserted (‘ligated’ or ‘spliced’).
Antibiotic resistance markers facilitated the artificial
transformation of E. coli recipients. In addition to a
gene encoding antibiotic resistance, another conveni-
ent marker involves a trait that, when expressed by the
transformed bacterium, causes a characteristic color
45
Sedgley & Clewell

Fig. 6. (A) Total DNA and (B) Plasmid DNA analysis of endodontic Enterococcus faecalis. (A) Pulsed field gel
electrophoresis (PFGE) of SmaI-digested genomic DNA from endodontic E. faecalis strains GS3–GS7, GS12, GS13,
GS21, GS22. Note similarities between GS3–GS7, GS12, and GS21. Reference standard: lambda phage DNA. (B)
Plasmid analysis of the same E. faecalis isolates. Lane M, molecular size marker (1 kb Plus DNA Ladder, Invitrogen); - -,
undigested; H, digested with HindIII. Strain designations are shown above the lane designations. Isolates classified
based on PFGE pattern as clonal, GS3, GS12, and GS21 are similar in plasmid content. GS4 and GS5 appear to be alike
in plasmid content. GS6 and GS7 each contain two similar small plasmids; however, GS6 has two additional plasmids.
Adapted from reference (91). Reproduced with permission from Blackwell Publishing.

46

change of colonies on a specific medium (e.g. blue/
white selection associated with the lacZ system).
Commercially available kits allow rapid extraction of
plasmids from Gram-negative bacteria. In contrast,
plasmid extraction from Gram-positive bacteria (e.g. E.
faecalis) is more problematic due in part to the greater
difficulty in lysing, or breaking open, the thick
peptidoglycan layer of the Gram-positive cell wall.
The Gram-negative bacterium E. coli is the most
commonly used ‘host’ with the gene(s) of interest
being ‘delivered’ by the plasmid. Commercially avail-
able plasmids have generally been sequenced in their
entirety and their genes are well characterized. A useful
vector has structural and segregational stability as well
as an appropriate plasmid copy number. Copy number
will determine the gene dosage and may therefore
influence the level of expression (94).
Plasmid vectors are involved in diverse applications
and also contribute to understanding basic details of
DNA replication, topology, partitioning, and the
movement of transposable elements. Plasmids have
been crucial tools for the construction of DNA libraries
that can in turn serve as a starting point for whole
genome sequencing. Isolated genes or gene systems
cloned on given plasmid vectors also can be conveni-
ently studied with respect to factors that control
expression; and mutants can be readily generated and
studied. Many protein expression systems in research
and industry use plasmids as carriers of genes for the
mass production of (recombinant) proteins or non-
proteinaceous substances.
Future applications for plasmids with far-reaching
clinical potential for human gene therapy may exploit
the ability of intra-cellular bacteria (i.e. prokaryotes) to
transfer eukaryotic expression plasmids to mammalian
host cells. For example, a plasmid can be placed into an
attentuated (non-virulent) bacterium capable of invad-
ing mammalian target cells after which it dies, thereby
releasing its DNA into the mammalian cell (95). Such
plasmids might be used to introduce vaccines (96).
While several practical issues have yet to be overcome,
use of the oral route for plasmid DNA vaccine delivery
systems for immunization is in development (97). In
the rat model, successful outcomes using intra-nasal
mucosal immunization against dental caries with
plasmid DNA encoding the pac gene of S. mutans
have already been reported (98). At this stage, it is
difficult to envision a vaccine against endodontic
infections in view of their diverse polymicrobial nature.
Bacterial plasmids
Concluding remarks and future
directions
Plasmids exhibit diverse mechanisms that enable their
survival and horizontal transfer, to the extent that, with
the advent of more sophisticated detection techniques,
the distinction between chromosome and plasmid has
become ‘blurred’ (99).
It has been suggested that medically relevant plasmid-
bearing strains are more likely to form a biofilm (100),
representing microbial societies with their own defense
and communication systems (101). This mode of
growth is associated with the chronic nature of the
subsequent infections and with an inherent resistance
to antibiotic chemotherapy, applicable to periodontal
and endodontic infections. For example, formation of
E. faecalis biofilms in root canals medicated with
calcium hydroxide was shown using scanning electron
microscopy (102). Even though the probability of
random meetings of cells in suspension is greater than
in biofilms, the relative spatial stability of bacteria in
biofilms should favor conjugation (103). A recent
review reported that gene transfer takes place within
biofilms, but with ‘bottlenecks’ in the process (104).
Nonetheless, when bacteria are dispersed from a
biofilm they usually rapidly become susceptible to
antibiotics, which suggests that resistance of bacteria in
biofilms is not acquired via mutations or mobile genetic
elements (105). The significance of these findings
regarding gene transfer between microorganisms in
endodontic infections, which typically exists in a non-
planktonic (i.e. not free-floating) state, remains to be
established.
In the endodontic literature, numerous recent studies
using molecular methods have demonstrated that the
flora associated with endodontic infections is much
more diverse than was previously found in studies using
conventional culture methods. These findings are not
unexpected, considering that less than 1% of environ-
mental microbial species enumerated by direct micro-
scopic counts are considered culturable (106, 107).
However, with the current trend toward exclusive use
of molecular methods to directly detect and identify
species in root canal samples, it is important to note
that these techniques analyze total DNA extracted from
the sample (i.e. comprising pooled chromosomal and
non-chromosomal DNA) and in the process destroy
the microoorganisms. Thereafter, being no longer
culturable, it is not possible to study phenotypic
47
Sedgley & Clewell
characteristics and potential pathogenicity associated
with individual strains recovered from the root canal. It
would seem that a combination of both molecular and
culture techniques will be required to provide a more
comprehensive understanding of the endodontic in-
fection process. The intriguing question remains as to
what is the impact of virulence factors expressed by
plasmids in microorganisms in endodontic infections.
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