Physical mapping of Mycobacterium bovis BCG pasteur reveals differences from the genome map of Mycobacterium tuberculosis H37Rv and from M. bovis.
ABSTRACT A Dral restriction map of the approximately 4.35 Mb circular chromosome of the vaccine strain Mycobacterium bovis BCG Pasteur was constructed by linking all 21 Dral fragments, ranging in size from 6 to 820 kb, using specific clones that spanned the Dral recognition sites as hybridization probes. The positions of 20 known genes were also established. Comparison of the resultant genome map with that of the virulent tubercle bacillus Mycobacterium tuberculosis H37Rv revealed extensive global conservation of the genomes of these two members of the M. tuberculosis complex. Possible sites of evolutionary rearrangements were localized on the chromosome of M. bovis BCG Pasteur by comparing the Asnl restriction profile with that of M. tuberculosis H37Rv. When selected cosmids from the corresponding areas of the genome of M. tuberculosis H37Rv were used as hybridization probes to examine different BCG strains, wild-type M. bovis and M. tuberculosis H37Rv, a number of deletions up to 10 kb in size, insertions and other polymorphisms were detected. In addition to the known deletions covering the genes for the protein antigens ESAT-6 and mpt64, other genetic loci exhibiting polymorphisms or rearrangements were detected in M. bovis BCG Pasteur.
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ABSTRACT: Genome maps have been constructed for the mycobacterial pathogens Mycobacterium leprae and Mycobacterium tuberculosis, as well as for the attenuated vaccine strain Mycobacterium bovis BCG Pasteur. While the chromosomes of M. tuberculosis and M. bovis BCG Pasteur show extensive conservation at the grosslevel, comparison with M. leprae revealed a high degree of diversification, with a mosaic-like pattern apparent. The ordered libraries of M. tuberculosis and M. leprae produced during the course of these studies played a central role in the genome sequencing projects of these two bacilli, showing the utility of this approach for systematic sequencing of bacterial genomes.Electrophoresis 04/1998; 19(4). · 3.16 Impact Factor
- Practical Approach to Tuberculosis Management, Edited by V.K. Arora, 07/2006: chapter Genetic mapping of tuberculus bacillus: clinical issues of future.: pages 577-583; Jaypee Brothers Medical Publishers (P) Ltd., New Delhi., ISBN: 81-8061-767-X
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ABSTRACT: We performed genotyping of 112 tuberculosis agent isolates from patients suffering from lung tuberculosis in Mongolia using the RD9, RD7, TbD1, RD105, and RD750 loci. Genotypes of all the obtained isolates were characterized by preservation of the RD9, RD7, and RD750 loci and by a deletion in the locus TbD1. A deletion of RD105 was found in 65 (58%) isolates. The isolates were classified into two groups, East Asian and European-American ones, by the results of genotyping.Molecular Genetics Microbiology and Virology 12/2012; 26(4). · 0.22 Impact Factor
Mkrobiology (1996), 142,3135-3145
Printed in Great Britain
Physical mapping of Mycobacterium bovis BCG
Pasteur reveals differences from the genome
map of Mycobacterium tuberculosis H37Rv
and from M. bovis
Wolfgang J. Philipp,’t Shamila Nair,’+ Gerard Guglielmi,2
Micheline Lagrande~-ie,~ Brigitte Gicquel2v3 and Stewart T. Cole’
Author for correspondence: Stewart T. Cole. Tel: +33 1 45688446. Fax: +33 1 40613583.
e-mail : stcole @ pasteur, fr
A DraI restriction map of the - 435 Mb circular chromosome of the vaccine
strain Mycobacterium bovis BCG Pasteur was constructed by linking all 21 DraI
fragments, ranging in size from 6 to 820 kb, using specific clones that spanned
the DraI recognition sites as hybridization probes. The positions of 20 known
genes were also established. Comparison of the resultant genome map with
that of the virulent tubercle bacillus Mycobacterium tuberculosis H37Rv
revealed extensive global conservation of the genomes of these two members
o f the M. tuberculosis complex. Possible sites of evolutionary rearrangements
were localized on the chromosome of M. bovis 6CG Pasteur by comparing the
Asnl restriction profile with that of M. tuberculosis H37Rv. When selected
cosmids from the corresponding areas of the genome of M. tuberculosis H37Rv
were used as hybridization probes to examine different BCG strains, wild-type
M. bovis and M. tuberculosis H37Rv, a number of deletions up to 10 kb in size,
insertions and other polymorphisms were detected. In addition to the known
deletions covering the genes for the protein antigens ESAT-6 and mpt64, other
genetic loci exhibiting polymorphisms or rearrangements were detected in M.
bovis BCG Pasteur.
Unit6 de GCnBtique
Unit6 de GCnBtique
Laboratoire du BCG3,
lnstitut Pasteur, 28 rue du
Docteur Roux, 75724 Paris
Cedex 15, France
Keywords : Mycobacteritlm bovk BCG Pasteur, tuberculosis, BCG vaccine, genomics
Mycobacterium bovis BCG (Calmette, 1927), an attenuated
culture of M. buvis, is the world’s most widely used
vaccine. It has been used to immunize more than two
billion people against tuberculosis (Bloom & Fine, 1994),
and a very limited number of major side-effects has been
reported (Bloom & Murray, 1992). However, protection
imparted by BCG against pulmonary tuberculosis is
highly variable (Clemens et al., 1983; Fine, 1995)
although it is generally accepted that it is efficacious in
protecting infants from the severe form of the disease,
tuberculous meningitis and miliary tuberculosis.
Since the isolation of the first attenuated culture, from
which the strain was never cloned (Calmette, 1927),
t Present address: Institute of Medical Microbiology, University of Berne,
3010 Berne, Switzerland.
*Present address: INSERM U 41 1, Facult6 Necker, 156 rue de Vaugirard,
75730 Paris Cedex 15, France.
different variants have emerged during the production of
the vaccine in different countries (Bloom & Fine, 1994).
Today, the most widely used vaccine strains are BCG
Pasteur, BCG Glaxo, BCG Copenhagen and BCG Japan
which show morphological, biochemical and immuno-
logical differences (Bloom & Fine, 1994; Lagranderie et
al., 1996). Although it is recommended that BCG be used
in areas where tuberculosis is endemic, a better vaccine is
required for the elimination of the disease. Analysis of the
BCG genome will provide us with important information
that will help in constructing BCG strains with improved
immunological properties and may lead to the rational
attenuation of Mycobacterizcm ttlberczllosis.
The construction of genomic maps of nearly 100 bacterial
chromosomes has led to improved understanding of their
general organization and evolution (Cole & Saint-Girons,
1994; Fonstein & Haselkorn, 1995; Krawiec & Riley,
1990). Data about genome size, architecture and topology
can be very rapidly generated by a combination of physical
and genetic analysis, and often enable regions of interest
0002-0918 0 1996 SGM
W. J. PHILIPP and OTHERS
Table 1. Different members of the M. tuberculosis
complex analysed in this study
M. tubercdosis H37Rv
M. tubercdosis H37Ra
M. bovis ATCC 19210
M. bovis BCG Pasteur
M. bovis BCG Denmark
M. bovir BCG Glaxo
M. bovis BCG Japan
M. bovis BCG Moreau
* GMB, Unit6 de Gtnttique Moltculaire Bacttrienne; LBCG,
Laboratoire du BCG, Institut Pasteur, Paris, France.
Institut Pasteur Collection
to be identified and analysed in detail. Information is
lacking about the genomic organization of M. bovis BCG
and its progenitor, but might be useful for the molecular
dissection of the genetic basis of the pathogenicity of the
tubercle bacillus. Extending this approach to the different
BCG substrains could explain some of the phenotypic and
immunological differences. Here, the construction of a
physical map of the genome of M. bovis BCG Pasteur is
described, and the results of global comparison with the
genome map of M. tt/bercdosis H37Rv, and local analysis
of selected polymorphic regions in four other BCG strains
and in M. bovis, are presented. The availablity of a high
resolution, integrated genomic map of M. tzibercdosis
H37Rv (Philipp etal., 1996) facilitated this task and led to
the localization of areas of possible rearrangements.
Preparation of agarose-embedded chromosomal DNA. BCG
strains (Table 1) were grown on Sauton medium, and M.
tzdberczllosis H37Rv and H37Ra were cultured in Dubos medium
(Pasteur Diagnostics) supplemented with oleic acid/albumin/
dextrose/catalase (OADC; Difco), and incubated for 10-12 d
at 37 OC. D-Cycloserine (1 mg ml-') was added and incubation
continued for a further 24 h prior to harvesting of cells at 4000
r.p.m. in a Sorvall RC-5B centrifuge. The cells were resuspended
in 1 x TE buffer (10 mM HCl, 1 mM EDTA, pH 7-6), enclosed
in 1% (w/v) low melting point agarose (BRL) and further
processed to release intact genomic DNA as described pre-
viously (Philipp et al., 1996).
PFCE and conventional gel electrophoresis. Chromosomal
DNA from the different strains was digested to completion after
incubating agarose plugs overnight in the appropriate buffers
containing 30 U DroI (Gibco BRL) in the presence of 0.1 %
Triton X-100 (Serva) and 30 U Am1 (New England Biolabs) or
30 U EcoRI (Boehringer Mannheim), then washed in 1 x TE
buffer before loading. In some experiments PmeI, or the intron-
encoded endonucleases I-Ced, I-PpoI and I-TluI (all from New
England Biolabs) or I-SceI (Boehringer Mannheim) were used
either alone or in conjunction with DraI.
Fig. 7. Comparison of the Dral profiles and linking analysis of M. bovis BCG Pasteur and M. tuberculosis H37Ra. (a)
Ethidium-bromide-stained pulsed field gel on which Dral restriction fragments of M. bovis BCG Pasteur and M.
tuberculosis H37Ra have been separated. (b), (c) Corresponding Southern blot with two linking clones of M. tuberculosis
H37Rv used as hybridization probes: probe Y2 that links fragmentsu and P or K and N (b) and probe T225 that links
fragments Q and H or 22 and G (c) in M. tuberculosis H37Ra or M. bovis BCG Pasteur, respectively. The positions of key
size markers are indicated.
Genome map of Mycobacteriztm bovis BCG Pasteur
Table 2. Restriction fragments of the chromosome of M. bovis BCG Pasteur and comparison with those of M.
BCG H37Rv BCG H37Rv
Fragment Size (kb)
Total 4346 4394
W. 1. PHILIPP and OTHERS
Fig- 2 . Ethidium-bromide-stained gel of an Asnl digestion of the chromosomes of M. bovis BCG Pasteur and M.
tuberculosis H37Rv after separation by PFGE. The left and right panels correspond to gels on which fragments up 100 kb
and up to 700 kb, respectively, were separated. The arrows indicate the principal differences in the restriction patterns
and the brackets the compressed zone.
, ...... . ............. ... .............................
, .... , ........ ..... .,.. . ,... . .........................................................................................................
DraI and Am1 restriction fragments were separated on 1.2%
agarose gels using a CHEF I1 Pulsaphor apparatus (Pharmacia)
with pulse times of 3 s for 20 h for fragments up to 100 kb, 10 s
for 20 h for fragments up to 300 kb, and 20 s for 20 h for the
largest fragments, up to 1 Mb. Electrophoretic runs were carried
out at 280 V and 10 OC. DNA was subsequently Southern-
blotted, transferred (Philipp et al., 1996; Sambrook et al., 1989)
to Hybond-C or Hybond-N membranes (Amersham) and then
used for hybridization experiments. EcoRI restriction fragments
were resolved on 0.7 % agarose gels using standard conditions
and then processed for hybridization experiments as described
Library construction and screening for linking clones.
Chromosomal DNA (2 pg) was partially digested with 0.07 U
Sau3AI (Boehringer Mannheim) and size-fractionated on a
1 YO agarose gel. Fragments of 1-3 kb in size were purified
by the Geneclean method (BiolOl), then ligated into the
dephosphorylated kanamycin-resistant vector pUC19GGK and
transformed into Escherichia coli DH5a (Sambrook et al., 1989).
Plasmid DNA was subjected to digestion with DraI and positive
clones sorted into groups for hybridization experiments. In
parallel, known DraI linking clones from the pYUB18 cosmid
library of M. tuberctrlosis H37Rv were used in hybridization
experiments together with probes for well-characterized genes
(Philipp & Cole, 1995; Philipp et al., 1996).
Hybridization. Cosmid or plasmid DNA (100 ng) was labelled
with [a-32P]dCTP (ICN) by nick translation and linear molecules
were labelled by random priming with the multiprime kit
(Amersham). Labelled probes were purified by low speed
centrifugation on P10 micro-columns (BioRad) then transferred
to a solution containing 5 x SSC (1 x SSC is 0.15 M sodium
chloride, 0.015 M sodium citrate) and 50 YO (w/v) formamide
(Philipp etal., 1996; Sambrook eta/., 1989). Filters were washed
after hybridization at 37 O C overnight in a final concentration of
0.1 x SSC at 65 O C or at room temperature as required,
The mapping strategy
In a previous study, a detailed physical map of the genome
of the tubercle bacillus M.
ttlberczlloszs H37Rv was con-
structed using a combined approach involving
hybridization analysis with linking clones to join macro-
restriction fragments obtained after digestion of genomic
DNA with AsnI or DraI, followed by verification and
extension by two-dimensional gel electrophoresis of
reciprocal AsnI and DraI digests (Philipp et a/., 1996). As
M. buvis BCG is highly related to M. tzibercdosis it seemed
probable that the M.
tuberczllosis linking probes, as well as
the unique coding sequences, would be useful for
mapping the BCG genome. However, since it was also
conceivable that some of the rare restriction sites might be
confined to one or other species, it was decided that a
second linking library should be constructed from M.
bovis BCG Pasteur.
Genome map of Mfcobacteritrm bovis BCG Pasteur
Since the genomes of species from the M. tzabercdosis
complex have a high G + C content, a series of restriction
enzymes recognizing A + T-rich sites was tested to
identify the most appropriate ones and, in parallel, a
battery of intron-encoded endonucleases was evaluated.
None of the latter enzymes, including the immensely
versatile I-CeuI which cleaves in the rdgene, encoding the
23s rRNA of most bacteria and lower eukaryotes (Liu et
al., 1993; Marshall & Lemieux, 1992), showed evidence of
cleavage in single or double digestions, Subsequent
inspection of the sequence of the M. ttlbercdusis rrl gene
(Bergh & Cole, 1994) revealed a single base deletion in the
sequence generally recognized by I-Ced (Marshall &
As found previously with M. tuberczclosis H37Rv (Cole &
Smith, 1994; Philipp et a/., 1996), digestion of the
chromosome of M. bovis BCG with DraI (TTTAAA)
yielded the lowest number of fragments, 21, ranging in
size between 6 and 820 kb (Fig. 1; Table 2). Thirteen of
these fragments displayed identical electrophoretic
mobilities to DraI fragments obtained from the chromo-
some of M. tzrberctrlosis indicating that they were probably
equivalent (Table 2). PmeI which recognizes an extended
DraI site (GTTTAAAC) was found to cleave the BCG
genome at a single site (data not shown) and the genome
of M. ttlbercdosis at two sites.
Digestion with AsnI (ATTAAT) generated 48 fragments
from BCG Pasteur and 47 from M. tzjbercdosis (Fig. 2).
Many of them (33 out of 48) were identical in size and the
sum of the resultant fragment sizes indicated that the
genome consisted of - 4370 kb. A similar value was
obtained by summing DraI fragment sizes (- 4346 kb,
Table 2) but in both cases the total BCG genome size was
- 40 kb smaller than that of M. tzaberctllosis. Because the
migration of some of the fragments resulted in the creation
of two zones of compression (around 87 kb, 3 fragments;
and between 210 and 250 kb, 5 fragments with DraI, and
in the 15-75 kb range with AsnI) it was necessary to use
several different running conditions for optimal sep-
aration. Typical gels giving optimal resolution of AsnI
fragments in the 100-700 kb and 1&100 kb ranges are
shown in Fig. 2. Despite these modifications, the DraI
fragments around 87 kb could not be separated to
completion, so their locations on the BCG map, and those
of the fragments I/H and F/G, which are located in the
second compression, were inferred by comparison with
the map of M. tabercdosis (Philipp et al., 1996) and
confirmed by hybridization analysis (see below).
Construction of a BCG linking library
Two libraries were constructed in different vectors to
isolate BCG DNA harbouring DraI restriction sites.
Clones with inserts bearing a DraI site were identified,
sorted into groups and then used in hybridization
experiments on total genomic DraI digests to find the
naturally-existing links between the DraI macrorestriction
fragments. Screening of 150 clones from these libraries
rable 3. Linking and localization analysis of
M. tuberculosis H37Rv and M. bovis BCG Pasteur
chromosomes using various probes
NT, Not tested.
resulted in the identification of 14 independent DraI
linking clones, the pGG series (Table 3), which were
subsequently used in linking analysis.
The genome map
To determine the order and orientation of the 21 DraI
restriction fragments, suitable Southern blots of BCG
DNA were hybridized with a panel of 7 BCG and 19 M.
W. J. PHILIPP and OTHERS
Fig. 3 . The circular Dral restriction map of the chromosome of
M. bovis BCG Pasteur. The positions of some genetic markers
(see Table 4) and of the insertion sequence 156110 are shown.
The position of the direct repeat region that harbours the sole
copy of IS61 10 in M, bovis, BCG strains and some clinical isolates
of M. tuberculosis is indicated by 'DR'.
tz/berczllosis linking clones (Table 3). A representative
linking analysis is shown in Fig. 1. It can be seen that
identically sized fragments of 125 and 80 kb (Table 2) in
the chromosomes of M.
bovis BCG Pasteur (K and N2,
respectively) and M. tuberczllosis H37Ra (U and P, re-
spectively) were contiguous (Fig. 1 b). By contrast,
hybridization with probe T225 (Fig. lc) shows that, while
one of two adjacent fragments (30 kb; Q in BCG, G in M.
tzlberczllosis; Table 2) was the same size in both myco-
bacteria, the other was about 20 kb smaller in the genome
of BCG (H in BCG, 22 in M. tzlberculosis; Table 2).
Linkage of fragments H and M was obtained by using
AsnI fragment Q from M. tzlbercdosis as a probe. In this
way all 21 DraI restriction fragments were linked, thereby
revealing a single circular chromosome (Fig. 3). Ad-
ditional proof, albeit indirect, of the circularity of the
chromosome of M. bovis BCG was obtained by using
optimized PFGE running conditions for resolving linear
megabase-sized DNA molecules since undigested circular
genomic DNA did not enter the gels. Under these
conditions, the linear chromosome of Agrobacterizlm
tzlmefaciens was resolved (Allardet et al., 1993; Fonstein &
Haselkorn, 1995) whereas that of BCG Pasteur remained
at the origin.
The above linking and restriction analysis suggested that
the chromosomes of M. tzlbeczlhsis H37Rv and M. bovis
BCG were indeed very similar. To determine whether the
positions of known genes were also conserved (Philipp et
al., 1996), probes corresponding to 18 different genetic
markers (see Table 4) were used in hybridization experi-
ments with DraI and AsnI fragments resolved by PFGE.
The locations of all markers tested were compared with
those of their homologues in the genome of M.
The DNA gyrase genes gyrAB, and oriC, the chromo-
somal origin of replication of M. tz-iberczllosis, hybridized to
Table 4 . Identity and source of known genetic markers
mapped in M. bovis BCG Pasteur
Cytochrome o ubiquinol oxidase
Gyrase, A and B subunits
Origin of replication
Ribosomal protein S12
RNA polymerase, /3 and
Urease, subunits A, B and C
Unique sequence for detection
of M. tuberculosis complex
Mt, T. Shinnick
Mt, H. E. Takiff
Mt, H. E. Takiff
Mb, R. A. Young
*Mt, M. tuberculosis; Mb, M. bovis; GMB, Unitt de Gtnttique
MolCculaire BactCrienne and UGM, Unite de GCnCtique
Mycobacttrienne, Institut Pasteur, Paris, France; T. Shinnick,
Centers For Disease Control, Atlanta, GAY USA; H. E. Takiff,
Instituto Venezolano de Investigaciones Cientificas, Caracas,
Venezuela; R. A. Young, Whitehead Institute for Biomedical
Research, Cambridge, MA, USA.
AsnI and DruI fragments of the same size in the
chromosomes of both species. This was also the case for
the genes rpsL, rpoB and ureC (Philipp & Cole, 1995). The
results of this analysis, summarized in Figs 3 and 4,
revealed extensive genome-wide conservation.
Detailed molecular comparison with M. tuberculosis
The linking clone analysis strongly suggested that all 21
DraI cleavage sites were conserved in both M. bovis BCG
and M. tuberczllosis H37Rv although the fragments varied
in size and number (Table 2). This variation can be
explained by differences in the copy number of the
insertion sequence ISGIIO, which carries a DraI site
(Thierry et al., 1990a, b), as a single copy is present in M.
bovis whilst 16 copies have been mapped in M. tnberdosis
(Hermans et a/., 1991; Philipp et al., 1996). Although
extensive conservation of Am1 sites and fragment lengths
were detected (at least 33 out of 47 were common), a
number of fragments appeared to differ significantly (see
Table 2). In BCG Pasteur, most of these AsnI fragments
corresponded to regions of the chromosome of M.
tzlbemdosis H37Rv where repetitive sequences are present
Genome map of Mycobacterizlm bovis BCG Pasteur
I I I -
Fig. 4. Schematic presentation of the chromosomes of M. bovis BCG Pasteur and M. tuberculosis H37Rv by alignment of
their Dral fragments. The approximate locations of mapped genes, linking clones and cosmids used as probes for
detection of variable regions are indicated. Cosmids that were only hybridized to blots of EcoRl digests are denoted by
an asterisk. Variable regions are highlighted in the central portion: Pm, polymorphisms; Add., additional DNA; Del.,
deletions. The positions of the regions deleted from the chromosome of BCG Connaught, RD1-RD3 (Mahairas et a/.,
1996), are indicated. Other details are as in the legend to Fig. 3.
(Fig. 4) and this suggested that these loci may be readily
susceptible to rearrangements, insertions and deletions.
To test this possibility, 15 cosmids were selected from the
ordered library of H37Rv clones for use as hybridization
probes to try and detect differences in the EcoRI restriction
patterns of the respective genomes. For control and
comparative purposes, DNA from M. bovis and BCG
substrains Copenhagen, Glaxo, Moreau and Tokyo was
examined and the combined results of this study are
summarized in Table 5 and Fig. 4. Eleven of the 15
cosmids used in hybridization detected genomic
differences due to insertions, deletions and restriction
fragment length polymorphisms in the comparative
genomic analysis. Certain probes detected fragments in
M. tzlberculosis that were also present in M. bovis but were
apparently missing from the genomes of some, or all, of
the various BCG strains (Y57, Y277, T59 and Y366) while
others only hybridized to sequences that were present in
M. tuberculosis, and not in M. bovis or BCG (Y22, Y98,
Y138, Y277, Y324, Y349 and Y366). In one case (Y57;
Fig. 5b), a polymorphism was detected only in M. bovis,
whereas with probe Y324 the pattern obtained with all
BCG strains resembled more closely that of M. tuberculosis
is of particular interest as it corresponds, in part, to the
locus encoding ESAT-6. The corresponding gene, esx, is
known to be present in M. tuberculosis and M. bovis on
a large (> 15 kb) EcoRI fragment and is missing from
BCG (Sorensen et al., 1995) as can be seen in (Fig.
5a). Strikingly, this region of the chromosome of M.
bovis (Fig. 5c). The region covered by probe Y277
tuberculosis H37Rv also contained three other EcoRI
fragments that do not appear to have counterparts in M.
bovis or BCG (Table 5). Similar results were obtained
with probe Y366 which detected two additional EcoRI
fragments in M. tuberculosis H37Rv compared to M. bovis,
and indicated that a deletion had occurred in the corre-
sponding region of the chromosome of BCG Pasteur as at
least two fragments seemed to be missing (Fig. 5e, Table
The principle objective of this work was to construct a
physical map of the chromosome of the classical vaccine
strain M. bovis BCG Pasteur and hence to obtain general
insight into its genomic organization. BCG has served as
a safe live vaccine for over 70 years (Bloom & Fine, 1994),
and the comparative dissection of its genome could help
to identify regions that differ extensively from the
corresponding segments of the chromosomes of the
pathogens M. bovis and M. tuberculosis. The recent com-
pletion of an integrated map of the circular chromosome
of the tubercle bacillus M. tubercdosis H37Rv, and the
tools generated therein (Philipp et al., 1996), considerably
facilitated this task in the absence of the parental M. bovis
strain originally isolated by Calmette, which was lost
during the war (Calmette, 1927).
Comparison of the genome maps of M. bovis BCG Pasteur
and the virulent reference strain M. tubercdosis H37Rv
revealed that there was extensive conservation of genes,
W. J. PHILIPP and OTHERS
Table 5. Comparison of patterns after hybridization of selected cosmids on complete
EcoRl digestions of the chromosomes of M. tuberculosis H37Rv, wild-type M. bovis and
different M. bovis BCG strains
Probe Myco bacterial
8, 7, 6, 4, 1.5
8, 7, 6, 4, 1.5
15, 12, 7, 5.5, 45, 3
15, 12, 7, 5.5, 45, -
15, 12, 7, 5.5, 45, -
15, 12, -, 55, 45, -
15, 8, 5, -, 3.5, 25, 1.8, 1.5, 1.3, 1
15, 8, 5, 4, -, 25, 1.8, 1.5, 1.3, 1
15, 8, 5, -, -, 25, 1.8, 1.5, 1.3, 1
12, 9, 6, 5.5, 4.5, 3.8, 3.3, 3, 2.5, 2,
1.5, 0.8, 0.6, 0.5, 0.3
12, 9, 6, 5.5, 45, 3.8, -, 3, 25, 2,
1.5, 0.8, 0.6, 0.5, -
12, 9, 6, 5.5,45, 3.8, -, 3, 25, 2,
1.5, 0.8, 0.6, 05, -
> 15, 15, -, 5.5, 4.5, 2.7, 1
> 15, 15, -, 5.5, 4.5, 2.7, 1
> 15, 15, -, 5.5, 4.5, 2.7, 1
> 15, 15, 6, 5.5, 4.5, 2.7, 1
> 15, 15, 6, 5.5, 45, 2-7, 1
> 15, 9, 5.5, 4, 3.5, 3, 25, 23, 075, 05
> 15, 9, -, -, 3.5, 3, 2.5, 2.3, 0.75, 0.5
-, 9, -, -, 3.5, 3, 2.5, 2.3, 0.75, 0.5
9.5, 8.5, 6, 5.1, 45, 4, 3.5, 2.9, -, 2.1,
1.3, 1.2, 0.8
9.5, 8.5, 6, -, 4.5, -, 3.5, 2.9, 2.2, 2.1,
1.3, 1.2, 0 8
9-5, 8.5, 6, 5.1, 45,-, 3*5,2.9, 22, 21,
1-3, 1.2, 0.8
> 8, 6, 5, 4, 3.5, 27, 2, 0.7, 0 5
> 8, 6, -, 4, 3.5, 2.7, 2, 0.7, 0.5
> 8, 6, -, 4, 3.5, 2.7, 2, 0.7, 0.5
> 11, 11, 55,4, 3.7, 3.2, 3.1, -, 2.1,
> 13, 9, -, 4, 3.7, 3.2, 3.1, 22, 21,
10, -, -, 4, 3.7, 3.2, 3.1, -, 2.1, -, 0.6
12.5, 12, 6, 5, 3.5, 2.5, 1.8, 1.1, 0.7, 0.5
12.5, 12, 6, 5, 3.5, 2.5, 1.8, 1.1, 0.7, 0.5
125, 12, -, 5, 3.5, 2.5, 1.8, 1.1, 07, 0.5
12.5, 12, 6, 5, 3.5, 2.5, 1.8, 1.1, 0.7, 0.5
6, 5, 4, 3.5, 3.2
ND, Not determined.
Genome map of Mycobacterizdm bovis BCG Pasteur
Fig. 5 . Analysis of variable regions of the chromosome of different M. bovis BCG strains by hybridization with selected
cosmids. Samples were complete €coRI digests from M. tuberculosis H37Rv (lane l), M. bovis (lane 2) and M. bovis BCG
Pasteur (lane 3). Hybridization was performed with probes prepared from cosmids Y277(a), Y57 (b), Y324 (c), Y98 (d) and
Y366 (e). The sizes of fragments that differ in M. tuberculosis and in M. bovis are labelled without and with an asterisk,
respectively. Owing to the use of nick-translated cosmids as probes some of the smaller fragments do not show up well in
these autoradiograms but were visible on longer exposure. The results of other hybridizations are summarized in Table 5
and Fig. 4.
marker order, restriction sites and fragment sizes, indi-
cating that the two genomes were essentially colinear
(Figs 3,4; Tables 2,3,5). The least divergent segment of
the genome is a 1500 kb stretch centred around the oriC
locus (Fig. 4). The finding of global conservation is
consistent with the fact that comparison of the sequences
of genes from these two organisms and their strains
reveals an identity of - 99 % (Kapur et al., 1994).
Nevertheless, comparison of the restriction patterns, the
AsnI fragment sizes and the respective genome maps led
to the identification of regions that appeared to differ in
their local organization. In several cases where differences
were suspected they were confirmed by hybridization
analysis using cosmids carrying segments of the M.
ttrberculosis chromosome as probes. The variable regions
could be grouped into three classes: the first, where M.
ttrbercdosis contains DNA that is not present in M. bovis or
BCG (Fig. 5a, d) ; the second, in which the arrangement in
M. tz/bercz/losis and M. bovis appears to be the same but
differs from that of BCG (Fig. 5a, c); the third where M.
tziberctrlosis and BCG are similar but M.
restriction fragment length polymorphism (Fig. 5c).
bovis displays a
W. J. PHILIPP and OTHERS
There are two likely explanations for the first class of
polymorphisms ; either they correspond to unique
genomic sequences that are confined to H37Rv and
possibly other isolates of M. tuberculosis, or they represent
an insertion event such as acquisition of an IS element or
expansion of a repetitive sequence. The latter explanation
may be correct in some cases but it is not true of all. For
instance, two and three additional EcoRI fragments were
detected with probes Y98 and Y277, respectively (Fig. 5a,
d), whereas only one polymorphic fragment would be
expected if any of the known insertion sequences in M.
tzderctriosis had transposed as none of these contains a site
for EcoRI. A number of important phenotypic differences
between M. bovis, or BCG, and M. tuberculosis are known
(Heifets & Good, 1994). Neither M. bovis or BCG is
capable of respiration with nitrate or able to produce
niacin, unlike M. tuberctrlosis. Both M. bovis and BCG are
resistant to pyrazinamide but susceptible to 2-thiophene-
carboxylic acid hydrazide (TCH) whereas M. ttrberculosis
displays the opposite phenotypes. While M.
infect humans efficiently, M.
for bovines. It is conceivable that some of these discrepant
phenotypes may have their origins in the divergent
genomic areas described here. The availability of well-
characterized shuttle cosmid clones (Philipp et a/., 1996),
will allow this hypothesis to be tested.
bovis is able to
ttrberculosis is not pathogenic
The second class of polymorphisms corresponds to three
loci that appear to have undergone deletion events during
the isolation of the original BCG Pasteur strain and
subsequent evolution of the various BCG substrains. In
two instances, regions which differ between the two
mapped mycobacterial genomes and appear to have
contracted in BCG Pasteur are known to harbour the
genes for M. tzlberculosis protein antigens, MPT64 and
ESAT-6. Other workers have demonstrated that these
coding sequences are missing from some (MPT64) or all
(ESAT-6) BCG strains (Harboe etal., 1996; Li etal., 1993;
Sorensen et al., 1995) but present in virulent tubercle
bacilli. Recently, by means of a genomic subtraction
approach involving M. bovis and BCG Connaught,
Mahairas et al. (1 996) identified and extensively charac-
terized three regions termed RD1-RD3, which had been
deleted from the chromosome of BCG Connaught. Two
of the RDs encoded the MPT64 and ESAT-6 antigens
while RD1 contained a regulatory locus that influences
production of several proteins. The findings of these
workers are consistent with the mapping data described
here and the positions of the RD regions are indicated in
Fig. 4. It is of interest that these regions susceptible to
deletions are located towards the putative mycobacterial
replication terminus as it is known from work with other
bacteria that the terminus is often a site of genetic
rearrangements (Krawiec & Riley, 1990). Furthermore,
RD1 and RD3 are situated within the same 100 kb
mapping interval (Fig. 4).
In a recent authoritative study (Lagranderie et al., 1996), it
was convincingly demonstrated that different BCG strains
could be classified in terms of their growth rates and of the
immune responses that they induced in mice. Two of the
strains examined, the Prague and Japanese BCG strains,
could not confer resistance against a second inoculation
with various recombinant BCG strains whereas
immunization with the Glaxo, Pasteur or Russian strains
could. The preliminary results presented in Table 5
indicate that polymorphisms exist between the various
BCG substrains and detailed analysis of the genomic
organization may well shed further light on the genetic
basis of this immunovariability by highlighting poly-
morphic segments of the chromosome. The genome map
of BCG Pasteur established in the present work will thus
be of great value for the interpretation of further
We wish to thank M. Gheorghiu, B. Heym and S. Gordon for
strains, help and useful discussions. This investigation received
financial support from the World Health Organisation, the
European Community biotechnology programme (BIO. CT92-
0520), the Association Frangaise Raoul Follereau and the Institut
Allardet, 5. A., Michaux, C. S., Jumas, B. E., Karayan, L. & Ramuz,
M. (1993). Presence of one linear and one circular chromosome in
the Agrobacterium tumefaciens chromosome C58 genome. J Bacteriol
Bergh, 5. & Cole, S.T. (1994). MycDB - an integrated myco-
bacterial database. Mol Microbiol 12, 51 7-534.
Bloom, B. R. & Fine, P. E. M. (1994). The BCG experience:
implications for future vaccines against tuberculosis. In Tubercufosis:
Pathogenesis, Protection, and Control, pp. 531-557. Edited by B. R.
Bloom. Washington, DC : American Society for Microbiology.
Bloom, B. R. & Murray, C. 1. L. (1992). Tuberculosis: commentary
on a reemergent killer. Science 257, 1055-1064.
Calmette, A. (1 927). La vaccination Preventive Contre la Tubercdose.
Paris: Masson et Cie.
Clemens, J. D., Jackie, J. H., Chuong, J. H. & Feinstein, A. R.
(1983). The BCG controversy : a methodological and statistical
reappraisal. JAMA 249, 2362-2369.
Cole, S.T. & Saint-Girons, I. (1994). Bacterial genomics. FEMS
Microbiol Rev 14, 139-160.
Cole, S. T. & Smith, D. R. (1994). Toward mapping and sequencing
the genome of Mycobacterizm tuberculosis. In Tuberczdosis : Pathogenesis,
Protection, and Control, pp. 227-238. Edited by B. R. Bloom.
Washington, DC : American Society for Microbiology.
Fine, P. (1995). Variation in protection by BCG: implications of
and for heterologous immunity. Lancet 346, 1339-1345.
Fonstein, M. & Haselkorn, R. (1995). Physical mapping of bacterial
genomes. J Bacterioll77, 3361-3369.
Harboe, M., Oetgger, T., Wiker, H. G., Rosenkrands, 1. &
Andersen, P. (1996). Evidence for occurrence of the ESAT-6
protein in Mycobacterium tuberculosis and virulent Mycobacterium bovis
and for its absence in Mycobacterium bovis BCG. Infect Immun 64,
Heifets, L. B. & Good, R. C. (1994). Current laboratory methods for
the diagnosis of tuberculosis. In Tuberculosis : Pathogenesis, Protection,
and Control, pp. 85-110. Edited by B. R. Bloom. Washington, DC:
American Society for Microbiology.
Genome map of Mycobacterizmz bovis BCG Pasteur
Hermans, P. W. M., van Soolingen, D . ,
P. E. W., Dale, J. W. & van Embden, J. D. A. (1991). Insertion
element IS987 from Mycobacterium bovis BCG is located in a hot-spot
region for insertion elements in Mycobacterium tuberculosis complex
strains. Infect Immm 59, 2695-2705.
Kapur, V . , Whittam, T. 5. & Musser, 1. (1994). Is Mycobacferium
tuberculosis 15 000 years old ? J Infect Dis 170, 1 348-1 349.
Krawiec, 5. & Riley, M. (1990). Organization of the bacterial
chromosome. Microbiol Rev 54, 502-539.
Lagranderie, M. R. R . , Balazuc, A.-M., Deriaud, E . , Leclerc, C. D. &
Gheorghiu, M. (1996). Comparison of immune responses of mice
immunized with five different Mycobacterimv bovis BCG vaccine
strains. Infect Immun 64, 1-9.
Li, H., Ulstrup, J. C., Jonassen, T. O., Melby, K., Nagai, S. &
Harboe, M. (1993). Evidence for absence of the MPB64 gene in
some substrains of Mycobacterium bovis BCG. Infect Immun 61,
Liu, S.-L., Hessel, A. & Sanderson, K. E. (1993). Genomic mapping
with I-CeuI, an intron-encoded endonuclease specific for genes for
ribosomal RNA, in Salmonella spp., Escbericbia coli, and other
bacteria. Proc Natl Acad Sci U S A 90, 6874-6878.
Mahairas, G. G . , Sabo, P. J . , Hickey, M. J . , Singh, D. C. & Stover,
C. K. (1 996). Molecular analysis of genetic differences between
Mycobacterium bovis BCG and virulent M. bovis. J Bacteriol 178,
Marshall, P. & Lemieux, C. (1992). The I-CeuI endonuclease
recognizes a sequence of 19 base pairs and preferentially cleaves the
Bik, E. M., de Haas,
coding strand of the Cblamydomonas moewasii chloroplast large
subunit rRNA gene. Nucleic Acids Res 20, 6401-6407.
Philipp, W. 1 . &Cole, 5. T. (1995). Local comparison of the genomes
of Mycobacteriaim tubercailosis and Mycobacteriunz leprae using the
polymerase chain reaction. FEMS Microbiol Lett 132, 263-269.
Philipp, W. J . , Poulet, 5.. Eiglmeier, K . ,
ramanian, B., Heym, B., Bergh, s., Bloom, B. R., Jacobs, W. R . , Jr
& Cole, S. T. (1996). An integrated map of the genome of the
tubercle bacillus, Mycobacterium tuberculosis H37Rv, and comparison
with Mycobacterium leprae. Proc Nafl Acad Sci U S A 93, 3132-3137.
Sambrook, J . , Fritsch, E. F . & Maniatis, T. (1989). Molecular Cloning:
a Laboratoory Manual, 2nd edn. Cold Spring Harbor, NY: Cold
Spring Harbor Laboratory.
Sorensen, A. L . , Nagai, S . , Houen, G., Andersen, P. & Andersen, A.
(1 995). Purification and characterization of a low molecular mass
T-cell antigen secreted by Mycobacterium tabercdosis. Infect Imnznn
Thierry, D., Brisson-Noel, A., Vincent-L&y-Fr&bault, V . , Nguyen,
S., Guesdon, J. & Gicquel, B. (1990a). Characterization of a
Mycobacterizlm tuberculosis insertion sequence, IS61 10, and its ap-
plication in diagnosis. J Clin Microbiol28, 2668-2673.
Thierry, D . , Cave, M. D . , Eisenach, K. D . , Crawford, J. T . , Bates,
J. H., Gicquel, B. & Guesdon, J. L. (199Ob). IS6110, an IS-like
element of Mycobacterium tuberculosis complex. Nucleic A c i d s Res 18,
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Received 29 April 1996; revised 16 July 1996; accepted 17 July 1996.
Pascopella, L . ,
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