Role of the pks15/1 Gene in the Biosynthesis of Phenolglycolipids in the Mycobacterium tuberculosisComplex
2002; Elsevier BV; Volume: 277; Issue: 41 Linguagem: Inglês
10.1074/jbc.m206538200
ISSN1083-351X
AutoresPatricia Constant, Esther Pérez‐Herrán, Wladimir Malaga, Marie‐Antoinette Lanéelle, Olivier Saurel, Mamadou Daffé, Christophe Guilhot,
Tópico(s)Antibiotic Resistance in Bacteria
ResumoDiesters of phthiocerol and phenolphthiocerol are important virulence factors ofMycobacterium tuberculosis and Mycobacterium leprae, the two main mycobacterial pathogens in humans. They are both long-chain β-diols, and their biosynthetic pathway is beginning to be elucidated. Although the two classes of molecules share a common lipid core, phthiocerol diesters have been found in all the strains of the M. tuberculosis complex examined although phenolphthiocerol diesters are produced by only a few groups of strains. To address the question of the origin of this diversity 8 reference strains and 10 clinical isolates of M. tuberculosis were analyzed. We report the presence of glycosylated p-hydroxybenzoic acid methyl esters, structurally related to the type-specific phenolphthiocerol glycolipids, in the culture media of all reference strains of M. tuberculosis, suggesting that the strains devoid of phenolphthiocerol derivatives are unable to elongate the putativep-hydroxybenzoic acid precursor. We also show that all the strains of M. tuberculosis examined and deficient in the production of phenolphthiocerol derivatives are natural mutants with a frameshift mutation in pks15/1 whereas a single open reading frame for pks15/1 is found in Mycobacterium bovis BCG, M. leprae, and strains of M. tuberculosis that produce phenolphthiocerol derivatives. Complementation of the H37Rv strain of M. tuberculosis,which is devoid of phenolphthiocerol derivatives, with the fusedpks15/1 gene from M. bovis BCG restored phenolphthiocerol glycolipids production. Conversely, disruption of thepks15/1 gene in M. bovis BCG led to the abolition of the synthesis of type-specific phenolphthiocerol glycolipid. These data indicate that Pks15/1 is involved in the elongation of p-hydroxybenzoic acid to givep-hydroxyphenylalkanoates, which in turn are converted, presumably by the PpsA-E synthase, to phenolphthiocerol derivatives. Diesters of phthiocerol and phenolphthiocerol are important virulence factors ofMycobacterium tuberculosis and Mycobacterium leprae, the two main mycobacterial pathogens in humans. They are both long-chain β-diols, and their biosynthetic pathway is beginning to be elucidated. Although the two classes of molecules share a common lipid core, phthiocerol diesters have been found in all the strains of the M. tuberculosis complex examined although phenolphthiocerol diesters are produced by only a few groups of strains. To address the question of the origin of this diversity 8 reference strains and 10 clinical isolates of M. tuberculosis were analyzed. We report the presence of glycosylated p-hydroxybenzoic acid methyl esters, structurally related to the type-specific phenolphthiocerol glycolipids, in the culture media of all reference strains of M. tuberculosis, suggesting that the strains devoid of phenolphthiocerol derivatives are unable to elongate the putativep-hydroxybenzoic acid precursor. We also show that all the strains of M. tuberculosis examined and deficient in the production of phenolphthiocerol derivatives are natural mutants with a frameshift mutation in pks15/1 whereas a single open reading frame for pks15/1 is found in Mycobacterium bovis BCG, M. leprae, and strains of M. tuberculosis that produce phenolphthiocerol derivatives. Complementation of the H37Rv strain of M. tuberculosis,which is devoid of phenolphthiocerol derivatives, with the fusedpks15/1 gene from M. bovis BCG restored phenolphthiocerol glycolipids production. Conversely, disruption of thepks15/1 gene in M. bovis BCG led to the abolition of the synthesis of type-specific phenolphthiocerol glycolipid. These data indicate that Pks15/1 is involved in the elongation of p-hydroxybenzoic acid to givep-hydroxyphenylalkanoates, which in turn are converted, presumably by the PpsA-E synthase, to phenolphthiocerol derivatives. dimycocerosates of phthiocerol homonuclear (1H-1H) chemical shift-correlated spectroscopy diphthioceranates of phthiocerol Heteronuclear (1H-13C) Multiple Bound Correlation Heteronuclear (1H-13C) Multiple Quantum Correlation matrix-assisted laser desorption-ionization time-of-flight nuclear Overhauser and exchange spectroscopy phenolglycolipid p-hydroxybenzoic acid open reading frame gas chromatography mass spectrometry Mycobacterial infections cause extremely serious infectious diseases and are responsible for millions of deaths annually worldwide. Based on the data accumulated during the last decades, the mycobacterial envelope appears to play a fundamental role in the physiology of these bacteria, both in terms of pathogenicity and resistance to antibiotics. This envelope is singular and differs from those of other bacteria by both its molecular composition and the architectural arrangement of its constituents (1Daffé M. Draper P. Adv. Microbiol. Phys. 1998; 39: 131-203Crossref PubMed Google Scholar). A key feature of the mycobacterial envelope is its high lipid content, up to 60% of the dry weight of the bacteria (2Goren M.B. Brennan P.J. Youmans G.P. Tuberculosis. WB Saunders, Philadelphia1979: 62-193Google Scholar). Another distinctive feature resides in the variety of lipid compounds with unusual structures, many of them being unique to mycobacteria (1Daffé M. Draper P. Adv. Microbiol. Phys. 1998; 39: 131-203Crossref PubMed Google Scholar). Consistent with this singularity, genome sequence data for several mycobacterial species reveal that a large portion of the genes may be involved in lipid metabolism (∼250 genes in Mycobacterium tuberculosis) (3Cole S.T. Brosch R. Parkhill J. Garnier T. Churcher C. Harris D. Gordon S.V. Eiglmeier K. Gas S. Barry 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 (6556) Google Scholar). Therefore there is a large avenue for future research toward the definition of the biological functions of specific lipids, some of them important for pathogenicity. Understanding the biosynthetic pathways leading to the production of virulence factors or essential components of the cell envelope may help to identify new targets for the development of antituberculous drugs and represents an important post-genomic challenge for the near future. Among the few genes that have been clearly associated with a defined metabolic pathway in M. tuberculosis and shown to play a role in the pathogenicity of the tubercle bacillus are the cluster of genes involved in the biosynthesis of phthiocerol and phenolphthiocerol dimycocerosates (4Camacho 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 (303) Google Scholar, 5Camacho L.R. Ensergueix D. Perez E. Gicquel B. Guilhot C. Mol. Microbiol. 1999; 34: 257-267Crossref PubMed Scopus (518) Google Scholar, 6Cox J.S. Chen B. McNeil M. Jacobs Jr., W.R. Nature. 1999; 402: 79-83Crossref PubMed Scopus (616) Google Scholar). These compounds and their relatives are constituents of the mycobacterial cell envelope produced by only some of the slow growers in particular the major pathogens such as Mycobacterium leprae, Mycobacterium ulcerans,Mycobacterium marinum, and members of the M. tuberculosis complex (7Daffé M. Lanéelle M.A. J. Gen. Microbiol. 1988; 134: 2049-2055PubMed Google Scholar). Phthiocerol and its relatives consist of a long-chain β-diol, which occurs naturally as diesters of polymethyl-branched fatty acids (Fig. 1). According to the mycobacterial strain, the asymmetric centers bearing the methyl branches in these fatty acids may be of the d- or the l-series and are known as mycocerosates and phthioceranates, respectively (7Daffé M. Lanéelle M.A. J. Gen. Microbiol. 1988; 134: 2049-2055PubMed Google Scholar). The mycobacterial species that produce phthiocerol dimycocerosates (DIMs)1 or phthiocerol diphthioceranates (DIPs) may also synthesize structurally related substances, called phenolphthiocerol and its relatives, in which the lipid core is ω-terminated by an aromatic nucleus, probably derived from p-hydroxybenzoic acid (see Ref. 1Daffé M. Draper P. Adv. Microbiol. Phys. 1998; 39: 131-203Crossref PubMed Google Scholar). In these substances the hydroxyl group of the phenol moiety is usually glycosylated by a type- or species-specific mono-, tri-, or tetra-saccharide unit (8Brennan P.J. Ratledge C. Wilkinson S.G. Microbial lipids. 1. Academic Press, London1988: 203-298Google Scholar, 9Daffé M. Lemassu A. Doyle R.J. Glycomicrobiology. Plenum Press, 2000: 225-273Google Scholar) leading to phenolphthiocerol glycolipids, also called phenolic glycolipids (PGLs) (Fig. 1). Numerous studies have established the usefulness of PGLs, notably PGL-1 from M. leprae, in the serodiagnosis of leprosy and tuberculosis (10Gaylord H. Brennan P.J. Am. Rev. Microbiol. 1987; 41: 645-675Crossref PubMed Scopus (73) Google Scholar, 11Torgal-Garcia J. David H.L. Papa F. Ann. Inst. Pasteur/Microbiol. 1988; 139: 289-294Crossref PubMed Scopus (29) Google Scholar, 12Martin Casabona N. Gonzalez Fuente T. Arcalis Arce L. Ortal Entraigos J. Vidal Pla R. Acta Leprologica. 1989; 7 Suppl. 1: S89-S93PubMed Google Scholar, 13Simonney N. Molina J. Molimard M. Oksenhendler E. Peronne C. Lagrange P. Eur. J. Clin. Microbiol. Infect. Dis. 1995; 14: 883-891Crossref PubMed Scopus (35) Google Scholar). In addition some of these glycolipids exhibit biological activities in vitrothat may be relevant to the pathogenesis of mycobacterial infections. For instance, PGL-1 from M. leprae has been reported to inhibit the proliferation of T lymphocytes after stimulation with concanavalin A (14Mehra V. Brennan P.J. Rada E. Convit J. Bloom B.R. Nature. 1984; 308: 194-196Crossref PubMed Scopus (138) Google Scholar); this observation has been extended to PGLs produced by other mycobacterial species (15Fournié J.-J. Adams E. Mullins R.J. Basten A. Infect. Immun. 1989; 57: 3653-3659Crossref PubMed Google Scholar). Furthermore, PGL-1 seems to be associated with resistance to intracellular killing by macrophages (16Neill M.A. Klebanoff S.J. J. Exp. Med. 1988; 167: 30-42Crossref PubMed Scopus (68) Google Scholar) and promotes phagocytosis of M. leprae by macrophages and Schwann cells through binding, respectively, to complement component C3 or laminin-2 (17Schlesinger L.S. Horwitz M.A. J. Exp. Med. 1991; 174: 1031-1038Crossref PubMed Scopus (67) Google Scholar, 18Ng V. Zanazzi G. Timpl R. Talts J.F. Salzer J.L. Brennan P.J. Rambukkana A. Cell. 2000; 103: 511-524Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Recently, the molecular basis of the interaction between M. leprae and Schwann cells was established: the saccharide moiety of PGL-1 binds the α2LG1, α2LG4, and α2LG5 modules of the peripheral nerve laminin α2 chain (18Ng V. Zanazzi G. Timpl R. Talts J.F. Salzer J.L. Brennan P.J. Rambukkana A. Cell. 2000; 103: 511-524Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). The structural similarities between DIMs, DIPs, phenolphthiocerol derivatives, and PGLs have led the proposal of a biosynthesis pathway for these diols involving a common branch in which two different precursors, either C22-C24 fatty acid orp-hydroxyphenylalkanoic acid, are elongated by three malonyl-CoA and two methylmalonyl-CoA units to yield a common lipid core (Fig. 1) (19Azad 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). This postulate was supported by the identification in the Mycobacterium bovis BCG and M. tuberculosis genomes of a group of 5 polyketide synthase genes (named ppsA-E) that contain the domains required for these sequential elongation steps. As predicted, disruption ofppsB and ppsC in M. bovis BCG led to a mutant strain unable to synthesize PGLs or DIMs (19Azad 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). Mycocerosic acids, the class of polymethyl-branched fatty acids that esterify the phthiocerol and phenolphthiocerol of members of the M. tuberculosis complex, M. leprae, Mycobacterium kansasii, and Mycobacterium gastri (7Daffé M. Lanéelle M.A. J. Gen. Microbiol. 1988; 134: 2049-2055PubMed Google Scholar), are synthesized by another polyketide synthase, Mas (20Mathur M. Kolattukudy P.E. J. Biol. Chem. 1992; 267: 19388-19395Abstract Full Text PDF PubMed Google Scholar, 21Azad 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). The mas andppsA-E genes are clustered on the M. tuberculosis chromosome in a region that has been later shown to contain 6 other genes encoding acyl-CoA synthase and transporters important for DIM biosynthesis and translocation (4Camacho 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 (303) Google Scholar, 5Camacho L.R. Ensergueix D. Perez E. Gicquel B. Guilhot C. Mol. Microbiol. 1999; 34: 257-267Crossref PubMed Scopus (518) Google Scholar, 6Cox J.S. Chen B. McNeil M. Jacobs Jr., W.R. Nature. 1999; 402: 79-83Crossref PubMed Scopus (616) Google Scholar, 22Fitzmaurice A.M. Kolattukudy P.E. J. Biol. Chem. 1998; 273: 8033-8039Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). In species that produce DIMs or DIPs, all the strains do so; however many of these strains do not contain phenolphthiocerol derivatives (1Daffé M. Draper P. Adv. Microbiol. Phys. 1998; 39: 131-203Crossref PubMed Google Scholar, 23Daffé M. Varnerot A. Vincent Lévy-Frébault V. J. Gen. Microbiol. 1992; 138: 131-137Crossref PubMed Scopus (33) Google Scholar). For instance, phenolphthiocerol derivatives are produced by only a few strains of M. tuberculosis, notably those belonging to the subspecies M. tuberculosisCanetti (24Daffé M. Lacave C. Lanéelle M.-A. Lanéelle G. Eur. J. Biochem. 1987; 167: 155-160Crossref PubMed Scopus (116) Google Scholar, 25Daffé M. Cho S.-N. Chatterjee D. Brennan P.J. J. Inf. Dis. 1991; 163: 161-168Crossref PubMed Scopus (24) Google Scholar). We investigated the intraspecies differences in the production of PGLs in M. tuberculosis. We demonstrate the occurrence of glycosylated p-hydroxybenzoic acid methyl esters structurally related to the type-specific PGLs in the culture media of all the strains of M. tuberculosis, suggesting that the strains devoid of phenolphthiocerol derivatives are unable to elongate the putative p-hydroxybenzoic acid precursor (p-HBA). We also provide evidence that the polyketide synthase gene pks15/1 is involved in the elongation of p-hydroxybenzoic acid derivatives (p-HBAD) to form p-hydroxyphenylalkanoates, which in turn are converted, presumably by the PpsA-E synthase, to phenolphthiocerol derivatives. This demonstration is based on the observation that: (i) p-hydroxybenzoate derivatives are produced in every tested strains of M. tuberculosis, (ii) that the disruption of the pks15/1 gene ofM. bovis BCG abolished the production of the M. bovis-specific PGL but not that of DIMs, and (iii) that the production of PGL-tb by M. tuberculosis H37Rv can be restored by introducing the pks15/1 gene ofM. bovis BCG. Consistent with this result is the finding that strains of M. tuberculosis devoid of PGL are natural mutants with a frameshift mutation inpks15/1. M. tuberculosis H37Rv (ATCC 27294), H37Ra (ATCC 25177), Erdman (ATCC 35801), R1Rv (ATCC 35818), R1Ra (ATCC 35819), Canetti (Collection Institut Pasteur 140010059), Mt103 (a clinical isolate from Institut Pasteur, Paris), and M. bovis BCG 1173P2 (the Pasteur strain) were grown at 37 °C on Sauton's medium as surface biofilm for biochemical analyses. M. tuberculosis H37Rv and M. bovis BCG were also grown on Middlebrook 7H9 medium (Difco) supplemented with ADC (0.2% dextrose, 0.5% bovine serum albumin fraction V, 0.0003% beef catalase) and 0.05% Tween 80 where indicated, or on solid Middlebrook 7H11 medium (Difco) supplemented with OADC (0.005% oleic acid, 0.2% dextrose, 0.5% bovine serum albumin fraction V, 0.085% NaCl, 0.0003% beef catalase). Kanamycin (km), hygromycin (Hyg), and sucrose were added when required to final concentrations of 40 μg/ml, 50 μg/ml, and 2% (w/v), respectively. Ten additional M. tuberculosis strains (kindly provided by Dr. V. Vincent, Pasteur Institute, Paris) were also grown on Lowenstein Jensen slants at 37 °C for sequencing analysis and PGLs production analysis. Mycobacterial DNA was extracted from 5 ml of saturated cultures as described previously (26Belisle J.T. Sonnenberg M.G. Parish T. Stoker N.G. Mycobacteria protocols. 101. Humana Press Inc., Totowa1998: 31-44Google Scholar). DNA pellets were resuspended in 100 μl of 10 mm Tris (pH 8) buffer. For sequencing of the pks15/1 junction in various mycobacterial strains, a loop full of bacteria from a Lowenstein Jensen slant was resuspended in 100 μl of deionized water and lysed by 5 cycles of freezing in liquid nitrogen and boiling. 5 μl of this crude extract was used to perform the PCR amplification. Primers used for PCR amplification of pks15/1 were pks1I (5′-GCAGGCGATGCGTCATGGGG-3′) and pks1J (5′-TCTTGCCCACCGACCCTGGC-3′). PCR reaction was performed in a final volume of 50 μl containing 2.5 units of Taq DNA polymerase (Roche Molecular Biochemicals, Meylan, FR), 10% Me2SO, and 1 mm each primer. The amplification program consisted of one cycle of 10 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 30 s at 57 °C, 30 s at 72 °C, and a final 10 min at 72 °C. The PCR products were analyzed by electrophoresis in 0.8% agarose gels. The 520-bp fragments were purified using the QiaQuick purification kit (Qiagen, Courtaboeuf, FR) and sequenced using the same primers (ESGS Qbiogene, Evry, FR). M. tuberculosis strain CDC1551 and strain 210 DNA sequences were obtained from the Institute for Genomic Research website at www.tigr.org and M. bovis strain AF2122/97 DNA sequence (spoligotype 9) from the Sanger Institute website at www.sanger.ac.uk. Multiple sequence alignments were performed using ClustalW (27Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56001) Google Scholar) at the Pasteur Institute Website www.pasteur.fr. Sequences in the pks15/1 regions of M. tuberculosis and M. leprae were compared using the Blast 2 program (28Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60228) Google Scholar) at the Pasteur Institute website www.pasteur.fr. A DNA fragment from pks15/1 was amplified by PCR using M. tuberculosis H37Rv genomic DNA and primers pks1A (5′-GCTCAGAGTTTAAACCGAGCATGCCTTGTTGGG-3′) and pks1B (5′-GCTCTAGAGTTTAAACGCGTTTGCCGCCGAGTA-3′). The PCR reaction was performed in a final volume of 50 μl containing 2.5 units ofTaq DNA polymerase (Roche Molecular Biochemicals, Meylan, FR), 10% Me2SO, and 1 mm each primer. The amplification program consisted of one cycle of 10 min at 95 °C followed by 30 cycles of 1 min at 95 °C, 1 min at 60 °C and 1 min at 72 °C, and a final 10 min at 72 °C. The PCR product was analyzed by electrophoresis in 0.8% agarose gel. The ∼2.7-kb fragment was gel-purified using the QiaQuick gel extraction purification kit and inserted into the pGEM-T easy vector according to the manufacturer's recommendations (Promega, Lyon, FR) to give pCG125. A kanamycin resistance cassette formed by the Ωkm cassette from pHP45Ωkm (29Fellay R. Frey J. Krisch H.M. Gene (Amst.). 1987; 52: 147-154Crossref PubMed Scopus (567) Google Scholar) flanked by two res site from transposon γδ was inserted between the two XhoI sites ofpks15/1 generating a 1068-bp deletion. The resulting plasmid was named pCG134. The 4.5-kb PmeI fragment from pCG134 containing the disrupted pks15/1construct was inserted at the SmaI site of pJQ200 (30Quandt J. Hynes M.F. Gene (Amst.). 1993; 127: 15-21Crossref PubMed Scopus (839) Google Scholar) leading to pWM08. M. bovis BCG was electrotransformed as previously described, and transformants were selected on 7H11+ADC+km (31Pelicic V. Reyrat J.-M. Gicquel B. Mol. Microbiol. 1996; 20: 919-925Crossref PubMed Scopus (136) Google Scholar). In three independent experiments a total of 76 kmR transformants were obtained and screened by PCR using primers pks1C (5′-CAGCGACGGCGGTTTTGGGA-3′), pks1D (5′-AGCCAGGACTGCAACACCTC-3′), pks1E (5′-ACCCCACGCCAGTGATATCC-3′), Ωkm1 (5′-GTGGATGACCTTTTGAATGACC-3′), Ωkm2 (5′-TTGGCGAAGTAATCGCAACATC-3′), res1 (5′-GCTCTAGAGCAACCGTCCGAAATATTATAAA-3′) or res2 (5′-GCTCTAGATCTCATAAAAATGTATCCTAAATCAAATATC-3′). The amplification program consisted of one cycle of 10 min at 95 °C followed by 30 cycles of 1 min at 95 °C, 1 min at 55 °C and 1 min at 72 °C, and a final 10 min at 72 °C. One clone gave the pattern corresponding to the insertion of pWM08 via a single homologous recombination event. This strain was renamed PMM1 and grown in 5 ml of 7H9, ADC, km, Tween at 37 °C until saturation. Dilutions of this culture were plated on 7H11, OADC, km, sucrose and incubated at 37 °C for 3 weeks. Four clones were then analyzed further. Saturated 5-ml liquid cultures of these four clones were prepared, and total DNA was extracted. The four DNA preparations were then analyzed using primers pks1C, pks1D, pks1E, res1, and res2. Plasmid pMIP12H is anEscherichia coli-mycobacteria shuttle vector derived from pAL5000. It contains an hygromycin resistance gene and a mycobacterial promotor, pBlaF*, upstream from a multicloning site itself upstream from a transcription terminator (32Le Dantec C. Winter N. Gicquel B. Vincent V. Picardeau M. J. Bacteriol. 2001; 183: 2157-2164Crossref PubMed Scopus (79) Google Scholar). To clone the genepks15/1 from M. bovis BCG, we used two different strategies. First the gene pks15/1 was amplified from M. bovis BCG as two fragments using primers pks15A (5′-GGACTAGTAGATCTATAGAGGAGCAACGGACGATG-3′) and pks1F (5′-GGACTAGTCCAGCAAAACGGCAGTCTCG-3′) for the 5′-end of the gene and pks1G (5′-GGACTAGTACCGAATGGGGATGCATCCG-3′) and pks1H (5′-GGACTAGTATGCAGGTTGATTGGATCACG-3′) for the 3′-end. The PCR was performed in a final volume of 50 μl containing 2.5 units ofPfu DNA polymerase (Promega, Lyon, FR), 10% Me2SO, and 1 mm of each primer. The amplification program consisted of one cycle of 10 min at 95 °C followed by 30 cycles of 1 min at 95 °C, 1 min at 57 °C and 5 min at 72 °C, and a final 10 min at 72 °C. The PCR products were analyzed by electrophoresis in 0.8% agarose gel. The ∼3.4-kb and ∼2.9-kb fragments were gel-purified using the QiaQuick gel extraction purification kit. The 5′-PCR fragment was digested withBglII and SpeI and inserted between theBamHI and SpeI of pMIP12H to give pWM15. The 3′-PCR fragment was digested with NsiI and SpeI and ligated to pWM15 cut with NsiI and SpeI. The resulting plasmid, pWM16, contains the entirepks15/1 gene from M. bovis BCG under the control of the pBlaF* promotor and 7-bp downstream from a Shine-Dalgarno sequence. In the second strategy, we used the M. bovis BCG BAC library constructed by Brosch et al.(33Brosch R. Gordon S.V. Billault A. Garnier T. Eiglmeier K. Soravito C. Barrel B.G. Cole S.T. Infect. Immun. 1998; 66: 2221-2229Crossref PubMed Google Scholar). An 8-kbp fragment was obtained from BAC X203 following digestion with enzymes NruI and StuI. This fragment containing the entire pks15/1 gene from M. bovisBCG was gel-purified using QiaQuick gel extraction kit and inserted into pBluescript KS leading to pWM11. This plasmid was digested withSpeI and AseI and the 8.3-kbp fragment containing the entire pks15/1 gene was gel-purified and cloned into pMIP12H digested with SpeI and NdeI. The resulting plasmid, named pPET1, contains the genepks15/1 in the same orientation as thepBlaF* promotor. Mycobacteria were separated from the culture media and both were kept. The media were sterilized by filtration through 0.2-μm pore size membrane and concentrated to one-tenth of the initial volumes. The cells were left in CHCl3/CH3OH 2:1, v/v, for 2 days at room temperature to kill bacteria, and lipids were extracted twice with CHCl3/CH3OH, 1:1, v/v, washed twice with water, and dried. Lipids from culture media were obtained by adding 2 volumes of CH3OH and 1 volume of CHCl3 to 0.8 volume of concentrated culture medium to yield a homogenous one-phase mixture (34Bligh E. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43126) Google Scholar). After a 2 h-period, the mixture was partitioned into two phases by the addition of 1 volume of H2O/CHCl3, 1:1, v/v. The organic phases were recovered, washed twice with water, and dried. The production of DIMs and glycolipids by the various strains was analyzed by thin-layer chromatography (TLC). Briefly, the various extracts were dissolved in CHCl3 to give a final lipid concentration of 20 mg/ml. Equivalent volumes of each extract were deposited on silica gel G 60 plates (20 × 20 cm, Merck), which were run in petroleum ether-diethylether (90:10, v/v) and CHCl3/CH3OH (95:5, v/v) for the detection of DIMs and PGLs, respectively. Glycolipids and DIMs were visualized by spaying the plates with 0.2% anthrone (w/v) in concentrated H2SO4 and H2SO4, respectively, following by heating. Glycolipids were purified as previously described (24Daffé M. Lacave C. Lanéelle M.-A. Lanéelle G. Eur. J. Biochem. 1987; 167: 155-160Crossref PubMed Scopus (116) Google Scholar). Crude lipid extracts from cells or culture medium were chromatographed on a Florisil (60–100 mesh) column and eluted with a series of concentrations of CH3OH (0, 1, 2, 3, 4, 5, 10, 50, and 100%) in CHCl3. Each fraction was analyzed by TLC on silica gel G 60 plates (0.3 mm, 20 × 20 cm, Merck) using CHCl3/CH3OH, 95:5, v/v as the solvent system. Glycolipids were visualized by spraying the plates with 0.2% anthrone (w/v) in concentrated H2SO4, followed by heating. When necessary, glycolipids were additionally purified by preparative chromatography on silica gel G 60 plates (0.3 mm, 20 × 20 cm, Merck) using CHCl3/CH3OH, 95:5, v/v as the developing solvent. Chemical shift obtained by NMR spectroscopy were assigned using two-dimensional homo- and heteronuclear experiments at 500.13 MHz for proton and 125.76 MHz for carbon, using chloroform as reference both for protons (7.27 ppm) and for carbon (77.0 ppm). Spectra were recorded at 295 K on a Bruker DMX 500. The double quantum-filtered chemical-shift correlated spectroscopy (DQF-COSY) experiment was performed using the Bruker standard pulse-field gradient program cosygpmf (35Ancian B. Bourgeois I. Dauphin J.-F. Shaw A.A. J. Magn. Res. 1997; 125: 348-354Crossref Scopus (48) Google Scholar), with 0.41-s acquisition time, 4096 data points in the F2 dimension and 512 increments in the F1 dimension. The data matrix was zero-filled in the F1 dimension to give a matrix of 4096 × 4096 points, and a shifted sine-bell apodization function was applied prior to Fourier transformation. Similarly, nuclear Overhauseur spectroscopy (NOESY) (36Kumar A. Ernst R.R. Wüthrich K. Biochem. Biophys. Res. Commun. 1980; 95: 1-6Crossref PubMed Scopus (2031) Google Scholar) was performed using the Bruker standard program nosesyst with a spin lock time of 200 ms. The heteronuclear Multiple Quantum Correlation (HMQC) and Multiple Bound Correlation (HMBC) were performed using the standard pulse-field gradient programs inv4gp and inv4gplrnd (37Bax A. Griffey R.H. Hawkins B.L. J. Magn. Res. 1983; 55: 301Google Scholar). MALDI mass spectrometry was performed using a voyager DE-STR MALDI-TOF instrument (PerSeptive Biosystems) equipped with a pulse nitrogen laser emitting at 337 nm as previously described (38Laval F. Lanéelle M.-A. Deon C. Montsarrat B. Daffé M. Anal. Chem. 2001; 73: 4537-4544Crossref PubMed Scopus (101) Google Scholar). Samples were analyzed in the Reflector mode using an extraction delay time set at 100 ns and an accelerating voltage operating in positive ion mode of 20 kV. The mass spectra were mass assigned by external calibration. Samples (1 μl of a 1 mg/ml solution in CHCl3) were directly applied onto the sample plate. The matrix solution (0.5 μl of 2,5-dihydroxybenzoic acid at 10 mg/ml in CHCl3/CH3OH (1:1, v/v)) was added. The samples were then allowed to crystallize at room temperature. Lipid samples were methanolized using CH3OH/HCl prepared by the acetyl chloride reaction on methanol in anhydrous conditions. Samples were dissolved in 500 μl of CH3OH/HCl (1 n) and incubated overnight at 80 °C under nitrogen atmosphere. The solvent was evaporated under nitrogen and then co-evaporated three times with anhydrous CH3OH. For trimethylsilyl (TMS) derivatization, samples were dissolved in 200 μl of anhydrous pyridine, followed by the addition of 100 μl of hexamethyldisilazane and 50 μl of trimethylchlorosilane (39Sweeley C.C. Bentley R. Makita M. Wells W.W. J. Am. Chem. Soc. 1963; 85: 2497-2507Crossref Scopus (2158) Google Scholar). The reaction was incubated at room temperature for 30 min. The mixture was dried under nitrogen, and the TMS derivatives were solubilized in either diethylether, for gas chromatography (GC) and GC-mass spectrometry (GC-MS), or in CHCl3 for MALDI-mass spectrometry. For transesterification, 200 μl of sodium ethanolate was added to dried samples (100 μg) under anhydrous conditions. The reaction was incubated at room temperature for 30 min and then stopped by adding acetic acid. The sample was dried, and lipids were extracted with CHCl3 and wa
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