Phosphatidylinositol Is an Essential Phospholipid of Mycobacteria
2000; Elsevier BV; Volume: 275; Issue: 39 Linguagem: Inglês
10.1074/jbc.m004658200
ISSN1083-351X
AutoresMary Jackson, Dean C. Crick, Patrick J. Brennan,
Tópico(s)Antibiotic Resistance in Bacteria
ResumoPhosphatidylinositol (PI) and metabolically derived products such as the phosphatidylinositol mannosides and linear and mature branched lipomannan and lipoarabinomannan are prominent phospholipids/lipoglycans of Mycobacterium sp. believed to play important roles in the structure and physiology of the bacterium as well as during host infection. To determine if PI is an essential phospholipid of mycobacteria, we identified the pgsA gene of Mycobacterium tuberculosis encoding the phosphatidylinositol synthase enzyme and constructed a pgsAconditional mutant of Mycobacterium smegmatis. The ability of this mutant to synthesize phosphatidylinositol synthase and subsequently PI was dependent on the presence of a functional copy of the pgsA gene carried on a thermosensitive plasmid. The mutant grew like the control strain under permissive conditions (30 °C), but ceased growing when placed at 42 °C, a temperature at which the rescue plasmid is lost. Loss of cell viability at 42 °C was observed when PI and phosphatidylinositol dimannoside contents dropped to ∼30 and 50% of the wild-type levels, respectively. This work provides the first evidence of the essentiality of PI to the survival of mycobacteria. PI synthase is thus an essential enzyme of Mycobacterium that shows promise as a drug target for anti-tuberculosis therapy. Phosphatidylinositol (PI) and metabolically derived products such as the phosphatidylinositol mannosides and linear and mature branched lipomannan and lipoarabinomannan are prominent phospholipids/lipoglycans of Mycobacterium sp. believed to play important roles in the structure and physiology of the bacterium as well as during host infection. To determine if PI is an essential phospholipid of mycobacteria, we identified the pgsA gene of Mycobacterium tuberculosis encoding the phosphatidylinositol synthase enzyme and constructed a pgsAconditional mutant of Mycobacterium smegmatis. The ability of this mutant to synthesize phosphatidylinositol synthase and subsequently PI was dependent on the presence of a functional copy of the pgsA gene carried on a thermosensitive plasmid. The mutant grew like the control strain under permissive conditions (30 °C), but ceased growing when placed at 42 °C, a temperature at which the rescue plasmid is lost. Loss of cell viability at 42 °C was observed when PI and phosphatidylinositol dimannoside contents dropped to ∼30 and 50% of the wild-type levels, respectively. This work provides the first evidence of the essentiality of PI to the survival of mycobacteria. PI synthase is thus an essential enzyme of Mycobacterium that shows promise as a drug target for anti-tuberculosis therapy. phosphatidylethanolamine phosphatidylserine phosphatidylinositol phosphatidylglycerol cardiolipin phosphatidylinositol mannoside phosphatidylinositol dimannoside lipomannan diacylglycerol polymerase chain reaction kilobase open reading frame 4-morpholinepropanesulfonic acid 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid kanamycin kanamycin-resistant sucrose sucrose-resistant streptomycin streptomycin-resistant The increased incidence of tuberculosis during the last decades and the emergence of multidrug-resistant strains have made it clear that there is a need for new chemotherapeutic agents (1Blanchard J.S. Annu. Rev. Biochem. 1996; 65: 215-239Crossref PubMed Scopus (231) Google Scholar). The mycobacterial cell envelope is the site of action of many of the first-line antimycobacterial agents (2Chopra I. Brennan P.J. Tubercle Lung Dis. 1997; 78: 89-98Abstract Full Text PDF PubMed Scopus (55) Google Scholar). A better understanding of the biochemistry and genetics of the pathways leading to the synthesis of envelope components required for cell viability or survival in the host will provide a basis for the rational design of new drugs. Among the potentially attractive drug targets are the enzymes involved in the synthesis of the main mycobacterial phospholipids: phosphatidylethanolamine (PE),1 phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), and cardiolipin (CL) (3Goren M.B. Kubica G.P. Wayne L.G. The Mycobacteria: A Sourcebook. Marcel Dekker, Inc., New York1984: 379-415Google Scholar). It is expected that a deficiency in some of these phosphoglycerides would affect the structural and functional organization of the mycobacterial plasma membrane, presumably resulting in the death of the bacterium. Contrary to PE, PG, PS, and CL, which are frequently encountered in all living organisms, PI is an essential phospholipid of eukaryotic cells (4Nikawa J.-I. Yamashita S. Eur. J. Biochem. 1982; 125: 445-451Crossref PubMed Scopus (42) Google Scholar, 5Nikawa J. Kodaki T. Yamashita S. J. Biol. Chem. 1987; 262: 4876-4881Abstract Full Text PDF PubMed Google Scholar, 6Nikawa J.-I. Yamashita S. Biochim. Biophys. Acta. 1997; 1348: 173-178Crossref PubMed Scopus (40) Google Scholar, 7Antonsson B. Biochim. Biophys. Acta. 1997; 1348: 179-186Crossref PubMed Scopus (54) Google Scholar), but has seldom been found in prokaryotic cells. Actually, the distribution of PI in prokaryotes seems to be confined to some actinomycetes (Mycobacterium,Corynebacterium, Nocardia,Micromonospora, Streptomyces, and Propionibacterium) (3Goren M.B. Kubica G.P. Wayne L.G. The Mycobacteria: A Sourcebook. Marcel Dekker, Inc., New York1984: 379-415Google Scholar, 8Brennan P.J. Lehane D.P. 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Chem. 1997; 272: 18460-18466Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) of which PI constitutes a lipid anchor to the cell envelope, such as phosphatidylinositol mannosides (PIMans) (16Brennan P.J. Ballou C.E. J. Biol. Chem. 1967; 242: 3046-3056Abstract Full Text PDF PubMed Google Scholar), linear lipomannan (LM), (15Besra G.S. Morehouse C.B. Rittner C.M. Waechter C.J. Brennan P.J. J. Biol. Chem. 1997; 272: 18460-18466Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), and mature branched LM and lipoarabinomannan (17Hunter S.W. Gaylord H. Brennan P.J. J. Biol. Chem. 1986; 261: 12345-12351Abstract Full Text PDF PubMed Google Scholar), are prominent and important phospholipids/lipoglycans. PI and PIMans are regarded as essential for membrane stability and thus for cell viability (3Goren M.B. Kubica G.P. Wayne L.G. The Mycobacteria: A Sourcebook. Marcel Dekker, Inc., New York1984: 379-415Google Scholar). Lipoarabinomannan is an important modulator of the immune response in the course of tuberculosis and leprosy (18Chatterjee D. Khoo K.-H. Glycobiology. 1998; 8: 113-120Crossref PubMed Scopus (297) Google Scholar) as well as a key ligand in the interactions between Mycobacterium tuberculosis and macrophages that ultimately may facilitate the survival of the tubercle bacillus within phagocytic cells (19Schlesinger L.S. Hull S.R. Kaufman T.M. J. Immunol. 1994; 152: 4070-4079PubMed Google Scholar). Finally, although PI is regarded as an essential component of the mycobacterial cell wall, positive proof of an essential role of this molecule was lacking. Previously,de novo synthesis of PI in mycobacterial cell wall extracts had been described involving the exchange of the CMP moiety of CDP-diacylglycerol (DAG) for inositol (20Salman M. Lonsdale J.T. Besra G.S. Brennan P.J. Biochim. Biophys. Acta. 1999; 1436: 437-450Crossref PubMed Scopus (43) Google Scholar). In the present study, we undertook the identification of the gene encoding the M. tuberculosis PI synthase and addressed the question of the essentiality of this gene through the construction of a conditional mutant of Mycobacterium smegmatis. Escherichia coli XL1-Blue, the strain used in this study for cloning experiments, was routinely propagated in LB-Lennox medium (10 g/liter peptone from casein, 5 g/liter yeast extract, and 5 g/liter sodium chloride; Life Technologies, Inc.) at 37 °C. M. smegmatisstrain mc2155 (21Snapper S.B. Melton R.E. Mustafa S. Kieser T. Jacobs Jr., W.R. Mol. Microbiol. 1990; 4: 1911-1919Crossref PubMed Scopus (1009) Google Scholar) was routinely grown at 30, 37, or 42 °C in LB-Lennox liquid medium supplemented with 0.05% Tween 80. LB-Lennox 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; and streptomycin, 20 μg/ml. When required, 10% sucrose was added to the solid medium. Electrocompetent cells of E. coli XL1-Blue and M. smegmatismc2155 were prepared as described (22Pelicic V. Reyrat J.M. Gicquel B. Mol. Microbiol. 1996; 20: 919-925Crossref PubMed Scopus (135) Google Scholar) and electrotransformed using a Gene Pulser unit (Bio-Rad). Purification of DNA restriction fragments and PCR fragments was performed using the QIAquick nucleotide removal kit, the QIAquick gel extraction kit, and the QIAquick PCR purification kit (QIAGEN Inc., Chatsworth, CA). Plasmids were isolated from E. coli XL1-Blue using the QIAprep miniprep kit (QIAGEN Inc.). Partial M. smegmatis chromosomal DNA libraries were constructed using PstI-, SalI-, and SmaI-digested and dephosphorylated "ready-to-clone" pUC18 vectors (Appligene, Illkirch, France). These libraries were transformed into E. coli XL1, and colonies harboring a plasmid carrying the M. smegmatis pgsA gene were identified by colony hybridization using the M. tuberculosis pgsA gene as a probe (PCR-amplified using primers A1.a/A1.b; see below). Three plasmids were isolated in this way: pUCpgsA.P carrying the M. smegmatis pgsA on a 2.0-kb PstI restriction fragment, pUCpgsA.S carrying the pgsA gene on a 2.6-kb SalI restriction fragment, and pUCpgsA.Sm carrying the pgsA gene on a 4.0-kb SmaI restriction fragment. They were used for DNA sequencing of the M. smegmatis pgsA gene and surrounding ORFs. Southern blot analysis and colony hybridization were performed as described (23Jackson M. Portnoı̈ D. Catheline D. Dumail L. Rauzier J. Legrand P. Gicquel B. Infect. Immun. 1997; 65: 2883-2889Crossref PubMed Google Scholar). Sequences of double-stranded plasmids were obtained by Macromolecular Resources (Colorado State University) using an ABI Prism 377 automated DNA sequencer. DNA sequence comparisons were done by BLAST analysis (National Center for Biotechnology Information) (24Altschul S.F. Gish W. Miller W. Myers E.M. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (70353) Google Scholar). Sequences were assembled and processed by using the DNA Strider program (Commissariat à l'Energie Atomique, Gif-sur-Yvette, France). Standard PCR strategies with Vent DNA polymerase (New England Biolabs, Inc.) were used to amplify the M. tuberculosis H37Rv pgsA,pgsA2, and pgsA3 genes. PCR amplifications consisted of one cycle of denaturation (95 °C, 10 min), followed by 30 cycles of amplification that included denaturation (95 °C, 1 min), annealing (61 °C, 1 min), and primer extension (72 °C, 1 min). The primers used were as follows: A1a (5′-cccccccccatatgagcaagctgcccttcctgtcc-3′) and A1b (5′-cccaagcttccggtcgccctttccaggaatc-3′) for the pgsA gene, A2a (5′-cccccccccatatggagccggtgctcacgcag-3′) and A2b (5′-cccaagcttgccacgttcaccagcgttctg-3′) for the pgsA2 gene, and A3a (5′-cccccccccatatgagcaggtcaacccgttattc-3′) and A3b (5′-cccaagcttgctggcggtctggcggatccc-3′) for the pgsA3 gene. They were designed to provide PCR-amplified fragments containing an NdeI and a HindIII restriction site, enabling direct cloning into the pVV16 expression vector, a derivative of pMV261 (25Stover C.K. de la Cruz V.F. Fuerst T.R. Burlein J.E. Benson L.A. Bennett L.T. Bansal G.P. Young J.F. Lee M.H. Hatfull G.F. Snapper S.B. Barletta R.G. Jacobs Jr., W.R. Bloom B.R. Nature. 1991; 351: 456-460Crossref PubMed Scopus (1199) Google Scholar) harboring a kanamycin resistance marker, a hygromycin resistance marker, the phsp60 promoter, and a six-histidine tag for the expression of C-terminal His6-tagged fusion proteins. The resulting expression vectors, named pVVpgsA, pVVpgsA2, and pVVpgsA3, were transformed into M. smegmatis mc2155, and transformants were selected on LB-Km-hygromycin plates. The expression of the PgsA, PgsA2, and PgsA3 proteins in the recombinant strains of M. smegmatis was checked by immunoblotting using mouse monoclonal anti-His antibodies (Penta-His antibody, QIAGEN Inc.). M. smegmatis crude extracts were prepared by harvesting cultures of the recombinant strains (A600 nm = 0.6–0.7), washing them twice with cold phosphate-buffered saline, subjecting them to probe sonication for 3 min in the form of 3 × 60-s pulses with 60-s cooling intervals between pulses, and removing the unbroken cells and bacterial debris by centrifugation of the sonicate at 10,000 × g for 15 min. Protein concentration was estimated using the BCA protein assay kit (Pierce). SDS-polyacrylamide gel electrophoresis and immunoblot experiments were carried out as described previously (23Jackson M. Portnoı̈ D. Catheline D. Dumail L. Rauzier J. Legrand P. Gicquel B. Infect. Immun. 1997; 65: 2883-2889Crossref PubMed Google Scholar), except that 100 μg of proteins were loaded onto the gels. The protocol used for immunodetection was as recommended by QIAGEN Inc. Transfer membranes were incubated with the anti-His antibody diluted 1:1000 and with an alkaline phosphatase-conjugated goat anti-mouse IgG secondary antibody diluted 1:2000 (Sigma). For radiolabeling of whole M. smegmatis cells with 32Pi,M. smegmatis was grown in LB-Lennox medium supplemented with 0.05% Tween 80, 200 μg/ml myo-inositol, 20 μg/ml glycerol, and kanamycin at 37 °C under agitation. 1 μCi/ml32Pi (carrier-free; specific activity of 1–60 Ci/mmol; NEN Life Science Products) was added to the medium when cultures reached A600 nm = 0.05 (or A600 nm = 0.01 for strain mc2/pVVpgsA3). The incubation was terminated after 24 h when cells reached mid-log phase (early log phase for mc2/pVVpgsA3). Cultures were then pelleted and washed once with phosphate-buffered saline prior to lipid extraction. For radiolabeling of the M. smegmatis conditional mutant with [14C]acetate, the conditional mutant strain was first grown at 30 °C in LB-Lennox medium supplemented with 0.05% Tween 80 and kanamycin. This culture was then diluted in fresh medium containing 0.5 μCi/ml [14C]acetate (specific activity of 54 mCi/mmol; NEN Life Science Products) to A600 nm < 0.1 and placed at 42 °C for different periods of time prior to lipid extraction. A pUC18 vector harboring the M. smegmatis pgsA gene on a 2.0-kb PstI restriction fragment (vector pUCpgsA.P) was isolated by colony hybridization using the M. tuberculosis pgsA gene (PCR-amplified with primers A1.a/A1.b) as a probe. The M. smegmatis pgsA gene and flanking regions were excised from this plasmid on a 1.8-kb SmaI restriction fragment and cloned into the HindIII-cut and blunt-ended pXYL4 vector (a pBluescript derivative carrying the xylE colored marker) (26Pelicic V. Jackson M. Reyrat J.M. Jacobs Jr., W.R. Gicquel B. Guilhot C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10955-10960Crossref PubMed Scopus (386) Google Scholar), yielding plasmid pPGSX. The Km cassette from pUC4K conferring kanamycin resistance and carried on a 1.2-kb HincII fragment was then cloned into the HindIII-cut and blunt-ended pPGSX plasmid, yielding plasmid pPGSXK. Finally, p27PGSXK, the construct used for allelic exchange, was obtained by transferring a 4.2-kb BamHI fragment from pPGSXK containing pgsA:: Km and xylE into BamHI-cut pPR27, a temperature-sensitive mycobacterial vector carrying the counter-selectable marker sacB (26Pelicic V. Jackson M. Reyrat J.M. Jacobs Jr., W.R. Gicquel B. Guilhot C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10955-10960Crossref PubMed Scopus (386) Google Scholar). pCG76, a Mycobacterium/E. coli shuttle plasmid derived from pCG63, harboring a mycobacterial thermosensitive origin of replication and a streptomycin resistance cassette (27Guilhot C. Gicquel B. Martin C. FEMS Microbiol. Lett. 1992; 98: 181-186Crossref Scopus (45) Google Scholar), was used to carry functional copies of the M. smegmatis pgsA gene in the M. smegmatis pgsA mutant. One of these complementing vectors, pCGpis.1, was constructed by inserting the 1.8-kb SmaI restriction fragment from pUCpgsA.P into BamHI-cut and blunt-ended pCG76. The second complementing vector, pCGpis.2, was constructed by cloning the 2.6-kb blunt-ended SalI fragment from pUCpgsA.S into BamHI-cut and blunt-ended pCG76. These temperature-sensitive vectors are able to replicate at 30 °C, but not at 42 °C. M. smegmatiscultures collected at A600 nm = 0.6–0.7 were washed, and cells were resuspended at 4 °C in buffer A (50 mm MOPS (pH 7.9), 5 μm β-mercaptoethanol, and 10 mm MgCl2). The cell suspension was subjected to probe sonication (1-cm probe, Soniprep 150, MSE, Sussex, United Kingdom) for 10 min in the form of 10 × 60-s pulses with 90-s cooling intervals between pulses. After the whole sonicate was centrifuged at 27,000 × g for 15 min at 4 °C, the pellet was resuspended in buffer A. The crude cell wall fraction was obtained upon centrifugation of this resuspended pellet at 3000 rpm for 10 min to remove cell debris and unbroken cells. Proteins were kept frozen in small aliquots at −70 °C. Crude cell wall fractions were assayed for PI synthase activity in a final volume of 400 μl (20Salman M. Lonsdale J.T. Besra G.S. Brennan P.J. Biochim. Biophys. Acta. 1999; 1436: 437-450Crossref PubMed Scopus (43) Google Scholar). Each reaction mixture contained crude cell wall preparation (∼1 mg of protein), 0.1 mm ATP, 5 mm glucose, 300 μm CDP-diacylglycerol (dipalmitoyl; Sigma), 0.4% CHAPS, 22 μCi of [3H]inositol (2.5 μm final concentration; specific activity of 22 Ci/mmol; NEN Life Science Products), and buffer A up to 400 μl. The mixture was incubated at 37 °C for the indicated times. At the end of incubations, the reactions were terminated by the addition of CHCl3/CH3OH (2:1, 3 ml/400 μl of reaction mixture), followed by centrifugation to separate the pellet. The pellet was extracted once more with 3 ml of CHCl3/CH3OH (2:1). The combined extracts were washed once with 0.9% NaCl (1.2 ml) and once with 1 ml of CHCl3/CH3OH/H2O (3:47:48) to yield the washed CHCl3/CH3OH (2:1) lipids. Since no labeled product other than PI was found in the washed lipid extract, quantification of the reaction was performed directly by scintillation counting of the washed lipid extract. Lipids from labeled and unlabeled cells were extracted by two consecutive overnight extractions in 4 ml of CHCl3/CH3OH (2:1). The combined CHCl3/CH3OH (2:1) extracts were washed once with 0.9% NaCl and once with CHCl3/CH3OH/H2O (3:47:48) to yield a fraction containing the phospholipids including PIMan2s. When phosphatidylinositol pentamannosides and linear LM were to be extracted, the insoluble pellet resulting from the CHCl3/CH3OH (2:1) extraction was further extracted overnight with CHCl3/CH3OH/H2O (10:10:3). Characterization of the various PIMans followed earlier work (28Khoo K.-H. Dell A. Morris H.R. Brennan P.J. Chatterjee D. Glycobiology. 1995; 5: 117-127Crossref PubMed Scopus (116) Google Scholar) and was based on fast atom bombardment mass spectrometry analysis, one- and two-dimensional thin-layer chromatographic patterns of the intact PIMans (16Brennan P.J. Ballou C.E. J. Biol. Chem. 1967; 242: 3046-3056Abstract Full Text PDF PubMed Google Scholar, 28Khoo K.-H. Dell A. Morris H.R. Brennan P.J. Chatterjee D. Glycobiology. 1995; 5: 117-127Crossref PubMed Scopus (116) Google Scholar), sugar analysis by gas chromatography, and fatty acid analysis by gas chromatography-mass spectrometry (29Hunter S.W. McNeil M.R. Brennan P.J. J. Bacteriol. 1986; 168: 917-922Crossref PubMed Google Scholar). PIMan2s were analyzed by fast atom bombardment mass spectrometry analysis either directly in the negative ion mode or as acetyl derivatives (100 μl of pyridine/acetic anhydride (1:1, v/v) for 24 h) in the positive ion mode. Fast atom bombardment mass spectrometry analysis was performed on a Fisons VG AutoSpec mass spectrometer with a cesium ion gun operating at 25 kV. Samples (30 μg) were applied to a m-nitrobenzyl alcohol matrix. Triacylated PIMan2 and lyso-PIMan2 were purified by preparative TLC in the solvent systems described below. TLC was conducted in one- and two-dimensions on aluminum-backed plates of Silica Gel 60 F254 (Merck, Darmstadt, Germany). Solvents used for the analysis of phospholipids and PIMan2s were CHCl3/CH3OH/CH3COOH/HCOOH/H2O (35:15:6:2:0.3) and CHCl3/CH3OH/NH4OH/H2O (65:25:0.5:3.6), respectively. Analysis of phosphatidylinositol pentamannosides and linear LM was performed in the same solvent as the one used for PIMan2s. Two-dimensional TLCs were performed using solvent CHCl3/CH3OH/H2O (60:30:6) in the first dimension and solvent CHCl3/CH3OH/CH3COOH/H2O (40:25:3:6) in the second dimension. An α-naphthol spray (1% α-naphthol in ethanol), a Dittmer-Lester spray (30Dittmer J.C. Lester R.L. J. Lipid Res. 1964; 5: 126-127Abstract Full Text PDF PubMed Google Scholar), and a cupric sulfate spray (10% CuSO4 in an 8% phosphoric acid solution) were used to detect carbohydrate-containing lipids, phosphorus-containing lipids, and all organic compounds, respectively. Autoradiograms were obtained by exposing chromatograms to Kodak X-Omat AR films at −70 °C usually for 1–4 days. Plates were also scanned for radioactivity using a Bio-Scan System 200 imaging scanner with Autochanger 3000, or relevant spots were scraped off for scintillation counting. l-α-Phosphatidylethanolamine (Sigma),l-α-phosphatidyl-l-serine (Sigma),l-α-phosphatidylinositol (Sigma),l-α-phosphatidyl-dl-glycerol (Sigma), and cardiolipin (ICN Pharmaceuticals Inc.) commercial standards were used to identify the mycobacterial phospholipids on thin-layer chromatograms. The search for the mycobacterial PI synthase gene was based on the fact that all the enzymes (with the exception of phosphatidylserine synthases of Gram-negative bacteria) capable of catalyzing the transfer of a free alcohol (inositol, serine, or glycerol) onto CDP-diacylglycerol share a common motif in their primary sequence, named the CDP-alcohol phosphatidyltransferase signature (Prosite accession number PS00379). Screening of the M. tuberculosis H37Rv genome (31Cole 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 (6506) Google Scholar) for this motif revealed that four ORFs, pgsA, pgsA2, pgsA3, and pssA, potentially encoded enzymes carrying this signature. The presence of the psd gene encoding a phosphatidylserine decarboxylase responsible for the synthesis of PE from PS adjacent to the pssA gene in addition to sequence similarities the M. tuberculosis PssA enzyme shares with known PS synthases (32Matsumoto K. Biochim. Biophys. Acta. 1997; 1348: 214-227Crossref PubMed Scopus (64) Google Scholar) (Fig. 1 a) strongly suggest that the pssA gene encodes a PS synthase. Therefore, this gene was not considered to be a PI synthase candidate gene. The three other genes, pgsA, pgsA2, and pgsA3, were PCR-amplified and placed under the control of the phsp60 promoter (25Stover C.K. de la Cruz V.F. Fuerst T.R. Burlein J.E. Benson L.A. Bennett L.T. Bansal G.P. Young J.F. Lee M.H. Hatfull G.F. Snapper S.B. Barletta R.G. Jacobs Jr., W.R. Bloom B.R. Nature. 1991; 351: 456-460Crossref PubMed Scopus (1199) Google Scholar) in the mycobacterial expression vector pVV16, yielding plasmids pVVpgsA, pVVpgsA2, pVVpgsA3. These three plasmids were electrotransformed into M. smegmatismc2155. The production of recombinant proteins in each recombinant strain was checked using anti-His antibodies (data not shown). Interestingly, in LB-Km liquid medium at 37 °C, the mc2/pVVpgsA and mc2/pVVpgsA2 strains exhibited the same growth rate as the mc2/pVV16 control strain, whereas mc2/pVVpgsA3 exhibited a much slower growth due to an extended lag period that lasted ≅24 h instead of 10–12 h for all the other strains (data not shown). On LB-Km solid medium at 37 °C, mc2/pVVpgsA3 colonies appeared after 8 days instead of 3 days for the other strains. The phospholipid composition of the three recombinant strains and that of the control strain were analyzed by measuring the distribution of labeled 32Pi among the major lipid classes.32Pi labeling was performed as described under "Experimental Procedures." After 24 h of labeling, when bacterial cultures reached mid-log phase (early log phase for mc2/pVVpgsA3), bacteria were pelleted, and their lipids were extracted and analyzed. This experiment was performed on two different mc2/pVVpgsA transformants, two different mc2/pVVpgsA2 transformants, and one mc2/pVVpgsA3 transformant. TableI shows the radiolabeled phospholipid composition of each strain in one typical experiment. The mc2/pVVpgsA2 strain exhibited the same pattern of32Pi distribution as the mc2/pVV16 control strain. The mc2/pVVpgsA recombinant strain exhibited a slightly higher quantity of PI (PI represented 34% of the total phospholipids as compared with 21–23% for the mc2/pVVpgsA2 and control strains), suggesting that pgsA may encode a PI synthase. The mc2/pVVpgsA3 strain had a completely different pattern of32Pi distribution, with the appearance of a new highly labeled compound that was barely detected in the other strains. This compound comigrated with the phosphatidylglycerol commercial standard, suggesting that pgsA3 encodes a phosphatidylglycerophosphate synthase. This result is consistent with the sequence similarities that the PgsA3 enzyme of M. tuberculosis shares with other known PG synthases (Fig.1 b). Overproduction of PG thus appeared to be the cause of the slow growth of strain mc2/pVVpgsA3. The distribution of 32Pi among other PI-containing compounds such as PIMans and linear LM was identical for all strains (data not shown).Table IPhospholipid composition of M. smegmatis control and recombinant strainsmc2pVV16mc2pVVpgsAmc2pVVpgsA2mc2pVVpgsA3%%%%Origin2.82.31.97.2PIMan24.53.94.76.5PI23.434.521.318.0PE31.629.131.724.1PG0.40.60.323.5CL37.329.640.120.7The 32P1-labeled lipids were extracted and separated by TLC as described under "Experimental Procedures," and radioactivity distribution among phospholipids was determined using a Bio-Scan System 200 imaging scanner. Open table in a new tab The 32P1-labeled lipids were extracted and separated by TLC as described under "Experimental Procedures," and radioactivity distribution among phospholipids was determined using a Bio-Scan System 200 imaging scanner. PI synthase cell-free assays were performed on crude cell wall preparations of each strain. Results are presented in Fig. 2 (a and b). After a 1-h incubation at 37 °C, the cell wall preparation of the mc2/pVVpgsA recombinant strain exhibited a PI synthase activity that was 2–2.5-fold greater than that of the other strains (Fig. 2 a). A comparative time course of PI synthesis by the crude cell wall preparations of mc2/pVVpgsA and mc2/pVV16 further reflected the overproduction of PI synthase in the recombinant strain mc2/pVVpgsA (Fig. 2 b), strongly suggesting that the mycobacterial PI synthase is encoded by the pgsAgene. Although no obvious function could be attributed to the PgsA2 enzyme from these experiments, it is likely that this enzyme is involved in the synthesis of cardiolipin. It has been suggested that mycobacteria possess a eukaryotic type of cardiolipin synthase activity involving the transfer of a phosphatidyl group from CDP-diacylglycerol to PG to form CL and CMP (33Mathur A.K. Suryanarayana-Murthy P. Saharia G.S. Venkitasubramanian T.A. Can. J. Microbiol. 1976; 22: 354-358Crossref PubMed Scopus (14) Google Scholar). In yeast, the Crd1p enzyme (34Schlame M. Greenberg M.L. Biochim. Biophys. Acta. 1997; 1348: 201-206Crossref PubMed Scopus (30) Google Scholar) that catalyzes this reaction carries the same CDP-alcohol phosphatidyltransferase signature as the one found in the M. tuberculosis PgsA, PgsA2, and PgsA3 proteins. Moreover, although it has been suggested that mycobacteria are also able to make CL from the usual prokaryotic reaction, PG + PG → CL + glycerol (33Mathur A.K. Suryanarayana-Murthy P. Saharia G.S. Venkitasubramanian T.A. Can. J. Microbiol. 1976; 22: 354-358Crossref PubMed Scopus (14) Google Scholar), no protein carrying the characteristic amino acid motifs of bacterial cardiolipin synthases (35Tropp B.E. Biochim. Biophys. Acta. 1997; 1348: 192-200Crossref PubMed Scopus (53) Google Scholar) was found in the genome of M. tuberculosis H37Rv. This suggests that the synthesis of CL from PG and CDP-diacylglycerol may be prevalent in mycobacteria and catalyzed by an enzyme of the same
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