Laboratory of Parasitic Diseases, National
Institute of Allergy and Infectious Diseases,1
and National Center for Biotechnology Information, National
Library of Medicine,2 National Institutes of
Health, Bethesda, Maryland 20894
Received 2 August 1999/Returned for modification 15 October
1999/Accepted 29 November 1999
A comparative analysis of the predicted protein sequences encoded
in the complete genomes of Borrelia burgdorferi and
Treponema pallidum provides a number of insights into
evolutionary trends and adaptive strategies of the two spirochetes. A
measure of orthologous relationships between gene sets, termed the
orthology coefficient (OC), was developed. The overall OC value for the
gene sets of the two spirochetes is about 0.43, which means that less
than one-half of the genes show readily detectable orthologous
relationships. This emphasizes significant divergence between the two
spirochetes, apparently driven by different biological niches.
Different functional categories of proteins as well as different
protein families show a broad distribution of OC values, from near 1 (a
perfect, one-to-one correspondence) to near 0. The proteins involved in
core biological functions, such as genome replication and expression,
typically show high OC values. In contrast, marked variability is seen
among proteins that are involved in specific processes, such as
nutrient transport, metabolism, gene-specific transcription regulation, signal transduction, and host response. Differences in the gene complements encoded in the two spirochete genomes suggest active adaptive evolution for their distinct niches. Comparative analysis of
the spirochete genomes produced evidence of gene exchanges with other
bacteria, archaea, and eukaryotic hosts that seem to have occurred at
different points in the evolution of the spirochetes. Examples are
presented of the use of sequence profile analysis to predict proteins
that are likely to play a role in pathogenesis, including secreted
proteins that contain specific protein-protein interaction domains,
such as von Willebrand A, YWTD, TPR, and PR1, some of which hitherto
have been reported only in eukaryotes. We tentatively reconstruct the
likely evolutionary process that has led to the divergence of the two
spirochete lineages; this reconstruction seems to point to an ancestral
state resembling the symbiotic spirochetes found in insect guts.
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INTRODUCTION |
Comparative analysis of the protein
products encoded in complete genomes provides a powerful tool for
evaluating evolutionary trends and adaptive strategies in pathogenic
microbes. Identification of metabolic and signaling pathways that are
unique to pathogens offers novel targets for therapeutic intervention.
Also, such unique determinants may prove useful in developing better
diagnostic and prognostic indicators of infectious diseases in humans.
Conclusions from such an analysis enhance understanding of the
host-parasite interactions that enable pathogens to carve out unique
ecological niches in nature.
Borrelia burgdorferi is the causative agent of Lyme disease,
whereas the related spirochete Treponema pallidum causes
syphilis. Both organisms are fastidious in their growth requirements,
have small genomes, and manifest clinically as distinct, chronic,
disseminated diseases. The clinical similarities (68)
include progression of disease in stages following local inoculation
either through a tick in Lyme disease or through sexual contact in
syphilis. Involvement of the central nervous system is seen in both
infections, with sequalae ranging from neurologic deficits to
neuropsychiatric abnormalities (19). Chronic, recurrent
joint involvement is classically associated with Lyme disease
(64). The protean clinical manifestations of these pathogens
and their propensity to cause chronic infection in the human host
contribute to the ongoing diagnostic and therapeutic challenges offered
by these spirochetes (22, 42).
The recent determinations of the complete genome sequences of B. burgdorferi (30) and T. pallidum
(31) have made a comprehensive computer-based comparison of
the two spirochete genomes a feasible exercise. Recent comparative
genomic studies include comparisons between two closely related
mycoplasma species (36) and between bacteria that belong to
related genera but encode vastly different numbers of proteins, namely,
Escherichia coli and Haemophilus influenzae
(72). The complete genomes of T. pallidum and
B. burgdorferi give us the first chance to compare two
moderately related pathogenic bacteria with approximately equivalent
coding capacities. In order to systematically compare the protein sets encoded in the two spirochete genomes, we devised a measure for evaluating evolutionary relationships between families and functional classes of proteins: the orthology coefficient. With regard to the two
spirochetes, it seemed likely that differences observed across families
and functional groups of proteins could be indicative of the distinct
evolutionary pressures of the host environment.
Completely automated computational analysis of genome sequences is
often plagued by errors arising from compositional bias in protein
sequences, difficulties in automatic assessment of the domain
organization of proteins, and the lack of in-depth predictive power
(16). Therefore, in comparative analysis of the two
spirochete genomes we used a semiautomated strategy that is based on
recently developed, powerful tools for sequence analysis, particularly
position-specific iterating BLAST (PSI-BLAST) (2, 3), and
also involves case-by-case examination of protein families and
individual protein sequences. Optimization of the functional annotation
of proteins accompanied by reconstruction of metabolic and signaling
pathways in the two spirochetes helped uncover a number of unique
features that likely play a role in host response-related functions. In
this report, we compare and contrast the gene complements of the two
spirochetes and provide examples of shared and unique genes and
functional systems.
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MATERIALS AND METHODS |
Databases.
The complete protein sets of B. burgdorferi (30) and T. pallidum
(31) were obtained from the genome division of the Entrez retrieval system at
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Genome. All
database searches were carried out against the nonredundant (NR)
sequence database maintained at the National Center for Biotechnology Information (National Institutes of Health, Bethesda, Md.).
Sequence analysis: detection and interpretation of varying levels
of sequence similarity.
Large-scale sequence analysis was handled
using the SEALS program package (76). The first step of the
analysis involved a search of the NR database of protein sequences at
the National Center for Biotechnology Information using the gapped
BLAST program (3), with the complete protein sets of
B. burgdorferi and T. pallidum as queries.
Compositionally biased regions in query sequences that tend to produce
spurious hits in database searches were masked using the SEG program
(81). Two different sets of parameters for SEG were used,
namely, mild masking (window length, 12; trigger complexity, 2.2;
extension complexity, 2.5) for routine searches and stringent masking
(window length, 45; trigger complexity, 3.4; extension complexity,
3.75) for delineating particular globular domains (81).
In-depth database searches were performed using the PSI-BLAST program
(3) whenever specific annotation required detection of
subtle relationships. Briefly, PSI-BLAST uses multiple alignments of
sequences that show expectation values, or e-values, lower than a
specified cutoff (in this case 0.01) in the first-pass gapped BLAST
search to construct a position-specific scoring matrix (PSSM). The PSSM
is then used as the input for further iterations of the database
searches, which results in increased detection of subtle sequence
similarities (2, 8). Statistical evaluation of the PSI-BLAST
result is based on the empirical version of Karlin-Altschul statistics,
modified for gapped alignments (3). The e-value indicates
the number of sequences which show a certain level of similarity by
chance alone for a database of the size equivalent to NR. The e-value
reported on the first instance that PSI-BLAST detects the given
sequence above the cutoff is a reliable indicator of statistical
significance. Under the condition that properly filtered globular
domains are used as queries, an e-value of less than 0.01 is generally
suggestive of homology (8).
Structural features of proteins were analyzed using the FAMASK program
of the SEALS package (76), which incorporates several prediction methods. Signal peptides were predicted using the SIGNALP program (49), transmembrane helices were predicted using the PHD topology program (57) or the TopPred II program
(20), and coiled-coil regions were predicted using the
COILS2 program with a window length of 21 (43).
The phyletic distribution of homologous proteins detected by gapped
BLAST searches was evaluated using the TAX_COLLECTOR program (76) of SEALS. The hits with an e-value below 0.001 between each protein encoded in the two spirochete genomes and the NR databases
were analyzed, and the best hits in the second spirochete as well as
those in other bacteria, archaea, and eukarya, with their respective
e-values, were tallied. This provided the starting point for
identifying orthologous genes in the two genomes as well as a
preliminary indication of the phyletic distribution of homologs for
most of the proteins encoded in each genome. An attempt was made to
delineate true orthologous relationships on the basis of symmetric best
hits between the proteins from the two spirochetes as well as between
the spirochete proteins and those from other bacteria, archaea, and
eukaryotes. In problematic cases, phylogenetic tree analysis using
multiple sequence alignments of conserved domains was carried out in
order to evaluate the relationships. Phylogenetic trees were
constructed using the PAUP3 or CLUSTALW programs (35) with
the neighbor-joining and least squares (Fitch-Margoliash) methods,
accompanied by bootstrap analysis (27).
A complementary approach was employed for case-by-case analysis of
those spirochete proteins that failed to show statistically significant
matches when used as queries for PSI-BLAST. These sequences were
screened for the presence of known conserved domains using the
previously developed libraries of PSSMs (18, 53) as well as
newly developed PSSMs. Domain-family PSSMs were created by running
PSI-BLAST against the NR database with the appropriate query and an
e-value cutoff of 0.01 and saving the profile using the "
C"
option. The resulting PSSM was rerun against the protein sequence sets
of T. pallidum and B. burgdorferi using the R
option of PSI-BLAST, and hits with significant scores were detected. To
test the robustness of this procedure, we prepared databases using
randomized protein sequences from each of the spirochete proteomes and
conducted similar searches with each of the PSSMs on them. No hits with
significant e-values (<0.01) were detected in these searches,
supporting the validity of the domains detected in the spirochete
proteomes with the PSSMs. Additional database searches were performed
with family-specific Hidden Markov models that were constructed and run
using the HMMER2 program package (66). Multiple alignments
of protein families were constructed using a combination of the m4
option of PSI-BLAST and the CLUSTALW program (35).
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RESULTS AND DISCUSSION |
Orthology coefficient: functional classes and protein
families.
The proteins encoded by the genomes can be broadly
categorized on the basis of either (predicted) cellular function
(functional class) or shared conserved domains (protein families).
Orthologs are genes (proteins) in different genomes that are related by vertical descent; i.e., they can be traced back to a common ancestor. Such orthologous proteins typically have similar, if not identical, biological functions in the respective organisms (71).
Paralogs, in contrast, are genes related by duplication within a
lineage (genome); paralogs are expected to possess generally similar
biochemical functions but distinct biological roles. Given these
differences in functional implications, careful delineation of
orthologous and paralogous relationships between genes is of primary
importance for predicting functions of proteins with an appropriate
degree of precision. We adopted a quantitative measure of orthology
that we termed the orthology coefficient (OC). This index can be
applied to different protein categories, namely, superfamilies related by descent as opposed to functional classes of proteins, such as those
involved in transcription or replication. In the simplest case of a
one-to-one correspondence between genes in two genomes, OC = 2No/(N1 + N2) where No is the
number of orthologs and N1 and
N2 are the numbers of members of the given
protein family or functional category in the two compared genomes (Fig.
1). If the gene complements in the two
genomes are completely conserved, then the OC is 1.00; by contrast, in
the hypothetical case where there are no orthologs, the OC is 0. If
there is one or more duplications in one or both of the species that
occurred after their divergence, then OC = (No1 + No2)/(N1 + N2) where No1 and
No2 are the numbers of members in
orthologous clusters from the two respective genomes (Fig. 1).
Criteria used to identify orthologs included symmetry of retrieval
in reciprocal BLAST searches, conservation of the domain organization
of proteins, and, in problematic cases, phylogenetic tree analysis
(71, 72).
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FIG. 1.
OC as a quantitative measure of orthology between
different types of protein categories. (A) In the case of a one-to-one
correspondence between genes in two genomes, OC = 2No/(N1 + N2) where No is the
number of orthologs and N1 and
N2 are the numbers of members of the given
protein family or functional category in the two compared genomes. (B)
If there is a duplication (two or more members) in one or both of the
species that occurred after divergence of the two species, then OC = (No1 + No2)/(N1 + N2) where No1 and
No2 are the numbers of members in orthologous
clusters from the two respective genomes.
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Conservation and diversity of functional classes of proteins
between the two spirochetes.
Functional classes of proteins include
core functions, such as replication and cell division, repair,
transcription, and translation, that are shared by all bacteria and to
a large extent also by archaea and eukaryotes, and more specialized
functions, such as metabolism, metabolite transport, host and
environment response, and signal transduction. Figure
2 shows the representation of the
functional classes in the two genomes and the level of orthology in
each class measured using OC. In order to assess the effects of
specific adaptations of each of the spirochetes to their distinct niches, investigation of classes with low OC values appears to be
critical. Below, we discuss the distribution of functional classes in
the two spirochetes along the gradient of OC values. A selection of
prominent differences in the functional systems of the two spirochetes
is shown in Table 1, and examples of apparent spirochete synapomorphies
are listed in Table 2.
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FIG. 2.
OCs for functional classes of proteins. The annotated
predicted open reading frames from the proteomes of B. burgdorferi and T. pallidum were grouped by the
predicted functional class of the protein. Orthologous proteins within
each functional class were identified as described, and the OC values
for each class were calculated as in Fig. 1. The vertical axis on the
left shows the absolute number of proteins, and the one on the right
shows OC values. OCs of >0.8 delineate highly conserved protein
classes, OCs in the range of 0.6 to 0.8 are considered intermediate,
and OCs of <0.6 represent significant divergence between the two
genomes.
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Highly conserved functional classes with OCs close to 1.
Not
surprisingly, the core functional classes, namely, DNA replication and
translation, had OCs of 0.94 and 0.98, respectively, which is
indicative of largely vertical inheritance (Fig. 2). The limited
differences observed in these conserved functional classes could be
attributed to interesting cases of horizontal gene transfer, gene loss,
or duplication with divergence.
Differences in the gene complement involved in DNA replication include
the lack of topoisomerase IV (parC, parE) in
T. pallidum, which is of interest given the conserved role
of these proteins in chromosome partitioning in other bacteria
(77). Conversely, the proofreading 3'-5' exonuclease
the
subunit of DNA polymerase III (e.g., the E. coli DnaQ
protein)whose role in DNA replication seems to be conserved in all
other bacteria (29), is missing in B. burgdorferi. The absence of this enzyme would result in error-prone replication, which seems to be compatible with the generation of new variants through mutation in the genes encoding variable antigens of Borrelia (58, 84). An
alternative is nonorthologous displacement (E. V. Koonin, A. R. Mushegian, and P. Bork, Letter, Trends Genet.
12:334-336, 1996), that is, an unrelated or distantly
related and so far unidentified exonuclease being recruited for the
proofreading role. Interestingly, T. pallidum encodes a
second copy of the single-stranded DNA-binding protein (TP0310), which
might have a distinct function in replication or recombination.
In addition to these striking differences, we detected an interesting
case of apparent displacement of an existing gene by its ortholog from
a distant species, probably via horizontal gene transfer. Sequence
searches showed that topoisomerase I (Topo I) from B. burgdorferi shares an insert within the TOPRIM domain (10) and a C-terminal repeat domain with actinomycetes and
cyanobacteria. These features are lacking in other bacterial lineages
and appear to be derived, shared characters (synapomorphies) within
this protein superfamily. Phylogenetic analysis using multiple
alignment of the conserved region of bacterial Topo I strongly
supported grouping of B. burgdorferi with actinomycetes and
cyanobacteria (Fig. 3
[bootstrap value, >90%]). This
phylogenetic affinity of Topo I is in stark contrast to the typical
clustering of the other spirochete proteins, particularly those from
the core functional classes, and suggests horizontal gene
transfer into the B. burgdorferi lineage followed by
loss of the ancestral form. This phenomenon, which may be termed
xenologous gene displacement, that is, displacement by an orthologous
gene from a phylogenetically distant source (J. P. Gogarten,
Letter and comment, J. Mol. Evol. 39:541-543, 1994), is
seen also in several other instances in the two spirochetes (see
below).
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FIG. 3.
Phylogenetic trees for representative examples of
xenologous gene displacement and horizontal gene transfer in the
spirochetes. The trees were constructed using alignments generated with
CLUSTALW, followed by manual adjustments based on PSI-BLAST outputs.
The neighbor-joining (NEIGHBOR program from Phylip) and least squares
(from the PAUP package) methods were used to construct the trees, and
1,000 bootstrap replications were performed. The nodes supported by
bootstrap values greater than 75% are indicated with the symbol
" ." All genes are shown with the mnemonic species name along
with the GenBank accession number. Species abbreviations are as
follows: Tp, T. pallidum; Bb, B. burgdorferi; Bs,
B. subtilis; Ec, E. coli; Hi, H. influenzae; Pa, Pseudomonas aeruginosa; Pdn,
Pseudomonas denitrificans; Rp, R. prowazekii; Zm,
Zymomonas mobilis; Acom, Actinobacillus
actinomycetemcomitans; Mtu, Mycobacterium tuberculosis;
Mle, Mycobacterium leprae; Ssp, Synechocystis
sp.; Ssc, Synechococcus sp.; Ana, Anabaena sp.;
Nos, Nostoc sp.; Cpn, Chlamydia pneumoniae; Ct,
Chlamydia trachomatis; Tm, T. maritima; Aae,
Aquifex aeolicus; Ap, Aeropyrum pernix; Af,
Archaeoglobus fulgidus; Mta, Methanobacterium
thermoautotrophicum; Mj, Methanococcus jannaschii; Ph,
Pyrococcus horikoshi; Um, Ustilago maydis; Poda,
Podospora anserina; Sp, Schizosaccharomyces
pombe; Sc, Saccharomyces cerevisiae; Ce,
Caenorhabditis elegans; Hs, Homo sapiens; Dm,
Drosophila melanogaster; Mm, Mus musculus; Oncvo,
Onchocerca volvulus; At, Arabidopsis thaliana;
Horvu, Hordeum vulgare; Soltu, Solanum tuberosum;
Gi, Giardia intestinalis; Tbr, Trypanosoma
brucei; En, Entamoeba histolytica.
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In spite of the high level of conservation in the translation
apparatus, we did detect a few interesting examples of evolutionary divergence between the two species. One notable case was the
prolyl-tRNA synthetase from B. burgdorferi, where
phylogenetic analysis strongly suggested that the enzyme in B. burgdorferi has been acquired from a eukaryotic source by the
xenologous displacement mechanism (78). The other
aminoacyl-tRNA synthetases are strongly conserved between the
spirochetes, but phylogenetic analysis provided evidence for likely
ancient horizontal transfers into the ancestor of spirochetes accompanied by displacement of the original gene. One well-studied case
in this category is class I lysyl-tRNA synthetase, which apparently was
acquired from the archaea (37, 78); similar evidence of
archaeal origin was obtained for the two subunits of phenylalanyl-tRNA
synthetase (78). By contrast, the synthetases for arginine,
glutamate, methionine, isoleucine, and serine showed evidence of likely
horizontal transfer from eukaryotic sources (78).
Functional classes with intermediate OCs (0.6 to 0.8).
The
gene complements involved in repair, recombination, and transcription
show significant differences between the two spirochetes, which is
reflected in OC values of less than 0.8 (Fig. 2). A number of important
repair genes are different in the two genomes. Thus, B. burgdorferi encodes the helicase-nuclease complex RecBCD, whereas T. pallidum lacks these genes but has instead a complement
of recFGNR genes. Another repair enzyme unique to T. pallidum is the ERCC3-like eukaryotic-type DNA repair helicase
that is also present in mycobacteria (54). It is likely that
this gene was horizontally transferred from eukaryotes into a certain
bacterial lineage and subsequently disseminated amidst the bacteria.
The triplication of the C-terminal BRCT domain of DNA ligase in
T. pallidum is a unique feature that is in contrast to the
similarly located single copy of this domain present in all other
bacteria (7, 15). The RecQ helicase gene is found in
T. pallidum but not in B. burgdorferi.
Conversely, B. burgdorferi but not T. pallidum retains the ancient duplication of the mutS gene, which is
also seen in other bacteria. Interestingly, B. burgdorferi
lacks RuvC, the endonuclease subunit of the Holliday junction resolvase
complex, which suggests either that the Ruv complex is nonfunctional or that a distinct nuclease complements this function. Both spirochetes possess a novel mismatched-base DNA glycosylase (TP0229/BB0013), a base
excision repair enzyme, orthologs of which are conserved in a number of
bacteria and in all archaea (60), but the B. burgdorferi version is highly divergent from all other bacterial orthologs. The major disparities in the repertoires of repair proteins
suggest a difference in the selective forces that act on this system in
the two spirochetes. The fact that B. burgdorferi undergoes
antigenic variation (58, 84) that may require both active
recombination and error-prone repair provides a possible explanation
for the retention of only certain of its repair pathways.
The intermediate OC of the transcriptional apparatus seems to reflect a
bimodal effect of selective evolutionary forces. The core
transcriptional machinery, which includes RNA polymerase subunits and
components of the elongation and termination complexes, is highly
conserved, whereas the repertoires of specific transcriptional regulators are largely different (Fig. 2). Within the shared heritage, there are some unique features that are likely to have been derived in
the common ancestor of these spirochetes and may be considered signatures of this clade. In particular, the spirochete NusA protein (a
part of the transcription termination complex) contains a C-terminal Zn
ribbon domain in place of the helix-hairpin-helix (HhH) domain that is
found in most other bacteria (data not shown). Similarly, the
transcription elongation factor GreA (40) in both
spirochetes possesses a long N-terminal extension which is shared only
with chlamydiae (69). An orthologous pair of spirochete
proteins (TP0511 and BB0355) are homologous to the transcription factor CarD from Myxococcus (48) and share a domain with
the transcription repair-coupling helicase (TRCF) (Fig.
4A).
Since the corresponding region in
TRCF interacts with the RNA polymerase holoenzyme (63), these proteins are likely to be novel transcription factors,
characteristic of the spirochete clade, that modulate transcription by
interacting directly with the RNA polymerase.
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FIG. 4.
Multiple alignments of previously uncharacterized
protein domains discovered in the course of comparative genome analysis
of the spirochetes. (A) Resolvase-type HTH domains identified in large
proteins containing an N-terminal signal peptide. (B) RNA
polymerase-interacting domain found in CarD-like proteins and TRCF. (C)
von Willebrand factor A domains in the spirochetes. (D) PR-1 domain in
B. burgdorferi. The alignments were constructed using
PSI-BLAST-derived pairwise alignments as input for the CLUSTALW program
and were further adjusted based on the PSI-BLAST output. Proteins are
designated with the protein name, species of origin, and GenBank
accession number. Numbers preceding and following the aligned regions
refer to the amino acid positions of the domain within each protein
sequence. Coloring is according to the consensus shown below the
alignment. Uppercase letters indicate conserved residues, by the
single-letter amino acid code. Lowercase letters indicate conserved
classes of amino acids that are highlighted using differential coloring
or shading as follows: violet, polar residues (p)
(C,D,E,H,K,N,Q,R,S,T); pink, charged residues (c) (D,E,H,K,R);
green background, tiny residues (u) (A,G,S); green shading, small
residues (s) (A,C,D,N,G,P,S,T,V); grey shading, bulky residues (b)
(E,F,I,K,L,M,Q,R,W,Y); yellow shading, hydrophobic residues (h)
(A,C,F,I,L,M,V,W,Y), aromatic residues (a) (F,H,W,Y), and aliphatic
residues (l) (I,L,V). Species abbreviations are as follows: Hp,
H. pylori; Mx, Myxococcus xanthus; Pf,
Plasmodium falciparum. Others are as indicated in the legend
to Fig. 3.
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Major differences between the two spirochetes were revealed even among
the sigma factors. While they share 
70 (RpoD) and
54 (RpoN), B. burgdorferi also encodes an
RpoS ortholog, whereas T. pallidum possesses
24 (RpoE), 28, and 43
(SigA). The three sigma factors present in T. pallidum but
not in B. burgdorferi are likely to be involved in
regulating multiple gene sets specific to the former. Of particular
interest in this context is 24, whose orthologs in other
bacteria regulate gene batteries associated with stress response and
pathogenesis (1). Consistent with the detection of multiple
sigma factors, T. pallidum encodes elements of the system of
sigma factor regulators similar to that seen in Bacillus,
Chlamydia, Synechocystis, and the actinomycetes. These regulators include two phosphatases of the PP2C family and an
anti-sigma factor antagonist (83), which suggests that
T. pallidum senses a distinct environmental signal
regulating the anti-sigma factor antagonist. However, T. pallidum does not encode an ortholog of the small serine kinase of
the histidine kinase class (RsbV protein), which is an essential
component of the anti-sigma factor regulatory system in other bacteria
(83). If this system is to be functional, it must include a
kinase other than conventional histidine or serine/threonine kinases,
as none of them, with the exception of CheA (which is not known to
participate in this pathway), are detectable in T. pallidum.
Each of the spirochetes encodes several predicted specific
transcriptional regulators of the helix-turn-helix (HTH) class of
DNA-binding proteins. In addition, B. burgdorferi encodes a member of the MetJ/Arc family of 
-sheet-containing transcription factors (BBD22) on its linear plasmid. With the exception of a single,
novel family of HTH proteins, none of these likely transcriptional regulators are orthologous in the two spirochetes, which is consistent with the diversification of the possible target metabolic pathway operons (see below). The shared HTH proteins are an unusual group of
two orthologous protein pairs (TP0711/BB0265 and TP0408/BB0512) that
contain a C-terminal HTH domain similar to the PAIRED domains of
resolvases (Fig. 4B). In addition, they have a hydrophobic N-terminal
region resembling a signal peptide that may be responsible for an
unexpected membrane association and processing associated with their
function. Orthologs of these proteins with the same domain arrangement
are detectable in H. pylori, Bacillus subtilis, and Thermatoga maritima. As an example of the diversified
transcriptional regulation for similar functions, T. pallidum encodes a winged-HTH protein, TroR (34),
whereas B. burgdorferi encodes the unrelated Fur protein
(75), both of which are predicted to negatively regulate
metal uptake. Two HTH-containing regulators of sugar metabolism, XylR-1
and XylR-2, that combine the HTH domain with the Hsp-70 like
sugar-kinase domain are present only in B. burgdorferi.
Functional classes with low OC (<0.6) signaling.
The signal
transduction systems in T. pallidum and B. burgdorferi show low OC values (Fig. 2), with only a few conserved
features that seem to be remnants of the signaling apparatus of the
common ancestor of the spirochetes. This
shared core includes components of chemotactic signaling based on the
histidine kinase CheA, which contains a C-terminal CheW domain
(Fig. 5), and associated downstream signaling components, such as methyl-accepting and methyltransferase chemotaxis proteins. Even within this system, however, B. burgdorferi possesses specific methylesterase, methyltransferase,
and methyl-accepting proteins with no counterparts in T. pallidum (Fig. 5). Furthermore, B. burgdorferi has a
larger representation of the two-component relay systems, with two
additional histidine kinases other than the two CheA paralogs and
multiple additional receiver domain proteins. These histidine kinases
are likely to function as sensors of different stimuli, since one of
them contains a PAS domain that has been implicated in redox sensing
and the other one contains a large, uncharacterized extracellular
(periplasmic) domain (Fig. 5). Cross-talk between these kinases and
other signaling systems is suggested by the fusion of the receiver
domain of the two-component system with other signaling domains, such
as methylesterase and diguanylate cyclase/phosphodiesterase. Of
additional interest is the presence, in both spirochetes, of CheX
proteins (TP0365/BB0671) (32). These proteins are homologs
of the FliM protein of E. coli, which appears to provide a
link between chemotactic signaling and the flagellar motor
(46).
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FIG. 5.
Differences and conservation in the domain architectures
of proteins involved in signaling in B. burgdorferi and
T. pallidum. Proteins unique to either organism are shown in
the outer panels; the middle panel represents proteins conserved in the
two organisms. Domain identification/organization is based on sequence
searches using PSI-BLAST-derived PSSMs and Hidden Markov models derived
using the HMMER2 package and was confirmed by detailed examination of
sequence alignments. The figure is drawn roughly to scale. The double
slash (//) indicates that a portion of the long sequence has been
omitted, blue vertical bars represent transmembrane helices predicted
using the TopPred II program, and the red horizontal bar represents a
signal peptide predicted using the SignalP program. Domain
abbreviations are as follows: MeTr, methyl transferase; MeAc, methyl
acceptor; MeEst, methyl esterase; cheW, two-component signaling adaptor
domain; Rec, receiver domain; AC, adenylyl cyclase; IIA/IIB/IIC,
components of phosphoenolpyruvate:sugar phosphotransferase (PTS)
system; PAS, Per/Arnt/Sim domain; HisK, histidine kinase; PhHis,
phosphohistidine; HPT, histidine phosphotransferase; TIM, TIM barrel
domain; DGC/P, diguanylate cyclase/phosphodiesterase domain; HAMP,
domain common to histidine kinase, adenylyl cyclase, methyl acceptor,
and PP2C phosphatase (11); HTH, helix-turn-helix; HD, HD
superfamily phosphodiesterase domain; GAF, domain present in
cGMP-specific phosphodiesterase (cyclic nucleotide binding); cAMP,
cyclic AMP binding domain; TPR, tetratricopeptide repeat; PP2C, PP2C
family phosphatase; ANK, ankyrin repeat; CBS, cystathionine-beta
synthase domain (a widespread domain implicated in signaling
[13]); HPT, histidine-containing phosphotransfer
domain; ACT, aspartokinase, chorismate mutase, TyrA domain
(8); TGS, threonyl-tRNA synthetase, GTPase, SpoT domain
(78); HPRK, HPR kinase; HPr, phosphocarrier protein; PhHis,
phosphohistidine; X, conserved domain of unknown function seen in the
SpoT protein; fliY, domain common to CheX and FliY/M proteins.
|
|
The phosphoenolpyruvate:sugar phosphotransferase system (PTS)
(59) is fully represented in B. burgdorferi (Fig.
5). It includes multiple EII enzymes with different sugar specificities
along with other PTS proteins, such as HPR and
phosphoenolpyruvate-protein-phosphotransferase. By contrast, T. pallidum encodes an unusual motley of proteins from the PTS system
which includes the phosphocarrier protein Hpr and its kinase, similar
to the one involved in catabolite repression in gram-positive bacteria
(56), and a single protein containing an EIIA domain but not
the other PTS components (Fig. 5). It is possible that these remnants
of the PTS system in T. pallidum are involved in a novel
signal transduction mechanism.
T. pallidum possesses a well-developed cyclic AMP (cAMP)
signaling system that is lacking in B. burgdorferi (Fig. 5).
The presence, in T. pallidum, of four cAMP-binding
domain-containing proteins and a classical "eukaryote-type"
adenylyl cyclase similar to those present in some other bacteria, such
as cyanobacteria, myxobacteria, and actinomycetes, indicates a
prominent role for this system in signal transduction. The domain
architectures of these proteins suggest functional diversitythese
include duplication of the cAMP-binding domain (TP0089) and fusions to
an HTH DNA-binding domain (TP0262) and to an alpha-helical domain
distantly related to the tetratricopeptide repeats (TPR) (TP0261) (Fig.
5). Furthermore, diguanylate cyclase/phosphodiesterase from T. pallidum contains a GAF domain that in some instances binds cyclic
nucleotides (12). This protein might participate in
cross-talk between cyclic nucleotide signaling and cyclic diguanylate
signaling. The adenylyl cyclase of T. pallidum (TP0485)
contains a HAMP (for histidine kinases, adenylyl cyclases, methyl
accepting chemotaxis receptors, and phosphatases) domain, a recently
described module that is associated with the cytoplasmic face of
several bacterial signaling proteins (11). This domain is
predicted to transmit extracellular stimuli sensed by these receptor
proteins to the respective intracellular signaling domains. All
bacteria that contain this eukaryotic-type cAMP-dependent signaling
system lack a counterpart to the eukaryotic cyclic nucleotide
phosphodiesterase and, in addition, lack the proteobacterial-type
phosphodiesterase Icc (44). However, T. pallidum
encodes a predicted membrane protein with six transmembrane helices
that contains a metal-dependent phosphodiesterase domain of the HD
superfamily, which is distantly related to eukaryotic phosphodiesterases (9). This protein is a candidate for the cAMP phosphodiesterase function. B. burgdorferi lacks
classical adenylyl cyclases and cAMP-binding proteins but encodes an
unrelated adenylyl cyclase (BB0723) that belongs to a recently
described class of adenylyl cyclases found in thermophilic archaea and
Aeromonas hydrophila (65). The function of this
protein in signal transduction remains unclear in the absence of any
detectable downstream effector molecules that would recognize cAMP. It
cannot be ruled out that a new class of cAMP-binding domains shared by
archaea, eukaryotes, and some bacteria, including B. burgdorferi, remains to be discovered.
Most bacteria encode the SpoT protein, which is a
guanosine-3',5'-bispyrophosphate (ppGpp)
3'-pyrophosphohydrolaseguanosine 3',5'-bispyrophosphohydrolase
(62). This key enzyme is responsible for cellular (p)ppGpp
degradation, which reverses ppGpp accumulation during the stringent
response to amino acid deprivation. While B. burgdorferi
possesses a SpoT ortholog (BB0198) (Fig. 5), with its characteristic
catalytic HD domain (9) and regulatory TGS (78)
and ACT (8) domains, T. pallidum surprisingly
lacks this protein. The parasitic lifestyle of T. pallidum
may differ from that of Borrelia, resulting in the loss of
this stringent response signaling system. Of the published genomes of
bacteria, only the two chlamydial species (38, 69) and
T. pallidum lack this enzyme, whereas Rickettsia
prowazekii encodes a degenerate and probably inactive version of
the enzyme (5). This may reflect the superfluous nature of
stringent response regulation in the nutrient-rich environments of
these pathogens.
Functional classes with low OCs (<0.6): metabolic pathways and
transport and other membrane components.
Consistent with the
picture emerging from other pathogenic bacteria, the genomes of the two
spirochetes are noticeably reduced in terms of genes coding for
metabolic enzymes, which suggests a general degeneration of metabolic
systems that most likely were represented in their common ancestor.
However, even in this degenerate state, there are striking differences
in the predicted metabolic pathways between T. pallidum and
B. burgdorferi, suggesting different adaptation strategies.
The common metabolic heritage is largely restricted to the trunk of the
glycolytic Embden-Meyerhof pathway, the phosphorylation steps of
nucleotide interconversion, and a system for membrane potential
generation, namely, the multisubunit V-type ATPase. The latter is the
principal energy-generating ATPase in archaea; in bacteria, the
V-ATPase subunits are encoded in a single operon that appears to be
evolutionarily mobile and shows a scattered distribution, being found,
in addition to in the spirochetes, in Chlamydia,
Enterococcus, Deinococcus, and Thermus
(55).
As already discussed for other, more conserved pathways, even within
the shared core of metabolism there are instances of likely horizontal
transfer and associated xenologous and nonorthologous (E. V. Koonin et al., Letter, Trends Genet. 12:334-336, 1996) gene
displacement. Apparent lateral gene transfer accompanied by
displacement is illustrated by fructose-1-6-bisphosphate aldolase and
the glyceraldehyde 3-phosphate dehydrogenase that belong to the central
glycolytic pathway. Phylogenetic analysis of the aldolases from the two
spirochetes showed that, while both of them are class II aldolases,
they belong to two entirely distinct, well-supported groups, which
suggests independent acquisition via lateral gene transfer from other
bacteria (Fig. 3). In phylogenetic trees, glyceraldehyde 3-phosphate
dehydrogenase from T. pallidum groups strongly with the
orthologs of this protein from kinetoplastid eukaryotes (Fig. 3). This
points to horizontal gene transfer from this eukaryotic lineage to
T. pallidum after the divergence of the two spirochetes,
followed by displacement of the original spirochete version. An even
more interesting case is T. pallidum uridine kinase, which
not only shows the unexpected grouping with the ortholog from
Thermotoga in phylogenetic trees but also possesses a unique
domain architecture, with two domains, including the TGS domain,
apparently acquired from threonyl-tRNA synthetases (78). In
the threonyl-tRNA synthetases, these domains play a role in RNA binding
(61), which suggests that these unique uridine kinases could
regulate their own expression by binding to mRNA. Another notable case
of likely horizontal transfer followed by gene displacement involves a
pair of closely linked genes in an operon, namely glucose 6-phosphate
dehydrogenase (G6-PDH) and the adjacent gene that encodes the DevB
protein. In T. pallidum, both of these proteins are closely
related to their counterparts from Haemophilus and
Actinobacillus rather than to those from B. burgdorferi. Phylogenetic analysis (Fig. 3) provides strong bootstrap support for this lineage and suggests that the entire operon
comprised of G6-PDH and DevB has been exchanged between T. pallidum and the proteobacterial lineage.
Even among the genes encoding components of metabolic pathways that
have been vertically inherited by the two spirochetes, there are
footprints of likely ancient horizontal transfer events. Horizontal
transfer from eukaryotes to spirochetes is attested by the presence of
a pyrophosphate-dependent phosphofructokinase (BB0020, TP0542) that
otherwise is characteristic of plants and several protists, such as
Entamoeba and Giardia. This enzyme is present in
the spirochetes along with the original bacterial ATP-dependent phosphofructokinase (BB0727, TP0108) in the glycolytic pathway, suggesting functional partitioning of the two versions.
Each of the spirochetes encodes two cytidylate kinases. One of these
appears to have been acquired by the common ancestor of the spirochetes
from the archaea. This is strongly suggested by their highly
significant relationship to the archaeal proteins in phylogenetic tree
analysis (Fig. 3) and the modified P-loop ATPase motif (data not shown)
that is uniquely shared by these proteins with their archaeal counterparts.
Both spirochetes encode an aminopeptidase M containing an N-terminal
insert sequence that is specifically shared with eukaryotes; phylogenetic analysis indicates that it might have been horizontally transferred into the ancestral spirochete from a eukaryote (Fig. 3).
Some of the drastic differences in the gene complements for metabolic
processes seem to reveal adaptive features unique to each spirochete.
The ability to utilize glycerol and chitin as energy sources through
the glycolytic pathway, which is unique to B. burgdorferi,
is consistent with the existence of an arthropod-specific stage in the
life cycle of this spirochete. This is supported by the presence of
genes for glycerol uptake and the FAD-dependent glycerol-3-phosphate dehydrogenase (BB0243), the latter
apparently being an unusual case of horizontal transfer from a
eukaryotic source (Fig. 3). Utilization of
N-acetylglucosamine polymers or chitin (available in the
arthropod host) as a substrate for cell wall biosynthesis (glucosamine
deacetylase, NagA; BB0151) and/or as an energy source (glucosamine
deaminase, NagB; BB0152) could represent an adaptation of B. burgdorferi for survival in its specific niche, in this case the
arthropod host. The presence of an additional pathway for conversion of
dihydroxyacetone phosphate into lactate via a methylglyoxal
intermediate also could be an adaptation for growth under certain
environmental conditions and carbohydrate sources (28, 74).
Another interesting metabolic feature of B. burgdorferi
inferred from sequence analysis is the presence of a pathway for the biosynthesis of isopentenyl pyrophosphate (IPP). All of the enzymes necessary for the formation of IPP from 3-hydroxymethyl
glutaryl-coenzyme A (HMG-CoA), along with a novel glycolate
oxidase-like, TIM barrel fold oxidoreductase (BB0684) that could
participate in the same process, are encoded in a single long operon.
With the exception of this oxidoreductase, the remaining proteins of
this pathway show eukaryotic or archaeal affinities in phylogenetic
analysis (data not shown), with no closely related proteins in
bacteria. Taken together with the fact that Pseudomonas
mevalonii is the only other bacterium to date that encodes an
orthologous HMG-CoA reductase (14), these observations
suggest that this pathway has been acquired by B. burgdorferi via horizontal transfer from either an archaeal or a
eukaryotic source. The enzymes encoded in this unusual operon are
likely to participate in the synthesis of still uncharacterized
membrane/lipoprotein components in B. burgdorferi.
The repertoires of transporters encoded by the two spirochetes are
significantly different and, with an OC of 0.38, are among the most
divergent functional classes revealed in our analysis; this suggests
distinct substrate requirements. In this class, it is notable that,
unlike B. burgdorferi, T. pallidum appears to
lack a transport mechanism for proline or glycine-betaine. Given the
proposed osmoprotective role of proline and glycine-betaine in
bacterial stress response (4, 23), the presence of a full complement of genes for proline biosynthesis in T. pallidum
likely serves as an adaptive response to osmotic stress.
Finally, the surface lipoproteins and other, uncharacterized membrane
proteins of the two spirochetes show very low OC values (Fig. 2), which
is compatible with the different modes of parasite-host interaction.
Individual protein families in the two spirochetes. (i) Conserved
and variable families.
As an alternative approach to assessing the
evolutionary forces acting on the spirochetes, we analyzed all of the
largest families of paralogous proteins in the two genomes and
calculated the OCs for each of them (Fig.
6). Like other bacteria and archaea (39, 80), the largest protein superfamily in the spirochetes includes P-loop-containing NTPases, with 86 members in B. burgdorferi and 69 in T. pallidum. Different families
within this large superfamily show substantial variability in the
degree of conservation. A number of ATPases, such as those involved in
DNA replication, V-type ATPases, and GTPases, show a largely vertical
inheritance pattern. By contrast, the ABC transporter ATPases show
considerable diversity in the two spirochetes, which is consistent with
the differences noted in nutrient transport and substrate specificity (see above). There is a specific expansion of the MinD family of
ATPases, which are involved in chromosome partitioning in B. burgdorferi (12 members); this is consistent with the presence of
several cosegregating plasmids that comprise parts of the B. burgdorferi genome (30).
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FIG. 6.
OCs for protein families and superfamilies identified in
the two spirochetes. Protein and domain (super)families within the two
spirochete proteomes were identified using transitive BLASTP searches
and family-specific PSSMs, as described in Materials and Methods. The
OC was computed for each of these (super)families as described in the
text.
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|
Compared to the P-loop ATPase superfamily, all other protein families
are much smaller and show widely varying degrees of orthology (Fig. 6).
Some of these families, such as the pseudouridine synthetases, the PDZ
domain proteins, and lysostaphin-like proteases, show OC values close
to 1, which suggests functional coherence between the two organisms.
The two classes of aminoacyl-tRNA synthetases and SAM-dependent
methyltransferases have slightly lower OC values, which is due to the
likely horizontal transfers discussed above or to relatively minor
lineage-specific gene losses. Other families, such as HD superfamily
hydrolases (9), metallo--lactamase fold hydrolases
(6), calcineurin-like phosphatases (7), and
peptidyl-prolyl cis-trans isomerases, show much lower OC
values, which indicates major effects of lineage-specific gene loss,
horizontal transfer, and rapid diversification. These families, unlike
those associated with translation and replication, appear to
participate in diverse roles that are under constant selection due to
variations in the niches occupied by the two spirochetes. Certain
protein families, such as the P-methylase/lipoate synthetase
family, are seen only in T. pallidum. This raises the
possibility of distinct metabolic options that are absent in B. burgdorferi.
(ii) Newly identified protein families.
We identified several
previously undetected protein families in the spirochetes by using
family-specific PSSMs (see Materials and Methods). These include some
secreted (or possibly periplasmic) and membrane proteins that could be
involved in pathogen-host interactions. The most interesting of these
are the von Willebrand A factor (vWA) domain-containing proteins
(52), which, until recently, had been detected only in
eukaryotes. The vWA domain is a Mg2+-binding protein module
with an 
/ fold that participates in adhesion and protein-protein
interactions in a wide range of eukaryotic proteins, such as integrins
(26). Using profile searches, we identified three vWA
domain-containing proteins in each of the spirochetes (Fig. 4C).
However, the vWA proteins from B. burgdorferi are not highly
similar to those from T. pallidum, suggesting that they
either have been independently acquired or have diverged rapidly due to
selective forces acting on extracellular proteins. These secreted or
membrane-associated vWA domain proteins are reminiscent of similar
domains present in the extracellular adhesion molecule (TRAP) of a
eukaryotic pathogen, the malarial genus Plasmodium (73). By analogy to TRAP, it is likely that the spirochete
vWA proteins are involved in adhesion of these bacteria to the
extracellular matrix or to cells of the host connective tissues. These
proteins could be potential targets for specific antispirochete
therapies. The similarity between the spirochete vWA domains and that
of the LFA-1 integrin is of interest given the recent report
identifying LFA-1 as an autoantigen in treatment-resistant Lyme
arthritis (33).
Another interesting protein of B. burgdorferi is BB0689, a
secreted or periplasmic protein containing a PR1 domain
(70). This domain hitherto has been detected only in
eukaryotes, namely in plant pathogenesis-related proteins (PR-1) and in
proteins expressed in the animal immune system such as GliPR
(70). One of the human proteins in this family has been
suggested to be a novel trypsin inhibitor (82), whereas
other members, such as helothermine, found in the toxins of Helodermid
lizards and certain snakes, act as inhibitors of potassium and calcium
channels (17, 50). Another protein of this family, TPX-1, is
implicated in the interaction between spermatogenic and Sertoli cells
(45). All of these observations indicate that PR-1
domain-containing proteins participate in diverse extracellular
protein-protein interactions, suggesting a role for BB0689 in adhesion
to host extracellular proteins and, accordingly, in pathogenesis.
Conversely, T. pallidum encodes an unusual, large, secreted
(periplasmic) protein (TP0544) that contains an OB-fold domain (47). This protein might interact with host cells and act as a virulence factor like the OB-fold-containing toxins from a wide range
of bacteria (25).
Both spirochetes encode proteins (BB0236 and TP0421) containing a
modified version of the YWTD domain (67) that is seen in
intracellular animal proteins, some of which also contain the RING
finger domain (L. Aravind, unpublished data). This relationship to a
class of animal proteins suggests lateral transfer from an animal host
into the ancestor of the two spirochetes. YWTD domains assume a
-propeller structure similar, for example, to that in low-density
lipoprotein receptors and in proteins from other parasites (e.g.,
Trypanosoma cruzi) that mediate extracellular adhesion (51). The presence of a predicted signal peptide suggests
that, like the vWA and PR-1 domain-containing proteins, the spirochete YWTD domain proteins are secreted and interact with host cell surface
molecules (52).
Another interesting family that is conserved between the two
spirochetes, some of whose members appear to be secreted, are the
tetratricopeptide repeat (TPR) domain-containing proteins (24). These domains form an alpha-helical superstructure and mediate protein-protein interactions. The use of TPR motifs in extracellular interactions might be a novel strategy of host
interaction employed by the spirochetes and is reminiscent of a family
of secreted Sel-1 repeat proteins in Helicobacter pylori
(52; L. Aravind, unpublished data).
To add to this repertoire of previously undetected proteins that could
be important for the pathogen's interaction with host cell surfaces,
T. pallidum encodes two proteins with multiple ankyrin
repeat motifs (TP0502, TP0835) (Fig. 5). At least TP0835, which
contains 20 ankyrin repeats, has a canonical signal peptide, indicating
that, unlike the eukaryotic ankyrin repeat proteins, this
Treponema protein is secreted.
Evolutionary implications of comparative analysis of the two
spirochete proteomes.
The use of the OC concept, along with a
case-by-case analysis of individual protein sequences, has provided
considerable information that allows us to address several evolutionary
issues: (i) the amount of shared heritage present in the spirochete
genomes, (ii) the likely gene repertoire and lifestyle of the common
ancestor, (iii) the role of horizontal transfer, and (iv) the
strategies and adaptations used by these organisms in their interaction
with the host, which are important for pathogenesis.
Likely orthologs comprise about 43% of the total number of proteins
encoded by the two spirochetes (39.5% [495 of 1,256] of the B. burgdorferi proteins and 47% [486 of 1,031] of the T. pallidum proteins). This fairly low fraction of orthologs
indicates that lineage-specific gene loss, horizontal gene transfer,
and rapid divergence account for more than one-half of the gene
repertoires of the two spirochetes. The relatively lower fraction of
orthologs in Borrelia is to a large extent due to the fact
that multiple plasmids harbored by this spirochete encode a number of
highly variable proteins without counterparts in Treponema
(30). Genome comparisons between two more closely related
bacteria, namely H. influenzae and E. coli, have
indicated that lineage-specific gene loss was a major force in the
evolution of the parasitic organism, in this case H. influenzae (72). Given that both spirochetes are
obligate parasites, it is likely that the two lineages have lost genes
largely independently, resulting in major differences in the complement
of genes eventually retained. However, the number of genes lost by the
spirochetes is lower than that seen in some other obligate parasites,
such as the mycoplasmas, chlamydiae, and rickettsiae (5, 69,
79).
The OC values for different functional classes of proteins (Fig. 2)
illustrate the spectrum of genes that have retained identical or
similar functions and were probably present in the common ancestor of
the spirochetes. As expected, certain core functions, such as
translation, RNA modification, and replication, are hardly affected by
gene loss. By contrast, the DNA repair systems and the transcription
systems show major differences, probably due to a combination of
lineage-specific loss and horizontal gene transfer driven by different
selective forces acting on the two organisms. The core of the
glycolytic pathway and associated metabolic steps show a predominantly
vertical inheritance. These systems with high OC values are very
similar to their counterparts in other bacteria, which suggests a
typical bacterial core physiology of the common ancestor. The presence
of V-type ATPase operons in both spirochetes indicates that, like
Thermus and Enterococcus (55), the
common ancestor of the spirochetes used H+/Na+
exchange for ion gradient generation. The signal transduction systems
in general show low OC values, but the elements involved in chemotactic
response are conserved. Together with the conservation of the flagellar
apparatus (Fig. 2), this suggests an actively motile chemoresponsive
ancestral spirochete that used a mode of locomotion similar to that of
the two extant species.
Even within the conserved systems, there are some striking cases of
xenologous and nonorthologous gene displacement that are suggestive of
horizontal transfer from distantly related bacteria and archaea. Some
of these appear to have occurred in the common ancestor of the
spirochetes, whereas others are lineage specific. Examples of apparent
ancient gene transfers include the acquisition of certain
aminoacyl-tRNA synthetases and cytidylate kinase from archaea. Likely
xenologous displacements in one of the spirochete lineages were seen in
a number of cases, such as fructose-1-6-bisphosphate aldolase,
glyceraldehyde 3-phosphate dehydrogenase, glucose 6-phosphate dehydrogenase subunits, 6-phosphogluconate dehydrogenase, and topoisomerase I (Fig. 3). Other examples of likely gene exchange with
distant bacteria include the small, primase-like TOPRIM domain protein
(10), which is otherwise seen only in low-GC gram-positive bacteria and in archaea, and the unusual HTH proteins shared with Bacillus, Thermotoga, and
Helicobacter. These observations suggest that, unlike the
extant forms that lead a relatively isolated existence due to their
specialized life cycles, the ancestors of B. burgdorferi and
T. pallidum, both before and after their divergence from
each other, existed in close proximity with other prokaryotes, which
favored gene exchange.
There is considerable evidence of likely acquisition of genes from
eukaryotic, and possibly animal, sources by the common ancestor of the
spirochetes; on most occasions, such gene acquisitions apparently have
been accompanied by displacement of the endogenous bacterial versions.
Several of the aminoacyl-tRNA synthetases exemplify this phenomenon
(78). Other striking cases include the YWTD domain proteins
and the secreted vWA domain proteins, which until now appeared to be
specific to animals, as well as predicted secreted proteins containing
TPR repeats. Thus, it appears that the common ancestor of
Treponema and Borrelia was already in contact
with animal hosts. However, the evidence of gene exchange with other
bacteria, archaea, and perhaps eukaryotic protists (see above) suggests
that they were not specialized obligate parasites isolated from the
rest of the microbial community. The lifestyle predicted for the common
ancestor of B. burgdorferi and T. pallidum is
consistent with that of the spirochetes in the microbial community in
invertebrate guts (21, 41). These spirochetes are not only in contact with an animal host but also share the niche with other bacteria, methanogenic archaea, and eukaryotic protists. The
alternative hypothesis, that the common ancestor of
Treponema and Borrelia was a free-living
spirochete and that the bacteria of these two lineages have
independently established symbiosis with the eukaryotic host, cannot be
ruled out but seems to be less strongly supported by the evidence.
Indeed, many of the likely cases of horizontal gene transfer from
eukaryotes are shared by the two spirochetes, which strongly suggests
that their ancestor was already in contact with a eukaryotic host.
Subsequent to the divergence from its common ancestor, each lineage
independently acquired the ability to infect vertebrate hosts. This is
indicated by the presence of very different variant antigens in the two
genera, a feature that might have enabled these organisms to evade the
acquired immunity of the vertebrate hosts. The possibility that
Borrelia has moved from arthropod parasitism to vertebrate
parasitism is suggested by the presence of specific carbohydrate
metabolism genes that provide for the utilization of arthropod chitin
by this bacterium. The presence of commensal treponemes in vertebrates
(21) suggests that the ancestors of T. pallidum
were well adapted for life in a vertebrate host before the final step
in the evolution of pathogenicity that might have been accompanied by
the acquisition of specific variant antigens to evade the host immune
system. T. pallidum lacks many of the carbohydrate
metabolism genes and a functional PTS system; these systems might have
been lost subsequent to its displacement from the carbohydrate-rich
niches occupied by the ancestral treponeme, such as the vertebrate oral cavity.
Conclusions.
By means of local sequence similarity searches,
profile searches, and analysis of individual domains and protein
families, we have conducted a detailed comparative analysis of the
genomes of the spirochetes B. burgdorferi and T. pallidum. The level of conservation between functional classes and
paralogous families of proteins was measured using the orthology
coefficient. Using this measure, it was possible to characterize, in
functional terms, the nature of the divergence between the two
spirochetes and the common and distinct aspects of their physiological
strategies. Protein profile searches resulted in the identification of
hitherto undetected components of the signal transduction machinery and novel proteins containing domains previously seen only in eukaryotes. Secreted proteins containing these domains might mediate interactions between the spirochetes and host cells or the extracellular matrix. It
appears possible to tentatively reconstruct the evolutionary steps
leading from a common ancestor that might have been an invertebrate gut
symbiont to the divergence of the two genera of spirochetes and
adaptation to their specific niches.
Availability of complete results.
The list of T. pallidum and B. burgdorferi orthologs classified by
functional categories and by protein families is available by anonymous
ftp at ftp://ncbi.nlm.nih.gov/pub/Koonin/Spirochetes.
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Abstract
Introduction
