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Molecular Dissection of the Role of Two Methyltransferases in the Biosynthesis of Phenolglycolipids and Phthiocerol Dimycoserosate in the Mycobacterium tuberculosis Complex

2004; Elsevier BV; Volume: 279; Issue: 41 Linguagem: Inglês

10.1074/jbc.m406134200

1083-351X

Esther Pérez‐Herrán, Patricia Constant, Françoise Laval, Anne Lemassu, Marie‐Antoinette Lanéelle, Mamadou Daffé, Christophe Guilhot,

Carbohydrate Chemistry and Synthesis

A few mycobacterial species, most of which are pathogenic for humans, produce dimycocerosates of phthiocerol (DIM) and of glycosylated phenolphthiocerol, also called phenolglycolipid (PGL), two groups of molecules shown to be important virulence factors. The biosynthesis of these molecules is a very complex pathway that involves more than 15 enzymatic steps and has just begun to be elucidated. Most of the genes known to be involved in these pathways are clustered on the chromosome of M. tuberculosis. Based on their amino acid sequences, we hypothesized that the proteins encoded by Rv2952 and Rv2959c, two open reading frames of this locus, are involved in the transfer of methyl groups onto various hydroxyl functions during the biosynthesis of DIM, PGL, and related p-hydroxybenzoic acid derivatives (p-HBAD). Using allelic exchange and site-specific recombination, we produced three recombinant strains of Mycobacterium tuberculosis carrying insertions in Rv2952 or Rv2959c. Analysis of these mutants revealed that (i) the protein encoded by Rv2952 is a methyltransferase catalyzing the transfer of a methyl group onto the lipid moiety of phthiotriol and glycosylated phenolphthiotriol dimycocerosates to form DIM and PGL, respectively, (ii) Rv2959c is part of an operon including the newly characterized Rv2958c gene that encodes a glycosyltransferase also involved in PGL and p-HBAD biosynthesis, and (iii) the enzyme encoded by Rv2959c catalyzes the O-methylation of the hydroxyl group located on carbon 2 of the rhamnosyl residue linked to the phenolic group of PGL and p-HBAD produced by M. tuberculosis. These data further extend our understanding of the biosynthesis of important mycobacterial virulence factors and provide additional tools to decipher the molecular mechanisms of action of these molecules during the pathogenesis of tuberculosis. A few mycobacterial species, most of which are pathogenic for humans, produce dimycocerosates of phthiocerol (DIM) and of glycosylated phenolphthiocerol, also called phenolglycolipid (PGL), two groups of molecules shown to be important virulence factors. The biosynthesis of these molecules is a very complex pathway that involves more than 15 enzymatic steps and has just begun to be elucidated. Most of the genes known to be involved in these pathways are clustered on the chromosome of M. tuberculosis. Based on their amino acid sequences, we hypothesized that the proteins encoded by Rv2952 and Rv2959c, two open reading frames of this locus, are involved in the transfer of methyl groups onto various hydroxyl functions during the biosynthesis of DIM, PGL, and related p-hydroxybenzoic acid derivatives (p-HBAD). Using allelic exchange and site-specific recombination, we produced three recombinant strains of Mycobacterium tuberculosis carrying insertions in Rv2952 or Rv2959c. Analysis of these mutants revealed that (i) the protein encoded by Rv2952 is a methyltransferase catalyzing the transfer of a methyl group onto the lipid moiety of phthiotriol and glycosylated phenolphthiotriol dimycocerosates to form DIM and PGL, respectively, (ii) Rv2959c is part of an operon including the newly characterized Rv2958c gene that encodes a glycosyltransferase also involved in PGL and p-HBAD biosynthesis, and (iii) the enzyme encoded by Rv2959c catalyzes the O-methylation of the hydroxyl group located on carbon 2 of the rhamnosyl residue linked to the phenolic group of PGL and p-HBAD produced by M. tuberculosis. These data further extend our understanding of the biosynthesis of important mycobacterial virulence factors and provide additional tools to decipher the molecular mechanisms of action of these molecules during the pathogenesis of tuberculosis. Bacteria of the Mycobacterium tuberculosis complex exhibit amazing capacities to infect their host, resist bactericidal responses, and subvert the host immune response. Thus, M. tuberculosis, the causative agent of tuberculosis, has colonized one-third of the population of the world and kills more than two million people annually (1Organization World Health Fact Sheet 104. World Health Organization, Geneva2002Google Scholar). This pathogenicity has been largely attributed to the unusual mycobacterial envelope. This complex structure has a high lipid content, up to 60% of the dry weight of the bacterium, and contains a large variety of lipids with unusual structures (2Daffé M. Draper P. Adv. Microb. Physiol. 1998; 39: 131-203Crossref PubMed Google Scholar). Schematically, from the cytoplasm to the external side of the bacterium, the cell envelope is formed by (i) a plasma membrane, (ii) a cell wall core composed of three covalently attached macromolecules, i.e. the peptidoglycan, the arabinogalactan, and the mycolic acids, which are long chain (C60-C90) fatty acids, capped with a layer of non-covalently linked lipids and glycolipids, and (iii) a capsule of polysaccharide, proteins and lipids (2Daffé M. Draper P. Adv. Microb. Physiol. 1998; 39: 131-203Crossref PubMed Google Scholar). Among the extractable constituents of the cell envelope are two structurally related families of lipids, the diesters of phthiocerol and phenolglycolipids. The former class of lipids is a mixture of long chain β-diols, called phthiocerol and relatives, which are esterified by multimethyl-branched fatty acids (3Asselineau J. Indian J. Chest. Dis. 1982; 24: 143-157Google Scholar). Depending on the stereochemistry of the chiral centers bearing the methyl branches, the fatty acids are called mycocerosic or phthioceranic acids (4Daffé M. Lanéelle M.A. J. Gen. Microbiol. 1988; 134: 2049-2055PubMed Google Scholar). To date, phthiocerol dimycocerosates (DIM) 1The abbreviations used are: DIM, dimycocerosates of phthiocerol; DIP, diphthioceranates of phthiocerol; km, kanamycin; MALDI-TOF, matrix-assisted laser desorption-ionization time-of-flight; ORF, open reading frames; PGL, phenolglycolipid; PGL-tb, PGL of M. tuberculosis; p-HBAD, p-hydroxybenzoic acid derivatives; TMS, trimethylsilyl; BCG, Bacille Calmette-Guérin; km, kanamycin. and diphthioceranates (DIP) have been identified in eight mycobacterial species; DIM have been found in M. tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium leprae, Mycobacterium gastri, and Mycobacterium kansasii, whereas DIP have been found in Mycobacterium marinum and Mycobacterium ulcerans (4Daffé M. Lanéelle M.A. J. Gen. Microbiol. 1988; 134: 2049-2055PubMed Google Scholar). With the exception of M. gastri, all the DIM- or DIP-containing species are pathogenic. The mycobacterial species that produce DIM or DIP may also synthesize structurally related substances, called phenolphthiocerols and relatives, in which phthiocerol is ω-terminated by an aromatic nucleus, usually glycosylated by a type- or species-specific mono-, tri-, or a tetrasaccharide unit leading to PGL (5Brennan P.J. Ratledge C. Wilkinson S.G. Microbial Lipids. Academic Press, Inc., London1988: 203-298Google Scholar, 6Daffé M. Lemassu A. Doyle R.J. Glycomicrobiology. Plenum Press, New York2000: 225-273Google Scholar). More recently, we identified a new group of molecules related to PGL, the glycosylated p-hydroxybenzoic acid methyl esters (p-HBAD), in the culture media of bacteria of the M. tuberculosis complex (7Constant P. Perez E. Malaga W. Lanéelle M.-A. Saurel O. Daffé M. Guilhot C. J. Biol. Chem. 2002; 277: 38148-38158Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). The genes involved in the biosynthesis of DIM, PGL, and p-HBAD are clustered on the chromosome of bacteria of the M. tuberculosis complex (Fig. 1) (8Cole S.T. Brosch R. Parkhill J. Garnier T. Churcher C. Harris D. Gordon S.V. Eiglmeier K. Gas S. Barrey III, C.E. Tekaia F. Badcock K. Basham D. Brown D. Chillingworth T. Connor R. Davies R. Devlin K. Feltwell T. Gentles S. Hamlin N. Holroyd S. Hornsby T. Jagels K. Krogh A. McLean J. Moule S. Murphy L. Oliver K. Osborne J. Quail M.A. Rajandream M.-A. Rogers J. Rutter S. Seeger K. Skelton J. Squares R. Squares S. Sulston J.E. Taylor K. Whitehead S. Barrell B.G. Nature. 1998; 393: 537-544Crossref PubMed Scopus (6522) Google Scholar, 9Camacho L.R. Ensergueix D. Perez E. Gicquel B. Guilhot C. Mol. Microbiol. 1999; 34: 257-267Crossref PubMed Scopus (517) Google Scholar, 10Cox J.S. Chen B. McNeil M. Jacobs Jr., W.R. Nature. 1999; 402: 79-83Crossref PubMed Scopus (615) Google Scholar, 11Camacho L.R. Constant P. Raynaud C. Lanéelle M.-A. Triccas J.-A. Gicquel B. Daffé M. Guilhot C. J. Biol. Chem. 2001; 276: 19845-19854Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). Five of these genes, ppsA–E, encode a type-I modular polyketide synthase responsible for the synthesis of phthiocerol and phenolphtiocerol by the elongation of a C20-C22 fatty acyl chain or an acyl chain containing a phenol moiety with three malonyl-CoA and two methylmalonyl-CoA units (12Azad A.K. Sirakova T.D. Fernandes N.D. Kolattukudy P.E. J. Biol. Chem. 1997; 272: 16741-16745Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). The pks15/1 gene encodes an iterative type-I polyketide synthase catalyzing the elongation of p-HBA to form p-hydroxyphenylalkanoates, which in turn are converted into phenolphthiocerol derivatives by the PpsA–E synthase (7Constant P. Perez E. Malaga W. Lanéelle M.-A. Saurel O. Daffé M. Guilhot C. J. Biol. Chem. 2002; 277: 38148-38158Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). This gene is mutated in most M. tuberculosis clinical isolates, explaining why most strains of M. tuberculosis are unable to synthesize PGL even though they produce the structurally related DIM and p-HBAD. The mas gene encodes another iterative type-I polyketide synthase responsible for the synthesis of mycocerosic acids after 2–4 rounds of extension of C18-C20 fatty acids with methylmalonyl-CoA units (13Azad A.K. Sirakova T.D. Rogers L.M. Kolattukudy P.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4787-4792Crossref PubMed Scopus (86) Google Scholar). Two additional genes, fadD26 and fadD28 (also named acoas), encode two acyl-adenylate synthases involved in the formation of DIM and PGL presumably by activating the various polyketide synthase substrates (11Camacho L.R. Constant P. Raynaud C. Lanéelle M.-A. Triccas J.-A. Gicquel B. Daffé M. Guilhot C. J. Biol. Chem. 2001; 276: 19845-19854Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 14Fitzmaurice A.M. Kolattukudy P.E. J. Biol. Chem. 1998; 273: 8033-8039Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 15Trivedi O.A. Arora P. Sridharan V. Tickoo R. Mohanty D. Gokhale R.S. Nature. 2004; 428: 441-445Crossref PubMed Scopus (228) Google Scholar). Finally, four genes (drrA, drrB, and drrC encoding an ABC transporter and mmpL7 encoding a transporter of the RND permease superfamily) are also located in this locus and are involved in the translocation of DIM from the cytoplasm to the bacterial cell surface (11Camacho L.R. Constant P. Raynaud C. Lanéelle M.-A. Triccas J.-A. Gicquel B. Daffé M. Guilhot C. J. Biol. Chem. 2001; 276: 19845-19854Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). Close examination of this genomic region revealed five putative open reading frames (ORF) encoding proteins possibly involved in the glycosylation and methylation of DIM and PGL (7Constant P. Perez E. Malaga W. Lanéelle M.-A. Saurel O. Daffé M. Guilhot C. J. Biol. Chem. 2002; 277: 38148-38158Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). In the accompanying paper (16Perez E. Constant P. Lemassu A. Laval F. Daffé M. Guilhot C. J. Biol. Chem. 2004; 279: 42574-42583Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) we demonstrated that three of these genes (Rv2957, Rv2958c, and Rv2962c) encode proteins with similarities to glycosyltransferases and that these proteins are involved in the sequential elongation of both phenolphthiocerol dimycocerosates and p-hydroxybenzoic acid to form PGL and p-HBAD, respectively. In the present paper we show that the other two genes (Rv2959c and Rv2952) encode methyltransferases. We demonstrate (i) that the protein encoded by Rv2952 catalyzes the methylation of phthiotriol and phenophthiotriol to form phthiocerol and phenophthiocerol, respectively, and (ii) that the Rv2959c product is involved in the O-methylation of the hydroxyl group located at position 2 of the first rhamnosyl residue found in PGL-tb and p-HBAD of M. tuberculosis. We also show that Rv2959c and Rv2958c, which encodes the glycosyltransferase involved in the transfer of the terminal disaccharide moiety of PGL-tb and p-HBAD II (16Perez E. Constant P. Lemassu A. Laval F. Daffé M. Guilhot C. J. Biol. Chem. 2004; 279: 42574-42583Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), form an operon. Most of the materials and methods used are described in the accompanying article (16Perez E. Constant P. Lemassu A. Laval F. Daffé M. Guilhot C. J. Biol. Chem. 2004; 279: 42574-42583Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Only the experimental procedures specific to the present work are detailed. Construction of M. tuberculosis H37Rv Mutants—PCR was carried out in a final volume of 50 μl containing M. bovis Bacille Calmette-Guérin (BCG) genomic DNA, 1 mm primer A (Rv2959A or Rv2952A), 1 mm primer B (Rv2959B or Rv2952B) (Table I), 2.5 units of Taq DNA polymerase (Roche Applied Science), and 10% Me2SO. The amplification program consisted of 1 cycle of 10 min at 95 °C followed by 35 cycles of 30 s at 95 °C, 30 s at 57 °C, and 3 min at 72 °C and a final extension at 72 °C for 10 min. The PCR product was analyzed by electrophoresis in 0.8% agarose gel. The resulting fragments, 2805 bp for Rv2952 and 2690 bp for Rv2959c, were gel-purified using the Qiaquick gel extraction purification kit (Qiagen, Courtaboeuf, France). The Rv2952 fragment was digested with SacI and XbaI, and the Rv2959c fragment was digested with NotI and XbaI. The pBlueScript vector was digested with SpeI and KpnI and religated yielding pPET2. The Rv2952c and Rv2959c PCR fragments were inserted into the pPET2 vector between the XbaI and SacI or between the XbaI and NotI restriction sites, respectively, to give pPET9 (Rv2952) and pPET5 (Rv2959c). A kanamycin resistance cassette (km) formed by the Ωkm cassette from pHP45Ωkm (17Fellay R. Frey J. Krisch H.M. Gene (Amst.). 1987; 52: 147-154Crossref PubMed Scopus (566) Google Scholar) flanked by two res sites from transposon γδ (18Malaga W. Perez E. Guilhot C. FEMS Microbiol. Lett. 2003; 219: 261-268Crossref PubMed Scopus (41) Google Scholar) was inserted between the KpnI and SalII sites of Rv2952, generating a 605-bp deletion, and the two MfeI and BglII sites of Rv2959c, generating a 290-bp deletion. The resulting plasmids were named pPET23 and pPET14, respectively. The PmeI fragments from the various plasmids, containing the disrupted gene constructs, were inserted at the XbaI site of pPR27 (19Pelicic V. Jackson M. Reyrat J.M. Jacobs W.R. Gicquel B. Guilhot C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10955-10960Crossref PubMed Scopus (386) Google Scholar), generating pPET34 (ΔRv2952::km) and pPET40 (Rv2959c::km). M. tuberculosis H37Rv was electrotransformed as previously described, and transformants were selected on 7H11 + oleic acid-albumin-dextrose-catalase + km at 32 °C (20Pelicic V. Reyrat J.-M. Gicquel B. Mol. Microbiol. 1996; 20: 919-925Crossref PubMed Scopus (136) Google Scholar). Two transformants obtained with each of the plasmids were grown in 5 ml of 7H9 + albumin-dextrose-catalase + km + Tween at 32 °C until saturation. Dilutions of this culture were plated on 7H11 + oleic acid-albumin-dextrose-catalase + km + sucrose and incubated at 39 °C for 4 weeks. The colonies were screened by PCR using primers C, D, E, res1, or res2. The amplification program consisted of 1 cycle of 10 min at 95 °C followed by 35 cycles of 30 s at 95 °C, 30 s at 55 °C, 3 min at 72 °C, and a final 10 min at 72 °C. Two clones giving the pattern corresponding to allelic exchange were retained for further analysis. These strains were renamed PMM28 (ΔRv2952::km) and PMM28 (ΔRv2959c::km).Table IPrimers used for the construction and the characterization of the M. tuberculosis mutant strainsGeneOligonucleotideSequenceRv2959Rv2959A5′-GCTCTAGAGTTTAAACCGGCCCCGGACATGGTTG-3′Rv2959B5′-GCGCGGCCGCGTTTAAACCGTGACGGGTAAATCGGCC-3′Rv2959C5′-ATGTGGAGAAATGCTCTGCGCC-3′Rv2959D5′-ACGTTCTTCAGGTGGTTCCGG-3′Rv2959E5′-AACTCGCTCAGGATCTCCTGG-3′Rv2959F5′-GGAATGGGGCTAGTGTGGCGC-3′Rv2959G5′-TAGGGTCCAGGCCCAAACCCG-3′Rv2952Rv2952A5′-GCTCTAGAGTTTAAACGACAGATTCCAGCGCCTC-3′Rv2952B5′-GCGAGCTCGTTTAAACGCGCTCCACTAATCGTCG-3′Rv2952C5′-TCCCATTGGATGCTCGACGCC-3′Rv2952D5′-CTCGAAGGTGTAATGGCCCCG-3′Rv2952E5′-CGGCGGATCTTCCTCATAGGC-3′resres15′-GCTCTAGAGCAACCGTCCGAAATATTATAAA-3′res25′-GCTCTAGATCTCATAAAAATGTATCCTAAATCAAATATC-3′ Open table in a new tab Construction of M. tuberculosis H37Rv ΔRv2959c::res Unmarked Mutant—To recover the res-Ωkm-res cassette from M. tuberculosis PMM18, we transferred the plasmid pWM19 containing the resolvase gene of transposon γδ under the control of the mycobacterial promotor pBlaF* into this strain (18Malaga W. Perez E. Guilhot C. FEMS Microbiol. Lett. 2003; 219: 261-268Crossref PubMed Scopus (41) Google Scholar). The PMM18:pWM19 transformation mixture was resuspended in 5 ml of 7H9 + albumin-dextrose-catalase and incubated for 48 h at 32 °C to allow the expression of hygromycin resistance. Transformants were selected directly in the liquid medium by adding hygromycin to the transformation mixture and incubating the culture at 32 °C for 12 days. Viable bacteria were then recovered by plating serial dilutions on 7H11 + oleic acid-albumin-dextrose-catalase plates without antibiotics and incubating at 39 °C, a non-permissive temperature for pWM19 replication. Twenty-one colonies picked randomly were then tested for their growth on km-containing plates. Thirteen of these colonies were unable to grow on km-containing plates but grew as the control on antibiotic-free plates. Three of these clones were analyzed by PCR using primers res1 + 2959C, 2959E + 2959C, and 2959F + 2959G. The amplification program consisted of 1 cycle of 10 min at 95 °C followed by 35 cycles of 30 s at 95 °C,30 s at 55 °C, 3 min at 72 °C, and a final extension of 10 min at 72 °C. The amplification pattern revealed that the res-Ωkm-res cassette had been excised in these three clones, leaving a copy of the 132-bp res site within the Rv2959c gene. One clone, named PMM18res, was retained for further analysis. Disruption of Rv2952 and Rv2959c Genes in M. tuberculosis H37Rv by Allelic Exchange—The biosynthesis of PGL, p-HBAD, and DIM in bacteria of the M. tuberculosis complex involves the transfer of several methyl groups onto the carbohydrate moieties of PGL and p-HBAD and onto the lipid domains of PGL and DIM. This implies that several unknown methyltransferases catalyze these reactions. Previous studies have shown that many genes involved in the biosynthesis of DIM and PGL are clustered on a 70-kilobase fragment of the M. tuberculosis chromosome; that is, the DIM and PGL locus (Fig. 1). Therefore, we looked for ORFs encoding putative proteins with similarities to methyltransferase in this locus. Two such ORFs, Rv2952 and Rv2959c, were identified downstream of pks15/1. Interestingly, the protein encoded by Rv2952, but not that encoded by Rv2959c, harbored an amino acid motif conserved in several other mycobacterial methyltransferases shown to be required for the transfer of methyl groups onto fatty acids (21Jeevarajah D. Patterson J.H. McConville M.J. Billman-Jacobe H. Microbiology. 2002; 148: 3079-3087Crossref PubMed Scopus (44) Google Scholar). Thus, we hypothesized that the Rv2959c product is involved in the methylation of the glycosyl moiety of PGL-tb and p-HBAD and that the Rv2952 product methylates the lipid domains of both DIM and PGL-tb. To establish the exact roles of the proteins encoded by Rv2952 and Rv2959c in the biosynthesis of DIM, PGL-tb, and p-HBAD, we constructed two M. tuberculosis H37Rv mutants in which these genes were disrupted by allelic exchange. Briefly, chromosomal fragments overlapping the 5′ or 3′ ends of Rv2952 or Rv2959c were cloned flanking a km resistance cassette into the vector pPR27 (19Pelicic V. Jackson M. Reyrat J.M. Jacobs W.R. Gicquel B. Guilhot C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10955-10960Crossref PubMed Scopus (386) Google Scholar) yielding plasmids pPET40 (Rv2959c) and pPET34 (Rv2952). These constructs were independently transferred by electroporation into M. tuberculosis H37Rv. Two transformants obtained with each plasmid were grown in liquid broth at 32 °C until stationary phase and allelic exchange mutants were selected by plating serial dilutions of the cultures on solid medium containing kanamycin and sucrose at 39 °C. PCR analysis of 10 colonies obtained with each construct on the counterselective plates using C + D, C + res2, or E + res1 primers revealed that several clones gave an amplification pattern consistent with an allelic replacement of the wild-type allele of the different genes by the disrupted allele (Fig. 2). Two clones, named PMM18 (ΔRv2959c::km) and PMM28 (ΔRv2952::km), were retained for further studies. Biochemical Phenotypes of Methyltransferase Mutants—To examine the effects of the mutations in the Rv2952 and Rv2959c genes on p-HBAD, DIM, and PGL production, we first transferred the plasmid pPET1 into the H37Rv strain and in the two mutants, PMM18 and PMM28. This plasmid carries a functional pks15/1 gene from M. bovis BCG because the wild-type H37Rv strain is unable to produce PGL-tb due to a frame-shift mutation in pks15/1 (7Constant P. Perez E. Malaga W. Lanéelle M.-A. Saurel O. Daffé M. Guilhot C. J. Biol. Chem. 2002; 277: 38148-38158Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). The p-HBAD compounds are mostly found in the culture supernatant of the H37Rv strain of M. tuberculosis, whereas DIM and PGL-tb remain associated with the bacterial cells. Therefore, the mutants and the wild-type strain (either transformed with the plasmid pPET1 or not transformed) were grown in liquid culture, and lipids were extracted from both the culture supernatant and the bacterial pellets. TLC analysis of the organic solvent extracts from the culture supernatants obtained with the various strains revealed the presence of one glycoconjugate spot in the lipid extract of H37Rv. It corresponds to the previously described p-HBAD II (tri-O-methyl-fucosyl-(α1→3)-rhamnosyl-(α1→3)-2-O-methyl-rhamnosyl-α-p-hydroxybenzoic acid methyl ester) (7Constant P. Perez E. Malaga W. Lanéelle M.-A. Saurel O. Daffé M. Guilhot C. J. Biol. Chem. 2002; 277: 38148-38158Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). This compound is the major p-HBAD produced by M. tuberculosis H37Rv. A minor compound p-HBAD I corresponding to 2-O-methyl-rhamnosyl-α-p-hydroxybenzoic acid methyl ester is sometime visible but was undetectable in this experiment. The same pattern of glycoconjugate production was observed with the PMM28 mutant strain (Fig. 3A). In contrast, no glycoconjugates were detected in the organic-solvent extracts from the PMM18 mutant (Fig. 3A). Hence, the putative methyltransferase encoded by Rv2959c, but not that encoded by Rv2952, plays a role in the biosynthesis of p-HBAD. To determine the roles of the putative methyltransferase in the biosynthesis of PGL-tb, lipids from the bacterial cells were examined. TLC analysis of these lipids showed that the PMM28:pPET1 mutant produced two glycoconjugates, products B and C (Fig. 3B). These molecules have slightly different mobilities to PGL from M. bovis BCG (2-O-methyl-rhamnosylphenolphthiocerol dimycocerosates) and from M. tuberculosis (tri-O-methyl-fucosyl-(α1→3)-rhamnosyl-(α1→3)-2-O-methyl-rhamnosyl-phenolphthiocerol dimycocerosates). In the case of PMM18:pPET1 mutant, a glycoconjugate (product A, Fig. 3B) with a lower mobility than that of PGL-tb was observed. These results strongly suggest that both Rv2952 and Rv2959c are involved in PGL-tb biosynthesis. Because PGL and DIM are structurally related, we investigated the roles of the putative methyltransferases in the biosynthesis of DIM. As expected, the control strain (H37Rv: pPET1) produced both DIM A (phthiocerol dimycocerosates) and DIM B (phthiodiolone dimycocerosate) (Fig. 3B). The mutation in the Rv2959c gene did not affect the production of DIM, as both forms were detected in similar amounts to those found in the wild-type strain (data not shown). In contrast, the mutation in the Rv2952 gene seemed to abolish the synthesis of DIM A completely but did not affect the production of DIM B both in PMM28 and PMM28:pPET1 (Fig. 3C). When large amounts of lipid extracts were loaded on TLC plates, an additional spot (product D) was detected in the organic solvent extracts of PMM28:pPET1 in comparison with that of the wild-type strain. Thus, these preliminary analyses demonstrated that mutations in Rv2959c and Rv2952 affected the production of p-HBAD, PGL-tb, and DIM but in different ways. The protein encoded by Rv2959c seems to be required for the biosynthesis of both p-HBAD and PGL-tb but not DIM, whereas that encoded by Rv2952 appears to be required for the biosynthesis of PGL-tb and DIM A in M. tuberculosis but not p-HBAD. Because p-HBAD and PGL-tb share a common glycosyl-phenolic domain absent from DIM, whereas DIM and PGL-tb exhibit a common lipid domain absent from p-HBAD, these results support our hypothesis, based on the amino acid sequences of the two putative methyltransferases, that the Rv2959c product is involved in the methylation of the glycosyl moiety and that the Rv2952 product is involved in the methylation of the lipid domain. Structural Analysis of the Compounds Accumulated in the M. tuberculosis H37Rv ΔRv2952::km Mutant—The mutations in both genes resulted in the accumulation of new compounds (Fig. 3) that may be biosynthetic intermediates of p-HBAD, PGL-tb, and DIM. To test this hypothesis, we purified these substances by chromatography on Florisil and analyzed them further. First, products B and C, which accumulated in the M. tuberculosis PMM28:pPET1 mutant, were analyzed by MALDI-TOF mass spectrometry. The spectrum of glycoconjugate B showed a series of pseudomolecular ion (M+Na+) peaks at 1850, 1864, 1878, 1892, 1906, 1920, 1934, 1948, 1962, 1976, 1990, and 2004m/z (Fig. 4B) (the major peaks are underlined). The same peaks were observed in the mass spectrum of the purified PGL-tb from the M. tuberculosis H37Rv:pPET1 strain (Fig. 4A), but the mass values of the major pseudomolecular ion peaks corresponding to glycolipid B from the PMM28:pPET1 mutant were 14 mass units lower than those in the spectrum of PGL-tb. This suggests that the PMM28:pPET1 mutant produces a PGL-like substance that may differ from PGL-tb by the absence of a methyl group. This hypothesis was supported by the 1H NMR analysis of the glycolipid B, which showed that the 1H NMR spectrum of the purified glycoconjugate B from PMM28:pPET1 was very similar to that of PGL-tb (Fig. 5A) (7Constant P. Perez E. Malaga W. Lanéelle M.-A. Saurel O. Daffé M. Guilhot C. J. Biol. Chem. 2002; 277: 38148-38158Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 22Daffé M. Lacave C. Lanéelle M.-A. Lanéelle G. Eur. J. Biochem. 1987; 167: 155-160Crossref PubMed Scopus (116) Google Scholar). Two deshielded doublets were observed at 6.97 and 7.10 ppm and corresponded to proton resonances of the phenolic group of PGLs (7Constant P. Perez E. Malaga W. Lanéelle M.-A. Saurel O. Daffé M. Guilhot C. J. Biol. Chem. 2002; 277: 38148-38158Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 22Daffé M. Lacave C. Lanéelle M.-A. Lanéelle G. Eur. J. Biochem. 1987; 167: 155-160Crossref PubMed Scopus (116) Google Scholar). Three anomeric proton resonances were seen in the 1H NMR spectrum of the glycolipid B from PMM28:pPET1 at 5.50 ppm (1H) and 5.15 ppm (2H) (Fig. 5A). Their chemical shift values were identical to those of PGL-tb (7Constant P. Perez E. Malaga W. Lanéelle M.-A. Saurel O. Daffé M. Guilhot C. J. Biol. Chem. 2002; 277: 38148-38158Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 22Daffé M. Lacave C. Lanéelle M.-A. Lanéelle G. Eur. J. Biochem. 1987; 167: 155-160Crossref PubMed Scopus (116) Google Scholar). In addition, four singlets were observed in the region of the resonances of sugar-linked methoxyl (OCH3) protons at 3.5–3.7 ppm. Again, the chemical shift values of the methoxyl proton resonances of product B (Fig. 5A) were identical to those found located on the trisaccharide portion of PGL-tb (7Constant P. Perez E. Malaga W. Lanéelle M.-A. Saurel O. Daffé M. Guilhot C. J. Biol. Chem. 2002; 277: 38148-38158Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 22Daffé M. Lacave C. Lanéelle M.-A. Lanéelle G. Eur. J. Biochem. 1987; 167: 155-160Crossref PubMed Scopus (116) Google Scholar). These data strongly suggest that compound B has the same carbohydrate moiety as PGL-tb. The occurrence of polymethylenic (CH2) units in the glycolipid was deduced from the presence of a broad signal resonance at 1.25 ppm. The resonances of the expected terminal methyl (CH3) protons were observed at 0.8–1.0 ppm, whereas those attributable to methyl branches located on carbons 2 of fatty acyl residues were seen at 1.14 ppm. The resonance of the methine (CH) proton of the esterified β-glycol was seen at 4.83 ppm (Fig. 5A, signal a). Interestingly, the proton resonance typical of the methoxyl groups of the phthiocerol and phenolphthiocerol moiety of DIM A and PGL, respectively, expected at 3.32 ppm (singlet 3H) (7Constant P. Perez E. Malaga W. Lanéelle M.-A. Saurel O. Daf