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Journal of Bacteriology, June 2000, p. 3394-3399, Vol. 182, No. 12
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
A Point Mutation in the mma3 Gene Is Responsible for
Impaired Methoxymycolic Acid Production in Mycobacterium
bovis BCG Strains Obtained after 1927
Marcel A.
Behr,1,*
Benjamin G.
Schroeder,2
Jacquelyn N.
Brinkman,1
Richard A.
Slayden,2 and
Clifton E.
Barry III2
McGill University Health Centre, Montreal, Canada H3G
1A4,1 and Tuberculosis Research
Section, Laboratory of Host Defenses, National Institute of Allergy
and Infectious Diseases, Rockville, Maryland 208522
Received 12 January 2000/Accepted 29 March 2000
 |
ABSTRACT |
BCG vaccines are substrains of Mycobacterium bovis
derived by attenuation in vitro. After the original attenuation (1908 to 1921), BCG strains were maintained by serial propagation in
different BCG laboratories (1921 to 1961). As a result, various BCG
substrains developed which are now known to differ in a number of
genetic and phenotypic properties. However, to date, none of these
differences has permitted a direct phenotype-genotype link. Since BCG
strains differ in their abilities to synthesize methoxymycolic acids
and since recent work has shown that the mma3 gene is
responsible for O-methylation of hydroxymycolate precursors to form
methoxymycolic acids, we analyzed methoxymycolate production and
mma3 gene sequences for a genetically defined collection of
BCG strains. We found that BCG strains obtained from the Pasteur
Institute in 1927 and earlier produced methoxymycolates in vitro but
that those obtained from the Pasteur Institute in 1931 and later all
failed to synthesize methoxymycolates, and furthermore, the
mma3 sequence of the latter strains differs from that of
Mycobacterium tuberculosis H37Rv by a point mutation at bp
293. Site-specific introduction of this guanine-to-adenine mutation
into wild-type mma3 (resulting in the replacement of
glycine 98 with aspartic acid) eliminated the ability of this enzyme to
produce O-methylated mycolic acids when the mutant was cloned in tandem
with mma4 into Mycobacterium smegmatis. These
findings indicate that a point mutation in mma3 occurred between 1927 and 1931, and that this mutant population became the
dominant clone of BCG at the Pasteur Institute.
| |
INTRODUCTION |
BCG vaccines are attenuated
substrains of Mycobacterium bovis that were grown in vitro
for much of the first half of the 20th century (9). In
clinical trials, the protective efficacy has varied considerably,
leading to speculation that prolonged growth in vitro has resulted in
overattenuated vaccines (3). Based on a recent analysis of
gene content of various BCG strains, it is now apparent that during
prolonged in vitro passage, BCG strains have lost polygenic regions
both at the Pasteur Institute and at other BCG laboratories
(5). However, the genetic deletions described have not been
directly linked to phenotypic changes; therefore, their implication in
the attenuation process remains unknown.
An important and defining characteristic of mycobacteria is their
capacity to synthesize long-chain 
-hydroxy, 
-alkyl fatty acids,
known as mycolic acids (2). One type of mycolic acid, containing an -methyl branched methyl ether, is known as
methoxymycolic acid. It has long been known that certain BCG strains,
such as BCG-Pasteur, are incapable of synthesizing methoxymycolates
(18). Recently, Yuan and colleagues were able to implicate
the mma3 gene in methoxymycolate synthesis by complementing
BCG-Pasteur with the wild-type mma3 gene from
Mycobacterium tuberculosis (27). Importantly,
hyperexpression of mma3 not only resulted in methoxymycolate production but also altered cell wall function and growth in
macrophages, the cells where BCG resides after vaccination.
Unfortunately, the results obtained by Yuan were difficult to interpret
in a phylogenetic context because of the choice of BCG strains analyzed
and the comparison of genetic sequences generated by different
laboratories (7). As well, because of the large number of
genetic differences observed between BCG strains and M. tuberculosis H37Rv, it was not possible to determine precisely the
genetic lesion associated with impaired methoxymycolate production. We
have therefore undertaken a blinded assessment of methoxymycolate production across a genetically characterized collection of BCG strains
and compared this phenotype to the mma3 gene sequence for
each of these strains. Because of previous work which enabled us to
document the historical propagation of BCG strains from the Pasteur
Institute (4), we are able to show here that a single
nucleotide polymorphism occurred in mma3 between 1927 and 1931, resulting in loss of methoxymycolic acid production in BCG substrains obtained after that period.
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MATERIALS AND METHODS |
Genetic analysis of the mma3 sequence from BCG
strains.
Mycobacteria listed in Table 1 were grown for 14 to 21 days in 7H9 medium supplemented with OADC enrichment (Difco), and whole
genomic DNA was extracted as previously described (24). To
produce high-quality sequence data for the entire 879-bp
mma3 gene (nucleotides 737271 to 738149 of the M. tuberculosis H37Rv genome, searchable at the TubercuList website
[http://bioweb.pasteur.fr/GenoList/TubercuList/]), we designed two
pairs of primers to obtain sequence spanning beyond the gene. Primers
5'-CGCGTTGTAGGCGAACTTGA-3' (forward) and
5'-GATGTGCCATGCACCGTGT-3' (reverse) amplified the 5' portion
of mma3, while primers 5'-CGGCCATTCTCGTCATGTTCT-3' (forward) and 5'-ACTGGGCCAACTTCAGCGAG-3' (reverse)
were used to amplify the 3' portion. PCR mixtures contained 50 ng of
genomic DNA, 20 mM Tris-acetate (pH 9.0), 10 mM ammonium sulfate, 75 mM potassium acetate, 0.05% Tween 20, 2.5 mM MgSO4, 4 nmol of
deoxynucleoside triphosphates, 25 pmol of each primer, and 1 U of
Tfl polymerase (Promega) in a final volume of 50 µl. The
PCR consisted of a 5-min denaturation at 94°C followed by 35 cycles
of 94°C for 1 min, 65°C for 1 min, and 72°C for 2 min, with a
final extension cycle of 72°C for 10 min. Amplification was confirmed
by agarose gel electrophoresis, and PCR products were purified with a
kit (Qiagen). Purified samples were sequenced using ABI Prism Big Dye
Terminator Cycle Sequencing (Perkin-Elmer Applied Biosystems).
For rapid identification of the single mutation detected, genomic DNA
was amplified by PCR for the 5' 562 bp of mma3. Purified PCR
products were then digested with 5 U of SalI (Promega) for 1 h at 37°C. Samples were run in duplicate on a 2% agarose gel and scored as wild type or mutant based on restriction pattern.
Analysis of mycolic acid profiles in BCG strains.
The 13 BCG
strains were grown in 7H9-ADC-0.05% Tween, and mycolic acid methyl
esters were prepared and analyzed by one-dimensional thin-layer
chromatography (TLC) as previously described (27). TLC
plates were visualized by immersion in ceric ammonium molybdate dip and
heating to 120°C for 20 min. Ceric ammonium molybdate dip is prepared
by dissolving 1 g of ceric sulfate monohydrate and 25 g of
ammonium molybdate in 450 ml of distilled water. With stirring, 50 ml
of concentrated sulfuric acid is carefully added, and the solution is
stirred for 1 h at room temperature.
Mutagenesis of mma3 at bp 293.
The
mma3 and mma4 genes of M. tuberculosis
H37Rv (TubercuList Rv0643c and Rv0642c) were PCR amplified from genomic
DNA using primers gctctagaGATGGCCACCTGCTGAAG (forward) and
gcgcaagcttGGGCTTATGC-GTCTGCTC (reverse), which contain
nonhomologous sequences at each 5' end (lowercase letters) that are
used to add XbaI and HindIII sites, respectively. This PCR product was digested with XbaI and
HindIII and cloned into the XbaI and
HindIII sites of pUC19 to make plasmid pBGS93. The entire
insert was sequenced to ensure that no mutations were introduced by PCR
amplification. In addition to the standard M13 forward and reverse
sequencing primers, the following custom primers were used for
sequencing the insert: 3R1, GCTTCGATGGTTACGATGCGG (R
indicates reverse; F indicates forward); mut3.1 (see below), 4F1
GAGACGATCGAGGAGCATGTG; 4R1, GGTAGCTGACGCTGCTCTGG;
and 4F2, GACCCGACCCGAACTTAC. A single-base G-to-A
mutation was introduced at base 293 of the mma3 coding
sequence using the QuikChange Site-Directed Mutagenesis Kit
(Stratagene) and mutagenic primers mut3.1
(CGACAGGGTCAAGtCGACGACGTTGACGTCAACGTCGTCGaCTTGACCCTGTCG) (forward) and mut3.2 (CGACAGGGTCAAGtCGACGACGTTGAC)
(reverse) (mutated bases are shown in lowercased). The resulting
pUC19 clone containing the mutated mma3 and wild-type
mma4 genes, designated pBGS95, was sequenced using the
primers listed above to ensure that the desired mutation was present
and that no unwanted mutations were introduced. The
XbaI-to-HindIII fragments were excised from
pBGS93 and pBGS95 and introduced into the same sites of the
Escherichia coli-mycobacterial shuttle vector pMV206_Hyg(11)
to produce pBGS94 and pBGS96, respectively. To boost expression of
mma3 and mma4 in Mycobacterium
smegmatis, the hsp60 promoter from pMV261 was introduced in front of mma3 (11). Plasmids pBGS94
and pBGS96 were digested with XbaI, the resulting overhangs
were filled in with Klenow fragment, and the products were digested
with KpnI and then ligated to the 418-bp
KpnI-PvuII fragment containing the
hsp60 promoter from pMV261 to produce plasmids pBGS99 and pBGS100, respectively.
The
mma4 gene alone was subcloned from
pMV206_
mma4 into pMV206_Hyg as a 1.7-kb
BamHI
fragment to make pBGS16 (
26). The orientation
of the
mma4 insert in pBGS16 is such that
PvuII
digestion yields
4.7- and 1.2-kb
fragments.
Plasmids pMV206_Hyg, pBGS16, pBGS99, and pBGS100 were introduced into
M. smegmatis mc
2155 by electroporation, cultures
were labeled with [1-
14C]acetate, and mycolic acid methyl
esters were prepared and analyzed
by two-dimensional argentation TLC as
described previously (
11).
| |
RESULTS |
Sequence analysis of mma3 in 13 different strains of
M. bovis BCG.
To elucidate the molecular basis for the
loss of production of methoxymycolic acids by certain strains of BCG,
we obtained an extensively characterized collection of BCG isolates
with a defined history (5) (Table
1). The mma3 gene from each of these 13 strains was obtained by PCR amplification of genomic DNA, and
these products were sequenced in their entirety (Fig. 1A). A comparison of the nucleotide
sequence of the mma3 gene between BCG strains, M. bovis 2122, and M. tuberculosis H37Rv revealed that all
nucleotides in the gene were identical across all strains tested with
the exception of position 293 of the 879-nucleotide open reading frame.
At this position, M. tuberculosis H37Rv, M. bovis
2122, and BCG strains obtained from the Institut Pasteur from 1924 to
1927 (strains Russia, Moreau, Tokyo, Sweden, and Birkhaug) had the same
sequence as that reported for M. tuberculosis H37Rv
(6). In the eight BCG strains obtained in 1931 or later, a
G-to-A base substitution was seen at position 293. This transition results in an amino acid change from glycine (Gly, encoded by GGC) to
aspartic acid (Asp, encoded by GAC) at codon 98. Unlike the findings of
previous studies (7, 27), no other differences were observed
in BCG strains. To confirm these results, SalI digestion of
the 562-bp PCR product representing the 5' portion of the gene resulted
in two clearly distinct DNA fragment patterns, based on the G-to-A base
substitution causing an extra SalI cleavage site (see Fig.
1A and B).
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FIG. 1.
Genetic and phenotypic analysis of BCG methoxymycolic
acid production. (A) Map of the gene locus responsible for the
production of methoxymycolates. The shaded bar represents genomic DNA,
with numerical locations on the chromosome of strain H37Rv indicated
(6). The heavy arrows indicate methyltransferase open
reading frames. Small arrows indicate primers used to amplify the 5'
and 3' regions of mma3. Thin solid lines represent the PCR
products. Open squares, SalI sites. Shown below the genomic
regions are the amplicons from wild-type and mutant mma3 5'
regions, with SalI sites indicated (note the additional site
in the mutant mma3). Scale marker, 1,000 bp. (B) Purified
562-bp PCR products containing the 5' end of the mma3 gene
from the 13 BCG strains were digested with SalI. Without
digestion, a 562-bp product is seen. After incubation, the wild-type
strains have three fragments of 303, 144, and 115 bp, while the mutant
strains have four fragments of 246, 144, 115, and 57 bp. (C) TLC
analysis of purified mycolic acids from various BCG substrains. The
same BCG strains shown in the PCR-RFLP gel were analyzed by TLC for
production of mycolic acids as described in Materials and Methods. The
three bands obtained represent (from bottom to top) ketomycolates,
methoxymycolates, and -mycolates. It is seen in this figure that
eight BCG strains lack methoxymycolates. These are the same strains
that gave the extra SalI band; they represent strains of BCG
obtained from the Pasteur Institute in 1931 or later.
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|
Analysis of mycolic acid production among BCG strains.
We
examined the mycolic acid subclass production among the collection of
13 BCG strains by simple TLC analysis of the methyl esters of
saponified total cell lysates as shown in Fig. 1C. Samples are resolved
based upon the polarity of the functional groups involved so that the
fastest-migrating band represents the -mycolate subclass (with a
single cis cyclopropane in the distal position); the middle,
more-variable band represents methoxymycolic acids containing a more
polar -methyl-methyl ether; and the slowest-migrating band
represents ketomycolates, which contain the most-polar functional group, an -methyl branched ketone. The strains analyzed fell into
two broad classes: those that produced all three mycolic acids and
those that produced approximately 50% -mycolate and 50%
ketomycolate. There was a perfect correlation between BCG strains that
produced MMAS-3 (G98D) and those that failed to produce the middle band
representing methoxymycolates. With the stain used in Fig. 1C, we did
not observe the production of hydroxymycolic acids, which would run
more slowly than ketomycolates in this TLC system. A very small amount
of hydroxymycolate could be detected in
[14C]acetate-radiolabeled mycolates; however, there was
no correlation between the amount present and the mma3
mutation (data not shown).
Mutagenesis of mma3 and functional analysis of
MMAS-3(G98D).
Glycine 98 lies in a region of MMAS-3 which is
highly conserved among all fatty acyl methyltransferases identified to
date (Fig. 2). Immediately N-terminal of
Gly98 is an S-adenosylmethionine (SAM)-binding motif shared
by a wide variety of SAM-dependent methyltransferases (13).
Sequence alignment of the cyclopropane fatty acid synthase from
E. coli and all six mycolic acid methyltransferase sequences
for which a function has been described revealed that Gly 98 was
absolutely conserved. We hypothesized that the mutation of this small,
neutral residue to negatively charged aspartic acid would be sufficient
to inactivate MMAS-3 activity.
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FIG. 2.
Alignment of the pertinent region of amino acid sequence
from various methyltransferases. The starting amino acid number is
shown at the left. An arrow indicates the G98D mutation observed in the
BCG MMAS-3 sequence that appears to correlate with production of
methoxymycolic acids by M. tuberculosis. Sequences are
MMAS-3 (TubercuList (http://bioweb.pasteur.fr/GenoList/TubercuList/)
accession number Rv0643c), CMAS-1 (Rv3392c), CMAS-2 (Rv0503c), MMAS-1
(Rv0645c), MMAS-2 (Rv0644c), and MMAS-4 (Rv0642c), as well as the
cyclopropane fatty acid synthase from E. coli (CFAS;
SwissProt [http://www.ebi.ac.uk/cgi-bin/swissfetch] accession number
P30010).
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|
In order to assess the functional relevance of this mutation, we
introduced this mutation specifically into the
mma3 gene
derived from
M. tuberculosis by site-directed mutagenesis
(see
Materials and Methods). Assessing MMAS-3 function is possible
only
in a heterologous background that produces the appropriate
hydroxymycolate precursor. In
M. tuberculosis this
hydroxymycolate
precursor is produced by the MMAS-4 protein
(
26). This background
can be created by expressing the
MMAS-4 protein in the heterologous
host
M. smegmatis (Fig.
3). Introduction of
mma4 alone
gives a
strictly hydroxymycolate profile showing H1 and H2 (Fig.
4B),
while introduction of both
mma4 and
mma3 simultaneously gives
a mixture of
two isomers of methoxymycolate (one derived from
the
1 series with a
proximal
cis-olefin, M1, and one derived
from the
2
series with a proximal
trans-olefin and allylic methyl
branch, M2) (Fig.
4C). Introduction of an identical construct
bearing
the mutation resulting in the G98D amino acid substitution
results in
failure of the
O-methyltransferase portion of the reaction,
and the mycolate profile looks identical to that resulting from
expression of MMAS-4 alone (Fig.
4D and B, respectively). This
result
supports the notion that the G98D mutation directly results
in loss of
the
O-methyltransferase function of MMAS-3.
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FIG. 3.
Structures and biosynthetic relationships of mycolic
acids produced by heterologous expression of MMAS-4 and MMAS-3 in
M. smegmatis. 1, 2, and ' are the normal complement
of mycolic acids. H1 and H2 result from the action of MMAS-4 on
precursors to the normal mycolic acids, and M1 and M2 result from the
action of MMAS-3 upon H1 and H2, respectively.
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FIG. 4.
Two-dimensional TLC of M. smegmatis
expressing various methyltransferases. (A) Wild-type mc2155
containing the empty vector pMV206_Hyg; (B) mc2155
expressing MMAS-4; (C) mc2155 expressing MMAS-4 and
wild-type MMAS-3; (D) mc2155 expressing MMAS-4 and
MMAS-3(G98D). Designations are as explained in the legend to Fig. 3.
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DISCUSSION |
In previous work it has been shown that BCG vaccines vary in terms
of genetic composition due to evolution both at the Pasteur Institute
and in other BCG laboratories. Differences documented to date include
deletions (5), variable restriction fragment length
polymorphism (RFLP) patterns when strains are probed for IS6110 (4) or direct repeats (12), and
variable numbers of intergenic repeats as determined by the length of
PCR amplicons (10, 16). To date, none of these genetic
differences among BCG strains has been specifically linked to a
phenotypic change. The work described in this report documents one more
genetic event that has occurred during the serial propagation of BCG
vaccine. In this case, an important phenotypic alteration has resulted, as BCG vaccines obtained from the Pasteur Institute after 1927 are
unable to synthesize methoxymycolic acids. This indicates that between
1927 and 1931, a mutation in mma3 occurred which permitted
the mutant population to overgrow the parent methoxymycolate-producing population. In previous studies, sequences were obtained from strains
of ill-defined history (27) or previously constructed cosmid
libraries (7). Unlike these investigators, we used a historically defined collection of strains which were minimally passaged after receipt, and we performed direct sequencing to demonstrate that only a single nucleotide polymorphism occurred during
serial propagation of BCG strains.
In light of the biosynthetic relationship between hydroxy- and
methoxymycolates, mma3 mutants might be expected to produce hydroxymycolic acids. However, in M. smegmatis,
overproduction of hydroxymycolates alters colony surface morphology
(26) and severely impairs growth (B. G. Schroeder,
unpublished data). This suggests that cells that acquire a mutation in
mma3 are under selective pressure to reduce levels of
hydroxymycolates. This could be accomplished either by reducing MMAS-4
activity or by increasing the rate of oxidation of hydroxymycolate to
ketomycolate. The fact that we do not observe an increase in
hydroxymycolate levels in mma3 mutants of BCG supports the
notion that one or both of these mechanisms may be in use.
From 1908 to 1921, BCG was grown on glycerinated potato medium with
bile, but from 1921 to 1932, the Pasteur Institute maintained three
lineages of BCG, one on the conventional medium, one on glycerinated
potato without bile, and one on potato Sauton medium (20).
After 1932, the potato Sauton medium became the standard means of
propagating BCG, with 2 to 3 passages per year on potato glycerine.
Thus, somewhere between 1921 and 1932, the principal lineage of BCG was
changed from a bile-containing medium to one without this natural
detergent. Although we have previously shown that uptake of
chenodeoxycholate (a bile component) was not appreciably different
between an organism that produces a wild-type complement of mycolic
acids and one that produces only - and methoxymycolates (27), there is good reason to think that production of only - and ketomycolates does significantly alter the permeability properties. The MIC of rifampin for an organism that produces only -
and ketomycolates is slightly lower than that for an organism that
produces the normal set of three mycolic acids (27).
Permeability to glucose and sensitivity to ampicillin are affected in
apparently opposite ways, with ketomycolate production contributing to
higher rates of glucose uptake but increased resistance to ampicillin (27). These results make it difficult to reconstruct the
original basis for selection of the mutant. Whatever the reason for the selection of the mma3 mutant, it is now clear that all BCG
strains obtained after the era in which it occurred do not produce
methoxymycolic acids.
After vaccination, it is thought that BCG vaccines become incorporated
in macrophages, where they survive and replicate for an unknown
duration. Reports of disseminated BCG-itis in AIDS patients 30 years
after vaccination suggest that, at least for some persons, the vaccine
remains viable in the host long after immunization (1). The
importance of the survival of BCG vaccine in the host was suggested in
the 1950s by Dubos, who observed that animal inoculation with M. tuberculosis H37Ra, which does not replicate in mice, is
associated with limited protection against subsequent challenge with
virulent M. tuberculosis (8). Thus, the survival
and growth of BCG vaccines in macrophages may in part contribute to
their ability to provide protective immunity. In previous work, it was
shown that changes in methoxymycolate production by BCG vaccines result
in impaired survival in macrophage cell culture. Although these
analyses employed overproducers of MMAS-3, with no isogenic
mma3 knockouts studied, the comparison of a strain which
produces only - and ketomycolates (designated "Pasteur" in that
report [27]) to one with all three classes of these
molecules (designated "Connaught") suggests the possibility that
the loss of the ability to produce methoxymycolic acid may be directly
related to the ability of these strains to grow in macrophages.
It is tempting to scrutinize the historical record from 1927 to 1931 in
order to determine whether a change in vaccine properties in vivo
occurred. However, it should be noted that during the era in question,
there was also a loss of a deletion region (RD2) (17), and
expression of secreted proteins MPB70 and MPB83 appears to be greater
in strains obtained prior to that era (19). Before 1931, there was considerable concern that the newly derived vaccine might
revert to virulence, with many investigators alleging that they could
dissociate virulent from avirulent forms of BCG (21). After
1931, these efforts could no longer be replicated, and in fact,
observational studies from that period suggested that BCG had instead
become less virulent (14). An autopsy study performed on
children who died of other causes found that, prior to 1929, live BCG
vaccine could be cultured out of mesenteric nodes up to 6 months after
vaccination, while autopsies from 1930 onwards were no longer able to
detect viable vaccine (28). Thus, the fact that BCG strains
changed between 1927 and 1931 was observed and recorded by
investigators from that era, although the reason for these observed
changes remains to be confirmed in light of the number of changes that
can be dated to this era. Whether these mutations have impacted on
vaccine efficacy and virulence is difficult to infer from the present
data. For instance, in countries which have experienced different
vaccines, BCG-Russia was reported to be more virulent than BCG-Prague
(25) and BCG-Sweden had higher rates of dissemination than
BCG-Glaxo (15). In both examples, the
methoxymycolate-producing strain was the more virulent of the two.
However, BCG-Pasteur and BCG-Glaxo are both mma3 mutants, and in a randomized trial between them, BCG-Pasteur was the more virulent and more protective vaccine (23). Thus, the ability of BCG strains to synthesize methoxymycolic acids is likely but one of
a number of determinants of how BCG vaccines behave in vivo. The
implications of the mma3 mutation for selection of BCG strains used in immunization programs remain to be determined.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: A5-156, Montreal
General Hospital, 1650 Cedar Ave., Montreal, Canada, H3G 1A4. Phone: (514) 937-6011, ext. 2815. Fax: (514) 934-8016. E-mail:
mbgq{at}musica.mcgill.ca.
| |
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Journal of Bacteriology, June 2000, p. 3394-3399, Vol. 182, No. 12
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Abstract
Introduction
