New Insights into the Early Steps of Phosphatidylinositol Mannoside Biosynthesis in Mycobacteria

Phosphatidyl-myo-inositol mannosides (PIMs) are key glycolipids of the mycobacterial cell envelope. They are considered not only essential structural components of the cell but also important molecules implicated in host-pathogen interactions. Although their chemical structures are well established, knowledge of the enzymes and sequential events leading to their biosynthesis is still incomplete. Here we show for the first time that although both mannosyltransferases PimA and PimB′ (MSMEG_4253) recognize phosphatidyl-myo-inositol (PI) as a lipid acceptor, PimA specifically catalyzes the transfer of a Manp residue to the 2-position of the myo-inositol ring of PI, whereas PimB′ exclusively transfers to the 6-position. Moreover, whereas PimB′ can catalyze the transfer of a Manp residue onto the PI-monomannoside (PIM1) product of PimA, PimA is unable in vitro to transfer Manp onto the PIM1 product of PimB′. Further assays using membranes from Mycobacterium smegmatis and purified PimA and PimB′ indicated that the acylation of the Manp residue transferred by PimA preferentially occurs after the second Manp residue has been added by PimB′. Importantly, genetic evidence is provided that pimB′ is an essential gene of M. smegmatis. Altogether, our results support a model wherein Ac1PIM2, a major form of PIMs produced by mycobacteria, arises from the consecutive action of PimA, followed by PimB′, and finally the acyltransferase MSMEG_2934. The essentiality of these three enzymes emphasizes the interest of novel anti-tuberculosis drugs targeting the initial steps of PIM biosynthesis.

PIMs 3 are unique mannolipids found in abundant quantities in the inner and outer membranes of the cell envelope of Mycobacterium spp. and a few other actinomycetes. 4 They are based on a phosphatidyl-myo-inositol (PI) lipid anchor carrying one to six Manp residues and up to four acyl chains (for review see Refs. 1,2). Based on a conserved mannosyl-PI anchor, they are also thought to be the precursors of the two major mycobacterial lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM) (1,2). PIMs, LM, and LAM are considered not only essential structural components of the mycobacterial cell envelope (3)(4)(5)(6), but also important molecules implicated in host-pathogen interactions in the course of tuberculosis and leprosy (1).
Although the chemical structure of PIMs is now well established, knowledge of the enzymes and sequential events leading to their biosynthesis is still fragmentary. According to the currently accepted model, the biosynthetic pathway is initiated by the transfer of two Manp residues and a fatty acyl chain to PI in the cytoplasmic leaflet of the plasma membrane. Based on genetic and biochemical evidence, Korduláková et al. (5) identified PimA (MSMEG_2935 in Mycobacterium smegmatis mc 2 155) as the enzyme that catalyzes the first mannosylation step of the pathway transferring a Manp residue most likely to the 2-position of the myo-inositol (myo-Ins) ring of PI. In contrast, the identity of PimBЈ, the enzyme responsible for the transfer of the second Manp to the 6-position of the myo-Ins ring of PIM 1 , still remains controversial. The Rv0557 protein of Mycobacterium tuberculosis H37Rv (PimB; MSMEG_1113 in M. smegmatis mc 2 155) was originally characterized as PimBЈ (7). However, the lack of an Rv0557 ortholog in the genome of Mycobacterium leprae and the fact that the disruption of this gene in M. tuberculosis Erdman did not significantly affect the biosynthesis of PIMs suggest that compensatory activities exist in the bacterium or that Rv0557 serves another primary function (8,9). Somewhat supporting the latter hypothesis, the ortholog of Rv0557 in Corynebacterium glutamicum (NCgl0452, renamed mgtA) was implicated in the mannosylation of a novel glycolipid (1,2-di-O-C 16 /C 18:1 -(␣-D-mannosyl)-(134)-(␣-D-glucopyranosyluronic acid)-(133)-glycerol), and Rv0557 from M. tuberculosis was reported to functionally complement for this enzyme in a C. glutamicum knock-out mutant (10). However, to our knowledge this mannosylated glycolipid has never been reported in mycobacteria, and it remains unclear whether PimB serves a similar physiological function in Mycobacterium spp.
More recently, Lea-Smith et al. (11) have shown that the biosynthesis of Ac 1 PIM 2 from Ac 1 PIM 1 in C. glutamicum is catalyzed by NCgl2106 (Cg-PimBЈ). Disruption of the NCgl2106 gene totally abolished Ac 1 PIM 2 production in the mutant, arguing against the existence of a compensatory activity associated with the corynebacterial PimB enzyme. Although Ac 1 PIM 2 production in Cg-pimBЈ and Cg-pimBЈ/Cg-pimB knock-out mutants was restored upon complementation with the M. tuberculosis Rv2188c gene (11,12), direct evidence that Rv2188c carried out the same physiological function in mycobacteria has been lacking. Moreover, in light of the recent work by Torrelles et al. (9) showing an involvement of pimB (Rv0557) in the synthesis of LM and LAM in M. tuberculosis Erdman and of the demonstrated relaxed substrate specificity of the M. tuberculosis PimB (Rv0557) and PimBЈ (Rv2188c) enzymes expressed in C. glutamicum (12), whether or not pimB and pimBЈ could compensate for one another in mycobacteria remained open to speculation.
Both PIM 1 and PIM 2 can be acylated with palmitate at position 6 of the Manp residue transferred by PimA by the acyltransferase MSMEG_2934 (orthologous to Rv2611c from M. tb) to form Ac 1 PIM 1 and Ac 1 PIM 2 , respectively (13). Ac 1 PIM 2 can further be acylated at position 3 of the myo-Ins ring by an as yet unidentified acyltransferase to yield Ac 2 PIM 2 . Importantly, Ac 1 PIM 2 and Ac 2 PIM 2 are among the most abundant forms of PIMs found in mycobacteria and are considered both metabolic end products and intermediates in the biosynthesis of more polar forms of PIMs (PIM 5 and PIM 6 ), LM, and LAM.
In this work, clear evidence is provided that PimBЈ (MSMEG_4253 in M. smegmatis mc 2 155) is the ␣-ManT responsible for the biosynthesis of PIM 2 from PIM 1 in mycobacteria and that no other ManT can compensate for a deficiency in this enzyme in M. smegmatis. Like PimA (5), PimBЈ is essential to the growth of M. smegmatis. Cell-free assays using purified PimA and PimBЈ and M. smegmatis membrane preparations provide new insights into the sequential events leading to the synthesis of the early forms of PIMs in mycobacteria.
E. coli BL21(DE3)pLysS cells transformed with pET29a-MspimBЈ were grown in 2ϫ YT medium supplemented with 25 g ml Ϫ1 kanamycin and 34 g ml Ϫ1 chloramphenicol at 37°C. MspimBЈ expression was induced by adding 0.5 mM isopropyl 1-thio-␤-D-galactopyranoside (MP Biomedicals). After 4 h at 37°C, cells were harvested and resuspended in solution A (50 mM Tris-HCl, pH 8.0) containing protease inhibitors (Complete EDTA-free, Roche Applied Science). Cells were disrupted by sonication (five cycles of 1 min), and the suspension was centrifuged for 20 min at 10,000 ϫ g. The supernatant was applied to a HisTrap chelating column (1 ml; GE Healthcare) equilibrated with solution B (50 mM Tris-HCl, pH 8.0, 500 mM NaCl). The column was then washed with solution B until no absorbance at 280 nm was detected. Elution was performed with a linear gradient of 0 -500 mM imidazole in solution B at 1 ml min Ϫ1 . The resulting preparation displayed a single protein band when run on a 10% SDS-polyacrylamide gel stained with Coomassie Brilliant Blue (supplemental Fig. 1S). The purified enzyme was concentrated to 10 mg ml Ϫ1 in solution A containing 20% glycerol and stored at Ϫ80°C until further use in enzyme assays.
Enzyme Assays-The enzymatic activity of MsPimA and MsPimBЈ was monitored using a radiometric assay. In some assays, membrane preparations from M. smegmatis mc 2 155 (0.5 mg of proteins) served as the source of lipid acceptors. Reactions were incubated for 2 h at 37°C and stopped with 1.5 ml of CHCl 3 /CH 3 OH (2:1, by volume). The PIM-containing organic phase was prepared and analyzed by TLC as described by Korduláková et al. (5). MsPimA was purified as described previously (14).
For structural analyses, 500 M cold GDP-Man replaced GDP- [C 14 ]Man in the assay mixture described above. The reactions were incubated overnight at 37°C and stopped by adding 1.5 ml of CHCl 3 /CH 3 OH (2:1, by volume). The nonradioactive mannolipid products from 15 reactions were isolated by preparative TLC as described (5).
Matrix-assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry-Compounds 1-5 were mixed with an equal volume of matrix (2,5-dihydroxylbenzoic acid dissolved in 10 mg ml Ϫ1 acetonitrile/water, 50:50, 0.1% trifluoroacetic acid), and the molecular mass was measured in the negative ion mode by MALDI-TOF MS on a Bruker Ultraflex TOF/TOF mass spectrometer (Bruker Daltonics, Billerica, MA). External calibration was performed using an eight component calibration mixture on a spot adjacent to the sample.
NMR Analysis-One-dimensional and two-dimensional NMR experiments were carried out at 25°C in a Varian Inova 500-MHz NMR spectrometer (Varian Inc., Palo Alto, CA) using an HCN probe head equipped with shielded z-gradient. Samples were dissolved in 0.6 ml of CHCl 3 /CD 3 OD (2:1, by volume) and spectra acquired using a 5-mm NMR probe. Typical parameters used for one-dimensional 1 H experiments were as follows: sweep width, 5500Hz; flip angle, 45°; time domain data points, 32,768; number of transients, 32 or 256; and relaxation delay, 1.5 s. For the complete structural analysis of PIM 1 , PIM 2 , and Ac 1 PIM 2 , two-dimensional experiments, including gradient-selected correlation spectroscopy, total correlation spectroscopy, heteronuclear single quantum coherence spectroscopy, and heteronuclear multiple bond correlation spectroscopy were carried out. Parameters used for two-dimensional correlation spectroscopy and total correlation spectroscopy were as follows: sweep width, 5500 Hz in both F2 and F1 dimensions; time domain data points, 2048; number of free induction decay with t 1 increment, 512; number of transients, 32 or 256; and relaxation delay, 1.5s. Parameters used for heteronuclear single quantum coherence and heteronuclear single quantum coherence spectroscopy were as follows: sweep width, 5500 Hz in F2 and 30,188 Hz in F1; time domain data points, 2048; number of free induction decays with t 1 increment, 256; number of transients, 32 or 256; and relaxation delay, 1.5 s. The acquired NMR data were processed using the TOPSPIN 2.1 software (Bruker GmbH, Karlsruhe, Germany).
Construction of M. smegmatis MspimBЈ Conditional Mutant-Essentially the same strategy was used to construct a conditional MspimBЈ mutant of M. smegmatis as was used earlier to generate an MspimA conditional mutant (5). The M. smegmatis MspimBЈ gene (MSMEG_4253) and flanking regions were amplified from genomic M. smegmatis mc 2 155 DNA by standard PCR strategies using oligonucleotide primers MspimBЈ_KO_ApaI_fwd (5Ј-ATAATGGGCCCGCAAAACT-GCGTGACCTGTACG-3Ј) and MspimBЈ_KO_SpeI_rev (5Ј-ATTATACTAGTGACCTCGGCGCCATCGACG-3Ј), and Phusion DNA polymerase (New England Biolabs). A disrupted allele of MspimBЈ, MspimBЈ::km, was constructed by cloning the kanamycin resistance cassette from pUC4K (GE Healthcare) into the AgeI and StuI sites of MspimBЈ, generating a 363-bp deletion within the coding sequence of MspimBЈ. MspimBЈ::km was then ligated to pJQ200xylE to yield pJQM-spimBЈKX, the vector used to achieve allelic replacement at the MspimBЈ locus (5). The temperature-sensitive pCG76 derivative (15), pCGMspimBЈ, was used as the rescue plasmid to carry a functional copy of the MspimBЈ gene in the conditional mutant.
Homology Modeling of MsPimBЈ-Homology modeling of MsPimBЈ was performed with MODELLER 9 Version 4 (16) using the atomic coordinates of MsPimA complexed with GDP-Man (Protein Data Bank code 2GEJ (17)) as a template. Sequence alignment was carried out manually to match functionally conserved residues, predicted secondary structures, and hydrophobicity profiles. Secondary structures were predicted using the Jpred program (18). The models were assessed by the VERIFY_3D program.

RESULTS AND DISCUSSION
MsPimBЈ Catalyzes in Vitro the Transfer of a Manp Residue to the 6-Position of the Myo-Ins Ring of PI-With the goal of determining the function of the mycobacterial PimBЈ enzyme, a recombinant form of the M. smegmatis protein (MsPimBЈ) with a C-terminal histidine tag was produced in E. coli BL21(DE3)pLysS and purified to near homogeneity (supplemental Fig. 1S). As had been the case with the M. tuberculosis PimA protein earlier (5,14,17), attempts to produce the PimBЈ enzyme from M. tuberculosis yielded relatively small amounts of soluble protein compared with the M. smegmatis version, and these efforts were thus not pursued further.
ManT assays were then run using different combinations of the purified MsPimA and MsPimBЈ enzymes. When commercial liver PI and GDP-[ 14 C]Man served as the acceptor and donor substrates in the assay, purified MsPimA (14) catalyzed the formation of PIM 1 (mannolipid 1, Fig. 1). Unexpectedly, the formation of a 14 C-labeled mannolipid with an R f similar to that of PIM 1 was also observed when purified MsPimBЈ was used as the source of enzyme in the assay (mannolipid 2, Fig. 1). To further characterize mannolipids 1 and 2, nonradioactive products were purified by preparative TLC from reaction mixtures in which cold GDP-Man replaced GDP-[C 14  A combination of one-dimensional and two-dimensional NMR was then used to determine the position at which the Manp residues were attached to myo-Ins in mannolipids 1 and 2 (for details see supplemental material and supplemental Figs. 3S-6S) (19 -21). As depicted in Fig. 2, the 1 H NMR spectra of mannolipid 1 shows one peak at 5.14 ppm assigned to the ␣-anomeric proton of the Manp residue attached to position 2 of myo-Ins. The 1 H and 13 C chemical shift values of mannolipid 3 is exactly comparable with that of mannolipid 1, and therefore compound 3 was also assigned to 2-linked PIM 1 . In the spectra of compound 2, the peak at 5.072 ppm was assigned as the ␣-anomeric proton of the Manp residue attached to position 6 of myo-Ins. The 1 H NMR spectra of mannolipid 4 shows two distinct peaks at 5.129 and 5.046 ppm assigned to ␣-anomeric protons of two Manp residues attached to the 2-and 6-positions of myo-Ins.
For the first time, direct evidence arising from the use of purified enzymes was thus provided that MsPimA catalyzes the transfer of a Manp residue from GDP-Manp to the 2-position of the myo-Ins ring of PI, and MsPimBЈ catalyzes the transfer of a Manp residue to the 6-position.  Fig. 2S). From this experiment, it can thus be concluded that MsPimA and MsPimBЈ are sufficient for the formation of PIM 2 from PI and GDP-Man to occur. The fact that no PIM 3 or more mannosylated products were formed in the reaction even after prolonged incubation times further indicated that MsPimA and MsPimBЈ are unable to mannosylate PIM products beyond PIM 2 . Thus MsPimA and MsPimBЈ appear to each catalyze the transfer of one single Manp residue.
To determine the sequence of the reactions leading to the formation of PIM 2 , two independent assays were carried out in which purified MsPimA and MsPimBЈ were added sequentially to the reaction mixture. In one of the assays, MsPimA was added first to a reaction mixture containing PI and GDP-[C 14 ]Man. After 2 h of incubation, one-half of the reaction was stopped by the addition of CHCl 3 /CH 3 OH (2:1), and the other half was incubated at 60°C for 15 min to inactivate the enzyme. Purified MsPimBЈ was then added to the heat-inactivated assay mixture, and the reaction allowed to proceed overnight at 37°C. In the second assay, MsPimBЈ was added first to the reaction mixture, then inactivated as described above, and MsPimA finally added. That both MsPimA and MsPimBЈ were inactivated by heat treatment was verified by running independent assays with each of the purified enzymes (supplemental Fig. 7S). Consistent with our previous results, both MsPimA and MsPimBЈ catalyzed the transfer of a Manp residue from GDP-Man to PI to form PIM 1 (Fig. 3A). The subsequent addition of MsPimBЈ to the MsPimA reaction mixture clearly led to the synthesis of 14 C-labeled PIM 2 (Fig. 3A). In striking contrast, the addition of MsPimA to the MsPimBЈ reaction mixture only resulted in the stimulation of PIM 1 production with no detectable formation of PIM 2 (Fig. 3A). We thus conclude from this experiment that although MsPimBЈ recognizes the PIM 1 product of MsPimA (with an ␣-1,2-linked Manp residue on the myo-Ins ring; Fig. 2) as an acceptor substrate, MsPimA is unable to transfer a Manp residue onto a PIM 1 product bearing an ␣-1,6linked Manp residue.
With the transfer of Manp from GDP-Man onto PI catalyzed by MsPimBЈ occurring 53 times slower than both the MsPimAdependent transfer of Manp onto PI and the MsPimBЈ-dependent addition of Manp onto PIM 1 (Fig. 3, B and C), it is clear that the different activities of the two enzymes with PI and, subsequently, PIM 1 acceptors dictate the order in which the mannosylation of PI and PIM is to occur under physiological conditions. In further support of this assumption, the PIM 1 product FIGURE 2. NMR analysis of purified mannolipids. 1 H NMR spectra of mannolipids 1-4. The 1 H NMR spectra of mannolipid 1 shows one peak at 5.14 ppm assigned to the ␣-anomeric proton of the Manp residue attached to position 2 of myo-Ins. The 1 H and 13 C chemical shift values of mannolipid 3 are exactly comparable with that of mannolipid 1, and therefore compound 3 was also assigned to 2-linked PIM 1 . In the spectra of compound 2, the peak at 5.072 ppm was assigned as the ␣-anomeric proton of the Manp residue attached to position 6 of myo-Ins. The 1 H NMR spectra of mannolipid 4 shows two distinct peaks at 5.129 and 5.046 ppm assigned to ␣-anomeric protons of two Manp residues attached to distinct positions of myo-Ins. Based on the combined two-dimensional NMR spectral analyses, the ␣-anomeric protons at 5.129 and 5.046 ppm are assigned to the peaks that are 2-and 6-linked to myo-Ins, respectively, and therefore, compound 4 is assigned to 2,6-linked PIM 2 (see supplemental material and supplemental Figs. 3S-6S).
MsPimBЈ Stimulates the Production of Ac 1 PIM 2 in M. Smegmatis Membrane Preparations-When membranes prepared from M. smegmatis mc 2 155 were used as a source of phospho-(glyco)lipid acceptor, the addition of purified MsPimA clearly stimulated the synthesis of PIM 1 , accompanied by the accumulation of small amounts of Ac 1 PIM 1 (Fig. 4, lane 2). The addition of MsPimBЈ to the membrane preparations, in contrast, led to an even greater accumulation of a compound (mannolipid 5) with R f properties similar to that of Ac 1 PIM 2 (Fig. 4, lane 4). MALDI-TOF MS and NMR analyses confirmed the identity of this product as Ac 1 PIM 2 containing two C 16  Overall, the abundant de novo synthesis of Ac 1 PIM 2 in the assay mixture containing purified MsPimBЈ (Fig. 4, lane 4) suggests that significant amounts of PIM 1 are available in the membranes of M. smegmatis or that the synthesis of this acceptor substrate is stimulated by the addition of purified MsPimBЈ to the reaction mixture. This observation and the fact that radiolabeled Ac 1 PIM 2 was on the contrary not detectable in the assay mixture in which only purified MsPimA was added (Fig. 4, lane  2) suggest that the physiological amounts of MsPimBЈ present in the membranes of M. smegmatis may be rate-limiting in the formation of PIM 2 /Ac 1 PIM 2 . On the other hand, with almost   SEPTEMBER 18, 2009 • VOLUME 284 • NUMBER 38 FIGURE 5. Proposed pathway for the early steps of PIM biosynthesis in mycobacteria. The two pathways originally proposed for the biosynthesis of Ac 1 PIM 2 in mycobacteria are shown. (i) PI is mannosylated to form PIM 1 . PIM 1 is then mannosylated to PIM 2 , which is acylated to form Ac 1 PIM 2 . (ii) PIM 1 is first acylated to Ac 1 PIM 1 and then mannosylated to Ac 1 PIM 2 . Our experimental evidence indicates that although both pathways might co-exist in mycobacteria (13), the sequence of events PI 3 PIM 1 3 PIM 2 3 Ac 1 PIM 2 is favored. As an important part of the literature concerning PIM studies refers to the nomenclature based on the M. tuberculosis H37Rv sequences, the Rv numbers of the proteins are also included. all of the PIM 2 product of MsPimBЈ being instantly converted to Ac 1 PIM 2 (Fig. 4, lane 4), the activity of the acyltransferase does not seem to be limiting in the membranes of M. smegmatis. In fact, saturation of this enzyme only became clearly visible when both purified MsPimA and MsPimBЈ were added to the reaction mixture, resulting in the accumulation of abundant quantities of PIM 1 and PIM 2 (Fig. 4, lane 3). Finally, the quasi-exclusive occurrence of PIM 2 s under their acylated form (Ac 1 PIM 2 ) in the assay where MsPimBЈ was added (Fig. 4, lane 4), whereas the product of the reaction catalyzed by MsPimA essentially occurred as PIM 1 (i.e. with no acylation on the Manp residue) (Fig. 4, lane 2), strongly suggests that the acyltransferase MSMEG_2934 preferentially acylates PIM 2 over PIM 1 . Thus, despite MSMEG_2934 displaying acyltransferase activity on both PIM 1 and PIM 2 in vitro (13), it is likely that under physiological conditions the preferred pathway to Ac 1 PIM 2 involves the transfer of both mannosyl residues onto PI prior to the acylation of the ␣-1,2-linked Manp residue.

Phosphatidylinositol Mannoside Biosynthesis
Revised Model for the Early Steps of PIM Biosynthesis-Based on present experimental evidence, a revised model for the early steps of PIM biosynthesis is presented in Fig. 5. As inferred from previous studies (5,(22)(23)(24) and now unambiguously demonstrated, MsPimA is the first enzyme engaged in the pathway. It is responsible for transferring a Manp residue from GDP-Man onto the 2-position of the myo-Ins ring of PI to form PIM 1 . MsPimBЈ then transfers a second Manp residue from the same sugar donor to the 6-position of the myo-Ins ring of PIM 1 yielding PIM 2 . Finally, the acyltransferase MSMEG_2934 acylates the Manp residue transferred by PimA to yield one of the major forms of PIM species found in mycobacteria, Ac 1 PIM 2 .
MsPimBЈ Is Essential for the Growth of M. smegmatis-To investigate the essentiality or, on the contrary, possible redundancy of the ManT PimBЈ in mycobacteria, a MspimBЈ (MSMEG_4253) conditional mutant of M. smegmatis mc 2 155 was constructed. The methodology employed relies upon a suicide plasmid harboring the counter-selectable marker sacB to achieve allelic replacement, and a replicative temperature-sensitive plasmid (pCG76) to express a rescue copy of the gene of interest. Briefly, clones having undergone single crossover at the MspimBЈ locus were first selected upon plating of mc 2 155/ pJQMspimBЈKX transformants on LB-Kan plates at 37°C. Single crossover recombinants were grown in LB-Kan broth and then plated onto sucrose containing plates at 30 or 37°C to select for allelic exchange mutants. No knock-out mutants were isolated at this stage strongly suggesting that MspimBЈ was essential for growth regardless of the temperature used. To confirm this assumption, a conditional mutant of M. smegmatis was constructed. A temperature-sensitive rescue plasmid carrying a wild type copy of the MspimBЈ gene, pCGMspimBЈ, was introduced in one of the single crossover recombinants, and the resulting merodiploids were plated onto LB-Kan-sucrose plates at 30°C. Candidate conditional mutants were obtained in which allelic replacement at the chromosomal MspimBЈ locus was confirmed by PCR (Fig. 6A). The conditional mutants grew normally at 30°C in liquid broth or on plates, a temperature at which pCGMspimBЈ replicates, but lost viability at 42°C where the rescue plasmid is lost (Fig. 6B). Results thus indicated that MspimBЈ is essential for the growth of M. smegmatis under the experimental conditions used. Therefore, despite the interchangeability of the M. tuberculosis PimB and PimBЈ enzymes expressed in C. glutamicum in cell-free assays (12), the function of  ily of glycosyltransferases, which includes more than 9800 proteins and at least 12 different enzymatic activities (see the Carbohydrate-Active enZymes data base). The GT4 family contains several enzymes of potential therapeutic significance and has been proposed as the ancestral "retaining" family from which enzymes with this type of stereochemistry have evolved (25,26). MsPimA is one of the few GT4 enzymes whose threedimensional structure has been solved. The enzyme displays the GT-B fold that consists of two Rossmann-like ␤-␣-␤ domains separated by a large cleft that includes the catalytic center (Fig. 7A). The GDP-Man-binding site is located mainly in the C-terminal domain, where it makes a number of hydrogen bonds with the protein. Docking calculations and site-directed mutagenesis recently provided clear insights into the position of the polar head of the acceptor substrate, PI. Structural and enzymatic evidence support a model of interfacial catalysis in which MsPimA recognizes PI with its polar head within the catalytic cleft and the fatty acid moieties only partially sequestered from the bulk solvent. Membrane association is mediated by an interfacial binding surface in the N-terminal domain of the protein, which likely includes a cluster of basic residues in the amphipathic ␣-helix 2 (17) (Fig. 7A).
A three-dimensional model of MsPimBЈ was generated by homology modeling using the crystal structure of the MsPimA-GDP-Man complex as a template. Given that the two enzymes share only 28% overall sequence identity, the alignment was manually corrected incorporating information such as secondary structure prediction and conservation of functional residues. The overall predicted structure of MsPimBЈ strongly resembles the experimental model of MsPimA (Fig. 7, A and B). Critical residues and their interactions are preserved in the two enzymes strongly supporting conserved catalytic and membrane association mechanisms (Fig. 7C) (17). Interestingly, the MsPimBЈ model predicts an amphipathic ␣-helix of the same length (14 residues) as the amphipathic ␣2 of MsPimA in which Arg 78 , Lys 80 , and Arg 81 are also conserved. However, some of the key residues involved in PI binding, most notably the connecting loop between ␤3 and ␣2, differ between the two proteins reflecting their different acceptor substrate specificity. Overall, the structural conservation of MsPimA and MsPimBЈ suggests that the two enzymes follow similar molecular mechanisms of substrate/ membrane recognition and catalysis.
Concluding Remarks-Altogether, the results of our cell-free assays support a revised model for the early steps of PIM biosynthesis wherein the major PIM product of mycobacteria, Ac 1 PIM 2 , is formed via the sequential activity of PimA followed by PimBЈ and, finally, the acyltransferase MSMEG_2934 (Fig.  5). Evidence is also provided for the first time that PimBЈ is the ManT responsible for the addition of the Manp residue linked to position 6 of the myo-Ins moiety of PI in mycobacteria, and that a deficiency in its activity cannot be compensated by any other ManT of M. smegmatis. Thus, despite PimB and PimBЈ having the potential to mannosylate the same substrates in in vitro assays (12), PimB and PimBЈ clearly do not have redundant physiological functions in whole mycobacterial cells.
After PgsA1 (MSMEG_2933, Rv2612c in M. tuberculosis H37Rv), PimA (MSMEG_2935, Rv2610c in M. tuberculosis H37Rv), and the acyltransferase MSMEG_2934 (Rv2611c in M. tuberculosis H37Rv), PimBЈ (Rv2188c in M. tuberculosis H37Rv) is now the fourth enzyme of the PIM pathway found to be essential in M. smegmatis and/or M. tuberculosis (5,13,27). 5 Although this finding implies that PI, PIM 1 , and PIM 2 are essential phospho(glyco)lipids, it is at present difficult to distinguish which of their roles as metabolic end products or as precursors for more mannosylated molecules (LM, LAM, and biosynthetic intermediates) specifically accounts for their essentiality. Ac 1 PIM 2 appears to be a metabolic end product that accumulates at high steady state levels in the cells as well as a precursor for more polar forms of PIMs, LM and LAM. Both the PIM 2 and the polar PIM contents of mycobacteria were found to directly impact on the permeability of the cell envelope (4, 5). 6 Moreover, polar PIMs have been implicated in the homeostasis of the plasma membrane (6). In contrast to apolar PIMs, the essentiality of LM, LAM, and biosynthetic intermediates to the physiology of mycobacteria appears to depend on the Mycobacterium species. For instance, whereas the arabinosylation of LM was found to be essential to the growth of M. tuberculosis (28), this process is not essential to the viability of M. smegmatis (29). An M. smegmatis knock-out mutant defective in some aspects of the elongation of the mannan backbone of LM was also found to be viable, although its colonial morphology and growth rates were altered (30). Clearly, PIMs, LM, and LAM are likely to be involved in more than one critical function in mycobacterial cells, each of which or the combination of which might account for their essentiality. From a drug development perspective, the essential character of PIM biosynthetic enzymes and their relative restriction to mycobacteria and a few other actinomycetes  emphasizes their interest as novel targets for anti-tuberculosis chemotherapeutic agents.