INFECTION AND IMMUNITY, Aug. 2011, p. 3273–3283
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 8
Analysis of the HD-GYP Domain Cyclic Dimeric GMP
Phosphodiesterase Reveals a Role in Motility and
the Enzootic Life Cycle of Borrelia burgdorferi?†
Syed Z. Sultan,1Joshua E. Pitzer,1Tristan Boquoi,1Gerry Hobbs,2
Michael R. Miller,3and M. A. Motaleb1*
Department of Microbiology and Immunology, Brody School of Medicine, East Carolina University, Greenville, North Carolina 278341;
Department of Statistics, West Virginia University, Morgantown, West Virginia 265062; and Department of
Biochemistry, Health Sciences Center, West Virginia University, Morgantown, West Virginia 265063
Received 31 March 2011/Returned for modification 5 May 2011/Accepted 1 June 2011
HD-GYP domain cyclic dimeric GMP (c-di-GMP) phosphodiesterases are implicated in motility and viru-
lence in bacteria. Borrelia burgdorferi possesses a single set of c-di-GMP-metabolizing enzymes, including a
putative HD-GYP domain protein, BB0374. Recently, we characterized the EAL domain phosphodiesterase
PdeA. A mutation in pdeA resulted in cells that were defective in motility and virulence. Here we demonstrate
that BB0374/PdeB specifically hydrolyzed c-di-GMP with a Kmof 2.9 nM, confirming that it is a functional
phosphodiesterase. Furthermore, by measuring phosphodiesterase enzyme activity in extracts from cells
containing the pdeA pdeB double mutant, we demonstrate that no additional phosphodiesterases are present in
B. burgdorferi. pdeB single mutant cells exhibit significantly increased flexing, indicating a role for c-di-GMP
in motility. Constructing and analyzing a pilZ pdeB double mutant suggests that PilZ likely interacts with
chemotaxis signaling. While virulence in needle-inoculated C3H/HeN mice did not appear to be altered
significantly in pdeB mutant cells, these cells exhibited a reduced ability to survive in Ixodes scapularis ticks.
Consequently, those ticks were unable to transmit the infection to naïve mice. All of these phenotypes were
restored when the mutant was complemented. Identification of this role of pdeB increases our understanding
of the c-di-GMP signaling network in motility regulation and the life cycle of B. burgdorferi.
The bacterial second messenger cyclic dimeric GMP (c-di-
GMP) [bis(3?,5?)-cyclic diguanylic acid] has been associated
with a wide range of adaptive processes, most notably, the
regulation of virulence-related gene products and motility (17,
36, 76, 83, 92, 96). C-di-GMP exerts its regulatory function at
the level of transcription, translation, protein activity, secre-
tion, and/or protein stability via its interaction with various
types of downstream effector molecules (36, 37, 80, 90). For
example, in response to c-di-GMP, the Escherichia coli PilZ
domain protein YcgR induces a counterclockwise (CCW) ro-
tational bias by interacting with flagellar motor switch protein
FliG, FliM, or MotA; however, in Caulobacter crescentus, a
PilZ domain receptor, DgrA, was reported to destabilize the
flagellar protein FliL, negatively affecting motility in response
to c-di-GMP (7, 14, 22, 66, 80).
The levels of c-di-GMP are controlled by the opposing ac-
tivities of diguanylate cyclase (DGC) and phosphodiesterase
(PDE) enzymes. DGC enzymes synthesize c-di-GMP from two
molecules of GTP, while PDEs hydrolyze c-di-GMP to pGpG
or GMP. Open reading frames encoding DGC activity are
characterized by the presence of a GGDEF protein domain.
Proteins with PDE activity typically harbor either EAL or
HD-GYP domains (36, 76, 83, 92). Although there are numer-
ous reports on the characterization of GGDEF and EAL do-
main-containing proteins in diverse species of bacteria, only a
few HD-GYP domain (HD-GYP; Pfam PF01966) proteins
have so far been characterized, despite the fact that the HD-
GYP domain proteins are also abundant in bacteria (28, 31).
Comparative genome analyses indicated that a number of bac-
terial genomes encode proteins with the GGDEF domain but
no identifiable EAL domain PDE (30, 31). A role for HD-GYP
domain proteins in c-di-GMP hydrolysis was then proposed
based on the distribution and numbers of GGDEF, EAL, and
HD-GYP domains encoded by different bacteria and on the
known activities of other members of the HD superfamily of
enzymes as metal-dependent hydrolases (30). Experimental
evidence of a role for the HD-GYP domain in c-di-GMP
hydrolysis was provided through studies of Xanthomonas sp.
RpfG (1, 77, 79). Two additional HD-GYP domain proteins,
PA4108 and PA4718, from Pseudomonas aeruginosa also have
been reported to be c-di-GMP PDEs (78). In each case, inac-
tivation of these putative HD-GYP PDEs resulted in reduced
motility and decreased virulence (1, 78, 79).
Synthesis of c-di-GMP in Borrelia burgdorferi, the Lyme dis-
ease spirochete, is controlled by the Hk1/Rrp1 two-component
signal transduction system (31, 75), with Hk1 serving as the
sensor histidine kinase component responsible for phosphory-
lating Rrp1, a diguanylate cyclase. Recent independent studies
by Caimano et al. (10a), He et al. (35a), and Kostick et al. (45)
indicate that these two gene products act cooperatively to
promote spirochete survival in feeding ticks following acquisi-
tion. Interestingly, neither gene was required to establish in-
* Corresponding author. Mailing address: Department of Microbi-
ology and Immunology, Brody School of Medicine, East Carolina
University, Greenville, NC 27834. Phone: (252) 744-3129. Fax: (252)
744-3535. E-mail: firstname.lastname@example.org.
† Supplemental material for this article may be found at http://iai
?Published ahead of print on 13 June 2011.
fection in mice by needle inoculation. Moreover, spirochetes
lacking Hk1 were able to migrate out of the bite site into
feeding nymphs but were killed within 36 h following ingestion.
These results indicate that the function of Hk1/Rrp1 and c-di-
GMP is likely to be restricted in the arthropod phase of the
enzootic cycle. The presence of two distinct PDEs, PdeA
(BB0363) and PdeB (BB0374), implies that the ability to mod-
ulate the intracellular levels of c-di-GMP is an important com-
ponent of the spirochete’s pathogenic strategy. Indeed, inacti-
vation of the EAL domain containing c-di-GMP-specific PDE
(pdeA) resulted in cells that were avirulent in mice, presumably
as a result of their altered swimming pattern (91).
The motility of B. burgdorferi results from coordinated rota-
tion of the periplasmic flagella residing between the outer
membrane and the cell cylinder (12, 13, 18, 34, 46, 50). The
motile behavior of B. burgdorferi and other spirochetes is
unique and complex (12, 13). In vitro, B. burgdorferi exhibits
three different swimming modes, i.e., running, flexing, and
reversing (5, 12, 50, 58), with running occurring when the
periplasmic flagellar motors at either end of the spirochete cell
body rotate in opposite directions. Thus, the periplasmic fla-
gella of the anterior ribbon rotates CCW and those of the
posterior end rotate clockwise (CW) (as a frame of reference,
a periplasmic flagellum is viewed from its distal tip along the
filament toward insertion into the motor) (12, 13, 18, 50). The
flex is a nontranslational mode that is often associated with
bending of the cell body (33, 34, 58). During a flex, the motors
at both ends rotate in the same direction; i.e., both rotate
either CW or CCW (12, 23, 34, 50, 58). The spirochete flex is
thought to be equivalent to the tumbling behavior displayed by
Escherichia coli and Salmonella enterica serovar Typhimurium
(8, 12, 23, 34, 50, 58). A reversal occurs in translating cells
when the motors at either end simultaneously reverse their
direction of rotation (12, 50). Phosphorylated CheY (CheY-P)
plays a major role in regulating the direction in which flagellar
motors rotate. Thus, a B. burgdorferi cheY3 mutant (no
CheY3-P) constantly runs whereas a CheY-P phosphatase
cheX mutant (elevated CheY3-P) constantly flexes (58, 61, 67).
Bacterial motility is important for the colonization/disease
process for numerous species of pathogenic bacteria, including
B. burgdorferi (9, 10, 42, 52, 55, 62). In vitro, B. burgdorferi is
able to traverse viscous gel-like media in which most other
flagellated bacteria slow down or stop (44). B. burgdorferi is
able to disseminate out of the site of inoculation and colonize
diverse host tissues, such as joints, the nervous system, and the
heart (33, 44, 86), due in part to its unique motility (12, 18, 34,
50–52, 91). However, motility is likely not required for spiro-
chetes to survive or replicate in ticks (our unpublished obser-
To better understand the importance of c-di-GMP signaling
in the life cycle of B. burgdorferi, we characterized the single
HD-GYP domain protein PdeB. We demonstrate that PdeB is
a functional c-di-GMP PDE that binds c-di-GMP with high
affinity and hydrolyzes it. By inactivating both pdeA and pdeB
and using cell extracts in PDE assays, we established that B.
burgdorferi encodes no additional PDE. Dark-field analysis of
spirochetes lacking PdeB demonstrated that these cells exhibit
significantly increased flexing, indicating a role for this PDE in
modulating motility. Furthermore, although loss of PdeB did
not significantly affect virulence in needle-inoculated mice, the
mutant displayed a reduced ability to survive in Ixodes scapu-
laris ticks. Ticks infected with the pdeB mutant were unable to
transmit the infection into naïve mice, resulting in a failure to
complete the mouse-tick-mouse infection cycle. Normal motil-
ity and survival in ticks were restored by complementation.
Together, these data suggest a role for PdeB in tick-mediated
transmission and imply that decreased levels of c-di-GMP may
be important for the spirochete’s survival postfeeding/
postrepletion. Inactivation of the c-di-GMP receptor PilZ pro-
tein PlzA in the pdeB mutant background suggests a role for
c-di-GMP/PlzA in chemotaxis signaling. Possible mechanisms
by which PdeB, PlzA, and c-di-GMP alter motility and infec-
tivity are discussed.
MATERIALS AND METHODS
Bacterial strains and growth conditions. Low-passage, virulent B. burgdorferi
strain B31-A3-K2?bbe002 (a kind gift from R. Rego and P. Rosa, Rocky Moun-
tain Laboratories [RML], NIH) (72) was used as the wild type throughout this
study. This strain is a derivative of A3-68 lacking circular plasmid 9 and linear
plasmid 56 (lp56), with the bbe002 gene inactivated using a PflgB-kan cassette to
increase the transformation frequency (43, 72). B31-A3-K2?bbe002 is a deriva-
tive of strain B31 (21). The genome of the virulent B31 strain has been sequenced
and was found to contain a total of 21 plasmids with 12 linear and 9 circular
plasmids, in addition to the 960-kb linear chromosome (11, 25). B. burgdorferi
was cultured in liquid Barbour-Stoenner-Kelly (BSK-II) medium; plating BSK-II
was prepared using 0.6% agarose (57).
Construction of a pdeB mutant. Targeted inactivation of pdeB (1,140 bp; gene
locus bb0374) was achieved by homologous recombination using a streptomycin
resistance gene (aadA) as a marker (24). The pdeB gene was inactivated by
replacing the first 1,040 bp of pdeB with the 792-bp aadA coding sequence using
overlapping PCRs, as we recently described in detail (59). PCR was used to
amplify 3 regions of DNA in 3 steps (not shown; see reference 59). In the first
step, each DNA region was amplified separately using PCR pairs P1-P2 (5?-
flanking DNA, bb0373), P3-P4 (aadA coding sequence from plasmid pKFSS1),
and P5-P6 (3?-flanking DNA, pfs). Primers P2, P3, P4, and P5 contain several
overlapping base pairs (see below). During step 2, a PCR product was obtained
using primers P1-P4 and the purified DNA products for bb0373 and aadA as
templates. In step 3, the final PCR product was obtained using primers P1-P6
and purified DNA products bb0373-aadA and pfs as templates. The primer
sequences (5?-3?) are as follows: P1, AGACTTTGCAAAAAAATAAT; P2, CG
CTTCCCTCATTTATTCCCTTGAAATTAAGCTC; P3, TCAAGGGAATAA
ATGAGGGAAGCGGTGATCGCCG; P4, TGGTATAGATTGTTATTTGCC
GACTACCTTGGTG; P5, GTCGGCAAATAACAATCTATACCAAATACA
AATAC; P6, AATCAATATTTGTATAGCCTATATC1. The final PCR yielded
a 2,891-bp product that was gel purified and cloned into the pGEM-T Easy
vector (Promega Inc.). The deletion-insertion was in frame. The integrity of the
pdeB inactivation plasmid was confirmed by PCR and restriction mapping. NotI-
digested DNA was electroporated into wild-type competent cells and plated on
plating BSK containing 200 ?g ml?1kanamycin plus 100 ?g ml?1streptomycin
as described previously (57). Resistant clones were analyzed by PCR for the
confirmation of homologous recombination. PCR-positive mutants were further
examined for their plasmid contents using 21 sets of primers to detect 21 linear
and circular plasmids (100).
Complementation of pdeB mutant. To complement the pdeB mutant, the
promoter of the pdeB operon (PpdeB; 345 bp upstream from the ATG start codon
of the pdeB gene) (74) and the coding sequence of pdeB were amplified by PCR
with primers Bb0373-BamHI-F (GGATCCGTGAATGCATCTTCCATCCCA)
and Bb0374-Pst-R (CTGCAGTTATATAATATCTATTAAAGAA). BamHI and
PstI sites are in bold font. The 1,499-bp PCR product (PpdeB-pdeB) was ligated
into pGEM-T Easy. The PpdeB-pdeB DNA was then inserted into shuttle vector
pBSV2G (41, 91) using PstI and BamHI restriction digestion, yielding
pBSbb0374. Fifty micrograms of the resultant plasmid was electroporated into
the pdeB::aadA competent cells. Transformants were selected on solid growth
medium containing 200 ?g ml?1kanamycin, 100 ?g ml?1streptomycin, and 40
?g ml?1gentamicin. Resistant transformants were analyzed by PCR for the
presence of Strr, Gmr, and pdeB. To confirm that the plasmid was complementing
in trans, the pBSVbb0374 shuttle vector was rescued from complemented pdeB?
cells, transformed, and then purified from E. coli and the integrity of PpdeB-pdeB
3274SULTAN ET AL.INFECT. IMMUN.
was verified by restriction digestion. Positive transformants were further exam-
ined for their plasmid content by PCR.
Dark-field microscopy and swarm plate assays. Exponentially growing B.
burgdorferi cells were observed under a dark-field microscope (Zeiss Axio Imager
M1) connected to an AxioCam digital camera and video recorded. The flex rate
was determined by determining the mean number of flexes per minute ? the
standard deviation. At least 12 cells of each strain were analyzed. A paired
Student t test was used to determine statistical significance (a P value for the
difference between the wild type and the mutant). Swarm plate assays were
performed as described previously (50, 52, 57, 58). Approximately 1 ? 106cells
in a 5-?l volume were spotted onto a 0.35% agarose plate containing plating
BSK-II medium diluted 1:10 in phosphate-buffered saline (PBS) without divalent
cations. Because B. burgdorferi is a slow-growing organism with an 8- to 12-h
generation time, swarm plates were incubated for 6 days at 35°C in a 2.5% CO2
humidified incubator (57, 91).
Reverse transcription (RT)-PCR. Exponentially growing cultures of B. burg-
dorferi were treated with RNAprotect, and then total RNA was isolated using the
RNeasy minikit (Qiagen Inc.). Contaminating DNA in the RNA samples was
removed by RNase-free Turbo DNase I (Ambion, Inc.) digestion for 3 h at 37°C,
followed by RNeasy mini purification. For RT-PCR, cDNA was prepared from
1 ?g of RNA using the Bio-Rad cDNA synthesis kit according to the manufac-
turer’s protocol. The iCycler detection system (Bio-Rad Inc.) was used to mea-
sure the transcript level of the test genes in wild-type, pdeB mutant, and com-
plemented cells according to the manufacturer’s instructions. The B. burgdorferi
gene for enolase was used as a reference (60). The gene-specific primers (5?-3?)
were (i) BB0374 qRT F (TTATTCATTCGGTAAATACAGCTATC) and
BB0374 qRT R (CTAGTTCTTCCTCAGTTAATGCTTCT) and (ii) RT-eno-
lase-F (TGGAGCGTACAAAGCCAACATT) and RT-enolase-R (TGAAAAA
CCTCTGCTGCCATTC). The relative level of expression was calculated using
the 2???CTmethod (54, 84).
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
Western blot analysis. SDS-PAGE and Western blotting with an enhanced
chemiluminescent detection method (GE Healthcare) were carried out accord-
ing to the manufacturer’s instructions. The concentration of proteins in the cell
lysates was determined by a Bio-Rad protein assay kit. Unless noted otherwise,
10 ?g of lysates was loaded in each lane for SDS-PAGE, transferred to polyvi-
nylidene fluoride membranes, and subjected to Western blotting using specific
antibodies. The following antibodies were kindly provided by other investigators:
polyclonal anti-Pfs by B. Stevenson (University of Kentucky, Lexington, KY),
monoclonal anti-FlaB (H9724) by A.G. Barbour (University of California, Irvine,
CA), monoclonal anti-DnaK by J. Benach (SUNY, Stony Brook, NY), polyclonal
anti-MotB by J. Carroll (NIH at RML, Hamilton, MT), polyclonal FliG1 and
FliG2 by C. Li (SUNY, Buffalo, NY), and polyclonal FliM by D. Blair (University
of Utah, Salt Lake City, UT). The specific reactivity of these antibodies to B.
burgdorferi CheX, CheY3, Pfs, FlaB, MotB, FliG1, FliG2, and FliM has been
reported previously (16, 50, 52, 56, 58, 60, 74, 82).
R-PdeB protein expression and PDE assays. For overexpression and purifi-
cation of recombinant PdeB (R-PdeB) protein, the coding sequence of pdeB was
amplified without the first ATG start codon by PCR using primers R-374-Bam-F
(GGATCCCAAAATTCTGAAAGCATTAT) and R-374-Pst R (CTGCAGTTA
TATAATATCTATTAAAGAATA), creating a BamHI and a PstI site (bold),
respectively. For overexpression and purification of the HD-GYP domain
(amino acids [aa] 90 to 305) of PdeB, the pdeB coding sequence was amplified
without the first 89 and the last 75 aa residues by PCR using primers R-374-
creating a BamHI and a PstI site (bold), respectively. The amplified DNA
fragments were ligated into E. coli expression vector pMAL-c2X (New England
BioLabs) using BamHI and PstI. The recombinant expression vectors con-
structed for PdeB and the HD-GYP domain were named pbb0374-MAL and
pbbHDGYP-MAL, respectively. The recombinant expression vectors were ana-
lyzed by restriction digestion to confirm their integrity. E. coli cells containing
pbb0374-MAL or pbbHDGYP-MAL were induced with 0.25 mM isopropyl-?-D-
thiogalactopyranoside at 30°C for 4 h and purified on amylose resin (New
England BioLabs). Purified proteins were concentrated and dialyzed in PDE
buffer (75 mM Tris-HCl [pH 8], 10 mM MgCl2, 25 mM KCl, 250 mM NaCl).
Purified R-PdeB or HD-GYP protein was resolved by SDS-PAGE to determine
protein concentration and purity.
PDE assays of wild-type, pdeB mutant, complemented pdeB?, and pdeA pdeB
double mutant cell extracts were performed as described previously (15, 91), with
minor modifications. Briefly, cell pellets were washed once with PBS and resus-
pended in PDE assay buffer (250 mM NaCl, 25 mM Tris-HCl [pH 8], 10 mM
MgCl2, 25 mM KCl, 10% glycerol). Cells were lysed by a combination of French
press and sonication. Lysed cells were centrifuged at 100,000 ? g for 1 h, and the
protein concentration in the supernatants was determined using a Bio-Rad
protein assay kit. Synthesis of radiolabeled c-di-GMP substrate for PDE assays
was performed as described previously (49, 91) and approved by the East Car-
olina University Radiation Safety Subcommittee. An E. coli clone carrying P.
aeruginosa His6-WspR, which has high diguanylate cyclase activity, was affinity
purified as described previously (47, 49, 91).33P-labeled c-di-GMP was prepared
by incubating 20 ?g of His6-WspR, 100 mCi of [?-33P]GTP (3,000 Ci mmol?1; 10
mCi ml?1; Perkin-Elmer) in 50 mM Tris-HCl (pH 7.5)–250 mM NaCl–10 mM
MgCl2–1 mM dithiothreitol for 4 h at 25°C. The reaction mixture was then
incubated with 20 units of calf intestine alkaline phosphatase (New England
BioLabs) for 1 h at 25°C to hydrolyze unreacted GTP. The reaction was stopped
with 0.5 M EDTA, and the product was passed through an Ultrafree MC-5000
column (Millipore Inc.). PDE assays were performed as described previously (91,
94). R-PdeB protein (2 ?g) or B. burgdorferi crude extract (4 ?g) was incubated
with33P-labeled c-di-GMP with or without 50 mM MnCl2for different periods of
time. A no-enzyme or unrelated-protein (maltose binding protein [MBP]-FlhF)
negative control and a Vibrio cholerae PDE His6-CdpA (1.5 ?g) positive control
were used (91, 93). Reaction products (1 ?l) were spotted and separated by
thin-layer chromatography (TLC) using polyethyleneimine-cellulose plates in 1.5
M KH2PO4(pH 3.65) buffer. The plates were air dried, and then phosphorim-
aging and volume analysis using Image Quant TL v2003 were done. To deter-
mine the Km, R-PdeB was incubated with 2 to 70 nM33P-labeled c-di-GMP for
up to 30 min, reactions were stopped every 5 min, the products were subjected
to TLC, and the results were analyzed using GraphPad Prism 5 (Michaelis-
Menten equation) as described previously (15, 91, 94). To determine the sub-
strate specificity of R-PdeB, 15 to 30 nM3H-labeled cGMP (Perkin-Elmer) was
incubated with 6 mg of R-PdeB for 30 min to overnight and the reaction product
was separated by TLC as described previously (91, 94).
Experimental mouse-tick-mouse infection model of B. burgdorferi. Five-week-
old female C3H/HeN mice were purchased from Charles River Laboratories,
Durham, NC, and housed in the East Carolina University animal facility at the
Brody School of Medicine according to the institutional guidelines for the care
and use of laboratory animals. For infection via needle, 5 ? 102to 5 ? 104in
vitro-grown spirochetes were injected subcutaneously as described previously (21,
40, 73). The number of spirochetes was determined using a Petroff-Hausser
chamber, and each clone was verified for retention of lp25, lp28-1, and lp36
plasmids. Mice were bled 2 weeks postinfection, and sera against B. burgdorferi
antigens were tested to determine infectivity as described previously (21, 85).
Reisolation of spirochetes from mouse ears, joints, and bladders was performed
at 4 weeks postinfection as previously described (91) to assess the ability of
spirochetes to infect mice. Mouse tissues were incubated in BSK-II growth
medium for up to 35 days, and the presence of spirochetes was determined by
dark-field microscopy. Reisolated clones were genotyped using PCR as described
For tick infection studies, naïve I. scapularis larvae were purchased from
Oklahoma State University. Two independent experiments were performed with
ticks. Ticks were kept at 23 to 24°C under a 14/10 light/dark photoperiod in a
humidified chamber with 85 to 90% relative humidity. Approximately 150 larval
ticks were fed to repletion on spirochete-infected mice for 5 to 7 days, allowed
to fall off, and collected. One subset of larvae was dissected 7 days after repletion,
and the isolated midguts were analyzed by indirect immunofluorescence assays
(IFA) for the presence of spirochetes (87, 91). A second subset of fed larvae were
surface sterilized using 3% H2O2, followed by 70% ethanol; crushed in BSK-II
medium; and plated to determine the number of CFU per tick. The remaining
fed larvae were allowed to molt to nymphs. Those nymphs were then fed to
repletion on naïve mice (five-week-old female C3H/HeN mice), allowed to fall
off, and collected. Subsets of nymphs were dissected 7 to 9 days after repletion,
and the isolated midguts were analyzed by IFA for the presence of spirochetes.
Fed nymphs were surface sterilized as described above, crushed in BSK-II me-
dium, and plated to determine the number of CFU per tick. Mice were bled 2
weeks after nymph infection for immunoblot analysis of mouse sera, and reiso-
lation of B. burgdorferi from mouse ear, bladder, and joint tissues was performed
4 weeks postinfection.
Determination of 50% infective dose (ID50) and statistical analysis. The dose
required to infect 50% of the mice inoculated was experimentally determined for
the wild-type and pdeB mutant strains as described previously (40, 68, 71). The
data from the ID50infection experiment and the single-dose infection experi-
ment for each strain were combined for estimation of the ID50. Comparison of
strain ID50values was done using a generalized linear model with a probit link
function. This method is also known as probit regression (40), and in it we
assume identical slopes in the response/log-dose relationship but different inter-
cepts for each strain. Graphically, those assumptions manifest themselves as
VOL. 79, 2011 A ROLE FOR THE HD-GYP PDE IN MOTILITY AND VIRULENCE3275
dose-response curves with lateral shifts corresponding to the changes in inter-
cept. Additionally, an overdispersion parameter was fitted in order to accommo-
date greater homogeneity of infection rates than would otherwise be permitted
by the model. All calculations were carried out using JMP V9 software (SAS
Institute Inc., Cary, NC).
Artificial inoculation of ticks by immersion. Approximately 150 larval ticks
were artificially infected by immersion (in duplicate) in equal-density exponen-
tial-phase B. burgdorferi cultures as described previously (6, 69, 91), except that
larval ticks were equilibrated to a lower relative humidity overnight before
immersion to enhance spirochete uptake. Ticks were fed to repletion on separate
naïve mice for 5 to 7 days, allowed to fall off, and collected. One subset of larvae
were crushed 7 days after repletion and analyzed by IFA for the presence of
spirochetes (87, 91). A second subset of fed larvae were surface sterilized using
3% H2O2, followed by 70% ethanol; crushed in BSK-II medium; and plated to
determine the number of CFU per tick. Serum was collected at 2 weeks after
repletion, and mouse tissues were harvested at 4 weeks after repletion as de-
scribed above. In determining the total number of spirochetes per fed tick, 5 ticks
were analyzed for each strain and the results are expressed as the mean ? the
IFA. Ticks were dissected in10 ?l PBS–5 mM MgCl2on Teflon-coated micro-
scope slides, mixed by pipetting, and then air dried (91). To avoid quenching by
hemin in the blood, dissected tick contents were 10-fold serially diluted (P.
Policastro, RML, NIH, personal communication). Slides were blocked with
0.75% bovine serum albumin in PBS–5 mM MgCl2and washed with PBS–5 mM
MgCl2. Spirochetes were detected using a 1:100 dilution of goat anti-B. burgdor-
feri antiserum labeled with fluorescein isothiocyanate (Kirkegaard & Perry Lab-
oratories). Images were captured using a Zeiss Axio Imager M1 microscope
coupled with a digital camera (91).
Inactivation and complementation of pdeB. B. burgdorferi
genome organization, as well as RT-PCR analyses, indicated
that pdeB is cotranscribed with other genes in an operon,
pdeB-pfs-metK-luxS (25, 38, 74). A ?70promoter has also been
located directly upstream of pdeB (74). In this operon, pdeB is
predicted to be a PDE (see below), pfs encodes a nucleosidase
which is essential for depleting toxic metabolic by-products,
metK is essential for normal cell growth and septation, and the
luxS-encoded enzyme synthesizes 4,5-dihydroxy-2,3-pentanedi-
one/autoinducer 2 (4, 38, 65, 74). A luxS mutant was con-
structed and reported to be not essential for infecting I. scapu-
laris ticks or mice or transmission between these hosts (38).
The expression of genes in the pdeB operon has also been
reported to be modulated as a function of the growth phase
(74). However, whether the HD-GYP domain-containing pro-
tein PdeB is a functional PDE has not been reported. To
determine if pdeB functions in the B. burgdorferi enzootic life
cycle, we inactivated this gene using allelic-exchange mutagen-
esis (Fig. 1) (59) by replacing a 1,040-bp fragment of the gene
with the streptomycin resistance aadA gene (24) without its
promoter. We found that this strategy prevents polar effects on
the expression of downstream genes (59). PCR analysis con-
firmed that the Strrmarker (aadA coding sequence) was cor-
rectly inserted within the pdeB gene, as expected (Fig. 2, left
panel). To assess whether insertion of aadA exerted an unex-
pected polar effect on the expression of pfs (immediately down-
stream from pdeB), Western blot analysis was conducted using
antiserum raised against Pfs (74). Western blotting results in-
dicated that the synthesis of Pfs was not altered in the pdeB
mutant (Fig. 2, right panel). These results indicated that the
in-frame insertion of the aadA/Strrmarker did not induce a
To demonstrate that the pdeB mutant phenotype (see be-
low) was due solely to the mutation and not due to a secondary
alteration elsewhere, we complemented the mutant in trans
using a shuttle vector, pBSV2G (Fig. 1, bottom) (20, 88, 91).
Real-time PCR analysis indicated that pdeB transcripts were
detected in wild-type cells but not in mutant cells, confirming
inactivation of pdeB; pdeB transcripts in complemented pdeB?
cells were elevated approximately 5-fold above the level in
wild-type cells, indicating overexpression from the multicopy
vector (data not shown; see below) (95). To confirm retention
of B. burgdorferi endogenous plasmids (63, 70) in the pdeB
mutant and complemented strains, PCR-based plasmid profil-
ing using each of the plasmid-specific primers was employed
(70, 100). The plasmid profiles of the wild-type, pdeB mutant,
and pdeB?strains were the same, confirming the retention of
all of the plasmids required for infectivity (not shown; see
Analysis of pdeB mutant phenotype. To determine if a mu-
tation in pdeB altered B. burgdorferi motility, the mutant cells
were analyzed using dark-field microscopy and swarm plate
FIG. 1. Construction and complementation of pdeB. Schematic
representation of pdeB and its neighboring genes in wild-type B. burg-
dorferi is shown at the top. The first 1,040 bp of the pdeB gene is
deleted to insert a 792-bp streptomycin resistance gene (aadA) in
frame (middle panel). The approximate locations of one of the primer
sets that were used to confirm pdeB inactivation (see Fig. 2) are shown
by the arrowheads. Complementation plasmid pBSVbb0374 contain-
ing the pdeB promoter (PpdeB) that drives the expression of the pdeB
gene was used to complement the pdeB mutant (bottom panel).
FIG. 2. Confirmation of pdeB inactivation and assessment of polar
effect. (Left panel) PCR was used to confirm the deletion of the pdeB
gene and integration of aadA (Strr). The locations of the PCR primers
used are shown in Fig. 1. A 792-bp DNA fragment containing the aadA
coding sequence was inserted after the deletion of a 1,040-bp fragment
from the pdeB gene. Marker lane, 1-kb DNA ladder (Fermentas Life
Sciences). Wild-type lane, wild-type DNA (2,333 bp); pdeB lane,
pdeB::aadA DNA (1,541 bp); control lane, pdeB::aadA DNA (1,541
bp) that was electroporated into wild-type cells (positive control).
(Right panel) Lack of a polar effect on BB0375 (Pfs; 27 kDa) protein
expression in pdeB mutant cells was determined by Western blotting
using anti-Pfs antiserum (74). FlaB (41 kDa) was used as a loading
control for the strains indicated.
3276SULTAN ET AL.INFECT. IMMUN.
motility assays (Table 1; see Fig. S1 in the supplemental ma-
terial). As shown in Table 1, the mutant cells ran, flexed, and
reversed as did the wild-type cells; however, their rate of flex-
ing was significantly higher than that of the wild-type cells (5
versus 10 flexes/min; P value, 0.002). When a B. burgdorferi cell
flexes, it becomes distorted or bent at the center (see videos S1
to S3 in the supplemental material for wild-type, pdeB mutant,
and complemented pdeB?cells) (33, 34, 58). In the swarm
plate assays, the mutant swarm diameters were not significantly
(P ? 0.074) different from those of wild-type cells (see Fig. S1).
As expected, the motility of the complemented pdeB?cells was
restored to the wild-type phenotype (Table 1; see Fig. S1 and
videos S1 to S3). Together, these results indicate a role for
pdeB in motility and that the mutant’s phenotype was due
solely to this mutation and not to a secondary alteration else-
Loss of PlzA and PdeB results in constant flexing motility.
In other species of bacteria, PilZ proteins were reported to
control motility in response to changes in the level of c-di-
GMP (3, 7, 14, 22, 66). B. burgdorferi possesses a c-di-GMP
binding PilZ protein, PlzA (26, 68). To determine if PlzA can
function in response to c-di-GMP to regulate B. burgdorferi
motility, we constructed a plzA mutant in the pdeB mutant
(plzA::Pl-kan) into the other mutant cells (pdeB::aadA). The
plzA single mutant exhibits a normal swimming pattern but
reduced swarming motility (68). The B. burgdorferi plzA pdeB
double mutant cells exhibited a constant-flexing motility phe-
notype similar to the phenotype of cheX mutant cells (see the
constant-flexing cheX mutant and plzA pdeB double mutant
cell videos) (58). To determine if the increased flexing motility
exhibited by the pdeB single mutant or the plzA pdeB double
mutant was due to diminished levels of a motility/chemotaxis
protein, we performed Western blotting using specific antise-
rum. As shown in Fig. 3, the expression of prominent motility
or chemotaxis protein FlaB, FliG2, FliG1, FliM, MotB,
CheY3, or CheX was not altered in the pdeB or plzA pdeB
mutant cells. These results suggest that the altered motility
exhibited by those mutants is not due to the reduced expres-
sion of major motility/chemotaxis proteins. However, it is pos-
sible that the constant-flexing phenotype exhibited by the dou-
ble mutant cells is due to inhibition of CheX activity.
PdeB is a functional c-di-GMP PDE, and PdeA and PdeB
are the only active PDEs in B. burgdorferi. Recently, we re-
ported that the EAL domain-containing protein PdeA is a
functional c-di-GMP-specific PDE (91). HD-GYP domain
proteins have been reported to possess c-di-GMP PDE activity
in Xanthomonas campestris and P. aeruginosa (77, 78). To de-
termine if PdeB is an active PDE, we performed PDE assays
using purified R-PdeB, as well as B. burgdorferi mutant cell
extracts, as we and others previously described (15, 91, 94).
Purified (greater than 90% pure; see Fig. 4B, right panel)
recombinant full-length PdeB (379 aa) or only the HD-GYP
domain (aa 90 to 305) and crude cell extracts prepared from
wild-type, pdeB mutant, and pdeB?complemented cells were
tested for the ability to hydrolyze33P-labeled c-di-GMP. As
shown in Fig. 4A, the wild-type crude extract hydrolyzed c-di-
GMP to GMP, indicating PDE activity (29 ? 3 fmol/mg/min;
wild-type lanes). Addition of 50 mM MnCl2to the reaction
mixture enhanced the PDE activity, which is indicative of met-
al-dependent PDE activity (all lanes). pdeB mutant extracts
also hydrolyzed c-di-GMP to GMP but at a lower rate (19 ?
1.9 fmol/mg/min) than the wild-type extracts (Fig. 4A, lanes
pdeB), indicating the presence of another PDE in these mu-
tants. pdeB?complemented cell extracts rapidly hydrolyzed
33P-labeled c-di-GMP (67 ? 1.7 fmol/mg/min; lanes pdeB?),
indicating that the reduction of33P-labeled c-di-GMP hydro-
lysis in pdeB mutant cells was restored upon complementation.
pdeB?cell extracts exhibited greater-than-wild-type PDE ac-
tivity, likely because pdeB was overexpressed in the comple-
mented cells. These results indicated that PdeB is a functional
PDE. To further confirm that PdeB is a c-di-GMP PDE, pu-
rified full-length PdeB or only the HD-GYP protein was as-
sayed for the ability to hydrolyze33P-labeled c-di-GMP (Fig.
4B and C). The full-length protein or only the functional do-
FIG. 3. The expression of prominent motility or chemotaxis pro-
teins was not altered in pdeB or plzA pdeB mutant cells. Cell lysates
from the indicated strains were probed with antibodies specific for the
proteins shown on the right side of each Western blot. The molecular
size (in kDa) of each protein is provided on the left side of each blot.
TABLE 1. pdeB mutant cells exhibit a significantly
increased flex ratea
Strain Swimming pattern
5.0 ? 1.7
9.7 ? 3.3
5.5 ? 1.6
aSee cell videos in the supplemental material.
bThe flex rate was determined as the mean number of flexes per minute ? the
standard deviation. For convenience, flexing is defined as cells bending in the
middle or exhibiting a twisted or circular morphology with no positive displace-
VOL. 79, 2011 A ROLE FOR THE HD-GYP PDE IN MOTILITY AND VIRULENCE3277
main hydrolyzed33P-labeled c-di-GMP to GMP (Fig. 4B and
C). The addition of MnCl2enhanced the PDE activity, estab-
lishing that PdeB is a metal-dependent c-di-GMP PDE (all
Full-length R-PdeB was assayed with different concentra-
tions of33P-labeled c-di-GMP, and the Kmvalue was calculated
to be 2.9 nM, indicating a high affinity for c-di-GMP. The
positive control, CdpA, rapidly hydrolyzed c-di-GMP, and neg-
ative controls without added protein or with an unrelated FlhF
protein failed to hydrolyze c-di-GMP (Fig. 4A and B) (91, 93).
PdeB is a c-di-GMP-specific PDE.3H-labeled cGMP was not
hydrolyzed by R-PdeB, even when the reaction mixture was
incubated for a prolonged period of time (overnight; data not
shown), suggesting that PdeB is a c-di-GMP-specific PDE.
3H-labeled cGMP was used to determine if
To determine if B. burgdorferi possesses only two PDEs
(PdeA and PdeB), we constructed a pdeA::Pl-kan-pdeB::aadA
double mutant by introducing one inactivated plasmid into the
other mutant cells. The pdeA pdeB double mutant cell motility
phenotype was indistinguishable from that of the pdeB single
mutant. Extracts from pdeA pdeB double mutant cells failed to
hydrolyze33P-labeled c-di-GMP, even when the reaction mix-
ture was incubated for extended times (up to 4 h) (Fig. 4A,
pdeA pdeB lanes; data not shown). Therefore, it is likely that
PdeB and PdeA are the only two active c-di-GMP PDEs in B.
pdeB mutant cells are not attenuated to establish an infec-
tion in mice. In order to evaluate the infection potential of the
pdeB mutant, groups of C3H/HeN mice were challenged by
subcutaneous injection with 10-fold increasing doses of wild-
type, pdeB mutant, or isogenic complemented pdeB?cells to
determine the ID50. At 2 weeks postinoculation, the mice were
bled and their sera were assessed for reactivity with B. burg-
dorferi antigens. B. burgdorferi membrane protein A, also
known as P39, was used as a marker of infection in animals (40,
85). To confirm the serological results, mice were sacrificed at
4 weeks postinoculation and tissue (ear, joint, and bladder)
samples were aseptically isolated and assessed for the pres-
ence/absence of B. burgdorferi using dark-field microscopy. Se-
rology results correlated well with reisolation of spirochetes
from the tissues examined (Table 2 and not shown). pdeB
mutant cells did not show a significant increase in the ID50
compared to parental wild-type cells (P ? 0.3976; Table 2).
These results indicate that the pdeB mutant is not significantly
attenuated in virulence relative to the parental wild-type strain
or the complemented pdeB?strain.
pdeB mutant cells are unable to complete the mouse-tick-
mouse infection cycle. The B. burgdorferi infectious life cycle
includes persistent infection of and survival within tick and
mammalian hosts. Because persistent infection of the mamma-
lian host represents only one facet of the spirochete’s enzootic
life cycle, a more comprehensive evaluation of the behavior of
pdeB mutant strains in the tick vector was warranted. To per-
form such an evaluation, naïve larval ticks were allowed to feed
on mice that were infected with the wild-type, mutant, or
complemented strain. Upon larval repletion, the majority of
the ticks were allowed to advance naturally through the month-
long molting process. At 7 days postfeeding, a subset of fed
larvae was assessed for the survivability of spirochetes using
IFA as well as squashing and plating of triturated ticks to count
viable spirochetes (Table 3 and data not shown; see Fig. S2 in
the supplemental material). While the numbers of spirochetes
in ticks that fed on the wild-type and complemented pdeB?
strains were similar, the number of viable spirochetes was
TABLE 2. pdeB mutant cells are not significantly attenuated in
virulence in needle-inoculated C3H/HeN mice
No. of infected mice/total
5 ? 102a
5 ? 103a
5 ? 104a
1.7 ? 103
2.9 ? 103
Not doneNot done
aNo. of spirochetes/mouse.
FIG. 4. pdeB exhibits PDE activity. (A) Representative TLC chro-
matogram of the PDE assay. cdG represents c-di-GMP. All lanes
contain 50 mM MnCl2. A V. cholerae PDE, CdpA, was used as a
positive control. Wild type, pdeB, pdeB?, and pdeA pdeB refer to
extracts of the corresponding cells. Cell extracts were incubated with
33P-labeled c-di-GMP for 20 min. (B) Representative TLC assay (20
min) of purified recombinant (lanes R-PdeB) or only the HD-GYP
domain (lanes HD-GYP) protein. All lanes contained MnCl2, except
the positive control, CdpA. The Rfvalues for the nucleotides in these
assays (c-di-GMP, 0.31; GMP, 0.55) agree well with published reports
(91, 93, 94). MBP-FlhF was used as a negative control (lane FlhF). The
right panel shows the purified R-PdeB (?87 kDa) and HD-GYP (?68
kDa) proteins. PageRuler plus protein markers were obtained from
Fermentas Life Sciences. (C) Representative time course (0 to 25 min)
TLC assay of the R-PdeB protein.
3278SULTAN ET AL.INFECT. IMMUN.
5-fold lower in ticks that fed on the mutant-infected mice
(Table 3). The percentage of ticks infected with the wild-type,
mutant, or complemented strain was approximately 50%; these
results indicated that a mutation in pdeB resulted in cells that
were deficient in survival in ticks (Table 3). The subset of fed
larvae that molted to nymphs was examined for the spiro-
chetes’ ability to migrate from the ticks to naïve mice to com-
plete the mouse-tick-mouse infection cycle (below).
Ticks infected with pdeB mutant cells are unable to transmit
the infection to naïve mice. In the normal B. burgdorferi infec-
tious cycle, mice are infected when spirochete-laden ticks bite
them. We determined whether infected nymphs are able to
transmit the spirochetes to naïve mice. Thirty nymphs of each
group of wild-type, pdeB mutant, and complemented pdeB?
strains were allowed to feed on a naïve mouse (n ? 4) to
determine spirochete transmission from ticks to the mamma-
lian hosts (Table 3). Although the percentage of ticks infected
with all 3 strains was approximately 50%, the mutant-infected
ticks failed to transmit the infection to any mice, whereas
wild-type-infected ticks were able to transmit the infection to
all mice, as confirmed by reisolation of spirochetes from sac-
rificed mouse tissue (ear, joint, and bladder) specimens (Table
3). The pdeB?spirochete-infected ticks were also able to trans-
mit the infection to 2/4 naïve mice (Table 3). The pdeB?cells’
partially restored virulence likely resulted from the overexpres-
sion of pdeB, or a small population of the complemented
pdeB?cells may have lost an endogenous plasmid(s). These
results indicated that pdeB mutant cells failed to complete the
mouse-tick-mouse infection cycle.
pdeB mutant cells are deficient in survival in artificially
infected ticks. Tick immersion studies were performed to con-
firm the extent to which wild-type and mutant B. burgdorferi
strains are able to infect, survive, and transmit the infection
from arthropod tick vectors to a mammalian host (87, 89, 91).
Tick immersion studies allow direct artificial tick infection and
serve two purposes, i.e., (i) to optimally infect naïve ticks with
the wild-type and mutant spirochetes and determine their col-
onization and the ability to survive within the tick vector and
(ii) to examine for the potential of spirochetes to migrate from
the arthropod host vector to the mammalian host. Three-
month-old tick larvae were artificially inoculated with expo-
nentially growing (5 ? 107cells/ml) cultures of wild-type, pdeB
mutant, or complemented pdeB?B. burgdorferi cells. As ticks
ingest B. burgdorferi cultures, their intestines are inoculated
with spirochetes. After tick immersion, approximately 150 spi-
rochete-laden ticks of each strain were allowed to feed on a
naïve mouse (n ? 2 or 4) (Table 4). Fed ticks were analyzed for
the presence of spirochetes by IFA (see Fig. S2 in the supple-
mental material), and crushed ticks were plated and viable
spirochetes were counted. While the spirochete burdens of the
ticks inoculated with wild-type or complemented pdeB?cells
were approximately the same, the mutant strain spirochete
burden was 5-fold lower when ticks were analyzed 7 days after
blood acquisition (Table 4). The percentage of fed ticks in-
fected with the wild-type, mutant, or complemented strain was
the same (?80%; Table 4). These data and the mouse-tick-
mouse results demonstrate that PdeB is likely required for B.
burgdorferi to colonize and/or survive in the tick vector. Mice
on which ticks fed were sacrificed to determine the extent to
which spirochetes could be isolated from tissue specimens.
Consistent with mouse-tick-mouse infection cycle studies (Ta-
ble 3), spirochetes were isolated only from tissue specimens
collected from mice on which ticks infected with the wild-type
or the complemented pdeB?strain fed. No spirochetes were
isolated from any of the tissue specimens from mice on which
ticks infected with the pdeB mutant cells fed (Table 4). These
results confirm that the pdeB mutant has a reduced ability to
colonize ticks and is unable to transmit an infection from ticks
to naïve mice.
HD-GYP domains belong to the HD superfamily of metal-
dependent hydrolases (2, 29–31) and are often encountered
fused to other signal transduction domains such as REC,
GGDEF, and PAS domains (27, 31). Based on these observa-
tions, Galperin et al (31) initially proposed that HD-GYP
domain-containing proteins may function as PDEs directed
toward either the dephosphorylation of phosphoproteins or
the hydrolysis of cyclic nucleotides, especially c-di-GMP. HD-
GYP domain-containing proteins subsequently have been
shown to indeed possess c-di-GMP-hydrolyzing activity (77,
78). Although more than 590 HD-GYP domains in over 140
genomes have been reported (www.ncbi.nlm.nih.gov/complete_
genomes/signalcensus.html), only a handful have been charac-
terized (1, 35, 77, 78). The genome of B. burgdorferi encodes a
single diguanylate cyclase and two PDEs, one of the EAL type
and one of the HD-GYP type (25, 31). Purified recombinant
forms of the diguanylate cyclase Rrp1 and the EAL domain-
containing PdeA have been shown to synthesize and hydrolyze
TABLE 3. pdeB mutant cells are unable to complete the mouse-tick-mouse infection cycle
% of mice
% of ticks
Mean no. of
spirochetes/nymph ? SD
No. of nymphs
No. of mice
5 ? 104
5 ? 105
5 ? 104
20,900 ? 11,172
4,466 ? 702
15,666 ? 757
TABLE 4. Tick immersion studies indicate the pdeB mutant is
defective in the ability to colonize ticks and in the ability to
transmit the infection from ticks to naı ¨ve mice
% of fed ticks
Mean no. of
spirochetes/tick ? SD
No. of mice
3,366 ? 1,750
618 ? 837
3,460 ? 1,975
VOL. 79, 2011 A ROLE FOR THE HD-GYP PDE IN MOTILITY AND VIRULENCE3279
c-di-GMP in vitro, respectively (81, 91). In PDE assays, extracts
from pdeA mutant cells exhibited a reduced ability to hydrolyze
33P-labeled c-di-GMP, relative to that of wild-type cells. How-
ever, pdeA mutant cell extracts retained considerable c-di-
GMP-hydrolyzing activity, leading us to propose that B. burg-
dorferi expresses more than one functional PDE (91). Several
lines of evidence led us to conclude that the HD-GYP domain-
containing protein PdeB functions as a c-di-GMP-specific PDE
(Fig. 4A to C). First, we constructed a pdeB mutant of B.
burgdorferi and compared the abilities of extracts from wild-
type, mutant, and complemented cells to hydrolyze radiola-
beled c-di-GMP in vitro. The wild-type and complemented cell
extracts readily hydrolyzed33P-labeled c-di-GMP, and this ac-
tivity was reduced in extracts from pdeB mutants (Fig. 4A).
Second, purified recombinant full-length protein and a trun-
cated form of PdeB containing the HD-GYP domain exhibit
c-di-GMP-specific PDE activity (Fig. 4B and C); as with other
HD-GYP PDEs, the activity of PdeB was enhanced by the
addition of Mn2?(77, 78, 94). It is notable that when the
HD-GYP domain of RpfG of X. campestris was mutated to
HA-GYP, the c-di-GMP-hydrolyzing activity was abolished
(77). B. burgdorferi PdeB contains an imperfect HK-GYP do-
main instead of HD-GYP; it is not known if a mutation in the
HD-GYP domain of RpfG to HK-GYP would alter enzyme
activity. However, our PDE assays clearly demonstrate that the
HK-GYP domain-containing PdeB hydrolyzes c-di-GMP and
exhibits a high affinity for c-di-GMP (Fig. 4A to C).
To date, only three HD-GYP-type PDEs have been shown
to hydrolyze c-di-GMP in bacteria; however, Kmvalues for
those PDEs have not been reported (77, 78). Here, we deter-
mined that the Kmfor PdeB is 2.9 nM, a value which is sub-
stantially lower than those previously reported for EAL-type
PDEs (60 nM to a few micromolar) (36, 91, 94). Bioinformatic
analysis suggests that PdeA and PdeB are the only PDEs in B.
burgdorferi (31). Consistent with this notion, cell extracts pre-
pared from the wild type, the single mutants (pdeA and pdeB),
or their complements hydrolyzed33P-labeled c-di-GMP within
2 to 20 min of incubation, while extracts prepared from spiro-
chetes lacking both PdeA and PdeB failed to hydrolyze the
nucleotide even when the reaction mixtures were incubated for
up to 4 h (Fig. 4A and reference 91).
Similar to their EAL-type counterparts (39, 94, 96), HD-
GYP-type PDEs have been implicated in motility regulation in
P. aeruginosa and X. campestris (77, 78). Specifically, mutations
in the P. aeruginosa PDEs PA4108 and PA4781 and X. camp-
estris pv. campestris PDE rpfG resulted in reduction of swarm-
ing motility (77, 78). Recently, we reported that inactivation of
the B. burgdorferi EAL-type PdeA rendered spirochetes unable
to reverse their direction of swimming (91). Interestingly, the
aberrant motility displayed by spirochetes lacking PdeB dif-
fered from that of the pdeA mutant; PdeB-deficient cells ex-
hibit significantly increased flexing (Table 1). This phenotype
was attributed solely to the pdeB mutation, since complemen-
tation in trans restored the wild-type phenotype (Table 1; see
Fig. S1 and videos in the supplemental material). Two condi-
tions might be expected to lead to increased flexing, (i) over-
expression of CheY3 and (ii) inhibition of CheX activity or
decreased expression of CheX (58, 61, 67). Based on Western
blotting, however, both CheY3 and CheX were expressed at
wild-type levels in lysates prepared from pdeB single and plzA
pdeB double mutants (Fig. 3), ruling out a direct role for
c-di-GMP in regulating the expression of these chemotaxis
proteins. Alternatively, c-di-GMP may regulate bacterial mo-
tility via an interaction with PilZ proteins (7, 14, 66, 91, 96). B.
burgdorferi possesses a c-di-GMP-binding PilZ domain protein,
PlzA (26, 68). Inactivation of plzA results in reduced swarming
motility, although the swimming pattern of this mutant was
indistinguishable from that of its parent (68). Recently, we
demonstrated that the alteration of motility displayed by the
pdeA mutant (91) is modulated by a PlzA-independent mech-
anism (68). However, spirochetes lacking PlzA in the pdeB
mutant background constantly flex, a phenotype that is indis-
tinguishable from that of a cheX mutant (see the cheX single
mutant and plzA pdeB double mutant cell videos in the sup-
plemental material) (58). These results suggest that c-di-GMP/
PlzA may interact with the B. burgdorferi chemotaxis signaling
pathway via CheX or CheY3, most likely by modulating the
activity of CheX. We hypothesize that when it is not bound to
c-di-GMP, PlzA may stimulate CheX activity. Thus, in the
pdeB mutant, c-di-GMP is elevated, more PlzA–c-di-GMP
complex is formed, and CheX activity is diminished, resulting
in elevated CheY3-P and increasing the rate of flexing. In the
latter scenario, the plzA pdeB double mutant would exhibit very
low CheX activity, greatly increasing CheY3-P and causing the
constant-flexing phenotype. It is possible to test these possibil-
ities using in vitro biochemical assays.
It is interesting that B. burgdorferi apparently has only two
functional c-di-GMP PDEs with disparate motility and viru-
lence phenotypes (91; this study). While both PDEs appear to
impact motility, it is unclear how they are able to alter motility
in such different ways. Because of their distinct motility phe-
notypes, it is tempting to speculate that PdeA or PdeB may
localize at different poles of a cell, thereby causing the
periplasmic flagellar motor rotation to be CW or CCW by
interacting with a flagellar protein or modulating the activity of
a chemotaxis protein(s) in a manner that is not yet understood.
A mutation in B. burgdorferi pdeA or E. coli/S. enterica PDE
(yhjH) resulted in a biased direction of rotation of flagellar
motors and/or decreased flagellar motor rotation. One possi-
bility is that PdeA functions analogously to the EAL-type PDE
YhjH in E. coli and S. enterica. Studies in different laboratories
indicated that YhjH alters motility via the c-di-GMP effector
protein YcgR, which, when complexed with c-di-GMP, acts as
a “brake” on the flagellar motor switch complex (7, 22, 66, 91).
However, to our knowledge, this is the first report to demon-
strate that pdeB mutant cells exhibit a flexing motility pheno-
type without altering the speed of motor rotations.
In addition to their role in regulating bacterial motility,
HD-GYP-type PDEs have also been linked to virulence (76,
79, 96). Recently, we demonstrated that spirochetes lacking
PdeA were unable to infect mice following needle or tick
inoculation, presumably as a result of their failure to reverse
swimming direction (91). Loss of PdeB, on the other hand, had
no significant effect on virulence (P ? 0.3976), further support-
ing our hypothesis that PdeA and PdeB exert their regulatory
effects by independent mechanisms. Recently, independent
studies by Caimano et al. (10a), He et al. (35a), and Kostick et
al. (45) demonstrated that hk1 and rrp1 are required for sur-
vival within fed tick midguts. Survival of the rrp1 mutant could
be partially restored in feeding ticks by constitutive expression
3280SULTAN ET AL.INFECT. IMMUN.
of genes involved in glycerol uptake and utilization (glp genes)
(X. F. Yang, personal communication) (35a). Interestingly,
spirochetes lacking pdeB also displayed a survival defect within
fed ticks, although the mechanism(s) underlying the phenotype
observed with our pdeB mutant differs from that of hk1 and
rrp1 mutants; unlike hk1/rrp1 mutants, which are killed early
(within 36 h) during the blood meal, spirochetes lacking PdeB
appear to be killed postrepletion. Not surprisingly, loss of pdeB
had no effect on glp gene (bb0241 and bb0243) expression in
vitro (our unpublished observation). Together, these data may
indicate that a precise modulation of c-di-GMP levels is central
to the tick phase of the spirochete’s enzootic cycle, with in-
creased and decreased levels of c-di-GMP being required for
adaptation to the fed and flat tick midgut environments, re-
spectively. Somewhat surprisingly, nymphal ticks infected with
the pdeB mutant failed to transmit the infection to naïve mice
during engorgement, despite containing substantial numbers
of viable spirochetes (Table 3). The latter data suggest a po-
tential role for decreased c-di-GMP levels in promoting the
migration of spirochetes out of the midgut during feeding. The
decreased ability of PdeB-deficient spirochetes to survive
within replete ticks may suggest that the degradation of c-di-
GMP controls the expression and/or activity of B. burgdorferi
gene products required to withstand growth within the flat
midgut environment. Recently, constructed bb0323, bb0365,
guaAB, dps, ospA/B, and bptA mutant strains exhibited defects
in the ability to survive in ticks (41, 53, 64, 68, 73, 98, 99).
Furthermore, the bba52, bba64, or bba07 mutant was reported
to be unable to transmit spirochetes from ticks to naïve mice,
although each of the mutants was able to infect mice by needle
inoculation (32, 48, 97). Whether c-di-GMP affected the ex-
pression of one or more of these or other B. burgdorferi genes
remains to be investigated. Clearly, additional studies are re-
quired to understand the unusual and complicated signaling
produced by c-di-GMP-metabolizing proteins.
We thank Melissa Caimano, Justin Radolf, and X. Frank Yang for
sharing their unpublished results and Nyles Charon, Justin Radolf, and
Melissa Caimano for comments on the manuscript. We thank C. Li, R.
Rego, P. Rosa, B. Stevenson, V. T. Lee, A. Camilli, D. Blair, A.
Barbour, J. Benach, and J. Carroll for providing reagents. We also
thank D. Akins for B. burgdorferi plasmid primer sequence informa-
This research is sponsored by an East Carolina University Research
and Development “start-up” fund to M.A.M.
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