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Tsuyoshi Tanaka, Yoshio
Tateno, and Takashi
Gojobori - Center for
Information Biology and
DNA Data Bank of Japan,
National Institute of
Genetics, Mishima, Japan
Vitamin B6 (VB6)
functions as a cofactor
of many diverse enzymes
in amino acid
metabolism. Three
metabolic pathways for
pyridoxal 59-phosphate (PLP;
the active form of VB6)
are known: the de novo
pathway, the salvage
pathway, and the fungal
type pathway. Most
unicellular organisms
and plants biosynthesize
VB6 using one or two of
these three biosynthetic
pathways. However,
animals such as insects
and mammals do not
possess any of the
pathways and, thus, need
to intake VB6 in their
diet to survive. It is
conceivable that
breakdowns of these
pathways occurred in the
evolutionary lineages of
insects and mammals, and
one of the major reasons
for this would be the
loss of pertinent genes.
We studied the evolution
of VB6 biosynthesis from
the view of the gain and
loss of 10 pertinent
genes in 122 species
whose genome sequences
were completely
determined. The results
revealed that each gene
in the pathways was lost
more than once in the
entire evolutionary
lineages of the 122
species. We also found
the following three
points regarding the
evolution of PLP
biosynthesis: (1) the
breakdown of the PLP
biosynthetic pathways
occurred independently
at least three times in
animal lineages, (2) the
de novo pathway was
formed by the generation
of pdxB in c-proteobacteria,
and (3) the order of the
gene loss in VB6
metabolism was conserved
among different
evolutionary lineages.
These results suggest
that the evolution of
VB6 metabolism was
subject to gains and
frequent losses of
related genes in the 122
species
examined. This dynamic
nature of the
evolutionary changes
must have been
responsible for the
breakdowns of the
pathways, resulting in
profound differentiation
of heterotrophy among
the species.
Introduction
Vitamin B6 (VB6)
functions as a cofactor
of many enzymes. In
particular, pyridoxal
59-phosphate (PLP),
which is the active form
of VB6, has multiple
roles as a versatile
cofactor of enzymes that
are mainly involved in
the metabolism of amino
acid compounds (Grogan
1988; Rottmann et al.
1991; Helmreich 1992;
Mihara et al. 1997; Kack
et al. 1999). Moreover,
VB6 appears to play an
important role against
photosensitization in
fungi (Bilski et al.
2000). Most unicellular
organisms and plants
biosynthesize PLP by
themselves.
Many studies of VB6
metabolism have been
conducted in Escherichia
coli (White and Dempsey
1970; Lam and Winkler
1990; Zhao et al. 1995;
Yang, Zhao, and Winkler
1996; Man, Zhao, and
Winkler 1996) and
fungi such as Cercospora
nicotianae, Neurospora
crassa, Aspergillus
nidulans, and
Saccharomyces cerevisiae
(Ehrenshaft et al. 1999;
Osmani, May, and Osmani
1999; Bean et al. 2001;
Ehrenshaft and Daub
2001). For PLP
biosynthesis, three
pathways have been
characterized, and a
total of 10 genes are
involved in the pathways
in bacteria and fungi
(fig. 1). In the case of
E. coli, both de novo
and salvage pathways
have been identified.
These two pathways
include enzymes encoded
by eight genes in total
(Mittenhuber 2001) and
share only one gene,
pdxH. In the de novo
pathway, the pyridine
ring of VB6 is generated
from
D-erythrose-4-phosphate
(E4P) and
glyceraldehyde-3-phosphate.
On the other hand, in
the salvage pathway, PLP
is synthesized without
pyridine ring
generation. A
corresponding de novo
pathway has not been
discovered in
fungi or plants.
Instead, fungi were
found to have another
biosynthetic pathway,
the fungal type pathway.
This pathway has two
genes, SNZ and SNO,
whose functions are
currently unknown.
Therefore, species that
synthesize PLP have been
reported to have at
least one of the three
PLP biosynthetic
pathways.
Animals such as insects
and mammals have to
intake VB6 compounds in
their diet. In
particular, humans
intake VB6 in meats and
vegetables (Manore
2000). Animal lineages,
therefore, have a
problem regarding VB6
metabolism, and it is
conceivable that their
PLP biosynthetic
pathways became
dysfunctional because of
the loss of some of the
pathway genes during
their evolution,
assuming that the
ancestor of animals had
the PLP biosynthetic
pathways. In fact, pdxA
and pdxJ, which are two
of the abovementioned 10
genes, have not been
found in animals
(Ehrenshaft et al.
1999). Moreover, SNZ and
SNO have been reported
to be lost in animals,
except for the marine
sponge Suberites
domuncula (Seack et al.
2001). Even though the
functions of SNZ and SNO
are unknown, they should
have an indispensable
role in the biosynthetic
pathway in fungi.
The loss of these two
genes has been
considered as a cause of
the inability of animal
lineages to
biosynthesize PLP,
especially the
Eumetazoan lineage.
To study the
evolutionary process of
VB6 metabolism, we
investigated the 10
genes involved in the
three pathways for PLP
biosynthesis. We were
particularly interested
in learning when the
individual genes were
gained or lost during
evolution. Therefore, we
focused on the gain and
loss of these 10 genes
in 122 species in the
three domains of life,
namely eubacteria,
archaebacteria, and
eukaryotes (Woese,
Kandler, and Wheelis
1990). We estimated the
pertinent gene set of
the common ancestor of
the 122 species on the
basis of their
genealogical
relationships. Next, we
identified the gain and
loss events for the
genes by comparing the
gene sets between the
ancestral and extant
species. On the basis of
the results obtained, we
report the evolutionary
features of the
formation and
dysfunction processes of
VB6 metabolism from the
view of the gains and
losses of the genes.
Materials and Methods
Genes Related to VB6
Metabolism
There are 10 genes
involved in the three
PLP biosynthetic
pathways: glyceraldehyde
3-P
dehydrogenaseA(gapA),deoxy-xylulose-P
synthase (dxs),
4-hydroxythreonine-4-
phosphate dehydrogenase
(pdxA),
erythronate-4-phosphate
dehydrogenase (pdxB),
phosphoserine
aminotransferase (pdxF),
pyridoxine-phosphate
oxidase (pdxH),
pyridoxalphosphate
biosynthetic protein (pdxJ)
and pyridoxal kinase
(pdxK) in E. coli, and
SNZ and SNO in yeast
(Yang et al. 1998a,
1998b; Mittenhuber
2001). To identify the
PLP biosynthetic pathway
genes in the complete
genome sequences of 122
species (nine
eukaryotes, 16
archaebacteria, and 97
eubacteria) (appendix A
of Supplemental Material
online), BlastP homology
searches for the 10
genes were performed
against each set of
their predicted protein
sequences (E value ,
1025) (Altschul et al.
1990). As query
sequences, we used the
protein sequences of
gapA (DDBJ/ EMBL/GenBank
accession number
AAC74849), dxs
(accession number
AAC73523), pdxA
(accession number
AAC73163), pdxB
(accession number
AAC75380), pdxF
(accession number
AAC73993), pdxH
(accession number
AAC74710), pdxJ
(accession number
AAC75617), and pdxK
(accession number
AAC75471) derived from
E. coli K-12 MG1655 and
those of SNZ (NP_013814)
and SNO (NP_013813)
derived from S.
cerevisiae. Next, we
compared our results
with the KEGG Orthology
(KO) data set in the
KEGG database (Bono et
al. 1998; Kanehisa et
al. 2002) for
confirmation. The KO
data set contains
orthologous gene
families that are
categorized on the basis
of experimental
information, sequence
homology, and gene order
in the genome. If a gene
were found to be related
to metabolic reactions
other than the PLP
biosynthetic pathways,
we removed it from our
data set. Finally, if
any of the 10 genes
could not be found by
the homology search
against the set of
predicted protein
sequences of a
particular species, we
conducted a TBlastN
search of its genome
sequence (E value ,
1025). If there was no
homologous sequence to
the query sequence in
the genome, we concluded
that the gene was absent
from the species.
Phylogenetic Tree
To estimate the gene
sets involved with the
PLP biosynthetic
pathways of ancestors of
the 122 species, we
basically employed the
eukaryotic lineages of
Baldauf et al. (2000)
and the eubacterial and
archaebacterial lineages
of Nelson et al. (2000).
For missing species in
the eubacterial and
archaebacterial
lineages, such as
proteobacteria,
firmicutes,
actinobacteria,
chlamydia, spirochete,
cyanobacteria,
euryarchaeota, and
crenarchaeota, we
constructed their
phylogenetic trees for
the same gene (16s rRNA)
as that used by Nelson
et al. (2000). To do
this, we applied their
16s rRNA sequences to
the ClustalW program
with 1,000 bootstrap
trials (Thompson,
Higgins, and Gibson
1994). We excluded the
positions with gaps and
corrected for multiple
substitutions.
Estimation of the Gene
Set of the PLP
Biosynthetic Pathways in
the Ancestor
We assumed that a single
gene was acquired only
once during the
evolution of the 122
species examined in
this model and ignored
horizontal gene transfer
and parallel evolution.
The method for
estimating the set of
genes in the ancestor
was as follows. As shown
in figure 2, we used the
following two states to
represent whether a
species had a particular
gene (Gene-A): ‘‘1’’ was
used when the species
had at least one
homologous gene to
Gene-A, and ‘‘0’’ was
used when the species
had no homologous genes
to Gene-A.
If the states of two
species were the same,
namely (0,0) or (1,1),
we assumed that the
ancestor had the same
state (patterns 1 and 2
in figure 2). If the
state was different
between the two closest
species compared, namely
(0,1) or (1,0), we used
the states for all the
other species as
outgroup species. For
example, let us
designate a group of
these species as group
C. If at least one
species in group C had
the state of ‘‘1,’’ we
regarded the ancestral
status as ‘‘1’’ (pattern
3 in figure 2). If all
the species in group C
had the status of ‘‘0,’’
we regarded the
ancestral state as ‘‘0’’
(pattern 4 in figure 2).
In this way, we
estimated the states for
all the given genes at
all internal nodes. The
Order of the Losses
Among the Genes
Using the results for
the estimation of the
gene losses, we
investigated the order
of the losses among the
10 genes. If the loss of
a gene occurred randomly
during the evolution of
the PLP biosynthetic
pathways, skewness of
the early loss of either
one of the two genes may
not be observed (null
hypothesis). Therefore,
we considered that the
frequency of the order
of gene loss between two
genes followed the
binominal distribution.
For all pairs among the
10 genes, we examined
which gene was lost
first during the
evolution of the 122
species and
statistically tested the
frequency of the order
of gene loss on the
basis of the binominal
distribution. If the
losses of two genes
occurred on the same
branch in the
phylogenetic tree, we
did not count them,
because we did not know
which gene was lost
first.
Results
Comparison of the Gene
Sets Among Species Three
PLP biosynthetic
pathways have been
reportedas described
earlier: the de novo and
salvage pathways in E.
coli and the fungal-type
pathway. When we
examined whether the 10
genes existed in the 122
species whose genome
sequences were
completely or almost
completely determined,
we found that no species
had all the genes
(appendix A of
Supplemental Material
online). In the cases of
gapA, pdxA, and pdxK, we
found several homologous
sequences among the
species. When we
constructed the
phylogenetic trees for
the 10 genes, we found
that duplication of the
genes existing in each
gene family may have
occurred not only in the
particular lineage
recently but also in the
ancestral lineage (data
not shown). This
observation suggests
that each of the 10 gene
families, especially
gapA, pdxA, and pdxK,
evolved from the common
ancestral sequence by
gene duplication and
gene loss. However, we
could not conclude that
all the genes listed
were involved in VB6
synthesis, because there
was no evidence for
their functions in the
experimental results.
Moreover, we found that
gene sets containing all
seven genes for the de
novo pathway (gapA, dxs,
pdxA,
pdxB, pdxF, pdxH, and
pdxJ) were only found in
18 eubacteria. These
bacterial species were
all c-proteobacteria,
indicating that the de
novo pathway may only
function in
c-proteobacteria.
Distribution of the
Genes in the Three
Domains of Life Based on
the gene sets of the 122
species, we compared the
gene sets among the
three domains of life
(fig. 3). SNZ, SNO, and
pdxF were observed in
all the domains,
indicating that they
existed before the
divergence into the
three domains. This
result also indicates
that the fungal type
pathway composed of SNZ
and SNO was formed
before the divergence
into the three domains.
The other four genes in
the de novo and salvage
pathways (gapA, dxs,
pdxH, and pdxK) were
discovered in eukaryotes
and eubacteria,
indicating that part of
these pathways was
present in eukaryotes
and eubacteria. Based on
our assumption that a
single gene was acquired
only once during the
evolution of the 122
species examined, we
interpret this result to
indicate that the
salvage pathway composed
of pdxH and pdxK was
formed before the
divergence into the
three domains. We also
note that pdxA, pdxB,
and pdxJ were only
observed in eubacteria.
When we focused on the
eubacteria, we found
that both pdxA and
pdxJ were observed not
only in proteobacteria
but also firmicutes,
cyanobacteria, chlorobi,
and aquificae, whereas
pdxB only existed in c-proteobacteria.
Therefore, we consider
that both pdxA and pdxJ
were generated in the
eubacterial lineage
after the divergence
from the other two
domains and that pdxB
was generated in the c-proteobacterial
lineage after the
divergence from the
other lineages. These
three genes are,
therefore, considered to
have contributed to the
formation of the de novo
pathway in the
eubacterial
lineage. In particular,
pdxB may be the most
important gene for the
completion of this
pathway because it was
only generated in c-proteobacteria.
Estimation of the Losses
of the Genes
Because the gene sets of
the PLP biosynthetic
pathways were different
among the 122 species
examined, it was
considered that loss as
well as gain of genes
had occurred for the 10
genes. We, therefore,
examined how many losses
of the 10 genes had
occurred during the
evolution of the 122
species. To estimate the
occurrence of the gain
or loss of a particular
gene in the evolutionary
lineage from the common
ancestor to each extant
species, we needed to
estimate whether the
gene had existed in the
common ancestor. Using
the phylogenetic tree,
we estimated the
ancestral gene set at
each node based on the
assumption that each of
the genes was acquired
only once during the
evolution of the entire
122 species. As a
result, we found that a
total of 132 gene losses
hadoccurred during the
evolution of the 122
species from the common
ancestor (table 1). The
numbers of losses of
pdxB, gapA, and pdxJ
were 2, 3, and 8,
respectively, and all
smaller than the numbers
of losses of the other
seven genes. In the case
of gapA, the lower
number of gene
losses may be explained
by the following
functional constraint:
GapA functions not only
in the PLP biosynthetic
pathways but also in
glycolysis (Seta et al.
1997), and, therefore,
gapA is not expected to
be lost because of
functional constraints.
Because we estimated
that pdxB had emerged in
the c-proteobacterial
lineage, we did not need
to count the number of
losses of the gene
before the divergence of
this lineage from the
other lineages, and,
thus, the total number
of losses of this gene
was smaller than those
of the other genes. In
the case of pdxJ, we
estimated that this gene
emerged in the
eubacterial lineage and
was lost during an early
period in the particular
lineages of the 97
eubacterial species. The
total number of losses
of pdxJ was, therefore,
smaller than those of
the other genes, because
it was only present in
the lineages for a
comparatively short
time.
Losses of SNO and SNZ in
Animal Lineages The SNO
and SNZ genes have only
been reported in the
sponge S. domuncula
among Metazoa. By
homology searches
against the genome
sequences of six animals
(Homo sapiens, Mus
musculus, Rattus
norvegicus, Ciona
intestinalis,
Caenorhabditis elegans,
and Drosophila
melanogaster), we found
that these two genes
also existed in the
genome of C.
intestinalis. The
positions of the two
genes in the genome of
C. intestinalis, in
which they aligned
head-to-head, were the
same as those in S.
domuncula and S.
cerevisiae (fig. 4).
However, the two genes
were aligned
head-to-tail in the
genomes of 33 eubacteria
and archaebacteria in
which SNO was adjacent
to SNZ. Moreover, when
we conducted BlastP
homology searches among
SNZ or SNO protein
sequence families, the
sequences in C.
intestinalis and S.
domuncula were
reciprocally the
best-hit sequences.
Therefore, it is
reasonable to conclude
that SNO and SNZ in C.
intestinalis were not
transferred from
bacterial species. From
these results, we
conclude that SNO and
SNZ existed in the
Eumetazoan lineage and
that losses of SNO and
SNZ occurred
independently at least
three times in C.
elegans, D.
melanogaster,and
vertebrate lineages
(fig. 5).
Correlation of Losses of
Genes Among Pyridoxine
Biosynthetic Pathways
We found that
simultaneous loss of SNO
and SNZ occurred more
often than loss other
pairs of genes (appendix
B of Supplemental
Material online) and
also that the loss of
SNZ occurred together
with that of SNO in the
same branch. As
mentioned above, the two
genes were often present
next to each other in
the genome. Moreover,
the two genes only
function in the fungal
type pathway. These
observations support
that the losses of these
two genes occurred at
the same time.
Next, we examined the
order of the losses for
the 10 genes. When we
compared the order of
the losses between two
genes, a significant
bias was observed in
nine particular
combinations of genes:
pdxH and dxs, pdxJ and
pdxH, pdxH and pdxF,
pdxA and dxs, pdxJ and
pdxA, pdxJ and dxs, pdxJ
and pdxK, pdxJ and SNO,
and pdxK and pdxF
(appendix B of
Supplemental Material
online). In every pair
of these nine
combinations, we
observed that the loss
of the latter gene
occurred more frequently
after the loss of the
former gene. These
biases were
statistically
significant against the
binominal distribution
(P , 0.05). From this
observation, we deduced
the patterns of losses
of five genes, as shown
in figure 6. The loss of
pdxJ caused
the loss of at least one
of pdxA, pdxH, and pdxK.
These four genes encode
enzymes whose reactions
are connected through
pyridoxine 59-phosphate
(PNP). Moreover, the
losses of these four
genes caused the loss of
at least one of pdxF and
dxs. These two genes
encode enzymes whose
reactions are connected
to pdxA and pdxJ through
4-phosphohydroxy-L-threonine
(4PHT) and
1-deoxyxylulose
5-phosphate (DXP),
respectively. If we
designate the two genes
encoding enzymes that
catalyze two consecutive
reactions the neighbor
genes, we can, thus,
conclude that the loss
of one gene accelerates
the loss of the neighbor
gene.
Discussion
To our knowledge, this
is the first attempt to
elucidate the evolution
of VB6 metabolism by
focusing on the gain and
loss of the 10 genes
involved in the PLP
biosynthetic pathways.
On the assumption that
each of these genes
wasacquired only once
during the evolution of
the entire 122 species,
we found that every gene
in the PLP biosynthetic
pathways had been lost
more than once in the
evolutionary lineages of
the 122 species. This
suggests that the
breakdown of the PLP
biosynthetic pathways by
gene losses may have
occurred in many
lineages, which should
be examined
experimentally. We also
revealed three aspects
to the evolution of the
PLP biosynthetic
pathways by estimating
the gain and loss of the
10 genes. The first
aspect is related to the
evolutionary order of
the generation of the
three PLP biosynthetic
pathways. From the
distribution of the 10
genes in the 122 species
examined, we found that
the fungal type and
salvage pathways were
probably older than the
de novo pathway on the
basis of the following
two results. First, pdxK
and pdxH of the salvage
pathway exist in
eubacteria and
eukaryotes, and SNO and
SNZ of the fungal type
pathway are found in all
the three domains,
namely, eubacteria,
archaebacteria, and
eukaryotes. Therefore,
we consider that the
fungal type and salvage
pathways both existed
before the separation of
the three domains of
life. Second, pdxB of
the de novo pathway only
exists in
cproteobacteria,
indicating that it was
generated in
cproteobacteria after
the divergence of the
three domains of life.
Applying the second
result mentioned above
to the existing model
for explaining the
evolution of the
metabolic networks, the
patchwork model and de
novo invention (Jensen
1976; Schmidt et al.
2003), we propose that
the process for the
formation of the de novo
pathway in c-proteobacteria
was as follows (fig. 7).
Originally, the common
ancestor of the 97
eubacterial species
studied had part of the
de novo pathway
involving five genes (dxs,
pdxA, pdxF, pdxH, and
pdxJ). Because gapA
functioned not only in
the PLP biosynthetic
pathways but also in
glycolysis (Seta et al.
1997), we think that
this gene also existed
in the common ancestor.
As shown in figure 7,
when pdxB was generated
in the lineage of c-proteobacteria,
the reaction catalyzed
by the product of pdxB
was connected to the two
metabolic reactions that
were separately
catalyzed by the
products of gapA and
pdxF. As a result, the
de novo pathway was
completed by the
presence of the seven
genes (dxs, gapA, pdxA,
pdxB, pdxF, pdxH, and
pdxJ) in c-proteobacteria.
We have, therefore,
reached the same
conclusion as
Mittenhuber (2001) but
via a different process.
Mittenhuber postulated
that the de novo pathway
was largely restricted
to c-proteobacteria on
the basis of the
functions of pdxA and
pdxJ and the requirement
of VB6 in the de novo
pathway. However, we
could not answer which
of the fungal type and
salvage pathways was
established as the first
PLP biosynthetic
pathway, because we
could not estimate the
times when pdxK, pdxH,
SNO, and SNZ originated.
In other words, we could
not determine which of
the gene sets of the two
pathways was generated
first. The second aspect
is related to the losses
of SNO and SNZ in animal
lineages. Among animals,
these two genes have
only been discovered in
the marine sponge S.
domuncula (Seack et al.
2001). Therefore, it is
plausible that the
losses of SNZ and SNO
occurred only once in
the Eumetazoan lineage
after its divergence
from the Poriferal
lineage. We found that
the two genes were
present in the complete
genome sequence of C.
intestinalis in the
present study. This
species is more closely
related to mammals than
D. melanogaster and C.
elegans, neither of
which have the two genes
(fig. 5). Therefore, we
consider that SNZ and
SNO existed in animals
after the divergence
between invertebrates
and vertebrates and that
their losses occurred
independently at least
three times in the
animal lineage, as shown
in figure 5. We reject
the possibility of
horizontal gene transfer
from the bacterial
lineage to C.
intestinalis not only by
the homology of the two
genes but also by the
order and orientation of
the two genes in the
genome (fig. 4).
The third aspect is
related to the
evolutionary order of
the gene loss. The
losses of five genes
occurred in the order
shown in figure 6 during
the evolution of the PLP
biosynthetic pathways.
Historically, five
models have been
proposed to explain the
formation of metabolic
pathways: the retrograde
model, the patchwork
model, de novo
invention,
specialization of a
multifunctional enzyme,
and pathway duplication
(Horowitz 1945; Jensen
1976; Schmidt et al.
2003). However, these
models only consider the
gene gain. Because there
are other reports that
gene losses have often
occurred in the
metabolic pathways in
bacterial lineages (Tatusov
et al. 1996; Shigenobu
et al. 2000), it is not
sufficient to only
consider the gain of
genes for the evolution
of metabolic pathways in
bacterial lineages.
Therefore, we propose a
new model based on our
results that explains
the evolution of
metabolic pathways
by gene loss. Once the
loss of a gene has
occurred in a metabolic
pathway, the neighboring
gene is more easily lost
than other genes in the
pathway. This can be
explained by functional
constraints. The
breakdown of a metabolic
pathway by gene loss
will decrease the
functional constraints
on the other genes of
the pathway. Our model
suggests that the
functional constraint on
the proximal genes to
the lost gene decreases
more extensively than
that on the distant
genes. Of course, it is
possible that the
functional constraint is
affected by other
pathways. For example,
if a gene is also
involved in another
metabolic pathway, as in
the case of gapA, its
functional constraint
may not be changed.
Our approach to
estimating the gain and
loss of genes is
affected by at least two
points. The first point
is the frequency of the
gain and loss of genes.
In this study, we
considered that gene
gain occurred only once,
even though gene loss
could have occurred more
than once in the
evolution of the 122
species examined. As a
result, we concluded
that there were seven
genes in the common
ancestor of the 122
species examined and
that a total of 132 gene
losses took place during
the evolution of the 122
species. However, when
we performed an
estimation based on the
parsimony method, there
were only three genes in
the common ancestor and
the number of gene
losses was
underestimated because
of overestimation of the
gene gain (data not
shown). These results
indicate that the
prediction of the gene
set in the ancestor and
the gain and loss of
genes are clearly
affected by the initial
assumption. However, we
can emphasize the low
possibility of gene gain
for the following
reasons. The cause of
gene gain is mainly
horizontal gene transfer
or parallel
evolution. Therefore, if
there is a difference in
the gene sets among
closely related species,
the number of gene
losses is expected to be
larger than that of gene
gains. If horizontal
gene transfer and
parallel evolution
occurred, then gene loss
would decrease and gene
gain would increase.
When we have evidence
for horizontal gene
transfer and parallel
evolution of the 10
genes in this study, it
will be possible to
estimate the times of
the gain and loss of the
genes more accurately.
In fact, we examined the
probability of
horizontal gene transfer
for the 10 genes between
eubacteria and
archaebacteria using the
method of
Nakamura et al. (2004)
and concluded that the
probability was
negligible.
The second point is that
our results are affected
by the topology of the
phylogenetic tree. If
the topology is
changed, the estimation
of the evolutionary
times of the gain and
loss of genes are
changed accordingly. As
a result, it is possible
to miscount the total
numbers of gains and
losses of the genes.
However, our results
showed that the sets of
genes were different
among the 122 species
examined (appendix A of
Supplemental Material
online). Because the
gain and loss of genes
cause the
differentiation of the
sets of genes in the
species, our conclusion
that the evolutionary
process of VB6
metabolism has been
quite dynamic regarding
the events of gain and
loss of genes, under
some constraints, is not
altered, even when the
topology of the
phylogenetic tree
changes. Studies using
comparative analysis
have often shown
differences in gene sets
involved in metabolic
pathways among species (Huynen,
Dandekar, and Bork
1999). By estimating the
gain and loss of genes,
we are able to learn not
only the differences in
a metabolic pathway
among the species
examined but also in
which lineage the change
in the metabolic pathway
occurred during
evolution. This means
that we will be able to
understand the
evolutionary processes
of the metabolic
networks by evaluating
the gains and losses of
genes. In some metabolic
pathways, dysfunctions
in particular lineages
have been reported
(Smirnoff 2001;
Meganathan 2001). By
applying our approach to
these metabolic
pathways, we will be
able to elucidate the
dysfunctions in these
pathways by the gain and
loss of genes. It is
also possible to further
extend our approach to
other metabolic networks
in the KEGG (Kanehisa et
al. 2002) and EcoCyc
(Karp et al. 2002)
databases, to more
clearly understand the
evolutionary processes
of the metabolic
pathways they contain.
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