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Journal of Bacteriology, September 1998, p. 4850-4855, Vol. 180, No. 18
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Transformation of the Lyme Disease Spirochete
Borrelia burgdorferi with Heterologous DNA
Brian
Stevenson,*
James L.
Bono,
Abdallah
Elias,
Kit
Tilly, and
Patricia
Rosa
Laboratory of Microbial Structure and
Function, Rocky Mountain Laboratories, National Institute of
Allergy and Infectious Diseases, National Institutes of Health,
Hamilton, Montana 59840
Received 6 May 1998/Accepted 22 July 1998
 |
ABSTRACT |
Studies of the spirochete Borrelia burgdorferi have
been hindered by the scarcity of genetic tools that can be used in
these bacteria. For the first time, a method has been developed by
which heterologous DNA (DNA without a naturally occurring B. burgdorferi homolog) can be introduced into and persistently
maintained by B. burgdorferi. This technique uses
integration of circular DNA into the bacterial genome via a
single-crossover event. The ability to transform B. burgdorferi with heterologous DNA will now permit a wide range of
experiments on the biology of these bacteria and their involvement in
the many facets of Lyme disease.
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INTRODUCTION |
Lyme disease is caused by
Borrelia burgdorferi and other closely related spirochetes
(3). Spread to mammals through the bites of infected ixodid
ticks, it is the most commonly reported arthropod-borne disease
affecting humans in the United States, and the number of cases is
increasing yearly (4). The disease can manifest in many
tissues and organs, including the skin, musculoskeletal, cardiac, and
neurologic systems. Furthermore, the causative bacteria can persist for
long periods in the mammalian body despite an active immune response
directed against bacterial proteins (26, 28).
First reported in 1982 (3), B. burgdorferi has
since been the focus of extensive research, yet little is known about
how these bacteria cause the many aspects of Lyme disease or how they persistently infect mammals. Infected humans and other mammals produce
antibodies directed against a number of B. burgdorferi proteins (1, 5, 7, 8, 10, 24, 29, 35), which indicates that
these proteins are produced by the bacteria during mammalian infection.
Several of the identified antigens appear to be located on the outer
surfaces of the bacteria, suggesting that these proteins may facilitate
interactions between the spirochetes and their environments. Synthesis
of the B. burgdorferi OspC protein is induced upon tick
feeding (25), which suggests that this protein is involved
in transmission between the tick and mammalian hosts. Despite these and
other studies, the actual functions of most identified Lyme disease
antigens remain unknown.
Protein functions in many other bacteria have been studied by using
recombinant genetic tools. For example, specific mutant bacteria can be
created and studied for altered phenotypes. Protein function is then
confirmed if the bacteria are restored to the wild-type phenotype by
reintroducing the wild-type gene encoding that protein (the
"molecular Koch's postulates" [18]). Gene expression patterns can also be determined through the use of transcriptional and translational fusions with reporter genes. Such
studies are currently impossible with B. burgdorferi, as there are no known methods by which heterologous DNA (DNA without a
naturally occurring B. burgdorferi homolog) can be
introduced into and stably maintained by these bacteria. A recent study
described the transient expression levels of a reporter gene under the
transcriptional control of B. burgdorferi promoters, but
these fusion constructs could not be maintained by the spirochetes
(27). Stable transformation with reporter fusions will be
critical for measuring gene expression during the B. burgdorferi infection cycle.
Previous studies have demonstrated that DNA can be introduced into
B. burgdorferi and integrate into the genomes of the
recipient cells by homologous recombination. Mutant forms of the
gyrB gene that encode a subunit of DNA gyrase that is
resistant to the antibiotic coumermycin have been developed
(23). DNA carrying these mutant gyrB genes can be
introduced into B. burgdorferi by electroporation and will
recombine with the chromosomal gyrB gene to produce
coumermycin-resistant bacteria (20-22). In an earlier
study, one of these mutant alleles, gyrBr, was
cloned into a segment of a native B. burgdorferi plasmid, cp26, and the recombinant DNA was used to transform B. burgdorferi by electroporation. Coumermycin-resistant colonies
were identified in which the gyrBr gene had
inserted via allelic exchange with the flanking DNA into the resident
cp26 (15). This technique has since been used to disrupt the
B. burgdorferi ospC, guaB, and oppAIV
genes (2, 32).
An additional result of these studies was the demonstration that
coumermycin resistance is a selectable phenotype in merodiploid bacteria. A problem faced when developing a novel genetic system is
that one cannot be certain that a foreign marker gene will be expressed
at levels adequate to confer a selectable phenotype. Consistent with
this, the gyrBr gene is the only selectable
marker that has been successfully used for gene disruptions by allelic
exchange (2, 15, 32), while attempts to transform B. burgdorferi with other antibiotic resistance markers have not been
successful (33).
In other bacterial systems, fragments of heterologous DNA have often
been introduced on small, independently replicating plasmids. However,
many plasmids have limited host ranges and are unlikely to be stably
maintained in B. burgdorferi. While some plasmids have wide
host ranges, the evolutionary distance between the spirochetes and
other eubacteria (12, 36) raises the possibility that even
"broad-host-range" plasmids may be unable to replicate and segregate efficiently in B. burgdorferi. To date, there are
no known plasmids from other bacteria that can replicate in B. burgdorferi (our unpublished results, this work, and reference
27). A technique is now presented by which
heterologous plasmids can be maintained in B. burgdorferi by
integration of circular DNA into the bacterial genome via a
single-crossover event, thus eliminating the requirement for
independent DNA replication and segregation.
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MATERIALS AND METHODS |
Bacteria.
A culture of B. burgdorferi isolate B31
that has been continuously cultivated for several years in laboratory
medium (22) was used for all transformations. A mutant of
isolate B31, B31-NGR (15), was the source of the
gyrBr gene used in the DNA constructs described
below. B. burgdorferi were grown at 35°C in solid
Barbour-Stoener-Kelly (BSK) medium (9, 15) or in liquid
BSK-H medium (Sigma, St. Louis, Mo.) supplemented with 6% rabbit serum
(Sigma).
Recombinant DNA.
Plasmid DNAs were purified from 100-ml
Escherichia coli cultures by using Qiagen midi-purification
kits (Qiagen, Chatsworth, Calif.) or from 1-ml cultures by a crude
boiling-lysis method (19). All restriction endonucleases, T4
DNA ligase, and appropriate buffers were obtained from New England
Biolabs (Beverly, Mass.).
As we described earlier (15), the
gyrBr gene, including the presumed
gyrB promoter, was PCR amplified from B31-NGR chromosomal DNA by using oligonucleotides that introduced a BglII
recognition site at the 5' end and a BclI site at the 3' end
and then cloned into the TA vector pCR2.1 (Invitrogen, San Diego,
Calif.). The DNA fragment containing the gyrBr
gene was cut from this plasmid with BglII and
BclI, separated by agarose gel electrophoresis, and purified
with a Qiaex II kit (Qiagen). The gyrBr fragment
was ligated into the BglII site of pOK12 (34),
and the ligation mix was used to transform E. coli Inv
F'
(Invitrogen) by selecting for kanamycin resistance. Plasmids were
purified from 1-ml cultures, and insertion of
gyrBr into pOK12 was assessed by digestion with
restriction endonucleases. DNA sequencing confirmed the insertion and
indicated the orientation of gyrBr. One such
plasmid, pBLS500, was used thereafter.
A fragment of DNA encoding the
B. burgdorferi oppAV gene
(
2) was subsequently cloned into pBLS500. The
oppAV gene was amplified
from purified
B. burgdorferi B31 plasmid DNA by using the oligonucleotide
primers
listed in Table
1 and cloned into pCR2.1
(Invitrogen).
The
oppAV insert was removed from the
recombinant plasmid by cleavage
at the flanking
EcoRI sites
and cloned into the
EcoRI site of
pBLS500 to generate pJLB5.
Transformation of B. burgdorferi.
Recombinant plasmids
were introduced into B. burgdorferi B31 via electroporation
by the previously described procedure (15, 20).
Approximately 7 µg of DNA dissolved in 1 µl of distilled water was
used in each transformation. Bacteria were resuspended in 5 ml of
liquid medium and incubated overnight at 35°C. Aliquots (100 µl) of
the transformed culture were each plated in solid BSK medium containing
0.5 µg of coumermycin A1 (Sigma) per ml and incubated at 35°C in a
1% CO2 environment.
Screening of transformed B. burgdorferi.
Coumermycin-resistant B. burgdorferi colonies were
individually stabbed with toothpicks, and bacteria were transferred to PCR tubes. The PCR amplification solution contained a pair of oligonucleotide primers specific to the introduced recombinant plasmid
(Table 1). Reaction conditions consisted of 25 cycles, with each cycle
consisting of 30 s at 94°C, 30 s at 50°C, and 1 min at
65°C in a GeneAmp 9600 (Perkin-Elmer, Norwalk, Conn.) with 96-well
PCR plates. A blank spot on the plate and solutions alone served as
negative controls, and either purified pBLS500 or pJLB5 was used as a
positive control. PCR products were separated by agarose gel
electrophoresis and visualized by ethidium bromide staining.
Southern blotting.
Potential recombinant B. burgdorferi clones were grown to a density of approximately
108 bacteria per ml in 100 ml of liquid medium, and total
bacterial DNA was purified as previously described (16). DNA
was digested with restriction endonucleases, separated by pulsed-field
agarose gel electrophoresis (30), and transferred to a nylon
membrane (ICN, Irvine, Calif.). Membranes with DNA from bacteria
transformed with pBLS500 were sequentially hybridized with the purified
DNA fragment containing gyrBr described above
and with purified pOK12. Membranes from bacteria transformed with pJLB5
were sequentially hybridized with the gyrBr
probe and with a previously described oppAV-specific probe
(2). Each probe was individually labeled with
[-32P]dATP (Du Pont, Boston, Mass.) by random priming
(Life Technologies, Gaithersburg, Md.) and incubated with membranes at
55°C (16). Membranes were washed in 0.2× SSC (1× SSC is
0.15 M NaCl plus 0.015 M sodium citrate) at 55°C, and hybridized
probes were detected by autoradiography. Radiolabeled probe was
stripped from the membrane by washing with boiling water, and probe
removal was confirmed by autoradiography before the membrane was
rehybridized with a second probe.
Rescue of integrated DNA.
Approximately 125 ng of total DNA
from B. burgdorferi transformant clone KS5 or AB1 was
subjected to BamHI digestion, followed by phenol extraction
and ethanol precipitation. The digested DNA was resuspended in 1× T4
ligase buffer and T4 DNA ligase. The self-ligated DNA was used to
transform E. coli XL-1 Blue (Stratagene, La Jolla, Calif.),
and the bacteria were plated on Luria-Bertani medium (19)
containing 40 µg of kanamycin per ml. Equivalent amounts of B. burgdorferi transformant DNA that had not been treated with either
BamHI or T4 DNA ligase were also used to transform E. coli XL-1 Blue.
| |
RESULTS |
Construction of a vector and transformation of B. burgdorferi.
The gyrBr gene was previously
amplified by PCR, using oligonucleotide primers that introduced
BglII-compatible restriction sites at both ends
(15). The 2-kb gyrBr fragment was
cloned into the BglII site of pOK12, a 2-kb, low-copy-number E. coli plasmid that confers resistance to kanamycin
(34) to produce a recombinant plasmid, pBLS500 (Fig.
1A). Most of the restriction endonuclease
cleavage sites present in the pOK12 multiple cloning site (MCS) are
unique in pBLS500 (Fig. 1B). The gyrBr gene
contains BamHI, HindIII, and MluI
recognition sites (data not shown; 15) which are
also present in the pOK12 MCS, so these enzymes are unsuitable for
cloning additional DNA fragments into pBLS500.
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FIG. 1.
Characteristics of pBLS500. (A) Construction of pBLS500.
kan is the kanamycin resistance gene. (B) Unique restriction
endonuclease cleavage sites within the MCS of pBLS500. Although the
pOK12 MCS contains BamHI, HindIII, and
MluI recognition sites, additional sites are present in the
gyrBr gene, making these restriction
endonucleases unsuitable for cloning DNA fragments into pBLS500.
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pBLS500 was introduced into
B. burgdorferi isolate B31 under
standard electroporation conditions (
15,
20) and plated in
solid medium containing coumermycin. As a control, another aliquot
of
bacteria was treated identically but without the addition of
DNA into
the electroporation cuvette. After 10 days of incubation,
many
coumermycin-resistant colonies were observed from the culture
that had
been transformed with pBLS500. Approximately 1,500 coumermycin-resistant
colonies were obtained per µg of pBLS500 DNA.
No colonies were
observed in the plates of mock-transformed bacteria
during this
time period.
Coumermycin-resistant colonies could arise from bacteria that stably
integrated pBLS500 into the chromosome (Fig.
2) or from
bacteria that had incorporated
only the
gyrBr gene into the chromosomal
gyrB locus via a double-crossover event.
The bacteria might
also resolve the integration of pBLS500 by
deleting the intervening DNA
through recombination between the
direct duplicating repeats.
Antibiotic-resistant colonies were
therefore screened for pBLS500
integration by PCR amplification
with oligonucleotide primers that
produce a DNA fragment only
from bacteria that contain pBLS500 DNA
(Table
1 and Fig.
2A).
The oligonucleotides used in this initial
screening direct the
synthesis of an approximately 500-bp PCR fragment
from pBLS500
but do not direct synthesis of PCR fragments from
wild-type
B. burgdorferi B31 (Fig.
2A and
3). Of 250 coumermycin-resistant
colonies
tested, 3 were found to contain pBLS500 DNA (a frequency
of 1.2%). One
of these bacterial clones, KS5, was chosen for additional
characterization.
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FIG. 2.
(A) Diagram of the integration of pBLS500 via a
single-crossover event into the gyrB locus of B31 to produce
KS5. Locations of oligonucleotide primers used in the identification of
pBLS500 integrants by PCR are indicated by the small numbered arrows.
Note that oligonucleotide primer number 2 can hybridize with the
gyrB genes of both B31 and KS5 but cannot produce a PCR
product from B31. The two gyrB genes of KS5 have not been
sequenced, so it is unknown which allele contains the coumermycin
resistance-conferring mutations. (B) Restriction map of the chromosomes
of B31 and KS5 near the gyrB locus. Abbreviations: B,
BamHI; E, EcoRI.
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FIG. 3.
Products obtained by PCR amplification of total DNA from
B31 and KS5 using oligonucleotide primers 1 and 2. Reaction products
were separated by agarose gel electrophoresis and stained with ethidium
bromide. The positions of molecular size markers (in kilobases) are
indicated to the left of the gel.
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Analysis of transformed B. burgdorferi.
KS5 was further
analyzed to confirm that pBLS500 had integrated into the chromosomal
gyrB locus as expected. Total genomic DNA (chromosomes and
plasmids) was isolated from B31 and KS5, digested with
EcoRI, Southern blotted, and sequentially incubated with
radiolabeled probes. A probe specific for the B. burgdorferi gyrB gene hybridized with a single B31 EcoRI DNA
fragment of the expected size (Fig. 2B and
4A) and with two KS5 EcoRI DNA
fragments, as expected (Fig. 2B and 4A). A probe specific for the
non-gyrBr region of pBLS500 hybridized to the
7.5-kb EcoRI fragment of KS5 (Fig. 4B). Similar results were
obtained from Southern blot analyses of B31 and KS5 DNA digested with
other restriction endonucleases (data not shown), confirming that
pBLS500 had integrated into the chromosome of KS5 at the
gyrB locus.
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FIG. 4.
Southern blots of B31 and KS5 DNAs cleaved by
EcoRI and hybridized with a probe specific for the B. burgdorferi gyrB gene (A) or with labeled pOK12 (B). The positions
of molecular size markers (in kilobases) are indicated to the left of
the Southern blots.
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KS5 was next analyzed to determine whether the heterologous, integrated
DNA could be rescued. The pBLS500 gene encoding kanamycin
resistance
(
kan) and the genes required for replication and segregation
in
E. coli are flanked by
BamHI sites in KS5
(Fig.
2B). Total
genomic DNA from KS5 was digested with
BamHI, self-ligated, and
used to transform
E. coli, with selection for kanamycin resistance.
Resistant
E. coli arose at a frequency of 550 colonies per µg
of total KS5
DNA. Six kanamycin-resistant
E. coli clones were
picked at
random, and each was found to contain a plasmid that
was identical in
size and restriction endonuclease cleavage pattern
to the corresponding
BamHI fragment of pBLS500 (data not shown).
Therefore,
neither the pBLS500 replication genes nor the kanamycin
resistance gene
were detectably altered while integrated into
the KS5 chromosome. For a
control,
E. coli was also transformed
with KS5 DNA that had
been neither cut nor ligated. No kanamycin-resistant
E. coli
colonies were obtained from this experiment, indicating
that there are
no detectable episomal copies of pBLS500 in KS5.
The pOK12-derived kan gene does not confer kanamycin
resistance to B. burgdorferi KS5.
pBLS500 contains a
gene that confers kanamycin resistance to E. coli;
experiments were performed to examine whether pBLS500 also permits
B. burgdorferi to grow in the presence of this antibiotic. Approximately fivefold-fewer colonies were formed when wild-type B31
was plated in medium containing 20 µg of kanamycin per ml than in
medium without antibiotic. No colonies arose in medium containing 40 µg/ml or higher concentrations of the antibiotic. Similar results
were obtained when KS5 was plated in medium containing kanamycin.
Additionally, B31 was subjected to electroporation with pBLS500 under
the same conditions previously used to generate KS5, except the
bacteria were plated in solid medium containing 40 µg of kanamycin
per ml rather than coumermycin. No colonies arose from these
transformation experiments. We conclude that the pBLS500 kan
gene, which encodes an aminoglycoside 3'-phosphotransferase (11,
34), does not confer detectable antibiotic resistance to KS5.
Integration of additional DNA into B. burgdorferi.
One
goal of these studies will be to complement mutant genes through the
introduction of wild-type alleles. To determine whether this will be
possible via the plasmid integration technique, a 2-kb fragment of
B. burgdorferi DNA containing the oppAV gene (2) was cloned into pBLS500. This plasmid, pJLB5, was
introduced into B31 by electroporation, and bacteria were plated in
medium containing coumermycin. A total of 276 antibiotic-resistant
colonies were screened by PCR for pJLB5 integration with the
oligonucleotide primers listed in Table 1, and one integrant was
identified. This recombinant clone, AB1, was grown further without
coumermycin selection, and extracted DNA was analyzed by Southern
blotting with probes specific for oppAV and
gyrBr, both of which confirmed that pJLB5 was
integrated into the chromosomal gyrB locus (data not shown).
As described above for KS5, the integrated pJLB5 DNA was rescued from
purified AB1 genomic DNA by digestion with BamHI and
self-ligation. It can be concluded, therefore, that at least 6 kb of
DNA can be incorporated into and maintained by B. burgdorferi by the plasmid integration method of transformation.
| |
DISCUSSION |
By utilizing site-directed integration of circular DNA, for the
first time, a method has been developed by which heterologous DNA can
be maintained in B. burgdorferi. At least 6 kb of DNA can be
introduced into B. burgdorferi by this technique.
Furthermore, DNA rescued from the transformed bacteria remained
functionally and structurally intact. Plasmid pJLB5 contains an
additional 2 kb of B. burgdorferi DNA (the oppAV
gene) that could result in homologous recombination, but approximately
equivalent transformation efficiencies were obtained when using either
pBLS500 or pJLB5 (3:250 versus 1:276, respectively). A possible
explanation is that pJLB5 is 50% larger than pBLS500 and is not as
efficiently taken up by the bacteria. One microgram of pBLS500 also
contains more molecules than a similar weight of pJLB5, which will also affect the determination of transformation efficiency.
To avoid the requirement for independent plasmid replication and
segregation, we introduced foreign DNA that can integrate into the
B. burgdorferi genome. This technique uses a recombinant plasmid carrying a DNA fragment that is also located in the genome of
the bacterium to be transformed. The introduced circular DNA can
integrate into the genomic copy of the DNA via a single-crossover event, resulting in a duplication of the target DNA that flanks the
remainder of the introduced DNA. This technique has been widely used to
introduce heterologous DNA into gram-positive cocci and, recently,
Neisseria gonorrhoeae (6, 13, 14, 31). The B. burgdorferi gyrBr gene has an advantage in
that it can serve as both the selectable marker and as the DNA segment
for targeting homologous recombination with the chromosomal
gyrB.
Clearly, the present method of circular plasmid integration can be
improved by the use of selectable markers other than the coumermycin
resistance gyrBr gene. Additional markers will
eliminate the need to screen colonies for DNA integration, permit the
integration of DNA at sites other than the gyrB locus, and
allow the introduction of additional DNA into previously transformed
bacteria. The mutant DNA gyrase in bacteria containing the
gyrBr gene may also alter DNA supercoiling,
which could affect expression of other genes. Identification of
additional selectable markers will also be a valuable first step in
constructing independently replicating shuttle vectors for B. burgdorferi. Recombinant DNAs based on pBLS500 can be useful in
identifying alternative selectable markers for use in B. burgdorferi. An antibiotic resistance gene can be cloned into
pBLS500 and transformed into B. burgdorferi, and integrants
can be selected by plating in medium containing coumermycin. Integrants
can then be assessed for the ability to grow in the presence of the
appropriate antibiotic. The kan gene carried by pBLS500 does
not confer kanamycin resistance to B. burgdorferi, but other
aminoglycoside 3'-phosphotransferase genes with different promoters or
codon usages may be functional in these bacteria. Aminoglycoside
resistance can also be conferred by enzymes with
aminoglycoside-N-acetyltransferase or
aminoglycoside-O-adenyltransferase activity (17).
A major obstacle to studying the biology of B. burgdorferi
and its role in Lyme disease has been a paucity of genetic tools that
can be used in these bacteria. The technique described herein should
open the doors to a number of important experiments. Phenotypes associated with known mutations can be confirmed by complementation with wild-type alleles to see if defects are corrected, although most
such studies will require other selectable markers in addition to
coumermycin resistance. The successful integration of pJLB5, which
contains a full-length copy of the oppAV gene, indicates that such experiments will be possible. Additionally, fusions between
B. burgdorferi genes and reporter genes encoding easily detectable products will be useful in determining the expression patterns of B. burgdorferi genes during infection and in the
transmission cycle between mammalian and arthropod hosts.
| |
ACKNOWLEDGMENTS |
We thank Scott Samuels for providing B31-NGR; Tom Schwan, Michael
Chausee, and Joseph Hinnebusch for comments on the manuscript; Gary
Hettrick and Robert Evans for graphic support; and Kelly Matteson for
secretarial assistance.
| |
FOOTNOTES |
*
Corresponding author. Present address: Department of
Microbiology and Immunology, University of Kentucky College of
Medicine, MS415 Medical Center, Lexington, KY 40536-0084. Phone: (606)
257-9358. Fax: (606) 257-8994. E-mail:
bstev0{at}pop.uky.edu.
| |
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Journal of Bacteriology, September 1998, p. 4850-4855, Vol. 180, No. 18
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
