# Genome-Based Construction

Authors: Chan-Ki Min; Jae-Seong Yang; Sanguk Kim; Myung-Sik Choi; Ik-Sang Kim; Nam-Hyuk Cho
Journal: Comparative and Functional Genomics
 (2008)

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## Abstract

Orientia
tsutsugamushi, the causative agent of
scrub typhus, is an obligate intracellular
bacterium that belongs to the order of
Rickettsiales. Recently, we have reported that
O. tsutsugamushi has a unique
genomic structure, consisting of highly
repetitive sequences, and suggested that it may
provide valuable insight into the evolution of
intracellular bacteria. Here, we have used
genomic information to construct the major
metabolic pathways of
O. tsutsugamushi and performed a
comparative analysis of the metabolic genes and
pathways of O. tsutsugamushi
with other members of the Rickettsiales order.
While O. tsutsugamushi has the
largest genome among the members of this order,
mainly due to the presence of repeated
sequences, its metabolic pathways have been
highly streamlined. Overall, the metabolic
pathways of O. tsutsugamushi
were similar to Rickettsia but
there were notable differences in several
pathways including carbohydrate metabolism, the
TCA cycle, and the synthesis of cell wall
components as well as in the transport systems.
Our results will provide a useful guide to the
postgenomic analysis of
O. tsutsugamushi and lead
to a better understanding of the virulence and
physiology of this intracellular pathogen.


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## Body

## 1. Introduction

O. tsutsugamushi, an
obligate intracellular bacterium, is the causative agent of scrub typhus [1] which is characterized
by fever, rash, eschar, pneumonitis, meningitis, and disseminated intravascular
coagulation that leads to severe multiorgan failure if untreated [2]. The mortality rate of
scrub typhus in untreated patients ranges from 1 to 40%, depending on the
patient condition, the endemic area, and the strain of O. tsutsugamushi [3]. Scrub typhus is
confined to a geographical region that extends from far eastern Russia and northern
Japan in the north, to northern Australia in the south, and Pakistan and
Afghanistan in the west [3]. The principal ecologic
feature that distinguishes scrub typhus from other enzootic rickettsiosis is
related to the distribution and life cycle of trombiculid mite vectors and
their vertebrate host [3]. Human infection by O. tsutsugamushi is mediated through the
bites of the larva of the trombiculid mite, which harbor the bacterium in their
salivary glands [4].Although
scrub typhus can be treated effectively with antibiotics such as doxycycline
and chloramphenicol, reinfection and relapse frequently occur due to the wide variety
of antigenically distinct serotypes [5]. Furthermore, decreased
effectiveness of antibiotic treatments was recently reported in several cases [6, 7]. While the number of
patients with scrub typhus and recurrent outbreaks has recently increased in
endemic areas [6, 8, 9], an effective vaccine has
yet to be developed, possibly due to the limited duration of the immune
response [10] and immunosuppression in
the infected host [11].Orientia belongs to α-proteobacteria and was
reclassified as a new genus distinct from Rickettsia based on phenotypic and genotypic differences [12]. Orientia differs from Rickettsia in the structure of the cell wall, antigenic profile, and genome size, which is
almost twice the size of the Rickettsia genome [13]. We have recently
completed sequencing of the genome of O. tsutsugamushi, and shown that it contains the highest
content of repeated sequences (approx. 40% of the genome) among bacterial
genomes sequenced to date. We also showed that the repeats are generated by the
massive proliferation of mobile genetic elements such as conjugative type IV
secretion system components and transposons [14].The
members of the Rickettiales order, which is divided into the Anaplasmataceae family
(Wolbachia, Anamplasma, Ehrlichia, and Neorickettsia) and the Rickettsiaceae
family (Rickettsia and Orientia) are associated with a diverse
set of hosts and vectors which exhibit a range of mutualistic and parasitic
relationships. Host switching and differences in the mode of transmission, from
transovarian to horizontal transmission, create additional diversity in the host-parasite
relationship [15]. Recently, the wealth
of genomic information for the Rickettsiales members including the agents of
scrub typhus, epidemic typhus, ehrlichioses, and heartwater disease has provided
valuable resources for exploring the effect of host association on the evolution
of intracellular pathogenic bacteria [14–19].The
characterization of the metabolic properties of intracellular bacteria as well
as the mechanisms by which these pathogens acquire nutrients from their host, is
important in understanding virulence and related diseases. Genome-based construction
of the metabolic pathways of intracellular pathogens may provide valuable
insights into their pathogenic properties as well as indicate potential targets for the development
of novel therapeutics. In the current study, we have generated a detailed map
of the metabolic pathways ofO. tsutsugamushi based on genomic information, and compared the metabolic features of O. tsutsugamushi to other members of the
Rickettsiales order.


## 2. Methods

Metabolic
and genetic analysis ofO. tsutsugamushi was based on previous published annotation data [14, 19]. Annotation of COGs of putative
functional genes was further confirmed by performing a BLAST search against the
COG database (e-value <10−10, multiple assignments per protein
allowed) [20]. Metabolic pathways
were subsequently analyzed using the Kyoto encyclopedia of genes and genomes
(KEGGs) metabolic database [21]. Each gene that was
implicated in a metabolic pathway was manually confirmed by a BLAST search of KEGG
genes using the web-based BLASTP program (e-value <10−20). Genes
encoding putative transporters were identified based on the TransportDB
database [22] and KEGG membrane transport
data. Annotated transporters of 9 Rickettsiales members in TransportDB were
collected and used to identify homologous transporters in Orientia. Additional putative transporters that were previously annotated
[14] were analyzed using the
web-based BLASTP program (e-value <10−20). Among 1249 genes of O. tsutsugamushi, which excluded the
genes for mobile genetic elements, 819 genes were annotated with functional COGs
and used for the metabolic construction. Ortholog searches (length ratio
criteria 80% and cutoff e-value <10−10) within the members of
Rickettsiales were performed by BLASTP searches using formatted data collected
from the genomes of Rickettsia conorii (NC003103), R. bellii (NC007940), R. felis (NC007109), R. typhi (NC006142), R. prowazekii (NC000963), Wolbachia endosymbiont
strain TRF of Brugia malayi (NC006833), Wolbachia endosymbiont of Drosophila melanogaster (NC002978), Anaplasma marginale (NC004824), A. phagocytophilum (NC007797), Ehrlichia canis (NC007354), E. ruminantium (NC006832, NC005295), E. chaffeensis (NC007799), Neorickettsia sennetsu (NC007798), and O. tsutsugamushi (NC 009488).


## 3. Results and Discussion

### 3.1. Carbohydrate and Energy Metabolism

Glycolysis
and the citric acid cycle are the major energy-producing catabolic pathways and
they are conserved in all kingdoms of life. Genome sequence analysis revealed
that some of the enzymes of these pathways are present inO. tsutsugamushi. Three enzymes of the glycolysis pathway, in which
glucose is oxidized to pyruvate, were present (gap, pgk, and tpiA; Figure 1). These enzymes may be
used to generate glycerol phosphate which is the starting material for the synthesis
of glycerophospholipids or used to generate energy from glycerol-3-phosphate by a reverse
reaction. Rickettsia do not possess these
enzymes [23], but they possess a
gene for glycerol phosphate transporter (glpT)
which imports the material from host cells (Table 1 and Figure 7). A limited
number of enzymes of glycolysis were identified in the genomes of Wolbachia, Anaplasma, and Ehrlichia, and it has been suggested that in
these organisms the synthesis of glyceraldehydes-3-phosphate may be the
mechanism used for the production of pentose, a cofactor that is required for nucleotide
biosynthesis [17]. The presence of the
gene for fructose bisphosphatase (glpX),
which is the key enzyme of gluconeogenesis, in these organisms provides further
evidence that the enzymes of the glycolysis pathway are used in gluconeogenesis
rather than glycolysis (Figures 1 and 8). Consistent with the limited capability
for carbohydrate metabolism, the genes for sugar phosphate transporters or
hexokinases were barely identified in all of the members of Rickettsiales, which
suggests that most of the energy in the members of this order is obtained from
amino acids, rather than hexose catabolism (see below). Pyruvate is the major
product of glycolysis and is used in multiple metabolic pathways. In O. tsutsugamushi, pyruvate would not be
synthesized by the glycolytic pathway (Figure 1). It is possible that it is acquired
from the host, or synthesized from malate by malate dehydrogenase (maeA), and then subsequently converted
to phosphoenol-pyruvate by pyruvate phosphate dikinase (ppdK), both of which are present in all of the Rickettsiales
members (Figures 1 and 8). The presence of a putative permease (OTBS_1312, Table 1) that is found only in Rickettsia and Orientia and may function in
transporting malate from the host cell [24] further supports this
idea.Figure 1
Glycolysis and the
TCA cycle inO. tsutsugamushi. Missing
components and the resultant metabolites that are not synthesized are indicated
in gray. Steps that generate reductive power or lead to the formation of ATP by
substrate-level phosphorylation are indicated in green. The genes and
corresponding CDSs present in the genome are indicated in blue, and pseudogenes
and genes that are absent are shown in gray.Table 1
Membrane transport systems found in theO. tsutsugamushi genome.
Transporter typeGene/CDSPutative substrateATP dependant (ABC family)ABCMembraneBinding proteinyqiX: OTBS_0447Amino acidyqiY: OTBS_0224GlutamineglnQ: OTBS_0223GlutaminemkI: OTBS_0496yrbE: OTBS_0495yrbD: OTBS_1584Toluene toleranceznuC: OTBS_1910znuB: OTBS_1909znuA:OTBS_0441Zinc, manganeseccmA: OTBS_1392ccmB: OTBS_1393HemeccmC: OTBS_1577HememsbA1: OTBS_0552*MultidrugmsbA2: OTBS_0995*MultidrugaprD: OTBS_0305*Alkaliine proteaseabcZ: OTBS_0269abcT3: OTBS_0695*abcT1: OTBS_0781Ion channelsOTBS_1945Mechanosensitive ionSecondary transportertlc: OTBS_0312, OTBS_0313,ATP/ADP antiporterOTBS_0547, OTBS_1035, OTBS_1636OTBS_1312, OTBS_0017?MalateatrC1: OTBS_1417Cationic aminio acidpotE: OTBS_1403Arginine/orinithinep34: OTBS_1715CationkefB: OTBS_1539PotassiumgltP: OTBS_0443Proton/glutamaterarD: OTBS_0203, OTBS_0407?S-adenosylmethionineproP: OTBS_0158, OTBS_0204,Proline/betaineOTBS_0844, OTBS_1493, OTBS_1955panF: OTBS_0515, OTBS_1379,Na+/proline symporterampG: OTBS_0516Muropeptidebcr: OTBS_1499,  OTBS_1891MultidurgmnhE: OTBS_0147, mnhG: OTBS_1421,Na+/H+ antiportermbhE: OTBS_1422mnhB: OTBS_1423, mnhC: OTBS_1424(multisubunit antiporter)UnclassifiedmgtE: OTBS_0470MagnesiumOTBS_0219?iron*Contains both an ABC and a membrane domain as one polypeptide.Figure 2
Nucleotide
biosynthesis. Components of purine and pyrimidine metabolism that are present
in the genome. As described in the text, it is likely that only the salvage
pathways are functional.It
seems likely that pyruvate does not fuel the citrate acid cycle inO. tsutsugamushi because the three
enzymes involved in the initial steps of the pathway are lacking [14, 19]. Among the members of Rickettsiales, O. tsutsugamushi is the only member that
lacks the functional pyruvate dehydrogenase complex and has to rely on the host
cell as a source of acetyl-CoA, which is an essential coenzyme in diverse
biosynthetic pathways. The only functional component of the pyruvate
dehydrogenase complex that we identified was dihydrolipoamide dehydrogenase (lpdA), whereas the other two subunits, pdhB and pdhC, were absent or present as a pseudogene. gltA and acnA which encode
citrate synthase and aconitate hydratase, respectively, are also present as
pseudogenes in O. tsutsugamushi, while
most of the components of the energy-yielding reactions between isocitrate
dehydrogenase (icd) and malate
dehydrogenase (mdh) are present [14, 19]. Considering the lack of
the initiating enzymes of the citric acid cycle, this pathway in O. tsutsugamushi would start with α-ketoglutarate and end with
oxaloacetate. Consistent with the presence of a glutamate transport system (GltP),
the putative glutamine ATP-binding cassette (ABC) transporter (ygiX, ygiY, and glnQ), and glutamine
synthase (glnA), it is possible that
glutamate is imported from the host cell or synthesized from glutamine by GlnA
(Figures 3, 7, and Table 1). Glutamine is converted to aspartate and α-ketoglutarate by AatA, using
oxaloacetate as a cosubstrate and could fuel the “minimal" citrate acid cycle
to generate energy [19].Figure 3
Amino acid
metabolism. The components that are absent from the genome are indicated in
gray. Lysine biosynthesis is partially present and may provide DAP for
peptidoglycan synthesis.ATP is
the universal energy source in all biological systems, and can be synthesized by
glycolysis and oxidative phosphorylation. As in otherRickettsia [23], O. tsutsugamushi possesses the majority of genes for oxidative
phosphorylation including three proton pumps, the succinate dehydrogenase
complex, and the ATP synthase complex. Another mechanism of acquiring ATP is to
import host ATP through ATP/ADP translocases which are present in Rickettsia and Chlamydia [25]. O. tsutsugamushi has five copies of the ATP/ADP translocase (Figure 7 and Table 1). These obligate intracellular pathogens may first exploit ATP that
is already present in the host cell cytoplasm through the function of translocases
and subsequently produce ATP via aerobic respiration when the host pool of ATP
has been consumed [23]. Recently, it was demonstrated
that the translocases of Rickettsia have differential transportation properties for nucleotides [26]. In addition to exchanging
bacterial ADP for host cell ATP as a source of energy, some of the translocases
are believed to function primarily in maintaining intracellular pools of
nucleotides for rickettsial nucleic acid biosynthesis. Consistent with this, Rickettsia and Orientia are generally deficient in the enzymes of de novo nucleotide
synthetic pathways.


### 3.2. Nucleotide Metabolism

Several components of the salvage pathways
of purine and pyrimidine biosynthesis were present inO. tsutsugamushi, similar
to other Rickettsia (Figure 2) [23]. The absence of enzymes
for the interconversion of adenine and guanine suggests that these bacteria
depend on the host for the both purines and may import them via different
subtypes of ATP/ADP translocases [26]. In contrast, the interconversion
of pyrimidine nucleotides is feasible in O. tsutsugamushi due to the presence of deoxycytidine triphosphate deaminase (dcd) and FAD-depedant
thymidylated synthase (thyX)
(Figure 2). The gene (codA, OTBS_1716)
for cytosine deaminase, which converts cytosine into uracil, is present in all
the members of Rickettsiales.Phosphorybosyl pyrophosphate (PRPP), which
is produced by the pentose phosphate pathway, is a key metabolite in the synthesis
of nucleotides.O. tsutsugamushi lacked
all the enzymes for this pathway with the exception of ribose-5-phosphate
isomerase (LacA) (Figure 1). A similar deficiency in the enzymes of this pathway
was reported for Rickettsia [23], while the gene for ribose-phosphate
diphosphokinase (prsA) has been identified
in R. felis and R. bellii. These bacteria may synthesize PRPP from
ribulose-5-phosphate which may be imported from the host cell. In contrast to Orientia and Rickettsia, the members of Anaplasmataceae are well equipped with
the enzymes for the de novo
nucleotide synthetic pathways and the pentose phosphate pathway (Figure 8).The guanosine nucleotides pppGpp and ppGpp
are important second messengers in the bacterial stringent response to cope
with nutritional starvation [27]. O. tsutsugamushi is the only bacterium of the Rickettsiales order
that encodes a fully bifunctional spoT/relA homologue [14, 15, 19]. Even though Orientia and some Rickettsia have several short ORFs encoding either the hydrolase or
synthase domains, the
biological significance of these is unknown [28].


### 3.3. Amino Acid Metabolism

Amino
acid metabolism appeared to be limited inO. tsutsugamushi (Figure 3). A similar level of deficiency in the amino acid metabolic
pathways of Rickettsia has also been
described [23]. The genes for converting
glutamine to glutamate (glnA),
glutamate to α-ketoglutarate
and aspartate (aatA), serine to
glycine (glyA), and branched-chain
amino acid aminotransferase (ilvE) were
present in the genome of O. tsutsugamushi.
In contrast, the members of Anaplasmataceae, particularly Ehrlichia, have a greater capacity to synthesize amino acids such
as proline, arginine, and lysine (Figure 8) [17]. Three genes that are
involved in the biosynthesis of lysine from aspartate, argD, dapF, and lysA, were missing in O. tsutsugamushi. Among the Rickettsiales, argD is absent in Orientia and Rickettsia, while dapF is missing only in Orientia. lysA, which encodes diaminopimelate decarboxylase, is present only
in the genus Ehrlichia. Given that these
genes are not present in Orientia,
they may be involved in the synthesis of diaminopimelate, which is an important
component of peptidoglycan, rather than lysine biosynthesis. Genes for the
biosynthetic pathways for aromatic amino acids (tryptophan, tyrosine, and
phenylalanine), and for histidine are missing in Orientia as well
as other Rickettsia species. Those
amino acids must be provided by the host cell or the culture medium. In
contrast to Rickettsia, Orientia lacks the alanine racemase (alr) which converts L-alanine to
D-alanine, a key component of peptidoglycan [19]. However, it has the
enzymes (ddl, murD, and murF) for incorporating
D variants of amino acids into peptidoglycan, which suggests that this bacterium
may obtain D-amino acids from the host cell, or use the L variants for murein
biosynthesis [29]. For aminoacyl-tRNA
synthesis, it is notable that two copies of tryptophanyl-tRNA synthetase and
two pseudogenes for phenylalanyl-tRNA synthetase alpha chain, in addition to
all 20 aminoacyl tRNA synthetases, were present in the genome of O. tsutsugamushi.


### 3.4. Lipid Metabolism and Cell Wall Structure

The
majority of genes for fatty acid biosynthesis were present inO. tsutsugamushi, as in other members of the Rickettsiales order (Figure 4). An
important exception was fabH which encodes
beta-ketoacyl-acyl carrier protein (ACP) synthase III of the fatty acid
elongation reaction. Interestingly, activation of long chain fatty acids with
ACP by acyl-ACP synthetase (aas) may
occur in Orientia and Rickettsia. The aas gene product catalyzes the synthesis of acyl-ACP from a long
chain fatty acid which may be imported from the host cell, and activated
acyl-ACP might subsequently play a role in the incorporation of fatty acids
into phospholipids [30]. The entire set of
genes for phospholipid synthesis (plsC, cdsA, pssA, psd, pgsA, and pgpA) with the exception of plsB which encodes an enzyme involved in the incorporation of the first acyl chain
into glycerol-3-phosphate, are present in all the members of Rickettsiales (Figure 4). The β-oxidation system of fatty
acids for energy generation was absent in Orientia, whereas this pathway is present in Rickettsia  [23].Figure 4
Phospholipid
synthesis. The pathways for the interconversion of phosphatidyl glycerol,
phosphatidyl serine, and phosphatidyl enthanolamine are complete as well as the
pathway for fatty acid biosynthesis.One of
the major constituents of the outer cell membrane in Gram-negative bacteria is
lipopolysaccharide (LPS). Genomic analysis ofO. tsutsugamushi indicated that it lacks the genes for the
biosynthesis of lipid A, as suggested by previous biochemical analyses [31]. Among the members of Rickettsiales, only Rickettsia are equipped with the genes required
for lipid A biosynthesis (Figure 8) [19]. Consistent with this,
the genes for the O-antigen export system (rfbA,
rfbE) are present only in Rickettsia.The genes for cell wall biosynthesis were present
in the genome ofO. tsutsugamushi (Figure 5), similar to Rickettsia and Wolbachia. Interestingly, A. marginale also has the same set of
genes for peptidoglycan synthesis with the exception of murC which is present as a pseudogene [32]. Other species of Anaplasma and Ehrlichia completely lack these enzymes (Figure 8). Furthermore, the
complete aminosugar metabolic pathway for the synthesis of UDP-N-acetyl
muramate which is an initiating material for peptidoglycan biosynthesis from fructose-6-phosphate
is present (glmS, glmM, glmU, murA, and murB) in A. marginale, whereas glmS, glmM, and glmU are missing in O. tsutsugamushi.
Penicillin-binding proteins are involved in the last stages of peptidoglycan
biosynthesis, and mediate the transglycosylation and transpeptidation reactions
[33]. Two penicillin-binding
proteins (OTBS_0700 and OTBS_2173) that were identified in O. tsutsugamushi are conserved in Rickettsia, Wolbachia,
and A. marginale. Another penicillin-binding
protein, pbpE, is found in Orientia (OTBS_0205) and Rickettsia. AmpC, a β-lactamase
which regulates cell shape [33], is present only in R. felis and R. conorii.Figure 5
Peptidoglycan
biosynthesis. The pathway for the synthesis of peptidoglycan from UDP-N-acetyl-D-glucosamine
is complete. Even though the D-amino acids such as D-glutamate and D-alanine
are not likely to be synthesized byO. tsutsugamushi, the genes for incorporating them into glycan are present.Given the differential distribution of genes for cell wall components in
the genomes of arthropod- and mammalian-associated pathogens, it is possible
that these bacteria may have evolved to modify their envelope structures
depending on the specificity of their host where they have adapted. The
components of the bacterial envelope, such as LPS and peptidoglycan, are strong
inducers of the innate immune responses through Toll-like receptors, which are
conserved from insects and mammals [34]. During the course of intracellular adaptation in higher eukaryotes, the
presence of immune-stimulating components in the bacterial envelope may have
been critical for a successful association with the host. The differential loss
of genes for envelope biogenesis has also occurred during the evolution of the
other insect-associated endosymbionts [29]. Although the common ancestor of Rickettsiales may have all the
components of the cell wall as in other free-living α-proteobacteria, it might
have gradually lost sets of genes for the macromolecular biosynthesis during
the course of reductive genomic evolution and intracellular adaptation.The antigenic
variability ofO. tsutsugamushi among
diverse strains is generated by several immunogenic proteins such as 110 kD,
56 kD, and 47 kD proteins, and it
has hampered the development of a protective vaccine [1, 35]. The 56 kD
strain-specific antigen, which is the main source of antigenic heterogeneity
among different strains [1], is present as a single
copy, and no homologous protein has been identified in other bacteria. Another
major antigen, 47 kD group-specific protein, has been identified, and close
homologues are found in diverse α-proteobacteria
including all the members of Rickettsiales. The conserved 47 kDa antigen may
not contribute to the antigenic variability of Orientia. We also identified two additional OmpA-like membrane
proteins (OTBS_0601, OTBS_1984) which may contribute to the surface
antigenicity of O. tsutsugamushi. It
is interesting to note that one of the OmpA-like proteins (OTBS_0601) is
located just downstream of the 56 kD protein, suggesting that these genes may
form an operon and be expressed coordinately [14]. Like other Rickettsia species, O. tsutsugamushi has multiple types of Sca family genes (OTBS_0102, OTBS_0864,
OTBS_1686, OTBS_1913, OTBS_2126, and OTBS_2137) containing autotransporter
domains [36]. Several of these
surface proteins in Rickettsia are
known as antigenic
determinants and may play a role in adhesion to host cells [37, 38]. Recently, It has been
shown that Sca5 (OmpB) of R. conorii is involved in adhesion to host cells via membrane-associated Ku70 [38]. We previously
identified 6 ORFs encoding genes containing autotransporter domains [14, 19]. None of them showed
close homology with other autotransporters of Rickettsia outside the autotranspoter
domain. Four of them have putative transmembrane domains in their N terminus.
Two autotransporters that do not contain a transmembrane domain are encoded by the
same gene (OTBS_0864 and OTBS_2137) which may have been duplicated through the
action of a transposase. The presence of multiple types of autotransporters in O. tsutsugamushi may contribute to
bacterial pathogenesis, in addition to surface antigenicity.


### 3.5. Metabolism of Cofactors

The
pathways for de novo
biosynthesis of the vitamins and cofactors were generally absent inO. tsutsugamushi. The bacterium retained only
parts of the biosynthetic pathways for heme (hemA, hemB, hemC, hemE, hemF, hemH, ctaB, and cox15) and ubiquinone (ubiA,
ubiD, ubiB, ubiG, ubiH, ubiE, and coq7)
(Figure 6). hemD, which encodes uroporphyrinogen-III
synthase, is present as a pseudogene in O. tsutsugamushi (OTBS_1020) and Rickettsia. The gene
for protoporphyrinogen oxidase (hemY,
hemG) is absent in all the Rickettsiales members. A few genes for the final
components of folate metabolism were also present in O. tsutsugamushi (Figure 6). The genes (pntA and pntB) for the NAD(P)
transhydrogenase complex are present only Rickettsia.Figure 6
Metabolism of Cofactors.
The biosynthetic pathways for porphyrin (heme) and ubiquinone are present inO. tsutsugamushi. Several genes for folate
metabolism were also present in the genome.Figure 7
Comparative
analysis of transporters and their putative substrates in five representative
members of the Rickettsiales order. The types and putative substrates of the
nutrient transporters are as indicated in the figure and legend box. The
figures were constructed using data from transportDB (http://www.membranetransport.org/).
The transporters of O. tsutsugamushi were predicted using a BLASTP search against the transporter database. It is
notable that there are more transporters for amino acids and nucleotides in Orientia and Rickettsia than in members of Anaplasmataceae. The downward
direction of the arrows indicates flow into the bacteria.Figure 8
Comparison of the metabolic
pathway maps of Rickettsiaceae and Anaplasmataceae. The overview of maps is
color coded to indicate pathways that are complete, partial, or absent in
Rickettsiaceae and Anaplasmataceae, based on the data from KEGG DB (http://www.genome.ad.jp/kegg/),
as described in Section 2. The upper panel showing the metabolic map of
Rickettsiaceae is slightly modified from the data reported by Fuxelius et al. [19].The
presence of several essential vitamin and cofactor biosynthetic pathways in the
members of Rickettsiales, particularly Anaplasmataceae, suggests that they do not compete with the host cell for these nutrients
and may even supply host cells with essential vitamins and cofactors [17, 39, 40]. However,
the ability to synthesize some cofactors is somewhat limited in Wolbachia, and even more so in Rickettsia, and Orientia. W. pipientis has completely lost the
biosynthetic pathways for biotin, thiamine, and NAD. R. prowazekii has lost the ability to synthesize these cofactors as
well, and in addition, cannot synthesize FAD, pantothenate, and
pyridoxine-phosphate, which can be produced by Anaplasma, Ehrlichia, and Neorickttsia [17]. O. tsutsugamushi possesses the least capability for vitamin and
cofactor synthesis among the sequenced Rickettsiales members.


### 3.6. Membrane Transporters

The
transporter systems ofRickettsia consist mainly of secondary transporters, in which transport activity is driven
by an ion gradient across the membrane and ABC-type transporters which are driven
by ATP hydrolysis [23, 24]. While the substrates
of these transporters in Rickettsiales members have not been well defined, putative
substrates have been identified by amino acid sequence-based BLAST searches and
domain predictions [24]. As shown in Table 1
and Figure 7, O. tsutsugamushi has a similar
level of transporters as Rickettsia [23]. Both genuses have multiple types of amino acids transporters, as
well as 4-5 copies of ATP/ADP transporters, which are absent in the members of
Anaplasmataceae. Deficiencies in the pathways for amino acid biosynthesis in Rickettsia and Orientia could be partly overcome by the presence of multiple amino
acid transporters (proP,atrC1, panF, and potE) (Figure 7
and Table 1). A putative sodium/alanine symporter (orthologs of AM882) is
present exclusively in Anaplasmataceae family members (Figure 8). In addition, two
ABC-type transporter systems for phosphate (pstA, pstB, pstC, and pstS) and iron
(afuA, afuB, afuC) were missing
only in Orientia and Rickettsia. A putative iron permease
(OTBS_0219) may function as an iron transporter in Orientia and Rickettsia.
Several transporters such as the lipoprotein export system (lolC, lolE, and lolD), lipid A
transporter (rfbA and rfbE), and glycerol-3-phosphate
transporter (glpT) were absent in O. tsutsugamushi, in contrast to other Rickettsia species. Protein secretion systems such as the Sec-mediated translocation
system and the type IV secretion system, other than the highly amplified
conjugative forms are conserved in all the members of Rickettsiales including Orientia.


## 3.1. Carbohydrate and Energy Metabolism

Glycolysis
and the citric acid cycle are the major energy-producing catabolic pathways and
they are conserved in all kingdoms of life. Genome sequence analysis revealed
that some of the enzymes of these pathways are present inO. tsutsugamushi. Three enzymes of the glycolysis pathway, in which
glucose is oxidized to pyruvate, were present (gap, pgk, and tpiA; Figure 1). These enzymes may be
used to generate glycerol phosphate which is the starting material for the synthesis
of glycerophospholipids or used to generate energy from glycerol-3-phosphate by a reverse
reaction. Rickettsia do not possess these
enzymes [23], but they possess a
gene for glycerol phosphate transporter (glpT)
which imports the material from host cells (Table 1 and Figure 7). A limited
number of enzymes of glycolysis were identified in the genomes of Wolbachia, Anaplasma, and Ehrlichia, and it has been suggested that in
these organisms the synthesis of glyceraldehydes-3-phosphate may be the
mechanism used for the production of pentose, a cofactor that is required for nucleotide
biosynthesis [17]. The presence of the
gene for fructose bisphosphatase (glpX),
which is the key enzyme of gluconeogenesis, in these organisms provides further
evidence that the enzymes of the glycolysis pathway are used in gluconeogenesis
rather than glycolysis (Figures 1 and 8). Consistent with the limited capability
for carbohydrate metabolism, the genes for sugar phosphate transporters or
hexokinases were barely identified in all of the members of Rickettsiales, which
suggests that most of the energy in the members of this order is obtained from
amino acids, rather than hexose catabolism (see below). Pyruvate is the major
product of glycolysis and is used in multiple metabolic pathways. In O. tsutsugamushi, pyruvate would not be
synthesized by the glycolytic pathway (Figure 1). It is possible that it is acquired
from the host, or synthesized from malate by malate dehydrogenase (maeA), and then subsequently converted
to phosphoenol-pyruvate by pyruvate phosphate dikinase (ppdK), both of which are present in all of the Rickettsiales
members (Figures 1 and 8). The presence of a putative permease (OTBS_1312, Table 1) that is found only in Rickettsia and Orientia and may function in
transporting malate from the host cell [24] further supports this
idea.Figure 1
Glycolysis and the
TCA cycle inO. tsutsugamushi. Missing
components and the resultant metabolites that are not synthesized are indicated
in gray. Steps that generate reductive power or lead to the formation of ATP by
substrate-level phosphorylation are indicated in green. The genes and
corresponding CDSs present in the genome are indicated in blue, and pseudogenes
and genes that are absent are shown in gray.Table 1
Membrane transport systems found in theO. tsutsugamushi genome.
Transporter typeGene/CDSPutative substrateATP dependant (ABC family)ABCMembraneBinding proteinyqiX: OTBS_0447Amino acidyqiY: OTBS_0224GlutamineglnQ: OTBS_0223GlutaminemkI: OTBS_0496yrbE: OTBS_0495yrbD: OTBS_1584Toluene toleranceznuC: OTBS_1910znuB: OTBS_1909znuA:OTBS_0441Zinc, manganeseccmA: OTBS_1392ccmB: OTBS_1393HemeccmC: OTBS_1577HememsbA1: OTBS_0552*MultidrugmsbA2: OTBS_0995*MultidrugaprD: OTBS_0305*Alkaliine proteaseabcZ: OTBS_0269abcT3: OTBS_0695*abcT1: OTBS_0781Ion channelsOTBS_1945Mechanosensitive ionSecondary transportertlc: OTBS_0312, OTBS_0313,ATP/ADP antiporterOTBS_0547, OTBS_1035, OTBS_1636OTBS_1312, OTBS_0017?MalateatrC1: OTBS_1417Cationic aminio acidpotE: OTBS_1403Arginine/orinithinep34: OTBS_1715CationkefB: OTBS_1539PotassiumgltP: OTBS_0443Proton/glutamaterarD: OTBS_0203, OTBS_0407?S-adenosylmethionineproP: OTBS_0158, OTBS_0204,Proline/betaineOTBS_0844, OTBS_1493, OTBS_1955panF: OTBS_0515, OTBS_1379,Na+/proline symporterampG: OTBS_0516Muropeptidebcr: OTBS_1499,  OTBS_1891MultidurgmnhE: OTBS_0147, mnhG: OTBS_1421,Na+/H+ antiportermbhE: OTBS_1422mnhB: OTBS_1423, mnhC: OTBS_1424(multisubunit antiporter)UnclassifiedmgtE: OTBS_0470MagnesiumOTBS_0219?iron*Contains both an ABC and a membrane domain as one polypeptide.Figure 2
Nucleotide
biosynthesis. Components of purine and pyrimidine metabolism that are present
in the genome. As described in the text, it is likely that only the salvage
pathways are functional.It
seems likely that pyruvate does not fuel the citrate acid cycle inO. tsutsugamushi because the three
enzymes involved in the initial steps of the pathway are lacking [14, 19]. Among the members of Rickettsiales, O. tsutsugamushi is the only member that
lacks the functional pyruvate dehydrogenase complex and has to rely on the host
cell as a source of acetyl-CoA, which is an essential coenzyme in diverse
biosynthetic pathways. The only functional component of the pyruvate
dehydrogenase complex that we identified was dihydrolipoamide dehydrogenase (lpdA), whereas the other two subunits, pdhB and pdhC, were absent or present as a pseudogene. gltA and acnA which encode
citrate synthase and aconitate hydratase, respectively, are also present as
pseudogenes in O. tsutsugamushi, while
most of the components of the energy-yielding reactions between isocitrate
dehydrogenase (icd) and malate
dehydrogenase (mdh) are present [14, 19]. Considering the lack of
the initiating enzymes of the citric acid cycle, this pathway in O. tsutsugamushi would start with α-ketoglutarate and end with
oxaloacetate. Consistent with the presence of a glutamate transport system (GltP),
the putative glutamine ATP-binding cassette (ABC) transporter (ygiX, ygiY, and glnQ), and glutamine
synthase (glnA), it is possible that
glutamate is imported from the host cell or synthesized from glutamine by GlnA
(Figures 3, 7, and Table 1). Glutamine is converted to aspartate and α-ketoglutarate by AatA, using
oxaloacetate as a cosubstrate and could fuel the “minimal" citrate acid cycle
to generate energy [19].Figure 3
Amino acid
metabolism. The components that are absent from the genome are indicated in
gray. Lysine biosynthesis is partially present and may provide DAP for
peptidoglycan synthesis.ATP is
the universal energy source in all biological systems, and can be synthesized by
glycolysis and oxidative phosphorylation. As in otherRickettsia [23], O. tsutsugamushi possesses the majority of genes for oxidative
phosphorylation including three proton pumps, the succinate dehydrogenase
complex, and the ATP synthase complex. Another mechanism of acquiring ATP is to
import host ATP through ATP/ADP translocases which are present in Rickettsia and Chlamydia [25]. O. tsutsugamushi has five copies of the ATP/ADP translocase (Figure 7 and Table 1). These obligate intracellular pathogens may first exploit ATP that
is already present in the host cell cytoplasm through the function of translocases
and subsequently produce ATP via aerobic respiration when the host pool of ATP
has been consumed [23]. Recently, it was demonstrated
that the translocases of Rickettsia have differential transportation properties for nucleotides [26]. In addition to exchanging
bacterial ADP for host cell ATP as a source of energy, some of the translocases
are believed to function primarily in maintaining intracellular pools of
nucleotides for rickettsial nucleic acid biosynthesis. Consistent with this, Rickettsia and Orientia are generally deficient in the enzymes of de novo nucleotide
synthetic pathways.


## 3.2. Nucleotide Metabolism

Several components of the salvage pathways
of purine and pyrimidine biosynthesis were present inO. tsutsugamushi, similar
to other Rickettsia (Figure 2) [23]. The absence of enzymes
for the interconversion of adenine and guanine suggests that these bacteria
depend on the host for the both purines and may import them via different
subtypes of ATP/ADP translocases [26]. In contrast, the interconversion
of pyrimidine nucleotides is feasible in O. tsutsugamushi due to the presence of deoxycytidine triphosphate deaminase (dcd) and FAD-depedant
thymidylated synthase (thyX)
(Figure 2). The gene (codA, OTBS_1716)
for cytosine deaminase, which converts cytosine into uracil, is present in all
the members of Rickettsiales.Phosphorybosyl pyrophosphate (PRPP), which
is produced by the pentose phosphate pathway, is a key metabolite in the synthesis
of nucleotides.O. tsutsugamushi lacked
all the enzymes for this pathway with the exception of ribose-5-phosphate
isomerase (LacA) (Figure 1). A similar deficiency in the enzymes of this pathway
was reported for Rickettsia [23], while the gene for ribose-phosphate
diphosphokinase (prsA) has been identified
in R. felis and R. bellii. These bacteria may synthesize PRPP from
ribulose-5-phosphate which may be imported from the host cell. In contrast to Orientia and Rickettsia, the members of Anaplasmataceae are well equipped with
the enzymes for the de novo
nucleotide synthetic pathways and the pentose phosphate pathway (Figure 8).The guanosine nucleotides pppGpp and ppGpp
are important second messengers in the bacterial stringent response to cope
with nutritional starvation [27]. O. tsutsugamushi is the only bacterium of the Rickettsiales order
that encodes a fully bifunctional spoT/relA homologue [14, 15, 19]. Even though Orientia and some Rickettsia have several short ORFs encoding either the hydrolase or
synthase domains, the
biological significance of these is unknown [28].


## 3.3. Amino Acid Metabolism

Amino
acid metabolism appeared to be limited inO. tsutsugamushi (Figure 3). A similar level of deficiency in the amino acid metabolic
pathways of Rickettsia has also been
described [23]. The genes for converting
glutamine to glutamate (glnA),
glutamate to α-ketoglutarate
and aspartate (aatA), serine to
glycine (glyA), and branched-chain
amino acid aminotransferase (ilvE) were
present in the genome of O. tsutsugamushi.
In contrast, the members of Anaplasmataceae, particularly Ehrlichia, have a greater capacity to synthesize amino acids such
as proline, arginine, and lysine (Figure 8) [17]. Three genes that are
involved in the biosynthesis of lysine from aspartate, argD, dapF, and lysA, were missing in O. tsutsugamushi. Among the Rickettsiales, argD is absent in Orientia and Rickettsia, while dapF is missing only in Orientia. lysA, which encodes diaminopimelate decarboxylase, is present only
in the genus Ehrlichia. Given that these
genes are not present in Orientia,
they may be involved in the synthesis of diaminopimelate, which is an important
component of peptidoglycan, rather than lysine biosynthesis. Genes for the
biosynthetic pathways for aromatic amino acids (tryptophan, tyrosine, and
phenylalanine), and for histidine are missing in Orientia as well
as other Rickettsia species. Those
amino acids must be provided by the host cell or the culture medium. In
contrast to Rickettsia, Orientia lacks the alanine racemase (alr) which converts L-alanine to
D-alanine, a key component of peptidoglycan [19]. However, it has the
enzymes (ddl, murD, and murF) for incorporating
D variants of amino acids into peptidoglycan, which suggests that this bacterium
may obtain D-amino acids from the host cell, or use the L variants for murein
biosynthesis [29]. For aminoacyl-tRNA
synthesis, it is notable that two copies of tryptophanyl-tRNA synthetase and
two pseudogenes for phenylalanyl-tRNA synthetase alpha chain, in addition to
all 20 aminoacyl tRNA synthetases, were present in the genome of O. tsutsugamushi.


## 3.4. Lipid Metabolism and Cell Wall Structure

The
majority of genes for fatty acid biosynthesis were present inO. tsutsugamushi, as in other members of the Rickettsiales order (Figure 4). An
important exception was fabH which encodes
beta-ketoacyl-acyl carrier protein (ACP) synthase III of the fatty acid
elongation reaction. Interestingly, activation of long chain fatty acids with
ACP by acyl-ACP synthetase (aas) may
occur in Orientia and Rickettsia. The aas gene product catalyzes the synthesis of acyl-ACP from a long
chain fatty acid which may be imported from the host cell, and activated
acyl-ACP might subsequently play a role in the incorporation of fatty acids
into phospholipids [30]. The entire set of
genes for phospholipid synthesis (plsC, cdsA, pssA, psd, pgsA, and pgpA) with the exception of plsB which encodes an enzyme involved in the incorporation of the first acyl chain
into glycerol-3-phosphate, are present in all the members of Rickettsiales (Figure 4). The β-oxidation system of fatty
acids for energy generation was absent in Orientia, whereas this pathway is present in Rickettsia  [23].Figure 4
Phospholipid
synthesis. The pathways for the interconversion of phosphatidyl glycerol,
phosphatidyl serine, and phosphatidyl enthanolamine are complete as well as the
pathway for fatty acid biosynthesis.One of
the major constituents of the outer cell membrane in Gram-negative bacteria is
lipopolysaccharide (LPS). Genomic analysis ofO. tsutsugamushi indicated that it lacks the genes for the
biosynthesis of lipid A, as suggested by previous biochemical analyses [31]. Among the members of Rickettsiales, only Rickettsia are equipped with the genes required
for lipid A biosynthesis (Figure 8) [19]. Consistent with this,
the genes for the O-antigen export system (rfbA,
rfbE) are present only in Rickettsia.The genes for cell wall biosynthesis were present
in the genome ofO. tsutsugamushi (Figure 5), similar to Rickettsia and Wolbachia. Interestingly, A. marginale also has the same set of
genes for peptidoglycan synthesis with the exception of murC which is present as a pseudogene [32]. Other species of Anaplasma and Ehrlichia completely lack these enzymes (Figure 8). Furthermore, the
complete aminosugar metabolic pathway for the synthesis of UDP-N-acetyl
muramate which is an initiating material for peptidoglycan biosynthesis from fructose-6-phosphate
is present (glmS, glmM, glmU, murA, and murB) in A. marginale, whereas glmS, glmM, and glmU are missing in O. tsutsugamushi.
Penicillin-binding proteins are involved in the last stages of peptidoglycan
biosynthesis, and mediate the transglycosylation and transpeptidation reactions
[33]. Two penicillin-binding
proteins (OTBS_0700 and OTBS_2173) that were identified in O. tsutsugamushi are conserved in Rickettsia, Wolbachia,
and A. marginale. Another penicillin-binding
protein, pbpE, is found in Orientia (OTBS_0205) and Rickettsia. AmpC, a β-lactamase
which regulates cell shape [33], is present only in R. felis and R. conorii.Figure 5
Peptidoglycan
biosynthesis. The pathway for the synthesis of peptidoglycan from UDP-N-acetyl-D-glucosamine
is complete. Even though the D-amino acids such as D-glutamate and D-alanine
are not likely to be synthesized byO. tsutsugamushi, the genes for incorporating them into glycan are present.Given the differential distribution of genes for cell wall components in
the genomes of arthropod- and mammalian-associated pathogens, it is possible
that these bacteria may have evolved to modify their envelope structures
depending on the specificity of their host where they have adapted. The
components of the bacterial envelope, such as LPS and peptidoglycan, are strong
inducers of the innate immune responses through Toll-like receptors, which are
conserved from insects and mammals [34]. During the course of intracellular adaptation in higher eukaryotes, the
presence of immune-stimulating components in the bacterial envelope may have
been critical for a successful association with the host. The differential loss
of genes for envelope biogenesis has also occurred during the evolution of the
other insect-associated endosymbionts [29]. Although the common ancestor of Rickettsiales may have all the
components of the cell wall as in other free-living α-proteobacteria, it might
have gradually lost sets of genes for the macromolecular biosynthesis during
the course of reductive genomic evolution and intracellular adaptation.The antigenic
variability ofO. tsutsugamushi among
diverse strains is generated by several immunogenic proteins such as 110 kD,
56 kD, and 47 kD proteins, and it
has hampered the development of a protective vaccine [1, 35]. The 56 kD
strain-specific antigen, which is the main source of antigenic heterogeneity
among different strains [1], is present as a single
copy, and no homologous protein has been identified in other bacteria. Another
major antigen, 47 kD group-specific protein, has been identified, and close
homologues are found in diverse α-proteobacteria
including all the members of Rickettsiales. The conserved 47 kDa antigen may
not contribute to the antigenic variability of Orientia. We also identified two additional OmpA-like membrane
proteins (OTBS_0601, OTBS_1984) which may contribute to the surface
antigenicity of O. tsutsugamushi. It
is interesting to note that one of the OmpA-like proteins (OTBS_0601) is
located just downstream of the 56 kD protein, suggesting that these genes may
form an operon and be expressed coordinately [14]. Like other Rickettsia species, O. tsutsugamushi has multiple types of Sca family genes (OTBS_0102, OTBS_0864,
OTBS_1686, OTBS_1913, OTBS_2126, and OTBS_2137) containing autotransporter
domains [36]. Several of these
surface proteins in Rickettsia are
known as antigenic
determinants and may play a role in adhesion to host cells [37, 38]. Recently, It has been
shown that Sca5 (OmpB) of R. conorii is involved in adhesion to host cells via membrane-associated Ku70 [38]. We previously
identified 6 ORFs encoding genes containing autotransporter domains [14, 19]. None of them showed
close homology with other autotransporters of Rickettsia outside the autotranspoter
domain. Four of them have putative transmembrane domains in their N terminus.
Two autotransporters that do not contain a transmembrane domain are encoded by the
same gene (OTBS_0864 and OTBS_2137) which may have been duplicated through the
action of a transposase. The presence of multiple types of autotransporters in O. tsutsugamushi may contribute to
bacterial pathogenesis, in addition to surface antigenicity.


## 3.5. Metabolism of Cofactors

The
pathways for de novo
biosynthesis of the vitamins and cofactors were generally absent inO. tsutsugamushi. The bacterium retained only
parts of the biosynthetic pathways for heme (hemA, hemB, hemC, hemE, hemF, hemH, ctaB, and cox15) and ubiquinone (ubiA,
ubiD, ubiB, ubiG, ubiH, ubiE, and coq7)
(Figure 6). hemD, which encodes uroporphyrinogen-III
synthase, is present as a pseudogene in O. tsutsugamushi (OTBS_1020) and Rickettsia. The gene
for protoporphyrinogen oxidase (hemY,
hemG) is absent in all the Rickettsiales members. A few genes for the final
components of folate metabolism were also present in O. tsutsugamushi (Figure 6). The genes (pntA and pntB) for the NAD(P)
transhydrogenase complex are present only Rickettsia.Figure 6
Metabolism of Cofactors.
The biosynthetic pathways for porphyrin (heme) and ubiquinone are present inO. tsutsugamushi. Several genes for folate
metabolism were also present in the genome.Figure 7
Comparative
analysis of transporters and their putative substrates in five representative
members of the Rickettsiales order. The types and putative substrates of the
nutrient transporters are as indicated in the figure and legend box. The
figures were constructed using data from transportDB (http://www.membranetransport.org/).
The transporters of O. tsutsugamushi were predicted using a BLASTP search against the transporter database. It is
notable that there are more transporters for amino acids and nucleotides in Orientia and Rickettsia than in members of Anaplasmataceae. The downward
direction of the arrows indicates flow into the bacteria.Figure 8
Comparison of the metabolic
pathway maps of Rickettsiaceae and Anaplasmataceae. The overview of maps is
color coded to indicate pathways that are complete, partial, or absent in
Rickettsiaceae and Anaplasmataceae, based on the data from KEGG DB (http://www.genome.ad.jp/kegg/),
as described in Section 2. The upper panel showing the metabolic map of
Rickettsiaceae is slightly modified from the data reported by Fuxelius et al. [19].The
presence of several essential vitamin and cofactor biosynthetic pathways in the
members of Rickettsiales, particularly Anaplasmataceae, suggests that they do not compete with the host cell for these nutrients
and may even supply host cells with essential vitamins and cofactors [17, 39, 40]. However,
the ability to synthesize some cofactors is somewhat limited in Wolbachia, and even more so in Rickettsia, and Orientia. W. pipientis has completely lost the
biosynthetic pathways for biotin, thiamine, and NAD. R. prowazekii has lost the ability to synthesize these cofactors as
well, and in addition, cannot synthesize FAD, pantothenate, and
pyridoxine-phosphate, which can be produced by Anaplasma, Ehrlichia, and Neorickttsia [17]. O. tsutsugamushi possesses the least capability for vitamin and
cofactor synthesis among the sequenced Rickettsiales members.


## 3.6. Membrane Transporters

The
transporter systems ofRickettsia consist mainly of secondary transporters, in which transport activity is driven
by an ion gradient across the membrane and ABC-type transporters which are driven
by ATP hydrolysis [23, 24]. While the substrates
of these transporters in Rickettsiales members have not been well defined, putative
substrates have been identified by amino acid sequence-based BLAST searches and
domain predictions [24]. As shown in Table 1
and Figure 7, O. tsutsugamushi has a similar
level of transporters as Rickettsia [23]. Both genuses have multiple types of amino acids transporters, as
well as 4-5 copies of ATP/ADP transporters, which are absent in the members of
Anaplasmataceae. Deficiencies in the pathways for amino acid biosynthesis in Rickettsia and Orientia could be partly overcome by the presence of multiple amino
acid transporters (proP,atrC1, panF, and potE) (Figure 7
and Table 1). A putative sodium/alanine symporter (orthologs of AM882) is
present exclusively in Anaplasmataceae family members (Figure 8). In addition, two
ABC-type transporter systems for phosphate (pstA, pstB, pstC, and pstS) and iron
(afuA, afuB, afuC) were missing
only in Orientia and Rickettsia. A putative iron permease
(OTBS_0219) may function as an iron transporter in Orientia and Rickettsia.
Several transporters such as the lipoprotein export system (lolC, lolE, and lolD), lipid A
transporter (rfbA and rfbE), and glycerol-3-phosphate
transporter (glpT) were absent in O. tsutsugamushi, in contrast to other Rickettsia species. Protein secretion systems such as the Sec-mediated translocation
system and the type IV secretion system, other than the highly amplified
conjugative forms are conserved in all the members of Rickettsiales including Orientia.


## 4. Conclusion

We
performed an analysis of the genomic sequence ofO. tsutsugamushi and have identified several fundamental properties of
the bacterium including metabolic properties and cell wall structure.
Genome-based metabolic construction of O. tsutsugamushi revealed that this organism has limited central metabolic and biosynthetic capability,
similar to other Rickettsia species.
Even though it has the largest genome among the members of Rickettsiales, it
lacks a majority of the components of the major biosynthetic pathways. Rather,
it has streamlined its metabolic pathways to a greater extent than other Rickettsia. Due to its limited metabolic
capabilities, O. tsutsugamushi most
likely relies on its host for many organic nutrients. In this regard, the
presence of a diverse set of transporters for various nutrients that is required
for bacterial growth within the host cell may compensate for the lack of
metabolic pathways (Table 1 and Figure 7). In light of its apparently extreme
dependence on the host cell for nutrients, the bacterium may have evolved to
retain sensors that react to environmental changes, and to regulate growth
within the host cell. This may be particularly true for arthropod hosts, in
which the bacterium has evolved for several hundreds of millions of years [15]. O. tsutsugamushi encodes a
fully functional stringent response regulator, spoT/relA, multiple types of two-component signal transduction
systems, and diverse host interaction genes, all of which may be involved in
the bacteria-host interaction [14]. Modification of the bacterial
envelope and the presence of multiple membrane proteins with antigenic potential
indicate that the bacterium may have been evolved under pressure from the host
immune system [41].In
addition to gaining a better understanding of the basic physiology and
virulence of intracellular pathogens, the development of improved diagnostics,
vaccines, and novel therapeutics is one of the major aims of bacterial genome
analysis. Postgenomic approaches such as expression profiling during growth
within a mammalian host cells have to be undertaken in order to achieve these ultimate
goals. In addition, functional studies of putative proteins of unknown
function, which comprise approximately 30% of the total CDSs of theO. tsutsugamushi genome, must also be carried
out and may provide additional therapeutic targets. In particular, potential cell
surface proteins and host interaction proteins could be placed as genes of priority
in future functional studies.



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*Source: 623145-2008-06-01.xml*