Department of Microbiology and Immunology,
Medical College of Virginia, Virginia Commonwealth University,
Richmond, Virginia 23298-0678
Received 17 April 1997/Returned for modification 2 June
1997/Accepted 31 December 1997
 |
INTRODUCTION |
Lyme disease develops upon infection
with pathogenic spirochete species of the Borrelia
burgdorferi sensu lato complex (9, 51). This complex
includes the pathogenic species B. burgdorferi, Borrelia garinii, and Borrelia afzelii (3,
9, 24, 28, 29, 31, 41, 42, 57, 59) and two species of uncertain pathogenic potential, Borrelia japonica and Borrelia
andersonii (17, 31). Recent work has suggested that
there may be additional genomic groups and species (2, 17-19, 24,
40), including the recently proposed Borrelia
valaisiana sp. nov. (58) and Borrelia
lusitaniae sp. nov. (16) (previously referred to as genomic groups VS116 and PotBi, respectively).
The Borrelia genome is unique (7), being composed
of a linear chromosome and a series of linear and circular plasmids
(LPs and CPs, respectively) (4, 14). Numerous genes have
been localized to these plasmids (6, 12, 13, 25, 32, 38, 53,
56). The Borrelia genome is variable among isolates in both the number and size of the plasmids present (4, 33, 47, 48,
60). Mechanisms associated with the generation of plasmid
variability include plasmid loss (38, 47), lateral exchange
of plasmids (23, 33), recombination (26, 30, 33,
43), and dimer formation (22, 27, 55). Some plasmids carry repeated sequences (11, 20, 21, 35, 39, 50, 52, 63)
which may allow for homologous recombination among plasmids. It has
been suggested that plasmid cp8.3 of B. afzelii (13) or similarly sized small CPs of other isolates may
carry genes that encode factors necessary for infectivity and/or
virulence (47). Coincident with the loss of plasmids in this
size range, it has been reported that some isolates lose their
infective potential (47). An 8.3-kb CP (cp8.3) from B. afzelii IP21 has been sequenced and carries nine open reading
frames (ORFs) and a 184-bp inverted repeat (IR) element
(13). However, the potential role of the ORFs of cp8.3
remains unclear, and these sequences do not exhibit significant
homology with the sequences of any known genes of other pathogenic
organisms. Assessment of their potential importance is further
complicated by the fact that some of the cp8.3 ORFs are multicopy and
are carried on plasmids other than cp8.3 (5, 11, 52, 63). It
has been suggested that the 184-bp IR elements on cp8.3 may have
triggered recombination and integration events that have resulted in
the multicopy state of cp8.3 ORFs found in some isolates
(63). Hence, an assessment of the distribution of the IR
elements may provide information as to the potential role of these
elements in generating organizational diversity of the ORFs of cp8.3.
In view of the extensive genomic variability of B. burgdorferi sensu lato isolates, it cannot, de facto, be concluded
that the ORFs of cp8.3 are carried by all B. burgdorferi sensu lato species or that they exhibit the same genetic organization as that seen in the few isolates that have been studied to date (5, 10, 63). An assessment of the organization and molecular variability of genes carried on these plasmids among the different B. burgdorferi sensu lato complex species will prove
important in elucidating the possible involvement of these ORFs in the
biology of these pathogens. The goals of this study were (i) to assess the distribution, molecular organization, and possible multicopy state
of cp8.3 ORFs 1, 2, and 4 among B. burgdorferi sensu lato species and isolates; (ii) to determine if B. burgdorferi sensu lato isolates carry sequences related to
the IR element of cp8.3; and (iii) to identify the genomic
elements that carry copies of these ORFs and IR elements.
| |
MATERIALS AND METHODS |
Bacterial cultivation and nucleic acid isolation.
Bacteria
(Table 1) were cultivated at 32°C in
BSK-H medium (Sigma) supplemented to 6% with rabbit serum (Sigma).
Cells were harvested by centrifugation and washed twice with
phosphate-buffered saline (pH 7.0). RNA and DNA were isolated as
previously described (33, 36).
PCR analyses.
PCR was performed by using isolated DNA and
Taq polymerase (Promega) as described previously
(33). Primers were used at a final concentration of 0.5 pmol/µl. In single-primer PCR analyses (spPCR), the final
concentration of the primer was 1.0 pmol/µl. Cycle conditions
consisted of 30 cycles of 95°C for 45 s, 50°C for 30 s,
and 72°C for 90 s. Amplicons were analyzed in 1% agarose gels
in 40 mM Tris-acetate-2 mM EDTA (TAE; pH 8.5). Primer binding sites
are depicted in Fig. 1, and the primer
sequences are as follows: primer O1F1, TTATCTAATGTTAACAAAACTCG;
primer O1R1, CGAGTTTTGTTAACATTAGATAA; primer O1R2,
GTAACAAATACATTATTGTATTC; primer O2F1,
CATGGAGAATTTATTGAAAAC; primer O2R2,
TTAGTCCCTTATCAGAAT; primer O4F1, CAAGCAGAAATTCACTTTATA; primer O4R1, TATAAAGTGAATTTCTGCTTG; primer O4R3,
TATCCATATCCTTTAAGA; primer O7R1,
TTTAAGCACTCTATTTACCAATT; and primer IRA-F1,
TTGATTAATTTCTTGTGGATT.
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 1.
Map of B. afzelii IP21 cp8.3 indicating
the location of ORFs, IR elements, and oligonucleotide binding sites.
The sequence of cp8.3 was determined by Dunn et al. (13).
The direction of possible transcription of each ORF is indicated by
arrows. Oligonucleotides with designations including the letter R
(reverse) that are not shown in the figure target the identical sites
as their forward (F) primer counterparts except that they bind to the
opposite strand. The map was generated with the MacPlasmap (Jindong
Liu, University of Utah) and Canvas (Deneba) programs.
|
|
Southern blot analyses.
DNA (4 µg) was digested to
completion with EcoRV, electrophoresed in 0.8%
genetic-technology-grade agarose gels, vacuum blotted onto a Hybond N
membrane (Amersham), and UV cross-linked to the membrane as described
previously (33). Oligonucleotides were labeled by using
polynucleotide kinase and [
-32P]ATP (6,000 Ci/mmol;
DuPont-NEN). PCR-generated probes were labeled by random primer methods
(Boehringer Mannheim) and with [
-32P]dATP (3,000 Ci/mmol; DuPont-NEN). Hybridizations with oligonucleotides and PCR
probes were conducted at 32 to 37°C and at 42 to 50°C, respectively. The hybridization buffer consisted of 0.2% bovine serum
albumin, 0.2% polyvinylpyrrolidone (molecular weight, 40,000), 0.2% Ficoll (molecular weight, 400,000), 50 mM Tris-HCl (pH
7.5), 0.1% sodium pyrophosphate, 1% sodium dodecyl sulfate (SDS),
10% dextran sulfate, 100 µg of herring sperm DNA per ml, and 1 M
NaCl. With PCR probes, formamide was added to the hybridization buffer at a final concentration of 50%. Washes were performed at temperatures ranging from 32 to 60°C, depending on the probe. A variety of wash
and hybridization temperatures were employed in these analyses since
interspecies sequence divergence in the target sequences was expected.
Two 10-min washes with 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M
sodium citrate)-0.1% SDS and a 60-min wash with 0.1× SSC-0.1% SDS,
followed by a 5-min wash with 0.1× SSC-0.1% SDS with vigorous
agitation, were performed. Membranes were exposed to film at 
70°C.
Two-dimensional (2D) CHEF-PFGE.
DNA from cells lysed in
agarose (27) was fractionated in 1%
genetic-technology-grade agarose gels by using the contour-clamped homogeneous electric field mapper-pulsed-field gel electrophoresis (CHEF-PFGE) system (Bio-Rad). The algorithm used to separate the DNA by
CHEF-PFGE in the first dimension was generated by using the
auto-algorithm program with the following parameters: run time, 20 h 16 min; buffer, 0.5× Tris-borate-EDTA (TBE; pH 8.0); temperature,
14°C; ramping constant, 1.107; initial switch time, 0.47 s;
final switch time, 35.46 s; angle, 120°; gradient, 6 V/cm. After
electrophoresis in the first dimension, the gels were rotated 90° and
electrophoresed for 3 h in 0.5× TBE buffer at 80 V (constant field).
The DNA was stained with 1.0 µg of ethidium bromide per ml for 30 min, UV irradiated with 60 mJ of energy, destained, and photographed. The gels were soaked in 0.4 N NaOH-1.5 M NaCl for 15 min. Transfer onto a Hybond N+ membrane (Amersham) was accomplished by
either capillary action with 0.4 N NaOH-1.5 M NaCl over a period of 2 to 3 days or by vacuum blotting with a 6-h transfer time. Membranes
were neutralized in 0.5 M Tris-HCl (pH 7.0) and rinsed in 2× SSC prior
to hybridization.
DNA sequence analysis.
Briefly, PCR-generated sequencing
templates were purified (Wizard PCR Prep system; Promega) and sequenced
with the fmol DNA cycle sequencing system (Promega). Reactions were run
in 6% acrylamide-8 M urea gels (85 W).
Nucleotide sequence accession numbers.
The GenBank accession
numbers for the O1R1-O7R1 amplicon sequences from B. andersonii 21038 and B. afzelii UMO1 and IP21 are AD001532, AD001533, and AD001534, respectively; the B. afzelii ECM1 and UMO1 ORF 2 amplicons generated with the O2F1-O2R2 primer set have been assigned the numbers AD001535 and AD001536, respectively.
| |
RESULTS AND DISCUSSION |
PCR analysis of ORFs originally identified on cp8.3.
cp8.3
from B. afzelii IP21 carries nine ORFs and a 184-bp IR
element (13). Although the functional role of the ORFs is
currently unknown, it has been suggested that plasmids in the 8- to
9-kb range may carry genes encoding factors necessary for infectivity or virulence (47). This study was undertaken to investigate the distribution of these putative virulence factors and their organization in other B. burgdorferi sensu lato species
and isolates. This information will aid in future studies designed to
assess the potential role of these ORFs in the overall biology of these bacteria. As a first step towards assessing the distribution of ORFs 1, 2, and 4 among B. burgdorferi sensu lato isolates, PCR analyses were conducted. These particular ORFs were analyzed since they
represent ORFs that reside on two different segments of cp8.3 whose
borders are defined by the presence of 184-bp IR elements (Fig. 1). The
reason for analyzing sequences derived from these two different
segments of cp8.3 stems from the influence that IR elements can have on
the stability and organization of surrounding sequences. All isolates
investigated except B. garinii VSBP carry plasmids in
the 7- to 9-kb size range that are visible by ethidium bromide staining
of DNA fractionated in 0.4% agarose gels (data not shown)
(61). To verify that all templates were free of inhibitors, a positive-control PCR primer set targeting the 16S rRNA gene (rrs) was used. rrs-derived amplicons were
obtained from all templates (PCR data are presented in Table 1). An ORF
4-directed primer set also yielded amplicons from all isolates,
demonstrating that ORF 4 is carried by all species of the B. burgdorferi sensu lato complex tested (B. burgdorferi, B. garinii, B. afzelii, B. japonica, B. andersonii, and B. valaisiana sp. nov).
B. garinii VSBP, which lacks a 7- to 9-kb CP, was also
PCR positive. In contrast to ORF 4, ORFs 1 and 2 were amplified from
only 2 of 14 and 3 of 14 isolates, respectively. PCR analyses of
additional B. burgdorferi sensu lato isolates revealed
that only 5 of 34 isolates were positive for ORF 2 while 28 of 34 were
positive for ORF 4 (data not shown). All isolates positive for ORF 2 were B. afzelii. These data suggest that either ORFs 1 and 2 are absent from the genomes of most isolates or they are less
conserved across species lines than ORF 4 and were therefore not
detected by PCR with the primers used. These analyses also suggest that
isolate VSBP may carry an ORF 4-related sequence on a genomic
element other than cp8.3.
With the exception of a truncated ORF 2 amplicon obtained from
B. afzelii ECM1, all ORF amplicons were of the
predicted size, indicating that these genes are conserved in size. The
ORF 2 amplicon from B. afzelii ECM1 was 173 bp instead
of the predicted 500-bp product (Table 1). Sequence analysis of the ORF
2 amplicon revealed an in-frame coding sequence deletion of 327 nucleotides (109 amino acids).
Southern blot analysis of cp8.3 ORFs.
Southern hybridization
analyses were performed to assess restriction fragment length
polymorphism (RFLP) patterns of each ORF and to determine if ORFs 1, 2, and 4 are multicopy. DNA, digested to completion with EcoRV,
was hybridized with PCR-generated ORF probes amplified from
B. afzelii IP21 or with ORF-specific oligonucleotide probes.
An ORF 2 PCR-generated probe hybridized with one or more DNA fragments
from some, but not all, B. burgdorferi, B. garinii, and B. afzelii isolates (Fig.
2). Similar results were obtained for ORF
1 (Southern hybridization data are summarized in Table 2). To determine if the multiple
hybridizing bands were due to incomplete digestion of the DNA, the
blots were stripped and probed with an rrs (16S rRNA
gene)-targeting oligonucleotide. Consistent with rrs being a
single-copy gene (49), a single hybridizing fragment was
detected in each isolate (data not shown). This demonstrates that
incomplete digestion is not responsible for the multiple ORF 1 and ORF
2 hybridizing fragments. Regarding hybridization specificity, it is
important to note that some isolates did not hybridize with either the
ORF 1 or the ORF 2 probe. This indicates that the hybridization
conditions were sufficiently stringent to prevent general nonspecific
binding of the probes to the AT-rich DNA of the Borrelia
species. This suggests that the multiple hybridizing bands observed do
in fact reflect specific binding of the probes to repeated ORF-related
sequences. The data presented here are consistent with a recent
report by Barbour et al., who detected two ORF 1 homologs on a 17-kb LP
of B. burgdorferi B31 (5).
View larger version (51K):
[in this window]
[in a new window]
|
FIG. 2.
RFLP pattern analysis of ORF 2 among B. burgdorferi sensu lato isolates. DNA from each isolate was
digested to completion with EcoRV, fractionated, blotted,
and probed with an ORF 2 PCR-generated probe as discussed above.
Isolates analyzed were B. burgdorferi LP7, CA9, and 297 (lanes 1 to 3, respectively); B. garinii FRG and N34
(lanes 4 and 5, respectively); B. valaisiana VS116
(lane 6); B. garinii VSBP (lane 7); B. afzelii IP21, ECM1, and UMO1 (lanes 8 to 10, respectively);
B. andersonii 19857 and 21038 (lanes 11 and 12, respectively); and B. japonica IKA2 and HO14 (lanes 13 and 14, respectively). Molecular size standards (in kilobases) are
indicated on the left.
|
|
Several isolates that hybridized with ORF 1 or ORF 2 PCR-generated
probes were PCR negative for these ORFs. When the ORF 1 and ORF 2 PCR
primers were used as probes, strong hybridization was observed only
with B. afzelii-derived DNA (Table 2). Weak hybridization with B. valaisiana VS116-derived DNA was
observed with the O2F1 oligonucleotide. The lack of hybridization of
the oligonucleotide probes with most isolates likely reflects sequence divergence in the primer binding sites among isolates and explains why
so few were PCR positive for ORF 1 or 2. The selective hybridization with B. afzelii isolates is not surprising since the
probes were designed based on the B. afzelii IP21 cp8.3
sequence.
Sequence analysis of the truncated B. afzelii ECM1 ORF
2 amplicon revealed a deletion in the coding sequence of 327 nucleotides. Oligonucleotides targeting the 5' and 3' ends of ORF 2 hybridized with an 8.3-kb fragment in ECM1 (Table 2). This was expected since the binding sites of these probes are not contained within the
deletion. In contrast, the ORF 2 PCR probe did not hybridize with the
ECM1 DNA (Fig. 2, lane 9). However, in view of the deletion, this is
not surprising since the deletion would destabilize hybridization of
the full-length gene probe. This result also indicates that ECM1 does
not carry a second, full-length, conserved copy of ORF 2 elsewhere in
its genome. It had been postulated, based on the homology of the amino
acid sequence deduced from ORF 2 with that of RepC, a protein involved
in rolling-circle plasmid replication (13), that ORF 2 may
also play a role in plasmid replication. However, in light of the fact
that a full-length copy of ORF 2 is not carried by all isolates, an
essential role in plasmid replication or during growth in vitro seems
unlikely.
Although most isolates were hybridization positive with ORF 4 PCR
probes, significant differences in the RFLP patterns and overall
intensities of hybridization were observed with ORF 4 amplicons derived
from different B. burgdorferi sensu lato isolates (Fig.
3A and B). The B. afzelii
IP21-derived probe hybridized with greater intensity to an 8.3-kb
fragment in B. afzelii isolates than did the probe
generated from B. burgdorferi CA9 (Fig. 3A and B, lanes
9 to 11). This likely reflects ORF 4 sequence divergence at the
interspecies level. Interspecies sequence divergence has been reported
for virtually all of the characterized genes of the B. burgdorferi sensu lato complex. Even the multiple ORF 4 homologs
carried by an isogeneic population of B. burgdorferi B31 exhibit significant sequence divergence (63). It is
interesting to note that while the IP21-derived probe hybridized with
multiple fragments in B. andersonii 19857 and 21038 DNA
(albeit faintly), the CA9-derived probe did not under identical
hybridization conditions. Hence, the B. andersonii ORF
4-related sequences are more related to the ORF 4 sequence of
B. afzelii than to that of B. burgdorferi. The lack of hybridization of the ORF 4 probes with
B. japonica IKA2 also serves to illustrate that the
probes are binding with specificity, since if general nonspecific
binding were occurring, it would have occurred with this isolate as
well. The ORF 4 hybridization analyses also demonstrate that there is
intraspecies genomic variability. While ORF 4 was not detected
by hybridization in B. japonica IKA2, hybridization of
the ORF 4 probes with B. japonica HO14 DNA was observed. This could reflect differences in plasmid content, plasmid copy number, or ORF 4 sequences among B. japonica
isolates.
View larger version (56K):
[in this window]
[in a new window]
|
FIG. 3.
ORF 4 RFLP pattern analyses. The blots were probed with
ORF 4 PCR probes obtained by amplification of ORF 4 from B. afzelii IP21 (A) or B. burgdorferi CA9 (B) or with
the O4R3 oligonucleotide probe (C). The isolates analyzed were
B. japonica HO14 (lane 1); B. burgdorferi LP7, CA9, and 297 (lanes 2 to 4, respectively);
B. garinii FRG and N34, B. valaisiana
VS116, and B. garinii VSBP (lanes 5 to 8, respectively); B. afzelii IP21, ECM1, and UMO1 (lanes 9 to 11, respectively); B. andersonii 19857 and 21038 (lanes 12 and 13, respectively); and B. japonica IKA2
(lane 14). Molecular size standards (in kilobases) are indicated on
the left.
|
|
Zuckert and Meyer recently identified an ORF 4 homolog (ORF G) in
B. burgdorferi B31 that exhibits 80% identity to ORF 4 of isolate IP21 (63). Alignment of these sequences allowed
for the design of a consensus oligonucleotide (O4R3) that could be used
to further assess the potential multicopy nature of ORF 4-related sequences. O4R3 hybridized strongly with multiple restriction fragments
in several B. burgdorferi sensu lato isolates (Fig. 3C). In many isolates, the same fragments that bound the PCR probes also bound the oligonucleotide probes. Hybridizing fragments greater than 8.3 kb in size were detected in isolates 297, FRG, and N34. The
detection of multiple hybridizing bands with the oligonucleotide probes
clearly demonstrates that there is more than one copy of ORF 4-related
sequences in some isolates. Furthermore, the sizes or sums of the sizes
of the hybridizing restriction fragments detected indicate that some
copies are carried on genomic components other than cp8.3. This
is consistent with the detection of ORF 4-related sequences on plasmids
larger than 8.3 kb in B. burgdorferi B31
(63).
Localization of the multiple copies of ORFs 1, 2, and 4 to specific
genomic elements through Southern blot analysis of
genomic DNA fractionated by 2D CHEF-PFGE.
To identify the
genetic elements that carry the ORF 1-, 2-, and 4-related sequences,
DNA fractionated by 2D CHEF-PFGE was hybridized with ORF-specific
PCR-generated probes. 2D CHEF-PFGE allows for the differentiation of
LPs and CPs and has been widely used in the study of
Borrelia plasmids (1, 11, 14, 15, 27, 33, 34, 45, 46,
52). CPs migrate with retarded mobility in PFGE systems and upon
electrophoresis in the second dimension lag behind the axis along which
the LPs migrate. An exception are small CPs whose mobility is not
significantly retarded upon electrophoresis in the second dimension and
thus migrate along the same axis as LPs (this suggestion was confirmed
through control experiments discussed below). DNA fractionated by this method is shown in Fig. 4A. A variety of
LPs and CPs, but not the chromosome, hybridized with the ORF 1, 2, or 4 probes. Not all probes hybridized with the same plasmids, indicating
that ORF 1-, 2-, and 4-related sequences are not always linked and that
the presence of these sequences on plasmids other than cp8.3 is not in
all cases due to a simple integration event of cp8.3. Consistent with
this, Barbour et al. detected only ORF 1 homologs on a 17-kb LP of
B. burgdorferi (5).
View larger version (42K):
[in this window]
[in a new window]
|
FIG. 4.
Determination of the size and conformation of the
genomic elements that carry ORF 1- and ORF 4-related sequences.
DNA was fractionated by 2D CHEF-PFGE as described in the text. The
direction of electrophoresis in each dimension is indicated. Arrowheads
indicate the locations of the linear plasmids. The gel shown in panel A
was blotted and probed with an ORF 1 probe (B), while a similar gel was
used in the ORF 4 analyses (C). Isolates analyzed were B. garinii FRG (lane 1), B. valaisiana VS116 (lane
2), and B. afzelii IP21 (lane 3). The migration
positions of the approximately 980-kb linear chromosome (lc)
(7), 50-kb ospAB-carrying LP (lp50)
(8), 80-kb LP of isolate VS116 (lp80) (27), and
cp8.3 are indicated. The migration positions of the CPs are not
indicated but can be seen just to the left of the LPs of isolate FRG in
panel A. It is important to note that lp80, the size of which was
determined in a previous study, is carried only by VS116
(27). The signal to the right of the FRG LPs in panel C
represents background.
|
|
The ORF 1 probe hybridized with several plasmids in isolates FRG and
IP21 (Fig. 4B). Only weak hybridization with VS116 DNA was observed. In
FRG, a CP hybridized with the probe. This plasmid is faintly visible in
the ethidium bromide-stained gel just to the left of the 50-kb
ospAB-carrying LP. Based on its migration characteristics,
this plasmid is likely one or more of the 32-kb CPs that carry members
of the UHB gene family (1, 11, 35). This was confirmed
through Southern analysis by using the uhb(+) oligonucleotide
(35) as a probe (data not shown). To ascertain if ORF 1 sequences are present on the ospC-carrying plasmid cp26, the
blots were probed with an ospC-targeting oligonucleotide
(32, 44) (data not shown). The probe hybridized with a CP
slightly smaller than the one that bound the ORF 1 probe, indicating
that ORF 1-related sequences are not present on cp26. Significant
hybridization of the ORF 1 probe to a CP was observed only with isolate
FRG. In the ethidium bromide-stained gels, two CPs are visible in
isolate FRG, while these bands are not visible in VS116 and IP21. It is unclear if these isolates lack conserved 32-kb CPs or if they are
carried by only a small percentage of the isolate population and thus
were not detected by Southern hybridization.
The ORF 1 probe also hybridized with what appeared to be an LP of 8 to
10 kb in isolates IP21 and FRG (faint hybridization was observed with
VS116). Linear plasmids of this size have not been observed in
B. burgdorferi sensu lato isolates, and in light of
this, we wanted to determine if this plasmid might be cp8.3. We
speculated that due to the size of this plasmid, its migration in the
second dimension might not be retarded to the same degree as that of
larger Borrelia CPs. To test this, we electrophoresed under
2D CHEF-PFGE conditions the following: (i) a purified 6-kb control CP
mixed with lambda DNA monocut molecular size standards (linear DNA),
(ii) purified plasmid alone, (iii) lambda monocut molecular size
standards alone, and (iv) Borrelia DNA. The 6-kb CP migrated
along the linear axis of migration and exhibited no retarded mobility
in the second dimension (data not shown). To verify that the plasmid
had not been linearized, we also performed electrophoresis under
constant field conditions. Under these conditions, the plasmid migrated
with characteristics indicative of a supercoiled conformation (i.e.,
with an apparent size of 3.5 kb). These experiments demonstrate that
the migration of small CPs are not significantly regarded in 2D
CHEF-PFGE and suggest that the 8- to 10-kb plasmid observed along the
linear plasmid axis is in fact cp8.3.
ORF 1-hybridizing LPs were also detected in some isolates.
B. garinii FRG carries two hybridizing LPs of
approximately 30 and 17 kb, while B. afzelii IP21
carries two hybridizing LPs of 50 and 25 kb. Weak hybridization with a
20-kb LP was observed with isolate VS116. This 20-kb LP was not visible
in the stained gels; hence, the weak signal likely reflects a low copy
number of the plasmid in the isolate population. The detection of ORF 1-related sequences on LPs is consistent with the detection of ORF 1 homologs on a 17-kb LP in B. burgdorferi B31
(5). However, the analyses presented here demonstrate that
ORF 1-carrying LPs vary in size and are not present in all isolates.
Hybridization analyses of ORF 2 detected this ORF on cp32 in isolates
FRG, VS116, and IP21. In VS116 and IP21, other hybridizing CPs that
migrated close to cp32 were also detected (data not shown). Since these
other CPs did not hybridize with the ORF 1 or UHB probes, it can be
concluded that they are not conformational variants of cp32 and are
distinct CPs. Weak hybridization with 50- and 20-kb LPs and with cp8.3
was observed with all three isolates. As with ORF 1, ORF 2-related
sequences appear to be present on variably sized plasmids.
The PCR-generated ORF 4 probe hybridized with both CPs and LPs (Fig.
4C). Five hybridizing plasmids were detected in IP21, and four were
detected in FRG. While hybridization with the 32-kb CPs of FRG was
detected, this was not the case for isolates IP21 and VS116. In FRG,
several LPs hybridized with the probe but only weakly. It is unclear if
this represents specific hybridization, and in light of this, we will
not focus on these plasmids in this discussion. In isolate IP21, the 8- to 10-kb plasmid that hybridized with the ORF 1 and 2 probes also
hybridized with the ORF 4 probe, supporting the identification of this
plasmid as cp8.3. Hybridization with a plasmid of equivalent size was
observed in FRG as well. In FRG and IP21, the ORF 4 probe also
hybridized with large CPs that migrated slower than the 32-kb CPs (Fig.
4C). Although these plasmids were not visible in stained gels, they
were readily detected by Southern blotting. Size determinations based
on 2D CHEF-PFGE alone are not sufficient for accurate size
determination of CPs; however, based on its relative migration, we
tentatively estimate this plasmid to be approximately 70 kb. CPs of
this size have not been previously described in B. burgdorferi sensu lato isolates. Since these plasmids did not
hybridize with the ospC, UHB, and ORF 1 and 2 probes, which
hybridize with either cp26, cp32, or cp8.3, it can be concluded that
they are not artifacts, altered conformations, or multimers of cp32,
cp26, or cp8.3 but are distinct plasmids.
To determine if these plasmids are present in other isolates,
one-dimensional CHEF-PFGE and Southern hybridization with the ORF 4 PCR
probe were performed. Hybridizing plasmids that migrated to positions
analogous to those of the large CPs of B. garinii FRG
and B. afzelii IP21 were detected in B. burgdorferi LP7, CA9, and 297 and in B. afzelii
ECM1 and UMO1 (data not shown). Other isolates listed in Table 1 do not
carry plasmids with similar migration characteristics that hybridize
with the ORF 4 probe. This does not preclude the possibility that they
carry a similarly sized plasmid which lacks ORF 4.
Southern blot and spPCR analyses of IRA.
In light of the
repeated nature of cp8.3-carried sequences, we wanted to determine if
the IR elements of cp8.3 may have contributed to the multicopy state of
these ORFs by promoting the integration of cp8.3 or portions thereof
into other plasmids. To determine if related IR elements exist in other
isolates that carry multiple copies of the ORFs, digested DNA was
probed with the IRA-F1 oligonucleotide. B. burgdorferi
LP7 and CA9, B. garinii FRG, B. valaisiana VS116, and B. afzelii IP21 and UMO1
were hybridization positive, and multiple bands were observed in some
isolates (Fig. 5). As predicted by the
restriction map of cp8.3, an 8.3-kb hybridizing fragment was detected
in isolates LP7, IP21, and UMO1. Smaller fragments were detected in FRG
(6.5 kb), VS116 (6.0 kb), and CA9 (5.6 kb), and larger fragments were
detected in LP7 (10 kb), CA9 (10 kb), and VS116 (15 kb). The sizes of
these larger restriction fragments indicate that they must be derived
from genomic elements other than cp8.3. The smaller fragments
could be derived from other plasmids or from cp8.3 variants which have
different EcoRV RFLP patterns.
View larger version (47K):
[in this window]
[in a new window]
|
FIG. 5.
RFLP pattern analyses of cp8.3-related IR elements. DNA
was digested to completion with EcoRV, fractionated in an
0.8% agarose gel, blotted, and probed with the IRA-F1 probe as
described in the text. For lane identification, see the legend to Fig.
2.
|
|
To identify the genomic elements carrying the IR element, the
IRA-F1 probe was hybridized with B. garinii FRG and
B. valaisiana VS116 DNA fractionated by 2D CHEF-PFGE
(data not shown). In both isolates, strong hybridization with cp32 was
detected. A weaker signal was associated with two slightly smaller CPs
in both isolates, although the precise sizes of these plasmids differed
in each isolate. The IRA-F1 probe also hybridized weakly with LPs of 25 and 17 kb in FRG and VS116, respectively. While the IR element is
distributed across species lines, it is not carried by all isolates,
and like the ORFs, its location on specific genomic elements
differs among isolates.
To determine if the IRA-F1 hybridizing sequences exist with IR
orientation, spPCR analyses were performed with the IRA-F1 primer
(Table 1). spPCR will yield product only if the primer binding sites
exist with an IR orientation and are within an amplifiable range of
each other. Amplicons of 3 kb were obtained from isolates CA9, N34,
IP21, and UMO1, demonstrating an IR organization. This size is
consistent with that predicted by the cp8.3 sequence of IP21. Isolates
LP7 and VS116, which hybridized with the IRA-F1 probe, did not yield
spPCR amplicons, indicating that multiple primer target sites in these
isolates either are not IRs, are located too far apart, are variable in
sequence such that PCR amplification is prevented, or are located on
different plasmids.
PCR and DNA sequence analysis of the regions flanking IRA.
IR
elements often flank mobile genetic elements, and as a consequence of
translocation and integration, regions adjacent to IRs tend to be
polymorphic. The IRA element of cp8.3 is flanked by ORFs 1 and 7, which have an opposite orientation to each other. To screen
for polymorphisms around the borders of IRA, IRA and its flanking
regions were amplified by using the O1R1 and O7R1 primers (Fig. 1).
Amplicons of 700 bp were predicted and obtained from isolates FRG, N34,
VS116, IP21, and UMO1 (Fig. 6A). However, the amplicon obtained from B. andersonii 21038 was only 300 bp in size. Sequence analyses of the
amplicons from isolates 21038, IP21, and UMO1 were performed to
identify the molecular basis for this truncated amplicon. The
B. afzelii UMO1 amplicon carries a conserved IR element
that differs from the IP21 sequence at only a few positions. The
amplicon from isolate 21038 exhibits 67% nucleotide identity with a
segment of the IP21 cp8.3 sequence extending from just upstream of ORF
7 through the majority of ORF 8 (with the exception of its 5' end),
while the other end of the amplicon exhibits identity with the 5' end
of ORF 1 (Fig. 6B). The IRA sequence and the start codons of ORFs 1 and
8 are absent from this amplicon. Hence, these copies of ORFs 1 and 8 can be considered to be pseudogenes. Relative to the IP21 cp8.3 sequence, there is a deletion of several hundred bases and a possible insertion of foreign sequence. The sequence analyses also explain why
the IRA-F1 probe did not hybridize with B. andersonii
21038 DNA and provide suggestive evidence for previous recombination events that may have involved the IR element.
View larger version (19K):
[in this window]
[in a new window]
|
FIG. 6.
PCR and sequence analyses of IRA and its flanking
regions. (A) Amplicons obtained with the O7R1 and O1R1 primers are
presented. The O7R1-O1R1 primer pair was designed to amplify across IRA
of cp8.3. For lane identification, see the legend to Fig. 2. (B)
Selected amplicons were partially sequenced. Gaps introduced by
alignment are indicated by dashes, conserved nucleotides are indicated
by periods, and the sequence corresponding to IRA is indicated by
boldface letters. The start codon of ORF 8 is underlined and indicated
with italic letters.
|
|
Conclusions.
The goals of this study were to assess the
distribution and molecular organization of the cp8.3 ORFs and IR
elements among species of the B. burgdorferi sensu lato
complex. RFLP pattern analyses of the ORFs revealed that their
organization varies widely among isolates and is of greater
complexity than previously appreciated. These ORFs (or closely
related sequences) exist in a multicopy state in some isolates, with
copies carried on both LPs and CPs. A significant body of evidence that
demonstrates pronounced redundancy in the plasmid component of the
Borrelia genome has now accumulated (5, 11, 21, 35, 39,
50, 52, 54, 62, 63). The presence of repeated sequences and IR
elements on some Borrelia plasmids could provide a molecular
basis for homologous recombination at the inter- and intraplasmid
level. Recombination among plasmids could lead to hypervariability in
plasmid composition and organization even among closely related
isolates. Consistent with this, the organization and copy number of
ORFs 1, 2, and 4 and the IR element differ among isolates and do not
appear to be evolutionarily stable traits. It is important to note that
not all plasmids of the Borrelia genome exhibit
hypervariability. The ospC-carrying plasmid cp26 is
conserved in size and is universal among isolates of the B. burgdorferi sensu lato complex (32, 55). This is not
surprising since cp26 carries housekeeping genes such as those involved
in purine biosynthesis (37). Hence, there may be selective
pressure to maintain conservation of cp26, while variation in other
plasmids can be tolerated.
In light of the multicopy nature of cp8.3 ORF-related sequences, we
looked for evidence of genetic recombination in these sequences and
sought to assess the possible contribution of the IR elements in these
processes. IRA-related sequences were detected in some isolates on both
LPs and CPs, and spPCR analyses confirmed their IR orientation. PCR and
sequence analyses revealed that the regions adjacent to the IR elements
of at least one isolate, B. andersonii 21038, are
polymorphic, possibly indicating earlier recombinational activity.
Interestingly, the first 39 nucleotides of the coding sequences of ORFs
8 and 4 are carried within the IR elements of cp8.3 (IRA and IRB,
respectively) (13). Hence, transposition or deletion of the
IR-flanked sequence of cp8.3 could conceivably regulate the expression
of these ORFs. In fact, many isolates lack the IR elements and
therefore may not carry the start codons for ORFs 4 and 8. Direct
evidence for the absence of IRA and some of its flanking sequence was
obtained for isolate 21038 through hybridization and DNA sequence
analyses. The IRA flanking sequence that is absent in isolate 21038 also carries the start codon for ORF 1. Hence, this isolate lacks the
start codon for both ORFs 1 and 8 and presumably for ORF 4 (as inferred from the absence of IRB), generating pseudogenes. However, since the IR
elements are absent from many isolates that carry multicopy ORFs, it
cannot be concluded that the IR elements are solely responsible for
generating the multicopy state of cp8.3 ORFs.
Hybridization analyses, conducted to identify the genetic elements that
carry cp8.3 ORF-related sequences, revealed that the ORFs are carried
on variable LPs and CPs but not on the chromosome. In addition, ORF
4-related sequences were detected on previously uncharacterized CPs
that are considerably larger than the largest known B. burgdorferi sensu lato CPs (i.e., cp32). Since these large CPs did
not hybridize with ORF 1, ORF 2, or IRA probes, it can be concluded (i)
that a complete copy of cp8.3 has not been integrated into these
plasmids and (ii) that these plasmids are not multimers or altered
conformations of cp32 or cp8.3. The origin, organization, and coding
capacity of the CPs identified here require further analysis.
The role of the putative cp8.3 ORF-encoded gene products and homologs
in the biology of the Lyme disease spirochetes remains unclear. While a
function for these ORFs has yet to be determined, an important
functional role seems likely in view of their wide distribution among
natural populations and their multicopy state. Database searches have
not proven helpful in identifying the possible functional role of these
proteins, since they exhibit no homology with sequences or sequence
motifs of known function. Preliminary analyses conducted in our
laboratory have demonstrated that these ORFs are not transcriptionally
expressed during in vitro cultivation, indicating that they do not play
an essential role under these environmental conditions (26).
Expression may require different environmental stimuli, perhaps those
encountered in the tick or mammalian host. In conclusion, the data
presented here demonstrate significant complexity and variability in
the organization of cp8.3 ORF-related sequences. Although the ORFs are
widely distributed among isolates, the organization of these sequence
elements is not evolutionarily stable and appears to have been
influenced by interplasmid recombination. Work in progress is focusing
on assessing the transcriptional patterns of expression of these ORFs
in different environments (i.e., in vitro, in the tick, and in the
mammalian host). These studies will provide clues as to the possible
biological role of the multicopy ORF 1-, 2-, and 4-related sequences in
the biology of the B. burgdorferi sensu lato complex.
This work was supported in part by grants from the Jeffress Trust
and the National Institutes of Health.
We thank Wolfram Zuckert, Jurg Meyer, Steve Porcella, Scott Samuels,
and the Virginia Commonwealth University molecular pathogenesis group
for helpful discussions and Todd Kitten for computer assistance.
| 1.
|
Akins, D.,
S. F. Porcella,
T. G. Popova,
D. Shevchenko,
S. I. Baker,
M. Li,
M. V. Norgard, and J. D. Radolf.
1995.
Evidence for in vivo but not in vitro expression of a Borrelia burgdorferi outer surface protein F (OspF) homologue.
Mol. Microbiol.
18:507-520[Medline].
|
| 2.
|
Assous, M. V.,
D. Postic,
G. Paul,
P. Nevot, and G. Baranton.
1994.
Individualization of two new genomic groups among American Borrelia burgdorferi sensu lato strains.
FEMS Microbiol. Lett.
121:93-98[Medline].
|
| 3.
|
Baranton, G.,
D. Postic,
I. Saint Girons,
P. Boerlin,
J.-C. Piffaretti,
M. Assous, and P. A. D. Grimont.
1992.
Delineation of Borrelia burgdorferi sensu stricto, Borrelia garinii sp. nov., and group VS461 associated with Lyme borreliosis.
Int. J. Syst. Bacteriol.
42:378-383[Medline].
|
| 4.
|
Barbour, A. G.
1988.
Plasmid analysis of Borrelia burgdorferi, the Lyme disease agent.
J. Clin. Microbiol.
26:475-478[Abstract/Free Full Text].
|
| 5.
|
Barbour, A. G.,
C. J. Carter,
V. Bundoc, and J. Hinnebusch.
1996.
The nucleotide sequence of a linear plasmid of Borrelia burgdorferi reveals similarities to those of circular plasmids of other prokaryotes.
J. Bacteriol.
178:6635-6639[Abstract/Free Full Text].
|
| 6.
|
Barbour, A. G., and C. F. Garon.
1988.
The genes encoding the major surface proteins of Borrelia burgdorferi are located on a plasmid.
Ann. N. Y. Acad. Sci.
539:144-153[Medline].
|
| 7.
|
Baril, C.,
C. Richaud,
G. Baranton, and I. Saint Girons.
1989.
Linear chromosome of Borrelia burgdorferi.
Res. Microbiol.
140:507-516[Medline].
|
| 8.
|
Bergström, S.,
V. G. Bundoc, and A. G. Barbour.
1989.
Molecular analysis of the linear plasmid encoded major surface proteins, OspA and OspB, of the Lyme disease spirochete Borrelia burgdorferi.
Mol. Microbiol.
3:479-486[Medline].
|
| 9.
|
Burgdorfer, W.,
A. G. Barbour,
S. F. Hayes,
J. L. Benach,
E. Grunwaldt, and J. P. Davis.
1982.
Lyme disease a tick-borne spirochetosis?
Science
216:1317-1319[Abstract/Free Full Text].
|
| 10.
|
Casjens, S.,
M. DeLang,
H. L. Ley III,
P. Rosa, and W. M. Huang.
1995.
Linear chromosome of Lyme disease agent spirochetes: genetic diversity and conservation of gene order.
J. Bacteriol.
177:2769-2780[Abstract/Free Full Text].
|
| 11.
|
Casjens, S.,
R. van Vugt,
K. Tilly,
P. A. Rosa, and B. Stevenson.
1997.
Homology throughout the multiple 32-kilobase circular plasmids in Lyme disease spirochetes.
J. Bacteriol.
179:217-227[Abstract/Free Full Text].
|
| 12.
|
Champion, C. I.,
D. R. Blanco,
J. T. Skare,
D. A. Haake,
M. Giladi,
D. Foley,
J. N. Miller, and M. A. Lovett.
1994.
A 9.0-kilobase-pair circular plasmid of Borrelia burgdorferi encodes an exported protein: evidence for expression only during infection.
Infect. Immun.
62:2653-2661[Abstract/Free Full Text].
|
| 13.
|
Dunn, J. J.,
S. R. Buchstein,
L.-L. Butler,
S. Fisenne,
D. S. Polin,
B. N. Lade, and B. J. Luft.
1994.
Complete nucleotide sequence of a circular plasmid from the Lyme disease spirochete, Borrelia burgdorferi.
J. Bacteriol.
176:2706-2717[Abstract/Free Full Text].
|
| 14.
|
Ferdows, M. S., and A. G. Barbour.
1989.
Megabase-sized linear DNA in the bacterium Borrelia burgdorferi, the Lyme disease agent.
Proc. Natl. Acad. Sci. USA
86:5969-5973[Abstract/Free Full Text].
|
| 15.
|
Ferdows, M. S.,
P. Serwer,
G. A. Griess,
S. J. Norris, and A. G. Barbour.
1996.
Conversion of a linear to a circular plasmid in the relapsing fever agent Borrelia hermsii.
J. Bacteriol.
178:793-800[Abstract/Free Full Text].
|
| 16.
|
Fleche, A. L.,
D. Postic,
K. Girardet,
O. Peter, and G. Baranton.
1997.
Characterization of Borrelia lusitaniae sp. nov. by 16S ribosomal DNA sequence analysis.
Int. J. Syst. Bacteriol.
47:921-925[Medline].
|
| 17.
|
Fukunaga, M.,
A. Hamase,
K. Okada,
H. Inoue,
Y. Tsuruta,
K. Miyamoto, and M. Nakao.
1996.
Characterization of spirochetes isolated from ticks (Ixodes tanukin, Ixodes turdus, and Ixodes columnae) and comparison of the sequences with those of Borrelia burgdorferi sensu lato strains.
Appl. Environ. Microbiol.
62:2338-2344[Abstract].
|
| 18.
|
Fukunaga, M.,
A. Hamase,
K. Okada, and M. Nakao.
1996.
Borrelia tanuki sp. nov. and Borrelia turdae sp. nov. found from ixodid ticks in Japan: rapid species identification by 16S rRNA gene-targeted PCR analysis.
Microbiol. Immunol.
40:877-881[Medline].
|
| 19.
|
Fukunaga, M.,
K. Okada,
M. Nakao,
T. Konishi, and Y. Sato.
1996.
Phylogenetic analysis of Borrelia species based on flagellin gene sequences and its application for molecular typing of Lyme disease borreliae.
Int. J. Syst. Bacteriol.
46:898-905[Medline].
|
| 20.
|
Hinnebusch, J., and A. G. Barbour.
1991.
Linear plasmids of Borrelia burgdorferi have a telomeric structure and sequence similar to those of a eukaryotic virus.
J. Bacteriol.
173:7233-7239[Abstract/Free Full Text].
|
| 21.
|
Hinnebusch, J.,
S. Bergstrom, and A. G. Barbour.
1990.
Cloning and sequence analysis of linear plasmid telomeres of the bacterium Borrelia burgdorferi.
Mol. Microbiol.
4:811-820[Medline].
|
| 22.
|
Hyde, F. W., and R. C. Johnson.
1988.
Characterization of a circular plasmid from Borrelia burgdorferi, etiologic agent of Lyme disease.
J. Clin. Microbiol.
26:2203-2205[Abstract/Free Full Text].
|
| 23.
|
Jauris-Heipke, S.,
G. Liegl,
V. Preac-Mursic,
D. Röbler,
E. Schwab,
E. Soutschek,
G. Will, and B. Wilske.
1995.
Molecular analysis of genes encoding outer surface protein C (OspC) of Borrelia burgdorferi sensu lato: relationship to ospA genotype and evidence of lateral gene exchange of ospC.
J. Clin. Microbiol.
33:1860-1866[Abstract].
|
| 24.
|
Kawabata, H.,
T. Masuzawa, and Y. Yanagihara.
1993.
Genomic analysis of Borrelia japonica sp. nov. isolated from Ixodes ovatus in Japan.
Microbiol. Immunol.
37:843-848[Medline].
|
| 25.
|
Lam, T. T.,
T.-P. K. Nguyen,
R. R. Montgomery,
F. S. Kantor,
E. Fikrig, and R. A. Flavell.
1994.
Outer surface proteins E and F of Borrelia burgdorferi, the agent of Lyme disease.
Infect. Immun.
62:290-298[Abstract/Free Full Text].
|
| 26.
| Marconi, R. T. Unpublished data.
|
| 27.
|
Marconi, R. T.,
S. Casjens,
U. G. Munderloh, and D. S. Samuels.
1996.
Analysis of linear plasmid dimers in Borrelia burgdorferi sensu lato isolates: implications concerning the potential mechanism of linear plasmid replication.
J. Bacteriol.
178:3357-3361[Abstract/Free Full Text].
|
| 28.
|
Marconi, R. T., and C. F. Garon.
1992.
Identification of a third genomic group of Borrelia burgdorferi through signature nucleotide analysis and 16S rRNA sequence determination.
J. Gen. Microbiol.
138:533-536[Medline].
|
| 29.
|
Marconi, R. T., and C. F. Garon.
1992.
Phylogenetic analysis of the genus Borrelia: a comparison of North American and European isolates of B. burgdorferi.
J. Bacteriol.
174:241-244[Abstract/Free Full Text].
|
| 30.
|
Marconi, R. T.,
M. E. Konkel, and C. F. Garon.
1993.
Variability of osp genes and gene products among species of Lyme disease spirochetes.
Infect. Immun.
61:2611-2617[Abstract/Free Full Text].
|
| 31.
|
Marconi, R. T.,
D. Liveris, and I. Schwartz.
1995.
Identification of novel insertion elements, restriction fragment length polymorphism patterns, and discontinuous 23S rRNA in Lyme disease spirochetes: phylogenetic analyses of rRNA genes and their intergenic spacers in Borrelia japonica sp. nov. and genomic group 21038 (Borrelia andersonii sp. nov.) isolates.
J. Clin. Microbiol.
33:2427-2434[Abstract].
|
| 32.
|
Marconi, R. T.,
D. S. Samuels, and C. F. Garon.
1993.
Transcriptional analyses and mapping of the ospC gene in Lyme disease spirochetes.
J. Bacteriol.
175:926-932[Abstract/Free Full Text].
|
| 33.
|
Marconi, R. T.,
D. S. Samuels,
R. K. Landry, and C. F. Garon.
1994.
Analysis of the distribution and molecular heterogeneity of the ospD gene among the Lyme disease spirochetes: evidence for lateral gene exchange.
J. Bacteriol.
176:4572-4582[Abstract/Free Full Text].
|
| 34.
|
Marconi, R. T.,
D. S. Samuels,
T. G. Schwan, and C. F. Garon.
1993.
Identification of a protein in several Borrelia species which is related to OspC of the Lyme disease spirochetes.
J. Clin. Microbiol.
31:2577-2583[Abstract/Free Full Text].
|
| 35.
|
Marconi, R. T.,
S. Y. Sung,
C. A. N. Hughes, and J. A. Carlyon.
1996.
Molecular and evolutionary analyses of a variable series of genes in Borrelia burgdorferi that are related to ospE and ospF, constitute a gene family, and share a common upstream homology box.
J. Bacteriol.
178:5615-5626[Abstract/Free Full Text].
|
| 36.
|
Marconi, R. T.,
J. Wigboldus,
H. Weissbach, and N. Brot.
1991.
Transcriptional start site and MetR binding sites on the Escherichia coli metH gene.
Biochem. Biophys. Res. Commun.
175:1057-1063[Medline].
|
| 37.
|
Margolis, N.,
D. Hogan,
K. Tilly, and P. Rosa.
1994.
Plasmid location of Borrelia purine biosynthesis gene homologs.
J. Bacteriol.
176:6427-6432[Abstract/Free Full Text].
|
| 38.
|
Norris, S. J.,
C. J. Carter,
J. K. Howell, and A. G. Barbour.
1992.
Low-passage-associated proteins of Borrelia burgdorferi B31: characterization and molecular cloning of OspD, a surface-exposed, plasmid-encoded lipoprotein.
Infect. Immun.
60:4662-4672[Abstract/Free Full Text].
|
| 39.
|
Porcella, S. F.,
T. G. Popova,
D. R. Akins,
M. Li,
J. R. Radolf, and M. V. Norgard.
1996.
Borrelia burgdorferi supercoiled plasmids encode multicopy open reading frames and a lipoprotein gene family.
J. Bacteriol.
178:3293-3307[Abstract/Free Full Text].
|
| 40.
|
Postic, D.,
M. V. Assous,
P. A. D. Grimont, and G. Baranton.
1994.
Diversity of Borrelia burgdorferi sensu lato evidenced by restriction fragment length polymorphism of rrf (5S)-rrl (23S) intergenic spacer amplicons.
Int. J. Syst. Bacteriol.
44:733-742.
|
| 41.
|
Postic, D.,
J. Belfazia,
E. Isogai,
I. Saint Girons,
P. A. D. Grimont, and G. Baranton.
1993.
A new genomic species in Borrelia burgdorferi sensu lato isolated from Japanese ticks.
Res. Microbiol.
144:467-473[Medline].
|
| 42.
|
Postic, D.,
C. Edlinger,
C. Richaud,
F. Grimont,
Y. Dufresne,
P. Perolat,
G. Baranton, and P. A. D. Grimont.
1990.
Two genomic species in Borrelia burgdorferi.
Res. Microbiol.
141:465-475[Medline].
|
| 43.
|
Rosa, P. A.,
T. Schwan, and D. Hogan.
1992.
Recombination between genes encoding major outer surface proteins A and B of Borrelia burgdorferi.
Mol. Microbiol.
6:3031-3040[Medline].
|
| 44.
|
Sadziene, A.,
B. Wilske,
M. S. Ferdows, and A. G. Barbour.
1993.
The cryptic ospC gene of Borrelia burgdorferi B31 is located on a circular plasmid.
Infect. Immun.
61:2192-2195[Abstract/Free Full Text].
|
| 45.
|
Samuels, D. S.,
R. T. Marconi, and C. F. Garon.
1993.
Variation in the size of the ospA-containing linear plasmid, but not the linear chromosome, among the three Borrelia species associated with Lyme disease.
J. Gen. Microbiol.
139:2445-2449[Medline].
|
| 46.
|
Samuels, D. S.,
R. T. Marconi,
W. M. Huang, and C. F. Garon.
1994.
gyrB mutations in coumermycin A1-resistant Borrelia burgdorferi.
J. Bacteriol.
176:3072-3075[Abstract/Free Full Text].
|
| 47.
|
Schwan, T. G.,
W. Burgdorfer, and C. F. Garon.
1988.
Changes in infectivity and plasmid profile of the Lyme disease spirochete, Borrelia burgdorferi, as a result of in vitro cultivation.
Infect. Immun.
56:1831-1836[Abstract/Free Full Text].
|
| 48.
|
Schwan, T. G.,
M. E. Schrumpf,
R. H. Karstens,
J. R. Clover,
J. Wong,
M. Daugherty,
M. Struthers, and P. A. Rosa.
1993.
Distribution and molecular analysis of Lyme disease spirochetes, Borrelia burgdorferi, isolated from ticks throughout California.
J. Clin. Microbiol.
31:3096-3108[Abstract/Free Full Text].
|
| 49.
|
Schwartz, J. J.,
A. Gazumyan, and I. Schwartz.
1992.
rRNA gene organization in the Lyme disease spirochete, Borrelia burgdorferi.
J. Bacteriol.
174:3757-3765[Abstract/Free Full Text].
|
| 50.
|
Simpson, W. J.,
C. F. Garon, and T. G. Schwan.
1990.
Analysis of supercoiled circular plasmids in infectious and non-infectious Borrelia burgdorferi.
Microb. Pathog.
8:109-118[Medline].
|
| 51.
|
Steere, A. C.,
R. L. Grodzicki,
A. N. Kornblatt,
J. E. Craft,
A. G. Barbour,
W. Burgdorfer,
G. P. Schmid,
E. Johnson, and S. E. Malawista.
1983.
The spirochetal etiology of Lyme disease.
N. Engl. J. Med.
308:733-740[Abstract].
|
| 52.
|
Stevenson, B.,
K. Tilly, and P. A. Rosa.
1996.
A family of genes located on four separate 32-kilobase circular plasmids in Borrelia burgdorferi B31.
J. Bacteriol.
178:3508-3516[Abstract/Free Full Text].
|
| 53.
|
Suk, K.,
S. Das,
W. Sun,
B. Jwang,
S. W. Barthold,
R. A. Flavell, and E. Fikrig.
1995.
Borrelia burgdorferi genes selectively expressed in the infected host.
Proc. Natl. Acad. Sci. USA
92:4269-4273[Abstract/Free Full Text].
|
| 54.
| Sung, S.-Y., C. LaVoie, J. A. Carlyon,
and R. T. Marconi. Evidence for extensive recombination among
ospE homolog members of the UHB gene family in isolates of
the Borrelia burgdorferi sensu lato complex. Submitted for
publication.
|
| 55.
|
Tilly, K.,
S. Casjens,
B. Stevenson,
J. L. Bono,
D. S. Samuels,
D. Hogan, and P. Rosa.
1997.
The Borrelia burgdorferi circular plasmid cp26: conservation of plasmid structure and targeted inactivation of the ospC gene.
Mol. Microbiol.
25:361-373[Medline].
|
| 56.
|
Wallich, R.,
C. Brenner,
M. D. Kramer, and M. M. Simon.
1995.
Molecular cloning and immunological characterization of a novel linear-plasmid-encoded gene, pG, of Borrelia burgdorferi expressed only in vivo.
Infect. Immun.
63:3327-3335[Abstract].
|
| 57.
|
Wallich, R.,
C. Helmes,
U. E. Schaible,
Y. Lobet,
S. E. Moter,
M. D. Kramer, and M. M. Simon.
1992.
Evaluation of genetic divergence among Borrelia burgdorferi isolates by use of OspA, fla, HSP60, and HSP70 gene probes.
Infect. Immun.
60:4856-4866[Abstract/Free Full Text].
|
| 58.
|
Wang, G.,
A. P. van Dam,
A. Le Flecha,
D. Postic,
O. Peter,
G. Baranton,
R. de Boer,
L. Spanjaard, and J. Dankert.
1997.
Genetic and phenotypic analysis of Borrelia valaisiana sp. nov. (Borrelia genomic groups VS116 and M19).
Int. J. Syst. Bacteriol.
47:926-932[Medline].
|
| 59.
|
Welsh, J.,
C. Pretzman,
D. Postic,
I. Saint Girons,
G. Baranton, and M. McClelland.
1992.
Genomic fingerprinting by arbitrarily primed polymerase chain reaction resolves Borrelia burgdorferi into three distinct phyletic groups.
Int. J. Syst. Bacteriol.
42:370-377[Medline].
|
| 60.
|
Xu, Y., and R. C. Johnson.
1995.
Analysis and comparison of plasmid profiles of Borrelia burgdorferi sensu lato strains.
J. Clin. Microbiol.
33:2679-2685[Abstract].
|
| 61.
|
Xu, Y.,
C. Kodner,
L. Coleman, and R. C. Johnson.
1996.
Correlation of plasmids with infectivity of Borrelia burgdorferi sensu stricto type strain B31.
Infect. Immun.
64:3870-3876[Abstract].
|
| 62.
| Zuckert, W. R., R. T. Marconi, J. A. Carlyon, and J. Meyer. 1997. Unpublished data.
|
| 63.
|
Zuckert, W. R., and J. Meyer.
1996.
Circular and linear plasmids of Lyme disease spirochetes have extensive homology: characterization of a repeated DNA element.
J. Bacteriol.
178:2287-2298[Abstract/Free Full Text].
|
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
Abstract
Introduction
