The Wolbachia Genome of Brugia malayi:
Endosymbiont Evolution within a Human
Jeremy Foster1, Mehul Ganatra1, Ibrahim Kamal1¤a, Jennifer Ware1, Kira Makarova2, Natalia Ivanova3¤b,
Anamitra Bhattacharyya3, Vinayak Kapatral3, Sanjay Kumar1, Janos Posfai1, Tamas Vincze1, Jessica Ingram1,
Laurie Moran1, Alla Lapidus3¤b, Marina Omelchenko2, Nikos Kyrpides3¤b, Elodie Ghedin4, Shiliang Wang4,
Eugene Goltsman3¤b, Victor Joukov3, Olga Ostrovskaya3¤c, Kiryl Tsukerman3, Mikhail Mazur3, Donald Comb1,
Eugene Koonin2, Barton Slatko1*
1 Molecular Parasitology Division, New England Biolabs, Beverly, Massachusetts, United States of America, 2 National Center for Biotechnology Information, National Library
of Medicine, National Institutes of Health, Bethesda, Maryland, United States of America, 3 Integrated Genomics, Chicago, Illinois, United States of America, 4 Parasite
Genomics, Institute for Genomic Research, Rockville, Maryland, United States of America
Complete genome DNA sequence and analysis is presented for Wolbachia, the obligate alpha-proteobacterial
endosymbiont required for fertility and survival of the human filarial parasitic nematode Brugia malayi. Although,
quantitatively, the genome is even more degraded than those of closely related Rickettsia species, Wolbachia has
retained more intact metabolic pathways. The ability to provide riboflavin, flavin adenine dinucleotide, heme, and
nucleotides is likely to be Wolbachia’s principal contribution to the mutualistic relationship, whereas the host
nematode likely supplies amino acids required for Wolbachia growth. Genome comparison of the Wolbachia
endosymbiont of B. malayi (wBm) with the Wolbachia endosymbiont of Drosophila melanogaster (wMel) shows that
they share similar metabolic trends, although their genomes show a high degree of genome shuffling. In contrast to
wMel, wBm contains no prophage and has a reduced level of repeated DNA. Both Wolbachia have lost a considerable
number of membrane biogenesis genes that apparently make them unable to synthesize lipid A, the usual component
of proteobacterial membranes. However, differences in their peptidoglycan structures may reflect the mutualistic
lifestyle of wBm in contrast to the parasitic lifestyle of wMel. The smaller genome size of wBm, relative to wMel, may
reflect the loss of genes required for infecting host cells and avoiding host defense systems. Analysis of this first
sequenced endosymbiont genome from a filarial nematode provides insight into endosymbiont evolution and
additionally provides new potential targets for elimination of cutaneous and lymphatic human filarial disease.
Citation: Foster J, Ganatra M, Kamal I, Ware J, Makarova K, et al. (2005) The Wolbachia genome of Brugia malayi: Endosymbiont evolution within a human pathogenic
nematode. PLoS Biol 3(4): e121.
Over 1 billion people in more than 90 countries are at risk
from filarial nematode infections, and 150 million people are
infected. The parasitic nematodes are insect-borne and are
responsible for lymphatic or cutaneous filariasis, leading to
medical conditions including elephantiasis or onchocerciasis
(African river blindness). Lymphatic filariasis is caused
predominantly by Wuchereria bancrofti and Brugia malayi and
affects 120 million individuals, a third of whom show
disfigurement, while onchocerciasis, caused by Onchocerca
volvulus, affects 18 million people of whom 500,000 have
visual impairment and 270,000 are blind [1,2]. Within these
filarial parasites are intracellular bacteria that were first
observed almost 30 y ago [3,4,5,6].
The establishment in 1994 of a Filarial Genome Project
funded by the World Health Organization (WHO/Tropical
Disease Research/United Nations Development Programme/
World Bank) contributed to the rediscovery of these endo-
symbiotic bacteria. In the analysis of cDNA libraries
generated from different life cycle stages of B. malayi, the
presence of rare non-Escherichia-coli-like, alpha-proteobacte-
rial sequences implicated the occurrence of endobacterial
DNA . Phylogenetic analyses subsequently identified the
bacteria as Wolbachia . These endosymbionts have now been
found in the vast majority of filarial nematode species, with
Received November 23, 2004; Accepted February 2, 2005; Published March 29,
Copyright: ? 2005 Foster et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original work is properly cited.
Abbreviations: BAC, bacterial artificial chromosome; COGs, clusters of orthologous
groups of proteins; kbp, kilobasepairs; LPS, lipopolysaccharide; meso-DAP, meso-
diaminopimelate; ORF, open reading frame; TCA, tricarboxylic acid; TLR4, toll-like
receptor 4; wBm, Wolbachia endosymbiont of Brugia malayi; wMel, Wolbachia
endosymbiont of Drosophila melanogaster
Academic Editor: Nancy A. Moran, University of Arizona, United States of America
*To whom correspondence should be addressed. E-mail: email@example.com
¤a Current address: Biochemistry Department, Faculty of Science Ain Shams
University, Abassiah, Cairo, Egypt
¤b Current address: Joint Genome Institute, Walnut Creek, California, United States
¤c Current address: Department of Molecular Biology and Microbiology, Case
Western Reserve University School of Medicine, Cleveland, Ohio, United States of
PLoS Biology | www.plosbiology.orgApril 2005 | Volume 3 | Issue 4 | e1210599
Open access, freely available online P PL Lo oS S BIOLOGY
notable exceptions [3,9,10,11,12,13,14,15,16,17,18,19]. Wolba-
chia appear to be absent in nonfilarial nematodes .
In nematodes that contain Wolbachia and which have been
well examined, the bacteria are located in the lateral chords
(invaginations of the body wall hypodermis that project into
the body cavity) in both sexes. They are also localized in
oocytes but not in the male reproductive tract. The endo-
symbionts appear to be present in 100% of individuals within
a population, when that species contains them, suggesting
that they are required for worm fertility and survival
[10,21,22]. They are therefore potential therapeutic targets
for filariasis control.
Certain antialpha proteobacterial agents, most notably
tetracycline and doxycycline, but also rifampicin and
azithromycin, show inhibitory effects on parasitic nematode
development and fertility [13,23,24,25,26,27,28,29,30,31,32,33].
After antibiotic treatment, immunogold staining, using Wolba-
chia-specific cell-surface probes, shows the absence of Wolbachia
in the female reproductive tract and the degeneration of
embryos, while Wolbachia remain in the lateral chords, albeit in
reduced numbers . Genchi et al.  have also shown that
Wolbachia are present at 1000X lower frequencies after
antibiotic treatment and can still be detected by PCR from
female hypodermis tissues, but not from female reproductive
tissue. No antibiotic effects are observed in filarial nematodes
that do not harbor Wolbachia, nor are they observed with other
antibiotics (e.g., penicillin, gentamicin, ciprofloxacin, or
erythromycin), suggesting that these effects correlate with
Wolbachia presence [11,12,13,36,37]. Human trials using dox-
ycycline, undertaken in Ghana, have shown that this antibiotic
interferes with embryogenesis in adult female filariae with a
concomitant depletion of Wolbachia from both adults and
microfilariae (first stage larvae) of O. volvulus and W. bancrofti
[38,39,40,41,42]. Thus, as in animal models, Wolbachia appears
to be a therapeutic target for human filarial parasitic
The use of anti-Wolbachia chemotherapy against filarial
parasites has initiated a novel approach for filarial disease
control and eradication. Previous strategies for elimination
of filariasis have included vector control in the presence or
absence of antiparasitic drugs [43,44,45,46,47]. Diethylcarba-
mazine, albendazole, and ivermectin have been the most
recent drugs of choice for prevention of filarial infections,
but they have little effect on adult worms. Thus repeated
doses in endemic areas are required to eliminate infections
that can arise again within months of treatment [39,44,48]. In
addition, the possibility of drug resistance, as observed with
intestinal helminths in animals is a concern [49,50,51]. No
new therapeutics have been developed in over 20 y, and there
is a need for better drugs that permanently sterilize or kill
Wolbachia play a role in the host immunological response to
filarial parasite invasion. Infection by filarial parasites results
in B-cell proliferation and the generation of antibodies
directed toward parasite- and Wolbachia-specific antigens,
including those to Wolbachia surface protein, heat shock
protein, aspartate aminotransferase, and Htr serine protease
[11,52,53,54,55,56,57]. Other Wolbachia-specific molecules also
play roles in the immune response to filarial infections
including the release of stimulatory and modulatory factors
from neutrophils and monocytes, which may be related to
Wolbachia release upon worm death [58,59,60,61]. One
component of the host immune response appears to mimic
a lipopolysaccharide (LPS)-like response, typically observed
as a host immune response to Gram-negative bacteria (such as
the alpha-proteobacterial Wolbachia) [22,58,62,63,64,65]. Fur-
ther, LPS-like products of Wolbachia appear to be involved in
the eye inflammation observed in African river blindness.
Leukocytes (neutrophils and eosinophils) infiltrate the cornea
as a result of microfilarial invasion and death within the eye,
leading to a loss of corneal transparency . LPS-like
molecules are implicated in this process due to activation of
the toll-like receptor 4 (TLR4) pathway by Wolbachia [61,67].
Release of filarial worm-associated molecules, especially
after drug treatments that cause worm death in the host, leads
to pathogenesis (‘‘Mazzotti Reaction’’) [68,69,70,71,72], and
Wolbachia has been associated with chronic and acute
infection states of filariasis (reviewed in ). Repetitive
exposures to LPS-like molecules due to release of Wolbachia
following death of microfilaria are thought to induce chronic
inflammation events giving rise to immune tolerance [65,73],
as hyporesponsiveness occurs with increasing parasite load
Wolbachia endosymbionts can be separated into six super-
groups based upon 16S rRNA, Wolbachia surface protein, and
ftsZ phylogenetics [8,11,15,77,78,79,80,81,82]. Four super-
groups contain Wolbachia from arthropods while supergroup
C contains Wolbachia from the nematodes O. volvulus and
Dirofilaria immitis, and supergroup D contains Wolbachia from
B. malayi, W. bancrofti, and Litomosoides sigmodontis [11,82]. In
nematodes, the evolution of Wolbachia parallels the phyloge-
netics of their hosts, while in the other supergroups,
horizontal transmission appears to have occurred
[11,14,15,79,82]. The closest bacterial relatives to the Wolba-
chia are in the Order Rickettsiales, including Rickettsia,
Ehrlichia, Cowdria, and Anaplasma, all parasites of mammals
that require arthropod vectors for transmission [83,84].
Up to 70% of all insect species appear to harbor Wolbachia
[85,86,87]. While parasitic and maternally inherited in insects,
they appear not to be required for host survival. But when
present in appropriate genetic backgrounds, they confer
developmental effects leading to sex ratio disturbances,
feminization of genetic males, parthenogenesis, cytoplasmic
incompatibilities and/or reciprocal-cross sterility
[79,88,89,90]. It has been suggested that endosymbionts,
including Wolbachia, might be of medical importance and
used for insect vector control to deliver antiparasitic
products to recipient hosts [91,92,93,94,95,96,97,98,99,100,101,
102]. For these reasons, a genome project was initiated and
completed on the Wolbachia endosymbiont of Drosophila
melanogaster (wMel) .
Identification of Wolbachia in parasitic nematodes, their
role in pathogenesis, their potential as a target for develop-
ment of antifilarial therapeutics, and their widespread
occurrence in arthropods triggered a meeting held in 1999
to initiate a consortium of Wolbachia researchers [104,105].
Three additional meetings have been held (see http://
www.wolbachia.sols.uq.edu.au/index.html), and eight addi-
tional Wolbachia genomes responsible for diverse phenotypes
are being sequenced.
We report the second complete genome sequence of
Wolbachia and the first from a parasitic nematode, B. malayi
(W. pipientis, BruMal TRS strain; Wolbachia endosymbiont of B.
malayi [wBm]). We also describe a comparative analysis of
PLoS Biology | www.plosbiology.orgApril 2005 | Volume 3 | Issue 4 | e1210600
Genome Sequence and Evolution of Wolbachia
reductive evolution in different lineages of endosymbiotic
bacteria, a major evolutionary trend in all intracellular
parasites and symbionts. Features of the wBm genome are
presented as a systematic comparison to wMel and Rickettsia
spp., the closest fully sequenced relatives of wBm and more
distant intracellular parasites and symbionts of the gamma-
proteobacterial lineage, such as Buchnera (aphid endosym-
biont), Blochmannia (ant endosymbiont), and Wigglesworthia
(tsetse fly endosymbiont) [106,107,108,109,110,111,112]. We
also delineate the metabolic pathways that might account for
the mutualistic relationship between Wolbachia and its
Genome Properties and General Comparison with the
Genomes of Other Parasites and Endosymbionts
The genome of wBm is represented by a single circular
chromosome consisting of 1,080,084 nucleotides and is 34%
GþC. The size agrees with the 1.1 Mb length previously
determined by both pulsed-field gel electrophoresis and
restriction mapping [113,114]. The origin of replication (oriC)
was tentatively mapped immediately upstream of the hemE
gene on the basis of GC- and AT-skew analyses  (Figure
1). The genome of wBm has an extremely low density of
predicted functional genes compared to all other bacteria,
with the exceptions of R. prowazekii (Table 1) and Mycobacte-
rium leprae. Both Wolbachia spp. and Rickettsia spp. have
undergone considerable gene loss in many metabolic path-
ways, relative to other alpha-proteobacteria (Table 2). A
comparison of predicted functional genes in wBm and
Rickettsia spp. reveals a large core set that is conserved among
these genomes, as well as smaller sets unique to each genome
(Figure 2). In contrast, nearly all observed pseudogenes are
unique to each genome (Figure 2), suggesting substantial
independent genome degradation. Wolbachia (wBm) and R.
conorii contain, in addition to many demonstrable pseudo-
genes, a considerable number of short open reading frames
(ORFs), which have no detectable orthologs in current
protein databases but are recognized as probable genes by
gene prediction programs. However, most of these sequences,
which comprise approximately 5% of the total predicted
gene number in wBm, are likely to be fragmented genes as
well (Table 1).
The wBm genome contains one copy of each of the
ribosomal RNA genes (16S, 23S, and 5S), which do not form
an operon, as also observed in wMel and Rickettsia but in
contrast to most other bacteria, and 34 tRNA genes that
include cognates for all amino acids. Probable biological
function was assigned to 558 (approximately 70%) of the 806
protein coding genes; a more general prediction of bio-
chemical function was made for an additional 49 ORFs. Most
of the predicted genes (617, 76%) could be included in
clusters of orthologous groups of proteins (COGs) with
orthologs not only in wMel and Rickettsia but also in more
A lack of flagellar, fimbrial or pili genes indicates that wBm
is probably nonmotile (Table 2). However, some intracellular
pathogens, including spotted fever group Rickettsia, exploit a
different motility mechanism that makes use of the host cell
actin polymerization to promote bacterial locomotion. Actin-
based motility of Rickettsia depends upon activation of the
host Arp2/3 complex by the WASP family protein RickA
[116,117]. A gene coding for WASP family protein (Wbm0076)
was identified in wBm suggesting that it might be able to
employ actin polymerization for locomotion and cell-to-cell
Informational and Regulatory Systems
Comparison with an obligatory gene set characteristic for
free-living alpha-proteobacteria (Table 2) shows that both
Wolbachia spp. and Rickettsia have retained an almost intact
gene set for translational processes (greater than 84%).
Several RNA metabolism genes are among the few shared
losses, including tRNA and rRNA modification enzymes
(LasT, RsmC, Sun, TrmA, CspR) and even pseudouridine
synthase, TruB (pseudogenes in both lineages). TruB is
present in all gamma-proteobacterial endosymbionts but
absent in other parasites and endosymbionts, including
Mycoplasma, Chlamydia, and spirochetes. It is likely that the
lack of these modifications affects reading frame mainte-
nance and translation efficiency in both Wolbachia spp. and in
Rickettsia. Further reduction of genes involved in RNA
modification occurs specifically in wBm and in wMel, which
have lost several genes involved in queuosine biosynthesis
(COG0809, COG603, COG702, COG0602, COG0780) 
and 16S rRNA uridine-516 pseudouridylate synthase. The
absence of RNA methylase (COG1189) highlights the loss of
RNA modification systems, which is a general trend in
evolution of endosymbionts among various lineages .
Although wBm retains most of the genes for DNA
replication and repair, the loss of several genes present in
other alpha-proteobacteria (except wMel) is notable. These
include the chi subunit of DNA polymerase III (HolC),
chromosome partitioning proteins ParB and ParA, repair
ATPase (RecN), exonuclease VII (XseAB), and the RNA
processing enzyme RNase PH (Rph).
Both Wolbachia spp. and Rickettsia have a complete
repertoire of UV-excision (UVR-ABCD-mediated), recombi-
national synaptic (RecA/RecFOR-mediated), and postsynaptic
(RuvABC-mediated) DNA repair pathways. In contrast,
Buchnera and Blochmannia are devoid of conventional homol-
ogous recombination and uvr pathways, although they
encode a putative phrB family photolyase [107,109,110,112,
120,121]. Wolbachia, Rickettsia, Buchnera, and Wigglesworthia
all encode enzymatic machinery to counter the deleterious
effects of various types of base oxidative damage, which
could be important for defense against mutagenic meta-
bolic by-products in the intracellular environment
Many proteins categorized as being involved in protein fate
in the two Wolbachia spp. and Rickettsia spp. (CcmF, CcmB,
CcmH, CcmE, CcmC, Cox11, CtaA), but which are absent in
the genomes of gamma-proteobacterial endosymbionts, are
involved in biogenesis of cytochrome c oxidase and c-type
cytochromes typical of alpha-proteobacterial aerobic respi-
ratory chains. Respiratory chains of gamma-proteobacterial
endosymbionts employ quinol oxidase rather than cyto-
chrome c oxidase.
A major loss of transcriptional regulators likely occurred
in the common ancestor of Wolbachia and Rickettsia spp.
(Table 2). Only a few of these genes have been additionally
lost in the wBm lineage, including those from COG1396,
COG1959, COG1329, COG1678, and COG1475. This is a
PLoS Biology | www.plosbiology.orgApril 2005 | Volume 3 | Issue 4 | e1210601
Genome Sequence and Evolution of Wolbachia
general trend in evolution of endosymbionts and parasites
[118,122,123], suggesting that most of their genes are likely
constitutively expressed. Those few regulators found in wBm
that are not present in other alpha-proteobacteria, includ-
ing two Xre-like regulators (COG5606), may be of interest
for future experimental characterization. Similarly, most
genes implicated in signal transduction systems are absent
in both Wolbachia and Rickettsia spp. Several regulatory
proteins that remain in the genome are involved in various
stress responses (Wbm0660, MerR/SoxR family; Wbm0707,
cold shock protein; Wbm0494, stress response morphogen;
Wbm0061, TypA-like GTPase) or in cell cycle regulation
(Wbm0184, PleD-like regulator; Wbm0596, cell cycle tran-
scriptional regulator CtrA).
Metabolic Capabilities of wBm are Key to Understanding
its Interaction with the Host
One of the roles of wBm as an obligate endosymbiont may
be to provide its host with essential metabolites. Although
wBm has retained more metabolic genes than Rickettsia spp.,
its biosynthetic capabilities appear to be rather limited.
Unlike Buchnera spp. [107,109,112,122,123], wBm is able to
make only one amino acid—meso-diaminopimelate (meso-
DAP), a major peptidoglycan constituent. In most bacteria,
it is produced as an intermediate in the pathway of lysine
Figure 1. Genogram of the Complete Circular Genome of wBm
The scale indicates coordinates in kilobase pairs (kbp) with the putative origin of replication positioned at 0 kbp. The outermost ring indicates
the GC-skew over all bases in the forward strand using a window size of 40 kbp and a step size of 1 kbp. Positive and negative skew are shaded
gold and blue, respectively. Features are shown as paired rings separated by a circular baseline. In each pair, the outer and inner rings represent
the forward and reverse DNA strands, respectively. Working inward from the scale, the features displayed are as follows: identified genes and
their broad functional classification (multihued, as listed); tRNA (blue)/rRNA (red) genes; putative pseudogenes (green); repeated sequences (red)
and transposon-related repeats (blue).
PLoS Biology | www.plosbiology.org April 2005 | Volume 3 | Issue 4 | e1210602
Genome Sequence and Evolution of Wolbachia
biosynthesis. Similar to Rickettsia spp. , wBm lacks meso-
DAP decarboxylase (LysA, COG0019), necessary for lysine
biosynthesis, such that the biochemical pathway ends with
Complete pathways for de novo biosynthesis of purines and
pyrimidines are found in wBm, as opposed to Rickettsia and
many other endosymbionts and parasites, including Buchnera,
Blochmannia, Mycoplasma, and Chlamydia (Table 3). The general
trend for nucleotide biosynthesis pathways to be lost in these
organisms appears to be independent of the presence of ADP/
ATP translocase (COG3202) (present only in Rickettsia and
Chlamydia), which facilitates the uptake of nucleotide-triphos-
phates from the hosts. This observation suggests that wBm
produces nucleotides not only for internal consumption but
also for supplementation of the nucleotide pool of the host
(Figure 3) when needed, such as during oogenesis and
embryogenesis, where the requirement for DNA synthesis is
likely very high .
All genes required for biosynthesis of fatty acids and all but
one gene for biosynthesis of phospholipids (phosphatidylgly-
cerol, phosphatidylserine, and phosphatidylethanolamine)
are present in the wBm genome. The absent gene in
phospholipid biosynthesis is glycerol-3-phosphate acyltrans-
ferase (COG2937), which catalyzes the transfer of the first
fatty acid to glycerol-3-phosphate. However, a ‘‘fatty acid/
phospholipid biosynthesis enzyme’’ PlsX is present, which can
Table 1. Comparison of Genome Features of Proteobacterial Endosymbionts and Endoparasites
wBmwMel R. conoriiR. prowazekii B. aphidicola B. floridanus W. glossinidia
GþC content (%)
Predicted functional protein-coding genes
Gene density (functional genes/1 kb)
Pseudogenes and fragmented genes
Various repeats, including IS (% of genome)
% of coding DNA (intact proteins and RNA genes)
1,080,0841,267,782 1,268,7551,111,523640,681 705,557 697,724
Not in operonNot in operon Not in operon Not in operonIn operon In operon In operon
aIndependent estimates obtained during this work. wBm, Wolbachia from B. malayi; wMel, Wolbachia from Drosophila melanogaster; R. conorii, Rickettsia conorii; R. prowazekii, Rickettsia prowazekii; B. aphidicola, Buchnera aphidicola; B.
floridanus, Blochmannia floridanus; W. glossinidia, Wigglesworthia glossinidia.
IS, insection element sequence
Table 2. Gene Loss and Decay in Wolbachia and Rickettsia
Functional GroupwBmwMelR. conorii R. prowazekiiShared Losses
Translation, ribosomal structure and biogenesis
DNA replication, recombination and repair
Posttranslational modification, protein turnover, chaperones
Signal transduction mechanisms
Amino acid transport and metabolism
Energy production and conversion
Nucleotide transport and metabolism
Sugar transport and metabolism
Secondary metabolites biosynthesis, transport, and catabolism
Cell division and chromosome partitioning
Cell envelope biogenesis, outer membrane
Inorganic ion transport and metabolism
Intracellular trafficking and secretion
General function prediction
Gene conservation and loss were determined with respect to the set of 1,177 genes that are represented by confidently identifiable orthologs in all free-living alpha-proteobacteria. For each category, the first number indicates retained genes,
the second number indicates lost genes, and the third number indicates pseudogenes. The sum of these numbers equals the total number of genes in this category in the alpha-proteobacterial core set. wBm, Wolbachia from B. malayi; wMel,
Wolbachia from Drosophila melanogaster; R. conorii, Rickettsia conorii; R. prowazekii, Rickettsia prowazekii.
PLoS Biology | www.plosbiology.orgApril 2005 | Volume 3 | Issue 4 | e1210603
Genome Sequence and Evolution of Wolbachia
Table 3. Differential Loss of Functionality and Differentially Preserved Functionality, if Only a Few Compared Alpha- and Gamma-Proteobacterial Parasite/Symbiont Genomes Have
Lost or Preserved This Functionality
Examples of differential loss of functionality
HemEBH, BioF, UbiEAX
TopA, MutT, Exo, RuvC, RecA, RecJ
TopA, MutT, Exo
TufB, SUA5, TrmUD, Tgt
NADH: ubiquinone oxidoreductase 11 subunits
Glycolysis and PPP
TktA, Gap, Pgk,
Eno, Rpe, TpiA
TktA, GapA, Pgk, Eno,
Examples of differentially preserved functionality
Amino acids biosynthesis
MetCEFK, CysEH, PabA, HisABCDGFE, TrpECD,
PheA, LeuACD, ThrC, IlvCD, ArgCHFG
MetCEFK, CysEH, PabA, HisABCDGFE,
TrpECD, PheA, LeuACD, ThrC, IlvCD
NadE, BioABD, ThiL, PanCB
NadE, ThiLD, PanCB
Udp, Upp, NupC, Tdk
PurDELFMK, PyrC, CoaA,
RfaGLJ, WecB, WcaA,
wBm, Wolbachia from B. malayi; wMel, Wolbachia from Drosophila melanogaster; R. conorii, Rickettsia conorii; R. prowazekii, Rickettsia prowazekii; B. aphidicola, Buchnera aphidicola; B. floridanus, Blochmannia floridanus; W. glossinidia, Wigglesworthia glossinidia.
PLoS Biology | www.plosbiology.orgApril 2005 | Volume 3 | Issue 4 | e1210604
Genome Sequence and Evolution of Wolbachia
complement the absence of glycerol-3-phosphate acyltrans-
ferase in E. coli . All but one gene for biosynthesis of
isoprenoids has been found in the genome. This absent gene
is 1-deoxy-D-xylulose-5-phosphate synthase (COG1154), an
essential gene in the nonmevalonate pathway. It is possible
that this biochemical function could be complemented by a
transketolase or transaldolase, two highly promiscuous
enzymes encoded by the wBm genome or, alternatively, 1-
deoxy-D-xylulose-5-phosphate must be supplied by the host.
Unlike Rickettsia, wBm contains all the enzymes for the
biosynthesis of riboflavin and flavin adenine dinucleotide
(Figure 3). wBm could be an important source of these
essential coenzymes for the host nematode. No genes for
riboflavin biosynthesis have been detected in the ongoing B.
malayi genome data (9X coverage) . Similar to most other
endosymbionts, wBm lacks complete pathways for de novo
biosynthesis of other vitamins and cofactors such as
Coenzyme A, NAD, biotin, lipoic acid, ubiquinone, folate,
and pyridoxal phosphate, retaining only a few genes for the
finals steps in some of these pathways. These incomplete
pathways may make wBm dependent upon the supply of those
precursors from the host.
Heme serves as a prosthetic group of cytochromes, catalase
and peroxidase, and may be another metabolite provided by
wBm to B. malayi. wBm has all but one gene for heme
biosynthesis and has maintained all genes for maturation of
c-type cytochromes. The absent gene in the heme biosyn-
thesis pathway encodes protoporphyrinogen oxidase, a gene
not identified in many alpha-proteobacteria. It is likely that
these bacteria contain a functional form of protoporphyri-
nogen oxidase, which is not yet known, or that the missing
function is complemented by another gene function, as in E.
Heme could play an important role in filarial reproduction
and development. It is possible that molting and reproduc-
tion are regulated by ecdysteroid-like hormones, since the
insect hormones ecdysone and 20-hydroxyecdysone and their
inhibitors affect molting and microfilarial release in D. immitis
and B. pahangi [128,129]. In Drosophila, five enzymatic
reactions in the pathway of ecdysteroid biosynthesis are
catalyzed by microsomal and mitochondrial cytochrome P450
mono-oxygenases . If similar enzymes participate in the
pathway of biosynthesis of filarial steroid hormones, heme
depletion caused by elimination of wBm could result in a
decreased activity of these enzymes, which might account for
the effects on nematode viability, larval development, and
reproductive output observed following antibiotic treatment
of filarial parasites.
There is currently no evidence of heme biosynthesis
enzymes in B. malayi (analysis of the draft genome sequence
of B. malayi does not identify any genes for heme biosynthesis
). These enzymatic activities have been detected in Setaria
digitata, a cattle filarial parasite, which is devoid of typical
cytochrome systems, yet has heme-containing enzymes, such
as microsomal cytochrome P450, catalase, and peroxidase
. It is not known whether S. digitata contains Wolbachia
and whether heme biosynthesis detected in this worm is due
to the presence of endosymbiotic bacteria. However the
closely related filarial parasites, S. equina, S. tundra, and S.
labiatopapillosa are devoid of endosymbiotic Wolbachia [15,16];
perhaps they have retained the genes for heme biosynthesis.
Genes for biosynthesis of glutathione are present in the
wBm genome (Wbm0556; Wbm0721). Two physiological roles
of glutathione in bacteria are known: one is detoxification of
methylglyoxal , and the other is protection against
oxidative stress through activation of the glutathione
peroxidase–glutathione reductase system [133,134]. Methyl-
glyoxal is accumulated in phosphate-limited environments,
such as those encountered by Salmonella inside macrophages
. It is possible that wBm encounters phosphate-limited
conditions inside the host and therefore needs glutathione as
a quencher of methylglyoxal. This view is supported by the
presence of the gene encoding the Kef-type potassium efflux
system, a participant in methylglyoxal detoxification through
acidification of cytosol . However, no homologs of E. coli
gloA–gloB genes responsible for glutathione-dependent meth-
ylglyoxal detoxification were found in the genome. Gluta-
thione peroxidase is also absent, hence the physiological role
of glutathione in wBm is unclear. Although genes for
glutathione biosynthesis are present in the B. malayi genome,
it is possible that wBm provides glutathione to the host, since
the latter needs high levels of this essential metabolite for
protection against oxidative stress  and detoxification
Intermediates for these biosynthetic pathways are likely
derived from gluconeogenesis, the nonoxidative pentose
phosphate shunt, and the tricarboxylic acid (TCA) cycle.
Glycolytic enzymes encoded by wBm probably function in a
gluconeogenesis pathway (Figure 3), since the genes coding
for two enzymes catalyzing irreversible glycolytic reactions, 6-
phosphofructokinase and pyruvate kinase, are absent. In-
stead, the gluconeogenic enzyme fructose-1,6-bisphosphatase
(Wbm0132) and pyruvate-phosphate dikinase (Wbm0209),
Figure 2. Venn Diagram Showing Comparison of Conserved and Unique
Genes and Pseudogenes in wBm (Wolbachia from B. malayi), Rickettsia
prowazekii, Rickettsia conorii, and in wBm and wMel (among Those
Assigned to COGs)
(A) Predicted functional protein-coding genes.
(C) Combined results for comparison between wBm and wMel.
G, intact gene; P, pseudogene.
PLoS Biology | www.plosbiology.org April 2005 | Volume 3 | Issue 4 | e1210605
Genome Sequence and Evolution of Wolbachia
which functions predominantly in gluconeogenesis in bac-
teria, are present suggesting that the pathway functions as
gluconeogenesis, albeit ending with fructose-6-phosphate
rather than glucose-6-phosphate. While fructose-6-phosphate
is necessary for biosynthesis of the peptidoglycan compo-
nents N-acetylglucosamine and N-acetylmuramate, no en-
zymes capable of utilizing glucose-6-phosphate as a substrate
are encoded in the wBm genome.
It is reasonable to suggest that the most likely growth
substrates for wBm would be those compounds that are highly
abundant in the worm. In adult B. malayi, B. pahangi, and
Dipetalonema viteae (Acanthocheilonema viteae), these include the
excretory metabolites lactate and succinate, which are the
principal products of glucose utilization under both aerobic
and anaerobic conditions, and a disaccharide trehalose,
which is used by the worms as a storage compound
[137,138]. Nuclear magnetic resonance studies of adult B.
malayi identified phosphoenolpyruvate as the major energy
reservoir . However, wBm is not predicted to be able to
utilize lactate due to the absence of genes coding for lactate
dehydrogenases and is likely unable to grow on sugars, as
evidenced by the lack of genes encoding sugar transporters or
sugar kinases. Thus, the most likely growth substrates for
wBm are pyruvate and TCA cycle intermediates derived from
amino acids, with enzymes present for amino acid degrada-
tion, a pyruvate dehydrogenase complex, a complete TCA
cycle, and a respiratory chain typical of alpha-proteobacteria
(Figure 3). Amino acids are likely imported from the
extracellular environment where they are obtained by
proteolysis of host proteins by proteases and peptidases.
Indeed, the genome of wBm encodes a variety of proteases,
including predicted metallopeptidases (at least seven Zn-
dependent proteases of four distinct families compared to
only one in Rickettsia) (Wbm0055, Wbm0153, Wbm0221,
Wbm0311, Wbm0419, Wbm0418, Wbm0742). In addition,
two Naþ/alanine symporters were found (Wbm0197,
Wbm0424), which are absent in Rickettsia.
Cell Wall Structure
A dramatic case of lineage-specific gene loss in both
Wolbachia spp. includes approximately 20 genes for enzymes
of cell-envelope LPS biosynthesis. It has been reported that
Figure 3. Metabolic Pathways Retained in wBm
Pathways shared by Wolbachia and Rickettsia are shown with black arrows. Pathways present in Wolbachia but not in Rickettsia are shown with green
arrows. Numbering alongside pathway arrows reflects enzyme annotation, a table of which is available at http://tools.neb.com/wolbachia/.
PLoS Biology | www.plosbiology.org April 2005 | Volume 3 | Issue 4 | e1210606
Genome Sequence and Evolution of Wolbachia
soluble endotoxin-like products of Wolbachia endosymbionts
of filarial nematodes, including B. malayi, B. pahangi, L.
sigmodontis, O. volvulus, and D. immitis, contribute to the
immunology and pathogenesis of filarial diseases through
induction of potent inflammatory responses, including
production of tumor necrosis factor alpha, interleukin-1-
beta, and nitric oxide by macrophages [22,58,59,60,71,72,140,
141]. Chemokine and cytokine responses to the sterile
extracts of Brugia and Onchocerca were dependent on signaling
through TLR4 and could be blocked by neutralizing anti-
bodies to CD14 and by the antagonistic lipid A analogs,
indicating that the inflammatory response was induced by an
LPS-like molecule. Recently the major surface protein of
Wolbachia spp. was implicated as the inducer of the immune
response acting in a TLR2- and TLR4-dependent manner
. However, it is not clear whether this protein is the only
Wolbachia-specific molecule eliciting a TLR4-dependent in-
nate immune response.
Analysis of the wBm genome indicates that, like Ehrlichia
chaffeensis and Anaplasma phagocytophilum , it lacks homo-
logs of the genes responsible for biosynthesis of lipid A.
Although lipid A structure can vary in different bacteria, it
always consists of a polysaccharide backbone carrying fatty
acid residues. The only predicted genes belonging to the
glycosyltransferase family were those participating in pepti-
doglycan biosynthesis, and one glycosyltransferase pseudo-
gene is present. Similarly, the only genes from the
acyltransferase family are those participating in fatty acid
and phospholipid biosynthesis. Thus, it is unlikely that the
cell wall of wBm contains LPS-like molecules. This idea is
supported by the absence of the gene products responsible
for maintaining the outer membrane structure in Gram-
negative bacteria, such as TolQ, TolR, TolA, and TolB.
Several lines of evidence suggest that the structure of the
wBm peptidoglycan is very unusual, and peptidoglycan
derivatives might be responsible in part for the observed
inflammatory responses. First, although all the genes neces-
sary for biosynthesis of lipid II are present in the wBm
genome, there are no homologs of alanine and glutamate
racemases responsible for synthesis of pentapeptide compo-
nents D-alanine and D-glutamate. While the genomes of
Rickettsia spp. contain L-alanine racemase that could catalyze
racemization of both alanine and glutamate, the only amino
acid racemase present in the genomes of both Wolbachia is
meso-DAP epimerase (Wbm0518), an enzyme catalyzing
interconversions of LL- and meso-isomers of diaminopimelate.
It is possible that meso-DAP epimerase is able to catalyze
racemization of alanine and glutamate, although this activity
has never been experimentally demonstrated. Alternatively,
instead of the usual D-isomers, wBm peptidoglycan might
contain L-isomers of alanine and glutamate.
Second, Gram-negative bacteria (including Rickettsia spp.)
usually contain two monofunctional transpeptidases. One of
them, FtsI (also known as PBP3), is localized to the septal ring
and is required for peptidoglycan biosynthesis in the division
septum, while the other, PBP2, is localized preferentially to
the lateral cell wall . FtsI and PBP2 are recruited to the
sites of their action by two membrane proteins, FtsW and
RodA, respectively. In the wBm genome, only functional
orthologs of E. coli RodA and PBP2 were found; the orthologs
of FtsW–FtsI are disrupted by multiple frameshifts.
Third, genomes of bacteria that have peptidoglycan in their
cell wall usually contain at least one gene coding for a high
molecular weight penicillin-binding protein responsible for
cross-linking of the murein sacculus. The transpeptidase and
transglycosylase domains of this protein catalyze transpepti-
dation and transglycosylation of the murein precursors,
respectively, to form the carbohydrate backbone of murein
and the interstrand peptide linkages. No homologs of
bifunctional transpeptidase/transglycosylase or monofunc-
tional biosynthetic transglycosylase were found in the
genomes of Wolbachia spp., although they are present in the
Rickettsial genomes. The homolog of lytic transglycosylase,
which is responsible for hydrolysis of the carbohydrate
backbone during bacterial growth and division, is also absent
from the genomes of both Wolbachia spp. Thus, their
peptidoglycan can be cross-linked by the interstrand peptide
linkages, but the carbohydrate backbone is not polymerized.
These observations suggest that peptidoglycan of wBm has
some features in common with the peptidoglycan-derived
cytotoxin produced by Neisseria gonorrhoeae and Bordetella
pertussis [144,145] and that muramyl peptides derived from
wBm peptidoglycan could elicit the inflammatory response
contributing to the pathogenesis of filarial infection.
Other Host Interaction Systems
As expected, functional Type IV secretion genes were
found in the wBm genome, including two operons:
Wbm0793–Wbm0798 and Wbm0279–Wbm0283. These sys-
tems are indispensable for successful persistence of endo-
symbionts within their hosts . Similar genes have been
observed in the sequence of wMel .
A role in the adaptation to the intracellular existence
seems likely for several genes that are present in wBm, wMel,
and Rickettsia. Thus, wBm encodes five ankyrin-repeat-
Figure 4. Organization of Direct and Palindromic Repeats in wBm
Circles represent the complete genomic sequence of wBm. Repeats
were identified using the REPuter program  and are connected
by line segments. Direct repeats are shown in the graphs in the top
row, while palindromic repeats are shown in the lower row of graphs.
The left column graphs display repeats of 50 to 500 bp in length. The
rightmost graphs display repeats of greater than 500 bp in length.
PLoS Biology | www.plosbiology.org April 2005 | Volume 3 | Issue 4 | e121 0607
Genome Sequence and Evolution of Wolbachia
containing proteins and, in addition, has at least seven
related pseudogenes, while wMel contains 23 ankyrin -repeat-
containing genes. Rickettsia contains two or three functional
ankyrin-repeat genes (and probably one pseudogene) . In
eukaryotes, ankyrins connect cell membranes, including
membranes of endosymbionts to the cytoskeleton , while
in bacteria the function of ankyrin-like proteins remains
largely unknown. One physiological function of bacterial
ankyrin-like proteins was demonstrated in Pseudomonas
aeruginosa, where ankyrin repeat AnkB is essential for optimal
activity of periplasmic catalase, probably serving as a
protective scaffold in the periplasm . Another ankyrin-
repeat protein, AnkA from E. phagocytophila, was detected in
association with chromatin in infected cells, suggesting its
possible role in regulation of host cell gene expression .
Another interesting protein is a member of the WASP
family and is conserved in Rickettsia and wBm (Wbm0076).
Eukaryotic homologs of these proteins are suppressors of the
cAMP receptor and regulate the formation of actin filaments
. The genes for an ankyrin-repeat protein and a WASP
protein might have been acquired from a eukaryotic host by
the common ancestor of Rickettsia and Wolbachia and could
have contributed to the evolution of the intracellular lifestyle
of these bacteria. wBm also encodes several proteins with
large nonglobular or transmembrane regions or internal
repeats, orthologs of which are present also in the wMel
Figure 5. Absence of Gene Order Colinearity between wBm and Rickettsia and Disruption of Gene Colinearity between wBm and wMel
Each dot represents a pair of probable orthologs defined as reciprocal BLAST best hits with E-value less than 0.001.
(A) Genome dot-plot comparison of wBm (Wolbachia from B. malayi) and Rpro (R. prowazekii).
(B) Genome dot-plot comparison of wBm (Wolbachia from B. malayi) and Rcon (R. conorii).
(C) Genome dot-plot comparison of Rpro (R. prowazekii) and Rcon (R. conorii).
(D) Genome dot-plot comparison of wBm and wMel.
PLoS Biology | www.plosbiology.orgApril 2005 | Volume 3 | Issue 4 | e1210608
Genome Sequence and Evolution of Wolbachia
genome (Wbm0010, Wbm0304, Wbm0362, Wbm0749, and
others). These proteins are likely to be surface proteins
interacting with host cell structures.
Further Comparisons of wBm and wMel
One of the most striking characteristics of the wMel
genome is a large amount of repetitive DNA and mobile
genetic elements, including three prophages, altogether
comprising more that 14% of genomic DNA (and about 134
ORFs). Despite the abundance of repeats in the wBm genome
(5.4%) (Figure 4), the percentage of repetitive DNA in wBm is
considerably less than in wMel. This may reflect a stronger
selection in wBm for repeat loss and, as no prophages were
identified in the wBm genome, little exposure to foreign
DNA. No plasmid maintenance genes were identified in the
Comparison of the repetitive elements between these two
genomes suggests the invasion of mobile genetic elements
occurred after the divergence of the two Wolbachia along the
wMel branch, or that the majority of the transposons and
phages were eliminated (degraded) specifically in the wBm
lineage. There is a similarly large difference in the amount of
repetitive DNA in the two Rickettsia species (Table 1). While
an appropriate outgroup would be useful in both compar-
isons, the apparent degradation of repetitive DNA in Buchnera
spp. [111,112,152,153,154,155] suggests the specific elimina-
tion of nonessential DNA is a result of reduced selection on
gene functions no longer necessary in the host cells in
Wolbachia spp. . The large number of repeats and an
apparently active system of DNA recombination suggest that
extensive genome shuffling within wBm and wMel has
eliminated colinearity between their genomes (Figure 5).
Frequent rearrangements in Wolbachia might be expected,
given the exceptionally high levels of repeated DNA and
mobile elements and the presence of several prophages in
wMel. It has been suggested that the surprisingly high
percentage of repetitive DNA in wMel might reflect a lack
of selection for its elimination . An alternative hypoth-
esis might be that in Wolbachia there is a selective benefit to
systems that maintain genetic diversity and that a high
percentage of repeats may contribute to genome plasticity, as
has been suggested for Helicobacter . It has been suggested
that the presence of a high level of repetitive DNA in wMel,
relative to wBm, might reflect recurrent exposures to mobile
elements and bacteriophages, as a result of its parasitic
Comparative analysis of the genes assigned to COGs in
both wMel and wBm shows that the genome of wBm is more
reduced (Figure 2; Table 2). In total, 696 individual proteins
from wBm have an ortholog in the wMel genome; 84 such
proteins are not assigned to COGs, and a considerable
fraction of them are specific for only these two genomes. At
least half of these predicted genes are larger than 100 amino
acids, and orthologs have a similar length and presumably
encode functional proteins. One of the important differences
between the two Wolbachia for which genomes are available is
that wBm is apparently a mutualistic symbiont of its host,
while wMel is parasitic. The smaller size of the wBm genome
might be related to this difference. wMel likely has to retain
genes required for infecting host cells and avoiding host
defense systems, whereas wBm may have lost many of these
genes, as has been seen in organelles and other mutualistic
symbionts such as the Buchnera symbionts of aphids.
Despite there being considerably fewer predicted genes in
wBm (Table 1), the metabolic capabilities of wMel and wBm
are very similar. Unlike wBm, wMel has retained some
enzymes for folate and pyridoxal phosphate biosynthesis,
two subunits of cytochrome bd-type quinol oxidase, and a few
additional enzymes for amino acid utilization (proline
dehydrogenase and threonine aldolase). Among the genes
unique to wBm, there are two extracellular metallo-pepti-
dases (Wbm0384, Wbm0742) that are only distantly related to
counterparts in the wMel genome. These results suggest a
basic common strategy used by wBm and wMel during the
evolution of their host symbiosis. In the case of wBm, the basis
of the interaction may be to provide essential vitamin
cofactors, heme biosynthesis intermediates, and nucleotides
while requiring amino acids and perhaps other nutrients
supplied by the host.
Both Wolbachia have lost a considerable number of
membrane biogenesis genes that make them apparently
unable to synthesize lipid A, the usual component of
proteobacterial membranes. However, a few differences do
exist. For example, in wMel there is a predicted gene
belonging to the family of GDSL-like lipases (WD1297),
similar to the major secreted phospholipase of Legionella
pneumophila , which also has phospholipid-cholesterol
acyltransferase activity. Its ortholog in wBm is disrupted by a
frameshift (Wbm0354 corresponds to the C-terminal portion
of the gene). However, it is still possible that, similar to E.
chaffeensis and A. phagocytophilum , wBm and wMel
incorporate cholesterol into their cell walls. Furthermore,
wMel retains several genes absent in wBm that might be
involved in cell wall biosynthesis. These include a small gene
cluster (WD0611–WD0613) and several other enzymes
(WD0620, WD0133, WD0431), suggesting that wMel might
produce peptidoglycan modified with an oligosaccharide
chain, while wBm makes unmodified peptidoglycan. Possible
differences in peptidoglycan structure may be additionally
predicted by the already mentioned loss of FtsW–FtsI genes in
wBm and their presence in wMel. These differences may
reflect the occurrence of a mutualistic lifestyle (wBm) in
contrast to a parasitic lifestyle (wMel).
Somewhat surprisingly, no recent apparent horizontally
transferred genes from hosts were found in either Wolbachia
genome. Moreover, an aforementioned WASP protein ho-
molog, apparently acquired by a common ancestor of
Wolbachia and Rickettsia from an animal host, is disrupted in
the wMel genome (WD0811). However, in wMel there are two
proteins encoded in the region of the prophages (WD0443,
WD0633) that have ‘‘eukaryotic’’ OTU-like protease domains
with their predicted catalytic residues apparently intact .
Proteases from this family are shown to be involved in
ubiquitin pathways . To our knowledge, this is a rare
appearance of these proteases in prokaryotic genomes,
although they are present in the genomes of C. pneumoniae
 and in a closely related genome, Chlamydophila caviae
Comparing the genomes of wBm and Rickettsia to those of
gamma-proteobacterial symbionts points to general similar-
ities and distinctions in the evolution of endosymbionts. The
PLoS Biology | www.plosbiology.org April 2005 | Volume 3 | Issue 4 | e121 0609
Genome Sequence and Evolution of Wolbachia
genomes of R. conorii and Wolbachia species contain numerous
repeats of various classes that are much more abundant than
in the gamma-proteobacterial endosymbionts (Table 1). This
correlates with the minimal gene colinearity between the
genomes of Wolbachia and Rickettsia [103,114,163] (Figure 5).
By contrast, gamma-proteobacterial endosymbionts share a
variety of operons with one another, and even with free-living
relatives, despite the dramatic gene loss. Furthermore,
gamma-proteobacterial endosymbionts (with the exception
of Wigglesworthia) have lost crucial genes involved in recom-
binational repair, whereas almost no gene loss in this
functional class was observed in Wolbachia or Rickettsia spp.
Active recombination between repeats might have led to both
gene loss and genome shuffling in Wolbachia and Rickettsia
spp., whereas other mechanisms of genome reduction were
probably involved in the evolution of gamma-proteobacterial
Comparative genome analysis highlights the different
metabolic capabilities that render endosymbionts indispen-
sable to their hosts [108,119,121]. For example, Buchnera and
Blochmannia retain a nearly complete repertoire of amino acid
biosynthesis pathways and supply amino acids to their insect
hosts [110,112]. In contrast, wBm, wMel, and Wigglesworthia
[103,108] have lost nearly all of these pathways but retain the
pathways for the biosynthesis of nucleotides and some
coenzymes (Table 3). Thus, endosymbiotic organisms in
different divisions of proteobacteria independently evolved
distinct strategies for symbiont–host interactions.
Genomic analysis of the alpha-proteobacterium wBm, the
first sequenced endosymbiont from a human parasitic
nematode, provides new insights into the evolution of intra-
the mutualistic relationship with the nematode. It is antici-
pated that continued genome analysis of nematodes and their
endosymbionts will provide novel targets for antimicrobials
aimed at the elimination of human filarial parasites.
Materials and Methods
B. malayi microfilaria worms were purchased from TRS Labs
(Athens, GA, United States) for preparation of DNA. Because of the
difficulties in obtaining purified Wolbachia DNA from the B. malayi
host, bacterial artificial chromosome (BAC) libraries were created
. From these libraries, a minimum tiling path of 21 Wolbachia
BACs was created and used for subcloning into plasmid vectors for
genomic sequencing. This ordered BAC approach was useful in the
assembly phase of the project because of the highly repetitive nature
of this genome.
For plasmid library generation, equal amounts of BAC DNAs were
pooled and 50 lg of DNA from the pool was sheared into 2.0–3.0 kb
fragments (HydroShear device, GeneMachines, Genomic Solutions,
Ann Arbor, Michigan, United States). Sheared DNA was purified from
a 0.7% agarose gel, blunted, and cloned into cleaved, dephosphory-
lated plasmid vectors. Libraries were generated containing DNA from
1 to 9 BACs.
Plasmid DNA was isolated by a modified alkaline lysis protocol.
Sequencing reactions were performed at Integrated Genomics
(Chicago, Illinois, United States) using the DYEnamic ET Dye
Terminator Cycle Sequencing Kit (Amersham Biosciences, Little
Chalfont, United Kingdom). Unincorporated dye was removed by
isopropanol precipitation as recommended by the manufacturer.
Samples were run on MegaBace 1000 (Amersham Biosciences)
sequencers; 87% of plasmid sequencing reactions were successful.
The genome was sequenced to an average coverage of 10.7X and at
2X minimum coverage (at least once in each direction) and
The sequence was assembled into contigs by using PHRED–
PHRAP–CONSED [165,166,167], and gaps were initially closed by
primer walking (1,766 reactions). Regions considered to be potential
frame shifts or sequencing errors after the first round of annotation
were resequenced from direct genomic PCR products. The completed
sequence was used to identify homologous sequences in the
independent ongoing B. malayi sequence project (TIGR parasites
genome database: http://www.tigr.org/tdb/e2k1/bma1/ ). The
sequence of one BAC had been previously determined . The
final assembly was in full agreement with the BAC physical map .
Integrated Genomics ERGO software  and other software
programs  were used for ORF calling, gene identification, and
feature recognition. Computational analysis of the genome sequence
was performed as previously described. Briefly, the tRNA genes were
identified using the tRNA-SCAN program , and the rRNA genes
were identified using the BLASTN program . For the identi-
fication of the protein-coding genes, the genome sequence was
conceptually translated in six frames to generate potential protein
products of ORFs longer than 100 codons. These potential protein
sequences were compared to the database of proteins from the COG
database using COGNITOR .
After manual verification of the COG assignments, the validated
COG members from wBm were called as protein-coding genes. The
COG assignment procedure was repeated with ORFs of greater than
60 codons from the intergenic regions. Additionally, the potential
protein sequences were compared to the nonredundant protein
sequence database using the BLASTP program  and to a six-
frame translation of unfinished microbial genomes using the
TBLASTN program , and those sequences that produced hits
with E (expectation) values less than 0.01 were added to the protein
set after an examination of the alignments. Finally, protein-coding
regions were predicted using the GeneMarkS program . After
manual refinement, the genes predicted with these methods in the
regions between evolutionarily conserved genes were added to
produce the final protein set. Protein function prediction was based
primarily on the COG assignments. In addition, searches for
conserved domains were performed using the Conserved Domain
Database (CDD) search option of BLAST (http://www.ncbi.nlm.nih.
gov/Structure/cdd/wrpsb.cgi) and the SMART system , and in-
depth, iterative database searches were performed using the
PSI-BLAST program . The KEGG database  (http://
www.genome.ad.jp/kegg/metabolism.html) and the Integrated Ge-
nomics ERGO database pathway collection  were used, in
addition to the COGs, for the reconstruction of metabolic pathways.
Paralogous protein families were identified by single-linkage cluster-
ing after comparing the predicted protein set to itself using the
BLASTP program . Signal peptides in proteins were predicted
using the SignalP program , and transmembrane helices were
predicted using the MEMSAT program . Gene orders in bacterial
genomes were compared using the Lamarck program .
Two closely related genome sequences were completed and
published since the above comparative analysis was undertaken
Data Access DNA sequence, ORF, as well as annotation and positional
information tables, are available at the following Web site: http://
The genome sequence was deposited in GenBank (http://
www.ncbi.nlm.nih.gov/) under accession number AE017321.
We gratefully acknowledge Drs. L. McReynolds, L. Raleigh, and R.
Roberts for intellectual discussions and encouragement throughout
this project. We thank the members of the Filarial Genome Project
and Wolbachia Consortium communities for their discussions and
support, in particular Drs. S. O’Neill, J. Werren, M. Blaxter, M. Taylor,
A. Scott, S. Williams, and C. Bandi. We also thank Drs. J. Eisen, H.
Ochman, J. Wernegreen, S. Bordenstein, A. Osterman and R.
Overbeek for insightful comments. We gratefully acknowledge
helpful comments from three anonymous reviewers. Financial
support was provided by internal funding from New England Biolabs,
Inc. Dedicated to the memory of Mikhail Mazur.
Competing interests. The authors have declared that no competing
Author contributions. J. Foster, A. Lapidus, E. Ghedin, V. Joukov, K.
Tsukerman, and B. Slatko conceived and designed the experiments. J.
PLoS Biology | www.plosbiology.org April 2005 | Volume 3 | Issue 4 | e1210610
Genome Sequence and Evolution of Wolbachia
Foster, M. Ganatra, I. Kamal, J. Ware, A. Bhattacharyya, V. Kapatral, J.
Ingram, L. Moran, A. Lapidus, E. Goltsman, V. Joukov, O. Ostrov-
skaya, K. Tsukerman, M. Mazur, and B. Slatko performed the
experiments. J. Foster, M. Ganatra, I. Kamal, J. Ware, K. Makarova,
N. Ivanova, A. Bhattacharyya, V. Kapatral, S. Kumar, J. Posfai, T.
Vincze, A. Lapidus, M. Omelchenko, N. Kyrpides, E. Ghedin, E.
Goltsman, V. Joukov, K. Tsukerman, M. Mazur, E. Koonin, S. Wang,
and B. Slatko analyzed the data. J. Foster, M. Ganatra, I. Kamal, J.
Ware, K. Makarova, N. Ivanova, A. Bhattacharyya, S. Kumar, J. Posfai,
J. Ingram, A. Lapidus, N. Kyrpides, E. Ghedin, E. Goltsman, D. Comb,
E. Koonin, and B. Slatko contributed reagents/materials/analysis tools.
J. Foster, M. Ganatra, J. Ware, K. Makarova, N. Ivanova, S. Kumar, J.
Ingram, L. Moran, D. Comb, E. Koonin, and B. Slatko wrote the
1. World Health Organization (1995) Onchocerciasis and its control. World
Health Organ Tech Rep Ser 852: 1–103.
Molyneux DH, Bradley M, Hoerauf A, Kyelem D, Taylor MJ (2003) Mass
drug treatment for lymphatic filariasis and onchocerciasis. Trends
Parasitol 19: 516–522.
McLaren DJ, Worms MJ, Laurence BR, Simpson MG (1975) Micro-
organisms in filarial larvae (Nematoda). Trans R Soc Trop Med Hyg 69:
Vincent AL, Portaro JK, Ash LR (1975) A comparison of the body wall
ultrastructure of Brugia pahangi with that of Brugia malayi. J Parasitol 63:
Kozek WJ (1977) Transovarially-transmitted intracellular microorganisms
in adult and larval stages of Brugia malayi. J Parasitol 63: 992–1000.
Kozek WJ, Marroquin HF (1977) Intracytoplasmic bacteria in Onchocerca
volvulus. Am J Trop Med Hyg 26: 663–678.
Williams SA, Lizotte-Waniewski MR, Foster J, Guiliano D, Daub J, et al.
(2000) The filarial genome project: Analysis of the nuclear, mitochondrial
and endosymbiont genomes of Brugia malayi. Int J Parasitol 30: 411–419.
Sironi M, Bandi C, Sacchi L, Di Sacco B, Damiani G, et al. (1995)
Molecular evidence for a close relative of the arthropod endosymbiont
Wolbachia in a filarial worm. Mol Biochem Parasitol 74: 223–227.
Plenge-Bonig A, Kromer M, Buttner DW (1995) Light and electron
microscopy studies on Onchocerca jakutensis and O. flexuosa of red deer show
different host–parasite interactions. Parasitol Res 81: 66–73.
Bandi C, Anderson TJ, Genchi C, Blaxter ML (1998) Phylogeny of
Wolbachia in filarial nematodes. Proc R Soc Lond B Biol Sci 265: 2407–
Bandi C, Trees S, Brattig N (2001) Wolbachia in filarial nematodes:
Evolutionary aspects and implications for the pathogenesis and treat-
ment of filarial disease. Vet Parasitol 98: 215–238.
Taylor MJ, Hoerauf A (1999) Wolbachia bacteria of filarial nematodes.
Parasitol Today 15: 437–442.
Hoerauf A, Nissen-Pahle K, Schmetz C, Henkle-Duhrsen K, Blaxter ML, et
al. (1999) Tetracycline therapy targets intracellular bacteria in the filarial
nematode Litomosoides sigmodontis and results in filarial infertility. J Clin
Invest 103: 11–18.
Casiraghi M, Anderson TJ, Bandi C, Bazzocchi C, Genchi C (2001) A
phylogenetic analysis of filarial nematodes: Comparison with the
phylogeny of Wolbachia endosymbionts. Parasitology 122: 93–103.
Casiraghi M, Bain O, Guerrero R, Martin C, Pocacqua V, et al. (2004)
Mapping the presence of Wolbachia pipientis on the phylogeny of filarial
nematodes: Evidence for symbiont loss during evolution. Int J Parasitol
Chirgwin SR, Porthouse KH, Nowling JM, Klei TR (2002) The filarial
endosymbiont Wolbachia sp. is absent from Setaria equina. J Parasitol 88:
Egyed Z, Sreter T, Szell Z, Nyiro G, Marialigeti K, et al. (2002) Molecular
phylogenetic analysis of Onchocerca lupi and its Wolbachia endosymbiont.
Vet Parasitol 108: 153–161.
Buttner DW, Wanji S, Bazzocchi C, Bain O, Fischer P. (2003) Obligatory
symbiotic Wolbachia endobacteria are absent from Loa loa. Filaria J 2(1): 10
Available: http://www.filariajournal.com/content/2/1/10. Accessed 11 Feb-
McGarry HF, Pfarr K, Egerton G, Hoerauf A, Akue JP, et al. (2003)
Evidence against Wolbachia symbiosis in Loa loa. Filaria J 2(1): 9 Available:
http://www.filariajournal.com/content/2/1/9. Accessed 11 February 2005.
Bordenstein S, Fitch D, Werren J (2003) Absence of Wolbachia in
nonfilariid nematodes. J Nematology 33: 266–270.
Taylor MJ, Bandi C, Hoerauf AM, Lazdins J (2000) Wolbachia bacteria of
filarial nematodes: A target for control? Parasitol Today 16: 179–180.
Taylor MJ, Cross HF, Bilo K (2000) Inflammatory responses induced by
the filarial nematode Brugia malayi are mediated by lipopolysaccharide-
like activity from endosymbiotic Wolbachia bacteria. J Exp Med 191: 1429–
Bosshardt SC, McCall JW, Coleman SU, Jones KL, Petit TA, et al. (1993)
Prophylactic activity of tetracycline against Brugia pahangi infection in
jirds (Meriones unguiculatus). J Parasitol 79: 775–777.
Genchi C, Sacchi L, Bandi C, Venco L (1998) Preliminary results on the
effect of tetracycline on the embryogenesis and symbiotic bacteria
(Wolbachia) of Dirofilaria immitis. An update and discussion. Parassitologia
Bandi C, McCall JW, Genchi C, Corona S, Venco L, et al. (1999) Effects of
tetracycline on the filarial worms Brugia pahangi and Dirofilaria immitis and
their bacterial endosymbionts Wolbachia. Int J Parasitol 29: 357–364.
26. Hoerauf A, Volkmann L, Hamelmann C, Adjei O, Autenrieth IB, et al.
(2000) Endosymbiotic bacteria in worms as targets for a novel chemo-
therapy in filariasis. Lancet 355: 1242–1243.
Langworthy NG, Renz A, Mackenstedt U, Henkle-Duhrsen K, de
Bronsvoort MB, et al. (2000) Macrofilaricidal activity of tetracycline
against the filarial nematode Onchocerca ochengi: Elimination of Wolbachia
precedes worm death and suggests a dependent relationship. Proc R Soc
Lond B Biol Sci 267: 1063–1069.
Rao R, Weil GJ (2002) In vitro effects of antibiotics on Brugia malayi worm
survival and reproduction. J Parasitol 88: 605–611.
Rao RU, Moussa H, Weil GJ (2002) Brugia malayi: Effects of antibacterial
agents on larval viability and development in vitro. Exp Parasitol 101: 77–
Smith HL, Rajan TV (2000) Tetracycline inhibits development of the
infective-stage larvae of filarial nematodes in vitro. Exp Parasitol 95: 265–
Casiraghi M, McCall JW, Simoncini L, Kramer LH, Sacchi L, et al. (2002)
Tetracycline treatment and sex-ratio distortion: A role for Wolbachia in
the moulting of filarial nematodes? Int J Parasitol 32: 1457–1468.
Chirgwin SR, Nowling JM, Coleman SU, Klei TR (2003) Brugia pahangi and
Wolbachia: The kinetics of bacteria elimination, worm viability, and host
responses following tetracycline treatment. Exp Parasitol 103: 16–26.
Rajan TV (2004) Relationship of anti-microbial activity of tetracyclines to
their ability to block the L3 to L4 molt of the human filarial parasite
Brugia malayi. Am J Trop Med Hyg 71: 24–28.
Kramer H, Passeri B, Corona S, Simoncini L, Casiraghi M (2003)
Immunohistochemical/immunogold detection and distribution of the
endosymbiont Wolbachia of Dirofilaria immitis and Brugia pahangi using a
polyclonal antiserum raised against wsp (Wolbachia surface protein).
Parasitol Res 89: 381–386.
Genchi C, Simoncini L, McCall JW, Venco L, Sacchi L, et al. (2001) The
Wolbachia endosymbionts of Dirofilaria immitis: Implications for pathology,
immunology and control. In: Steward, RL, editor. Recent advances in
heartworm disease: Symposia 01. Davidson (Illinois): American Heart-
worm Society. pp. 27–33.
Taylor MJ, Hoerauf A (2001) A new approach to the treatment of
filariasis. Curr Opin Infect Dis 14: 727–731.
Hoerauf A, Volkmann L, Nissen-Paehle K, Schmetz C, Autenrieth I, et al.
(2000) Targeting of Wolbachia endobacteria in Litomosoides sigmodontis:
Comparison of tetracyclines with chloramphenicol, macrolides and
ciprofloxacin. Trop Med Int Health 5: 275–279.
Hoerauf A, Mand S, Adjei O, Fleischer B, Buttner DW (2001) Depletion of
Wolbachia endobacteria in Onchocerca volvulus by doxycycline and micro-
filaridermia after ivermectin treatment. Lancet 357: 1415–1416.
Hoerauf A, Buttner DW, Adjei O, Pearlman E (2003) Onchocerciasis. BMJ
Hoerauf A, Mand S, Fischer K, Kruppa T, Marfo-Debrekyei Y, et al. (2003)
Doxycycline as a novel strategy against bancroftian filariasis-depletion of
Wolbachia endosymbionts from Wuchereria bancrofti and stop of micro-
filaria production. Med Microbiol Immunol (Berl) 192: 211–216.
Hoerauf A, Mand S, Volkmann L, Buttner M, Marfo-Debrekyei Y, et al.
(2003) Doxycycline in the treatment of human onchocerciasis: Kinetics of
Wolbachia endobacteria reduction and of inhibition of embryogenesis in
female Onchocerca worms. Microbes Infect 5: 261–273.
Hoerauf A (2003) Control of filarial infections: Not the beginning of the
end, but more research is needed. Curr Opin Infect Dis 16: 403–410.
Hougard JM, Alley ES, Yameogo L, Dadzie KY, Boatin BA (2001)
Eliminating onchocerciasis after 14 years of vector control: A proved
strategy. J Infect Dis 184: 497–503.
Richards FF, Boatin BA, Sauerbrey M (2001) Control of onchocerciasis
today: Status and challenges. Trends Parasitol 17: 558–563.
Mackenzie CD, Malecela M, Mueller I, Homeida MA (2002) Approaches to
the control and elimination of the clinically important filarial diseases.
In: Klei TR, Rajan, TV, editors. The filaria. Boston: Kluwer Academic. pp.
Ottesen EA (2003) Towards eliminating lymphatic filariasis. In: Nutman
TB, editor. Lymphatic filariasis. London: Imperial College Press. pp. 201–
Dadzie Y, Neira M, Hopkins D (2003) Final report of the Conference on
the Eradicability of Onchocerciasis. Filarial J 2(1): 2. Available: http://
www.filariajournal.com/content/2/1/2. Accessed 11 February 2005.
Campbell WC (1991) Ivermectin as an antiparasitic agent for use in
humans. Annual Rev Microbiol 45: 445–474.
Grant W (2000) What is the real target for ivermectin resistance selection
in Onchocerca volvulus? Parasitol Today 16: 458–459; discussion, 501–452.
PLoS Biology | www.plosbiology.org April 2005 | Volume 3 | Issue 4 | e1210611
Genome Sequence and Evolution of Wolbachia
Prichard R (1994) Anthelmintic resistance. Vet Parasitol 54: 259–268.
Prichard R (2001) Genetic variability following selection of Haemonchus
contortus with anthelmintics. Trends Parasitol 17: 445–453.
Jolodar A, Fischer P, Butner DW, Brattig NW (2004) Wolbachia endo-
symbionts of Onchocerca volvulus express a putative periplasmic HtrA-type
serine protease. Microbes Infect 6: 141–149.
Bazzocchi C, Ceciliani F, McCall JW, Ricci I, Genchi C, et al. (2000)
Antigenic role of the endosymbionts of filarial nematodes: IgG response
against the Wolbachia surface protein in cats infected with Dirofilaria
immitis. Proc R Soc Lond B Biol Sci 267: 2511–2516.
Fischer P, Bonow I, Buttner DW, Kamal IH, Liebau E (2003) An aspartate
aminotransferase of Wolbachia endobacteria from Onchocerca volvulus is
recognized by IgG1 antibodies from residents of endemic areas. Parasitol
Res 90: 38–47.
Lamb TJ, Le Goff L, Kurniawan A, Guiliano DB, Fenn K, et al. (2004) Most
of the response elicited against Wolbachia surface protein in filarial
nematode infection is due to the infective larval stage. J Infect Dis 189:
Punkosdy GA, Dennis VA, Lasater BL, Tzertzinis G, Foster JM, et al.
(2001) Detection of serum IgG antibodies specific for Wolbachia surface
protein in rhesus monkeys infected with Brugia malayi. J Infect Dis 3: 385–
Chirgwin SR, Coleman SU, Porthouse KH, Nowling JM, Punkosdy GA, et
al. (2003) Removal of Wolbachia from Brugia pahangi is closely linked to
worm death and fecundity but does not result in altered lymphatic lesion
formation in Mongolian gerbils (Meriones unguiculatus). Infect Immun 71:
Cross H, Haarbrink M, Egerton G, Yazdanbakhsh M, Taylor M (2001)
Severe reactions to filarial chemotherapy are associated with the release
of Wolbachia endosymbionts into the blood. Lancet 358: 1873–1875.
Brattig NW (2004) Pathogenesis and host responses in human oncho-
cerciasis: Impact of Onchocerca filariae and Wolbachia endobacteria.
Microbes Infect 6: 113–128.
Hise AG, Gillette-Ferguson I, Pearlman E (2003) Immunopathogenesis of
Onchocerca volvulus keratitis (river blindness): A novel role for TLR4 and
endosymbiotic Wolbachia bacteria. J Endotoxin Res 9: 390–394.
Hise AG, Gillette-Ferguson I, Pearlman E (2004) The role of endosym-
biotic Wolbachia bacteria in filarial disease. Cell Microbiol 6: 97–104.
Haarbrink M, Terhell AJ, Abadi GK, Mitsui Y, Yazdanbakhsh M (1999)
Inflammatory cytokines following diethylcarbamazine (DEC) treatment
of different clinical groups in lymphatic filariasis. Trans R Soc Trop Med
Hyg 6: 665–672.
Haarbrink M, Abadi GK, Buurman WA, Dentener MA, Terhell AJ (2000)
Strong association of interleukin-6 and lipopolysaccharide-binding
protein with severity of adverse reactions after diethylcarbamazine
treatment of microfilaremic patients. J Infect Dis 182: 564–569.
Taylor MJ, Cross HF, Ford L, Makunde WH, Prasad GB, et al. (2001)
Wolbachia bacteria in filarial immunity and disease. Parasite Immunol 7:
Taylor MJ (2002) Wolbachia endosymbiotic bacteria of filarial nematodes.
A new insight into disease pathogenesis and control. Arch Med Res 33:
Hall LR, Pearlman E (1999) Pathogenesis of onchocercal keratitis (River
blindness). Clin Microbiol Rev 3: 445–453.
Saint Andre A, Blackwell NM, Hall LR, Hoerauf A, Brattig NW, et al.
(2002) The role of endosymbiotic Wolbachia bacteria in the pathogenesis
of river blindness. Science 295: 1892–1895.
Francis H, Awadzi K, Ottesen EA (1985) The Mazzotti reaction following
treatment of onchocerciasis with diethylcarbamazine: Clinical severity as
a function of infection intensity. Am J Trop Med Hyg 3: 529–536.
Ottesen EA (1992) The Wellcome Trust Lecture: Infection and disease in
lymphatic filariasis: An immunological perspective. Parasitology 104:
Pearlman E (1996) Experimental onchocercal keratitis. Parasitol Today
Freedman D (1998) Immune dynamics in the pathogenesis of human
lymphatic filariasis. Parasitol Today 14: 229–234.
Keiser PB, Reynolds SM, Awadzi K, Ottesen EA, Taylor MJ, et al. (2002)
Bacterial endosymbionts of Onchocerca volvulus in the pathogenesis of
posttreatment reactions. J Infect Dis 6: 805–811.
Taylor M (2002) Wolbachia bacterial endosymbionts. In: Klei TR, Rajan
TV, editors. The filaria. Boston: Kluwer Academic. pp. 143–153.
Ottesen EA (1995) Immune responsiveness and the pathogenesis of
human onchocerciasis. J Infect Dis 171: 659–671.
MacDonald AS, Maizels RM, Lawrence RA, Dransfield I, Allen JE (1998)
Requirement for in vivo production of IL-4, but not IL-10, in the
induction of proliferative suppression by filarial parasites. J Immunol
King C (2002) Immune regulation and the spectrum of filarial disease. In:
Klei TR, Rajan TV, editors. The filaria. Boston: Kluwer Academic. pp.
O’Neill SL, Giordano R, Colbert AM, Karr TL, Robertson HM (1992) 16S
rRNA phylogenetic analysis of the bacterial endosymbionts associated
with cytoplasmic incompatibility in insects. Proc Natl Acad Sci U S A 89:
78. Bandi C, Sironi M, Nalepa CA, Corona S, Sacchi L (1997) Phylogenetically
distant intracellular symbionts in termites. Parassitologia 39: 71–75.
Werren JH (1997) Biology of Wolbachia. Ann Rev Entomol 42: 587–609.
Vandekerckhove T, Watteyne S, Willems A, Swings JG, Mertens J, Gillis M
(1999) Phylogenetic analysis of the 16S rDNA of the cytoplasmic
bacterium Wolbachia from the novel host Folsomia candida (Hexapoda,
Collembola) and its implications for wolbachial taxonomy. FEMS Micro-
biol Lett 180: 279–286.
Bazzocchi C, Jamnongluk W, O’Neill S, Anderson TJC, Genchi C, et al.
(2000) wsp sequences from the Wolbachia of filarial nematodes. Curr
Microbiol 4: 96–100.
Lo N, Casiraghi M, Salati E, Bazzocchi C, Bandi C (2002) How many
Wolbachia supergroups exist? Mol Biol Evol 3: 341–346.
Roux V, Raoult D (1995) Phylogenetic analysis of the genus Rickettsia by
16S rDNA sequencing. Res Microbiol 146: 385–396.
Dumler JS, Barbet AF, Bekker CP, Dasch GA, Palmer GH, et al. (2001)
Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae
in the Order Rickettsiales: Unification of some species of Ehrlichia with
Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descrip-
tions of six new species combinations and designation of Ehrlichia equi
and ‘‘HGE agent’’ as subjective synonyms of Ehrlichia phagocytophila. Int J
Syst Evol Microbiol 51: 2145–2165.
Werren JH, Windsor DM, Gao L (1995) Distribution of Wolbachia among
neotropical arthropods. Proc R Soc Lond B Biol Sci 265: 1447–1452.
Jeyaprakash A, Hoy MA (2000) Long PCR improves Wolbachia DNA
amplification: wsp sequences found in 76% of sixty-three arthropod
species. Insect Mol Biol 9: 393–405.
Werren JH, Windsor DM (2004) Wolbachia infection frequencies in insects:
Evidence of a global equilibrium? Proc R Soc Lond B Biol Sci 267(1450):
Werren JH (1997) Wolbachia run amok. Proc Natl Acad Sci U S A 94:
Bourtzis K, Braig HR (1999) The many faces of Wolbachia. In: Raoult D,
Brouqui P, editors. Rickettsiae and Rickettsial diseases at the turn of the
third millennium. Paris: Elsevier. pp.199–219.
Bourtzis K, Braig H, Karr T (2003) Cytoplasmic incompatibility. In:
Bourtzis K, Miller T, editors. Insect symbiosis. New York: CRC Press. pp.
Laven H (1967) Eradication of Culex pipiens fatigans through cytoplasmic
incompatibility. Nature 216: 383–384.
Durvasula RV, Gumbs A, Panackal A, Kruglov O, Aksoy S, et al. (1997)
Prevention of insect-borne disease: An approach using transgenic
symbiotic bacteria. Proc Natl Acad Sci U S A 94: 3274–3278.
Hoffman A, Turelli M (1997) Cytoplasmic incompatibility in insects. In:
O’Neill S, Hoffman A, Werren J, editors. Influential passengers: Inherited
microorganisms and arthropod reproduction. Oxford: Oxford University
Press. pp. 42–80.
Sinkins SP, Curtis CF, O’Neill S (1997) Potential application of
endosymbiont systems to pest control. In: O’Neill S, Hoffman A, Werren
J, editors. Influential passengers: Inherited microorganisms and inverte-
brate reproduction. Oxford: Oxford University Press. pp. 271–287.
Curtis CF, Sinkins SP (1998) Wolbachia as a possible means of driving
genes into populations. Parasitology 116: 111–115.
Sinkins SP, O’Neill SL (2000) Wolbachia as a vehicle to modify insect
populations. In: Handler AM, James AA, editors. Insect transgenesis:
Methods and applications. Boca Raton (Florida): CRC Press. pp. 271–287.
Sinkins SP, Godfray HC (2004) Use of Wolbachia to drive nuclear
transgenes through insect populations. Proc R Soc Lond B Biol Sci 271:
Aultman KS, Beaty BJ, Walker ED (2001) Genetically manipulated vectors
of human disease: A practical overview. Trends Parasitol 17: 507–509.
Beard CB, Dotson EM, Pennington PM, Eichler S, Cordon-Rosales C, et al.
(2001) Bacterial symbiosis and paratransgenic control of vector-borne
Chagas disease. Int J Parasitol 31: 620–626.
100. Townson, H (2002) Wolbachia as a potential tool for suppressing filarial
transmission. Ann Trop Med Parasitol 96: S117–S127.
101. Dobson SL (2003) Reversing Wolbachia-based population replacement.
Trends Parasitol 19: 128–133.
102. Zabalou S, Riegler M, Theodorakopoulou M, Stauffer C, Savakis C,
Bourtzis K (2004) Wolbachia-induced cytoplasmic incompatibility as a
means for insect pest population control. Proc Natl Acad Sci U S A. 101:
103. Wu M, Sun LV, Vamathevan J, Riegler M, Deboy R, et al. (2004)
Phylogenomics of the reproductive parasite Wolbachia pipientis wMel: A
streamlined genome overrun by mobile genetic elements. PLoS Biol 2(3):
104. Bandi C, Slatko B, O’Neill SL (1999) Wolbachia genomes and the many
faces of symbiosis. Parasitol Today 15: 428–429.
105. Slatko BE, O’Neill SL, Scott AL, Werren JL, Blaxter ML (1999) The
Wolbachia Genome Consortium. Microb Comp Genomics 4: 161–165.
106. Moran NA, Wernegreen JJ (2000) Lifestyle evolution in symbiotic
bacteria: Insights from genomics. Trends Ecol Evol 15: 321–326.
107. Shigenobu S, Watanabe H, Hattori M, Sakaki Y, Ishikawa H (2000)
Genome sequence of the endocellular bacterial symbiont of aphids
Buchnera sp. APS. Nature 407: 81–86.
PLoS Biology | www.plosbiology.org April 2005 | Volume 3 | Issue 4 | e1210612
Genome Sequence and Evolution of Wolbachia
108. Akman L, Yamashita A, Watanabe H, Oshima K, Shiba T, et al. (2002) Download full-text
Genome sequence of the endocellular obligate symbiont of tsetse flies,
Wigglesworthia glossinidia. Nat Genet 32: 402–407.
109. Tamas I, Klasson L, Canback B, Naslund AK, Eriksson AS, et al. (2002) 50
million years of genomic stasis in endosymbiotic bacteria. Science 296:
110. Gil R, Silva FJ, Zientz E, Delmotte F, Gonzalez-Candelas F, et al. (2003)
The genome sequence of Blochmannia floridanus: Comparative analysis of
reduced genomes. Proc Natl Acad Sci U S A 16: 9388–9393.
111. Moran NA (2003) Tracing the evolution of gene loss in obligate bacterial
symbionts. Curr Opin Microbiol 6: 512–518.
112. van Ham RC, Kamerbeek J, Palacios C, Rausell C, Abascal F, et al. (2003)
Reductive genome evolution in Buchnera aphidicola. Proc Natl Acad Sci U
S A 100: 581–586.
113. Sun LV, Foster JM, Tzertzinis G, Ono M, Bandi C, et al. (2001)
Determination of Wolbachia genome size by pulsed-field gel electro-
phoresis. J Bacteriol 183: 2219–2225.
114. Foster JM, Kumar S, Ganatra MB, Kamal IH, Ware J, et al. (2004)
Construction of bacterial artificial chromosome libraries from the
parasitic nematode Brugia malayi and physical mapping of the genome
of its Wolbachia endosymbiont. Int J Parasitol 34: 733–746.
115. Grigoriev A (1998) Analyzing genomes with cumulative skew diagrams.
Nucleic Acids Res 26: 2286–2290.
116. Jeng RL, Goley ED, D’Alessio JA, Chaga OY, Svitkina TM, et al. (2004) A
Rickettsia WASP-like protein activates the Arp2/3 complex and mediates
actin-based motility. Cell Microbiol 6: 761–769.
117. Gouin E, Egile C, Dehoux P, Villiers V, Adams J, et al. (2004) The RickA
protein of Rickettsia conorii activates the Arp2/3 complex. Nature 427:
118. Reader JS, Metzgar D, Schimmel P, de Crecy-Lagard V (2004) Identi-
fication of four genes necessary for biosynthesis of the modified
nucleoside queuosine. J Biol Chem 279: 6280–6285.
119. Klasson L, Andersson SG (2004) Evolution of minimal gene-sets in host
dependent bacteria. Trends Microbiol 12: 237–243.
120. Tamas I, Andersson S (2003) Comparative genomics of insect endo-
symbionts. In: Bourtzis K, Miller T, editors. Insect symbiosis. New York:
CRC Press. pp. 39–52.
121. Wernegreen JJ, Lazarus AB, Degnan PH (2002) Small genome of
Candidatus Blochmannia, the bacterial endosymbiont of Camponotus, implies
irreversible specialization to an intracellular lifestyle. Microbiology 148:
122. Andersson SG, Zomorodipour A, Andersson JO, Sicheritz-Ponten T,
Alsmark UC, et al. (1998) The genome sequence of Rickettsia prowazekii
and the origin of mitochondria. Nature 396: 133–140.
123. Wernegreen JJ (2002) Genome evolution in bacterial endosymbionts of
insects. Nat Rev Genet 11: 850–861.
124. Kuhn O, Tobler H (1978) Quantitative analysis of RNA, glycogen and
nucleotides from different developmental stages of Ascaris lumbricoides
(var. suum). Biochim Biophys Acta 521: 251–66.
125. Larson TJ, Ludtke DN, Bell RM (1984) sn-Glycerol-3-phosphate auxo-
trophy of plsB strains of Escherichia coli: Evidence that a second mutation,
plsX, is required. J Bacteriol 160: 711–717.
126. Ghedin ES, Wang S, Foster JM, Slatko BE (2004) First sequenced genome
of a parasitic nematode. Trends Parasitol 20: 151–153.
127. Narita S, Taketani S, Inokuchi H (1999) Oxidation of protoporphyri-
nogen IX in Escherichia coli is mediated by the aerobic coproporphyri-
nogen oxidase. Mol Gen Genet 261: 1012–1020.
128. Barker GC, Mercer JG, Rees HH, Howells RE (1991) The effect of
ecdysteroids on the microfilarial production of Brugia pahangi and the
control of meiotic reinitiation in the oocytes of Dirofilaria immitis.
Parasitol Res 77: 65–71.
129. Warbrick EV, Barker GC, Rees HH, Howells RE (1993) The effect of
invertebrate hormones and potential hormone inhibitors on the third
larval moult of the filarial nematode, Dirofilaria immitis, in vitro.
Parasitology 107: 459–463.
130. Warren JT, Petryk A, Marquies G, Jarcho M, Parvy J-P, et al. (2002)
Molecular and biochemical characterization of two P450 enzymes in the
ecdysteroidogenic pathway of Drosophila melanogaster. Proc Natl Acad Sci
USA 99: 11043–11048.
131. Kumar RA, Raj RK (1998) Presence and formation of heme and
occurrence of certain heme proteins in the filarial parasite Setaria
digitata. Biochem Biophys Res Commun 253: 49–52.
132. Booth IR, Ferguson GP, Miller S, Li C, Gunasekera B, et al. (2003)
Bacterial production of methylglyoxal: A survival strategy or death by
misadventure? Biochem Soc Trans 31: 1406–1408.
133. Li Y, Hugenholtz J, Abee T, Molenaar D (2003) Glutathione protects
Lactococcus lactis against oxidative stress. Appl Environ Microbiol 69:
134. Brenot A, King KY, Janowiak B, Griffith O, Caparon MG (2004)
Contribution of glutathione peroxidase to the virulence of Streptococcus
pyogenes. Infect Immun 72: 408–413.
135. Selkirk ME, Smith VP, Thomas GR, Gounaris K (1998) Resistance of
filarial nematode parasites to oxidative stress. Int J Parasitol 28: 1315–
136. Rao UR, Salinas G, Mehta K, Klei TR (2000) Identification and
localization of glutathione S-transferase as a potential target enzyme in
Brugia species. Parasitol Res 86: 908–915.
137. Powell J W, Stables JN, Watt RA (1986) An investigation of the glucose
metabolism of Brugia pahangi and Dipetalonema viteae by nuclear magnetic
resonance spectroscopy. Mol Biochem Parasitol 18: 171–182.
138. Powell JW, Stables JN Watt RA (1986) An NMR study on the effect of
glucose availability on carbohydrate metabolism in Dipetalonema viteae and
Brugia pahangi. Mol Biochem Parasitol 19: 265–271.
139. Shukla-Dave A, Degaonkar M, Roy R, Murthy PK, Murthy PS, et al. (1999)
Metabolite mapping of human filarial parasite, Brugia malayi, with nuclear
magnetic resonance. Magn Reson Imaging 17: 1503–1509.
140. Brattig NW, Rathjens U, Ernst M, Geisinger F, Renz A, et al. (2000)
Lipopolysaccharide-like molecules derived from Wolbachia endobacteria
of the filaria Onchocerca volvulus are candidate mediators in the sequence
of inflammatory and antiinflammatory responses of human monocytes.
Microbes Infect 2: 1147–1157.
141. Brattig NW, Bazzocchi C, Kirschning CJ, Reiling N, Buttner DW, et al.
(2004) The major surface protein of Wolbachia endosymbionts in filarial
nematodes elicits immune responses through TLR2 and TLR4. J Immunol
142. Lin M, Rikihisa Y (2003) Ehrlichia chaffeensis and Anaplasma phagocytophilum
lack genes for lipid A biosynthesis and incorporate cholesterol for their
survival. Infect Immun 71: 5324–5331.
143. den Blaauwen T, Aarsman ME, Vischer NO, Nanninga N (2003)
Penicillin-binding protein PBP2 of Escherichia coli localizes preferentially
in the lateral wall and at mid-cell in comparison with the old cell pole.
Mol Microbiol 47: 539–547.
144. Cloud KA, Dillard JP (2002) A lytic transglycosylase of Neisseria gonorrhoeae
is involved in peptidoglycan-derived cytotoxin production. Infect Immun
145. Locht C (1999) Molecular aspects of Bordetella pertussis pathogenesis. Int
Microbiol 3: 137–144.
146. Sexton JA, Vogel JP (2002) Type IVB secretion by intracellular pathogens.
Traffic 3: 178–185.
147. Amiri H, Davids W, Andersson SG (2003) Birth and death of orphan
genes in Rickettsia. Mol Biol Evol 20: 1575–1587.
148. Rubtsov AM, Lopina OD (2000) Ankyrins. FEBS Lett 482: 1–5.
149. Howell ML, Alsabbagh E, Ma JF, Ochsner UA, Klotz MG, et al. (2000)
AnkB, a periplasmic ankyrin-like protein in Pseudomonas aeruginosa, is
required for optimal catalase B (KatB) activity and resistance to hydrogen
peroxide. J Bacteriol 182: 4545–4556.
150. Caturegli P, Asanovich KM, Walls JJ, Bakken JS, Madigan JE, et al. (2000)
ankA: An Ehrlichia phagocytophila group gene encoding a cytoplasmic
protein antigen with ankyrin repeats. Infect Immun 68: 5277–5283.
151. Suetsugu S, Tezuka T, Morimura T, Hattori M, Mikoshiba K, et al. (2004)
Regulation of actin cytoskeleton by mDab1 through N-WASP and
ubiquitination of mDab1. Biochem J 384: 1–8.
152. Mira A, Ochman H, Moran NA. (2001) Deletional bias and the evolution
of bacterial genomes. Trends Genet 17: 589–596.
153. Moran NA, Mira A (2001) The process of genome shrinkage in the
obligate symbiont Buchnera aphidicola. Genome Biol 2: research0054.1–
0054.12. Available: http://genomebiology.com/2001/2/12/RESEARCH/0054.
Accessed 11 February, 2005.
154. Moran NA (2002) Microbial minimalism: Genome reduction in bacterial
pathogens. Cell 108: 583–586.
155. Daubin V, Moran NA, Ochman H (2003) Phylogenetics and the cohesion
of bacterial genomes. Science 301: 829–832.
156. Moran N, Plague G (2004) Genomic changes following host restriction in
bacteria. Curr Opin Genet Dev 14: 627–633.
157. Aras RA, Kang J, Tschumi AI, Harasaki Y, Blaser MJ (2003) Extensive
repetitive DNA facilitates prokaryotic genome plasticity. Proc Natl Acad
Sci U S A 100: 13579–13584.
158. Bordenstein S, Wernegreen J (2004) Bacteriophage flux in endosymbionts
(Wolbachia): Infection frequency, lateral transfer, and recombination
rates. Mol Biol Evol. 21: 1981–1991.
159. Flieger A, Neumeister B, Cianciotto NP (2002) Characterization of the
gene encoding the major secreted lysophospholipase A of Legionella
pneumophila and its role in detoxification of lysophosphatidylcholine.
Infect Immun 70: 6094–6106.
160. Lin M, Rikihisa Y (2003) Obligatory intracellular parasitism by Ehrlichia
chaffeensis and Anaplasma phagocytophilum involves caveolae and glycosyl-
phosphatidylinositol-anchored proteins. Cell Microbiol 5: 809–812.
161. Makarova KS, Aravind L, Koonin EV (2000) A novel superfamily of
predicted cysteine proteases from eukaryotes, viruses and Chlamydia
pneumoniae. Trends Biochem Sci 25: 50–52.
162. Balakirev MY, Tcherniuk SO, Jaquinod M, Chroboczek J (2003) Otubains:
A new family of cysteine proteases in the ubiquitin pathway. EMBO Rep
163. Ware J, Moran L, Foster J, Posfai J, Vincze T, et al. (2002) Sequencing and
analysis of a 63 kb bacterial artificial chromosome insert from the
Wolbachia endosymbiont of the human filarial parasite Brugia malayi. Int J
Parasitol 32: 159–166.
164. Andersson SG, Kurland CG (1998) Reductive evolution of resident
genomes. Trends Microbiol 6: 263–268.
PLoS Biology | www.plosbiology.org April 2005 | Volume 3 | Issue 4 | e121 0613
Genome Sequence and Evolution of Wolbachia