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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: mjackson@pasteur.fr.

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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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(1997) Proc. Natl. Acad. Sci. USA 94, 10955-10960

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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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