4272±4277 Nucleic Acids Research, 2002, Vol. 30 No.
19 ã 2002 Oxford University Press
Synonymous codon usage is subject to selection in
thermophilic bacteria
David J. Lynn, Gregory A. C. Singer and Donal A. Hickey*
Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, ON K1N 6N5, Canada
Received May 14, 2002; Revised July 24, 2002; Accepted August 1, 2002
ABSTRACT
The patterns of synonymous codon usage, both
within and among genomes, have been extensively
studied over the past two decades. Despite the
accumulating evidence that natural selection can
shape codon usage, it has not been possible to link
a particular pattern of codon usage to a speci®c
external selective force. Here, we have analyzed
the patterns of synonymous codon usage in 40
completely sequenced prokaryotic genomes. By
combining the genes from several genomes (more
than 80 000 genes in all) into a single dataset for
this analysis, we were able to investigate variations
in codon usage, both within and between genomes.
The results show that synonymous codon usage is
affected by two major factors: (i) the overall G+C
content of the genome and (ii) growth at high tem-
perature. This study focused on the relationship
between synonymous codon usage and the ability
to grow at high temperature. We have been able to
eliminate both phylogenetic history and lateral gene
transfer as possible explanations for the character-
istic pattern of codon usage among the thermo-
philes. Thus, these results demonstrate a clear link
between a particular pattern of codon usage and an
external selective force.
INTRODUCTION
The 20 amino acids that commonly occur in proteins are
encoded by 61 different codons. This redundancy in the
genetic code means that several `synonymous' codons may
encode the same amino acid. Consequently, one might argue
that mutational changes affecting these codons would not be
subject to natural selection, since the encoded protein
sequence would be unaffected by such changes. A large
body of indirect molecular evidence has accumulated, how-
ever, against such a simple assumption. First, it has been
shown that different genomes each have their own character-
istic patterns of synonymous codon usage (1). Secondly, and
more convincingly, it has been shown that within genomes,
highly expressed genes have shifted their codon usage toward
a more restricted set of `preferred' synonymous codons than
other, less highly expressed genes (2±8). In many cases, it has
*To whom correspondence should be addressed. Tel: +1 613 562 5800; Fax: +1 613 562 5744; Email: dhickey@uottawa.ca
been shown that codon usage mirrors the distribution of tRNA
abundances (2±4), indicating that the `preferred' codons are
those that tend to match the more abundant anticodons. This
correlation between the abundance of codons and their
matching anticodons suggests that relative tRNA abundance
is the selective force that determines synonymous codon usage
(2±4). Although the relative tRNA abundances may well be
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the short-term determinants of codon usage, it has been
suggested that over the course of long-term evolutionary
change the tRNA abundances themselves may also evolve to
match the genomic patterns of codon and nucleotide frequen-
cies (9). In other words, it is not clear if, in the long term, the
codon usage pattern is selected to match the relative
abundances of the isoaccepting tRNAs or vice versa. In any
case, there is strong evidence for a co-adaptation of the
relative frequencies of codons and their respective anticodons
within a genome. Despite this evidence, however, we cannot
explain why a particular codon±anticodon combination might
have a selective advantage over alternative synonymous
codon±anticodon pairs that are also perfectly matched. Thus,
although we have ample indirect evidence that a particular
pattern of synonymous codon usage has biological signi®-
cance, it is not as clear why that particular pattern is favored by
selection in a given genome. Despite the accumulation of data
on non-random patterns of synonymous codon usage, both
between and within genomes, it has been dif®cult to identify
an external selective force acting on synonymous codon
usage.
Here, we present evidence that the pattern of synonymous
codon usage within thermophilic prokaryotes is different from
that within the mesophilic prokaryotes and that this difference
is the result of natural selection linked to thermophily.
Moreover, we show that this phenomenon affects all of the
genes within the genome, that the pattern cannot be explained
by a simple accident of phylogenetic history and that it is not
due to horizontal gene transfer between mesophiles and
thermophiles. This result indicates that natural selection acting
through external environmental factors can indeed shape the
genomic pattern of synonymous codon usage.
MATERIALS AND METHODS
We analyzed the patterns of synonymous codon usage in a
total of 40 completely sequenced bacterial genomes (listed in
Table 1). This set of genomes includes 32 eubacteria and eight
archaea. Although the majority of the eubacterial species are
mesophiles and the majority of the archaea are thermophiles,
 Nucleic Acids Research, 2002, Vol. 30 No. 19 4273

Table 1. Total genomic G+C contents and optimal growth temperatures for the 40 genomes analyzed in our study

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Note that the variation in G+C contents is only weakly correlated with phylogenetic relatedness. Although the
majority of thermophilic organisms are Archaea, there are two exceptions (A.aeolicus and T.maritima). In addition,
there is one mesophilic archaeal species in our dataset (Halobacterium sp.).

the list does include two eubacterial thermophiles (Aquifex
aeolicus and Thermotoga maritima) and one mesophilic
archaeal species (Halobacterium sp.). These three genomes
have enabled us to distinguish between the effects of
environmental selection and phylogenetic history.
In our analysis, we combined the genes from all 40 genomes
(a total of 83 985 coding sequences) and calculated the relative
synonymous codon usage (10) for each gene. We used
correspondence analysis (11) to characterize the patterns of
codon usage among this large set of genes and to map this
pattern onto the distribution of codons on which the pattern is
based (Fig. 1A and B). Correspondence analysis was carried
out using the program CodonW1.4.2 (J. Peden, 2000; http://
www.molbiol.ox.ac.uk/cu/). This `transgenomic' analysis
allowed us to gain information on both the intra-genomic
and inter-genomic patterns of codon usage simultaneously.
4274 Nucleic Acids Research, 2002, Vol. 30 No. 19
Figure 1. Correspondence analysis of the relative synonymous codon usage
in 83 985 genes from 40 bacterial genomes (see Table 1). (A) Genes from
thermophilic bacteria are shown in red while those from mesophilic bacteria
are colored blue. For each of these two sub-groups, means and standard
deviations are shown. Note that there is extensive overlap between thermo-
philic and mesophilic genes along the horizontal axis, whereas there is
relatively little overlap between the two groups along the vertical axis. The
difference between mesophiles and thermophiles along this latter axis is
highly signi®cant (P < 0.0001). (B) The distribution of synonymous codons
along the ®rst and second axes of the correspondence analysis. Here, we see
that the GC-ending codons (shown in red) cluster to the right and the
AT-ending codons (shown in green) cluster to the left.
Moreover, it allowed us to directly compare the magnitude of
the within-genome and between-genome variations in codon
usage.
RESULTS
The genes from all 40 genomes were combined for the
correspondence analysis of relative synonymous codon usage.
Although all of the genes were combined, each gene could be
identi®ed in the output. Thus, we could, a posteriori, identify
genes by genome or by type. For instance, in Figure 1, genes
are identi®ed based on whether they came from thermophilic
or mesophilic species. Figure 1A shows the distribution of all
of the genes on the ®rst two axes of inertia of the
correspondence analysis. Genes from thermophiles are
Figure 2. Variation in codon usage within and between genomes. Genes
shown are identi®ed by genome and the means (699.99% con®dence
intervals) for each genome are shown (the abbreviations for each organism
are shown in Table 1). From this plot it is clear that the among-genome
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variation far exceeds the within-genome variation along both axes. Amongst
the thermophiles we have circled the two eubacterial genomes (T.maritima
and A.aeolicus). We have also circled the single mesophilic archaeal species
(Halobacterium sp.).
shown in red, whereas those from mesophiles are shown in
blue. Both mesophilic and thermophilic genes show a broad
distribution along the horizontal axis (the ®rst axis of inertia).
It is clear from Figure 1A, however, that these groups of
genes are signi®cantly different with respect to their position
along the vertical axis (the second axis of inertia). By looking
at the corresponding distribution of codons (Fig. 1B), we see
that the ®rst axis of inertia is due to the separation of codons
ending in A or T (shown in green) from codons ending in G or
C (shown in red). Thus, the separation of genes along the
horizontal axis is highly correlated with the overall G+C
content of the genome to which they belong (Table 1). This
can be seen more clearly in Figure 2, where we have grouped
the genes by genome. Genes from GC-rich genomes, such as
Mycobacterium tuberculosis and Pseudomonas aeruginosa
cluster to the right of Figure 2, whereas genes from the
AT-rich species, such as Methanococcus janaschii and
Borellia burgdorferi, appear on the far left. Species with an
intermediate G+C content, such as Escherichia coli and
T.maritima, appear near the middle of the distribution. While
variations in genomic G+C content explain most of the
variation along the ®rst axis of inertia, simple changes in
nucleotide content do not explain the separation of the
thermophilic and mesophilic genomes on the vertical axis. In
Figure 2, it can be seen that all of the thermophilic genomes,
including the two eubacterial thermophiles, are clearly
separated along the second axis of inertia (vertical axis
Fig. 2). Moreover, we can quantify this effect by comparing
the position of each species on the second axis of inertia with
its optimal growth temperature (see Table 1). The results of
our regression analysis showed that this relationship is highly
statistically signi®cant (P << 0.00001). By examining the
distribution of codons in Figure 1B, we can see that the major
contributors to this pattern are the arginine (AGR and CGN)
and isoleucine (ATH) codons, although many other codon
groups also contribute to the separation between the thermo-
philes and the mesophiles (see Discussion below).

Figure 3. Evidence for selection on synonymous codon usage. Synonymous
codon bias in highly expressed genes. Each arrow represents one genome,
with the base of the arrow at the mean position for the whole genome and
the arrowhead ending at the mean for the ribosomal genes within that
genome. Note that the arrows tend to point away from the division between
the thermophiles and the mesophiles; in other words, the ribosomal protein
genes have a greater degree of synonymous codon bias than the genomes as
a whole.
Since the difference in synonymous codon usage between
mesophiles and thermophiles is not due to a simple difference
in the nucleotide content of the genomes, we investigated the
possibility that it might be due to natural selection. To date,
the best evidence for selection acting on codon usage among
prokaryotes comes from the work of Ikemura and his
colleagues, who demonstrated that highly expressed genes
tend to have signi®cantly different codon frequencies than
other genes in the same genomes (2,3). Selection for optimal
codon usage is not, however, the only evolutionary force
acting on these genes. As stated by Gouy and Gautier (12), for
each gene within the genome there is a balance between
selection for optimal codons and other evolutionary forces
such as mutation and genetic drift. These other forces are
expected to affect all genes equally, whereas there is a
predicted correlation between the strength of selection and the
level of expression of each gene. Thus, although all genes are
subject to some degree of selection, it is only among the most
highly expressed genes that selection is strong enough to
constitute the dominant evolutionary force (12). This, in turn,
leads to a testable hypothesis: it has been proposed that if
selection is the underlying cause of synonymous codon usage
bias, then the bias should be more pronounced in the highly
expressed genes than in the rest of the genome (13). To test
this prediction, we compared the average codon usage of all
genes within a genome with the average for the ribosomal
protein genes from the same genome (Fig. 3). Among the
thermophiles, the highly expressed ribosomal protein genes
had a more extreme value on the second axis of inertia (the
vertical axis) for all nine species. The same was true for a
majority of the mesophilic genomes as well. These trends were
statistically highly signi®cant (P = 1 3 10±7 for the mesophiles
and P = 2.6 3 10±3 for the thermophiles in paired t-tests).
Essentially, the data show that the force responsible for the
difference in codon usage between thermophiles and meso-
philes acts more strongly upon highly transcribed genes than
other genes within the genome.
Nucleic Acids Research, 2002, Vol. 30 No. 19 4275
Figure 4. Summary of phylogenetic analyses based on the concatenated
sequences of 10 ribosomal protein genes from each of the 40 genomes used
in this study. Two possible phylogenetic groupings were compared. The
grouping shown on the left side of the ®gure represents the accepted
organismal tree. The tips of the branches are color coded (red for
thermophiles and blue for mesophiles); it shows that both thermophiles and
mesophiles are polyphyletic. The alternative hypothesis (shown on the right)
describes the horizontal gene transfer hypothesis, whereby the thermophilic
Eubacteria (A.aeolicus and T.maritima) would have acquired portions of
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their genomes, including the ribosomal protein genes, from thermophilic
Archaea. In this case, the tree based on the ribosomal protein sequences
would indicate that all thermophilic species would be monophyletic (again,
as indicated by the color coding on the tips of the branches). Our results,
using both distance-based and maximum likelihood methods of phylogenetic
reconstruction (J. Felsenstein, PHYLIP v.3.6a2.1; http://evolution.genetics.
washington.edu/phylip.html), greatly favor the ®rst grouping (left) over the
second (right), with 100% bootstrapping support.
The fact that the difference in codon usage between
thermophiles and mesophiles is more pronounced in the
highly expressed genes provides strong evidence for selection.
Nevertheless, we wanted to eliminate the possibility that such
a pattern was simply due to the fact that most of the
thermophiles studied are Archaea rather than Eubacteria. We
can eliminate phylogenetic history as an explanation because
the two eubacterial thermophiles (T.maritima and A.aeolicus)
show a typically thermophilic pattern of codon usage in both
their genomes as a whole (Fig. 2) and in their highly expressed
genes (Fig. 3). Likewise, the mesophilic archaeal species,
Halobacterium, shows a typically mesophilic pattern of codon
usage. It should be noted that three `exceptional' genomes
represent more than 1000 individual genes in this analysis.
Therefore, we can conclude that the separation in codon usage
is between thermophiles and mesophiles, and not between
eubacteria and archaea.
One might still argue that the common pattern of codon
usage in eubacterial and archaeal thermophiles could be
explained by horizontal gene transfer between the archaea and
the thermophilic eubacteria (14±16). Indeed, codon usage has
been used as an indicator of gene transfer between bacterial
lineages (16,17) and there is evidence for such transfers
between thermophiles (14,18). We addressed this question in
two ways. First, we used the concatenated amino acid
sequences of 10 ribosomal genes in both distance-based and
maximum likelihood phylogenetic analyses (Fig. 4). We
deliberately chose ribosomal proteins since they are included
among the class of highly expressed genes that show the most
pronounced differences between thermophiles and mesophiles
in the patterns of synonymous codon usage (Fig. 3). The
results of the phylogenetic test show very clearly that the
Halobacterium ribosomal protein sequences, despite their
mesophilic pattern of synonymous codon usage, group with
4276 Nucleic Acids Research, 2002, Vol. 30 No. 19
Figure 5. Frequency distributions of synonymous codon usage among
thermophilic (red) and the mesophilic genes (blue) along the second axis of
inertia. We have also plotted the A.aeolicus gene frequencies as a proportion
of all thermophilic genes (shown in green). This genome, despite being
eubacterial, shows a unimodal distribution that ®ts the distribution pattern
of the other thermophilic genomes. The `shoulder' in each curve is due to
the AT-rich genomes in each category.
the other archaea based on the amino acid sequences of the
encoded proteins. Likewise, the two eubacterial thermophiles
group with the Eubacteria. This is consistent with other recent
reports based on whole genome analyses of sequence data
(19,20). In other words, the synonymous codon usage patterns
of these ribosomal protein genes identify them unambiguously
as mesophiles or thermophiles, while the amino acid
sequences encoded by these same genes group them into a
conventional taxonomic arrangement of Eubacteria and
Archaea. This provides convincing evidence that the nucleo-
tides at the synonymous sites have undergone convergent
evolutionary change and it shows that the nature of this change
is directly related to thermophily or mesophily.
In addition to studying the ribosomal protein genes, we
wished to do an independent and more general test of the
possibility that horizontal gene transfer might be a signi®cant
factor in determining the codon usage of the thermophilic
Eubacteria. For instance, we wondered if the genes within
these genomes might show a bimodal distribution, re¯ecting
the fact that a fraction of their genome had been derived from
archaeal thermophiles by horizontal gene transfer. First, we
plotted the frequency distributions for the values of the second
axis of inertia for all genes from thermophiles and mesophiles
(Fig. 5). This ®gure shows quite dramatically how separate
both sets of genes are in terms of their codon usage. Our
primary interest, however, was in looking for evidence of
bimodality within a single genome, particularly within the
genomes of the eubacterial thermophiles. The results show no
trace of such bimodality. To illustrate this, we have plotted the
distribution of genes for the A.aeolicus genome in Figure 5. As
can be seen in Figure 5, the entire gene set for this eubacterial
thermophile is unimodal and matches almost exactly with that
of the entire set of non-AT-rich thermophile genes. This
means that all of the genes within this genome have converged
to a single pattern of codon usage regardless of their long-term
evolutionary history.
DISCUSSION
By combining the genes from all 40 genomes into a single data
set, we were able to make a direct comparison between the
intra-genomic and inter-genomic variations in codon usage.
These results show very clearly that the inter-genomic
differences can be very large relative to the variations between
genes within a particular genome. This is illustrated in
Figure 2, where we can see that the distribution of values for
all genes within a genome is relatively tightly clustered around
the mean of that genome. Examination of these results can also
give us an insight into how rapidly codon usage patterns may
change over the course of evolution. For instance, by
exploiting the fact that these 40 species represent a wide
range in divergence times, we can ask if codon usage is an
evolutionarily conserved character. From Figure 2, it is clear
that very closely related species, e.g. different species of
Chlamydia, have similar patterns of codon usage. When we
consider broader phylogenetic groupings such as the
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Proteobacteria, however, we see that this clustering of related
taxa no longer holds. In fact, P.aeruginosa (Paer) and
Buchnera sp. (Buch), both Proteobacteria, are found on
opposite extremes of the scale for the ®rst axis of inertia in
Figure 2. We also see some dramatic cases of evolutionary
convergence along the horizontal axis in Figure 2. For
example, the codon usage pattern of the GC-rich archaeal
species Halobacterium is very similar to that of the GC-rich
gram-positive eubacterium M.tuberculosis and the gram-
negative P.aeruginosa. This indicates that codon usage,
while stable in the short term, is a labile character over the
longer evolutionary term. It is particularly obvious that
the codon usage of genes within a genome can `track'
the evolutionary changes in nucleotide content of the
entire genome (compare Table 1 and Fig. 2). Given that
codon usage is responsive to evolutionary changes in
nucleotide composition, it is not surprising that it should
also be responsive to other evolutionary pressures, such as the
action of temperature-dependent selection.
Essentially, our results show that codon usage among these
40 genomes is determined by two major factors: nucleotide
content and optimal growth temperature. Of these two factors,
the G+C content of the genome explains more than 25% of the
variation between genomes, whereas optimal growth tem-
perature explains a further 10% of the variation. In this
analysis, there are more than 50 axes in all, and the remaining
variation is spread over a large number of the remaining axes.
No other single axis explains even 5% of the variation in
codon usage.
While it is clear that the second major factor affecting
codon usage on a genome-wide scale is optimal growth
temperature, it is not obvious what the nature of the selective
force might be. At ®rst glance, it seemed that the difference
between thermophiles and mesophiles lay solely in their usage
of arginine and isoleucine codons (Fig. 1B). However, when
we recalculated the codon frequencies in the absence of these
two codon groups, the difference between thermophiles and
mesophiles remained. We also re-analyzed the data using
2-fold, 4-fold and 6-fold degenerate codons groups separately.
In all cases, there was a difference between the genomes of the
thermophiles and the mesophiles. This means that the effect is
very pervasive. This pervasiveness, in turn, leads one to

wonder if the selection is for some general property of the
mRNAs that is particularly important under conditions of high
temperature, rather than for speci®c codon±anticodon
pairings. One possibility is that the process is driven by
selection for increased mRNA stability at high temperature,
rather than selection for translational ef®ciency. Increased
mRNA stability would result in increased levels of translated
protein per mRNA molecule. Thus mRNA stability could be
subject to similar selection pressures as translational ef®-
ciency. Interestingly, both forms of selection would be more
pronounced for highly expressed genes. It has been suggested
that thermophilic genomes are purine rich (21) and such a
purine preference could affect both mRNA stability and the
frequency of synonymous codons within these genomes.
In summary, we have shown that the patterns of synonym-
ous codon usage within a genome can change dramatically
during the course of evolution. Our results show that the two
major forces affecting the broad patterns of codon usage
among prokaryote genomes are (i) the nucleotide composition
of the genome and (ii) some form of natural selection linked to
optimal growth temperature. It will be of interest to ask if
those genomes that have changed their synonymous codon
usage in response to these evolutionary forces have undergone
a corresponding change in the relative abundances of
isoaccepting tRNAs. A second question that merits further
study is the biochemical basis of the selective advantage of
certain codons under high temperature conditions and, in
particular, if such selective forces are related to the selection
on non-synonymous sites among thermophiles (22). The main
conclusion that can be drawn from the results presented here is
that synonymous codon usage patterns can be subject to
natural selection and, speci®cally, that a particular environ-
mental factor such as high temperature can underlie selection
for a speci®c subset of codons in both eubacterial and archaeal
lineages.
ACKNOWLEDGEMENTS
This work was supported by a Research Grant from NSERC
Canada (D.A.H.) and graduate scholarships from the
University of Ottawa (D.J.L.) and NSERC (G.A.C.S.).
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