 |
INTRODUCTION |
Infection with Mycobacterium tuberculosis
(Mtb)1 remains one of the
world's great public health problems (1). New drugs active against Mtb
or vaccines that prevent Mtb disease are urgently needed but will only
come from broader and more detailed understanding of the pathogenic
strategies of this persistent microbe. Recently, dramatic advances in
the understanding of Mtb pathogenesis have come from the creation of
defined Mtb mutants that display altered pathogenesis in experimental
animals (2). A distinct subset of these mutants have established the
pathogenic importance of specific chemical structures in the Mtb cell
envelope (3-6). The cell envelope of Mtb is a complex structure that
contains many unique lipids and glycolipids including mycolic acids,
lipoarabinomannan, trehalose dimycolate, and phthiocerol dimycocerosate
(7, 8). Many of these compounds are suspected virulence effectors based on their in vitro activities, but the genetics of their
biosynthesis and exact role in pathogenesis has been unclear due to the
lack of defined Mtb mutants lacking specific cell envelope structures.
Mycolic acids are very long chain
-alkyl,
-hydroxy fatty acids
that are unique to mycobacteria and are greater than 80 carbons in Mtb
(9). These lipids form a thick hydrophobic layer in the cell envelope
in their covalently linked form and form the lipid groups in Trehalose
Dimycolate, an immunomodulatory glycolipid that is noncovalently
associated with the Mtb cell envelope (10-12). In pathogenic
mycobacteria, but not in non-pathogenic species, mycolic acids are
modified with cyclopropyl groups at relatively conserved positions in
the meromycolate chain (see Fig. 1 for structures). These cyclopropane
residues are added to Mtb mycolic acids by a family of
S-adenosylmethioninedependent
methyltransferases that exhibit exquisite substrate specificity for
their lipid substrates (9, 13). One such cyclopropane synthase,
pcaA, was recently shown to be crucial to Mtb persistence
and virulence in vivo (5).
Because of its pathogenic importance, we are systematically studying
the cyclopropane modification of mycolic acids in Mtb by deleting each
putative cyclopropane synthase from M. tuberculosis and
studying these mutant strains for alterations in cyclopropane content
of mycolic acids and alterations in pathogenesis. This approach has
been highly informative in elucidating the biosynthetic role of two
members of this gene family. PcaA was shown to synthesize the proximal
cyclopropyl group of the
-mycolate molecule (see Fig. 1) (5),
whereas cmaA2 was shown to be the
trans-cyclopropane synthase of the oxygenated mycolates
(14). Based on these insights, the structural basis for the substrate
specificity of these enzymes is being elucidated (15). In this study we
report the role of mmaA2 in mycolic acid modification
through the characterization of an Mtb mmaA2 null mutant. In
contrast to its previously assigned function in methoxymycolate
modification (16-18), mmaA2 has an unexpected and
non-redundant role in
-mycolate modification and a partially
redundant role in methoxymycolate modification.
 |
EXPERIMENTAL PROCEDURES |
Bacterial Strains and Media--
Mtb Erdman EF2 is an
animal-passaged strain previously described (5). Mycobacterium
smegmatis mc2155 has been previously described (19).
Mtb strains were grown in 7H9 broth or 7H10 agar with 10% oleic
acid-albumin-dextrose-catalase supplement (OADC), 0.5% glycerol,
0.05% Tween 80, and where required, hygromycin B (Roche Molecular
Biochemicals) (50 µg/ml) and kanamycin (20 µg/ml).
Construction of an mmaA2 Null Mutant by Allelic Exchange and
Complementation--
The mmaA2 and cmaA1 null
mutants were constructed by allelic exchange using hygromycin-marked
null alleles designed to delete the entire open reading frame. An
mmaA2 null allele was constructed by PCR amplifying the
5'-flanking region of mmaA2 from Mtb genomic DNA using omsg
143/144 as primers. This 634-bp PCR product includes the first 15 nucleotides of the mmaA2 open reading frame and includes SpeI and HindIII sites at the 5' and 3' ends of
the PCR product. The 3'-flanking region of mmaA2 was
amplified using omsg 145/146 as a 522-bp PCR product that includes the
last 14 nucleotides of the mmaA2 open reading frame and
Asp718I/XbaI sites at the ends of the PCR
product. Both of these PCR products were cloned, sequenced, and
inserted into pmsg 284 flanking the hygromycin resistance gene
(14). This final plasmid, pmsg 243, was used to construct a specialized
transducing phage as previously described (14, 20).
Hygromycin-resistant transductants were screened for allelic
exchange by southern blotting. The mmaA2 null mutant (Mtb Erdman mmaA2::Hyg) is mgm 104.
An identical strategy was employed to construct the cmaA1
null mutant. The PCR primers employed were omsg 37/38 and omsg 39/40, and the final cmaA1 targeting phage is phmsg 105.
For complementation of the mmaA2 mutant, a wild type copy of
mmaA2 under its putative promoter was cloned from the Mtb
cosmid MTY20H10. The mma locus was first isolated as a 6864 HindIII fragment. The mmaA2 gene with its
putative promoter (16) was subcloned as a 1618-bp
StuI/PshAI fragment into pmv306kan, a
site-specific integrating vector to create pmsg256. This single copy
complementation plasmid was used to transform mgm 104, and
kanamycin-resistant transformants were evaluated for restoration of
wild type mycolic acid patterns by radio thin layer chromatography
(TLC) of mycolic acids as described below.
Preparation and Analysis of Mycolic Acids--
Mycolic acids
were labeled in logarithmic phase cultures of Mtb with 50 µCi of
[14C]acetic acid (PerkinElmer Life sciences) for
18 h. Mycolic acids were prepared as described previously from
whole bacilli (14). For preparation of mycolic acid classes,
total mycolic acids prepared from 1 liter of Mtb strains were applied
to a 1 mm preparative silica gel TLC plate and developed 7 times with
hexanes/ethyl acetate 95:5. Lipids were visualized by rhodamine 6G
staining, scraped from the plate, and the silica extracted three times
with ethyl ether. Mycolates were then reprecipitated with
toluene/acetonitrile before analysis. The purity of each isolated
mycolic acid class was evaluated by TLC before structural characterization.
Structural Characterization of Mycolic Acids--
For NMR
analysis, mycolates were dissolved in Deuterochloroform (Cambridge
Isotope Laboratories) and analyzed on a Bruker 500 MHz
spectrophotometer. For mass spectroscopy, lipids were dissolved in
dichloromethane/methanol (8:2) and analyzed by electrospray ionization
mass spectroscopy on a PE SCIEX API100 mass spectrometer in positive
and negative ion mode. Mass spectrometry was performed after
regeneration of the free acid from the mycolic acid methyl ester by
saponification. Permanganate cleavage of mmaA2 mutant
-mycolate was performed as previously described (5) on the mycolic
acid methyl ester.
 |
RESULTS |
Construction of an Mtb mmaA2 Deletion Mutant--
We used a
genetic approach to understand the biosynthetic role of each member of
the mycolic acid methyltransferase gene family. Specifically, we
constructed M. tuberculosis null mutants in each methyltransferase and deduced the mycolic acid modification function of
each gene through examination of the mutant strains. This approach has
been highly informative, defining the biosynthetic role of pcaA as the proximal cyclopropane synthase of the
-mycolate molecule (5) and cmaA2 as the
trans-cyclopropane synthase of the oxygenated mycolates
(14). Given this prior data, the biosynthetic origin of the proximal
cis-cyclopropyl group of the oxygenated mycolates and the
distal cyclopropyl group of the
-mycolates remained unclear (see
Fig. 1). To investigate these
modifications, we attempted to disrupt mmaA2 by specialized
transduction.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 1.
Chemical structures of the major mycolic
acids of M. tuberculosis. Pictured are the
-mycolate, methoxymycolate, and ketomycolate. By convention, the
cyclopropyl nearest the -hydroxyl group is proximal. -Mycolates
contain two cis-cyclopropyl groups. Oxygenated mycolates
contain a proximal cis- or trans-cyclopropyl
group and a distal oxygen function. When known, individual cyclopropyl
or methyl groups are labeled with the genes involved in their
synthesis. Also pictured is the structure of the mmaA2
mutant -mycolate ( mmaA2 ) as determined in
this study (see "Results" for details).
|
|
A targeting construct for mmA2 was designed to replace the
mmaA2 open reading frame with a hygromycin resistance
cassette. Although a polar effect on the downstream genes
(mmaA3 and mmaA4) is possible, previous
experimentation (16) has shown that mmaA3 can be expressed
in M. smegmatis from a promoter 3' of mmaA2. In
addition, a polar effect on mmaA3 would be easily detectable by the loss of both methoxymycolates and ketomycolates that would result (6). To disrupt mmaA2, wild type M. tuberculosis Erdman was infected with a specialized transducing
phage carrying an mmaA2 knockout construct designed to
delete the entire mmaA2 open reading frame.
Hygromycin-resistant transductants were screened for allelic exchange
at mmaA2 by Southern blotting. All of the hygromycin-resistant transductants contained the mmaA2
disruption (Fig. 2). This Mtb
[mmaA2::Hyg] strain was designated
mgm 104 and characterized further.

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 2.
Disruption of mmaA2 in
M. tuberculosis. Southern blot of genomic DNA
digested with SmaI and probed with the indicated fragment.
Probe locations and restriction sites are indicated. All six
hygromycin-resistant transductants contain the mmaA2
disruption.
|
|
MmaA2 Is Required for
-Mycolate Cyclopropanation--
To
determine the function of mmaA2 in mycolic acid
modification, we prepared total mycolic acids from the mmaA2
null mutant and wild type cells and examined these lipids by
two-dimensional argentation TLC. We have used this system previously to
assign functions to pcaA and cmaA2 (5, 14). In
this system, lipids are separated by polarity in the first dimension
and then separated by degree of unsaturation in the second dimension
through the use of silver impregnation of the TLC plate. In the absence
of a mycolic acid cyclopropane modification, the affected mycolic acids
acquire a double bond at the site of the missing cyclopropane ring.
These unsaturated mycolic acid derivatives are retarded in their
migration in the silver dimension of the TLC plate but maintain the
polarity of the parent mycolate.
Wild type Mtb contains three major classes of mycolic acids,
-mycolates, methoxymycolates, and ketomycolates (see Fig. 1 for
structures and Fig. 3A for TLC
pattern). In the mmaA2 null mutant, the
-mycolate was
replaced by a new mycolic acid species that was retarded by silver ions
(Fig. 3B, arrow). A small amount of intact
-mycolate is synthesized in the absence of mmaA2. In addition, an unsaturated derivative of the methoxymycolates accumulates in the mmaA2 null mutant (Fig. 3B,
arrowhead). To confirm that these mycolic acid derivatives
are secondary to the mmaA2 mutation, we transformed the
mmaA2 mutant with a complementing single copy plasmid
expressing mmaA2 under its putative promoter. Both the
-
and methoxymycolic acid defects are attributable to the
mmaA2 mutation as the complemented strain displays wild type
mycolic acid patterns (Fig. 3C). These results establish a
role for mmaA2 in both
- and methoxymycolate
modification.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 3.
MmaA2, but not
cmaA1, is required for and
methoxymycolate modification. Each panel is a
two-dimensional radio 2D TLC analysis of purified mycolic acids from
the indicated strain. The mycolic acids are spotted in the
lower left corner of the TLC plate and developed in the
first dimension, which lacks silver impregnation. This dimension
separates the lipids on the basis of polarity. The plate is then
developed in the second dimension, which is impregnated with silver
nitrate. This dimension retards lipids with double bonds in relation to
saturated or fully cyclopropanated lipids. A, wild type
mycolates; B, mmaA2 mutant mycolates. The
arrow and arrowhead highlight new mycolic acid
species with the polarity of and methoxymycolates but retarded in
the silver dimension. C, mmaA2 mutant
complemented with wild type mmaA2. D, cmaA1
deletion mutant.
|
|
CmaA1 Is Not Required for
-Mycolate Modification--
Previous
examination of the other members of the mma gene cluster by
overexpression in M. smegmatis or M. tuberculosis
had suggested that the mma gene cluster was involved in methoxymycolate synthesis (16, 18, 21). Thus, the defective
-mycolate
cyclopropanation in the mmaA2 mutant was surprising. The
mycolic acid cyclopropane synthetase pcaA cyclopropanates
the proximal position of the Mtb
-mycolate (5), and cmaA1
was suspected to cyclopropanate the distal position of the
-mycolate
based on its activity when overexpressed in M. smegmatis
(22). To clarify the role of cmaA1 in
-mycolate modification, we constructed a cmaA1 null mutant and
examined the mycolic acid phenotype of this strain. The
-mycolate of
the cmaA1 null mutant is unaffected by silver impregnation
TLC, demonstrating that cmaA1 is not required for
-mycolate modification under the conditions tested (Fig.
3D). These results suggest that cmaA1 may modify
lipids other than mycolic acids or that the mycolic acid modification
function of cmaA1 is not evident in vitro.
MmA2 Mutant
-Mycolate Contains a Double Bond and a Cyclopropyl
Group--
To specifically define the function of mmaA2 in
mycolic acid modification, we purified each mycolic acid class by
preparative thin layer chromatography from mmaA2 mutant and
wild type cells. These mycolic acid classes were examined first by 500 MHz 1H NMR. The
-mycolate from the mmaA2
mutant contained both cis-cyclopropyl protons and a
resonance at 5.33 ppm, consistent with vinyl protons (Fig.
4). The coupling constant of these vinyl
protons is 5 Hz, consistent with cis geometry. Vinyl
resonances were absent from wild type
-mycolate (Fig. 4).
Integration of cyclopropyl proton resonances at
0.33 ppm 1H,
and the terminal methyl ester protons at 3.7 ppm 3H revealed a ratio of
1.58:1 (predicted 3:2 or 1.5:1 for dicyclopropanated
-mycolate) in
wild type and 2.46:1 (predicted 3:1 in monocyclopropanated mutant
-mycolate). The slight apparent excess of cyclopropyl protons in the
mutant
-mycolate likely represents the small amount of intact
-mycolate synthesized in the mutant strain (see Fig. 3). These
findings confirm that the mmaA2 mutant
-mycolate is
lacking a single cyclopropyl group and contains a double bond. To
confirm this structure in more detail, we performed two-dimensional
1H COSY NMR of the mmaA2 mutant
-mycolate.
This study revealed that the vinyl protons are adjacent to protons at
2.02 ppm, consistent with previously reported resonances of vinyl
protons in meromycolate chains (data not shown) (23). These findings
are similar to our previous NMR findings when examining the
-mycolate of the pcaA mutant and establish that the
mmaA2 mutant
-mycolate lacks either a proximal or distal
cyclopropyl group. However these findings do not establish which
cyclopropane ring is absent.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 4.
NMR analysis of mmaA2
mutant -mycolate. One-dimensional
500 MHz 1H NMR analysis of purified -mycolate from wild
type Mtb (A) and the mmaA2 null mutant
(B). In the absence of mmaA2, the -mycolate
has acquired a double bond visible at 5.3 ppm (panel B,
arrow and inset).
|
|
To further define the structural variation in the mmaA2
mutant mycolic acids, we examined each mycolic acid class from wild type and mutant cells by electrospray ionization mass spectroscopy. Wild type
-mycolate contained a series of compounds differing by 28 atomic mass units reflecting chain length variation of two methylene units, as is expected for Mtb mycolic acids (9). The
predominant
-mycolate of wild type Mtb Erdman appeared as a sodium
adduct in positive ion mode corresponding to a molecular mass of 1151 atomic mass units (Fig. 5). This mass
corresponds to a dicyclopropanated
-mycolate of 79 total carbons
with a 24-carbon
branch as pictured in Fig. 1 and as reported
previously for other strains of Mtb (24, 25). In addition, peaks at
1123 and 1179 reflect smaller and longer chain length variants by two methylene units. Spectra of purified methoxy-and ketomycolates from
wild type strains were as reported previously for M. tuberculosis (21).

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 5.
Mass spectroscopy of wild type and
mmaA2 mutant purified
-mycolate. Peaks represent sodium adducts
(+23) of free acid mycolic acids. The top panel is wild type
-mycolate, and the lower panel is mmaA2 mutant
-mycolate. The peaks differing by 28 atomic mass units represent
lipid chain length variation of 2 methylene units characteristic of all
mycolic acids.
|
|
mmaA2 mutant
-mycolate contained a similar series of
peaks differing by 28 atomic mass units, but were smaller by 14 atomic mass units compared with wild type lipids (Fig. 5). Consequently, the
two most abundant
-mycolates in the mmaA2 mutant have
molecular masses of 1137 and 1165 atomic mass units with lower
abundance isomers at 1132 atomic mass units. These compounds are
consistent with a monocyclopropanated, monounsaturated
-mycolate of
the same chain lengths as the parent wild type
-mycolate. In
addition, mmaA2 mutant
-mycolate contains a variant with
a molecular mass of 1179 atomic mass units, consistent either with some
intact
-mycolate of the longest chain length seen in wild type
lipids, or a monounsaturated
-mycolate with a methyl branch. The
latter is unlikely because NMR of mmaA2 mutant
-mycolate
did not reveal a doublet at 0.95-1.05 characteristic of methyl
branches in meromycolate chains (16). In addition, the mmaA2
mutant
-mycolate contains a series of peaks of higher molecular
weight at 1208, 1236, and 1264 of unclear identity (Fig. 5).
MmaA2 Is a Distal Cyclopropane Synthase of the
-Mycolate--
Based on the assigned function of pcaA in
proximal cyclopropane modification of the
-mycolate, the normal
-mycolate in the cmaA1 mutant, and the data presented
above, we hypothesized that mmaA2 synthesizes the distal
cyclopropane ring of the
-mycolate molecule. To examine this
hypothesis, we oxidized the purified
-mycolate from the
mmaA2 mutant with potassium permanganate and examined the
cleavage products of this reaction by mass spectroscopy. Potassium
permanganate oxidizes lipids at the site of double bonds and therefore
can be used to determine the position of unsaturations in lipid chains.
This strategy is similar to that used previously to determine the
position of the unsaturation in the M. tuberculosis pcaA
mutant
-mycolate (5). Cleavage of the mmaA2 mutant
-mycolate was performed alongside cleavage of the wild type
-mycolate, and both reactions were analyzed by electrospray
ionization mass spectroscopy. Cleavage of the mmaA2 mutant
-mycolate produced a base peak of 349 atomic mass units, consistent
with the sodium adduct of oxidative cleavage at a distal double bond
situated 21 carbons from the end of the meromycolate chain (data not
shown). This spacing of the cyclopropyl group/double bond is consistent with the known structure of Mtb
-mycolate as pictured in Fig. 1
(25). This fragment was not detected in wild type
-mycolate cleaved
with potassium permanganate. The proximal fragment of cleavage at a
distal double bond was detected but of lower abundance than the distal
fragment. Neither of the possible fragments from proximal cleavage was
detected. This experiment was repeated with similar results. Thus, the
mutant
-mycolate in the mmaA2 mutant lacks a distal
cyclopropane group, and therefore mmaA2 is necessary for the
distal cyclopropane modification of the
-mycolate, an enzymatic
function previously attributed to cmaA1 (22).
MmaA2 May Be the Preferred cis-Cyclopropane Synthetase of the
Methoxymycolates--
The thin layer chromatography analysis of the
mmaA2 mutant mycolic acids presented in Fig. 3 shows
accumulation of an unsaturated derivative of the methoxymycolates in
addition to apparently mature methoxymycolates. These intact
methoxymycolates could either represent an intact mixture of
cis- and trans-cyclopropanated methoxymycolate, or only trans-cyclopropanated methoxymycolates. To determine
the function of mmaA2 in methoxymycolate modification we
examined purified methoxymycolates of mutant and wild type strains by
NMR. In the absence of mmaA2, intact methoxymycolates with
either cis- or trans-cyclopropyl groups are
present, showing that mmaA2 is not absolutely required for
proper methoxymycolate modification (data not shown). In addition, a
resonance at 5.33 in purified mmaA2 mutant methoxymycolate
is consistent with a double-bonded methoxymycolate. Thus,
methoxymycolates from the mmaA2 mutant contain both intact
and unsaturated methoxymycolates. By comparing the cis- and
trans-cyclopropyl resonances from purified methoxymycolates we estimated that wild type methoxymycolates contain a
cis/trans ratio of 10:1, whereas mmaA2
mutant methoxymycolates contain a 5:1 ratio. As a control, we performed
the same examination of cis/trans ratios in
purified ketomycolates from wild type and mutant strains and found
these ratios unchanged by the mmaA2 mutation. Thus, in the
absence of mmaA2, the relative cis-cyclopropane
content of methoxymycolates is reduced by half. These results suggest that in the absence of mmaA2, intact methoxymycolates are
synthesized, but the efficiency of cis-methoxymycolate
cyclopropanation is reduced, leading to accumulation of a
cis double-bonded methoxymycolate derivative and a relative
overabundance of trans-cyclopropanated methoxymycolate.
 |
DISCUSSION |
The cyclopropane modification of mycolic acids in M. tuberculosis is a unique lipid structure that has been linked
to the pathogenesis of this infection. Mtb uses a family of
S-adenosylmethionine-dependent methyltransferases to modify the mycolic acids of its cell envelope with a variety of stereochemistries and positions of cyclopropyl groups. Given the pathogenic importance of this lipid modification, we
are systematically studying the biochemical and pathogenetic function
of this gene family through the creation of Mtb null mutants in each
synthase. Through this approach, we have defined the pathogenetic role
and biochemical function of pcaA as a proximal cis-cyclopropane synthase
-mycolate molecule essential
for Mtb virulence in mice (5). In addition, we have defined
cmaA2 as the trans-cyclopropane synthase of both
the methoxy- and ketomycolates (14). This study establishes the
biosynthetic specificity of a third mycolic acid cyclopropane synthase,
mmaA2.
Role of mmaA2 in
-Mycolate Modification--
Previous
examinations of mmaA2 from M. tuberculosis and
its homologue from Mycobacterium bovis BCG had been
by overexpression of the gene in M. smegmatis, a
nonpathogenic mycobacteria that does not produce cyclopropanated
mycolic acids (16, 18). These studies revealed that mmaA2
cyclopropanated both the epoxymycolate and
-mycolate of M. smegmatis at the proximal position. When mmaA2 was
expressed along with the other genes in the mma gene cluster, an intact
methoxymycolate was produced. Thus, in M. smegmatis, the
activity of mmaA2 was nonspecific, similar to the activity of other cyclopropane synthases in this host (22, 26). As has been
shown previously for pcaA and cmaA2, the
construction of null mutants in each cyclopropane synthase is a
powerful method to deduce the specific biosynthetic function of this
gene family. For mmaA2, deletion of the gene from M. tuberculosis revealed an unexpected non-redundant role in the
distal cyclopropane modification of the
-mycolate. This function was
previously ascribed to cmaA1 (22), a cyclopropane synthase
that has no discernible role in mycolic acid modification as determined
in this study by construction of a cmaA1 null mutant. This
surprising role for mmaA2 in distal
-mycolate
modification means that mmaA2 is biosynthetically closely linked to pcaA, the proximal cyclopropane synthase of the
-mycolate. Interestingly, the mycolic acid phenotypes of the
mmaA2 and pcaA mutant differ in ways other than
distal versus proximal cyclopropanation. For reasons that
are unknown, the pcaA mutant accumulates large amounts of
ketomycolates (5), a phenotype that is absent from the mmaA2
mutant (Fig. 3). Although the protein structure of MmaA2 has not yet
been solved, comparison of this structure to PcaA may provide further
insight into the exquisite catalytic specificity of this gene family
(15). Careful examination of the mass spectrum of the mmaA2
mutant
-mycolate revealed that the small amount of intact
-mycolate synthesized in the mutant was only one chain length. This
81-carbon intact
-mycolate is the longest major
-mycolate
synthesized in wild type cells, and this result may indicate that some
unidentified cyclopropane synthase can modify the distal position in
the absence of mmaA2, but that this redundancy is limited to
longer chain length lipids.
Role of mmaA2 in Methoxymycolate Modification--
The
role of mmaA2 in methoxymycolate modification is less clear
due to probable redundancy with other cyclopropane synthases. The
mmaA2 null mutant accumulates unsaturated derivatives of
methoxymycolates but still synthesizes intact methoxymycolates.
Characterization of the mmaA2 mutant methoxymycolate by
NMR revealed a 2-fold reduction in the relative abundance of
cis-cyclopropanated methoxymycolate. This data is most
consistent with a role for mmaA2 as the preferred cis-cyclopropane synthase of the methoxymycolates, as had
been suggested by its function in M. smegmatis and its
genomic organization alongside other putative methoxymycolate
biosynthetic genes. However, the production of
cis-cyclopropanated methoxymycolate in the mmaA2 mutant demonstrates that another enzyme can perform this function. The
most likely candidate for this redundant function is cmaA2, the trans-cyclopropane synthase of the methoxy- and
ketomycolates. Although cis-cyclopropanated
methoxymycolates are produced in the cmaA2 mutant (14),
cmaA2 does have nonspecific cis-cyclopropanating activity in M. smegmatis (26), suggesting that it could
serve a cis synthase in the absence of mmaA2.
The new insights into mmaA2 function presented here expand
our knowledge of the biosynthesis of the complex M. tuberculosis cell envelope and the biosynthetic specificity of the
mycolic acid cyclopropane synthases. In addition, the mmaA2
null mutant provides another defined mutant in cell envelope
biosynthesis that can be tested in animal models of pathogenesis. It
will be particularly interesting to compare the pathogenesis phenotype of the mmaA2 mutant to the pcaA mutant phenotype
as these two mutants differ predominantly in the position of the
missing cyclopropyl group in the
-mycolate. Characterization of
these mutant strains will provide further insight into the relationship
between the fine chemical structure of the Mtb cell envelope and
specific pathogenesis phenotypes. This information may help validate
the mycolic acids modification system as an attractive drug target for
new antituberculosis drugs and may reveal novel mechanisms by which the
host immune system recognizes the fine structure of mycobacterial lipids.