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Identification of the Required Acyltransferase Step in the Biosynthesis of the
Phosphatidylinositol Mannosides of Mycobacterium species
Jana Korduláková1,3, Martine Gilleron2, Germain Puzo2, Patrick J. Brennan4,
Brigitte Gicquel1, Katarína Mikusová3, and Mary Jackson1∗∗
From the 1 Unité de Génétique Mycobactérienne, Institut Pasteur, 25 rue du Dr. Roux, 75724
Paris Cedex 15, France, 2 the Institut de Pharmacologie et de Biologie Structurale du CNRS,
205 route de Narbonne, 31077 Toulouse Cedex, France, 3 the Department of Biochemistry,
Comenius University in Bratislava, Faculty of Natural Sciences, Mlynska dolina CH-1, 84215
Bratislava, Slovak Republic and the 4 Department of Microbiology, Immunology and
Pathology, Colorado State University, Fort Collins, Colorado 80523
Running title: Acylation of phosphatidylinositol mannosides in mycobacteria
Abbreviations: GPI, glycosylated phosphatidylinositol; ORF, open reading frame; LB, Luria
Bertani culture medium, TLC, thin-layer chromatography; PI, phosphatidyl-myo-inositol;
PIM, phosphatidyl-myo-inositol mannosides; LM, lipomannan; LAM, lipoarabinomannan;
PCR, polymerase chain reaction; Kb, kilobase; Km, kanamycin; Hyg, hygromycin; Suc,
sucrose; KmR, kanamycin-resistant; HygR, hygromycin-resistant; SucR, sucrose-resistant;
MIC, minimal inhibitory concentration; Man, mannose; myo-Ins, myo-inositol; Ara,
Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on July 9, 2003 as Manuscript M303639200 by guest on A
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arabinose; HABA, 2-(4-hydroxy-phenylazo)-benzoic acid; MALDI, Matrix-Assisted Laser
desorption/ionization; Tof, time of flight; ESI, electrospray ionization; C16, palmitate; C19,
tuberculostearate (10-methyloctadecanoate);
PIM is used to describe the global family of phosphatidylinositol mannosides that carries one
to four fatty acids (attached to the glycerol, inositol and/or mannose) and one to six mannose
residues. In AcXPIMY, x refers to the number of acyl groups esterified to available hydroxyls
on the mannose or myo-inositol residues, y refers to the number of mannose residues; e.g.
Ac1PIM1 corresponds to the phosphatidylinositol mono-mannoside PIM1 carrying two acyl
groups attached to the glycerol (the diacylglycerol substituent) and one acyl group esterified
to the mannose residue.
∗∗To whom correspondence should be addressed. Tel.: 33 1 45 68 88 77; Fax: 33 1 45 68 88
43, E-mail: [email protected].
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SUMMARY
The fatty acyl functions of the glycosylated phosphatidylinositol (GPI) anchors of the
phosphatidylinositol mannosides (PIM), lipomannan (LM) and lipoarabinomannan (LAM) of
mycobacteria play a critical role in both the physical properties and biological activities of
these molecules. In a search for the acyltransferases that acylate the GPI anchors of PIM, LM
and LAM, we examined the function of the mycobacterial Rv2611c gene that encodes a
putative acyltransferase involved in the early steps of phosphatidylinositol mannoside
synthesis. A Rv2611c mutant of Mycobacterium smegmatis was constructed which exhibited
severe growth defects and contained an increased amount of phosphatidylinositol mono- and
di-mannosides and a decreased amount of acylated phosphatidylinositol di-mannosides
compared to the wild-type parental strain. In cell-free assays, extracts from M. smegmatis
overexpressing the M. tuberculosis Rv2611c gene incorporated [14C]palmitate into acylated
phosphatidylinositol mono- and di-mannosides, and transferred cold endogenous fatty acids
onto [14C]-labeled phosphatidylinositol mono- and di-mannosides more efficiently than
extracts from the wild-type strain. Cell-free extracts from the Rv2611c mutant of M .
smegmatis were greatly impaired in these respects. This work provides evidence that Rv2611c
is the acyltransferase that catalyzes the acylation of the 6-position of the mannose residue
linked to position 2 of myo-inositol in phosphatidylinositol mono- and di-mannosides, with
the mono-mannosylated lipid acceptor being the primary substrate of the enzyme. We also
provide the first evidence that two distinct pathways lead to the formation of acylated PIM2
from PIM1 in mycobacteria.
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INTRODUCTION
Phosphatidylinositol (PI) and metabolically-derived products such as the phosphatidylinositol
mannosides (PIM), lipomannan (LM) and lipoarabinomannan (LAM) are prominent
phospholipids/lipoglycans of Mycobacterium species believed to play important roles in the
physiology of the bacterium (1-3) as well as during host infection. Lipoarabinomannan
(LAM) is for instance an important modulator of the immune response in the course of
tuberculosis and leprosy (4-6) as well as a key ligand in the interactions between
Mycobacterium tuberculosis and phagocytic cells (7-12).
Although the structures of PIM, LM and LAM have been well documented (for a review,
4,6,13), little is known about the biosynthesis of these complex molecules. PIM and their
multiglycosylated counterparts, LM and LAM, all share a conserved glycosylated
phosphatidylinositol (GPI) anchor, suggesting that they are metabolically related (13-18). A
large number of different acyl forms of this GPI anchor exist, depending on the number,
location and nature of the acylating fatty acids. This diversity leads to a wide spectrum of PIM
species and acyl forms of LAM (15,19-24). Four acylation sites have been identified in
mannosylated LAM, i. e., positions 1 and 2 of glycerol, position 6 of the mannose (Man)
linked to position 2 of myo-inositol (myo-Ins) (22-23) and position 3 of myo-Ins (15). Given
the crucial role of fatty acyl functions in both the physical properties and biological activities
of PIM and LAM (12,16,25-29), the characterization of the enzymes catalyzing the acylation
of the GPI anchor would represent an important step towards a full understanding of the
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biosynthesis of these molecules. The first step in PIM synthesis involves the transfer of a
mannose residue from GDP-Man to the 2-position of the myo-inositol ring of PI to form the
phosphatidylinositol monomannoside, PIM1 (3,30). This lipid or its acylated counterpart
(Ac1PIM1) then accepts another mannose residue (Man) at the 6-position of myo-Ins to form
phosphatidylinositol dimannoside (PIM2) or acylated phosphatidylinositol dimannoside
(Ac1PIM2), respectively (20,30-32). It is thought that PIM2 or Ac1PIM2 then undergo several
glycosylation steps with Man and then with Ara to form higher PIM (PIM3-PIM6) and the
highly branched lipoglycans, LM and LAM (18). It may be inferred from the studies of
Takayama and Goldman (31) that acylated PIM1 (Ac1PIM1) is a more potent substrate for the
second mannosylation step than is PIM1. Therefore, the acylation reaction responsible for the
formation of Ac1PIM1 may constitute a key regulatory event in determining the synthesis of
the final PIM, LM and LAM products.
Acylating activities have been reported by Brennan and Ballou (20,33) in the membrane
fraction of Mycobacterium phlei. Acylated PIM2 (Ac1PIM2) were the main products formed in
the reaction when endogenous or crude mycobacterial phospholipids were used as the lipid
acceptors and acyl-CoA derivatives of fatty acids (myristic, palmitic and oleic acids) were
used as the [14C]-labeled substrates. These results, together with the previous observation of
the formation of PIM1 from PI in M. phlei (30), led the authors to propose two models for the
early steps of PIM synthesis in Mycobacterium spp. (20,33) (Fig. 1). In the first proposed
pathway, PI is mannosylated to form PIM1. PIM1 is then mannosylated to PIM2 which is
acylated to form Ac1PIM2. In the second pathway, which appears to be more consistent with
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the observations subsequently published by Takayama and Goldman (31), PIM1 is first
acylated to Ac1PIM1 and then mannosylated to Ac1PIM2. Since these early studies, no further
work on the acylation steps of PIM, LM and LAM has been undertaken, and the genes
underlying the acyltransferase activities described by Brennan and Ballou (20,33) have not
been identified.
Recently, we identified a cluster of five ORF apparently dedicated to the early steps of PIM
synthesis in Mycobacterium spp. (2). The first ORF of this cluster (Rv2613c) encodes a
protein of unknown function. The second ORF encodes PgsA, the previously characterized
phosphatidylinositol synthase (2). The third ORF (Rv2611c) encodes a protein with
similarities to bacterial acyltransferases. The fourth ORF encodes PimA, the α -
mannosyltransferase responsible for the formation of PIM1 from PI and GDP-Man (3), and the
fifth ORF, Rv2609c, encodes a putative GDP-Man hydrolase carrying a mutT domain
signature (PS00893). This genetic organization suggests that Rv2611c encodes an
acyltransferase involved in the acylation of phosphatidylinositol mono- and di-mannosides.
Rv2611c is present in all of the mycobacterial genomes sequenced so far and has a homolog
in Streptomyces coelicolor (43% identity for a 296 amino acid overlap), an actinomycete that
shares the ability to synthesize PIM with mycobacteria (34).
In this report, we provide evidence that Rv2611c is the acyltransferase responsible for the
acylation of the 6-position of the Manp residue linked to position 2 of myo-Ins. PIM1 appeared
to be the main lipid acceptor in the reaction although the enzyme could also acylate PIM2. We
show that the Rv2611c gene is not essential for growth of M. smegmatis although its
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disruption induces dramatic changes in the PIM content of this strain accompanied by severe
growth defects in both solid and liquid media. The dispensability of Rv2611c appears to be
related, at least partly, to the ability of the mutant to synthesize Ac1PIM2 from PIM2.
Altogether, these observations provide evidence that the two pathways leading to the
synthesis of phosphatidylinositol di-mannosides originally proposed by Brennan & Ballou
(20,33) co-exist in mycobacteria.
EXPERIMENTAL PROCEDURES
Bacterial strains and growth conditions
E. coli XL1-blue, the strain used for cloning experiments was propagated in Luria Bertani
(LB) broth (pH 7.5) (Bactotryptone, 10 g/l, BactoTM yeast extract, 5 g/l, NaCl, 5 g/l) (Becton
Dickinson, Sparks, MD) at 37°C. Mycobacterium smegmatis strain mc2155 (35) was routinely
grown at 37°C in LB broth supplemented with 0.05% Tween 80. LB medium was used as the
solid medium for all bacteria. Antibiotics were added at the following concentrations:
ampicillin, 100 µg/ml; kanamycin, 20 µg/ml; hygromycin, 50 µg/ml. When required, 10 g/l
NaCl was added to the liquid LB medium and 10% sucrose was added to the solid medium.
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Cloning procedures, mycobacterial genomic DNA extraction and Southern blotting analysis
Molecular cloning, restriction endonuclease digestions and DNA purification were performed
by standard techniques according to the manufacturer’s recommendations. Electrocompetent
E. coli XL1-blue and M. smegmatis cells were prepared and transformed as previously
described (3). Isolation of mycobacterial genomic DNA, labeling of DNA probes with α-
32P[dCTP] and Southern blot analyses were performed as previously described (3).
Construction of the M. smegmatis Rv2611c mutant
The Rv2611c gene of M. smegmatis was disrupted by use of a two-step homologous
recombination procedure. This method relies upon the use of a suicide vector harboring the
counterselectable marker sacB and a kanamycin cassette-disrupted copy of the gene of
interest. In the first step of the experiment, a single crossover strain is isolated and cultured at
37°C in the presence of kanamycin. In the second step of the experiment, the single crossover
strain culture is plated out on sucrose-Km plates to select clones that underwent a second
intra-chromosomal crossover event leading to the excision of the body of the vector and
allelic replacement.
The M. smegmatis Rv2611c gene and flanking regions were excised from the plasmid
pUCpgsA.Sm (2) on a 3.7-Kb BamHI restriction fragment and inserted into the BamHI site of
pUC18, yielding pUCacylT. A disrupted allele of the Rv2611c gene, Rv2611c::Km, was then
constructed by cloning the kanamycin resistance cassette from pUC4K (Amersham Pharmacia
Biotech), carried on a 1.2-Kb HincII restriction fragment, into the AgeI-cut and blunt-ended
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pUCacylT. This digestion by AgeI resulted in the deletion of 121-bp of the Rv2611c coding
sequence. Rv2611c::Km was then excised from the resulting plasmid on a 4.8-Kb BamHI
restriction fragment, blunt-ended and inserted into the SmaI site of pXYL4 (a pBlueScript
derivative carrying the xylE colored marker) (36) yielding pX4acylTK. Finally, pJQacylT, the
construct used for allelic replacement, was obtained by transferring a 5.8-Kb BamHI fragment
from pX4acylTK containing Rv2611c::Km and XylE, into the BamHI site of pJQ200, an E.
coli cloning vector carrying the counterselectable marker sacB.
Overexpression of the M. tuberculosis Rv2611c gene in M. smegmatis and purification of the
recombinant His-tagged Rv2611c protein
Standard PCR strategies with Taq DNA polymerase (Applied Biosystems, Roche) were used
to amplify the M. tuberculosis Rv2611c gene. PCR amplification consisted of one
denaturation step (95°C, 6 min) followed by 40 cycles of denaturation (95°C, 1 min),
annealing (68°C, 1 min) and primer extension (72°C, 2 min), and a final extension step at
72°C for 10 minutes. The primers were AcylT1 (5'- ctttaaccatatgacactttccggccgcatcccg -3')
and AcylT2 (5'- cccaagcttggttcccaaccgtgcgcggcgc -3'). The primers were designed to generate
a PCR product corresponding to the entire Rv2611c gene devoid of its stop codon and
harboring NdeI and HindIII restriction sites (underlined in the primers sequences) to enable
direct cloning into the pVV16 expression vector (2). pVV16 harbors a kanamycin and a
hygromycin resistance marker. In this vector, genes are constitutively expressed under the
control of the hsp60 transcription and translation signals. Recombinant proteins produced
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with this system carry a six-histidine tag at their carboxyl terminus. Wild-type M. smegmatis
mc2155 and its Rv2611c mutant, MYC1573, were transformed with the resulting expression
vector, pVVacylT, and transformants were selected on LB-Km-Hyg plates. The production of
recombinant Rv2611c protein in M. smegmatis was analyzed by western blotting, as
previously described (3). For the purification of recombinant His-tagged Rv2611c protein, M.
smegmatis mc2155/pVVacylT cells (11 g wet weight) were washed and resuspended in 30 ml
of buffer A (20 mM Tris-HCl buffer, pH 7.45) before probe sonication for 7 min at 4°C in the
form of 7 x 60-s pulses with 90-s cooling intervals between pulses. The unbroken cells and
bacterial debris were removed by centrifugation of the sonicate at 10,000 x g for 30 min.
Recombinant His-tagged Rv2611c protein was purified from the supernatant of this
centrifugation using the BD TALON™ Resin (BD Biosciences Clontech, Palo Alto, CA)
according to the supplier’s recommendations. Unbound proteins were removed by washing
the resin with buffer A containing 500 mM NaCl. Proteins bound to the resin were then
gradually eluted with buffer A containing 500 mM NaCl and increasing concentrations of
imidazole (10, 50, 100, 200 and 500 mM). Recombinant Rv2611c protein was detected in the
fractions eluted with 100, 200 and 500 mM imidazole. These fractions were pooled and
desalted using a PD-10 column (Amersham Pharmacia Biotech). The resulting protein
preparation was significantly enriched in recombinant Rv2611c.
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Whole cell radiolabeling experiment
Radiolabeling of whole M. smegmatis mc2155/pVV16, mc2155/pVVacylT, MYC1573 and
MYC1573/pVVacylT cells with [1,2-14C]acetic acid (specific activity 113 mCi/mmol, ICN) or
myo-[2-3H]inositol (specific activity 17.0 Ci/mmol, Amersham Pharmacia Biotech) was
performed in LB medium supplemented with 10 g/l NaCl, kanamycin and hygromycin;
radiolabeled precursors (0.5 µCi/ml) were added to mid-log cultures, and cultures were
incubated at 37°C for a further 8 or 24 hours, respectively, with shaking.
Cell-free assays using [1-14C]palmitic acid, [14C]GDP-Man, and [14C]-labeled putative lipid
substrates
Membrane, cytosol and cell wall-membrane (P60) fractions of M. smegmatis mc2155,
MYC1573 and mc2155/pVVacylT cells were prepared as described previously (3). For an
initial comparison of PIM production in each strain, the whole cell lysate obtained by
sonication was used in a reaction containing 4 mg of protein, 0.25 µCi [14C]GDP-Man,
(specific activity, 305 mCi/mmol, Amersham Biosciences), 62.5 µM ATP, 10 mM MgCl2 and
buffer A (20 mM Tris-HCl buffer, pH 7.45) in a final volume of 250 µl. In the assays using
[1-14C]-palmitic acid as the substrate, [14C]GDP-Man was replaced by 0.0625 µCi of [1-
14C]palmitic acid (specific activity, 40-60 mCi/mmol, ICN) and the reaction mixtures were
supplemented with 100 µM CoA (Sigma) and 10 µM GDP-Man (Sigma). Membrane and cell
wall–membrane (P60) fractions (0.25 mg of protein in both cases) were used as enzyme
sources. After 1 hr at 37°C, the reactions were stopped by the addition of 1.5 ml of
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CHCl3/CH3OH (2:1). The samples were rocked for 30 min at room temperature and then
centrifuged at 2,000 x g for 10 min. The upper aqueous phase was discarded and the bottom
organic phase was washed with CHCl3/CH3OH/H2O (1:47:48, v/v). The final organic phase
was dried under a stream of nitrogen and dissolved in 100 µl of CHCl3/CH3OH (2:1, v/v)
before scintillation counting and thin-layer chromatography (TLC) analysis.
Radiolabeled putative lipid substrates for the acyltransferase reaction, namely [14C]PIM1 and
[14C]PIM2, were purified by preparative TLC. [14C]PIM1 was generated in vitro in a reaction
containing [14C]GDP-Man and M. smegmatis mc2155 membranes supplemented with crude
extracts from E. coli overproducing PimA (E. coli BL21/pETpimA) (3). [14C]PIM2 was
obtained from total lipid extracts of MYC1573 (20 ml culture) labeled in vivo with [1,2-
14C]acetate (the mutant strain produced more of this lipid than the wild-type strain). For cell-
free assays, [14C]PIM1 or [14C]PIM2 were transferred to Eppendorf tubes and dried under
nitrogen. 50 µM palmitic acid (Sigma), 100 µM CoA (Sigma), 62.5 µM ATP, membrane
fraction (0.1 mg of protein), cell wall–membrane (P60) fraction (0.1 mg of protein) and buffer
A were added to a final volume of 50 µl, and the reaction mixtures were bath sonicated for
one minute. After 3 hr at 37°C, the reactions were stopped by the addition of 300 µl of
CHCl3/CH3OH (2:1). The lipids were extracted as described above and analyzed by TLC.
In situ formation of the putative radioactive substrate of Rv2611c, [14C]PIM1, was achieved by
including crude extracts of E. coli overexpressing pimA (3) in the assays. Reaction mixtures
contained membrane fractions from wild-type M. smegmatis mc2155, MYC1573 or
mc2155/pVVacylT (0.1 mg of protein), crude extracts of E. coli BL21/pETpimA (0.3 mg of
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protein), 0.0125 µCi of [14C]GDP-Man, 62.5 µM ATP, 10 mM MgCl2 and buffer A in a final
volume of 50 µl. Reactions were incubated for 5, 30 and 180 min at 37 °C and stopped by the
addition of 300 µl of CHCl3/CH3O H (2:1). Lipids were extracted as described above,
separated by TLC and plates were subjected to autoradiography. The bands identified as
PIM1, Ac1PIM1 and Ac1PIM2 were scraped off the TLC plate and quantified by scintillation
counting. Reactions containing partially purified recombinant Rv2611c (see above) instead of
crude membrane fractions were performed in two steps. Firstly, crude extracts of E. coli
BL21/pETpimA (0.5 mg of protein), 0.0125 µCi [14C]GDP-Man, 0.25 mM PI (Sigma), 62.5
µM ATP, 10 mM MgCl2 and buffer A in a final volume of 50 µl were incubated for one hour
at 37°C to form [14C]PIM1. Secondly, palmitoyl-CoA dissolved in 1µl of DMSO was added to
a final concentration of 0.12 mM in the reaction mixture, along with purified recombinant
Rv2611c (60 µg in 30 µl), and the incubation was continued for another hour. Lipids were
extracted and analyzed as described above.
Analytical procedures
Lipids from labeled and unlabeled cells were extracted as described previously (3) and
routinely subjected to TLC in CHCl3/CH3OH/NH4OH/H2O (65:25:0.5:4) using Silica Gel 60
F254 plates (Merck). Radiolabeled lipids were visualized by exposure of TLC to Kodak
BIOMAX MR films at -70°C for 1 to 20 days. Analysis and characterization of the various
PIM was based on one- and two-dimensional TLC patterns and mass spectrometry (MS) as
described previously (3).
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Sample preparation and MALDI-TOF and ESI Mass Spectrometry
MALDI-Tof-MS in the negative ion mode was performed as previously described (3). The
MALDI mass spectra are dominated by peaks assigned to (M-H)- ions allowing both the
degrees of acylation and glycosylation to be determined. HABA (2-(4-hydroxy-phenylazo)-
benzoic acid) was used as the matrix at a concentration of 10 mg/ml in ethanol/water (1:1,
v/v). ESI-MS analysis of PIM2 and Ac1PIM2 acetolysis products dissolved in CHCl3/CH3OH
(9:1, v/v) was carried out on a Finnigan LCQduo ion trap mass spectrometer (Finnigan Mat,
San Diego, CA) system. The spray potential was set at 4.5 kV and the temperature of the
heated capillary was set at 200°C. The mass spectra were obtained by direct infusion using a
syringe pump (Harvard Instruments) at a flow rate of 5 µl min-1. Full scan spectra were
acquired in the ion peak centroid mode over the mass/charge range of 250-2000. No sheath
liquid or sheath gas was used. All data were collected on Xcalibur Software.
Acetolysis of PIM2 and Ac1PIM2
Purified PIM2 and Ac1PIM2 were acetolysed as previously described (16).
Purification of PIM and NMR analysis
Lipid fractions were applied to a QMA-Spherosil M (BioSepra SA, Villeneuve-la-Garenne,
France) column (1.0 x 14 cm) that had been irrigated successively with 8 ml of CHCl3,
CHCl3/CH3OH (1:1, v/v) and CH3OH to elute neutral compounds. Phospholipids were eluted
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using organic solvents containing ammonium acetate. 0.1 M ammonium acetate in
CHCl3/CH3OH (1:2, v/v) allowed the elution of phosphatidylinositol di-mannosides. Repeated
freeze-drying cycles were necessary to eliminate ammonium acetate salts. 20.4 mg and 49.7
mg of wild-type and mutant phosphatidylinositol di-mannosides were collected, respectively.
They were subjected to chromatography on silicic acid Sep-pak using CHCl3 (1.5 ml),
CHCl3/CH3OH/H2O (80:20:2) (35 ml), CHCl3/CH3OH/H2O (60:35:6) (7.5 ml) successively as
eluents. Purity was checked by TLC, on aluminum-backed Silica Gel plates (Alugram Sil G,
Macherey-Nagel, Duren, Germany) using CHCl3/CH3OH/H2O (60:35:6) as the migration
solvent.
NMR spectra were recorded with an Avance DMX500 spectrometer (Bruker GmbH,
Karlsruhe, Germany) equipped with an Origin 200 SGI using Xwinnmr 2.6. Samples were
dissolved in CDCl3/ CD3OD/ D2O (60:35:8) and analyzed in 200 x 5-mm 535-PP NMR tubes
at 343 K. Proton chemical shifts are expressed in ppm downfield from the signal of the methyl
of CDCl3 (δH/TMS 7.27 and δC/TMS 77.7). The one-dimensional phosphorus (31P) spectra
were measured at 202 MHz with phosphoric acid (85%) as the external standard (δp 0.0). All
the details concerning the NMR sequences and experimental procedures used have been
described in previous studies (14,16).
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RESULTS
Overexpression of the M. tuberculosis Rv2611c gene in M. smegmatis
For clarity, the homolog of the M. tuberculosis Rv2611c gene in M. smegmatis is given the
same name as its M. tuberculosis counterpart throughout this report. The product of the M.
tuberculosis Rv2611c gene is 316 amino acids long (35 kDa) and has homologs in all of the
mycobacterial genomes sequenced so far. It shares sequence similarity with acyltransferases
from Corynebacterium glutamicum (49 % identity over a 292 amino acid overlap),
Campylobacter jejuni (20 % identity over a 159 amino acid overlap) and a putative
acyltransferase from Streptomyces coelicolor (43 % identity over a 296 amino acid overlap).
Rv2611c from M. tuberculosis was PCR-amplified and placed under the control of the phsp60
promoter in the mycobacterial expression vector pVV16, yielding pVVacylT. Following the
electro-transformation of M. smegmatis mc2155 with this construct, colonies of
mc2155/pVVacylT were obtained that grew at a similar rate to the control strain
mc2155/pVV16 in LB-NaCl-Km-Tween80 broth and on LB-Km-Hyg plates at 37°C (data not
shown). The recombinant Rv2611c protein produced by mc2155/pVVacylT was checked by
western blot using a mouse monoclonal anti-His antibody. A protein of the expected size
(approximately 35 kDa) was detected in the membrane fraction (data not shown). The
association of Rv2611c with the membrane fraction is consistent with the prediction of one
putative transmembrane segment from amino acids 120 to 139, by the TMpred
(http://www.ch.embnet.org/) and DAS (http://www.sbc.su.se/~miklos/DAS/) transmembrane
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prediction programs. The uneven distribution of basic amino acid residues responsible for the
higher predicted pI of the N-terminal half of the protein (theoretical pI = 10.26 from residues
1 to 158) than that of the C-terminal half (theoretical pI = 5.65 from residues 159 to 316) may
also reflect the ability of some N-terminal domains of Rv2611c to interact with anionic
phospholipids of the membrane. The association of Rv2611c with the membrane fraction is
also consistent with the detection of acyltransferase activities associated with the membrane
fraction of M. phlei by Brennan and Ballou (33).
A comparative analysis of the cold and [1,2-14C]acetate-labeled total lipids from
mc2155/pVV16 and mc2155/pVVacylT by MALDI-MS and TLC showed no difference
between the PIM compositions of the two strains (data not shown). Thus, the over-production
of the putative acyltransferase Rv2611c did not affect the acylation of PIM in M. smegmatis.
This might be due to tight regulation of acyltransferase activities in M. smegmatis or to the
limited availability of the substrate of Rv2611c in this species rather than to the lack of
activity of the recombinant protein (see below).
Construction and analysis of a Rv2611c mutant of M. smegmatis
Disruption of Rv2611c by allelic exchange in M. smegmatis
Essentially the same strategy was used to construct this mutant as was used to disrupt the
pimA gene in M. smegmatis (3). This strategy consists of a two-step homologous
recombination procedure that results in allelic replacement at the Rv2611c locus. A
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kanamycin cassette-disrupted copy of Rv2611c from M. smegmatis, Rv2611c::Km, and the
xylE colored marker were inserted into the sacB suicide vector pJQ200, yielding pJQacylT.
pJQacylT was introduced into the wild-type M. smegmatis mc2155 by electroporation and
kanamycin-resistant transformants were selected on LB-Km plates at 37°C. Clones arising
from a single homologous recombination event at the Rv2611c locus were selected, grown in
LB-Km broth and then plated out onto LB-Km-Suc to select for clones that had undergone a
second intra-chromosomal cross-over event leading to the excision of the body of vector and
to allelic replacement. Allelic exchange mutants were expected to carry the Rv2611c::Km
disrupted allele and to have lost the sacB and xylE markers carried by pJQacylT. Therefore,
allelic exchange mutants should be resistant to kanamycin and sucrose and remain white upon
spraying with catechol (i. e. xylE-negative). Interestingly, two types of colony grew on LB-
Km-Suc plates after four days at 37°C; thirty percent of them were of the normal expected
size, whereas the other 70 % were unusually small. Spraying with catechol revealed that only
the small colonies exhibited the expected phenotype for allelic exchange mutants. The big
colonies all exhibited a XylE positive phenotype and probably resulted from mutations in the
sacB gene that rendered them resistant to sucrose. Genomic DNA from four mutant
candidates was analyzed by Southern blotting using a 2775-bp StuI restriction fragment
encompassing the entire pgsA and Rv2611c genes and the partial Rv2613c and pimA genes as
a probe. In the case of allelic replacement at the Rv2611c locus, the wild-type 2775-bp StuI
restriction fragment should be replaced by two StuI restriction fragments of 1850 and 2065-
bp, due to the presence of a StuI restriction site in the Km gene. All the mutant candidates
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gave hybridization signals consistent with allelic replacement at the Rv2611c locus (data not
shown). One M. smegmatis Rv2611c mutant was selected for further experiments and named
MYC1573.
Growth characteristics of the M. smegmatis Rv2611c mutant
As mentioned above, the MYC1573 mutant grew more slowly than the wild-type M.
smegmatis on LB-Km plates (with and without sucrose) and failed to grow in regular LB-Km
liquid medium at 30 or 37°C. Interestingly, normal growth was restored in LB-Km broth
following the addition of NaCl (10 g/l). The addition of 0.05% Tween 80 to LB broth
supplemented with NaCl completely abolished the growth of the mutant but did not affect that
of the wild-type strain mc2155. Therefore, LB-NaCl broth (without Tween 80) was used as the
culture medium in all subsequent experiments.
Complementation of MYC1573 with the wild-type M. tuberculosis Rv2611c gene carried on
pVVacylT restored the growth of the mutant in LB liquid medium supplemented with Tween
80 and suppressed its requirement for high NaCl concentrations. Altogether, these findings
suggested that Rv2611c is expressed in M. smegmatis and that a null mutation in this gene
causes loss of viability unless Tween 80 is omitted from the culture medium and NaCl is
added.
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Analysis of the PIM composition of the Rv2611c mutant of M. smegmatis
The PIM composition of the M. smegmatis wild-type and MYC1573 strains was analyzed
during exponential growth phase by metabolic labeling with myo-[2-3H]inositol and [1,2-
14C]acetic acid and by MALDI-MS in the negative ion mode. Both approaches revealed
comparable changes in PIM profiles. MYC1573 contained more PIM2 (m/z = 1175.6 for PIM2
with C16/C19) and PIM1 (m/z = 1013.6 for PIM1 with C16/C19), and less acylated form of PIM2
(Ac1PIM2) (m/z = 1413.8 for Ac1PIM2 with 2C16/C19) relative to PI than the wild-type strain
(Fig. 2). Complementation of the MYC1573 mutant with the pVVacylT vector carrying a
wild-type copy of the M. tuberculosis Rv2611c gene restored a wild-type PIM profile (Fig. 2).
Quantitative differences between the phosphatidylinositol mono- and di-mannosides
compositions of wild-type mc2155 and MYC1573 tended to become less obvious with the age
of the cultures, and totally disappeared in cells grown to late log- or stationary phase. At these
late time points, the mutant no longer accumulated PIM1 and PIM2, and produced wild-type
levels of Ac1PIM2 (data not shown). The accumulation of PIM1 and PIM2 and the decreased
amounts of acylated PIM2 in MYC1573 suggested that Rv2611c is involved in the transfer of
an additional acyl group, i.e. palmitic or tuberculostearic acyl chains, onto PIM1 or PIM2.
To determine whether MYC1573 produced different Ac1PIM2 isomers to the wild-type strain,
the Ac1PIM2 produced by the two strains were purified and characterized. The location of the
fatty acids was deduced by NMR studies and the nature of the fatty acyl chains substituting
the different acyl sites was determined by ESI-mass spectrometry analyses of the Ac1PIM2
acetolysis products (16). 2D 1H-31P HMQC-HOHAHA of Ac1PIM2 from both bacterial strains
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exhibited correlations of the phosphorus with the H2 of Gro at 5.04 ppm and with the H3 of
myo-Ins at 3.23 ppm. Thus, the glycerol moiety was diacylated and position 3 of the inositol
residue was not substituted by any fatty acyl chain (Fig. 3A). Moreover, H6/H6’ of the Manp
unit at the 2-position of myo-Ins at 4.14/4.03 ppm and C6 at 63.6 ppm deduced from the 1H-
13C HMQC data proved that this position is acylated (data not shown). The nature of the fatty
acids esterifying the different sites was investigated by mass spectrometry using ESI-MS
analysis of the acetolysis reaction products of Ac1PIM2 in the negative and positive modes as
previously described (16). The positive ESI-MS spectrum showed an intense peak at m/z
675.6, assigned to a sodium adduct of the di-acylated C16/C19 Gro (Fig. 3B). The negative
mass spectrum showed one main peak at m/z 1283.5, corresponding to the (M-H)- of the
Man2-Ins-P moiety acylated with one C16 (Fig. 3C).
Therefore, both strains produced the same Ac1PIM2 isomer, indicating that compensating
enzymatic activities probably exist in M. smegmatis that account for the production of the
wild-type Ac1PIM2 in MYC1573.
Similar analyses on the PIM2 that accumulated in MYC1573 indicated that the glycerol
moiety of this molecule is diacylated with C16/C19, and revealed no fatty acyl substituents on
the inositol or mannose residues (data not shown).
Rv2611c stimulates the production of acylated forms of phosphatidylinositol mono- and di-
mannosides in cell-free assays
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Cell-free assays in the presence of [14C]GDP-Man and [1-14C]-palmitic acid as
substrates
Based on our previous findings that a full spectrum of PIM species is synthesized in cell-free
assays containing mycobacterial enzyme fractions and [14C]GDP-Man (3), we examined the
acyltransferase mutant (MYC1573) and overproducing strain (mc2155/pVVacylT) along with
the wild-type M. smegmatis mc2155 in similar conditions, relying on the endogenous source
for transferable acyl moieties. The whole cell lysates of the studied strains were used as an
enzyme source to ensure that all of the cofactors that may be important for the reaction were
present. The radiolabeled lipid profiles of the individual strains were clearly different, mainly
revealing changes in the amounts of Ac1PIM1 and PIM1 (Fig. 4A). MYC1573 contained less
Ac1PIM1 and more PIM1 than the wild-type strain, suggesting that the activity of the
acyltransferase responsible for the formation of Ac1PIM1 from PIM1 was impaired in the
mutant. Conversely, the strain overproducing Rv2611c synthesized more Ac1PIM1 and
contained less PIM1 than the wild-type strain. The accumulation of PIM2 in the mutant strain
(Fig. 4A) suggested that PIM1 is mannosylated rather than acylated in this situation where the
acyltransferase activity is disturbed. PIM2 can then be acylated to form Ac1PIM2, which is a
major PIM species in mycobacteria. Thus, alternate pathways for the production of Ac1PIM2
probably co-exist (Fig. 1).
Given the nature of the fatty acid substituting the mannose residue at position 2 of myo-Ins
(Fig. 3) in Ac1PIM2, the third acyl group to be transferred onto PIM1 (or PIM2) is probably
palmitate. Cell-free assays were thus performed with [1-14C]palmitic acid as the radioactive
substrate and with membranes and cell wall-membrane (P60) fractions as the enzyme sources.
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The products formed in these reactions were similar to those formed when [14C]GDP-Man
was used as the radiolabeled substrate. Disruption of Rv2611c resulted in the decreased
synthesis of Ac1PIM1, whereas the overproduction of Rv2611c stimulated the synthesis of this
lipid (Fig. 4B). Moreover, the amount of PIM1 was slightly reduced in the strain
overexpressing the acyltransferase gene. A dramatic increase in Ac1PIM2 production was also
observed in this strain, probably resulting from the increased synthesis of its direct precursor,
Ac1PIM1. Taken together, these data suggest that Rv2611c is an acyltransferase involved in
the palmitoylation of PIM1 and PIM2.
Substrate specificity of the acyltransferase Rv2611c
To study the substrate specificity of Rv2611c, radiolabeled putative lipid acceptors for the
acyltransferase reaction ([14C]PIM1 and [14C]PIM2) were prepared and used in cell-free assays
containing membrane and cell wall–membrane (P60) fractions from M. smegmatis mc2155,
MYC1573 and mc2155/pVVacylT. In all three strains, [14C]PIM1 was converted to
[14C]Ac1PIM1. However, the amount of product was lower in the mutant and significantly
higher in the overproducing strain compared to in the wild-type (Fig. 5A), suggesting that
PIM1 is a true substrate of Rv2611c. The small amount of [14C]Ac1PIM1 produced in the
mutant strain suggests that compensating activities that account for the acylation of [14C]PIM1
exist in M. smegmatis.
Wild-type M. smegmatis mc2155, MYC1573 and mc2155/pVVacylT were also tested for their
ability to acylate [14C]PIM2. The wild-type and the overproducing strains produced
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[14C]Ac1PIM2 and, to a lesser extent, [14C]Ac2PIM2 (Fig. 5B). Slightly more [14C]Ac1PIM2 was
produced in the latter strain, which also consumed the [14C]PIM2 substrate more efficiently.
The considerable accumulation of PIM2 in MYC1573 (Fig. 2) significantly reduced the
specific activity of the radiolabeled substrate meaning that the amounts of products formed in
the reaction could not be compared to those formed when using mc2155 and
mc2155/pVVacylT extracts. However, [14C]Ac1PIM2 was produced at detectable levels in
MYC1573 (data not shown). These results indicated that Rv2611c can catalyze the acylation
of PIM2, and that other acyltransferase activities probably exist in M. smegmatis that account
for the formation of [14C]Ac1PIM2 from [14C]PIM2 in MYC1573. Therefore, Rv2611c can
acylate both PIM1 and PIM2. Given that differences between strains were more marked when
[14C]PIM1 was used as the substrate, this PIM species is probably the preferred lipid acceptor
of Rv2611c. When purified [14C]Ac1PIM2 was used as the substrate, small amounts of
[14C]Ac2PIM2 were produced, and no significant differences were observed between the three
strains (data not shown). Thus, Ac1PIM2 does not appear to be a potent substrate of Rv2611c.
The acyltransferase reaction catalyzed by Rv2611c was further examined in cell-free assays in
which the substrate of the reaction, [14C]PIM1, was formed in situ by including [14C]GDP-Man
and E. coli extracts overproducing the recombinant mannosyltransferase PimA (3) into the
reaction mixtures. The conversion of [14C]PIM1 into [14C]Ac1PIM1 and [14C]Ac1PIM2 by
enzyme fractions of M. smegmatis mc2155, MYC1573 and mc2155/pVVacylT was monitored
after different incubation times. The amount of radiolabeled PIM species produced by the
three individual strains over time was strikingly different (Fig. 6). [14C]PIM1 was rapidly
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synthesized in all three strains (due to the presence of the recombinant PimA protein),
however, it was gradually consumed only in the wild-type and overproducing strains (Fig.
6A). The overall amount of [14C]PIM1 in the overproducing strain was lower because it was
rapidly converted into Ac1PIM1 (Fig. 6B). After 30 minutes, the mutant produced virtually no
[14C]Ac1PIM1, whereas at the later time point (3 hr), this product accounted for almost 60% of
the wild-type production, which confirms the presence of compensatory acyltransferase
activities. The production of [14C]Ac1PIM2 correlates with that of [14C]Ac1PIM1; the greatest
amounts of [14C]Ac1PIM2 were produced in the overproducing strain, and [14C]Ac1PIM2 was
produced more slowly and at later time points in MYC1573 (Fig. 6C). These data provide
additional evidence for the direct involvement of Rv2611c in the acylation of [14C]PIM1.
Finally, the conversion of [14C]PIM1 into [14C]Ac1PIM1 was also observed when recombinant
Rv2611c enzyme partially purified from mc2155/pVVacylT instead of M. smegmatis
membranes was added to the E. coli extracts overproducing PimA (Fig 7). This conversion
was strongly stimulated by the addition of palmitoyl-CoA to the reaction mixture (Fig. 7).
These findings strongly suggest that Rv2611c is the structural gene for the enzyme catalyzing
the acylation of PIM1.
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DISCUSSION
The fatty acyl functions of the GPI anchor of PIM and LAM are crucial for the physical
properties and biological activities of these molecules (12,16,25-29). However, little is known
about the enzymes catalyzing the acylation of PIM, LM and LAM, and none of the genes
encoding such enzymes had been characterized. Brennan and Ballou (20,33) detected
acylating activities in the membrane of M. phlei responsible for the formation of Ac1PIM2 and
Ac2PIM2 from acyl-CoA derivatives of fatty acids (myristic, palmitic, stearic, tuberculostearic
and oleic acids) and PIM2 or Ac1PIM2 as the glycolipid acceptors. These observations,
together with previous results published by the same group on the mannosylation of PI (30),
led to the proposal of two models for the synthesis of phosphatidylinositol di-mannosides in
mycobacteria (20,33) (Fig. 1).
Our results provide evidence that Rv2611c is the acyltransferase that catalyzes the acylation
of the 6-position of the Man residue linked to position 2 of myo-Ins in PIM1 and PIM2.
Although this study was performed in M. smegmatis, the fact that over-expression of the M.
tuberculosis Rv2611c gene stimulated the production of Ac1PIM1 and Ac1PIM2 in cell-free
assays and restored a normal PIM composition in MYC1573 strongly suggests that the
enzyme responsible for the acylation of PIM1 and PIM2 is conserved among mycobacterial
species. Palmitic acid was a potent fatty acid substrate of the acyltransferase in our assays.
Given the occurrence of tuberculostearic acid on the same Man residue of PIM and LAM in
M. bovis BCG (16,23), it is likely that Rv2611c also catalyzes the transfer of this fatty acid. In
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all our assays, Ac1PIM1 was the product the synthesis of which was the most decreased in the
M. smegmatis Rv2611c mutant. The synthesis of Ac1PIM2 was also reduced, but to a lesser
extent. Conversely, the overexpression of the M. tuberculosis Rv2611c gene in M. smegmatis
primarily stimulated the synthesis of Ac1PIM1 from PIM1 and palmitate from both
endogenous and exogenous sources. Taken together, these data strongly suggest that PIM1 is
the main lipid acceptor in the reaction catalyzed by Rv2611c. Interestingly, the
overexpression of Rv2611c also stimulated the acylation of purified radiolabeled PIM2 but not
that of Ac1PIM2 in cell-free assays. The fact that Rv2611c can acylate PIM1 and PIM2 but not
acylated PIM2 (Ac1PIM2) suggests that the specificity of this enzyme is essentially directed
towards the acylation state of its substrates, not towards their degree of mannosylation.
The ability of the MYC1573 mutant to synthesize wild-type Ac1PIM2 from endogenous fatty
acids when PIM1 or PIM2 act as the lipid acceptors in cell-free assays indicates that M.
smegmatis has other acyltransferases that compensate partially for the loss of Rv2611c
activity. More importantly, these results and the fact that PIM2 accumulates in MYC1573
cells prove that the mannosylation of phosphatidylinositol mono-mannosides in vivo can
occur directly on PIM1 without the formation of an acylated PIM1 intermediate (Ac1PIM1).
Therefore, the two phosphatidylinositol di-mannosides pathways originally proposed by
Brennan and Ballou (20,33) appear to co-exist in mycobacteria (Fig.1). Given the major
changes in the PIM composition of MYC1573 and the severe growth defects exhibited by this
mutant, the compensating acyltransferase activities in the absence of Rv2611c are clearly not
sufficient for the optimal synthesis of Ac1PIM2 and its derivatives. These other
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acyltransferases may be insufficiently produced in M. smegmatis or have very low affinities
for PIM2 and PIM1 substrates. A computer search for acyltransferases in the genome of M.
tuberculosis H37Rv (37) revealed the existence of several putative enzymes, some of which
share (like Rv2611c) sequence similarities with LPS biosynthesis acyltransferases (Rv0111,
Rv1565c) or with bacterial glycerol 3-phosphate acyltransferases (PlsB1, PlsB2, Rv2483c,
Rv3026c, Rv2182c, Rv3814c, Rv3815c, Rv3816c). However, none of them shares significant
sequence similarity with Rv2611c. The existence of two pathways for the synthesis of
Ac1PIM2 in mycobacteria is also consistent with the finding that PimB, the
mannosyltransferase that catalyzes the formation of Ac1PIM2 from Ac1PIM1 and GDP-Man
(32), is not essential for the growth of M. tuberculosis (38) and M. smegmatis (M. Schaeffer,
J. Inamine, personal communication). In the absence of PimB, mycobacteria probably
mannosylate PIM1 to PIM2, which in turn is acylated by Rv2611c or other acyltransferases to
yield Ac1PIM2 (Fig. 1).
The M. smegmatis Rv2611c mutant could only be grown in the presence of a high
concentration of NaCl (15 g/l) and in the absence of Tween 80. Such a requirement for NaCl
has been described in phosphatidylserine (PS)/ phosphatidylethanolamine (PE)-deficient
mutants of E. coli. The addition of certain cations (such as Na+, K+, NH4+, Mg2+) to the culture
medium restored the normal growth of these otherwise lethal mutants, without correcting the
defect in PS/PE synthesis (39). By analogy with what was proposed for the E. coli mutants,
sodium salts may assert their remedial effects on MYC1573 by protecting its weakened
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membrane by osmotic pressure, or by neutralizing the surface charges of its membrane to
enforce the hydrophobic association of its membrane components. This phenotypic
suppression by NaCl suggests that the physiological roles of phosphatidylinositol mono- and
di-mannosides in the cell envelope are primarily structural. Consequently, the disruption of
Rv2611c dramatically alters the permeability of the cell envelope of M. smegmatis, rendering
MYC1573 highly sensitive to Tween 80, even in the presence of high concentrations of NaCl.
In light of the present results, it is now clear that a polar effect in the expression of Rv2611c
was responsible for the growth defect of the single cross-over strain mc2CSU01 used to
construct a pgsA (Rv2612c) conditional mutant of M. smegmatis (2). Previous data suggested
that Rv2613c (an ORF of unknown function) and Rv2612c (p g s A , encoding a
phosphatidylinositol synthase) were co-transcribed (2). It now seems likely that Rv2611c
belongs to the same transcriptional unit. Given the demonstrated acyltransferase activity of
Rv2611c, we re-examined the PIM composition of the single cross-over strain mc2CSU01 by
MALDI-MS in the negative ion mode (data not shown). Consistent with the data presented
herein, these analyses confirmed that the compound that was the most dramatically reduced in
mc2CSU01 was Ac1PIM2 (m/z = 1413.8 for Ac1PIM2 with 2C16/C19), not lyso-PIM2 as
previously reported (2). mc2CSU01 also displayed decreased levels of Ac2PIM2.
In conclusion, the involvement of the Rv2611c acyltransferase in key steps of PIM, LM and
LAM biosynthesis and its importance for mycobacterial growth make it an attractive drug
target for the development of novel anti-tuberculosis drugs. The availability of Rv2611c
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mutants of M. tuberculosis and M. bovis BCG exhibiting reduced rates of acylation of their
PIM, LM and LAM could also be useful for investigations of the biological functions
associated with the acyl forms of these molecules in cellular or animal models.
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Acknowledgments
J.K. was the recipient of a Marie Curie Fellowship (CT-2000-00058) from the European
Economic Community. This work was supported in part by the Slovak Republic/ USA Joint
Research Grant 032/2001 from APVT Slovakia. J. K. and K. M. acknowledge Prof. Marta
Kollarova for overall support.
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1, pp. 203-298, Academic Press Ltd., London
35. Snapper, S. B., Melton, R. E., Mustafa, S., Kieser, T., and Jacobs, W. R. (1990) Mol.
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37. Cole, S. T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S.
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FIGURE LEGENDS
Figure 1: Proposed pathway for the early steps of PIM synthesis in mycobacteria.
CDP-DAG, CDP-diacylglycerol; (R1, R2) = (C19, C16) fatty acyl groups; R3 = C16/C19, R4 =
C16/C18/C19 fatty acyl groups. (PIM3-PIM6), LM and LAM are extended forms of
phosphatidylinositol di-mannosides in which Man residues are attached to the Man linked to
position 6 of myo-Ins.
Figure 2: Effect of disrupting the Rv2611c gene on the PIM composition of M. smegmatis.
MALDI-MS analysis of the total lipids from wild-type mc2155, the Rv2611c mutant of M.
smegmatis (MYC1573) and the complemented mutant strain MYC1573/pVVacylT. Lipids
from exponentially growing cells were extracted and subjected to MALDI-MS analysis in the
negative ion mode. The peaks observed are m/z 851.6, PI with C16/C19 ; m/z 1013.6, PIM1 with
C16/C19; m/z 1175.6, PIM2 with C16/C19 ; m/z 1413.8, Ac1PIM2 with 2C16/C19 ; m/z 1652.1 and
1694.2, Ac2PIM2 with 3C16/C19 and 2C16/2C19, respectively ; m/z 2062.1, Ac1PIM6 with
2C16/C19 ; m/z 2462.4 and 2504.4, Ac2PIM6 with 3C16/C19 and 2C16/2C19, respectively.
Figure 3: Structural characterization of the Ac1PIM2 produced by wild-type M. smegmatis and
the MYC1573 mutant.
A) NMR analysis of the Ac1PIM2 phosphate substituents in CDCl3/CD3OD/D2O (60:35:8,
v/v/v) at 308 K. Expanded region (a) (δ 1H: 2.90 - 5.40) of the 1H 1D spectrum. Expanded
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region (b) (δ 1H: 2.90 - 5.40 and 31P: 0.50 - 3.50) of the 1H-31P 55 ms HMQC-HOHAHA
spectrum. Numerals with Ins correspond to the proton number of the myo-Ins unit and
numerals with Gro, to the proton number of the glycerol unit.
B) Positive ESI mass spectra of Ac1PIM2 acetolysis products
The peak at m/z 675.6 corresponds to a sodium adduct (M+Na)+ of the di-acylated C16/C19
Gro.
C) Negative ESI mass spectra of Ac1PIM2 acetolysis products.
The peak at m/z 1241.5 corresponds to the (M-H)- of the Man2-Ins-P moiety acylated with one
C16, with no acetate on the phosphate, whereas the peak at m/z 1283.5 corresponds to the (M-
H)- of the Man2-Ins-P moiety acylated with one C16, with one acetate on the phosphate.
Figure 4: Cell-free assays using [14C]GDP-Man and [1-14C]-palmitic acid and enzyme
fractions from M. smegmatis mc2155, MYC1573 and mc2155/pVVacylT
TLC autoradiograph of lipids derived from [14C]GDP-Man (A) or [1-14C]palmitic acid (B). In
vitro enzyme assays were performed as described under Experimental Procedures. One fifth
of the lipids extracted from each reaction were applied to TLC plates and developed in
CHCl3/CH3OH/NH4OH/H2O (65:25:0.5:4). x = 1,2.
Figure 5: Cell-free assays using [14C]-labeled lipid acceptors and enzyme fractions from M.
smegmatis mc2155, MYC1573 and mc2155/pVVacylT
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[14C]PIM1 purified from cell-free reactions and [14C]PIM2 purified from [14C]acetic acid-
labeled MYC1573 were used as the lipid acceptors in the assays. The whole lipid samples
from each reaction were applied to TLC plates and developed in CHCl3/CH3OH/NH4OH/H2O
(65:25:0.5:4).
Figure 6: Enzyme assays with in situ formed [14C]PIM1 and membrane fractions from M.
smegmatis
Reactions were carried out as described under Experimental Procedures using membrane
fractions from M. smegmatis mc2155 (solid line, diamonds), MYC1573 (dotted line, squares)
and mc2155/pVVacylT (dashed line, triangles) supplemented with crude extracts from E. coli
BL21/pETpimA and [14C]GDP-Man as the radioactive label. Lipids were extracted from the
reaction mixtures at the indicated time points, applied to TLC plates and developed in
CHCl3/CH3OH/NH4OH/H2O (65:25:0.5:4). Following autoradiography, bands corresponding
to PIM1, Ac1PIM1 and Ac1PIM2 were scraped off the plates and quantified by scintillation
counting.
Figure 7: Enzyme assays with in situ formed [14C]PIM1 and partially purified recombinant
Rv2611c
Reactions were carried out as described under Experimental Procedures using recombinant
Rv2611c partially purified from M. smegmatis mc2155/pVVacylT supplemented with crude
extracts from E. coli BL21/pETpimA and [14C]GDP-Man as the radioactive label. Lipids were
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extracted from the reaction mixtures, applied to TLC plates and developed in
CHCl3/CH3OH/NH4OH/H2O (65:25:0.5:4). Lane 1, recombinant Rv2611c protein was
omitted from the reaction mixture; Lane 2, standard assay (recombinant Rv2611c protein and
cold palmitoyl-CoA were added to the reaction mixture); Lane 3, palmitoyl-CoA was omitted
from the reaction mixture.
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Figure 1 :
OH
O
OR2
OR1
O
HO
HOHO
OH
O
OHOHO
OH
P
O O
OHO
HOHO
OH
O
O
OR2
OR1
O
HO
HOHO
OH
O
OHOHO
OH
P
O O
PI
PimA
OHO
HOHO
OH
O
O
OR2
OR1
O
R3O
HOHO
OH
O
OHOHO
OH
P
O O
OH
O
OR2
OR1
OH
OHOHO
OH
P
O O
OH
O
OR2
OR1
O
R3O
HOHO
OH
O
OHOHO
OH
P
O O
OHO
HOHO
OH
O
O
OR2
OR1
O
R3O
HOHO
OH
O
OHOHO
R4O
P
O O
PIM2
Ac1PIM2
Ac2PIM2
Higher forms of PIM, LM and LAM
PIM1
Ac1PIM1
Rv2611c ?Mannosyltransferase
Rv2611c ?
PimB
CDP-DAG + myo-InsPgsA
Acyltransferase
Acyltransferase
Acyltransferase by guest on August 11, 2020
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800 1 120 14 40 1760 2080 2400
M ass (m/z )
0
6.5E+4
0
10
20
30
40
50
60
70
80
90
100
% In tensity
Voyag er Spec #1=>NF0.7=>BC[BP = 850.9, 64635]
80
40
20
60
100
Inte
nsity
(%
)
1120 1440 1760 2080 2400800
851.6PI
1175.6
PIM2
1413.8Ac1PIM2
1652.1 1694.2
Ac2PIM2 2062.1
Ac1PIM6
2504.42462.4
Ac2PIM6
Mass (m/z)
800 1120 1440 1760 2080 2400
Mass (m/z)
0
6.4E+4
0
10
20
30
40
50
60
70
80
90
100
% Intensity
Voy ager Spec #1=>NF0.7=>BC[BP = 851.0, 63677]
80
40
20
60
100
Inte
nsity
(%
)
1120 1440 1760 2080 2400800Mass (m/z)
851.6PI
1175.6
PIM21413.8Ac1PIM2
1652.1 1694.2
Ac2PIM2
2062.1
Ac1PIM62504.4
2462.4
Ac2PIM6
1013.6
PIM1
800 1120 1440 1760 2080 2400
Mass (m/z)
0
4.2E+4
0
10
20
30
40
50
60
70
80
90
1 00
% I ntensity
Vo yager Spec #1=>NF0.7=>BC[BP = 851.1, 41581]
80
40
20
60
100
Inte
nsity
(%
)
1120 1440 1760 2080 2400800Mass (m/z)
851.6PI
1175.6
PIM2
1413.8Ac1PIM2
1652.1 1694.2
Ac2PIM2
2062.1
Ac1PIM6
2504.42462.4
Ac2PIM6
mc2155
MYC1573
MYC1573/pVVacylT
Figure 2:
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ppm
3.03.13.23.33.43.53.63.73.83.94.04.14.24.34.44.54.64.74.84.95.05.15.25.3 ppm
1.0
1.5
2.0
2.5
3.0
3.5
Ins-2 Ins-6Ins-1Ins-4
Ins-3
Ins-5
Gro-1Gro-2 Gro-3Gro-1'
MG1 # 515-575 RT: 12,71-13,80 AV: 61 NL: 3,44E5T: + c Full ms [ 150,00-2000,00]
300 400 500 600 700 800 900 1000
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
MG1 #443-458 RT: 11,31-11,66 AV: 16 NL: 6,62E6T: - c Full ms [ 150,00-2000,00]
1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
Mass (m/z)400 600 800 1000
Mass (m/z)1000 1200 1400 16001100 1300 1500
675.6
1283.5
1241.5
500 700 900
80
40
20
60
100
Inte
nsity
(%
)
80
40
20
60
100
Inte
nsity
(%
)
300
A
B
C
Figure 3:
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Ac1PIM1
Polyprenyl-P-Man
Ac1PIM2PIM1PIM2
Ac1PIM1Ac2PIM2PIAc1PIM2
mc2 15
5
MY
C15
73
pVV
acyl
T
AcxPIM3-6
mc2 15
5
MY
C15
73
PIM1
pVV
acyl
T
A. B.Figure 4:
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Ac1PIM1
Ac1PIM2
PIM1
Ac2PIM2
Ac1PIM2
PI
PIM2m
c2 155
pVV
acyl
T
mc2 15
5
MY
C15
73
pVV
acyl
T
A. B.Figure 5 :
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0
500
1000
1500
2000
2500
3000
3500
0 50 100 150 200
0
500
1000
1500
2000
2500
3000
3500
0 50 100 150 200
0
100
200
300
400
500
600
0 50 100 150 200
A. PIM1
B. Ac1PIM1
C. Ac1PIM2
Time (min)
Time (min)
Time (min)
dpm
dpm
dpm
Figure 6 :
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PIM1
Ac1PIM1
1 2 3
Figure 7:
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Katarina Mikusova and Mary JacksonJana Kordulakova, Martine Gilleron, Germain Puzo, Patrick J. Brennan, Brigitte Gicquel,
phosphatidylinositol mannosides of Mycobacterium speciesIdentification of the required acyltransferase step in the biosynthesis of the
published online July 8, 2003 originally published online July 8, 2003J. Biol. Chem.
10.1074/jbc.M303639200Access the most updated version of this article at doi:
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