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J Bacteriol, May 1998, p. 2373-2378, Vol. 180, No. 9
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Cloning and Characterization of a Gene Encoding the
Major Surface Protein of the Bacterial Endosymbiont
Wolbachia pipientis
Henk R.
Braig,1
Weiguo
Zhou,1,2
Stephen L.
Dobson,1 and
Scott L.
O'Neill1,*
Section of Vector Biology, Department of
Epidemiology and Public Health, Yale University School of Medicine,
New Haven, Connecticut 06520,1 and
Institute of Genetics, Fudan University, Shanghai 200433, People's Republic of China2
Received 7 November 1997/Accepted 24 February 1998
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ABSTRACT |
The maternally inherited intracellular symbiont Wolbachia
pipientis is well known for inducing a variety of reproductive
abnormalities in the diverse arthropod hosts it infects. It has been
implicated in causing cytoplasmic incompatibility, parthenogenesis, and
the feminization of genetic males in different hosts. The molecular mechanisms by which this fastidious intracellular bacterium causes these reproductive and developmental abnormalities have not yet been
determined. In this paper, we report on (i) the purification of one of
the most abundantly expressed Wolbachia proteins from infected Drosophila eggs and (ii) the subsequent cloning
and characterization of the gene (wsp) that encodes it. The
functionality of the wsp promoter region was also
successfully tested in Escherichia coli. Comparison of
sequences of this gene from different strains of Wolbachia
revealed a high level of variability. This sequence variation
correlated with the ability of certain Wolbachia strains to
induce or rescue the cytoplasmic incompatibility phenotype in infected
insects. As such, this gene will be a very useful tool for
Wolbachia strain typing and phylogenetic analysis, as well
as understanding the molecular basis of the interaction of Wolbachia with its host.
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INTRODUCTION |
The genus Wolbachia
comprises a group of maternally inherited intracellular bacteria that
have been identified in a wide range of arthropod hosts. Indeed, some
surveys suggest that around 16% of all insect species may be naturally
infected with this agent (35). Wolbachia strains
are best known for the reproductive distortions they generate in the
arthropods they infect. These phenotypes include the feminization of
genetic males, induction of parthenogenetic development, and most
commonly the expression of cytoplasmic incompatibility (CI)
(34). CI expression usually results in embryonic death in
crosses in which the male insect parent is infected with a
Wolbachia strain and the female parent is either uninfected
or infected with a different Wolbachia strain. It appears
that a Wolbachia strain is able to imprint the sperm of
insects it infects through an unknown mechanism and that this imprint
is rescued only in eggs that are infected with the same Wolbachia strain.
Through the action of these various reproductive manipulations,
Wolbachia is able to efficiently invade host populations
without being infectious or moving horizontally between individuals at an appreciable rate. It has been suggested that the ability of Wolbachia to actively invade populations could be used as a
vehicle to drive desirable genotypes into wild insect populations,
e.g., genes that prevent insect disease vectors from transmitting
pathogens to humans, livestock, or plants (1, 30).
While much is known about the phenomenology and population genetics of
Wolbachia infections, very little is known about the molecular mechanisms that underlie the interaction of this agent and
the insect. In previous studies, we have identified by metabolic labeling the major proteins that Wolbachia synthesizes in
vivo (27). One of these proteins showed size polymorphism
between Wolbachia strains that correlated with the ability
of a given Wolbachia strain to induce the CI phenotype in
Drosophila species. In this paper, we report the
purification of this protein and the cloning and characterization of
its gene from a number of different Wolbachia strains. In
addition, we show that the 5' noncoding region can serve as a
functional promoter in Escherichia coli.
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MATERIALS AND METHODS |
Insect strains.
The following strains of
Drosophila species which harbor Wolbachia
infections were used: D. melanogaster CantonS (DMCS)
(13), D. melanogaster Harwich (DMHarwich)
(13), D. simulans Coffs Harbour (DSCoffs)
(10), D. simulans Hawaii (DSH) (23),
D. simulans Riverside (DSR) (11),
tetracycline-treated DSR cured of Wolbachia (DSRT), and
D. simulans Watsonville(Mauritiana) (DSW/Mau) carrying the
transinfected Wolbachia strain from D. mauritiana (9). In addition to these Wolbachia strains known
to naturally infect Drosophila spp., the strain from the
Wolbachia-infected almond moth Cadra
(Ephestia) cautella was used (4). Of
the various Wolbachia strains, DMCS, DMHarwich, DSCoffs,
and DSW/Mau have been reported previously as being incapable of
expressing the CI phenotype (9, 10, 13), while DSR, DSH, and
the Wolbachia strain C. cautella have all been
reported as strong CI expressors (4, 11, 23).
Purification of Wolbachia.
Drosophila eggs
from 2- to 4-h collections were dechorionated for 2 min with 2.6%
sodium hypochlorite, washed with water, and packed under water in a
1.5-ml microcentrifuge tube with repeated spins for 10 s so as to
accumulate 50 µl of packed eggs. Excess water was removed, and the
eggs were homogenized by hand with a tight-fitting pestle (Kontes Co.,
Vineland, N.J.) in 100 µl of homogenization buffer (250 mM sucrose,
90 mM potassium chloride, 30 mM sodium chloride, 15 mM magnesium
sulfate, 5.5 mM calcium chloride, 0.1% [wt/vol] Lubrol; ICN Inc.,
Costa Mesa, Calif.). After homogenization, an additional 1 ml of
homogenization buffer was added and the tube was vortexed for 3 s.
Cellular debris was pelleted for 5 min with 80 × gmax at 20°C. The supernatant was centrifuged
for 5 min at 4,000 × gmax. The resulting
pellet was carefully resuspended with 100 µl of homogenization
buffer, an additional 1 ml of homogenization buffer was then added, and
the tube was vortexed for 3 s. After a 5-min spin at 300 × gmax, the supernatant was loaded onto a
13-mm-diameter filter cassette containing a 0.8- to 8-µm-pore-size
glass fiber prefilter (AP 20, 13 mm; Millipore Corp., Bedford, Mass.)
and a strong protein binding 3-µm-pore-size mixed cellulose membrane
(SSWP; Millipore) and filtered under unit gravity. The filter cassette
was washed with homogenization buffer until 1.5 ml of filtrate was
obtained; the filtrate was then spun for 5 min at 5,000 × gmax, and the pellet was saved.
Purification of Wolbachia was monitored by comparing the
protein profiles of DSR and DSRT preparations by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by silver staining. If no difference could be detected in protein profiles, the amount of
Wolbachia was estimated by DAPI
(4',6-diamidine-2-phenylindole dihydrochloride) staining and/or PCR.
DAPI staining for DNA was performed overnight by mixing equal volumes
of sample preparation and a 1-µg/ml solution of DAPI in 100%
methanol so that mitochondrial DNA could also be detected. Because the
size of the mitochondrial DNA is only a fraction of that of
Wolbachia, the two could easily be differentiated.
Wolbachia DNA quantities in preparations were also estimated
by PCR of serial dilutions with
Wolbachia-specific primers
for
the 16S rRNA gene (99F and 994R) (
22) and for the
ftsZ gene
(
29). To estimate whether nuclear DNA
of the host was present
in preparations, the single-copy gene
suppressor of sable [
su(s)]
(
33) was
PCR amplified. For the
su(s) gene, forward (5'-TCA
GTA CCG
CGA ACG CAG CAA ATA-3') and reverse (5'-GCC GCC ACG TAC
GTT CAT CAT
CTC-3') primers were designed; for mitochondria, 12S
rRNA primers
(12SAI and 12SBI) were used (
22).
Protein purification and protein sequencing.
Protein samples
were desalted and concentrated by the method of Wessel and Flügge
(36) except that acetone replaced methanol in the final
step. For screening purposes, SDS-12% gels were used (15).
Proteins were blotted from 10% (percentage of cross-linking monomer
over total amount of monomer C = 3.3%) tricine gels
(28), without a spacing gel, onto polyvinylidene difluoride
membranes (Immobilon-P; Millipore) under semidry conditions, using the
buffer described by Bjerrum and Schafer-Nielsen (2) with
20% methanol and 0.004% SDS. The molecular weight of the protein was
estimated in 10% (C = 2.6%) tricine gels without a spacing gel.
The isoelectric point was estimated in the matrix-free Rotofor system
(Bio-Rad Laboratories, Hercules, Calif.) with samples prepared as
described by O'Farrell (20). The protein was N terminally
sequenced and, after trypsin digestion, internally sequenced at the
Keck Foundation, Yale University, New Haven, Conn.).
Gene cloning and Southern blotting.
Degenerate
oligonucleotide primers were designed from regions of the N-terminal
and internal peptide sequences (forward, 5'-TAY GTI GTI YTI CAR TAY AAY
GGI GAR AT-3'; reverse, 5'-GTA IAG ICC ITC IAC ATC NAC-3'), and PCR
amplification with an annealing temperature of 45°C was done on total
DNA extracted from Drosophila DSR and DSRT eggs. A PCR
product was obtained from strain DSR but not strain DSRT. This product
was directly cloned into the EcoRV site of the pBluescript
vector (Stratagene, La Jolla, Calif.) after tailing the vector with
ddTTP, using terminal transferase (Boehringer Mannheim, Indianapolis,
Ind.) (13a). Sequencing confirmed that the cloned fragment
was derived from the gene encoding the protein, since putative
translation of the cloned DNA yielded a sequence identical to the
peptide sequence internal to the forward PCR primer.
The following strategies were used to clone the 5' and 3' coding
regions of the gene as well as flanking DNA. For the 3' end,
total DNA
of DSR was digested with
EcoRI and ligated to similarly
digested pBluescript. PCR was done with the primer
wsp 115F
(5'-GTG
GTG CTG CAA TAC AAC-3') and either the T3 or T7 primer which
recognizes
pBluescript. Then nested PCR was performed with the internal
primer
wsp 169F (5'-ATT GAA TAT AAA AAG GCC ACA GAC A-3').
The resulting
PCR product of 900 bp was cloned and sequenced to confirm
that
it represented the 3' end of the gene.
The 5' flanking fragment was obtained by means of ligation-mediated PCR
(LM-PCR) (
17). Primer extension was performed with
wsp 247R (5'-TGT AAC CAA ATG CAC CAC CAC CAG-3') on
PstI-digested
total DNA from DSR and DSRT, using
Pfu polymerase to produce blunt
ends. The products were
ligated to partially double-stranded oligonucleotides
formed by
annealing LM-PCR 1 (5'-GCG GTG ACC CGG GAG ATC TGA ATT
C-3') and LM-PCR
2 (5'-GAA TTC AGA TC-3'). The PCR product generated
with the primers
LM-PCR 1 and
wsp 247R was subjected to a nested
amplification with LM-PCR 1 and the internal primer
wsp 198R
(5'-ATG
AAT GTC TGT GGC CTT TTT AT-3'), of which the product (~1,200
bp)
was cloned and partially sequenced (500 bp). Once the 5' and 3'
ends were sequenced, PCR primers were designed to amplify the
entire
gene from infected DSR insects. Several primers were designed
on the
basis of the gene sequence from DSR and tested on different
strains of
Wolbachia. The primer combination
wsp 81F (5'-TGG
TCC
AAT AAG TGA TGA AGA AAC-3') and
wsp 691R (5'-AAA AAT TAA
ACG CTA
CTC CA-3') was found to be able to amplify
wsp gene
fragments
from different strains of
Wolbachia.
To determine copy number of the gene, 5 µg of total fly DNAs from
DSR, DSRT, DSH, and DSW/Mau were digested with
EcoRI. DNA
was transferred to a Zeta-Probe membrane (Bio-Rad) by vacuum blotting
after separation on a 1% agarose gel. The membrane was baked for
2 h at 80°C. Prehybridization was done overnight at 65°C with
0.5 M phosphate buffer (pH 7.2) containing 7% SDS, 1% bovine serum
albumin, and 1 mM EDTA. The probe spanning the coding region of
wsp was obtained by PCR with primers
wsp 81F and
wsp 691R and
labeled with [
-
32P]dATP, using
a random-primed DNA labeling kit (Boehringer Mannheim).
The labeled
probe was added directly into the prehybridization
solution and left
overnight for hybridization. The membrane was
washed twice with
low-stringency buffer (5% SDS, 40 mM phosphate)
for 5 min, then twice
in high-stringency buffer (1% SDS, 40 mM
phosphate), both at 65°C.
Additional Southern blots of total fly
DNA from DSR were performed with
SspI,
TaqI, and
Tth111I (all
cutting
in the 5' noncoding region), with
BsrI and
NlaIII
(both
cutting in the 3' noncoding region), and as a double digest with
XbaI and
BbsI (cutting in the 5' and 3' regions,
respectively).
Functional testing of the wsp promoter region in
E. coli.
The upstream wsp sequence (nucleotides
305 to 1) was PCR amplified by using specific primers with
incorporated SacI and SacII restriction sites
5'-GAG CTC AAG ATG GTA CTT GGA TAA GA-3' and 5'-CCG CGG AAT TGT CCT CGT
AA-3'. To introduce additional restriction sites adjacent to the
wsp sequence, this amplification product was double digested
with SacI and SacII and ligated into the pEGFP-N1
vector (Clontech Laboratories Inc., Palo Alto, Calif.). To examine the
wsp upstream region's promoting capability, it was cloned
into the pKK232-8 vector (Pharmacia Biotech Inc., Piscataway, N.J.).
For comparison, a lac promoter was similarly introduced into
pKK232-8. To accomplish this, the wsp construct was digested with SacII, blunted with T4 polymerase, and digested with
BglII. This was then ligated to the pKK232-8 vector which
had been digested with HindIII, blunted with T4
polymerase, and digested with BamHI. The lac
promoter was excised from pGFP (Clontech) by digestion with
SapI, blunting with T4 polymerase, and digestion with
HindIII. It was then ligated to the pKK232-8 vector
carrying an ampicillin resistance gene which had been digested with
SalI, blunted with T4 polymerase, and digested with
HindIII. Transformants were first selected on ampicillin
plates for the pKK232-8 vector-encoded resistance and then on
chloramphenicol (100 µg/ml) plates for chloramphenicol
acetyltransferase (CAT) activity. Each construct was verified by
restriction analysis.
The promoter strength of the
wsp upstream sequence was
determined by using a Flash CAT 1-deoxyCAM assay (Stratagene) for CAT.
As a comparison, the
lac promoter was also examined. For the
assay,
a 1:160 dilution of an overnight culture was grown for 3 h
under
selection with ampicillin. In one culture, 1 mM
isopropyl-
-
D-thiogalactopyranoside
(IPTG) was added. At
the end of this growth, an equivalent concentration
of each culture was
verified by plating a serial dilution. The
cells in 1 ml of each
culture were pelleted and washed with phosphate-buffered
saline. Each
pellet was resuspended in 1 ml of 100 mM KCl-25 mM
HEPES-0.1 mM
EDTA-12.5 mM MgCl
2-10% glycerol, 0.1% Nonidet P-40-1
mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride-500 µg
of
lysozyme per ml (pH 8.0). This suspension was then shaken on
ice for 30 min and sonicated. Following centrifugation for 5 min
at 21,000 ×
gmax, 55-µl aliquots of the supernatants were
assayed
as recommended in the Flash CAT protocol. The results were
quantified
with the NIH Image software package (
25). After
the assay, the
gene construct was reisolated and sequenced; it was
identical
to the original construct.
Nucleotide sequence accession numbers.
The sequences
reported in this paper have been deposited in the GenBank database
under accession no. AF020070, AF020065, AF020066, AF020067, AF020068,
AF020069, and AF020075.
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RESULTS |
Initial attempts to purify Wolbachia strains from
Drosophila eggs were based on protocols for the purification
of Rickettsia species from cultured cells, which rely on
isopycnic gradient separations. These protocols did not result in a
sufficiently complete separation of Wolbachia from host
material. The main reason for this failure may lie in the fact that
Wolbachia resides in a host vacuole that is intimately
associated with host cytoskeletal elements. We chose instead to use a
method utilizing a detergent and filtration membranes. Critical
parameters in the purification were the detergent concentration and the
pore size of the filtration membranes. The use of Lubrol at 0.1%
(range, 0.01 to 1%) and a 3-µm-pore-size membrane provided an
optimal trade-off between yield and purity. The major contaminants were
mitochondria, as judged by DAPI staining and PCR analysis, and host
membrane material. A higher purity with a lower yield could be achieved
by omitting the detergent and using a 5-µm-pore-size membrane
instead. The detergent removes host vacuolar material from
Wolbachia and facilitates the passage of
Wolbachia through the filtration membrane by preventing an
interaction between Wolbachia and the filtration membrane. This makes the operational filtration size of Wolbachia
smaller under detergent, but at the same time, the detergent generates membrane aggregates from host material that contaminate the
Wolbachia fractions. In the absence of detergent, a larger
pore size is needed. Since no aggregates of host membranes are
generated in detergent-free samples, the larger-pore-size filtration
results in preparations with less host contamination.
When purified samples of Wolbachia from DSR eggs were run on
silver-stained SDS-gels, a dominant protein with an apparent molecular
mass of around 28 kDa was resolved. In tricine-based gels, the same
protein had an apparent size of around 22 kDa, which is closer to the
size predicted from the gene sequence (Fig. 1). Under denaturing conditions, this
protein focused at a pI between 4.5 and 4.7. After cell lysis with
either water or treatment with detergents, the protein stayed
associated with membrane fractions. Its behavior on treatment with the
detergent Sarkosyl, which selectively solubilizes inner membrane
proteins, suggested that the protein is located in the outer membrane
of the bacterium (5, 7): the Wolbachia protein
stays membrane associated with 1% Sarkosyl but is solubilized with
2.75%. These data and the homology to other bacterial outer membrane
proteins as shown later indicate that this Wolbachia protein
is a surface protein; therefore, we named it Wolbachia
surface protein (WSP).
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FIG. 1.
WSP is the dominant protein in silver-stained
SDS-polyacrylamide gels of Wolbachia fractions from DSR
eggs; DSRT, uninfected control; left lane, marker proteins.
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Sequencing of the N terminus of WSP revealed the following
30-amino-acid sequence: N'-(A, S, G [V])-P-I
(P)-S-D-E-E-T-X-Y-Y-V-(V)-L-Q-Y (Q)-N-G-Q-I-L-P-X-F-X-K-(I)-C'. After
trypsin digestion, the sequence of an internal fragment was obtained:
N'-X-P-V (I)-X-P (I, A, D)-(I)-(I)-(D)-C'. These sequences were
sufficient to generate a nested PCR protocol that led to the cloning of
the complete gene.
The cloned gene from DSR Wolbachia (wRi) contains
an open reading frame (ORF) that codes for a protein of 230 amino acids (690 bp, 24,633 Da) starting with an N-terminal methionine (Fig. 2). However, sequencing of the purified
mature protein showed that the N-terminal amino acid was aspartic acid.
The 24 additional coded amino acids appear to constitute a
well-recognized gram-negative signal secretion sequence with a cleavage
site in front of the aspartic acid as predicted by the von Heijne
algorithm (18).
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FIG. 2.
wsp gene sequence and deduced amino acid
sequence from Wolbachia harbored by DSR. Several putative
regulatory regions are indicated.
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On Southern blots of total D. simulans DNA from DSR, DSH,
and DSW/Mau, digested with EcoRI, and DSR, digested with
either BsrI, NlaIII, SspI,
TaqI, Tth111I, or
XbaI-BbsI, and probed with a 610-bp fragment (bp
81 to 691) of wsp revealed only one hybridizing fragment,
indicating that wsp is a single-copy gene (Fig.
3). Southern blots of total fly DNA from
uninfected DSRT, digested with EcoRI, did not show any
hybridization when probed with the same wsp fragment.
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FIG. 3.
Southern blot showing a single hybridizing fragment in
total fly DNA digested with EcoRI and probed with 610 bp of
wsp from DSR, indicating a single-copy gene. Lanes: 1, DSR;
2, DSH; 3, DSW/Mau.
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Because WSP is an abundant Wolbachia protein, its expression
is likely to be driven by a very strong promoter. To examine whether
the DNA sequence immediately upstream of the wsp ORF could function as a promoter, 305 bp of 5' flanking DNA was cloned into the
pKK232-8 vector in front of the CAT gene. For comparison, the lac promoter was similarly cloned into this vector.
Selection of transformants on the basis of their resistance to
chloramphenicol indicated that a functional promoter was driving
CAT gene expression. The level of CAT activity induced by
the wsp promoter was quantified and compared with levels
induced by the lac promoter. Calculations based on
densitometry scores of the data in Fig. 4
revealed that the wsp sequence induced 33% more CAT
activity than the lac promoter with IPTG induction and 116%
more than the lac promoter without IPTG induction. The
pKK232-8 vector without a promoter insert did not show any CAT
activity. These results demonstrate that the upstream wsp
sequence not only is recognized in the unrelated bacterium E. coli, but also contains sequences that function as a stronger
promoter than the lac promoter.
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FIG. 4.
E. coli strains transformed with different
plasmid constructs were used to assay the wsp upstream
sequence for its ability to drive expression of a CAT gene.
Shown is a reverse negative of an autofluorogram of a thin-layer
chromatogram of CAT activity revealed by the cleavage of a CAT
substrate. Presence of CAT is demonstrated by cleavage of the
fluorescently labeled 1-deoxyCAM substrate (lower band) to generate the
higher band. Lanes 5 to 8 show 10-fold dilutions of lanes 1 to 4. The
unmodified pKK232-8 vector (lanes 1 and 5) shows no CAT activity. The
insertion of the wsp upstream sequence at the 5' end of the
CAT gene induced its expression (lanes 2 and 6). Similarly,
the insertion of the lac promoter induced expression of the
CAT gene in the presence (lanes 3 and 7) and in the absence
(lanes 4 and 8) of IPTG.
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DISCUSSION |
WSP is a membrane protein, and its solubilization behavior in
Sarkosyl suggests that it is an outer membrane protein. The sequence-based predicted protein localization site according to Klein
et al. (14) is equally probable for either the outer
membrane or periplasmic space. In addition, the protein contains a
carboxy-terminal phenylalanine that is considered to be essential for
the correct assembly of bacterial outer membrane proteins
(31). The TMpred algorithm (12) predicts two
membrane-spanning regions, surprisingly, both with the same strong
preference for an outside-inside orientation: 88 to 107 (20 amino
acids; score, 551) and 111 to 128 (18 amino acids; score, 575) (Fig.
2).
The gene encoding WSP shows homology with genes encoding outer membrane
proteins of the closely related rickettsiae. The greatest homology is
shared with major surface protein 4 (MSP4) of Anaplasma marginale (31% similarity to WSP from DSR)
(19), major antigenic protein 1 (MAP1) of Cowdria
ruminantium (28% similarity) (26), and the tia
invasion determinant of an enterotoxigenic strain of E. coli
(24% overall similarity; high similarity is found mainly in the middle
third of the sequence) (Fig. 5). The tia
invasion determinant is thought to be responsible for both epithelial
adherence and invasion of enteropathogenic and enteroaggregative
strains of E. coli and Shigella sonnei
(8). In addition, fragments of the wsp gene show
similarity with (i) the scrub typhus antigen (TSA, STA56) from
Orientia (Rickettsia) tsutsugamushi,
where the protein may function as an adherence factor potentiating
rickettsial adsorption to the host cell surface and as a virulence
determinant of individual rickettsial strains (21, 32), (ii)
MSP2 from A. marginale (6), and (iii) a highly
conserved outer membrane protein from Neisseria gonorrhoeae
(16). While many bacterial outer surface proteins (e.g.,
MSP2 from A. marginale [24]) are encoded by
multicopy gene families, Southern blot analyses indicate that
wsp, like its closest known relative, MSP4, is a single-copy gene.
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FIG. 5.
Alignment of the deduced amino acid sequence of WSP from
DSR with sequences of homologous outer surface proteins: MSP4 from
A. marginale (L01987), MAP1 from Cowdria
ruminantium (U50832), and the tia invasion (inv.) determinant from
E. coli (U20318). Only the second putative transmembrane
domain shown in Fig. 2 is shared by all homologs. Amino acids identical
or similar (3 distance units out of 22, using the PAM250 table of the
MegAlign software program, version 3.11 [DNASTAR Inc., Madison,
Wis.]) to those in the WSP sequence are in boldface.
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Alignment of the putative amino acid sequences of the wsp
gene from different strains of D. simulans, D. melanogaster, and the moth C. cautella show a high
similarity between these proteins. Nevertheless, there are small
regions that are highly variable. Two strains of D. melanogaster, DMCS and DMHarwich, and one strain of D. simulans, DSCoff, carry Wolbachia strains that are
incapable of inducing the phenotype of CI. These strains have been
extensively studied to exclude epiphenomena as the reason for the lack
of expression of CI (reference 10 and unpublished
data). Interestingly, in the three strains of Wolbachia we
investigated that were incapable of expressing the CI phenotype, all
shared similar sequences that were not conserved in the strains that
were capable of expressing the phenotype (Fig.
6). The conserved differences that we see in this alignment between expressing and nonexpressing strains might
reflect a functional difference related to the CI phenotype. The
protein composition in the outer membrane may reflect an adaptation and
specialization to the intimate interaction of this intracellular bacterium with the insect host cell. Alternatively, the sequence variability may reflect strain differences which correlate with the
phenotypic differences of these strains. In either case, the wsp gene is an excellent candidate for strain typing
different Wolbachia strains as well as providing characters
for a fine-scale phylogeny of Wolbachia strains
(37). At the present time, no other Wolbachia
gene which can be used to adequately resolve the evolutionary
relationships between different Wolbachia strains has been
cloned. In addition, the variability observed between wsp
sequences of different Wolbachia strains suggests that this gene should be able to be used to predict reproductive phenotypes generated by different strains (3) as well as be used as a marker to track multiple Wolbachia infections within
individual hosts. This, in turn, should greatly enhance our ability to
study the biology of this fastidious microorganism.
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FIG. 6.
The amino acid sequence alignment of a segment of WSP of
three Wolbachia strains capable of expressing CI (DSR, DSH,
and C. cautella) compared to four strains incapable of
expressing the phenotype (DSW/Mau, DS Coffs, DM Harwich, and DM
CantonS). Differences are in boldface.
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ACKNOWLEDGMENTS |
Weiguo Zhou and Henk R. Braig contributed equally to this report.
We gratefully acknowledge A. Hoffmann for supplying D. simulans Coffs Harbour S20, strain, R. Carde for supplying the
strain of almond moth C. cautella, and M. Turelli for the
suppressor of sable primers.
This work was supported by grants from the National Institutes of
Health (AI 34355, AI 07404, and AI40620), the McKnight Foundation, and
the UNDP/World Bank/WHO Special Programme for Research and Training in
Tropical Diseases.
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FOOTNOTES |
*
Corresponding author. Mailing address: Yale University,
60 College St., New Haven, CT 06520-8034. Phone: (203) 785 3285. Fax: (203) 785 4782. E-mail: scott.oneill{at}yale.edu.
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Abstract
Introduction
