From the Division of Infectious Diseases, Montefiore
Medical Center, Albert Einstein College of Medicine, the
¶ Department of Biochemistry, Structural NMR Resource, Albert
Einstein College of Medicine, and the
Howard Hughes Medical
Institute, Department of Microbiology and Immunology, Albert
Einstein College of Medicine, Bronx, New York 10461
Received for publication, September 18, 2000, and in revised form, November 9, 2000
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ABSTRACT |
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Infection with Mycobacterium
tuberculosis remains a major global health emergency. Although
detailed understanding of the molecular events of M. tuberculosis pathogenesis is still limited, recent genetic
analyses have implicated specific lipids of the cell envelope as
important effectors in M. tuberculosis pathogenesis. We
have shown that pcaA, a novel member of a family of
M. tuberculosis S-adenosyl methionine
(SAM)-dependent methyl transferases, is required for
Mycobacterium tuberculosis infection continues to
overwhelm the populations of the developing world. It has been
estimated that in 1997 there were 8 million new cases of active
tuberculosis that were added to the already existing 16 million cases
(1). In the same year, 2 million people died of tuberculosis as a
result of an astonishing case fatality rate of 23-50% (1). This high death rate for a disease treatable with available antibiotics reflects
the geographic superimposition of
HIV1 and M. tuberculosis infection, and the logistical and economic burden of
at least 6 months of multidrug therapy required to treat the disease.
New drugs to shorten therapy and vaccine candidates to prevent M. tuberculosis infection are badly needed but will only come with a
more thorough understanding of the mechanisms of M. tuberculosis pathogenesis.
The cell envelope of M. tuberculosis is a highly complex
array of distinctive lipids and glycolipids that has been intensely scrutinized as a potential effector in the interaction of M. tuberculosis with the human host (2-4). Investigation into the
role of the cell envelope in virulence has been hampered by a lack of
defined mutants of M. tuberculosis that fail to synthesize
specific components of the cell surface. Recently, advances in the
genetic manipulation of M. tuberculosis have allowed
isolation of several mutants with defined cell envelope deficiencies
and altered virulence (5-7). M. tuberculosis synthesizes
three classes of mycolic acids, very long chain Bacterial Strains and Growth Conditions--
Wild-type M. tuberculosis Erdman is a stock of an animal-passaged strain that
has been passaged once in vitro. M. tuberculosis strain
Erdman was grown at 37 °C in 7H9 (broth) or 7H10 (agar) (Difco)
media with OADC enrichment (Becton Dickinson), 0.5% glycerol, 0.05%
Tween 80 (broth), and where appropriate, hygromycin (Roche Molecular
Biochemicals) at 50 µg/ml or kanamycin (Sigma) 20 µg/ml. The
M. tuberculosis Erdman strain with the
Disruption of cmaA2 and Complementation--
A
For complementation, an M. tuberculosis Erdman cosmid
library was screened for cmaA2-containing clones by PCR.
Cosmid 3E4 was digested with XbaI/NcoI and a
2093-bp fragment containing Rv504c, cmaA2, and
part of Rv502 was cloned into pmv206 hyg to create pMSG129.
To create an inframe deletion of Rv504c, the 1522-bp BstEII/HindIII fragment, the 2279-bp
BstEII/MluI fragment, and the 2203-bp
MluI/HindIII fragment from pMSG129 were isolated
after creating blunt ended BstEII ends with the Klenow
fragment and were ligated in a three piece ligation to create pMSG133.
Using this strategy, Rv504c was reduced to a truncated
fusion protein of 81 amino acids. The reading frame of the fusion joint
was confirmed by DNA sequencing. To create single copy complementation
constructs, the inserts of pMSG129 and pMSG133 were subcloned into
pmv306kan, a site-specific integrating mycobacterial vector (12) to
create pMSG134 and pMSG136, respectively.
Expression of cmaA2 in M. smegmatis--
M. smegmatis
strain mc2 155 was transformed with pMSG129 or vector
control and total mycolic acids that were prepared as described below.
Total mycolic acids were examined by proton NMR for the presence of
cis- or trans-cyclopropane residues. For
coexpression of cmaA2 with mmaA1, the
mmaA1 open-reading frame with its putative promoter (13) was
cloned as a 1056-bp NgoMIV/AvrII fragment into
pMSG137 digested with NgoMIV/NheI to create pMSG148.
Preparation and Analysis of Mycolic Acids--
For radiolabeled
mycolic acids, 50 ml of mid-log phase liquid cultures were incubated
with 50 µCi of [14C]acetate (PerkinElmer Life Sciences)
for 12-18 h. Total mycolic acid methyl esters were prepared as
described previously (6) and precipitated with toluene/acetonitrile.
Analytical and preparative TLC was performed as previously described
(6), and radio TLCs were analyzed on a phosphorimager cassette
(Molecular Dynamics).
NMR Spectroscopy--
One-dimensional 1H NMR spectra
were acquired at 27 °C on either a Bruker DRX300 or DRX600
spectrometer in deuterochloroform (Cambridge Isotope Labs) and were
referenced to the chloroform peak. Two-dimensional DQF-COSY and TOCSY
NMR experiments were performed at 27 °C on a Bruker DRX600
spectrometer equipped with a 5 mm TXI probe. Typically, 256 T1 increments, each with 64 scans and 4000 data
points over a spectral width of 5 kHz, were collected for each
spectrum. The two-dimensional TOCSY experiment employed a 100 ms MLEV17
mixing sequence with a 9kHz spinlock field. Data processing and
analysis was performed using Bruker XWINNMR software.
Sequence Analysis--
Sequence alignment and phylogenetic tree
construction was performed as described (14) on the Multalin server.
Inactivation of cmaA2 in M. tuberculosis by Allelic Exchange and
Complementation with Wild-type cmaA2--
To define the function of
cmaA2 in M. tuberculosis, we sought to delete
cmaA2 from the chromosome of the Erdman strain of M. tuberculosis by allelic exchange. We constructed a substrate for
allelic exchange at cmaA2 by replacing the coding region
with a hygromycin resistance gene as described under "Experimental Procedures." We packaged this knockout construct into a specialized transducing mycobacteriophage and infected wild-type M. tuberculosis as previously described. (6,
16).2 Antibiotic-resistant
M. tuberculosis clones were screened for allelic exchange at
cmaA2 by Southern blotting. Three hygromycin-resistant clones contained the cmaA2 disruption (Fig.
2B), and one was designated mc23120 and used for further studies.
To show that any phenotype observed for the cmaA2 mutant was
attributable to the cmaA2 mutation, we complemented the
cmaA2 mutant with cmaA2 in single copy under its
own promoter. Inspection of the genomic sequence surrounding
cmaA2 suggests that this gene is transcribed as the second
gene in a two gene operon with Rv504c, a gene of unknown
function (see Fig. 2A for diagram). To complement the
cmaA2 mutant with only cmaA2 under its native
promoter, we reconstructed the cmaA2 operon with an inframe
deletion in Rv504c and complemented the cmaA2
mutant in single copy with this inframe deletion construct (pMSG136).
The strains mc23120 and mc23120 (pmsg136) were
analyzed in the subsequent experiments.
Inactivation of cmaA2 Alters the Oxygenated Mycolic Acids of M. tuberculosis--
As shown previously for pcaA (6) in the
absence of a cyclopropane synthetase, the mycolic acids of M. tuberculosis would likely acquire an unsaturation. Therefore, we
examined [14C]acetate-labeled mycolic acids of the
cmaA2 mutant by two-dimensional argentation TLC. This TLC
system has been described previously for the analysis of M. tuberculosis mycolic acids (6, 11). Briefly, the TLC plate is
impregnated with silver nitrate leaving an unimpregnated strip along
the left edge. The sample is developed first along the unimpregnated
strip to separate the mycolates by polarity (Fig.
3A, arrow 1). The plate is
then developed in the silver dimension (Fig. 3A, arrow 2),
separating the mycolates by degree of unsaturation. Silver nitrate
retards the migration of unsaturated lipids relative to saturated or
cyclopropanated lipids. Therefore, in the absence of a cyclopropane
synthetase, an unsaturated mycolic acid retarded in the second
dimension might appear.
The TLC pattern of wild-type M. tuberculosis mycolates is
shown in Fig. 3A. The Inactivation of cmaA2 Abolishes trans-Cyclopropanated
Mycolates--
Because the cmaA2 mutant has defects in a
subpopulation of oxygenated mycolates, we reasoned that
cmaA2 may be involved in either the cis or
trans cyclopropanation of these molecules. To define the
mycolic acid alteration in the cmaA2 mutant, we examined total mycolic acids from wild-type and the cmaA2 mutant by
1H NMR, a technique that can clearly distinguish between
cis- and trans-cyclopropane residues. The
cis- and trans-cyclopropane proton resonances
contributed by the three mycolic acid classes of wild-type M. tuberculosis are visible in the region of the NMR spectrum shown
in Fig. 4A, top
panel (3). In this expansion of the region from
The cmaA2 mutant lacks trans-cyclopropane rings,
as evidenced by the complete absence of the complex mutiplets at 0.15 and 0.45 ppm in the spectrum shown in Fig. 4B. Importantly,
the cis-cyclopropane resonances are unaffected. The TLC data
presented above demonstrates that the oxygenated mycolates in the
cmaA2 mutant contain a subpopulation of unsaturated
mycolates. Accordingly, the NMR spectrum of the total mycolates from
the cmaA2 mutant contains a complex multiplet at 5.33 ppm
that is not present in wild-type mycolates (Fig. 4, A and
B), consistent with the presence of the unsaturated
mycolates in the mutant strain.
To further investigate the structure of the altered oxygenated mycolic
acids in the cmaA2 mutant, we examined the mycolic acids of
wild-type and mutant strains by two-dimensional COSY and TOCSY proton
NMR spectroscopy. We first confirmed the previously reported structure
of the cyclopropyl groups and their surrounding functional groups in
total mycolic acids from wild type (Fig. 4D). According to
the two-dimensional TOCSY spectrum, the cis-cyclopropyl hydrogen resonances at
Two-dimensional TOCSY spectroscopy of purified methoxymycolates from
the cmaA2 mutant confirmed the lack of
trans-cyclopropyl protons demonstrated on the
one-dimensional spectrum (Fig. 4E). In addition, the
unsaturated derivatives of the methoxymycolates seen on TLC contain
predominantly trans double bonds, as evidenced by the TOCSY
cross-peak between the vinyl proton resonance centered at 5.33 ppm and
the methyl branch resonance at 0.95 ppm (Fig. 4F and Ref. 8)
and a COSY cross-peak between the vinyl protons and a methine proton
resonance at 2 ppm (data not shown). cis-Cyclopropanes and
cis double bonds in mycolic acids are not adjacent to methyl branches. Accordingly, a weak resonance at 5.39 ppm does not show a
TOCSY cross-peak with the methyl branch at 0.95 ppm, indicating a
small amount of cis-unsaturated methoxymycolate (Fig.
4F, box at 0.95 ppm) in the cmaA2
mutant methoxymycolates.
The trans cyclopropanation defect in the cmaA2
mutant was somewhat surprising as cmaA2 had previously been
shown to catalyze the formation of cis-cyclopropane rings
when overexpressed in M. smegmatis (11). Therefore, we
considered whether the lack of trans-cyclopropane residues
in the cmaA2 mutant could be an indirect effect on another,
as yet undefined, cyclopropane synthetase. To investigate this
possibility, we purified individual mycolate classes of the
cmaA2 mutant by preparative TLC and examined them by proton
NMR. Individual mycolate classes were examined for the presence of
cyclopropane and methyl branch resonances known or likely to be added
by the SAM-dependent methyl transferases of M. tuberculosis. The Expression of cmaA2 in M. smegmatis--
The data presented above
show that cmaA2 is the trans-cyclopropane
synthetase of M. tuberculosis. To confirm that
cmaA2 produces cis-cyclopropane rings in M. smegmatis as had been previously reported (11), we introduced
cmaA2 into M. smegmatis on a multicopy plasmid
under its own promoter. NMR examination of total mycolic acids from
this strain revealed cis-cyclopropane proton resonances but
not trans-cyclopropane proton resonances (data not shown). Mmas1 appears to catalyze the isomerization of the proximal
cis double bond in oxygenated mycolates with the
introduction of an allylic methyl branch (13). As this isomerization is
necessary for trans-cyclopropane formation, we investigated
whether cmaA2 would produce trans-cyclopropane
rings in M. smegmatis when introduced with mmaA1.
When coexpressed with mmaA1, cmaA2 still
catalyzed only cis-cyclopropane formation (data not shown).
The mycolic acid methyl transferases of M. tuberculosis
are a large family of highly homologous proteins that modify the
mycolic acids of the cell wall with cyclopropane rings and methyl
branches. Although cyclopropanated fatty acids are found in many
bacteria (17), M. tuberculosis has evolved an elaborate
enzymatic system of cyclopropane synthetases not found in any other
bacteria. In this work we have shown that one of these transferases,
cmaA2, is a trans-cyclopropane synthetase for
oxygenated mycolates and that the other members of this gene family
cannot compensate for the loss of cmaA2.
All of the members of this gene family share striking amino acid
sequence similarity. The sequence alignment of these proteins shown in
Fig. 5 demonstrates that the individual
cyclopropane synthetases share substantial amino acid identity over
most of their length and that the sequence divergence between the
members is limited to several distinct regions. Despite this striking sequence conservation, each member of this gene family appears to have
a distinct catalytic function that cannot be compensated by another
member of the family. Specifically, we have shown previously that
inactivation of pcaA abolishes proximal cyclopropanation of
the -mycolic acid cyclopropanation and lethal chronic persistent
M. tuberculosis infection. To examine the apparent redundancy between pcaA and cmaA2, another
cyclopropane synthetase of M. tuberculosis thought to be
involved in
-mycolate synthesis, we have disrupted the
cmaA2 gene in virulent M. tuberculosis by specialized transduction. Inactivation of cmaA2 causes
accumulation of unsaturated derivatives of both the methoxy- and
ketomycolates. Analysis by proton NMR indicates that the mycolic acids
of the cmaA2 mutant lack
trans-cyclopropane rings but are otherwise intact with respect to cyclopropane and methyl branch content. Thus, cmaA2 is required for the synthesis of the
trans cyclopropane rings of both the methoxymycolates and
ketomycolates. These results define cmaA2 as a
trans-cyclopropane synthetase and expand our knowledge of
the substrate specificity of a large family of highly homologous
mycolic acid methyl transferases recently shown to be critical to
M. tuberculosis pathogenesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-alkyl,
-hydroxyl
fatty acids (Fig. 1) in its cell envelope. These three classes of mycolic acids,
-, methoxy-, and
ketomycolates, are modified with cyclopropane rings and methyl branches
through the combined action of a large family of S-adenosyl methionine (SAM)-dependent methyl transferases that modify
double bonds in the meromycolate chain. The oxygenated mycolic acids contain either cis- or trans-cyclopropane rings
at their proximal position. Whereas the putative
cis-cyclopropane synthetase of the methoxymycolates has been
identified (8, 9), the trans-cyclopropane synthetase is
unknown. pcaA, one of the members of this distinctive gene
family, has been established as essential for M. tuberculosis pathogenesis because a mutant of pcaA
cannot establish a chronic persistent M. tuberculosis
infection in mice (6). Biochemically, pcaA is required for
the synthesis of the proximal cyclopropane ring of the
-mycolate
molecule (Fig. 1). The finding that pcaA was required for
proximal cyclopropanation of the
-mycolate molecule was surprising
because this function had been previously attributed to
cmaA2, another cyclopropane synthetase of M. tuberculosis (3, 10, 11). When introduced into
Mycobacterium smegmatis on a multicopy plasmid,
cmaA2 introduces cis-cyclopropane rings at the
proximal position of the
-mycolate and the epoxymycolate, a position
occupied by a double bond in the wild-type mycolates of this strain
(11). Despite this lack of substrate specificity in M. smegmatis, the function of cmaA2 in M. tuberculosis was thought to be proximal cyclopropanation of the
-mycolate molecule. Thus, the functions of pcaA and
cmaA2 appeared to overlap. To define the function of
cmaA2 and to more completely explore the substrate
specificity of the SAM-dependent methyl transferases of
M. tuberculosis, we have inactivated cmaA2 in
M. tuberculosis and shown here that cmaA2 is the
trans-cyclopropane synthetase for both the methoxy- and
ketomycolates.
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Fig. 1.
Structures of the mycolic acids of M. tuberculosis. -Mycolate contains two cyclopropane
residues whereas methoxy- and ketomycolate contain a proximal
cis- or trans-cyclopropane ring and a distal
oxygenated functional group.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cmaA2::hyg allele is designated
mc23120. For mycolic acid analysis, the wild-type strain
was wild-type Erdman transformed with pmv306 hygro, an integrating
vector that supplies a single copy hygromycin resistance gene.
cmaA2::hyg allele was constructed by
amplifying the flanking regions of the cmaA2 gene and
inserting these fragments on either side of the hygromycin resistance
gene. Specifically, a 619-bp flanking region of cmaA2 5' to
the start codon was amplified by PCR using primers omsg33 and omsg34,
which contain XbaI and Asp7181 sites at
their respective 5'-termini. A 646-bp flanking region 3' to the stop
codon was amplified using primers omsg35 and omsg36, which introduce
HindIII and SpeI sites, respectively. The PCR
products were cloned, sequenced, and inserted flanking the hygromycin
cassette in pMSG284, a cloning vector containing a bacteriophage lambda
cos site, a PacI site, and the hygromycin resistance gene flanked by resolvase sites. The final knockout construct (pMSG104) was packaged into phAE87 as previously described (6) to create phMSG104. PhMSG104 was used to transduce wild-type M. tuberculosis to hygromycin resistance as previously
described (6).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
Construction of a deletion mutation of
cmaA2 in M. tuberculosis by
specialized transduction. A, map of the
cmaA2 genomic region in both wild-type and cmaA2
mutant strains. B, Southern blot of SmaI-digested
genomic DNA from the indicated strain probed with the fragment
indicated in A. All three hygromycin-resistant strains
contain the cmaA2 disruption.
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Fig. 3.
Radio two-dimensional TLC analysis of
cmaA2 mutant mycolic acids. A, TLC
system is described in detail in the text. The sample is developed
along the left edge without silver impregnation (arrow 1 in
A) and then in the second dimension with silver impregnation
(arrow 2 in A). 14C-labeled mycolates
from wild-type M. tuberculosis Erdman (A),
M. tuberculosis
cmaA2::hyg (B), and
M. tuberculosis
cmaA2::hyg
attB::pMSG136 (cmaA2) (C) are
shown. The arrows in B point to new mycolic acid
species with the polarity of methoxy- and ketomycolates that are
retarded in the silver dimension
-, methoxy-, and ketomycolates are
labeled and correspond to the structures given in Fig. 1.
cis- and trans-Cyclopropanated oxygenated
mycolates are not distinguished in this TLC system. Inactivation of
cmaA2 alters the oxygenated mycolic acids. Specifically, two
new mycolic acid species are visible in the cmaA2 mutant
with the polarity of methoxy- and ketomycolates but which are retarded by silver impregnation (Fig. 3B). The
-mycolate of the
cmaA2 mutant is identical to that from wild-type in its
mobility. To demonstrate that this phenotype is due specifically to the
loss of cmaA2, we examined the mycolic acids from the
complemented mutant. Wild-type mycolic acid patterns were restored in
the complemented strain, demonstrating that the altered oxygenated
mycolates are secondary to the cmaA2 mutation (Fig.
3C). Thus, inactivation of cmaA2 causes the
accumulation of an unsaturated subpopulation of oxygenated mycolates,
demonstrating that cmaA2 is required for the proper
cyclopropanation of these lipids.
0.4 ppm to 0.8 ppm, the characteristic resonances of cis-cyclopropane hydrogens (
0.33 ppm 2H, 0.56 ppm 1H) and
trans-cyclopropane hydrogens (0.15 ppm 2H, 0.45 ppm 1H) can
be distinguished (Fig. 4A, cis- and
trans-cyclopropane structures label corresponding peaks). The cis-cyclopropane proton peak at 0.67 ppm (1H) and the
trans-cyclopropane proton peak at 0.70 ppm are overlapping.
In the wild-type Erdman strain used in this study, the ratio of
cis/trans cyclopropane hydrogens is 8:1, lower than in
previously examined laboratory strains (13).
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Fig. 4.
1H NMR analysis of wild-type and
cmaA2 mutant mycolic acids. One-dimensional
1H NMR from wild type (A), cmaA2
mutant (B), and complemented mutant (C). The
lower panels show the entire spectrum whereas the
upper panels are expansions of the region from 0.4 to 1 ppm, demonstrating the cis- and trans-cyclopropyl
protons that are labeled with structures in A. D,
two-dimensional TOCSY spectrum showing the region from
0.4 to 1 ppm
demonstrating the cis- and trans-cyclopropyl
structures from wild-type total mycolic acids. E and
F, two-dimensional TOCSY spectra on purified
methoxymycolates from the cmaA2 mutant. E shows
the same region as D and demonstrates the lack of
trans-cyclopropyl protons in the mutant. F shows
the TOCSY connections between the trans-vinyl protons
centered at 5.33 ppm and the allylic methyl branch at 0.95 ppm, the
neighboring methine proton (2.0 ppm), and the adjacent CH2
groups (1.3 ppm). The vinyl proton resonance at 5.39 ppm does not show
a connection with the methyl branch (boxed area in
F), demonstrating that the unsaturated methoxymycolates in
the cmaA2 mutant contain a subpopulation with a
cis double bond.
0.33, 0.56, and 0.67 ppm all belong to the
coupled spin network, as do the trans-cyclopropyl hydrogen resonances at 0.15, 0.45, and 0.7 (Fig. 4D). In addition,
the trans-cyclopropyl group protons are adjacent to a methyl
branch, as evidenced by a TOCSY cross-peak between the
trans-cyclopropane proton resonances and a doublet at 0.95 ppm (Fig. 4D).
-mycolate of the cmaA2 mutant was
identical to wild-type
-mycolate (data not shown). As detailed
above, the methoxymycolate of the cmaA2 mutant exhibited
characteristic resonances of cis-cyclopropane protons,
methyl branch protons adjacent to a methoxyl group (0.85 ppm, doublet),
and the allylic methyl branch of the proximal trans double
bond (0.95 ppm, doublet, Ref. 8). The ketomycolate also contained all
predicted resonances except for the trans-cyclopropane
residues. Therefore, as assessed by proton NMR of individual mycolate
classes from the cmaA2 mutant, the only cyclopropane or
methyl branch missing from the mycolic acids of the mutant is the
trans-cyclopropane ring.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mycolate molecule despite intact cmaA2,
mmaA2, and cmaA1 genes. It is interesting to note
in the sequence alignment that CmaA2 contains an 8-amino acid
segment at amino acids 152-160 that is not present in any of the other
methyl transferases. As CmaA2 is the only trans-cyclopropane
synthetase of the group, this eight amino acid segment may be important
for catalysis or substrate binding. In addition, a phylogenetic tree
derived from these sequences demonstrates that there are three distinct
groups within this gene family that are consistent with the known or suspected functions of these proteins (Fig. 5). The first group contains MmaA3 and MmaA4, two proteins that introduce the methoxy group
in the distal position of the methoxymycolates (7-9, 18, 19). The
second group contains CmaA2, MmaA1, and UmaA1. MmaA1 is likely
responsible for the isomerization of the proximal cis double
bond to a trans double bond in the meromycolate chain with simultaneous introduction of an allylic methyl branch (13). Because
overexpression of MmaA1 in M. tuberculosis produced an excess of both trans unsaturated and
trans-cyclopropanated mycolic acids, MmaA1 action is
presumably an early step in trans-cyclopropane synthesis. It
is therefore logical that CmaA2 is within the same phylogenetic
subfamily. UmaA1 has no known function at present. The last group
contains PcaA, CmaA1, and MmaA2. All of these enzymes are known or
putative cis-cyclopropane synthetases. PcaA synthesizes the
proximal cis-cyclopropane ring of the
-mycolates (6), CmaA1 produces a distal cis-cyclopropane ring in the
-mycolate of M. smegmatis (15), and MmaA2 likely
synthesizes the proximal cis-cyclopropane ring of the
methoxymycolates (8, 9). Three-dimensional structural studies of these
proteins may help elucidate the basis for their substrate
specificity.
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Fig. 5.
Amino acid sequence alignment and
phylogenetic tree of the putative mycolic acid methyl transferases of
M. tuberculosis. Black or shaded
residues, identical amino acids.
Several explanations are possible for the ability of cmaA2 to produce cis-cyclopropanes in M. smegmatis. Given the high sequence identity within this gene family, it is possible that cmaA2 can inefficiently catalyze cis-cyclopropane synthesis when highly overexpressed. Alternatively, the substrate specificity of these enzymes may be determined in part by physical association in multienzyme complexes. Although this possibility has not been examined experimentally, these enzymes catalyze the sequential modification of the meromycolate chain of mycolic acids and therefore could associate in multienzyme complexes to achieve efficient modification of a mycolic acid subclass. Therefore, it is possible that in M. smegmatis, CmaA2 cannot associate with other methyl transferases and the correct CmaA2 substrate is not available.
The significance of trans-cyclopropanated oxygenated mycolic
acids for M. tuberculosis pathogenesis is unknown. However,
previous work has shown that clinical strains of M. tuberculosis have higher trans-cyclopropane content
than extensively passaged laboratory strains, suggesting that in
vivo growth either dynamically enhances trans-cyclopropane formation or favors subpopulations of
M. tuberculosis with higher trans-cyclopropane
content (13). These results are consistent with the high proportion of
trans-cyclopropane rings in the wild-type M. tuberculosis strain used in this study as this strain was recently
passaged through animals and has not been passaged significantly
in vitro. The results presented here define cmaA2
as the trans-cyclopropane synthetase of M. tuberculosis. Further examination of the cmaA2 mutant
in animal models of infection will broaden our understanding of the
role of individual cyclopropane residues in general, and of
trans-cyclopropane residues in particular, in M. tuberculosis pathogenesis.
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ACKNOWLEDGEMENT |
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We thank Jeff Cox for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants AI01534 (to M. S. G.) and AI27160 (to W. R. J.). The NMR facility was supported in part by grants from the National Science Foundation and Howard Hughes Medical Institute-Biomedical Research Support Program for Medical Schools.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Div. of Infectious Diseases, 610 Belfer Bldg., Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-2886; Fax 718-518-0366; E-mail: glickman@aecom.yu.edu.
Published, JBC Papers in Press, November 22, 2000, DOI 10.1074/jbc.C000652200
2 S. Bardarov, M. Larsen, M. Pavelka, S. S. Bardarov, Jr., and W. R. Jacobs, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: HIV, human immunodeficiency virus; SAM, S-adenosyl methionine; bp, base pair(s); PCR, polymerase chain reaction.
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1. |
Dye, C.,
Scheele, S.,
Dolin, P.,
Pathania, V.,
and Raviglione, R. C.
(1999)
J. Am. Med. Assoc.
282,
677-686 |
2. | Brennan, P. J., and Nikaido, H. (1995) Annu. Rev. Biochem. 64, 29-63[CrossRef][Medline] [Order article via Infotrieve] |
3. | Barry, C. E., III, Lee, R. E., Mdluli, K., Sampson, A. E., Schroeder, B. G., Slayden, R. A., and Yuan, Y. (1998) Prog. Lipid Res. 37, 143-149[CrossRef][Medline] [Order article via Infotrieve] |
4. | Daffe, M., and Draper, P. (1998) Adv. Microb. Physiol. 39, 131-203[Medline] [Order article via Infotrieve] |
5. | Cox, J. S., McNeil, M., and Jacobs, W. R., Jr. (1999) Nature 402, 79-83[CrossRef][Medline] [Order article via Infotrieve] |
6. | Glickman, M. S., and Jacobs, W. R. (2000) Mol. Cell 5, 717-727[Medline] [Order article via Infotrieve] |
7. | Dubnau, E., Raynaud, C., Mohan, V. P., Laneelle, M. A., Yu, K., Quemard, A., Smith, I., and Daffe, M. (2000) Mol. Microbiol. 36, 630-637[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Yuan, Y.,
and Barry, C. E., III
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
12828-12833 |
9. | Dubnau, E., Laneelle, M. A., Soares, S., Benichou, A., Vaz, T., Prome, D., Prome, J. C., Daffe, M., and Quemard, A. (1997) Mol. Microbiol. 23, 313-322[Medline] [Order article via Infotrieve] |
10. | Cole, S. T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S. V., Eiglmeier, K., Gas, S., Barry, C. E., III, Tekaia, F., Badcock, K., Basham, D., Brown, D., Chillingworth, T., Connor, R., Davies, R., Devlin, K., Feltwell, T., Gentles, S., Hamlin, N., Holroyd, S., Hornsby, T., Jagels, K., Barrell, B. G., et al.. (1998) Nature 393, 537-544[CrossRef][Medline] [Order article via Infotrieve] |
11. |
George, K. M.,
Yuan, Y.,
Sherman, D. R.,
and Barry, C. E., III
(1995)
J. Biol. Chem.
270,
27292-27298 |
12. | Lee, M. H., Pascopella, L., Jacobs, W. R., Jr., and Hatfull, G. F. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3111-3115[Abstract] |
13. |
Yuan, Y.,
Crane, D. C.,
Musser, J. M.,
Sreevatsan, S.,
and Barry, C. E., III
(1997)
J. Biol. Chem.
272,
10041-10049 |
14. | Corpet, F. (1988) Nucleic Acids Res. 16, 10881-10890[Abstract] |
15. | Yuan, Y., Lee, R. E., Besra, G. S., Belisle, J. T., and Barry, C. E., III (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6630-6634[Abstract] |
16. | Carriere, C., Riska, P. F., Zimhony, O., Kriakov, J., Bardarov, S., Burns, J., Chan, J., and Jacobs, W. R., Jr. (1997) J. Clin. Microbiol. 35, 3232-3239[Abstract] |
17. | Grogan, D. W., and Cronan, J. E., Jr. (1997) Microbiol. Mol. Biol. Rev. (Washington, D. C.) 61, 429-441[Abstract] |
18. | Dubnau, E., Marrakchi, H., Smith, I., Daffe, M., and Quemard, A. (1998) Mol. Microbiol. 29, 1526-1528[Medline] [Order article via Infotrieve] |
19. | Yuan, Y., Zhu, Y., Crane, D. D., and Barry, C. E., III (1998) Mol. Microbiol. 29, 1449-1458[CrossRef][Medline] [Order article via Infotrieve] |