Evidence for symmetric chromosomal inversions around the replication origin in bacteria

http://genomebiology.com/2000/1/6/research/0011.1

Research
Evidence for symmetric chromosomal inversions around the

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replication origin in bacteria
Jonathan A Eisen, John F Heidelberg, Owen White and Steven L Salzberg
Address: The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD 20850, USA.

Correspondence: Jonathan A Eisen. E-mail: jeisen@tigr.org

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Published: 4 December 2000 Received: 7 August 2000
Revised: 25 September 2000
Genome Biology 2000, 1(6):research0011.1–0011.9
Accepted: 19 October 2000
The electronic version of this article is the complete one and can be
found online at http://genomebiology.com/2000/1/6/research/0011
© GenomeBiology.com (Print ISSN 1465-6906; Online ISSN 1465-6914)

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Abstract

Background: Whole-genome comparisons can provide great insight into many aspects of biology.
Until recently, however, comparisons were mainly possible only between distantly related species.
Complete genome sequences are now becoming available from multiple sets of closely related
strains or species.

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Results: By comparing the recently completed genome sequences of Vibrio cholerae, Streptococcus
pneumoniae and Mycobacterium tuberculosis to those of closely related species - Escherichia coli,
Streptococcus pyogenes and Mycobacterium leprae, respectively - we have identified an unusual and
previously unobserved feature of bacterial genome structure. Scatterplots of the conserved
sequences (both DNA and protein) between each pair of species produce a distinct X-shaped
pattern, which we call an X-alignment. The key feature of these alignments is that they have
symmetry around the replication origin and terminus; that is, the distance of a particular conserved
feature (DNA or protein) from the replication origin (or terminus) is conserved between closely

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related pairs of species. Statistically significant X-alignments are also found within some genomes,
indicating that there is symmetry about the replication origin for paralogous features as well.

Conclusions: The most likely mechanism of generation of X-alignments involves large chromosomal
inversions that reverse the genomic sequence symmetrically around the origin of replication. The
finding of these X-alignments between many pairs of species suggests that chromosomal inversions
around the origin are a common feature of bacterial genome evolution.

interactions
Background ability to detect large-scale genomic changes is dependent
Large-scale genomic rearrangements and duplications are in large part on which genomes are available. Such studies
important in the evolution of species. Previously, these in bacteria, for example, have been limited by the availabil-
large-scale genome-changing events were studied through ity of genomes only from distantly related sets of species.
genetic or cytological studies. With the availability of many Recently, however, the genomes of sets of closely related
complete genome sequences it is now possible to study such bacterial species have become available. We have compared
information

events through comparative genomics. The publication of these closely related bacterial genomes and have discovered
the yeast genome has led to much better insight into the an unusual phenomenon - alignments of whole genomes
duplication events that have occurred in fungal and eukary- that show an X-shaped pattern (which we refer to as
otic evolution (for example, see [1]). Large chromosomal X-alignments). Here we present the evidence for these
duplications have also been found from analysis of com- X-alignments and discuss mechanisms that might have pro-
pleted chromosomes of Arabidopsis thaliana [2,3]. The duced them.

2 Genome Biology Vol 1 No 6 Eisen et al.

Results and discussion at the same distance from the origin but not necessarily on
Whole-genome X-alignments between species at the the same side of the origin. The X-alignment between
DNA level V. cholerae and E. coli was found to be statistically signifi-
We compared the DNA sequences of the two chromosomes cant using a test based on the number of matches found in
of Vibrio cholerae [4] with the sequence of the Escherichia diagonal strips in the alignment (see the Materials and
coli chromosome [5] using a suffix tree alignment algorithm methods section). Specifically, when V. cholerae chrI is
[6]. The analysis revealed a significant alignment at the DNA aligned in the forward direction against E. coli, there are 459
level between the V. cholerae large chromosome (chrI) [4] maximal unique matching subsequences (MUMs; see the
and the E. coli chromosome [5] spanning the entire length of Materials and methods section), of which 177 occurred in a
these chromosomes (Figure 1a). Analysis of the reverse com- diagonal strip covering 10% of the total area (compared to
plement of V. cholerae chrI with E. coli also produced a sig- the expected value of 46). The probability of observing this
nificant alignment (Figure 1b). When superimposed, the two high a number of MUMs by chance is 4.7 x 10–59. The align-
alignments produce a clear ‘X’ shape (Figure 1c) that is sym- ment of V. cholerae chrI in the reverse direction against E. coli
metric about the origin of replication of both genomes. This (which corresponds to the MUMs on the anti-diagonal) has a
symmetry indicates that matching sequences tend to occur probability of 1.8 x 10–90. As a control, we compared the

(a) (b) (c)
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(d) (e)
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S. pyogenes

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S. pneumoniae M. leprae

Figure 1
Between-species whole-genome DNA alignments. Plots of maximally unique matching subsequences (MUMs) between
genomes as identified by the MUMmer program. (a) V. cholerae chrI forward strand versus E. coli forward strand. (b) E. coli
forward versus V. cholerae chrI reverse. (c) V. cholerae chrI versus E. coli, forward and reverse overlaid. (d) S. pneumoniae
forward versus S. pyogenes forward and reverse overlaid. (e) M. tuberculosis forward versus M. leprae forward and reverse
overlaid. A point ( x,y) indicates a DNA sequence that occurs once within each genome, at location x in one genome and at
location y in the other genome. The matching sequences may occur on either the forward or the reverse strand; in either case,
the locations indicate the 5’ end of the sequences. The point (0,0) corresponds to the origin of replication for each genome.


Table 1 species (see the Materials and methods section). Figure 2a
shows a scatterplot of chromosome positions of all proteins
Whole-genome DNA alignments using MUMmer
homologous between V. cholerae chrI and E. coli. The pres-

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Organism 1 Organism 2 Total Matches Probability* ence of many large gene families causes a great deal of noise
MUMs within in this comparison. This noise can be reduced by considering
diagonal only the best matching homolog for each open reading frame
(10%)
(ORF), rather than all protein homologs (Figure 2b). This fil-
V. cholerae E. coli 459 177 4.7 x 10-59 tered protein comparison results in an X-alignment that is
statistically significant (Table 2).
V. cholerae (rev) E. coli 467 217 1.8 x 10-90
V. cholerae (rev) V. cholerae 342 86 8.2 x 10-16 Whole-genome X-alignments within species

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E. coli (rev) E. coli 1,128 225 1.5 x 10-23 The finding of the X-alignment pattern between species led
S. pyogenes S. pneumoniae 706 259 4.5 x 10-80 us to search for similar patterns within species; that is, global
alignments of a genome with its own reverse complement. Of
S. pyogenes (rev) S. pneumoniae 626 255 2.3 x 10-90
the genomes for which we found between-species X-align-
S. pyogenes (rev) S. pyogenes 367 96 1.1 x10-18 ments (M. tuberculosis, M. leprae, S. pyogenes, S. pneumo-
S. pneumoniae (rev) S. pneumoniae 1,054 154 1.5 x 10-6 niae, E. coli and V. cholerae), statistically significant
self-alignments are detected for all except M. tuberculosis
M. leprae (rev) M. leprae 449 89 3.5 x 10-10
(Figure 3; probabilities shown in Table 1). Interestingly, these

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M. tuberculosis (rev) M. tuberculosis 2,476 268 0.092 self-alignments are not as strong as those between species.
E. coli M. tuberculosis 81 13 0.06 Proteome analysis also shows an X-alignment within species
E. coli (rev) M. tuberculosis 70 5 0.84 (shown for V. cholerae chrI in Figure 2d; probabilities shown
in Table 2). The X-alignment of proteins within V. cholerae
*Statistical significance was estimated as described in the text; rev, chrI is statistically significant only for recently duplicated-
reverse complement sequence. genes, but disappears when all paralogs are included. The
importance of filtering for recent duplications is discussed

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genomes of distantly related species, such as E. coli and below.
Mycobacterium tuberculosis. These do not show a signifi-
cant X-alignment (Table 1). Model I: whole-genome inverted duplications
One possible explanation for an X-alignment within and
We have found that X-alignments of whole genomes are not between species is an ancestral inverted duplication of the
limited to the V. cholerae versus E. coli comparison. For whole genome, as has been suggested for E. coli [10]. The
example, a whole-genome comparison of two bacteria in the weak or missing X-alignment within species could be
genus Streptococcus - S. pyogenes [7] and S. pneumoniae

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(H. Tettelin, personal communication) - reveals a global Table 2
X-alignment similar to that of V. cholerae versus E. coli Whole-genome protein-level comparisons
(Figure 1d) which is also statistically significant (Table 1). In
addition, an X-alignment is found between two species in Organism 1 Organism 2 Total Matches Probability*
the genus Mycobacterium - M. tuberculosis [8] and matches within
10%
M. leprae [9] (Figure 1e) - as well as between two strains of
diagonal
Helicobacter pylori (data not shown). The X-alignments
observed between any two pairs of genomes are not identical Top matches
in every aspect. For example, in the alignment between the
interactions
V. cholerae E. coli 1,797 369 3.2 x 10-40
two Mycobacterium species, each conserved region is much
V. cholerae (rev) E. coli 1,797 441 2.3 x 10-70
longer than in the other genome pairs. We believe this is due
to different numbers of evolutionary events between the V. cholerae V. cholerae 701 145 3.6 x 10-17
species (see below). Whole-genome X-alignments were not V. cholerae (rev) V. cholerae 701 70 0.52
found between any other pairs of species, although a related
E. coli E. coli 1,985 286 3.6 x 10-10
pattern was seen between some of the chlamydial species
(see below). E. coli (rev) E. coli 1,985 210 0.20
information

Recent duplications†
Whole-genome X-alignments between species are V. cholerae V. cholerae 195 60 1.0 x 10-15
also found at the proteome level
V. cholerae (rev) V. cholerae 195 26 8.4 x 10-4
To test whether the X-alignments found in the DNA analysis
could also be found at the level of whole proteomes, we con- *Statistical significance was estimated as described in the text. †Best
ducted comparisons of homologous proteins between match to another V. cholerae ORF versus any other complete genome.


(a) (b)
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T R
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Figure 2
Whole-genome proteome alignments. Plots show the chromosome locations of pairs of predicted proteins that have
significant similarity (on the basis of fasta3 comparisons). (a) V. cholerae chrI versus E. coli. All significant matches for each
V. cholerae ORF are shown. (b) V. cholerae chrI versus E. coli, top matches. Only the best match for each V. cholerae ORF is
shown. The filtering for top matches for each ORF removes noise due to the presence of many large multigene families. This
X-alignment is highly statistically significant (Table 2). (c) Chlamydia trachomatis versus C. pneumoniae, top matches. Only the
best match for each C. trachomatis ORF is shown. The position of the origins (R) and termini (T) of replication are slightly
shifted to see the inversions better. This pattern is consistent with the occurrence a small number of inversions around the
origin and terminus in the two lineages since their divergence from a common ancestor (see Figure 4). (d) V. cholerae chrI
versus V. cholerae chrI, self-alignment. Only recently duplicated pairs of genes are shown. Recent duplications were
operationally defined as those genes that were more similar to another gene in V. cholerae than to any gene in any other
complete genome sequence. The faint X-alignment is statistically significant (Table 2). No significant X-alignment was detected
when all pairs of paralogs were included.

explained by gene loss of one of the two duplicates of many between species, the member of the gene pair lost in a par-
of the pairs of genes in the different lineages. Gene loss has ticular lineage should be essentially random. If an ancient
been found to follow large chromosomal or genome duplica- inverted duplication followed by differential gene loss is the
tions [11-13]. This gene loss is thought to stabilize large correct explanation for the observed X-alignments, one
duplications by preventing recombination events between would expect the genes along one diagonal to be orthologous
duplicate genes. If gene loss is responsible for the weak between species (related to each other by the speciation
X-alignment within species, then to maintain the X-alignments event), while the genes along the other diagonal should be


(a) 3,000,000 (b)
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E. coli
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S. pneumoniae
S. pyogenes

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S. pyogenes S. pneumoniae

Figure 3
Within-genome DNA alignments. Plots of exactly matching sequences within four genomes as identified by the MUMmer
program: (a) V. cholerae; (b) E. coli; (c) S. pyogenes; (d) S. pneumoniae. A point (x,y) indicates a DNA sequence that is
repeated within the genome, occurring once at location x and again at location y. Points near the diagonal (y = x) correspond
to tandem repeats. Points near the anti-diagonal (y = L -x, where L is genome length) correspond to repeats that occur at
symmetric locations about the origin of location. The point (0,0) corresponds to the origin of replication in each plot.
Statistically significant X-alignments occur in all four species (Table 1).

interactions
paralogous (related to each other by the genome duplication possible mechanisms for such movement, but we believe the
event before the speciation of the two lineages). However, most likely explanation is the occurrence of large chromoso-
the evidence appears to contradict this model: likely ortholo- mal inversions that pivot around the replication origin
gous gene pairs are equally distributed on each diagonal and/or terminus. Large chromosomal inversions, including
(data not shown). those that occur around the replication origin and terminus,
have been shown to occur in E. coli and Salmonella
information

Model II: chromosomal inversions about the origin typhimurium in the laboratory (see, for example, [14-18]).
and/or terminus The occurrence of such inversions over evolutionary time
A second possible explanation for the X-alignments is that an scales was first suggested by comparative analysis of the com-
underlying mechanism allows sections of DNA to move plete genomes of four strains in the genus Chlamydia [19]. In
within the genome but maintains the distance of these sec- that study, we found that the major chromosomal differ-
tions from the origin and/or terminus. There are a variety of ences between C. pneumoniae and C. trachomatis (shown in


A2 A3

31 32 1 2 31 32 1 2 2 1 32 31
30 3 30 3 3 30
29 4 29 4 4 29
28 5 28 5 5 28
27 6 27 6
A2 *
27 *
6
26 7 A1 26 7 26 7
25
24 A1 8
9
25
24
A2 8
9
25
24 A3 8
9
23 10 23 10 23 10
22 11 22 11 22 11
21
20
12
13 Inversion 21
20
12
13 Inversion 21
20
12
13
19 14 14 19 14 19
18 17 16 15
around * 15 16 17 18 * around 15 16 17 18

terminus (*) origin (*)
32 1 2
30 31 3
29 4
28 5
27 6
26 Common 7
25
24 ancestor of
8
9 B1 B2 B3
23 10
22 A and B 11
21 12
20 13
19 14
18 17 16 15

A1 A2 A3

30 31
32 1 2
3
Inversion 30 31
32 1 2
3
Inversion 3 2
1 32 31
30
29 4 29 4 *
29 4*
28 5 around 28 5 around 28 5
27 6 27 6 27 6
26 7 terminus (*) 26 7 origin (*) 26 7
25 8 * 8 *
25 8 25
24
23
B1 10
9 9
10
B2 24
23
9
10
B3 24
23
22 11 11 22 11 22
21 12 12 21 12 21
20 13 13 20 13 20
19 14 14 19 14 19
18 17 16 15 15 16 17 18 15 16 17 18

B2 B3

B1 B2

Figure 4
Schematic model of genome inversions. The model shows an initial speciation event, followed by a series of inversions in the
different lineages (A and B). Inversions occur between the asterisks (*). Numbers on the chromosome refer to hypothetical
genes 1-32. At time point 1, the genomes of the two species are still co-linear (as indicated in the scatterplot of A1 versus
B1). Between time point 1 and time point 2, each species (A and B) undergoes a large inversion about the terminus (as
indicated in the scatterplots of A1 versus A2 and B1 versus B2). This results in the between-species scatterplot looking as if
there have been two nested inversions (A2 versus B2), similar to that seen for C. trachomatis versus C. pneumoniae (see
Figure 2). Between time point 2 and time point 3 each species undergoes an additional inversion (as indicated in the
scatterplots of B2 versus B3 and A2 versus A3). This results in the between-species scatterplots beginning to resemble an
X-alignment, similar to that seen in M. tuberculosis versus M. leprae (see Figure 2).

Figure 2c) were consistent with the occurrence of large could produce patterns very similar to those seen in the
inversions that pivoted around the origin and terminus Chlamydia, Mycobacterium and Helicobacter comparisons.
(including multiple inversions of different sizes). In Figure 4 The continued occurrence of such inversion over longer time
we present a hypothetical model showing how a small scales would result in an X-alignment similar to that seen in
number of inversions centered around the origin or terminus the V. cholerae versus E. coli and S. pneumoniae versus


S. pyogenes comparisons. Thus the different between- within the bacterial chromosome that inverts the sequence
species X-alignments could be the result of different from the origin up to that point. As with inversion events,
numbers of inversions between particular pairs of species. recombination and replication have been found to be tightly

comment
coupled [25].
Inversions about the origin and terminus could also produce
an X-alignment within species, through the splitting of
tandemly duplicated sequence. Many sets of tandemly dupli- Conclusions
cated genes are found in most bacterial genomes [19,20] We present here a novel observation regarding the conserva-
(also see Figure 3a,c). As tandem duplications are inherently tion between bacterial species of the distance of particular
unstable (one of the duplicates can be rapidly eliminated by genes from the replication origin or terminus. The initial
slippage and/or recombination events [21]), the fact that observation was only possible due to the availability of com-

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many tandem pairs are present within each genome suggests plete genome sequences from pairs of moderately closely
that tandem duplications occur frequently. Thus, it is rea- related species (for example, V. cholerae and E. coli). This
sonable to assume that occasionally a large inversion will shows the importance of having genome pairs from many
split a pair of tandemly duplicated genes. An inversion that levels of evolutionary relatedness. Comparisons of distantly
pivots about the origin and also splits a tandem duplication related species enable the determination of universal fea-
will result in a pair of paralogous genes spaced symmetri- tures of life as well as of events that occur very rarely. Com-
cally on opposite sides of the origin. parison of very closely related species allows the
identification of frequent events such as transitional changes

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If our inversion model is correct, then the genes along both at third codon positions or tandem duplications. To eluci-
diagonals in the between-species alignments should be date all other events in the history of life, genome pairs cov-
orthologous, which is the case (see above). In contrast, genes ering all the intermediate levels of evolutionary relatedness
along the anti-diagonal in the within-species X-alignments will be needed.
should be recent tandem duplicates that have been sepa-
rated by inversions. This also appears to be the case - in the
within-species analysis of V. cholerae chrI ORFs, the

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Materials and methods
X-alignment shows up best when only recent duplicates are Genomes analyzed
analyzed (Figure 2d). The splitting of tandem duplicates by Complete published genome sequences were obtained from the
inversions may be a general mechanism to stabilize the coex- National Center for Biotechnology Information website [26] or
istence of duplicated genes, as it will prevent their elimina- from the TIGR Comprehensive Microbial Resource [27]. These
tion by unequal crossing-over or replication slippage events. included Aeropyrum pernix [28], Aquifex aeolicus [29],
Archaeoglobus fulgidus [30], Bacillus subtilis [31], Borrelia
What could cause inversions that pivot around the origin burgdorferi [32], Campylobacter jejuni [33], Chlamydia
and terminus of the genome to occur more frequently than pneumoniae AR39 [19], Chlamydia pneumoniae CWL029

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other inversions? One possibility is that many inversions [34], Chlamydia trachomatis (D/UW-3/Cx) [35], Chlamydia
occur, but there is selection against those that change the trachomatis MoPn [19], Deinococcus radiodurans [36],
distance of a gene from the origin or terminus. Such a possi- Escherichia coli [5], Haemophilus influenzae [37], Helicobac-
bility has been suggested by experimental work in E. coli ter pylori [38], Helicobacter pylori J99 [39], Methanobac-
[14,15]. Additional studies have, however, suggested that terium thermoautotrophicum [40], Methanococcus jannaschii
there is little selective difference between inversions and that [41], Mycobacterium tuberculosis [8], Mycoplasma genital-
instead there may be certain regions that are more prone to ium [42], Mycoplasma pneumoniae [43], Neisseria meningi-
inversion than others [16-18,22,23]. Alternatively, the inver- tidis MC58 [20], Neisseria meningitidis serogroup A strain
sion events could be linked to replication, as has been sug- Z2491 [44], Pyrococcus horikoshii [45], Rickettsia prowazekii
gested for small local inversion events [24]. Whatever the [46], Synechocystis sp. [47], Thermotoga maritima [48], Tre- interactions
mechanisms, the fact that we find evidence for such inver- ponema pallidum [49], and Vibrio cholerae [4]. In addition, a
sions between many pairs of species suggests that they are a few unpublished genomes were analyzed: Streptococcus pyo-
common feature of bacterial evolution. Many aspects of the genes (obtained from the Oklahoma University Genome Center
X-alignments require further exploration. For example, to website [7]), Streptococcus pneumoniae (H. Tettelin, personal
split a tandem duplication, an inversion must fall precisely communication), and Mycobacterium leprae (obtained from
on the boundary between two duplicated genes. This would the Sanger Centre Pathogen Sequencing Group website [9]).
information

appear to be unlikely, requiring a large number of inversions
in order to generate a sufficient number of split gene pairs. If Whole-genome DNA alignments
the mechanisms of gene duplication are somehow related to DNA alignments of the complete genomic sequences of all
the mechanisms of inversion, however, then this model is bacteria used in this study were accomplished with the
more plausible. The process of duplicating a gene, if it occurs MUMmer program [6]. This program uses an efficient suffix
during replication, might promote a recombination event tree construction algorithm to rapidly compute alignments


of entire genomes. The algorithm identifies all exact matches termini of S. pneumoniae and S. pyogenes were determined
of nucleotide subsequences that are contained in both input by the authors of the present study using GC-skew analysis
sequences; these exact matches must be longer than a speci- and the locations of characteristic genes, particularly the
fied minimum length, which was set to 20 base pairs for this chromosome replication initiator gene dnaA.
comparison. To search for genome-scale alignments within
species, complete bacterial and archaeal genomes (25 in
total including all published genomes) were aligned with Acknowledgements
their own reverse complements. To search for between- We thank S. Eddy, M.A. Riley, T. Read, A. Stoltzfus, M-I Benito and I.
Paulsen for helpful comments, suggestions and discussions. S.L.S. was sup-
species alignments, all genomes were aligned against all ported in part by NSF grant IIS-9902923 and NIH grant R01 LM06845. S.L.S.
others in both orientations. and J.A.E were supported in part by NSF grant KDI-9980088. Data for all
published complete genome sequences were obtained from the NCBI
genomes database [26] or from The Institute for Genomic Research (TIGR)
Whole-genome protein comparisons Microbial Genome Database [27]. The sequences of V. cholerae, S. pneumo-
The predicted proteome of each complete genome niae, and M. tuberculosis (CDC 1551) were determined at TIGR with
support from NIH and the NIAID. The M. leprae sequence data were pro-
sequence (all predicted proteins in the genome) was com- duced by the Pathogen Sequencing Group at the Sanger Centre. Sequencing
pared to the proteomes of all complete genome sequences of M. leprae is funded by the Heiser Program for Research in Leprosy and
(including itself) using the fasta3 program [50]. Matches Tuberculosis of The New York Community Trust and by L’Association
Raoul Follereau. The M. tuberculosis CDC 1551 genome sequence was
with an expected score (e-value) of 10–5 or less were con- obtained from TIGR. The source of the S. pyogenes genome sequence was
sidered significant. the Streptococcal Genome Sequencing Project funded by USPHS/NIH grant
AI38406, and was kindly made available by B. A. Roe, S.P. Linn, L. Song, X.
Yuan, S. Clifton, R.E. McLaughlin, M. McShan and J. Ferretti, and can be
Statistical significance of X-alignments obtained from the website of the Oklahoma University Genome Center [7].
To calculate the statistical significance of the X-alignments,
the maximal unique matching subsequences (MUMs) for
unrelated genomes were examined and found to be uni- References
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