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• W2077888858 abstract "Distinct from other spirochetes, cells of Leptospira interrogans contain orthologues of all the Escherichia coli lpx genes required for lipid A biosynthesis, but they synthesize a modified form of lipopolysaccharide that supposedly activates toll-like receptor 2 (TLR2) instead of TLR4. The recent determination of the L. interrogans lipid A structure revealed an unprecedented O-methylation of its 1-phosphate group (Que-Gewirth, N. L. S., Ribeiro, A. A., Kalb, S. R., Cotter, R. J., Bulach, D. M., Adler, B., Saint Girons, I., Werts, C., and Raetz, C. R. H. (2004) J. Biol. Chem. 279, 25420-25429). The enzymatic activity responsible for selective 1-phosphate methylation has not been previously explored. A membrane enzyme that catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to the 1-phosphate moiety of E. coli Kdo2-[4′-32P]lipid A has now been discovered. The gene encoding this enzyme was identified based on the hypothesis that methylation of a phosphate group is chemically analogous to methylation of a carboxylate moiety at a membrane-water interface. Database searching revealed a candidate gene (renamed lmtA) in L. interrogans showing distant homology to the yeast isoprenylcysteine carboxyl methyltransferase, encoded by sterile-14, which methylates the a-type mating factor. Orthologues of lmtA were not present in E. coli, the lipid A of which normally lacks the 1-phosphomethyl group, or in other spirochetes, which do not synthesize lipid A. Expression of the lmtA gene behind the lac promoter on a low copy plasmid resulted in the appearance of SAM-dependent methyltransferase activity in E. coli inner membranes and methylation of about 30% of the endogenous E. coli lipid A. Inactivation of the ABC transporter MsbA did not inhibit methylation of newly synthesized lipid A. Methylated E. coli lipid A was analyzed by mass spectrometry and NMR spectroscopy to confirm the location of the phosphomethyl group at the 1-position. In human cells, engineered to express the individual TLR subtypes, 1-phosphomethyl-lipid A purified from lmtA-expressing E. coli potently activated TLR4 but not TLR2. Distinct from other spirochetes, cells of Leptospira interrogans contain orthologues of all the Escherichia coli lpx genes required for lipid A biosynthesis, but they synthesize a modified form of lipopolysaccharide that supposedly activates toll-like receptor 2 (TLR2) instead of TLR4. The recent determination of the L. interrogans lipid A structure revealed an unprecedented O-methylation of its 1-phosphate group (Que-Gewirth, N. L. S., Ribeiro, A. A., Kalb, S. R., Cotter, R. J., Bulach, D. M., Adler, B., Saint Girons, I., Werts, C., and Raetz, C. R. H. (2004) J. Biol. Chem. 279, 25420-25429). The enzymatic activity responsible for selective 1-phosphate methylation has not been previously explored. A membrane enzyme that catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to the 1-phosphate moiety of E. coli Kdo2-[4′-32P]lipid A has now been discovered. The gene encoding this enzyme was identified based on the hypothesis that methylation of a phosphate group is chemically analogous to methylation of a carboxylate moiety at a membrane-water interface. Database searching revealed a candidate gene (renamed lmtA) in L. interrogans showing distant homology to the yeast isoprenylcysteine carboxyl methyltransferase, encoded by sterile-14, which methylates the a-type mating factor. Orthologues of lmtA were not present in E. coli, the lipid A of which normally lacks the 1-phosphomethyl group, or in other spirochetes, which do not synthesize lipid A. Expression of the lmtA gene behind the lac promoter on a low copy plasmid resulted in the appearance of SAM-dependent methyltransferase activity in E. coli inner membranes and methylation of about 30% of the endogenous E. coli lipid A. Inactivation of the ABC transporter MsbA did not inhibit methylation of newly synthesized lipid A. Methylated E. coli lipid A was analyzed by mass spectrometry and NMR spectroscopy to confirm the location of the phosphomethyl group at the 1-position. In human cells, engineered to express the individual TLR subtypes, 1-phosphomethyl-lipid A purified from lmtA-expressing E. coli potently activated TLR4 but not TLR2. The outer membrane of Gram-negative bacteria is an asymmetric lipid bilayer. The inner monolayer consists of glycerophospholipids, whereas the outer monolayer consists of lipopolysaccharide (LPS). 1The abbreviations used are: LPS, lipopolysaccharide; BSA, bovine serum albumin; ICMT, isoprenylcysteine carboxyl methyltransferase; Kdo, 3-deoxy-d-manno-oct-2-ulosonic acid; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; SAM, S-adenosylmethionine; TLR, toll-like receptor; UDP-GlcNAc3N, UDP-2-acetamido-3-amino-2,3-dideoxy-α-d-glucose. LPS is composed of a saccharolipid anchor (1Fahy E. Subramaniam S. Brown H.A. Glass C.K. Merrill Jr., A.H. Murphy R.C. Raetz C.R.H. Russell D.W. Seyama Y. Shaw W. Shimizu T. Spener F. van Meer G. Vannieuwenhze M.S. White S.H. Witztum J.L. Dennis E.A. J. Lipid Res. 2005; 46: 839-862Abstract Full Text Full Text PDF PubMed Scopus (1141) Google Scholar) termed lipid A, a nonrepeating oligosaccharide core, and a distal polysaccharide (or O-antigen). The minimal LPS required for growth in Escherichia coli consists of lipid A and two 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) residues, designated Kdo2-lipid A. Lipid A is a potent immunostimulant in animals that activates the toll-like receptor 4 (TLR4) (2Beutler B. Hoebe K. Du X. Ulevitch R.J. J. Leukocyte Biol. 2003; 74: 479-485Crossref PubMed Scopus (501) Google Scholar, 3Gioannini T.L. Teghanemt A. Zhang D. Coussens N.P. Dockstader W. Ramaswamy S. Weiss J.P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4186-4191Crossref PubMed Scopus (297) Google Scholar, 4Miller S.I. Ernst R.K. Bader M.W. Nat. Rev. Microbiol. 2005; 3: 36-46Crossref PubMed Scopus (768) Google Scholar), and it is implicated in Gram-negative septic shock (5Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3382) Google Scholar). The Lpx enzymes responsible for the assembly of Kdo2-lipid A have been fully characterized in E. coli (5Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3382) Google Scholar). Several of these enzymes are attractive targets for the design of new antibiotics (6Onishi H.R. Pelak B.A. Gerckens L.S. Silver L.L. Kahan F.M. Chen M.-H. Patchett A.A. Galloway S.M. Hyland S.A. Anderson M.S. Raetz C.R.H. Science. 1996; 274: 980-982Crossref PubMed Scopus (357) Google Scholar, 7Coggins B.E. Li X. McClerren A.L. Hindsgaul O. Raetz C.R.H. Zhou P. Nat. Struct. Biol. 2003; 10: 645-651Crossref PubMed Scopus (93) Google Scholar). The lpx genes are well conserved among Gram-negative bacteria, despite some variations in lipid A structure. Additional modifying enzymes, not present in E. coli, are responsible for generating most of the structural diversity, and they generally function late in the biosynthetic pathway (8Brozek K.A. Kadrmas J.L. Raetz C.R.H. J. Biol. Chem. 1996; 271: 32112-32118Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 9Basu S.S. Karbarz M.J. Raetz C.R.H. J. Biol. Chem. 2002; 277: 28959-28971Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 10Que-Gewirth N.L.S. Karbarz M.J. Kalb S.R. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 2003; 278: 12120-12129Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 11Karbarz M.J. Kalb S.R. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 2003; 278: 39269-39279Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 12Sweet C.R. Ribeiro A.A. Raetz C.R.H. J. Biol. Chem. 2004; 279: 25400-25410Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 13Wang X. Karbarz M.J. McGrath S.C. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 2004; 279: 49470-49478Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). One example of a bacterium with an unusual, modified lipid A is Leptospira interrogans, a spirochete responsible for causing leptospirosis in humans. This disease is a problem in highly populated, tropical urban centers, and its clinical presentations range from flu-like symptoms to fatal kidney, liver, or pulmonary damage (14Pereira M.M. Matsuo M.G.S. Bauab A.R. Vasconcelos S.A. Moraes Z.M. Baranton G. Saint Girons I. J. Clin. Microbiol. 2000; 38: 450-452PubMed Google Scholar, 15Levett P.N. Clin. Microbiol. Rev. 2001; 14: 296-326Crossref PubMed Scopus (2231) Google Scholar). Typical O-antigen gene clusters and orthologues of nearly all of the E. coli lpx genes are present in the genomes of various Leptospira (16Bulach D.M. Kalambaheti T. de la Pena-Moctezuma A. Adler B. J. Mol. Microbiol. Biotechnol. 2000; 2: 375-380PubMed Google Scholar, 17Ren S.-X. Fu G. Jiang X.-G. Zeng R. Miao Y.-G. Xu H. Zhang Y.-X. Xiong H. Lu G. Lu L.-F. Jiang H.-Q. Jia J. Tu Y.-F. Jiang J.-X. Gu W.-Y. Zhang Y.-Q. Cai Z. Sheng H.-H. Yin H.-F. Zhang Y. Zhu G.-F. Wan M. Huang H.-L. Qian Z. Wang S.-Y. Ma W. Yao Z.-J. Shen Y. Qiang B.-Q. Xia Q.-C. Guo X.-K. Danchin A. Saint Girons I. Somerville R.L. Wen Y.-M. Shi M.-H. Chen Z. Xu J.-G. Zhao G.-P. Nature. 2003; 422: 888-893Crossref PubMed Scopus (484) Google Scholar). The presence of LPS is a major distinguishing feature that sets L. interrogans apart from the other spirochetes (such as Treponema pallidum, Treponema denticola, and Borrelia burgdorferi), perhaps explaining why L. interrogans is easily cultivated outside of its host. The structure of L. interrogans lipid A has been elucidated recently using mass spectrometry, NMR spectroscopy, and biochemical analysis (18Que-Gewirth N.L.S. Ribeiro A.A. Kalb S.R. Cotter R.J. Bulach D.M. Adler B. Saint Girons I. Werts C. Raetz C.R.H. J. Biol. Chem. 2004; 279: 25420-25429Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Fig. 1 illustrates the key structural differences between E. coli and L. interrogans lipid A. E. coli lipid A is a β,1′-6-linked disaccharide of glucosamine that is phosphorylated at the 1- and 4′-positions and is acylated with (R)-3-hydroxymyristate at the 2-, 3-, 2′-, and 3′-positions (Fig. 1A) (5Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3382) Google Scholar). The 2′- and 3′-linked fatty acyl chains are further esterified with secondary laurate and myristate chains, respectively. The structure of L. interrogans lipid A is a β,1′-6-linked disaccharide, consisting of the glucosamine analogue 2,3-diamino-2,3-dideoxy-d-glucopyranose (Fig. 1B) (18Que-Gewirth N.L.S. Ribeiro A.A. Kalb S.R. Cotter R.J. Bulach D.M. Adler B. Saint Girons I. Werts C. Raetz C.R.H. J. Biol. Chem. 2004; 279: 25420-25429Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). L. interrogans lipid A is acylated with R-3-hydroxylaurate at the 3- and 3′-positions and with R-3-hydroxypalmitate at the 2- and 2′-positions. The secondary acyl chains most likely are 12 or 14 carbons in length, and each contains one double bond. As in many strains of Rhizobium and Francisella, the 4′-phosphate group is missing in L. interrogans lipid A. However, the most unusual property of L. interrogans lipid A is the presence of a methylated 1-phosphate moiety. The proposed biosynthetic pathway for the assembly of L. interrogans Kdo2-lipid A is diagrammed in Fig. 2. For the most part, the pathway is catalyzed by orthologues of the E. coli lpx gene products. However, there are at least four additional genes that are required in the L. interrogans system. The first two, gnnA and gnnB, were originally discovered in Acidithiobacillus ferrooxidans because of their location between lpxA and lpxB (12Sweet C.R. Ribeiro A.A. Raetz C.R.H. J. Biol. Chem. 2004; 279: 25400-25410Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Together, these gene products function to synthesize the sugar nucleotide UDP-2-acetamido-3-amino-2,3-dideoxy-α-d-glucose (UDP-GlcNAc3N). GnnA catalyzes the oxidation of the glucosamine 3-OH of UDP-GlcNAc, and GnnB catalyzes the subsequent transamination to form UDP-GlcNAc3N. LpxA from L. interrogans is absolutely specific for UDP-GlcNAc3N versus UDP-GlcNAc (19Sweet C.R. Williams A.H. Karbarz M.J. Werts C. Kalb S.R. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 2004; 279: 25411-25419Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). E. coli LpxA uses both UDP-GlcNAc and UDP-GlcNAc3N in vitro, but it cannot synthesize the latter, because it lacks the gnnA and gnnB genes. Consequently, L. interrogans lipid A contains four N-linked acyl chains, whereas E. coli has only two. The chemical structure of L. interrogans lipid A (Fig. 1B) implies that two additional lipid A-processing enzymes must be present in this organism. A 1-methyltransferase and a 4′-phosphatase are proposed to methylate the 1-phosphate group and dephosphorylate the 4′-position, respectively (Fig. 2). Methylated phosphate residues are relatively uncommon in biology (20Freeze H.H. Hindsgaul O. Ichikawa M. J. Biol. Chem. 1992; 267: 4431-4439Abstract Full Text PDF PubMed Google Scholar, 21Shimba S. Reddy R. J. Biol. Chem. 1994; 269: 12419-12423Abstract Full Text PDF PubMed Google Scholar). There is only one well characterized example of a methylated phospholipid, a methylated phosphatidylglycerophosphate analogue found in the halophile Halobacterium salinarium (22Kates M. Moldoveanu N. Stewart L.C. Biochim. Biophys. Acta. 1993; 1169: 46-53Crossref PubMed Scopus (60) Google Scholar). In the field of lipid A biochemistry, a methylated phosphate moiety is without precedent (5Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3382) Google Scholar). The significance of the distinct lipid A structure seen in L. interrogans is unknown. A recent study suggested that leptospiral LPS might activate an alternative TLR as compared with the LPS from E. coli and most other Gram-negative bacteria (23Werts C. Tapping R.I. Mathison J.C. Chuang T.-H. Kravchenko V. Saint Girons I. Haake D.A. Godowski P.J. Hayashi F. Ozinsky A. Underhill D.M. Kirschning C.J. Wagner H. Aderem A. Tobias P.S. Ulevitch R.J. Nat. Immunol. 2001; 2: 346-352Crossref PubMed Scopus (573) Google Scholar). In the case of E. coli (2Beutler B. Hoebe K. Du X. Ulevitch R.J. J. Leukocyte Biol. 2003; 74: 479-485Crossref PubMed Scopus (501) Google Scholar, 3Gioannini T.L. Teghanemt A. Zhang D. Coussens N.P. Dockstader W. Ramaswamy S. Weiss J.P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4186-4191Crossref PubMed Scopus (297) Google Scholar), LPS first interacts with the LPS-binding protein, which delivers the LPS to the GPI-linked peripheral membrane protein, CD14. LPS is then brought into contact with the integral membrane protein, TLR4, and the accessory protein, MD-2. Upon activation, MyD88 is recruited to the cytoplasmic tail of TLR4, which in turn triggers a series of events that culminates in the translocation of NF-κB to the nucleus and the transcriptional activation of numerous cytokine genes. However, Werts et al. (23Werts C. Tapping R.I. Mathison J.C. Chuang T.-H. Kravchenko V. Saint Girons I. Haake D.A. Godowski P.J. Hayashi F. Ozinsky A. Underhill D.M. Kirschning C.J. Wagner H. Aderem A. Tobias P.S. Ulevitch R.J. Nat. Immunol. 2001; 2: 346-352Crossref PubMed Scopus (573) Google Scholar) reported that L. interrogans LPS instead activates TLR2. It is tempting to speculate that the apparent differences in TLR activation between L. interrogans and E. coli are due to the structural characteristics of their respective lipid A molecules. Identification of the L. interrogans genes and enzymes responsible for some of these structural variations should provide helpful tools for investigating this hypothesis. Here, it is reported that a novel L. interrogans membrane enzyme, designated LmtA, catalyzes the selective transfer of a methyl group in vitro from SAM to the 1-phosphate residue of Kdo2-lipid A. When LmtA is expressed in E. coli, a modified lipid A species is synthesized in vivo, which is shown to be the 1-phosphomethyl derivative of E. coli lipid A. Lipid A methylation probably occurs on the cytoplasmic face of the inner membrane, since it is independent of MsbA function. The addition of the methyl group to E. coli lipid A does not alter its potent, TLR4-specific bioactivity. Materials—[γ-32P]ATP, 32Pi, [glycerol-U-14C]phosphatidic acid, and [glycerol-U-14C]glycerol-3-phosphate were purchased from PerkinElmer Life Sciences, and Silica Gel 60 (0.25-mm) TLC plates were obtained from Merck. Tryptone and yeast extract were from Difco, whereas chloroform, ammonium acetate, and sodium acetate were from EM Science. Triton X-100 and the BCA protein determination kit were purchased from Pierce. LPS derived from E. coli strain O111:B4 was purchased from Sigma and reextracted by phenol chloroform as described (24Hirschfeld M. Ma Y. Weis J.H. Vogel S.N. Weis J.J. J. Immunol. 2000; 165: 618-622Crossref PubMed Scopus (971) Google Scholar). Pam2CysK4 was from EMC Microcollection GmbH (Tubingen, Germany), and human IL-1β was purchased from Peprotech (Rocky Hill, NJ). All other reagent grade chemicals were obtained from Sigma or Mallinckrodt. Bacterial Strains and Growth Conditions—All bacterial strains used in this study are described in Table I. L. interrogans serovar icterohemeorrhagiae (strain Verdun) cell pellets were kindly provided by Catherine Werts (Institut Pasteur) (18Que-Gewirth N.L.S. Ribeiro A.A. Kalb S.R. Cotter R.J. Bulach D.M. Adler B. Saint Girons I. Werts C. Raetz C.R.H. J. Biol. Chem. 2004; 279: 25420-25429Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). E. coli strains were grown at 37 °C in LB medium containing 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl per liter (25Miller J.R. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar). Overnight cultures were grown from a single colony and used to inoculate LB cultures of varying volume (initial A600 = 0.01) containing 1 mm isopropyl-1-thio-β-d-galactopyranoside. Cultures were grown to A600 = 1 before harvesting. Ampicillin was added at 100 μg/ml when required for plasmid selection.Table IRelevant bacterial strains and plasmidsStrain/PlasmidDescriptionSource or referenceStrainsL. interrogans Strain VerdunAvirulent variant of serovar icterohaemorrhagiaeRef. 23Werts C. Tapping R.I. Mathison J.C. Chuang T.-H. Kravchenko V. Saint Girons I. Haake D.A. Godowski P.J. Hayashi F. Ozinsky A. Underhill D.M. Kirschning C.J. Wagner H. Aderem A. Tobias P.S. Ulevitch R.J. Nat. Immunol. 2001; 2: 346-352Crossref PubMed Scopus (573) Google ScholarE. coli XL1 Blue-MRmcrABC recA1 endA1 gyrA96 relA1 supE44 thi-1 lacStratagene W3110Wild-type, F-, λ-E. coli Genetic Stock Center (Yale) W3110AWild-type, F-, λ-, aroA::Tn10Ref. 37Doerrler W.T. Gibbons H.S. Raetz C.R.H. J. Biol. Chem. 2004; 279: 45102-45109Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar WD2W3110, aroA::Tn10 msbA (A270T)Ref. 36Doerrler W.T. Reedy M.C. Raetz C.R.H. J. Biol. Chem. 2001; 276: 11461-11464Abstract Full Text Full Text PDF PubMed Scopus (182) Google ScholarPlasmidspET23aExpression vector, T7lac promoter, amprNovagenpMBH8pET23a expressing lmtAThis workpWSK29Low copy expression vector, lac promoter, amprRef. 27Wang R.F. Kushner S.R. Gene (Amst.). 1991; 100: 195-199Crossref PubMed Scopus (1011) Google ScholarpLmtApWSK29 expressing lmtAThis work Open table in a new tab Molecular Biology Protocols—Plasmids were isolated using the Qia-gen Spin Prep Kit, and DNA fragments were recovered from agarose gels using the QIAquick Gel Extraction Kit. Pfu DNA polymerase (Stratagene), T4 DNA ligase (Invitrogen), restriction endonucleases (New England Biolabs), and shrimp alkaline phosphatase (U. S. Biochemical Corp.) were used according to the manufacturers' instructions. The Duke University DNA Analysis Facility sequenced double-stranded DNA with an ABI Prism 377 instrument. All primers were obtained from MWG-Biotech. Chemically competent cells for transformations were prepared by the method of Inoue et al. (26Inoue H. Nojima H. Okayama H. Gene (Amst.). 1990; 96: 23-28Crossref PubMed Scopus (1569) Google Scholar). Cloning of lmtA from L. interrogans Genomic DNA—The L. interrogans lmtA gene was cloned into pET23a (Novagen) behind the T7lac promoter to generate pMBH8. First, the gene was amplified by PCR from L. interrogans serovar icterohemeorrhagiae (strain Verdun) genomic DNA, kindly provided by Catherine Werts (Institut Pasteur). The primers were designed based on the DNA sequence of L. interrogans serovar lai (strain 56601), which is 99.7% identical to serovar icterohemeorrhagiae (strain Verdun) at the DNA level and 100% at the protein level for LmtA. The forward primer consisted of a clamp region and an NdeI site (underlined) that overlaps with the first 28 base pairs of the lmtA gene (start codon in boldface type). The reverse primer contained a clamp region, a BamHI site (underlined), and the last 24 base pairs of lmtA (stop codon in boldface type). Sequences of the forward and reverse primers were 5′-GGCCATATGGCTTTGATCGAAGAATTTGAATCTC-3′ and 5′-GGCGGATCCTTAACGACCATCTACATGTAAAAG-3′, respectively. The PCR consisted of 100 ng of genomic DNA template, 250 ng of each primer, 200 μm each of dNTPs, 1× Pfu buffer (20 mm Tris-HCl, pH 8.8, 2 mm MgSO4, 10 mm KCl, 10 mm (NH4)2SO4, 0.1 mg/ml bovine serum albumin (BSA), 0.1% Triton X-100), and 5 units of Pfu DNA polymerase in a total reaction volume of 100 μl. The reaction conditions were as follows: 94 °C denaturation for 1 min followed by 25 cycles of 94 °C (denaturation) for 1 min, 50 °C (annealing) for 1 min, and 72 °C (extension) for 1 min. This was followed by a 10-min run-off at 72 °C. The gel-purified PCR product was digested with NdeI and BamHI and ligated into a NdeI/BamHI-digested and shrimp alkaline phosphatase-treated pET23a vector. The resulting pMBH8 was transformed into XL-1 Blue cells (Stratagene). The accession number for the lmtA DNA coding sequence is DQ097086. The lmtA gene was also cloned into the lac-inducible, low copy expression vector, pWSK29 (27Wang R.F. Kushner S.R. Gene (Amst.). 1991; 100: 195-199Crossref PubMed Scopus (1011) Google Scholar). Using XbaI and BamHI, the fragment containing lmtA and the upstream ribosome-binding site was excised. This fragment was ligated into XbaI/BamHI-digested and shrimp alkaline phosphatase-treated pWSK29. This plasmid, pLmtA, was then transformed into E. coli W3110. Preparation of Cell-free Extracts and Washed Membranes—L. interrogans was provided as a frozen cell pellet. E. coli W3110 cells, harboring either pLmtA or pWSK29, were grown in 100-ml cultures that were harvested by centrifugation (3500 × g, 20 min, 4 °C). Cells were resuspended in 4 ml of ice-cold 50 mm HEPES, pH 7.5, and lysed by two passages through a French pressure cell at 10,000 p.s.i. The lysate was cleared by centrifugation at 10,000 × g for 20 min at 4 °C. A small portion of the resulting supernatant (cell-free extract) was saved at -80 °C. Membranes were prepared from the remaining supernatant by ultracentrifugation at 100,000 × g for 60 min at 4 °C, and the resulting high speed supernatant (cytosol) was saved at -80 °C. The membranes were washed in 8 ml of 50 mm HEPES, pH 7.5, and subjected to an additional ultracentrifugation step. The final pellet was resuspended in 750 μl of 50 mm HEPES, pH 7.5, and stored at -80 °C. The BCA assay was used to determine protein concentration. Preparation of Lipid Substrates—The radiolabeled substrates, [4′-32P]Kdo2-lipid A, [4′-32P]lipid IVA, and [4′-32P]-Kdo2-lipid IVA, were prepared in vitro following a published procedure (28Reynolds C.M. Kalb S.R. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 2005; 280: 21202-21211Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 29Basu S.S. York J.D. Raetz C.R.H. J. Biol. Chem. 1999; 274: 11139-11149Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Phosphatidyl-[U-14C]glycerophosphate was prepared by enzymatic synthesis using CDP-diacylglycerol and [glycerol-U-14C]-glycerol-3-phosphate (30Karbarz M.J. Biochemistry of Endotoxin in Rhizobium leguminosarum: Characterization of a Family of Lipid Phosphatases Specific for the 1-Position of Lipid A. Duke University Medical Center, Durham, NC2004Google Scholar). Unlabeled Kdo2-lipid A was isolated from the heptose-deficient E. coli strain WBB06, as described (31Doerrler W.T. Raetz C.R.H. J. Biol. Chem. 2002; 277: 36697-36705Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Unlabeled lipid IVA and Kdo2-lipid IVA were obtained following a published procedure (32Brozek K.A. Hosaka K. Robertson A.D. Raetz C.R.H. J. Biol. Chem. 1989; 264: 6956-6966Abstract Full Text PDF PubMed Google Scholar). Methyltransferase Assay—The activity of LmtA in either cell-free extracts, cytosol, or membranes was assayed under optimized conditions in a 10-35-μl reaction volume with the substrates Kdo2-lipid A and SAM. Assays included 50 mm HEPES, pH 7.5, 0.1% Triton X-100, 5 mm EDTA, 1 mg/ml BSA, 10 mm SAM, 500 cpm/μl [4′-32P]Kdo2-lipid A, and 1 μm unlabeled Kdo2-lipid A. Assays conducted with E. coli membranes did not include 5 mm EDTA in order to minimize the formation of the PagP product, palmitoyl-Kdo2-lipid A (33Bishop R.E. Gibbons H.S. Guina T. Trent M.S. Miller S.I. Raetz C.R.H. EMBO J. 2000; 19: 5071-5080Crossref PubMed Scopus (280) Google Scholar). Reactions were incubated at 30 °C for varying times and terminated by spotting 4-μl portions onto Silica Gel 60 TLC plates. Substrates and products were separated in chloroform/methanol/water/acetic acid (25:15:4:4, v/v/v/v) and detected using a Amersham Biosciences PhosphorImager system (STORM 840) equipped with ImageQuant software. Mild Acid Hydrolysis of Methyltransferase Products—The assay was performed as described above for varying times. However, to terminate the reaction, 10-μl aliquots were added to 170 μl of 12.5 mm sodium acetate, pH 4.5, containing 1% SDS. The mixture was then boiled at 100 °C for 30 min to hydrolyze the Kdo residues. The resulting solution was converted into a two-phase Bligh/Dyer system by adding 200 μl each of chloroform and methanol, and it was centrifuged for 5 min. The lower phase (containing the free lipid A species) was removed, dried under vacuum, and resuspended in 10 μl of chloroform/methanol (2:1, v/v). The entire sample was spotted onto a TLC plate, and the lipid A molecules were resolved in chloroform, pyridine, 88% formic acid, water (50:50:16:5, v/v/v/v) and analyzed as described above. Preparation of Protein Samples for SDS-PAGE Analysis—Cell-free extracts or membrane samples (20 μg of protein) were incubated at 40 °C for 30 min in 50 mm Tris-HCl, pH 6.8, 12.5% glycerol, 3% SDS, 50 mm dithiothreitol, and 0.02% bromphenol blue. The samples were loaded onto a 12% SDS-polyacrylamide gel and subjected to electrophoresis at 150 V for 60 min. Inner and Outer Membrane Separation—Membranes derived from either W3110/pLmtA or W3110/pWSK29 were separated by isopycnic sucrose gradient centrifugation (34Osborn M.J. Munson R. Methods Enzymol. 1974; 31: 642-653Crossref PubMed Scopus (227) Google Scholar). A 2-ml membrane suspension, containing 5 mg of protein, was layered on top of a seven-step gradient (30-60% sucrose, w/w) (35Guy-Caffey J.K. Rapoza M.P. Jolley K.A. Webster R.E. J. Bacteriol. 1992; 174: 2460-2465Crossref PubMed Google Scholar) and ultracentrifuged for 18 h at 35,000 rpm in a Beckman SW40.1 rotor at 4 °C. A set of 22 0.5-ml fractions were collected and analyzed for protein concentration, NADH oxidase activity (inner membrane marker), and phospholipase A activity (outer membrane marker) (36Doerrler W.T. Reedy M.C. Raetz C.R.H. J. Biol. Chem. 2001; 276: 11461-11464Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 37Doerrler W.T. Gibbons H.S. Raetz C.R.H. J. Biol. Chem. 2004; 279: 45102-45109Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 38Zhou Z. White K.A. Polissi A. Georgopoulos C. Raetz C.R.H. J. Biol. Chem. 1998; 273: 12466-12475Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). Each fraction was also assayed for LmtA activity. In Vivo Labeling of E. coli Cells Expressing LmtA—Lipid A species were radiolabeled in 20-ml cultures of W3110/pLmtA or W3110/pWSK29 containing 5 μCi/ml 32Pi. Cells were collected by centrifugation and washed once with 5 ml of phosphate-buffered saline, pH 7.4. Next, the cells were resuspended in 3 ml of a single-phase Bligh/Dyer solution (chloroform/methanol/water, 1:2:0.8, v/v/v) and incubated at room temperature for 60 min in order to extract the phospholipids. The sample was centrifuged, and the pellet (containing the LPS, proteins, and DNA) was washed two times with 3 ml of a single-phase Bligh/Dyer mixture. Then the pellet was boiled at 100 °C in 3 ml of 12.5 mm sodium acetate, pH 4.5, containing 1% SDS, for 30 min to release the lipid A from the LPS core residues. The lipid A molecules were extracted by conversion into a two-phase Bligh/Dyer system (chloroform/methanol/water, 2:2:1.8, v/v/v). The lower phase, containing the lipid A, was removed after centrifugation, and the remaining upper phase was washed once with preequilibrated lower phase. Lower phases were pooled and dried under a stream of N2. The dried lipid was redissolved in 200 μl of chloroform/methanol (2:1, v/v), and 5000 counts were spotted onto a TLC plate. The lipid A species were separated in chloroform, pyridine, 88% formic acid, water (50:50:16:5, v/v/v/v) and detected using a PhosphorImager system as described above. The labeling experiment performed with the temperature-sensitive mutant WD2 (36Doerrler W.T. Reedy M.C." @default.
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• W2077888858 date "2005-08-01" @default.
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• W2077888858 title "A Leptospira interrogans Enzyme with Similarity to Yeast Ste14p That Methylates the 1-Phosphate Group of Lipid A" @default.
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• W2077888858 doi "https://doi.org/10.1074/jbc.m506103200" @default.
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