APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 2011, p. 4573–4578
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 77, No. 13
Disruption of a Type II Endonuclease (TDE0911) Enables
Treponema denticola ATCC 35405 To Accept an
Unmethylated Shuttle Vector?
Jiang Bian and Chunhao Li*
Department of Oral Biology, The State University of New York at Buffalo, Buffalo, New York 14214
Received 24 February 2011/Accepted 5 May 2011
The oral spirochete Treponema denticola is associated with human periodontal disease. T. denticola ATCC
35405 and ATCC 33520 are two routinely used laboratory strains. Compared to T. denticola ATCC 33520, ATCC
35405 is more virulent but less accessible to genetic manipulations. For instance, the shuttle vectors of
ATCC 33520 cannot be transformed into strain ATCC 35405. The lack of a shuttle vector has been a barrier
to study the biology and virulence of T. denticola ATCC 35405. In this report, we hypothesize that T. denticola
ATCC 35405 may have a unique DNA restriction-modification (R-M) system that prevents it from accepting the
shuttle vectors of ATCC 33520 (e.g., the shuttle plasmid pBFC). To test this hypothesis, DNA restriction
digestion, PCR, and Southern blot analyses were conducted to identify the differences between the R-M systems
of these two strains. DNA restriction digestion analysis of these strains showed that only the cell extract from
ATCC 35405 was able to digest pBFC. Consistently, PCR and Southern blot analyses revealed that the genome
of T. denticola ATCC 35405 encodes three type II endonucleases that are absent in ATCC 33520. Among these
three endonucleases, TDE0911 was predicted to cleave unmethylated double-stranded DNA and to be most
likely responsible for the cleavage of unmethylated pBFC. In agreement with this prediction, the mutant of
TDE0911 failed to cleave unmethylated pBFC plasmid, and it could accept the unmethylated shuttle vector. The
study described here provides us with a new tool and strategy to genetically manipulate T. denticola, in
particular ATCC 35405, and other strains that may carry similar endonucleases.
More than 80% of the adult population at some time in their
lives suffers from periodontal disease, which is primarily
caused by polymicrobial infections (11, 37). More than 700
different microorganisms have been suggested to colonize the
oral flora (13, 27, 36). A body of studies has shown that the
presence and burden of oral spirochetes is associated with
the severity of periodontal disease (16, 36, 42). In the oral
flora, more than 60 different spirochete species have been
identified, and they all belong to the genus Treponema (13, 36).
Due to their fastidious growth requirements, very few oral
treponemes can be reliably cultivated (6, 16). Treponema den-
ticola, an oral spirochete that can be readily cultured, has been
used as a model bacterium to study the biology and virulence
of oral treponemes because its genome has been sequenced
and it can be genetically manipulated (16, 23, 25, 28, 43, 50).
T. denticola ATCC 35405 and ATCC 33520 are two geneti-
cally related reference strains that are often used to study the
genetics and virulence of spirochetes (17, 21, 25). T. denticola
ATCC 33520 shares more than 76% DNA similarity with
ATCC 35405 (7). However, these two strains possess many
physiological and genetic differences, such as serotype (7, 8),
oxygen tolerance (46), and biofilm formation capability (24, 48,
49). In addition, four plasmids have been isolated from several
oral treponemes, including ATCC 33520, but none of these
plasmids has been isolated from ATCC 35405 (4, 5). More-
over, three shuttle vectors (pKMR4PE, pKMCou, and pBFC)
that were derived from the plasmid pTS1 have been success-
fully transferred into ATCC 33520 but not ATCC 35405 (5, 9,
10, 44). Thus far, there has been no shuttle vector available for
the genetic complementation of mutants derived from T. den-
ticola ATCC 35405. ATCC 35405 is more virulent than ATCC
33520, and its genome has been sequenced (3, 12, 14, 43). The
lack of a shuttle vector has compromised our efforts to use
ATCC 35405 and its genetic information to study the biology
and virulence of T. denticola.
DNA restriction and modification (R-M) systems have been
described as “immune systems” to defend against invading for-
eign DNA (33, 34). In many prokaryotes, R-M systems serve as
genetic barriers for gene transformation, conjugation, and trans-
fection (1, 15, 33, 47). The majority of R-M systems consist of a
DNA methyltransferase (MTase) and a restriction endonuclease
(REase). The MTase enables recognition of self DNA by meth-
ylation of specific nucleotides within particular DNA sequences,
and the REase cleaves invading unmodified DNA. R-M systems
can be divided into four types (I to IV), based on their enzyme
compositions, cofactors, and active modes (40). Among these
R-M systems, the type II R-M system often protects bacteria and
archaea against invading DNA (38). R-M systems were recently
identified in the Lyme disease spirochete Borrelia burgdorferi, and
disruptions of these systems were able to increase the transfor-
mation efficiency of foreign DNA (22, 26, 39). The genome of T.
denticola ATCC 35405 encodes three putative type II R-M
systems: TDE0227 (MTase)/TDE0228 (REase), TDE0909
(MTase)/TDE0911 (REase), and TDE1268 (REase) (41). In this
report, we hypothesize that the existence of these R-M systems
may prevent ATCC 35405 from accepting foreign DNA, such as
the shuttle vectors of ATCC 33520. To test this hypothesis, DNA
* Corresponding author. Mailing address: Department of Oral
Biology, SUNY at Buffalo, 3435 Main St., Buffalo, NY 14214-3902.
Phone: (716) 829-6014. Fax: (716) 829-3942. E-mail: email@example.com.
?Published ahead of print on 20 May 2011.
restriction digestion, PCR, and Southern blot analyses were con-
ducted to compare the differences between the R-M systems of
ATCC 33520 and ATCC 35405. It was found that these R-M
systems were absent in ATCC 33520 and that the inactivation of
TDE0911, a gene encoding a type II restriction endonuclease,
allowed the mutant to accept the unmethylated pBFC shuttle
MATERIALS AND METHODS
Bacterial strains, culture conditions, and oligonucleotide primers. T. denticola
ATCC 35405 and ATCC 33520 strains were grown in oral bacterial growth
medium (OBGM) (35) with 10% heated-inactivated rabbit serum at 37°C in an
AS-580 anaerobic chamber (Anaerobe Systems, Morgan Hill, CA) with an at-
mosphere of 80% nitrogen, 10% carbon dioxide, and 10% hydrogen, as previ-
ously described (50). The Escherichia coli TOP10 strain (dam?/dcm?; Life Tech-
nologies, Carlsbad, CA) was used for routine plasmid constructions and
preparations. A E. coli dam/dcm-deficient strain (New England BioLabs,
Ipswich, MA) was used to prepare unmethylated plasmids. The oligonucleotide
primers used in this study are listed in Table 1, and all primers were synthesized
by Integrated DNA Technologies, Inc. (Coralville, IA).
Genomic DNA preparation, DNA probe labeling, and hybridizations. Total
genomic DNAs from T. denticola wild-type strains and the isogenic mutant were
prepared with the Illustra bacteria genomic prep kit (GE Healthcare, Little
Chalfont, Buckinghamshire, United Kingdom). Southern blot analysis was car-
ried out following a standard procedure. Briefly, the purified genomic DNAs
were first digested with the restriction enzymes ClaI or HindIII, separated on
1.0% agarose gel, and blotted to a Hybond-N?membrane (GE Healthcare). To
prepare DNA probes for Southern blot assays, TDE0228 (925 bp), TDE0911 (766
bp), TDE1268 (659 bp), and ermB, an erythromycin resistance gene (696 bp)
(19), were amplified by PCR. Then, the obtained amplicons were labeled with
digoxigenin (DIG). To label pBFC, the plasmid was first linearized with NotI and
then labeled with DIG. The DNA labeling, hybridization, and detection were
carried out using DIG High Prime DNA labeling and detection starter kit I
(Roche Diagnosis GmbH, Mannheim, Germany), according to the manufacturer’s
Construction of a deletion mutant of TDE0911. A recently described nonpolar
gene inactivation method was applied to inactivate the TDE0911 gene of T.
denticola (32). The vector (TDE0911::ermB) for the targeted mutagenesis was
constructed by multiple-step PCR as illustrated in Fig. 1. In the first step, the
flanking regions of TDE0911 and the erythromycin resistance gene (ermB) were
amplified by PCR with three pairs of primers: P1/P2(flanking region 1), P3/P4
(ermB), and P5/P6(flanking region 2). The P2, P3, P4, and P5primers contain
several engineered overlapping base pairs (underlined in Table 1). In the second
step, flanking region 1 and ermB were fused by PCR using P1and P4primers. In
the final step, the constructed region 1-ermB fragment and flanking region 2 were
further merged by PCR using primers P1and P6. The final PCR product (flank-
ing region 1-ermB-flanking region 2) was cloned into the pGEM-T-Easy vector
(Promega, Madison, WI), generating the construct TDE0911::ermB, in which the
entire open reading frame of TDE0911 was deleted and replaced with the
promoterless ermB gene. To inactivate TDE0911, 10 ?g of TDE0911::ermB
plasmid was linearized with NotI and then electroporated into 80 ?l of T.
denticola ATCC 35405 competent cells. The transformants were selected on
OBGM semisolid plates containing erythromycin (60 ?g ml?1), and the mutation
was confirmed by PCR and Southern blot assays.
Preparation of methylated and unmethylated pBFC. The pBFC plasmid is a
shuttle vector between E. coli and T. denticola ATCC 33520 (44), and it was
kindly provided by R. Limberger (Wadsworth Center). To prepare methylated
pBFC, the plasmid was transformed into E. coli TOP10, which contains the dam
gene encoding a DNA methyltransferase that methylates the N6position of the
adenine residues in the sequence GATC (18, 20). To prepare unmethylated
TABLE 1. Oligonucleotide primers used in this study
Primer Sequence (5?–3?)a
5?-flanking region of TDE0911; F
5?-flanking region of TDE0911; R
ermB cassette; F
ermB cassette; R
3?-flanking region of TDE0911; F
3?-flanking region of TDE0911; R
TDE0911 probe; F
ermB probe; F
ermB probe; R
TDE0228 probe; F
TDE0228 probe; R
TDE1268 probe; F
TDE1268 probe; R
aacC1 site mutagenesis; F
aacC1site mutagenesis; R
aacC1 cassette; F
3?-flanking region of P6; R
aUnderlined portions show the engineered overlapping base pairs.
bPrimer orientation: F, forward; R, reverse.
FIG. 1. Schematic of construction of TDE0911::ermB for targeted
mutagenesis of TDE0911. The method illustrated here was used to
delete the entire reading frame of TDE0911 and to replace it with the
ermB cassette. Arrows indicate the approximate positions of the prim-
ers used for multiple-step PCR amplifications. The sequences of these
primers are listed in Table 1. Thin black and gray rectangles represent
the engineered adaptors for the fusion of individual amplicons.
4574 BIAN AND LIAPPL. ENVIRON. MICROBIOL.
pBFC, the plasmid was transformed into an E. coli dam/dcm mutant strain.
The plasmids were purified using the PureYield plasmid midiprep system
(Promega). The plasmid concentrations were measured with a NanoDrop
2000 spectrophotometer (Thermo Scientific, Wilmington, DE) and diluted to
a final concentration of 1 ?g ?l?1.
Site-directed mutagenesis. The site-directed mutagenesis of aacC1, a genta-
micin resistance gene (45), was carried out using the QuikChange II site-directed
mutagenesis kit (Stratagene, La Jolla, CA) with primers P15and P16, according
to the manufacturer’s instructions. The resultant mutation was confirmed by
DNA sequence analysis. The wild-type and mutated aacC1 genes were amplified
by PCR with primers P17and P18, and the obtained DNA fragments (534 bp)
were used for DNA digestion analysis as described below.
DNA digestion analysis. To prepare the cell lysates, 5 ml of stationary-phase
T. denticola cultures (approximately 1 ? 109cells ml?1) was centrifuged, and the
cell pellets were suspended in 1 ml of phosphate-buffered saline (PBS; pH 7.5)
buffer. The collected cells were lysed by sonication with the Branson Sonifier 450
(Branson Ultrasonics, Danbury, CT) for 150 s on ice. After centrifugation at
6,000 ? g for 5 min, the supernatants were collected and stored at 4°C as crude
cell extracts. These extracts were used to detect restriction digestion activity of
different T. denticola strains on the methylated and unmethylated plasmids and
on the PCR products. For the DNA digestion analysis, the reaction mixtures (40
?l) contained 5 ?g DNA, 20 ?l crude cell extract, and 4 ?l 10? NEB buffer 4
(New England BioLabs). The reaction mixtures were incubated at 37°C for 2 h.
The reaction mixtures were analyzed by agarose gel electrophoresis or further
detected by Southern blot analysis.
Measurement of DNA transformation efficiency. The transformation efficiency
of pBFC in T. denticola was measured as previously described (39). Briefly, three
T. denticola strains (ATCC 35405, ATCC 33520, and the TDE0911 mutant) were
grown to early logarithmic phase (optical density at 600 nm of 0.3 to 0.4), and the
cells were enumerated using Petroff-Hausser counting chambers. The cell num-
bers of these three strains were adjusted to similar amounts before the prepa-
ration of competent cells. The preparation of T. denticola competent cells and
electrotransformation were conducted as described before (28, 30). To measure
the transformation efficiency, 10 ?g of methylated or unmethylated pBFC plas-
mid was electroporated into 80 ?l of T. denticola competent cells. The transfor-
mants were selected on OBGM semisolid plates containing chloramphenicol (10
?g ml?1). The presence of pBFC in the obtained transformants was confirmed by
extracting the plasmid followed by restriction digestion analysis with NotI. The
data are expressed as the mean transformation efficiency (transformants/?g
pBFC) from three independent experiments.
RESULTS AND DISCUSSION
The shuttle vector pBFC was digested by T. denticola ATCC
35405 but not by ATCC 33520. The shuttle vector pBFC can be
transferred into ATCC 33520 but not the ATCC 35405 strain
(44). We hypothesized that this plasmid may be disrupted in
ATCC 35405 after the transformation. To test this hypothesis,
the pBFC plasmid purified from E. coli TOP10 strain was
coincubated with the crude cell extracts of ATCC 35405 and
ATCC 33520. As hypothesized, the DNA restriction digestion
and Southern blot analyses showed that the crude cell extract
of ATCC 35405 cleaved the methylated pBFC into at least 10
visible fragments (Fig. 2, lane 2), whereas ATCC 33520 was
unable to cleave the plasmid (Fig. 2, lane 4). These results
suggest that ATCC 35405 may contain a unique R-M system(s)
that is absent in the ATCC 33520 strain.
Lack of methylation partially protects pBFC from the cleav-
age mediated by T. denticola ATCC 35405. The main function
of R-M systems is to defend their hosts against foreign DNA,
which is often achieved by recognition of self DNA via meth-
ylation at defined sites and disrupting unmethylated invading
DNA (33). To test the influence of methylation on the cleavage
of pBFC, an experiment similar to the one described above was
conducted by using the unmethylated plasmid. The unmethyl-
ated pBFC was cleaved by the crude cell extract of ATCC
33520 (Fig. 2, lane 5). Interestingly, the lack of methylation
protected the plasmid from cleavage by ATCC 35405 to some
extent, since fewer fragments were detected (Fig. 2, lane 3)
than when ATCC 35405 extract was incubated with the meth-
ylated pBFC (Fig. 2, lane 2). Collectively, these results imply
that both strains ATCC 33520 and ATCC 35405 have R-M
systems that are able to disrupt the unmethylated DNA and
that ATCC 35405 may encode a unique restriction endonu-
clease(s) that cleaves methylated pBFC at defined sites.
The genes encoding three type II restriction endonucleases
are absent in T. denticola ATCC 33520. The genome of ATCC
35405 was sequenced, and it encodes three putative type II
TDE0909 (MTase)/TDE0911 (REase), and TDE1268 (REase)
(41, 43). Since the type II R-M systems typically protect bac-
teria against invading DNA (38), we reasoned that some of
these systems may be absent in ATCC 33520. To test if ATCC
33520 contains the above systems, PCR and Southern blot
analyses were conducted to detect the genes encoding three
endonucleases (TDE0228, TDE0911, and TDE1268). Both
PCR (data not shown) and Southern blot analyses revealed
that these three genes were present in the genome of ATCC
35405 (Fig. 3, lanes 3 and 4) but were absent in the ATCC
FIG. 2. pBFC digested by the crude cell extracts of ATCC 35405
and ATCC 33520. pBFC was first linearized by SpeI and then coincu-
bated with the crude cell extracts of strain ATCC 35405 or ATCC
33520, as described below. Lane 1, pBFC (methylated) treated with
boiled crude cell extract of ATCC 35405; lanes 2 and 4, pBFC (meth-
ylated) treated with ATCC 35405 extract (lane 2) or ATCC 33520
extract (lane 4); lanes 3 and 5, pBFC (unmethylated) treated with
extracts of ATCC 35405 (lane 3) and ATCC 33520 (lane 5). After the
digestions, the samples were analyzed by 1% agarose gel electropho-
resis and visualized with ethidium bromide staining (a); results were
further confirmed by Southern blotting with DIG-labeled pBFC (b).
M, DNA marker.
VOL. 77, 2011DNA RESTRICTION-MODIFICATION SYSTEM IN T. DENTICOLA4575
33520 strain (Fig. 3, lanes 1 and 2). These results further
demonstrate that ATCC 35405 and ATCC 33520 contain dif-
ferent type II R-M systems.
Isolation and characterization of the TDE0911 deletion mu-
tant. The functions of the above three type II endonucleases
were recently described in REBASE (41). It was predicted that
TDE1268 (TdeI) belongs to the methylation-dependent re-
striction enzymes and recognizes GmATC at the N6position of
methylated adenine. TDE0228 (TdeII) was predicted to be-
long to a family of nicking endonucleases that recognize the
asymmetric sequence 5?-CTCTTC-3? and cleave one strand of
DNA. REBASE also predicted that TDE0911 (TdeIII) be-
longs to a family of REases that recognize unmethylated
GGNCC. The predicted digestion profile of the pBFC plasmid
sequence (44) by TDE0911 corresponds to the above DNA
restriction digestion pattern (Fig 2, lane 3), suggesting that
TDE0911 is responsible for the cleavage of unmethylated
pBFC. If this is the case, we would expect the inactivation of
TDE0911 may block the cleavage of unmethylated pBFC and
the mutant may be able to accept the unmethylated plasmid.
To test if TDE0911 is involved in pBFC cleavage, the cor-
responding gene was inactivated by targeted mutagenesis with
the vector of TDE0911::ermB. A total of 63 Ermrcolonies
appeared 10 days after plating, and PCR analysis with a pair of
primers specific to ermB (P9and P10) showed that only 8
colonies contained ermB. One clone (Td?911) was further
confirmed by Southern blotting and PCR analysis. As ex-
pected, for the TDE0911-specific probe, two bands (3.3 kb and
5.8 kb) were detected in the chromosomal DNA of ATCC
35405 treated with ClaI (Fig. 4a, lane 1) and one band (4.8 kb)
when treated with HindIII (Fig. 4a, lane 2). In contrast, no
positive band was detected in the Td?911 mutant (Fig. 4a,
lanes 3 and 4), indicating that the TDE0911 gene was deleted
in the mutant. For the ermB-specific probe, one positive band
was detected in the chromosomal DNA of Td?911 treated with
ClaI (9.1 kb) and one with HindIII (6.1 kb) (Fig. 4b, lanes 3
and 4), but none of them appeared in ATCC 35405 (Fig. 4b,
lanes 1 and 2). The sizes of detected fragments corresponded
to the one with which ermB is integrated to the locus of
TDE0911. These results showed that the whole TDE0911 gene
was deleted and replaced with ermB as expected. This conclu-
sion was further confirmed by PCR analysis with a pair of
ermB-specific primers (P3/P4) in combination with one primer
(P19) flanking the mutant allele (Fig. 4c).
Identification of the recognition and cleavage site of
TDE0911. It was predicted that TDE0911 recognizes and
cleaves targeted DNAs containing the sequence GGNCC (41).
To confirm this prediction, aacC1, which contains one putative
recognition site (G97GCCC) of TDE0911 (45), was treated
with the crude cell extracts of ATCC 35405 and the Td?911
mutant. As shown in Fig. 5, the crude cell extract of ATCC
35405 cleaved aacC1 into two fragments (lane 2), as expected,
but the extract of the Td?911 mutant did not (lane 4). In
addition, a mutation in the recognition site (G97GCCC mu-
tated to A97GCCC) blocked the cleavage (lanes 1 and 3). The
cleaved fragments were further cloned and sequenced, and it
was determined that the cleavage occurred at the site 5?-
GGCC?C3?/3?-CŒCGGG5? (where the triangles represent
the cleavage sites).
FIG. 3. Detection of TDE0228, TDE0911, and TDE1268 by Southern blot analysis. The purified chromosomal DNAs of ATCC 33520 and
ATCC 35405 were digested with ClaI and HindIII and then probed with three different DIG-labeled DNA fragments: TDE0228 (a), TDE0911 (b),
and TDE1268 (c). Lanes 1 and 2, genomic DNA of ATCC 33520 digested with ClaI and HindIII; lanes 3 and 4, genomic DNA of ATCC 35405
digested with ClaI and HindIII. The numbers to the right of the gels present the sizes of detected genes.
FIG. 4. Characterization of the Td?911 mutant. The purified chromosomal DNAs of ATCC 35405 and the Td?911 mutant were treated with
ClaI and HindIII and then detected by Southern blotting with probes for TDE0911 (a) and ermB (b). Lanes 1 and 2, genomic DNA of ATCC 35405
treated with ClaI (lane 1) or HindIII (lane 2); lanes 3 and 4, genomic DNA of Td?911 mutant treated with ClaI (lane 3) or HindIII (lane 4). The
numbers to the right of the gels represent the sizes of the detected DNA fragments. (c) PCR analysis of the Td?911 mutant with two pairs of
primers, P3/P19and P3/P4. The PCR samples were loaded in the following order: ATCC 35405 amplified with P3/P19(lane 1); Td?911 amplified
with P3/P19(lane 2); ATCC 35405 amplified with P3/P4(lane 3); Td?911amplified with P3/P4(lane 4). The sequences and descriptions of these three
primers are listed in Table 1.
4576 BIAN AND LIAPPL. ENVIRON. MICROBIOL.
The Td?911 mutant fails to digest unmethylated pBFC. As
mentioned above, TDE0911 recognizes and digests unmethyl-
ated DNA. If this is a sole endonuclease that cleaved the
unmethylated pBFC, we would expect that the Td?911 mutant
would fail to cleave the unmethylated DNA. To test this hy-
pothesis, the DNA restriction digestion analysis described
above was carried out. As expected, the crude cell extract of T.
denticola ATCC 35405 cleaved the unmethylated pBFC into 4
fragments (Fig. 6, lane 3); however, there was no cleaved frag-
ment observed in the sample treated with the crude cell extract of
Td?911 (Fig. 6, lane 5). These results suggest that TDE0911 is a
sole endonuclease that cleaves unmethylated pBFC.
The Td?911 mutant accepts unmethylated pBFC plasmid.
The failure to cleave the unmethylated DNA suggests that the
Td?911 mutant may be able to accept unmethylated pBFC. To
test this speculation, the methylated and unmethylated pBFC
plasmids were electroporated into the Td?911 mutant. In addi-
tion, the strains ATCC 33520 and ATCC 35405 were included as
controls. Consistent with the previous reports (44), the methyl-
ated pBFC plasmid could be transferred into ATCC 33520 (Fig.
7c) but not the ATCC 35405 strain (Fig. 7a). As speculated, it was
found that the unmethylated plasmid was successfully transferred
into the Td?911 mutant (Fig. 7f), but not the ATCC 33520 strain
(Fig. 7d). The observed phenotype is consistent with the above in
vitro DNA digestion analyses, in which the unmethylated pBFC
was digested by ATCC 33520 but not the Td?911 mutant. The
transformation efficiency of the unmethylated pBFC in the
Td?911 mutant (6.0 ? 1.7 transformants/?g DNA [mean ? stan-
dard error of the mean]) was similar to that of the methylated
plasmid in the ATCC 33520 strain (7.3 ? 2.1 transformants/?g
DNA) (Table 2).
FIG. 6. The Td?911 mutant failed to cleave the unmethylated
pBFC. The SpeI-treated methylated or unmethylated pBFC was coin-
cubated with the crude cell extract of ATCC 35405 or Td?911 and
then analyzed on a 1.0% agarose gel (a), followed by Southern blot
analysis (b). The samples are in the following lane order: methylated
pBFC treated with boiled crude cell extract of ATCC 35405 (lane 1);
methylated (lane 2) and unmethylated (lane 3) pBFC treated with
ATCC 35405 extract; methylated (lane 4) and unmethylated (lane 5)
pBFC treated with Td?911 extract. M, DNA marker.
FIG. 5. Identification of the recognition and cleavage site of
TDE0911 (TdeIII). PCR-amplified 534 bp of the wild-type (G97GCCC)
and mutated aacC1 (A97GCCC) fragments were treated with the crude
cell extract of ATCC 35405 and Td?911. The obtained samples were then
analyzed by 2% agarose gel electrophoresis and visualized with ethidium
bromide staining. The samples were loaded in the following order: the
mutated aacC1 (lane 1) and the wild-type aacC1 (lane 2) treated with
ATCC 35405 extract, and the mutated aacC1 (lane 3) and the wild-type
aacC1 (lane 4) treated with Td?911 extract. M, DNA marker. The
cleaved fragments in lane 2 were cloned and sequenced to determine the
FIG. 7. Transformation of pBFC into different T. denticola strains.
Ten micrograms of methylated (mpBFC, top panels) or unmethylated
pBFC plasmid (umpBFC, bottom panels) was electroporated into
ATCC 35405, ATCC 33520, and the Td?911 mutant. Images were
taken 7 days after plating. A total of 20 antibiotic-resistant colonies
were picked from the positive transformants (c and f), and the pres-
ence of pBFC in these colonies was identified by extracting plasmid
followed by DNA restriction digestion. All examined colonies con-
tained the plasmid.
TABLE 2. pBFC transformation efficiency in different
T. denticola strains
Methylated pBFC Unmethylated pBFC
7.3 ? 2.1
6.0 ? 1.7
aNumber of transformants/?g of plasmid DNA. Data are the means ? stan-
dard errors of the means from three independent experiments.
VOL. 77, 2011 DNA RESTRICTION-MODIFICATION SYSTEM IN T. DENTICOLA4577
Conclusion. The lack of sufficient genetic tools is a bottle-
neck to study the biology and virulence of the oral spirochete
T. denticola. Although targeted and transposon mutagenesis
are currently available, their efficiencies are very low (2, 29, 31,
50). Thus far, only a few mutants have been constructed and
genetically complemented. It has been speculated the R-M sys-
tem, which has been referred to as a microbial “immune system,”
may prevent T. denticola from accepting foreign DNA. Consis-
tently, the genome of ATCC 35405 encodes several R-M systems
(41). The functions of these systems remain elusive. The study
reported here is the first step to investigate the roles of these R-M
systems in T. denticola, and it provides us with a new tool and
strategy to genetically manipulate T. denticola.
We thank R. Limberger for providing T. denticola strains and shuttle
This research was supported by Public Health Service grants
DE018829 and DE019667 to C. Li.
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