(Received for publication, July 7, 1995; and in revised form, August 25, 1995)
From the
The major mycolic acid produced by Mycobacterium
tuberculosis contains two cis-cyclopropanes in the
meromycolate chain. The gene whose product cyclopropanates the proximal
double bond was cloned by homology to a putative cyclopropane synthase
identified from the Mycobacterium leprae genome sequencing
project. This gene, named cma2, was sequenced and found to be
52% identical to cma1 (which cyclopropanates the distal double
bond) and 73% identical to the gene from M. leprae. Both cma genes were found to be restricted in distribution to
pathogenic species of mycobacteria. Expression of cma2 in Mycobacterium smegmatis resulted in the cyclopropanation of
the proximal double bond in the series of mycolic
acids. Coexpression of both cyclopropane synthases resulted in
cyclopropanation of both centers, producing a molecule structurally
similar to the M. tuberculosis
-dicyclopropyl mycolates.
Differential scanning calorimetry of purified cell walls and mycolic
acids demonstrated that cyclopropanation of the proximal position
raised the observed transition temperature by 3 °C. These results
suggest that cyclopropanation contributes to the structural integrity
of the cell wall complex.
An estimated 8 million persons develop tuberculosis each year,
and over 30 million people are expected to die from the disease in this
decade(1) . Mycobacterium tuberculosis, the causative
agent of tuberculosis, is an intracellular pathogen that establishes an
infection in oxygen-rich alveolar macrophages of the lung(2) .
Mycolic acids are long chain -alkyl-
-hydroxy fatty acids
unique to mycobacteria and related taxa and represent major components
of the cell wall(3) . Mycolic acids are thought to contribute
to both drug resistance and survival in the hostile intracellular
environment of the macrophage by the formation of an impermeable
asymmetric lipid bilayer (4) . The biosynthetic pathway for
these complex lipids is also thought to be the target for several
clinically useful chemotherapeutics, including isoniazid(5) .
With the increasing incidence of multidrug-resistant bacilli, alternate
chemotherapeutic targets are urgently needed.
Mycolic acids have
been proposed to be biosynthesized via a diversion of normal fatty acid
metabolism in which short chain fatty acids are extended and modified
to form lipids of exceptional length(6) . Mycobacterium
smegmatis synthesizes three different series of -mycolates
(which lack oxygen functionalities in the meromycolate chain outside
the
-hydroxy acid) shown in Fig. 1(7) . The
and
series are full-length mycolic
acids extending to an average of 78 and 79 carbons,
respectively(8) .
contains two cis-olefins in
the meromycolate chain, while
contains only a single
cis-olefin and a trans-olefin with an adjacent methyl group. In
addition to these three mycolates, M. smegmatis also produces
a shorter
` mycolic acid, which is 64 carbons in length as well as
a full-length epoxy mycolate(9) . M. tuberculosis contains only one series of
-mycolic acids that averages
78-80 carbons in length(4, 10, 11) .
The tubercle bacilli also produces two oxygenated mycolic acid series,
ketomycolates and methoxymycolates (not shown), which are generally of
lower abundance than the
series(12) . Pathogenic
mycobacteria cyclopropanate a majority of their mycolic acids, whereas
in saprophytic organisms, this modification is unusual(3) . The
functions of the various classes of mycolic acids in each of these
organisms is unknown. However, we have recently shown that
cyclopropanation at the distal position confers increased resistance to in vitro killing by hydrogen peroxide(13) . The
present studies were initiated to expand our understanding of the
relationship between mycolic acid structure and function.
Figure 1:
Structure of
the major mycolic acids of M. tuberculosis and M.
smegmatis. M. smegmatis produces three olefinic
-mycolates, which average 78-79 carbons in length in
and
and 64 carbons in
`. The
mycolic acid contains a cis-olefins in both the
proximal and distal positions. The
mycolic acid
contains a distal cis-olefin and a proximal trans-olefin with an
adjacent methyl branch. The sole
-mycolic acid (4) from M. tuberculosis contains two cis-cyclopropane moieties and
averages 78-80 carbons in length. The expoxy mycolic acid from M. smegmatis contains an epoxy group at the distal position
and a cis-olefin at the proximal position.
We have
previously reported the identification of cma1, a gene whose
protein product (cyclopropane mycolic acid synthase-1, CMAS-1) ()catalyzed the introduction of a cyclopropane at the distal
position in the meromycolate chain(13) . In the course of these
studies, we discovered that an unannotated related sequence had been
deposited in Genbank as part of the Mycobacterium leprae genome sequencing project (accession number U00018)(14) .
In this paper we report that this M. leprae sequence
represents a second cyclopropane synthase with a homolog in M.
tuberculosis whose protein product functions distinctly from
CMAS-1 to cyclopropanate the proximal cis-olefin in mycolic acid
biosynthesis.
M. smegmatis cell walls were prepared as follows: M.
smegmatis (250 ml A = 1.0)
culture was harvested by centrifugation and resuspended in 4 ml of 1
mM phenylmethylsulfonyl fluoride. This suspension was divided
equally among eight 1.5-ml tubes containing 0.5 g of 0.1-mm glass
beads. These were then placed in a Mini Beadbeater-8 (BioSpec Products,
Bartlesville, OK) and lysed at maximum speed for 3 min. After briefly
spinning at 5,000
g, the supernatants were removed,
and fresh phenylmethylsulfonyl fluoride was added (0.4 ml/tube). The
pellets were again lysed for 3 min, and the beads were allowed to
settle, after which the supernatants were combined (
5 ml), cooled
on ice, and 2.25 ml of 10% Triton X-114 (CalBiochem) was added. After
mixing on ice for several minutes, cell walls were removed by
centrifugation at 100,000
g for 1 h. These were washed
again in fresh 2% Triton X-114 followed by an additional wash in
phosphate-buffered saline to remove excess detergent. Monoclonal
antibodies used to establish purity of these cell wall preparations in
Western blots (Hsp60 (IT-13), the 19-kDa lipoprotein (IT-12), and the
32-kDa
-antigen (IT-49)) were provided by the UNDP/World
Bank/World Health Organization Special Program for Research and
Training in Tropical Diseases.
To test this hypothesis, PCR primers were designed to amplify one region of high homology and one region of low homology between the two sequences. These two probes were used sequentially to screen colony lifts of E. coli tranformed with an M. tuberculosis cosmid library(13) . Out of approximately 300 colonies, four clones were positive with the more homologous probe while only one of these was also positive with the less homologous probe. Cosmid DNA isolated from the positive clone was digested with BamHI and probed in a Southern blot with the same probes. The clone that reacted with both probes showed a strong band at 4.2 kb as well as weaker hybridization to the 7.2-kb fragment known to contain cma1(13) . Sequences homologous to cma2 were found by Southern blot analysis to be absent from the saprophytic M. smegmatis and present in pathogenic strains of Mycobacterium avium, Mycobacterium bovis BCG, and Mycobacterium marinum (data not shown).
A comparison of the deduced amino acid sequence of CMAS-2 (the cma2 gene product) with the other known cyclopropane synthases is shown in Fig. 2. As expected, the M. tuberculosis CMAS-2 sequence is more closely related to the M. leprae CMAS-2 sequence (73% identity) than either of the CMAS-2 are to CMAS-1 from M. tuberculosis (52% identity for each). The four proteins share the highest homology in the region corresponding to the N terminus of CMAS-1 (amino acids 14-96 of CMAS-1). Interestingly, the three cyclopropane synthases (including cyclopropane fatty acid synthase from E. coli(24) ) have variable N-terminal extensions, the significance of which is unknown. Functionally, one of the most important areas of homology shared by these four proteins spans amino acids 171-179 (using the cyclopropane fatty acid synthase numbering) and corresponds to a consensus motif of (V/L)L(E/D)XGXGXG, which has been proposed to play a role in binding of the enzyme cofactor S-adenosyl-L-methionine(25) . Cysteine 354 is also absolutely conserved and has been proposed to have catalytic function(24) .
Figure 2: Amino acid comparison of the four known cyclopropane synthases. This figure shows a multiple alignment of the deduced amino acid sequence from the M. tuberculosis cma2 gene with the M. leprae cyclopropane synthase homolog, the CMAS-1 protein from M. tuberculosis(13) and the amino acid sequence of cyclopropane fatty acid synthase from E. coli(24) . The putative S-adenosyl-L-methionine binding domain (25) spans amino acids 171-179 (using cyclopropane fatty acid synthase numbering), and cysteine 354 has been proposed to have catalytic function(24) .
To confirm that this transformation was due
to the putative cma2 sequence as well as to improve the extent
of conversion, a 1.2-kb NruI to BamHI fragment
containing the cma2 reading frame was subcloned into
pMH29_Hyg. pMH29_Hyg is a derivative of pMV261 that contains a
hygromycin resistance marker in place of kanamycin. Hygromycin
selection has much lower background and allows much faster recovery
times than kanamycin for transformed mycobacteria. ()This
vector also contains a synthetic MOP in place of the Hsp60 promoter
region. CMAS-2 produced from this construct was capable of converting
25% of the total mycolates to the cyclopropanated type as determined by
NMR.
Figure 3:
Two dimensional-TLC of M. smegmatis transformed with cma-expressing constructs.
[1-C]Acetate-labeled mycolic acids were purified
as described under ``Experimental Procedures.'' A, M. smegmatis (pYUB18). The epoxy (e),
,
, and
` are marked (see Fig. 1for structures). B, M. smegmatis (pMH29H_cma1). Spot 1 is the result of cyclopropanation
of the distal olefin of
(13) . C, M. smegmatis (pMH29H_cma2). Spots 2 and 3 are the result of cyclopropanation of the proximal olefin of
and the epoxy mycolic acid, respectively. D, M. smegmatis (pMV206H_cma1+2). Spot 4 is the
dicyclopropanated mycolic acid, which is not retarded by the silver ion
impregnation. Left to right development is in unmodified silica gel,
while up to down represents the silver ion impregnated
dimension.
Introduction of both cma genes
into M. smegmatis resulted in a pronounced change in the
radio-TLC profile, which contained both the CMAS individual products as
well as a unique MAME, which was unaffected by silver ion impregnation (Fig. 3D). This MAME exactly co-elutes with the major
-mycolic acid from M. tuberculosis (data not shown). To
confirm these structural predictions, MAMEs were purified from 1 liter
of M. smegmatis containing
pMV206_Hyg-cma1+cma2 and separated by
preparative argentation TLC. 500 MHz of
H NMR analysis of spot 4 (Fig. 4A) showed that this spot had no
olefinic resonances but had cyclopropane resonances (
-0.33,
0.56, and 0.65 ppm). These were present in a 4:6 ratio with terminal
methyl groups, indicating that this M. smegmatis mycolate
corresponded to structure 4, which is the major mycolate from M. tuberculosis (Fig. 1). The mycolate corresponding to spot 1 in Fig. 3D showed
H NMR
resonances (Fig. 4B) corresponding to a trans-olefin at
5.3 ppm (J = 15 Hz) as well as a doublet
corresponding to an
-methyl group at
0.93 ppm and
cyclopropane resonances as above. The olefinic and cyclopropane
resonances integrated for two and four protons, respectively; thus this
mycolate corresponds to the previously described structure containing a
distal cyclopropane and a methyl-branched trans-olefin. Spot 2 in Fig. 3D showed
H NMR resonances (Fig. 4C) consistent with a cis-olefin at
5.36
ppm (J = 10 Hz) as well as the cis-cyclopropane
resonances. This mycolate also displayed no
-methyl branch and is
produced by CMAS-2 alone, consistent with a proximally-cyclopropanated
-mycolic acid.
Figure 4:
500 MHz Proton NMR spectra of the three
cyclopropanated -mycolates produced in recombinant M.
smegmatis. Spectrum A shows the dicyclopropyl mycolate
corresponding to structure 4 (Fig. 1), which represents
the major
mycolate from M. tuberculosis. By integration,
the cyclopropane resonances (
-0.33, 0.56, 0.65 ppm) are
present in a ratio of 4:6 with terminal methyl groups. Spectrum B shows the mycolic acid corresponding to spot 1 in Fig. 3D, which contains a trans double bond (
5.36
ppm, J = 15 Hz) and a single cyclopropane. By
integration the olefinic and cyclopropyl protons represent two and four
protons with respect to terminal methyl groups. Spectrum C shows the mycolic acid corresponding to spot 2 in Fig. 3D, which contains a single cis-olefin as well as
a single cis-cyclopropane. This mycolate also lacks the
-methyl
branch since there is no doublet at
0.95ppm. Spectra were
recorded in deuterochloroform and are referenced to internal
tetramethylsilane.
Radio-TLC data for M. smegmatis transformants containing cma genes were quantitated by
PhosphorImaging analysis (Table 1). Introduction of CMAS-1
decreases the series from which it is derived, while
the
series decreases when CMAS-2 is present.
Interestingly, the total amount of mycolate cyclopropanated at the
distal position in the coexpressing construct (where cma1 is
expressed from its own promoter) is twice that produced when the gene
is expressed from the MOP promoter. The total amount of mycolic acids
cyclopropanated at the proximal position is the same between the
coexpressor and the overexpressor, suggesting that CMAS-1 activity is
affected by the presence of CMAS-2 but not vice versa.
Figure 5: Differential scanning calorimetry of cell walls and purified mycolic acids from recombinant M. smegmatis. A, purified mycolic acids from M. smegmatis (pYUB18) (solid line), M. smegmatis (pYUB18_cma1) (dashed line), and M. smegmatis (pYUB18_cma2) (dotted line) were analyzed by DSC as described under ``Experimental Procedures.'' Introduction of a cyclopropane at the proximal position by CMAS-2 results in consistently higher transition temperatures than control or distally cyclopropanated (CMAS-1) material. B, Triton X-114-extracted cell wall material was prepared and analyzed by DSC. M. smegmatis (pYUB18) (solid line), M. smegmatis (pYUB18_cma2) (dotted line), M. smegmatis (pMV206_Hyg-cma1+cma2) (dashed line).
In this work, we have used a homologous sequence from the M. leprae genome sequencing project to identify the protein
involved in construction of the proximal cyclopropane from M.
tuberculosis. This enzyme is the fourth identified member of a
family of proteins to catalyze the transfer of a methylene group from S-adenosyl-L-methionine to the double bond of a fatty
acid substrate. The three mycobacterial members of this family are
closely related to one another, with the cma2 genes from M. leprae and M. tuberculosis being more closely
related to one another (73% identity) than to the cma1 gene of M. tuberculosis (52% identity). Heterologous expression of cma2 in M. smegmatis results in a proportion of the
-mycolates becoming cyclopropanated at the proximal position.
Expression of cma1 results in cyclopropanation at the distal
position, while coexpression of both genes results in the production of
a dicyclopropyl mycolate nearly identical to the major mycolic acid
produced by M. tuberculosis.
The cyclopropanation of the
epoxy mycolates by CMAS-2 in M. smegmatis suggests that the
enzyme is either insensitive to substituents occurring toward the
end of the chain or that CMAS-2 acts on a precursor
meromycolate, which can become either cyclopropanated to form the
dicyclopropyl mycolate or further oxidized to form the epoxy series.
CMAS-2 activity is unchanged upon co-expression of both cyclopropane
synthases with about 30% of the total mycolates cyclopropanated at the
proximal position (Table 1). Total CMAS-1 activity, however,
increases upon coexpression from 30 to 50% cyclopropanation of the
distal position. One interpretation of this result is that the distal
cyclopropane is formed after the proximal cyclopropane with CMAS-1
preferentially recognizing the proximally cyclopropanated precursor as
a substrate.
The biological significance of lipid cyclopropanation
has been most extensively studied in E. coli; however, the
lack of any dramatic phenotype associated with either cyclopropane
fatty acid synthase null mutants or cyclopropane fatty acid synthase
overexpressors has left the role cyclopropanation plays in cellular
metabolism unclear(28, 29) . A large increase in the
synthesis of cyclopropane-containing plasma membrane fatty acids has
been shown to accompany the transition from log to stationary phase,
which suggests that cyclopropanation offers some protective advantage
to stationary cultures(27) . E. coli, which have been
grown on cyclopropane fatty acids, are more resistant to killing by
hyperbaric oxygen treatment, suggesting that cyclopropanes do have a
stabilizing or rigidifying effect on the membrane(30) . This is
confirmed by the increased susceptibility to killing by freezing
observed in cyclopropane fatty acid synthase mutants of E.
coli(29) . It has also been shown by examining the H NMR of specifically deuterated cyclopropane-containing
lipids, that cyclopropanated membranes enhance stability by suppressing
segmental mobility of hydrocarbon chains, thus providing increased
rigidity with respect to external shock(31) . These studies
consistently support the position that cyclopropanation of membrane
lipids, although a rather subtle modification, does contribute to
increased structural integrity of membranes containing short chain
fatty acids(32) . In addition, cyclopropanation is intermediate
in fluidity effects between the more fluid cis-olefin and the less
fluid trans-olefin as measured by DSC (33) .
Recent work on
the structure of the mycobacterial cell wall suggests that the proximal
cyclopropane lies at the boundary of what Minnikin (3, 26) has referred to as the structural permeability
barrier. A dramatic high temperature phase transition has recently been
demonstrated to occur at 60 °C in purified cell walls of M.
chelonei by DSC(4) . The temperature of this transition
suggests that at physiologically relevant temperatures, much of the
cell wall exists in a state of exceptionally low fluidity.
Cyclopropanation of mycolic acids, in addition to rendering lipids less
susceptible to peroxidation, may decrease the actual fluidity even
more, thus contributing to the overall impermeability of the cell wall.
We examined the effect of substitution of a cis-olefin with a
cis-cyclopropane in mycolic acids on cell wall thermochemistry and
showed, with either purified cell walls or MAMEs, that proximal
cyclopropanation increased the observed temperature of the transition
by approximately 3 °C. The magnitude of this change seems quite
reasonable since substitution of a cis-cyclopropane for a cis-olefin in
the much shorter palmitoleate (C16:1), raises the observed temperature
of phase transition by 15.6 °C(33) , and only about 30% of
the mycolates are converted to the cyclopropanated form. The distal
cyclopropane had no such effect, possibly reflecting the role of this
cyclopropane in interacting with other lipids that form a less tightly
associated region that is not observed by DSC of detergent-extracted
cell walls. In fact, our M. smegmatis cell wall preparations
gave significantly lower melting temperatures than purified cell walls
from M. smegmatis prepared without detergent extraction ()presumably due to the loss of ancillary lipids during the
Triton X-114 extraction.
The impermeability of the mycobacterial cell wall is a hallmark of the organism. In the case of slow growing and pathogenic mycobacteria such as M. tuberculosis, it seems likely that high durability of mycolic acids would be essential, especially in the face of environmental and host-initiated oxidative stress in its intracellular habitat(13, 16) . Dicyclopropyl mycolic acids are the major species found in many slow growing and pathogenic strains of mycobacteria including M. avium, Mycobacterium kansasii, M. marianum, M. leprae, Mycobacterium paratuberculosis, and M. tuberculosis(3) . In contrast fast growing saprophytic mycobacteria such as M. smegmatis, Mycobacterium phlei, and Mycobacterium chelonae appear to possess primarily diunsaturated mycolic acids with an abundance of cis-olefins(34) . In the case of the distal cyclopropane, we have previously demonstrated that expression in M. smegmatis results in significant protection from hydrogen peroxide (13) . In the case of the proximal cyclopropane, we have been unsuccessful in demonstrating a similar role in protection from oxidative stress (data not shown). This may be related to the largely internal and less accessible location of the proximal cyclopropane.
Cyclopropanation of fatty acids only occurs in a small number of related taxa of bacteria. Among mycobacteria, this modification is limited to the slow growing pathogens. Mammals do not cyclopropanate unsaturated lipids. Thus, enzymes catalyzing this unique modification constitute a viable target for the design of new chemotherapy against pathogenic mycobacteria, as well as providing the tools for understanding the biosynthesis, regulation, and function of these complex lipids.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U34637[GenBank].