Use of the Cre-lox recombination system to investigate the lp54 gene requirement in the infectious cycle of Borrelia burgdorferi.
ABSTRACT 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 unknown. 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.
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ABSTRACT: Borrelia burgdorferi, the vector-borne bacterium that causes Lyme disease, was first identified in 1982. It is known that much of the pathology associated with Lyme borreliosis is due to the spirochete's ability to infect, colonize, disseminate, and survive within the vertebrate host. Early studies aimed at defining the biological contributions of individual genes during infection and transmission were hindered by the lack of adequate tools and techniques for molecular genetic analysis of the spirochete. The development of genetic manipulation techniques, paired with elucidation and annotation of the B. burgdorferi genome sequence, has led to major advancements in our understanding of the virulence factors and the molecular events associated with Lyme disease. Since the dawn of this genetic era of Lyme research, genes required for vector or host adaptation have garnered significant attention and highlighted the central role that these components play in the enzootic cycle of this pathogen. This chapter covers the progress made in the Borrelia field since the application of mutagenesis techniques and how they have allowed researchers to begin ascribing roles to individual genes. Understanding the complex process of adaptation and survival as the spirochete cycles between the tick vector and vertebrate host will lead to the development of more effective diagnostic tools as well as identification of novel therapeutic and vaccine targets. In this chapter, the Borrelia genes are presented in the context of their general biological roles in global gene regulation, motility, cell processes, immune evasion, and colonization/dissemination.Advances in applied microbiology 01/2014; 86:41-143. · 2.24 Impact Factor
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ABSTRACT: Analysis of the transcriptome of Borrelia burgdorferi, the causative agent of Lyme disease, during infection has proven difficult due to the low spirochete loads in the mammalian tissues. To overcome this challenge, we have developed an In Vivo Expression Technology (IVET) system for identification of B. burgdorferi genes expressed during an active murine infection. Spirochetes lacking linear plasmid (lp) 25 are non-infectious yet highly transformable. Mouse infection can be restored to these spirochetes by expression of the essential lp25-encoded pncA gene alone. Therefore, this IVET-based approach selects for in vivo-expressed promoters that drive expression of pncA resulting in the recovery of infectious spirochetes lacking lp25 following a three week infection in mice. Screening of approximately 15,000 clones in mice identified 289 unique in vivo-expressed DNA fragments from across all 22 replicons of the B. burgdorferi B31 genome. The in vivo-expressed candidate genes putatively encode proteins in various functional categories including antigenicity, metabolism, motility, nutrient transport and unknown functions. Candidate gene bbk46 on essential virulence plasmid lp36 was found to be highly induced in vivo and to be RpoS-independent. Immunocompetent mice inoculated with spirochetes lacking bbk46 seroconverted but no spirochetes were recovered from mouse tissues three weeks post inoculation. However, the bbk46 gene was not required for B. burgdorferi infection of immunodeficient mice. Therefore, through an initial IVET screen in B. burgdorferi we have identified a novel in vivo-induced virulence factor critical for the ability of the spirochete to evade the humoral immune response and persistently infect mice.PLoS Pathogens 08/2013; 9(8):e1003567. · 8.06 Impact Factor
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ABSTRACT: ABSTRACT The Lyme disease spirochete Borrelia burgdorferi senses and responds to environmental cues as it transits between the tick vector and vertebrate host. Failure to properly adapt can block transmission of the spirochete and persistence in either vector or host. We previously identified BBD18, a novel plasmid-encoded protein of B. burgdorferi, as a putative repressor of the host-essential factor OspC. In this study, we investigate the in vivo role of BBD18 as a regulatory protein, using an experimental mouse-tick model system that closely resembles the natural infectious cycle of B. burgdorferi. We show that spirochetes that have been engineered to constitutively produce BBD18 can colonize and persist in ticks but do not infect mice when introduced by either tick bite or needle inoculation. Conversely, spirochetes lacking BBD18 can persistently infect mice but are not acquired by feeding ticks. Through site-directed mutagenesis, we have demonstrated that abrogation of spirochete infection in mice by overexpression of BBD18 occurs only with bbd18 alleles that can suppress OspC synthesis. Finally, we demonstrate that BBD18-mediated regulation does not utilize a previously described ospC operator sequence required by B. burgdorferi for persistence in immunocompetent mice. These data lead us to conclude that BBD18 does not represent the putative repressor utilized by B. burgdorferi for the specific downregulation of OspC in the mammalian host. Rather, we suggest that BBD18 exhibits features more consistent with those of a global regulatory protein whose critical role occurs during spirochete acquisition by feeding ticks. IMPORTANCE Lyme disease, caused by Borrelia burgdorferi, is the most common arthropod-borne disease in North America. B. burgdorferi is transmitted to humans and other vertebrate hosts by ticks as they take a blood meal. Transmission between vectors and hosts requires the bacterium to sense changes in the environment and adapt. However, the mechanisms involved in this process are not well understood. By determining how B. burgdorferi cycles between two very different environments, we can potentially establish novel ways to interfere with transmission and limit infection of this vector-borne pathogen. We are studying a regulatory protein called BBD18 that we recently described. We found that too much BBD18 interferes with the spirochete's ability to establish infection in mice, whereas too little BBD18 appears to prevent colonization in ticks. Our study provides new insight into key elements of the infectious cycle of the Lyme disease spirochete.mBio 07/2014; 5(2). · 6.88 Impact Factor
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: firstname.lastname@example.org.
?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
Strain Description Reference
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
A3-A07/A14 This study
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
2398 BESTOR 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. BURGDORFERI2399
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.
2400 BESTOR 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. BURGDORFERI 2401