JOURNAL OF BACTERIOLOGY,0021-9193/97/$04.0010
May 1997, p. 3244–3249 Vol. 179, No. 10
Copyright © 1997, American Society for Microbiology
Genome Structure and Phylogeny in the Genus BrucellaSYLVIE
MICHAUX-CHARACHON, GISELE BOURG, ESTELLE JUMAS-BILAK, PATRICIA
ANNICK ALLARDET-SERVENT, DAVID O’CALLAGHAN, AND MICHEL
Unité 431, Faculté de Médecine, Institut National de la
Santé et de la Recherche Médicale,30900 Nı̂mes, France
Received 21 October 1996/Accepted 7 March 1997
PacI and SpeI restriction maps were obtained for the two
chromosomes of each of the six species of the genusBrucella: B.
melitensis, B. abortus, B. suis, B. canis, B. ovis, and B.
neotomae. Three complementary techniqueswere used: hybridization
with the two replicons as probes, cross-hybridization of
restriction fragments, and anew mapping method. For each type
strain, a unique I-SceI site was introduced in each of the two
replicons,and the location of SpeI sites was determined by
linearization at the unique site, partial digestion, and
endlabeling of the fragments. The restriction and genetic maps of
the six species were highly conserved. However,numerous small
insertions or deletions, ranging from 1 to 34 kb, were observed by
comparison with the mapof the reference strain of the genus, B.
melitensis 16M. A 21-kb SpeI fragment specific to B. ovis was found
inthe small chromosome of this species. A 640-kb inversion was
demonstrated in the B. abortus small chromo-some. All of these data
allowed the construction of a phylogenetic tree, which reflects the
traditional pheneticclassification of the genus.
Brucella is a small gram-negative bacterium pathogenic
foranimals and occasionally for humans. The genus is divided
intosix species on the basis of cultural, metabolic, and
antigeniccharacteristics: B. melitensis, B. abortus, B. suis, B.
ovis, B. canis,and B. neotomae (6). However, it was shown by
DNA-DNAhybridization that the genus is more probably
monospecific(42). In a previous work (1), we found that each of the
so-called species exhibited a specific macrorestriction pattern
bydigestion of genomic DNA with XbaI, with only minor varia-tions
between biovars within a species. Recently, a physicalmap was
proposed for the genus type strain, B. melitensis 16M,by a
cross-hybridization method (30). We showed that theBrucella genome
was constituted by two circular chromosomeswith sizes of about 2.05
and 1.15 Mb.
In this study, we established the PacI and SpeI restrictionmaps
of the two chromosomes of the other reference strains inthe genus
Brucella: B. abortus 544, B. suis 1330, B. canis 62/290,B. ovis
63/290, and B. neotomae 5K33. Three different butcomplementary
techniques were employed: hybridization withthe two replicons as
probes, cross-hybridization of restrictionfragments, and a new
mapping strategy using I-SceI. I-SceI isan endonuclease encoded by
a group I intron of the Saccha-romyces cerevisiae mitochondrial 21S
rRNA gene (31). Re-cently, by using artificially inserted I-SceI
sites, a physical mapof chromosome XI of S. cerevisiae (40) and a
physical map ofYAC inserts (5) were made. Since bacterial genomes
lacknatural I-SceI sites, we showed that the introduction of
aunique restriction site recognized by I-SceI could be a usefultool
for the study of the genome organization (20). In thisstudy, we
used an indirect end-labeling method to localize theSpeI sites,
derived from a previously described procedure in-volving partial
digestion of linearized DNA, followed by South-ern blotting and
hybridization with two end probes (37).
Comparison of the six physical maps showed a rather
highconservation of restriction sites among the six Brucella
speciesgenomes, even though a large inversion was revealed in the
B.abortus 544 small chromosome.
MATERIALS AND METHODS
Bacterial strains. Bacterial strains used in the study are
listed in Table 1.Brucella strains were grown on a low-salt 2YT
medium (10 g of yeast extracts,10 g of tryptone, 5 g of NaCl per
liter) and Escherichia coli on LB medium.
Conjugation experiments. Introduction of a unique restriction
I-SceI site ineach replicon of the different Brucella species was
described elsewhere (20).Briefly, the suicide vector pGF2,
containing the Tn5Map with the 18-bp I-SceIrecognition site (18),
was transferred from E. coli SM10 lpir into Brucella mu-tants
resistant to either streptomycin or nalidixic acid. Transposition
events wereselected by kanamycin (25 mg/ml) and streptomycin
resistance or kanamycin andnalidixic acid resistance according to
the resistant Brucella mutant strain used.For mapping experiments,
we used five insertion mutants for B. melitensis, twofor B.
abortus, five for B. suis, and eight for B. ovis.
Endonuclease digestion of Brucella genomic DNA and pulsed-field
gel electro-phoresis (PFGE) conditions. Intact Brucella genomic DNA
was prepared inagarose plugs and digested with rare-cutting
endonucleases as previously de-scribed (30). For SpeI partial
digestion, a 120-min diffusion period at 4°C fol-lowed by a 10-min
digestion at 25°C gave optimal results with 3 U of endonu-clease
per plug (2 mg of DNA). For double digestions, plugs were first
partiallydigested with SpeI, incubated overnight at 37°C in 0.5 M
EDTA (pH 8) contain-ing proteinase K (1 mg/ml) and lauroyl
sarcosine (1%), and then washed for 1 hin
Tris-EDTA-phenylmethylsulfonyl fluoride (TE-PMSF) and three times
in TE.Digestion with I-SceI (Boehringer Mannheim) was then
performed as describedelsewhere (20). PFGE of intact and digested
DNA was performed in a contour-clamped homogeneous electric field
(CHEF) apparatus (CHEF-DRII; Bio-Rad)as previously described (30).
Separation of double-digested DNA was carried outfor 40 h in 1%
agarose gels at 170 V by using two sets of pulse conditions,
inseparate runs, in order to estimate the fragment sizes more
precisely: 100 to 12 sfor separating the large bands and 50 to 10 s
for separating the small bands.
Hybridization experiments. Southern blots of PFGE agarose gels
were pre-pared on Nytran membranes (Schleicher & Schuell) by
using the VacuGene XLBlotting System (Pharmacia LKB) with 203 SSC
(13 SSC is 0.15 M NaCl plus0.015 M sodium citrate) as the eluant.
Preparation of probes for cross-hybrid-ization of restriction
fragments or bands separated from intact DNA was done aspreviously
described (30). To obtain the 1.1- and 2.7-kb probes, corresponding
tothe ends of the I-SceI-linearized Tn5Map, plasmid ColE1::Tn5Map
DNA (18)was successively digested by SphI, NotI, and then I-SceI.
After separation on 1%low-melting-temperature agarose (SeaPlaque;
FMC), the 1.1- and 2.7-kb restric-tion fragments were excised from
the gel and labeled by a nonisotopic method(digoxigenin-DNA
labeling and detection kit; Boehringer). The Brucella DNAsize
markers were revealed with a total genomic DNA probe. Several genes
werelocated on the maps by hybridizing SpeI restriction fragments
with cloned oramplified (PCR) genes as described previously (30).
Probes for pal, bfr, and P16.5(8, 16, 41) were kindly provided by
J. J. Letesson; for omp19 by A. Tibor; for brrP(phoP homolog), brrN
(ntrC homolog), htrA (38), and recA (39) by B. Wren; forery (36) by
J. Aguerro; and for galE by L. Petrovska. A probe for dnaA
wasobtained by amplification using oligonucleotides calculated from
the sequence ofthis gene in Rhizobium meliloti (29). brrA is the
gene of a response regulatoramplified in our laboratory which is
similar to ctrA described in Caulobactercrescentus (unpublished
* Corresponding author. Phone: (33) 66 23 46 79. Fax: (33) 66 23
on October 21, 2020 by guest
Phylogenetic analysis. Similarity between each pair of strains
was calculated asthe Dice coefficient as described by Grothues and
Tümmler (13), with one mod-ification: two fragments with the same
size were considered as equivalent only ifthey cohybridized.
Strains were clustered by using the Phylip program (10).
Comparative PFGE-based macrorestriction analysis
anddetermination of genome size. The genomic DNA from refer-ence
strains of the six Brucella species (Table 1) was digestedwith the
rare-cutting endonucleases PacI (giving 9 to 10 frag-ments) and
SpeI (26 to 28 fragments) and subjected to PFGE(Fig. 1). It could
be seen that each species exhibited a specificrestriction pattern
with the two endonucleases, except for B.suis 1330 and B. canis,
which had the same patterns. PFGE ofintact genomic DNA revealed, as
for B. melitensis 16M (30),the presence of two chromosomes in the
other species (datanot shown). To recognize to which replicon
belong the PacIand SpeI restriction fragments, the bands
corresponding to thetwo chromosomes of each Brucella species
undigested genomicDNA were excised from the PFGE gel, labeled, and
used asprobes for hybridizing Southern blots of the same DNA
di-gested with PacI or SpeI. The addition of restriction
fragmentsizes allowed a good estimation of the two replicon sizes
foreach Brucella species, and these were always close to 2.05
and1.15 Mb, except for the small chromosomes of B. suis 1330,
B.canis, and B. neotomae, which were 50 kb longer. Then, foreach
species, the PacI restriction fragments were used as link-ing
probes on Southern blots of genomic DNA digested withSpeI. As the
large PacI restriction fragments, ranging from 900to 260 kb,
hybridized with more than two SpeI restrictionfragments, another
technique was needed to localize all theSpeI sites within each PacI
SpeI physical mapping by introducing a unique I-SceI site ineach
replicon. We used a Tn5 derivative, Tn5Map, to intro-duce a unique
I-SceI site into both replicons of each of the sixBrucella species,
as described previously (20). For each species,Tn5Map insertion
mutant clones were selected and the geno-mic DNA was digested with
SpeI, separated by PFGE, andtested by Southern hybridization for
the presence of Tn5Mapusing labeled ColE1::Tn5Map as a probe (data
For mapping experiments, agarose plugs of genomic DNAfrom each
Tn5Map insertion mutant were first partially di-gested with SpeI
and then I-SceI digestion was performed.DNA products of double
digestion were separated by PFGE induplicate, on both sides of the
Brucella DNA size markers.After Southern blotting of the gel, each
half of the nylonmembrane, containing a sample of I-SceI-SpeI
digested DNA,was hybridized with one of the two probes containing
the endsof Tn5Map. The SpeI restriction sites were directly
localizedon each side of the insertion of the I-SceI site, so that
thelabeled fragments read from the bottom to the top of
themembrane, allowing the construction of the SpeI physical
We show an example of the two hybridization patterns inFig. 2
for the B. ovis clone 63/290v7, with the I-SceI siteinsertion in
the 130-kb SpeI fragment on the small chromo-some. The sizes of the
DNA fragments labeled with the 1.1-kbprobe (Fig. 2, lane 1) were,
from the bottom to the top, 80, 85,105, 250, 300, 480, and 500 kb,
and with the 2.7-kb probe (lane2) they were 45, 65, 80, (85),
(105), (250), (300), 325 and 440 kb(parentheses indicate fragments
with fainter signals). In fact,all the restriction fragments
labeled with the 1.1-kb probe werealso found to hybridize with the
2.7-kb probe, although somehad fainter signals. The 2.7-kb probe
hybridized weakly withbands labeled by the 1.1-kb probe. We
therefore needed toignore the bands which also hybridized with the
1.1-kb probe.Starting from this 130-kb fragment, the order of the
SpeI re-striction fragments was 5, 20, 145, 50, 180, and 20 kb on
the1.1-kb end probe side and 20, 15, 245, and 115 kb on the
2.7-kbend probe side.
From these results, when constructing the SpeI restrictionmap of
the B. ovis 63/290 small chromosome, the main diffi-culty
encountered in aligning the fragments was the existenceof fragments
of slightly different sizes. To separate by PFGEthe fragments
obtained by partial digestion, large pulses wereneeded. In these
conditions, the determination of the sizes ofthe restriction
fragments from the hybridization patterns wasnot sufficiently
accurate to identify the fragments precisely,particularly in the
higher part of the run. To overcome thisproblem, we used Brucella
genomic DNA digested with SpeI orPacI instead of the usual markers
such as lambda concatemerDNA or S. cerevisiae chromosomes. This
gave a ladder rangingfrom 900 to 3 kb (Fig. 2, lanes S and P).
Furthermore, whennecessary, we used two different pulse ramps in
separate runsfor a better size calculation. Data from
cross-hybridizationexperiments between SpeI and PacI restriction
fragments andthe use of several mutants with different Tn5Map
insertionsites further confirmed the map.
Using these methods, we constructed physical maps of the
FIG. 1. PFGE. SpeI digestion of genomic DNA from six Brucella
speciesreference strains. Lane 1, B. melitensis 16M; lane 2, B.
abortus 544; lane 3, B. ovis63/290; lane 4, B. suis 1330; lane 5,
B. canis RM/666; lane 6, B. neotomae 5K33.Size markers in kilobases
are at the left of the figure.
TABLE 1. Brucella strains used in the study
Species Straina Host Geographicorigin
B. melitensis 16M (ATCC 23456) Goat United StatesB. abortus 544
(ATCC 23448) Cattle EnglandB. suis 1330 (ATCC 23444) Swine United
StatesB. canis RM/666 (ATCC 23365) Dog United StatesB. ovis 63/290
(ATCC 25840) Sheep AfricaB. neotomae 5K33 (ATCC 23459) Desert wood
a Type strains and biovar reference strains are listed by their
FAO/OMSdesignations and their American Type Culture Collection
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small and large chromosomes for the different Brucella
species:B. abortus 544, B. suis 1330, B. canis 62/290, and B. ovis
63/290.The B. neotomae 5K33 map was deduced from
cross-hybrid-ization of restriction fragments (Fig. 3). The
construction ofthe B. melitensis 16M physical map by this method
allowed usto test the validity of the new mapping technique using
I-SceIand to confirm the existing map (30). The restriction map of
B.suis 1330 was also confirmed by hybridization of SpeI frag-ments
with linking clones from a B. suis 1330 DNA cosmidlibrary: one to
three of 68 clones linked two adjacent SpeIfragments, except for
Comparison of the physical maps. Since a physical map hasbeen
established for the genus type strain, B. melitensis 16M(biovar 1)
(M1), we arbitrarily decided it was the referencemap and we
compared the three other maps to it. The sixphysical maps are shown
in Fig. 3.
The most frequent differences are small insertions or dele-tions
(indels) ranging from 1 to 34 kb. These events are mostnoticeable
in the small chromosome. We cannot, however,exclude the possibility
that small indels exist in the large SpeIfragments, which are
predominantly found in the large chro-mosome, since size
determination is less accurate for the largefragments.
A 21-kb fragment was found between two SpeI sites in thesmall
chromosome of B. ovis which did not hybridize to DNAs
from the other Brucella species (data not shown). Althoughother
strain-specific nucleotide sequences were not found,their existence
cannot be ruled out.
For B. abortus 544, a 640-kb inversion with respect to M1was
found in the small chromosome between two SpeI restric-tion sites.
The comparison of cross-hybridization results be-tween SpeI and
PacI restriction fragments of M1 DNA andthose of these B. abortus
strains confirmed this large inversion.However, this inversion was
found only in biovars 1, 2, 3, and4, the small chromosome of the
other biovars being very sim-ilar to that of M1 (unpublished
The map of B. suis 1330 was again related to that of M1.However,
modifications of restriction fragment length and thecreation or
loss of restriction sites were more often observed inthis species
than in the other. The small chromosome is 50 kblonger than that of
M1. B. canis 62/690 had a physical mapidentical to that of B. suis
1330. B. neotomae 5K33 has a smallchromosome that is very similar
to that of B. suis 1330, while itslarge chromosome is more similar
to that of M1.
Genomic organization. In order to compare the
genomicorganization of the two chromosomes of the four
Brucellaspecies, each of the SpeI restriction fragments of B.
melitensis16M was taken as a probe and hybridized with those of
theother species. At this macrorestriction level, the genetic
infor-mation seemed to be in the same order in the four
genomesexcept for the B. abortus 544 small chromosome, showing
alarge inversion (see below). Only a few genes have been clonedin
Brucella, and some of them were located on the maps byhybridization
with SpeI restriction fragments (Fig. 3). The lo-calization of rrn
loci, omp1 and omp2, BCSP 31, dnaK, groE,and IS6501 copies has
already been published for B. melitensis(30) and was confirmed for
the other species. The localizationof the other genes was obtained
as described in Materials andMethods. All the genes were found in
the corresponding SpeIrestriction fragments in the different
species. We also identi-fied the SpeI restriction fragments
containing at least one copyof the insertion sequence IS6501
described in the Brucellagenome (33). We found 16 of them in B.
ovis 63/290 and only5 in the three other strains (Fig. 4). But, by
hybridization withEcoRI-digested DNA of these Brucella strains, it
was shownthat the number of IS6501 copies was around 30 in the B.
ovisgenome and between 5 and 10 in the three others (33).
Finally,three rrn loci were found in all the strains, located in
thecorresponding SpeI fragments.
Phylogeny within the Brucella genus. Restriction fragmentlength
polymorphism has been used to study the phylogenyof Pseudomonas
aeruginosa (13). We employed a relatedmethod to study the phylogeny
within the genus Brucella, using16 type strains representing all
the species and biovars. Thedendrogram is shown in Fig. 4. This
tree is unrooted, sincemacrorestriction patterns are very specific
for Brucella anddefinition of a related extragroup to place the
root is artificial.The isolates were scattered in several groups.
The two B.abortus groups (biovars 1 to 4 and biovars 5 to 9) form a
tightlyrelated branch. The three B. melitensis biovars are
regroupedto the latter but are more remote from one another. B.
ovis ismore or less related to B. melitensis biovar 1 by the
methodused for the construction of the tree. The four B. suis
biovarsare localized at the opposite extremities of the tree and
arerelatively separated from one another. B. canis is not
repre-sented in a separate branch, since its physical map is
thesame as that of B. suis biovar 1. Finally, B. neotomae is
anintermediate between the B. melitensis-B. abortus group andthe B.
suis biovar group.
FIG. 2. Hybridization of I-SceI-linearized and partially
SpeI-digested geno-mic DNA of B. ovis clone 63/290v7 with the end
probes. The electrophoreticconditions were optimized for the
fragments below 450 kb. Lane 1, hybridizationwith the 1.1-kb probe;
lane 2, hybridization with the 2.7-kb probe; lanes S and P,SpeI
digestion and PacI digestion, respectively, of B. ovis DNA labeled
with atotal genomic DNA probe. The sizes of the PacI restriction
fragments are givenin kilobases.
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DISCUSSIONThe first striking observation concerning our results
the restriction maps of the six Brucella species are very
similar.Genome mapping of several isolates belonging to the
samebacterial species or genus has been performed for few
bacteria,and comparison showed different patterns of genome
conser-vation. First, chromosomal mapping could reveal a high
con-servation of the restriction site or gene order (2, 22, 24,
34).The second pattern associated the conservation of gene
orderwith a high restriction polymorphism (4, 25, 32). In a
thirdpattern, comparison of genome mapping revealed a
mosaicstructure showing the presence of conserved and
modifiedregions (3, 11, 23). In the most extreme case, the
restriction siteor gene order differed markedly (19, 43). The close
relation-ship found among the six physical maps in our study allows
usto assign the Brucella genus to the first group. Small indels
creation or loss of restriction sites are frequently
observed,most often in the small chromosome. Variability is
localized tocertain regions, similar to the polymorphic hot spots
observedin Clostridium perfringens (2).
A 640-kb inversion was found in the small chromosome of
B.abortus 544 (biovar 1). PacI restriction polymorphism showedthat
this inversion is also present in biovars 2, 3, and 4 of thesame
species but not in biovars 5, 6, and 9. It is unlikely that
thephenotypic differences between the two groups of biovars canbe
explained by this chromosomal rearrangement. Similar in-versions
with a size of several hundred kilobases have also beendescribed in
other bacterial species (21, 22, 25–28, 35, 43).
It has been demonstrated that large genomic rearrange-ments in
E. coli and in Salmonella serovars occur by homolo-gous
recombination at the rrn loci (17, 27, 28) or repeatednucleotide
sequences, such as insertion sequences (7, 12, 21).
FIG. 3. Physical maps of the two chromosomes of B. melitensis
16M (B. m), B. abortus 544 (B. a), B. suis 1330 (B. s), B. canis
RM/666, B. ovis 63/290 (B. o), andB. neotomae (B. n). The two
circular chromosomes are represented in linear form, each one
starting from a conserved SpeI fragment. (A) large chromosomes; (B)
smallchromosomes. For each chromosome map, SpeI sites are located
above and PacI sites are located below. Sizes are given in
kilobases for all the restriction fragmentsof the B. melitensis
chromosomes, and only for those different from B. melitensis for
the other species. Several genes are located (see Materials and
Methods). rrn lociare represented by open squares, IS6501 copies by
stars, and Tn5Map insertions by open circles. The inversion in the
small chromosome of B. abortus is shown by twolines.
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An insertion sequence, IS711 (15) or IS6501 (33), was de-scribed
in Brucella species. These mechanisms cannot explainthe
rearrangement in the small chromosome of B. abortus,since rrn loci
or IS copies were not located at the edges of theinversion. It is
also noteworthy that the B. ovis genome con-tains three to four
times more copies of IS6501 than the otherBrucella species, but its
restriction map is very similar to that ofthese species, without
evidence of major chromosomal rear-rangements. Other repeated
sequences exist in the Brucellagenome which may also have played a
role in the genomicrearrangements observed (14).
The dendrogram deduced from the maps grossly reflects
thetraditional phenetic classification of the genus Brucella,
con-firming a previous study in which we found that Brucella
spe-cies exhibited different XbaI restriction patterns, that
biovarswithin a species are less polymorphic, and that, except for
B.ovis isolates (33), field strains within a biovar have exactly
thesame pattern (1). This result was not expected since this
divi-sion of the genus into species and biovars is based solely
upona small number of metabolic traits, two
lipopolysaccharideepitopes, and sensitivity to a set of phages
which are host-rangevariants of the same ancestor phage (6). A
recent study, usingsequence data from the omp2 locus from different
Brucellaspecies, confirms the existence of species-specific
lineages (9).As in our study, a close relationship was found
between thesame strains (between B. suis and B. canis or between
B.melitensis and B. abortus, the latter being divided into the
sametwo subgroups), but an extreme divergence of B. ovis and
B.neotomae from the other species was also observed. However,the
authors suggested the existence of a gene conversion forthis locus
in these two species, explaining the divergence withthe other
The nucleotide sequence similarity between all the
Brucellastrains, measured by DNA-DNA hybridization, is so high
thatVerger et al. (42), following the international
recommenda-tions for bacterial classification, proposed that the
genus ismonospecific. However, although virulence is not
as a taxonomic criterion, the division of Brucella into
severalspecies has been always more or less influenced by the
restric-tion of the virulence of each so-called species to one or a
smallnumber of mammalian hosts. With view to a
phylogeneticclassification, such a proposal can be made only if the
different“species” are evolutive lineages restricted to a narrow
niche.This is the case for Brucella for three reasons. First,
because oftheir fastidious growth requirements, Brucella organisms
can-not actively multiply in the environment but only in
infectedanimals which harbor huge numbers of bacteria in their
tissues.Second, since mechanisms of genetic exchange, such as
plas-mid, temperate bacteriophage, or transformation, have
neverbeen demonstrated to occur naturally in Brucella, each
speciesis genetically isolated, despite the frequent coexistence of
dif-ferent strains infecting different animals in the same
farm.Third, virulence of the different species is restricted to a
smallnumber of hosts. For example, B. melitensis infects goats
andsheep while B. abortus infects cattle. While both species
arealso pathogenic for humans and B. melitensis occasionally
in-fects cattle, the “abnormal” host is always contaminated via
thenatural host and the infection is an epidemiological dead end.A
narrower restriction of the virulence is observed for B. canis,B.
ovis, and B. neotomae, which are, respectively, pathogenicfor dogs,
sheep, and desert wood rats. Finally, B. suis is essen-tially
virulent for swine. The similarity of the maps argues fora
monospecific genus, as suggested by Verger et al., and thetree for
the existence of subspecies corresponding to evolutivelineages
(pathovars) adapted to a particular host.
Liu and Sanderson have recently compared the physical andgenetic
maps of several different Salmonella serovars (27). Themaps of the
different serovars of Salmonella enterica (e.g., S.typhimurium and
S. enteritidis) are closely related. S. entericainfections in
humans range from gastroenteritis to fatal septi-cemia, but the
organism also infects a wide range of warm- andcold-blooded
animals. The genomic organization of the ubiq-uitous E. coli was
also similar (but not identical) to that of S.enterica. However,
the physical map of S. typhi, the agent of
FIG. 4. Phylogenetic tree of the six Brucella species.
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typhoid fever in humans, is very different in both its
organiza-tion and content. Moreover, when four wild-type S. typhi
iso-lates were tested, all were found to have identical
physicalmaps. Since S. typhi is pathogenic only for humans and has
noother animal hosts, the authors proposed that the restrictedhost
range has exerted specific selective constraints, conduciveto the
selection of a particular clone.
The very close homology between the different species,shown by
the comparison of macrorestriction maps, requires astill more
precise analysis of the Brucella genome in order tounderstand the
differences in the virulence restriction of thedifferent species.
High-resolution maps are now being con-structed in our
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