INFECTION AND IMMUNITY, June 2010, p. 2397–2407
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 78, No. 6
Use of the Cre-lox Recombination System To Investigate the lp54
Gene Requirement in the Infectious Cycle of Borrelia burgdorferi?†
Aaron Bestor,* Philip E. Stewart, Mollie W. Jewett, Amit Sarkar, Kit Tilly, and Patricia A. Rosa
Laboratory of Zoonotic Pathogens, National Institute of Allergy and Infectious Diseases, National Institutes of Health,
Rocky Mountain Laboratories, Hamilton, Montana 59840
Received 16 September 2009/Returned for modification 15 October 2009/Accepted 5 March 2010
Borrelia burgdorferi, the causative agent of Lyme disease, has a complex genome consisting of a linear
chromosome and up to 21 linear and circular plasmids. These plasmids encode numerous proteins critical to
the spirochete’s infectious cycle and many hypothetical proteins whose functions and requirements are un-
known. The conserved linear plasmid lp54 encodes several proteins important for survival in the mouse-tick
infectious cycle, but the majority of the proteins are of unknown function and lack homologs outside the
borreliae. In this study we adapted the Cre-lox recombination system to create large deletions in the B.
burgdorferi genome. Using Cre-lox, we systematically investigated the contribution of 14 adjacent genes on the
left arm of lp54 to the overall infectivity of B. burgdorferi. The deletion of the region of lp54 encompassing bba07
to bba14 had no significant effect on the infectious cycle of B. burgdorferi. The deletion of bba01 to bba07 resulted
in a slight growth defect but did not significantly affect the ability of B. burgdorferi to complete the infectious
cycle. This study demonstrated the utility of the Cre-lox system to efficiently explore gene requirements in B.
burgdorferi and surprisingly revealed that a large number of the highly conserved proteins encoded on lp54 are
not required to complete the infectious cycle.
The genome of Borrelia burgdorferi strain B31 consists of a
?900-kb linear chromosome and more than 20 linear and
circular plasmids (11, 18). Within the chromosome are many
genes that encode basic housekeeping proteins for DNA rep-
lication, transcription, translation, solute transport, and energy
metabolism, while the majority of open reading frames (ORFs)
on the plasmids encode proteins of unknown function (11, 18).
Genetic studies of B. burgdorferi are inefficient and hampered
by the loss of critical virulence plasmids during in vitro prop-
agation, making functional characterizations of these plasmid
ORFs difficult (20, 25, 30, 31, 38, 48, 59).
Among the plasmids shown to be critical for the overall
fitness of Lyme disease spirochetes is the linear plasmid lp54
(3, 39, 60), which is present in all characterized B. burgdorferi
isolates and is stably maintained during in vitro propagation
(24, 35, 40, 54, 58). In contrast, other linear plasmids, such as
lp25 and lp28-1, carry important virulence factors but are rel-
atively unstable during in vitro cultivation (11, 19, 31, 38, 39,
59). Relatively few of the lp54-encoded proteins have been
characterized in vitro or in vivo, and most lack homologs out-
side the borreliae (11, 18).
Although direct evidence is limited, analyses of environmen-
tally regulated genes and proteins suggested that lp54 encodes
proteins important to the spirochete in its natural infectious
cycle. Many of the hypothetical ORFs on lp54 are regulated by
temperature (33), pH (10), or both (41), which correlates with
the environmental conditions that distinguish the tick vector
and mammalian host. Array studies indicate that 32 of the 76
ORFs on lp54 are temperature regulated, a higher percentage
of regulated genes than on the chromosome or any other
plasmid in the genome (33). As most of these environmentally
regulated ORFs are hypothetical and gene inactivation in in-
fectious B. burgdorferi remains inefficient, the prospect of in-
dividually inactivating all 76 genes on lp54 is daunting.
In order to more efficiently manipulate the B. burgdorferi
genome, we adapted the Cre-lox recombination system to de-
lete large segments of lp54. This system utilizes the P1 bacte-
riophage Cre protein (51), which is a tyrosine site-specific
DNA recombinase (1), and two 34-bp recognition sites called
loxP (22, 23). The loxP sites are inserted into the genome in a
directly repeated orientation flanking the targeted DNA. The
sequential breaking and rejoining of the loxP sites by the Cre
recombinase result in the functional deletion of one site and
the intervening target sequence. The Cre-lox system requires
no accessory proteins or host cofactors and proceeds efficiently
with both supercoiled and linear DNA. After demonstrating
that Cre-loxP functioned efficiently in B. burgdorferi, we under-
took a systematic deletion mapping of the left arm of lp54,
from bba01 to bba14. The resulting B. burgdorferi mutants were
assessed for their in vitro growth phenotypes and in vivo com-
petence in the mouse-tick infectious cycle.
MATERIALS AND METHODS
Bacterial strains and culture conditions. B. burgdorferi strains used in this
study are described in Table 1. Spirochetes were grown in Barbour-Stoenner-
Kelly II (BSKII) medium (2) supplemented with 6% rabbit serum (Pel Freez
Biologicals, Rogers, AZ) at 35°C or in solid BSK medium incubated at 35°C
under 2.5% CO2(42). All DNA manipulations in Escherichia coli were per-
formed with TOP10 cells (Invitrogen, Carlsbad, CA).
Construction of the GFP expression vector. The green fluorescent protein
(GFP) expression vector containing flanking loxP sites (Fig. 1) was constructed as
follows. Primers 1 and 2 (Table 2), containing 5? and 3? loxP sites, were used to
* Corresponding author. Mailing address: Laboratory of Zoonotic
Pathogens, National Institute of Allergy and Infectious Diseases, Na-
tional Institutes of Health, Rocky Mountain Laboratories, 903 S. 4th
Street, Hamilton, MT 59840. Phone: (406) 375-7467. Fax: (406) 363-
9681. E-mail: email@example.com.
?Published ahead of print on 15 March 2010.
† The authors have paid a fee to allow immediate free access to this
amplify flaBp-gfp from pBSV?-flaBp-gfp (9) with Expand high-fidelity polymer-
ase (Roche, Indianapolis, IN), and the resulting PCR fragment was cloned into
pCR2.1-TOPO (Invitrogen). loxP-flaBp-gfp was removed from the pCR2.1-
TOPO vector by digestion with KpnI and PstI and ligated into pBSV2G (14),
yielding pBSV2G-loxP-flaBp-gfp (Fig. 1 and Table 3).
Construction of pBSV25-flgB-cre. The gene encoding Cre recombinase was
kindly provided by Michael Culbertson (University of Wisconsin-Madison) and
was amplified with Taq polymerase (New England Biolabs, Ipswich, MA) by
using primers 3 and 4 (Table 2) and cloned into pBSV2. The cre gene was
digested from pBSV2 with NdeI and XbaI and cloned into pBSV2ex (21) to
create the cre expression construct pBSV2ex-flgBp-cre. The flgBp-cre fragment
was removed from pBSV2ex by digestion with NotI and cloned into pBSV25
(52), yielding pBSV25-flgBp-cre (Table 3).
Suicide vector construction. The pABA01 suicide vector, used to introduce a
loxP site with a kanamycin resistance cassette (6) into bba01, was constructed as
follows. The region at positions 229 to 1228 (GenBank) of lp54 was amplified
with Expand high-fidelity polymerase (Roche) from B31-A3 by using primers 5
and 6 (Table 2) and cloned into pCR-XL-TOPO (Invitrogen). A KpnI site was
engineered into the 5? end of primer 5, allowing the removal of the BamHI site
within pCR-XL-TOPO by digestion with KpnI and religation. The flgBp-driven
kanamycin marker with a 5? loxP site was amplified from shuttle vector pBSV2
(53) by using primers 7 and 8 (Table 2) and ligated into the cloned fragment of
lp54 at positions 229 to 1228 by using a native BamHI site at position 761,
yielding pABA01 (Table 3).
The pABA07 suicide vector, used to introduce a loxP site with the streptomy-
cin resistance cassette aadA (17) into bba07, was constructed as follows. The
region at positions 4297 to 5391 of lp54 was amplified with Expand high-fidelity
polymerase (Roche) from B31 clone A3 by using primers 9 and 10 (Table 2) and
cloned into pCR-XL-TOPO. A KpnI site was engineered into the 5? end of
primer 9, allowing the removal of the SacI site within pCR-XL-TOPO by diges-
tion with KpnI and religation. The flaBp-driven spectinomycin-streptomycin
resistance cassette (aadA), with a 5? loxP site, was amplified by using primers 11
and 12 (Table 2). This fragment was inserted into the cloned region of lp54 at
positions 4297 to 5391 by using a native SacI site at position 4754 to yield
pABA07 (Table 3).
The pABA14 suicide vector, used to introduce a loxP site with a kanamycin
resistance cassette into bba14, was constructed as follows. The region of lp54 at
positions 8398 to 9198 was amplified with Expand high-fidelity polymerase
(Roche) from B31 clone A3 by using primers 13 and 14 (Table 2) and cloned into
pCR-XL-TOPO (Invitrogen). An MluI site was engineered into the 5? end of
primer 13 to allow the removal of the HindIII site in pCR-XL-TOPO by diges-
tion with MluI and religation. The loxP-flgBp-kan cassette in pABA01 was am-
plified by using primers 15 and 16 (Table 2) and ligated into the cloned region of
positions 8398 to 9198 of lp54 by using a native HindIII site at position 8915,
yielding pABA14 (Table 3).
Construction of the pBSV2* shuttle vector. Shuttle vector pBSV2* was con-
structed by altering the sequence of the second EcoRI site located outside the
multiple-cloning site on pBSV2 (53) by PCR-based mutagenesis. Primers 17 and
18 (Table 2) include the targeted mutation GAGTTC for the EcoRI site found
at bp 1852 on pBSV2 as well as 16 overlapping bases on the 3? terminus of each
primer to avoid primer self-extension. Reaction mixtures and parameters for the
PCR followed the protocol described previously by Zheng et al., with the slight
modifications of 20 cycles at 94°C for 1 min and an extension time of 16 min at
68°C (61). The PCR product was purified by use of a QIAquick PCR purification
kit (Qiagen, Valencia, CA) and treated with DpnI (New England Biolabs) to
digest the template DNA. The resulting modified pBSV2 PCR product was used
to transform E. coli Top10F? cells (Invitrogen) and selected on LB plates with
kanamycin. Plasmid DNA was digested with EcoRI and sequenced to ensure that
there were no mutations other than the disruption of the targeted EcoRI site.
The resulting modified vector, termed pBSV2*, was found to be stable during the
in vitro propagation of B. burgdorferi through 58 generations (the longest time
period tested) in the absence of selection.
Construction of the pBSV2G-pncAp-pncA-cre vector. A 679-bp DNA fragment
containing the pncA open reading frame and its ribosome binding site with
EcoRI and XbaI ends was PCR amplified from wild-type B. burgdorferi genomic
DNA by using Taq polymerase (Invitrogen) and primers 19 and 20 (Table 2). A
1,066-bp DNA fragment containing the cre open reading frame with XbaI and
SphI ends was PCR amplified from pBSV25-flgBp-cre by using Taq polymerase
(Invitrogen) and primers 21 and 22 (Table 2). Primer 17 also included the 24-bp
intergenic sequence between the guaA and guaB genes on the B. burgdorferi cp26
plasmid, which provided a ribosome binding site for the cre open reading frame.
The pncA and cre PCR products were digested with EcoRI/XbaI and XbaI/SphI,
TABLE 1. B. burgdorferi strains used in this study
Attenuated B. burgdorferi strain B31 clone lacking all linear plasmids, including lp54
Transformable, noninfectious B31 clone lacking lp5, lp25, lp28-1, lp28-4, lp56, lp36, cp-9, and cp32-6,
used for testing the Cre-lox system
Transformable, infectious B31 clone lacking cp9; background of lp54 mutants listed below
Streptomycin resistance cassette and loxP insertion into bba07
Streptomycin resistance cassette and loxP insertion into bba07, kanamycin resistance cassette and
loxP insertion into bba01
Streptomycin resistance cassette and loxP insertion into bba07, kanamycin resistance cassette and
loxP insertion into bba14
Deletion of bba01 to bba07 loci and retention of only the streptomycin resistance cassette
Deletion of bba07 to bba14 loci and retention of only the kanamycin resistance cassette
FIG. 1. Schematic diagram illustrating the loxP/Cre-mediated de-
letion of the gene encoding GFP. The introduction of Cre recombinase
into B. burgdorferi containing flaBp-gfp flanked by loxP sites should
result in recombination between loxP sites, the excision and loss of gfp
from shuttle vector pBSV2G (15) due to the absence of replication
factors in this region of the plasmid, and a loss of fluorescence by the
2398BESTOR ET AL.INFECT. IMMUN.
respectively, and ligated into pBSV2*, which was digested with EcoRI/SphI. The
structure and sequence of the pBSV2* pncA-cre plasmid were verified by restric-
tion digestion and sequence analysis. The pBSV2* pncA-cre plasmid was digested
with EcoRI, treated with Antarctic phosphatase (NEB), and gel purified by using
the Qiagen Mini-Elute gel purification kit. A 906-bp DNA fragment containing
the pncA promoter region with EcoRI ends was PCR amplified from B. burg-
dorferi genomic DNA by using Taq polymerase (Invitrogen) and primers 23 and
24 (Table 2). This DNA fragment was digested with EcoRI and cloned into the
prepared pBSV2*pncA-cre plasmid. The structure and sequence of plasmid
pBSV2*pncAp-pncA-cre were verified by restriction digestion and sequence anal-
ysis. The synthesis of Cre by both E. coli and B. burgdorferi harboring plasmid
pBSV2* pncAp-pncA-cre was detected by immunoblot analysis of bacterial lysates
using an anti-Cre polyclonal antibody (Covance, Madison, WI). Because a Cre
expression vector carrying gentamicin resistance was required for this study, the
pncAp-pncA-cre fragment was cut from the pBSV2* vector by using the NotI sites
flanking the multiple-cloning site and ligated into NotI-digested pBSV2G (14),
yielding pBSV2G-pncAp-pncA-cre (Table 3).
B. burgdorferi transformations. Strain B31-A34 was transformed with 10 ?g
pBSV2G-loxP-flaBp-gfp (Table 3) under standard electroporation conditions
(47), immediately resuspended in 5 ml BSKII liquid medium, and allowed to
recover for 24 h at 35°C. Cells were then plated onto solid BSKII medium
containing 40 ?g/ml gentamicin and grown at 35°C. Positive transformants were
confirmed by PCR for the presence of gfp and sequenced to ensure that the loxP
sites were intact. Fluorescence microscopy confirmed the expression of GFP in
all PCR-positive clones. A B31-A34 pBSV2G-loxP-flaBp-gfp clone was randomly
selected for the introduction of the Cre expression vector pBSV25-flgBp-cre
(Table 3). Transformations with 20 ?g of pBSV25-flgBp-cre or the pBSV25
empty vector control were conducted as described above, and cells were plated
in solid BSKII medium containing 200 ?g/ml kanamycin. Positive transformants
were investigated by PCR and sequencing for the presence of cre, gfp, and the
gentamicin resistance cassette aacCI. Fluorescence microscopy was used to as-
sess the presence or loss of GFP expression in the clones.
The loxP insertion mutants were created in the infectious clone B31-A3 (15) by
using standard electroporation conditions (47). The A3-A07 loxP insertion mu-
tant was obtained by electroporation with 20 ?g of pABA07 DNA (Table 3), and
transformants were selected in solid BSKII medium containing 50 ?g/ml strep-
tomycin and grown at 35°C. Positive transformants were confirmed by PCR for
the presence of the streptomycin resistance cassette aadA and sequenced to
confirm that the loxP site was intact. A single transformant containing all the
plasmids present in parental strain B31-A3 was randomly chosen and designated
A3-A07. This clone was electroporated with 20 ?g of either pABA01 or pABA14
DNA (Table 3), and transformants were plated in the presence of 50 ?g/ml
streptomycin and 200 ?g/ml kanamycin. Positive transformants were confirmed
by PCR for the presence of the kanamycin resistance cassette and sequenced to
confirm that both loxP sites were intact.
Twenty micrograms of methylated vector pBSV2G-pncAp-pncA-cre (Table 3)
(13) was electroporated into the double-loxP insertion mutants, and bacteria
were plated in the presence of 40 ?g/ml gentamicin. Colonies positive by PCR for
pncA-cre were grown in 5 ml BSKII medium, and total genomic DNA was isolated.
PCR for bba01 to bba07 and bba07 to bba14 in the respective mutants and Southern
blot analysis of genomic DNA confirmed the loss of targeted regions of lp54. The
plasmid profiles of all transformants were identical to that of parental clone B31-A3
as determined by PCR using the Purser-Norris primer set (39).
Fluorescence microscopy. One milliliter of a mid-log-phase B. burgdorferi
culture was pelleted at 8,000 rpm for 10 min, washed once in phosphate-buffered
saline (PBS), resuspended in GTE (50 mM glucose–20 mM Tris-HCl [pH 7.5]–10
TABLE 2. Oligonucleotides used in this study
GFP-loxP 3? PstI
Cre 5? NdeI
Cre 3? XbaI
pncA?RBS EcoRI 5?
pncA XbaI 3?
Cre?RBS XbaI 5?
cre SphI 3?
pncA prom 5? EcoRI
pncA prom 3? EcoRI
aRestriction enzyme sites are indicated in boldface type, and the loxP sequences are in uppercase type.
TABLE 3. Plasmid constructs used in this study
GFP expression vector with loxP sites flanking flaBp-gfp
Cre expression vector used to excise flaBp-gfp in pilot expt
Suicide vector used for inserting loxP-Strrinto bba07
Suicide vector used for inserting loxP-Kanrinto bba01
Suicide vector used for inserting loxP-Kanrinto bba14
Created by disruption of the EcoRI site found at bp 1852 on pBSV2
Cre expression vector used to excise targeted regions of lp54
10, 15; this study
58; this study
59; this study
15; this study
VOL. 78, 2010Cre-lox RECOMBINATION SYSTEM FOR B. BURGDORFERI 2399
mM EDTA) with a 1:100 dilution of FM4-64 membrane stain (Invitrogen), and
incubated for 15 min. Cells were placed onto a glass slide, and images of GFP
fluorescence and FM4-64 membrane staining were taken with a Nikon E800
microscope and a Photometrics CoolSnap HQ camera.
In vitro growth analysis. Strains inoculated from frozen stocks were grown to
mid-log phase and then diluted in triplicate to 105spirochetes/ml (time zero) in
5 ml BSKII medium and incubated at 35°C. Triplicate cultures were counted at
24-h intervals in Petroff-Hauser chambers to assess the growth rate.
Mouse-tick infection studies. Mouse studies were carried out in accordance
with guidelines of the National Institutes of Health. All infection studies were
done according to protocols approved by the Rocky Mountain Laboratories
Animal Care and Use Committee. The Rocky Mountain Laboratories are ac-
credited by the International Association for Assessment and Accreditation of
Laboratory Animal Care (AAALAC). These studies were done by using 6- to
8-week-old female RML mice, an outbred strain of Swiss-Webster mice reared at
the Rocky Mountain Laboratories breeding facility. Mice were retro-orbitally
bled and needle inoculated with 5 ? 103B. burgdorferi spirochetes (4 ? 103
spirochetes intraperitoneally and 1 ? 103spirochetes subcutaneously). At 3
weeks postinjection, mice were bled again to assess seroreactivity to B. burgdor-
feri proteins. Two ear punches (3 mm) were taken from seropositive mice, placed
into BSKII medium, and incubated at 35°C to confirm infection by the reisolation
of B. burgdorferi. Approximately 100 Ixodes scapularis larvae (Oklahoma State
University) were fed to repletion on each infected mouse. Some fed larvae were
ground and plated in solid BSKII medium at 10 days postfeeding to assess the
acquisition of B. burgdorferi from infected mice. The remaining fed I. scapularis
larvae were allowed to molt to nymphs and recover (approximately 10 weeks
after larval feeding) before feeding on naïve mice to assess the persistence of B.
burgdorferi mutant strains in ticks and transmission to mice.
Cre recombinase functions in B. burgdorferi. To test the
feasibility of using the Cre-lox system in B. burgdorferi, Cre
recombinase was introduced into a B. burgdorferi strain con-
taining a shuttle vector encoding GFP with flanking loxP sites.
Cre-mediated recombination should result in the excision and
loss of gfp from the shuttle vector (Fig. 1), which could easily be
observed by a loss of fluorescence.
The GFP expression vector pBSV2G-loxP-flaBp-gfp was
transformed into high-passage strain B31-A34 by electropora-
tion as described above. Positive B. burgdorferi transformants
containing the GFP shuttle vector were clearly visible by flu-
orescence microscopy (Fig. 2), and sequence analysis con-
firmed that intact loxP sites flanked flaBp-gfp in the transfor-
mants. The subsequent transformation of a GFP-containing
clone with the Cre expression vector pBSV25-flgBp-cre yielded
only four transformants. All pBSV2G-loxP-flaBp-gfp/pBSV25-
flgBp-cre double transformants no longer fluoresced (Fig. 2),
and sequencing of the rescued pBSV2G-loxP-flaBp-gfp shuttle
vector confirmed that flaBp-gfp had been excised, leaving only
a single loxP site. This pilot experiment demonstrated that Cre
functioned efficiently in B. burgdorferi and could be used to
engineer mutations in the spirochete. We noted, however, that
the transformation of B. burgdorferi with pBSV25-flgBp-cre was
relatively difficult compared to transformation with the empty
pBSV25 shuttle vector. Control transformations with pBSV25
resulted in several hundred transformants, compared to only
four with pBSV25-flgBp-cre. These results suggested that the
strong expression of Cre by the flgB promoter might be dele-
terious, although Cre activity within wild-type B. burgdorferi
should not produce any rearrangements or deletions, as there
are no sequences resembling intact loxP sites present within
the genome. However, subsequent experiments indicated that
the low transformation frequency was not due to an overex-
pression of Cre, leading us to believe that the flgBp-cre insert
sequence might be recognized by endogenous B. burgdorferi
restriction enzymes and thus represent a barrier to transfor-
mation (27, 32).
Cre-mediated deletions of lp54. Following the demonstra-
tion of Cre-lox recombination in B. burgdorferi with GFP, we
FIG. 2. Loss of fluorescence by B. burgdorferi carrying loxP-flanked GFP after introduction of Cre recombinase. Strain B31-A34 was first
transformed with shuttle vector pBSV2G-loxP-flaBp-gfp (Fig. 1) and subsequently transformed with the compatible shuttle vector pBSV25 or
pBSV25-flgBp-cre, which encodes Cre recombinase. (Top) Presence or absence of GFP fluorescence in these strains as visualized by fluorescence
microscopy. (Bottom) Cells were counterstained with the membrane stain FM4-64 to visualize all spirochetes in the same field.
2400BESTOR ET AL.INFECT. IMMUN.
utilized this system to investigate the in vivo requirement for
the region encompassing bba01 to bba14 of lp54 in the infec-
tious clone B31-A3. Although several of the proteins encoded
by genes in this region of lp54 were described in previous
studies (10, 16, 36, 37), their requirement during the mouse-
tick cycle has not been investigated. We engineered the dele-
tions in two parts, bba01 to bba07 and bba07 to bba14, which
encompass all genes on the left arm of lp54, between the
telomere and the ospAB locus (bba15 and bba16) (Fig. 3).
A loxP site carrying the streptomycin resistance cassette
flaBp::aadA was introduced into the bba07 gene by allelic ex-
change using the pABA07 suicide vector in the B31-A3 back-
ground (Fig. 4). PCR of the bba07 locus with primers 9 and 10
(Table 2) showed the expected increase in size associated with
a successful insertion of the aadA cassette in the gene (Fig.
4B), and sequencing confirmed that the loxP site was intact.
Analysis of plasmid content by PCR confirmed that the clone
retained all B31-A3 (wild-type)-associated plasmids.
The subsequent insertion of a second loxP site with a kana-
mycin resistance marker by allelic exchange into the bba01 or
bba14 gene using the suicide vector pABA01 or pABA14,
respectively, resulted in the successful disruption of these tar-
geted loci with loxP-kan (Fig. 4). PCR for bba01 or bba14
confirmed the size increase associated with the kanamycin cas-
sette (Fig. 4b), and sequencing confirmed that the loxP sites
were intact. The resulting double-loxP insertion mutants, A3-
A01/A07 and A3-A07/A14, retained all plasmids associated
with the parental clone B31-A3.
The lp54 loxP insertion mutants A3-A01/A07 and A3-A07/
A14 were subsequently transformed with in vitro-methylated
Cre expression vector pBSV2G-pncAp-pncA-cre to create de-
letions of these regions of lp54 (Fig. 5). Read-through from the
pncA promoter was used instead of flgBp-cre to limit Cre pro-
duction. Electroporation with the methylated Cre expression
vector yielded three positive transformants in the A3-A01/A07
insertion mutant and two positive transformants in the A3-
A07/A14 loxP insertion mutant. All pBSV2G-pncAp-pncA-cre
transformants were positive by PCR for pncA-cre and negative
by PCR for the targeted regions of lp54, indicating the suc-
cessful excision of the loxP-flanked regions by the Cre recom-
binase (Fig. 5C). Southern blot analysis of the resulting lp54
deletion mutants, A3?A1-7 and A3?A7-14, confirmed the loss
of the targeted regions of lp54 within the respective mutants
(Fig. 6). Deletion mutants with plasmid profiles identical to
that of wild-type clone B31-A3 were chosen for use in subse-
quent studies. Taken together, these results indicate that the
Cre-lox system was used successfully to delete multiple loci on
lp54 in infectious B. burgdorferi.
Growth phenotype of mutants lacking bba01 to bba07. The
requirement for the deleted regions of lp54 during in vitro
growth was assessed by counting triplicate cultures of mutant
and wild-type strains at 24-h intervals during growth in liquid
media. None of the mutants displayed a significant growth
phenotype when grown in liquid BSKII, and Cre production
alone did not impair growth relative to that of wild-type B.
burgdorferi (Fig. 7). Although there was no detectable effect on
spirochete morphology or growth rate in liquid media, the
A3?A1-7 mutant displayed a 2- to 3-day lag in colony forma-
tion compared to wild-type B31-A3 and other mutants, sug-
gesting that genes within the region of bba01 to bba07 of lp54
may contribute to bacterial replication under specific environ-
Genes in targeted lp54 regions are not essential for mouse
infectivity. The contributions of genes encompassing bba01 to
bba14 in establishing infection in a mammalian host were in-
vestigated by injecting mice with a standard inoculum of 5 ?
103organisms of either B31-A3, the lp54 loxP insertion mu-
tants A3-A01/A07 and A3-A07/A14, and the lp54 deletion
mutants A3?A1-7 and A3?A7-14. The 50% infectious dose
(ID50) of wild-type clone B31-A3 is ?103organisms by this
route (26, 55). At 3 weeks postinoculation, all mice were se-
ropositive for B. burgdorferi antigens, and reisolates were ob-
tained from all harvested tissues for all strains (Tables 4 and 5).
These results indicate that the genes encompassing bba01 to
bba14 are not critical to the spirochete’s ability to infect and
disseminate in a mammalian host.
Tick acquisition, survival, and transmission of B. burgdorferi
are not affected by lp54 deletions. To complete the analyses of
the infectious cycle of the lp54 deletion mutants, we investi-
gated their acquisition by feeding ticks, replication and persis-
tence within the tick midgut through the molting stage, and the
ability to infect naïve hosts by tick bite. Approximately 100
naïve larval I. scapularis ticks were fed to repletion (3 to 5 days)
on mice persistently infected with A3 (wild-type), A3?A1-7, or
A3?A7-14 spirochetes. At 10 days postfeeding, ticks from each
mouse were ground and plated in solid BSK medium to quan-
titatively assess spirochete numbers in the ticks. Spirochetes
from mice infected with either mutant colonized larval ticks at
FIG. 3. Schematic diagram of the targeted region encompassing bba01 to bba14 of lp54, extending from the left telomere to the ospAB operon.
Boxed numbers beneath the diagram indicate previously identified members of paralogous gene families (11, 18). Protein designations above the
diagram and gene designations below are based on previous studies and annotations (10, 11, 16, 18, 36, 37).
VOL. 78, 2010 Cre-lox RECOMBINATION SYSTEM FOR B. BURGDORFERI2401
FIG. 4. InsertionofloxPintotargetedlp54loci.(A)SchematicdiagramshowinghowaloxPsite(filledarrowheads)andanadjacentselectablemarker
conferring resistance to streptomycin or kanamycin (aadA and Kanr, respectively) were introduced by allelic exchange into bba07 and subsequently into
either bba01 or bba14 on lp54. The relevant restriction enzyme sites used in cloning allelic exchange constructs are indicated. Small arrows beneathbba07,
bba01, and bba14 indicate the positions of oligonucleotides (Table 2) used with PCR to confirm loxP insertions into these loci, as shown below (B). The
designations of the resulting strains (Table 1) are shown on the left. (B) PCR amplification of targeted lp54 loci demonstrating an increase in fragment
size after loxP insertion. The gene target and PCR primers are indicated above the lanes, and the source of template DNA is shown below the lanes. PCR
primer positions and sequences are shown above (A) and in Table 2, respectively. The relative mobility of DNA size standards (kb) is shown on the left.
2402 BESTOR ET AL.INFECT. IMMUN.
densities similar to that of wild-type B. burgdorferi (Table 5),
demonstrating no requirement for these lp54 genes for spiro-
chete acquisition by feeding ticks.
We next investigated the contributions of genes in the deleted
region of bba01 to bba14 of lp54 for spirochete survival through
the tick molt and transmission to a new host. Cohorts of 20 I.
scapularis nymphs infected with the wild type, A3?A1-7, or
A3?A7-14 were fed to repletion on naïve mice. Ten days later,
FIG. 5. Deletion of loxP-flanked regions of lp54 after introduction of Cre recombinase. (A and B) Schematic diagrams showing the excision
of the intervening DNA between loxP sites (filled arrowheads) present in the lp54 loci bba07 and bba01 (A) or bba07 and bba14 (B) as a result
of Cre-mediated recombination. A recombined loxP site and the adjacent resistance cassette are present on a nonreplicating circular DNA
fragment. Small arrows beneath bba07, bba01, and bba14 indicate the positions of oligonucleotides (Table 2) used with PCR to confirm the deletion
of the intervening sequences in Cre transformants, as shown below (C). The designations of the resulting strains (Table 1) are shown on the left.
(C) PCR amplification of targeted lp54 regions in the wild type and deletion mutants. Smaller products spanning the deleted regions were amplified
from mutant strains, whereas the larger lp54 fragments were not efficiently amplified from wild-type A3. PCR amplification of the kan and aadA
genes demonstrates the presence or absence of these antibiotic resistance cassettes as a consequence of the lp54 deletions. The gene target and
PCR primers are indicated above the lanes, and the template DNA is indicated below the lanes. Primer positions are indicated above (A and B),
and sequences are shown in Table 2. The relative mobility of DNA size standards (kb) is shown on the left.
VOL. 78, 2010 Cre-lox RECOMBINATION SYSTEM FOR B. BURGDORFERI 2403
nymphs were ground and plated to determine the number of
viable spirochetes in these ticks. As shown in Table 6, spirochete
burdens were similar between the mutant- and wild-type-infected
not required for spirochete survival or replication within the tick.
At 3 weeks after tick feeding, mouse infection with the lp54
mutants was assessed. Two out of three mice fed on by A3?A1-
7-infected nymphs did not become infected, whereas all three
mice fed on by ticks carrying either the wild-type or the
A3?A7-14 strain were infected. To extend this finding, we re-
peated the tick challenge of A3?A1-7-infected nymphs with four
additional naïve mice, and three became infected, resulting in a
total of four of seven mice acquiring A3?A1-7 infection following
a tick bite (Table 6). Although there may be a slight attenuation
in the A3?A1-7 mutant’s ability to infect mice by tick bite, these
differences were not statistically significant.
The genome of B. burgdorferi contains a number of plasmids,
including cp26, lp25, lp28-1, lp36, and lp54, that encode factors
essential for the spirochete’s survival at different points during
its infectious cycle. The persistence of B. burgdorferi in nature
depends upon the completion of an entire infectious cycle
through the tick vector and mammalian host; thus, the pres-
ence of critical gene functions on extrachromosomal elements
in B. burgdorferi contradicts the standard definition of plasmids
as nonessential replicons. The largest of the B. burgdorferi
strain B31 plasmids, lp54, encodes proteins important for both
mammalian and tick infection. These proteins include outer
surface protein A (OspA) (BBA15), which is important for
persistence within the tick (3, 34), and decorin binding protein
A (DbpA) and DbpB (BBA24 and BBA25, respectively),
which contribute to mammalian infection (5, 49, 50, 57). Other
lp54-encoded proteins that may play a significant role in vivo
include CspA or Crasp1 (BBA68), which was shown to bind
complement regulatory proteins and confer serum resistance
(28, 29), and OppAV (BBA34), which is a peptide binding
component of oligopeptide permease that could contribute to
nutrient acquisition (7).
Additional observations underline the importance of lp54 to
the life cycle of B. burgdorferi. In a comparative genomic study,
Qiu and colleagues reported previously that only two plasmids,
cp26 and lp54, appear to be universally conserved among Lyme
disease species (40); other studies reached the same conclusion
(24, 35, 54). Unlike the other linear plasmids, lp54 is remark-
ably stable during the in vitro propagation of B. burgdorferi. The
loss of this plasmid has been reported as an escape mechanism
only when selective pressure was exerted against it (44, 45),
suggesting the presence of genes encoding proteins or regula-
tory RNAs that are important to the spirochete’s fitness in
vitro. Interestingly, the loss of lp54 correlated with the inability
of these strains to form colonies on solid medium (44, 46).
Finally, microarray analyses in which environmental conditions
were adjusted to mimic mammalian or tick conditions revealed
that lp54 encodes the highest percentage of regulated proteins
of any B. burgdorferi replicon, including the chromosome (8,
33, 41, 56). The conserved nature of lp54, its stability during in
vitro propagation, and the indication that many of its genes are
differentially expressed in either the tick or the mammal sug-
gest that lp54 is likely to be essential for the spirochete’s
FIG. 6. Southern blots confirming the deletion of multigene seg-
ments of lp54 by loxP/Cre-mediated recombination. Genomic DNAs
prepared from the wild type (B31-A3), a strain lacking lp54 (B314),
and the lp54 deletion mutants (A3?A1-7 and A3?A7-14) were sub-
jected to Southern blot analysis with probes specific to bba03 and
bba10 to confirm the loss of targeted regions of lp54 containing these
loci in the respective loxP/Cre deletion mutants. The source of DNA is
identified above the lanes, and the probe is specified below.
FIG. 7. In vitro growth curves. Wild-type B. burgdorferi (A3) and
loxP insertion mutants (A3-A07, A3-A01/A07, and A3-A07/A14)
(A) or lp54 deletion mutants (A3?A1-7 and A3?A7-14) and wild-type
B. burgdorferi containing Cre on a shuttle vector (A3-Cre) (B) were
grown at 35°C in BSKII medium from a starting concentration of 105
spirochetes/ml. Spirochetes were counted at 24-h intervals in Petroff-
Hauser chambers, and the mean number of spirochetes per ml was
determined from triplicate cultures of each strain, as shown.
2404BESTOR ET AL.INFECT. IMMUN.
survival throughout the infectious cycle and also contributes to
efficient growth in vitro.
Although data indicate that lp54 is important, the determi-
nation of precisely which genes are responsible is challenging
because there are 76 predicted ORFs on lp54, the majority of
which encode proteins of unknown function (11, 18). There-
fore, we adapted the Cre-lox system to function in B. burgdor-
feri as an efficient screening tool with which to focus on loci
relevant to fitness and survival in vivo. The use of Cre to
engineer large deletions within the chromosome or a plasmid
presents unique advantages over other available methods for
genetic manipulation, such as allelic exchange or deletion
walking by telomere insertion (4, 12, 43). The low transforma-
tion efficiency of infectious B. burgdorferi precludes the tar-
geted inactivation of each individual gene as a practical means
to investigate the roles of all lp54 gene products in vivo (15).
Telomere insertion can be used to delete large segments from
the ends of linear DNAs in B. burgdorferi (4, 12), whereas the
Cre-lox system permits the introduction of internal deletions
on both linear and circular replicons.
Based on a pilot study of loxP-flanked GFP as a reporter for
Cre activity (Fig. 1 and 2), we modified the Cre-loxP system for
use in the infectious clone B31-A3. First, we adjusted Cre
expression by replacing the strong flgB promoter with that of
pncA, which is expressed at a lower level (33, 38). Although we
initially thought that Cre overexpression was toxic, resulting in
poor transformation frequencies, read-through from the
weaker pncA promoter did not increase the yield of transfor-
mants. In addition, Cre functioned efficiently upon the success-
ful introduction into the B31-A3 background without observ-
able adverse effects. Cre expression did not impede growth
(Fig. 7B) or attenuate the spirochete’s ability to complete the
infectious cycle (Tables 5 and 6), and the shuttle vector encod-
ing Cre was stably maintained by B. burgdorferi in infected mice
and during in vitro growth in the absence of antibiotic selection
(data not shown). Subsequently, we concluded that the cre
gene sequence, and not the gene product, likely diminished the
transformation efficiency, perhaps because it carries a site(s)
recognized by an endogenous restriction enzyme of B. burg-
dorferi (27, 32). This limitation was addressed by the in vitro
methylation of cre-containing plasmid DNA prior to the trans-
formation of the loxP insertion mutants (13). Methylation was
shown previously to increase the transformation efficiency in B.
burgdorferi strain B31 by bypassing the restriction barriers en-
coded by lp56 (13). This proved to be an essential step in
obtaining Cre transformants in the infectious clone B31-A3, as
attempts to introduce the cre locus on unmethylated shuttle
vector DNA into low-passage strains were unsuccessful.
With these modifications to the Cre-loxP system, we engi-
neered two sets of deletions in infectious B. burgdorferi, each
removing seven genes at a time from the left arm of lp54 (Fig.
3). The resulting deletion mutants, A3?A1-7 and A3?A7-14,
maintained all the plasmids present in the parental wild-type
clone and were assessed for both in vitro and in vivo pheno-
types. All strains grew comparably in liquid medium (Fig. 7),
but the A3?A1-7 strain took several days longer to form col-
onies in solid media than the wild-type and other mutant
strains. This phenotype is consistent with the previously re-
TABLE 4. Mouse infectivity of loxP insertion mutants by
of mice injected
with 5 ? 103
of injected mice
3/3, 3/3, 3/3
3/3, 3/3, 3/3
3/3, 3/3, 3/3
3/3, 3/3, 3/3
aNumber of infected mice/number of mice injected with 5 ? 103spirochetes
(4 ? 103spirochetes intraperitoneally and 1 ? 103spirochetes subcutaneously).
Seroconversion was determined by immunoblot analysis with B. burgdorferi lysate
3 weeks postinoculation.
bNumber of tissue reisolates/number of injected mice, for ear, bladder, and
joint. Mice were euthanized and tissues were cultured at 4 weeks postinjection.
TABLE 5. Mouse infectivity of lp54 deletion mutants by
No. of infected
with 5 ? 103
of injected mice
tick (mouse 1;
3/4, 3/4, 3/4
4/4, 4/4, 4/4
4/4, 4/4, 4/4
aNumber of infected mice/number of mice injected with 5 ? 103spirochetes
(4 ? 103spirochetes intraperitoneally and 1 ? 103spirochetes subcutaneously).
Seroconversion was determined by immunoblot analysis with B. burgdorferi lysate
3 weeks postinoculation.
bNumber of tissue reisolates/number of injected mice, for ear, bladder, and
joint at 5 weeks postinoculation. All three tissues from infected mice were
cLarval ticks were fed to repletion on two seropositive mice for each strain.
Ten days postfeeding, three fed larvae from each mouse were pooled, crushed,
and plated to determine the number of viable spirochetes. The average number
of spirochetes per tick was calculated from the total number of colonies obtained
per pool of three larvae for each mouse. Mean numbers of spirochetes per tick
for the A3?A1-7 and A3?A7-14 mutants were not significantly different from
those of the wild type (A3) as determined by a two-tailed, unpaired t test (P ?
0.3 and P ? 0.7, respectively), performed by use of GraphPad Prism 5 software.
TABLE 6. Mouse infectivity of lp54 deletion mutants by
No. of infected
mice fed on by
No. of tissue
mice fed on by
No. of spirochetes/nymphal
tick (mouse 1; mouse 2;
3/3, 3/3, 3/3
4/7, 4/7, 4/7
3/3, 3/3, 3/3
100,000; 130,000; 140,000
100,000; 140,000; 130,000
140,000; 120,000; 140,000
aSeroconversion was determined by immunoblot analysis with B. burgdorferi
lysate 3 weeks postfeeding. The number of mice infected by either A3- or
A3?A1-7-infected ticks was not significantly different using 20 ticks per mouse
(P ? 0.48, performed by Fisher’s two-tailed exact-probability test).
bNumber of tissue reisolates/number of mice fed on by infected ticks, for ear,
bladder, and joint. All three tissues from infected mice were positive.
cTwenty infected nymphal ticks were fed to repletion on three naı ¨ve mice per
strain. Ten days postfeeding, three fed nymphs from each mouse were pooled,
crushed, and plated to determine the number of viable spirochetes. The average
number of spirochetes per tick was calculated from the total number of colonies
obtained per pool of three nymphs. Mean numbers of spirochetes per tick were
not significantly different between strains as determined by one-way analysis of
variance (ANOVA) (P ? 0.75), performed by use of GraphPad Prism 5 software.
VOL. 78, 2010Cre-lox RECOMBINATION SYSTEM FOR B. BURGDORFERI 2405
ported observation that B. burgdorferi escape mutants lacking
lp54 had simultaneously lost the ability to form colonies in
solid media (44, 46). Although we have not identified the gene
product(s) responsible for this phenotype, the loxP insertions
in bba01 and bba07 rule out those genes as candidates, since
the respective mutants did not display any delay in colony
formation. This finding suggests that at least one of the genes
within the segment of bba02 to bba06 of lp54 contributes to
growth under specific environmental conditions.
All B. burgdorferi mutant strains (both deletion and loxP
insertion mutants) were able to infect mice at wild-type levels
by needle inoculation (Tables 4 and 5). Furthermore, all mu-
tant strains were acquired by naïve ticks that fed on infected
mice, and these strains were maintained by ticks through the
molting stage to the nymphal stage at wild-type levels (Table 5
and 6). The subsequent feeding of the infected nymphs on
naïve mice demonstrated no defect in the A3?A7-14 mutant’s
ability to cause infection by tick bite. However, the A3?A1-7
mutant’s ability to cause infection by tick bite may be slightly
attenuated under our standard challenge of 20 ticks per mouse,
as three of the seven mice did not become infected (Table 6).
Although these findings are not statistically significant, they do
suggest that a minor contribution might be made by one or
more genes within the region of bba01 to bba07 at this stage of
the infectious cycle.
Considering the highly conserved and regulated nature of
lp54, we found it surprising that the first 14 genes of the
plasmid could be deleted without significantly affecting the
overall fitness of B. burgdorferi. Although little is known about
the genes encompassing bba08 to bba14, previously character-
ized genes within the region of bba01 to bba07 suggested pos-
sible contributions by one or more of these genes during the
infectious cycle. Although our data demonstrate that this re-
gion of lp54 does not encode any proteins or small regulatory
RNAs that are required for mouse or tick infectivity, this study
demonstrates the efficiency and usefulness of the Cre-lox sys-
tem as a screening tool to scan the B. burgdorferi genome for
important loci. The stability of lp54 coupled with its highly
conserved and differentially expressed gene content argue that
the continued investigation of lp54 with Cre-lox and other
genetic tools is essential for a better understanding of the
biology of the Lyme disease spirochete.
We thank Michael Culbertson, University of Wisconsin, for provid-
ing the cre gene. We thank John Leong for sharing unpublished data
regarding in vitro methylation of E. coli DNA. We thank Jonah Cullen
for technical assistance. We gratefully acknowledge the expert assis-
tance of Anita Mora and Gary Hettrick in figure preparation. We
thank Paul Beare and Chris Bosio for critical reading of the manu-
script and helpful comments.
This research was supported by the Intramural Research Program of
the NIH NIAID.
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Editor: S. M. Payne
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