Expression Cloning and Biochemical Characterization of a Rhizobium leguminosarum Lipid A 1-Phosphatase
2003; Elsevier BV; Volume: 278; Issue: 41 Linguagem: Inglês
10.1074/jbc.m305830200
ISSN1083-351X
AutoresMark J. Karbarz, Suzanne R. Kalb, Robert J. Cotter, Christian R.H. Raetz,
Tópico(s)Microbial Metabolites in Food Biotechnology
ResumoLipid A of Rhizobium leguminosarum, a nitrogen-fixing plant endosymbiont, displays several significant structural differences when compared with Escherichia coli. An especially striking feature of R. leguminosarum lipid A is that it lacks both the 1- and 4′-phosphate groups. Distinct lipid A phosphatases that attack either the 1 or the 4′ positions have previously been identified in extracts of R. leguminosarum and Rhizobium etli but not Sinorhizobium meliloti or E. coli. Here we describe the identification of a hybrid cosmid (pMJK-1) containing a 25-kb R. leguminosarum 3841 DNA insert that directs the overexpression of the lipid A 1-phosphatase. Transfer of pMJK-1 into S. meliloti 1021 results in heterologous expression of 1-phosphatase activity, which is normally absent in extracts of strain 1021, and confers resistance to polymyxin. Sequencing of a 7-kb DNA fragment derived from the insert of pMJK-1 revealed the presence of a lipid phosphatase ortholog (designated LpxE). Expression of lpxE in E. coli behind the T7lac promoter results in the appearance of robust 1-phosphatase activity, which is normally absent in E. coli membranes. Matrix-assisted laser-desorption/time of flight and radiochemical analysis of the product generated in vitro from the model substrate lipid IVA confirms the selective removal of the 1-phosphate group. These findings show that lpxE is the structural gene for the 1-phosphatase. The availability of lpxE may facilitate the re-engineering of lipid A structures in diverse Gram-negative bacteria and allow assessment of the role of the 1-phosphatase in R. leguminosarum symbiosis with plants. Possible orthologs of LpxE are present in some intracellular human pathogens, including Francisella tularensis, Brucella melitensis, and Legionella pneumophila. Lipid A of Rhizobium leguminosarum, a nitrogen-fixing plant endosymbiont, displays several significant structural differences when compared with Escherichia coli. An especially striking feature of R. leguminosarum lipid A is that it lacks both the 1- and 4′-phosphate groups. Distinct lipid A phosphatases that attack either the 1 or the 4′ positions have previously been identified in extracts of R. leguminosarum and Rhizobium etli but not Sinorhizobium meliloti or E. coli. Here we describe the identification of a hybrid cosmid (pMJK-1) containing a 25-kb R. leguminosarum 3841 DNA insert that directs the overexpression of the lipid A 1-phosphatase. Transfer of pMJK-1 into S. meliloti 1021 results in heterologous expression of 1-phosphatase activity, which is normally absent in extracts of strain 1021, and confers resistance to polymyxin. Sequencing of a 7-kb DNA fragment derived from the insert of pMJK-1 revealed the presence of a lipid phosphatase ortholog (designated LpxE). Expression of lpxE in E. coli behind the T7lac promoter results in the appearance of robust 1-phosphatase activity, which is normally absent in E. coli membranes. Matrix-assisted laser-desorption/time of flight and radiochemical analysis of the product generated in vitro from the model substrate lipid IVA confirms the selective removal of the 1-phosphate group. These findings show that lpxE is the structural gene for the 1-phosphatase. The availability of lpxE may facilitate the re-engineering of lipid A structures in diverse Gram-negative bacteria and allow assessment of the role of the 1-phosphatase in R. leguminosarum symbiosis with plants. Possible orthologs of LpxE are present in some intracellular human pathogens, including Francisella tularensis, Brucella melitensis, and Legionella pneumophila. Lipopolysaccharide (LPS) 1The abbreviations used are: LPS, lipopolysaccharide; MES, 2-(N-morpholino)-ethanesulfonic acid; Kdo, 3-deoxy-d-manno-octulosonic acid; MALDI/TOF, matrix-assisted laser-desorption/time of flight; TLR, toll-like receptor.1The abbreviations used are: LPS, lipopolysaccharide; MES, 2-(N-morpholino)-ethanesulfonic acid; Kdo, 3-deoxy-d-manno-octulosonic acid; MALDI/TOF, matrix-assisted laser-desorption/time of flight; TLR, toll-like receptor. is a macromolecular glycolipid found in the outer membranes of Gram-negative bacteria (1Raetz C.R.H. Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Google Scholar, 2Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. Vol. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar, 3Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (2928) Google Scholar, 4Brade H. Opal S.M. Vogel S.N. Morrison D.C. Endotoxin in Health and Disease. Marcel Dekker, Inc., New York1999Google Scholar). The structure of LPS consists of three domains: the lipid A moiety that serves as the hydrophobic anchor, a nonrepeating core oligosaccharide, and a highly immunogenic, distal O-antigen polysaccharide (1Raetz C.R.H. Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Google Scholar, 2Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. Vol. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar, 3Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (2928) Google Scholar, 4Brade H. Opal S.M. Vogel S.N. Morrison D.C. Endotoxin in Health and Disease. Marcel Dekker, Inc., New York1999Google Scholar). LPS acts as an efficient barrier to antibiotics (5Nikaido H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. Vol. 1. 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Carlson R.W. J. Bacteriol. 1994; 176: 4646-4655Crossref PubMed Google Scholar). Several additional enzymes exist in R. leguminosarum that catalyze the further conversion of Kdo2-lipid IVA to R. leguminosarum lipid A. We have previously identified a 4′-phosphatase (38Price N.J.P. Jeyaretnam B. Carlson R.W. Kadrmas J.L. Raetz C.R.H. Brozek K.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7352-7356Crossref PubMed Scopus (52) Google Scholar), a 1-phosphatase (39Brozek 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), a long chain acyltransferase (22Brozek K.A. Carlson R.W. Raetz C.R.H. J. Biol. Chem. 1996; 271: 32126-32136Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 40Basu S.S. Karbarz M.J. Raetz C.R.H. J. Biol. Chem. 2002; 277: 28959-28971Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), a mannosyl transferase (39Brozek K.A. Kadrmas J.L. Raetz C.R.H. J. Biol. 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Allaway D. Studholme R.E. Sullivan J.T. Ronson C.W. Poole P.S. Raetz C.R.H. J. Biol. Chem. 1998; 273: 26432-26440Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) that are involved in the distinct metabolism of Kdo2-lipid IVA in extracts of R. leguminosarum but not in E. coli. Recently, we have also discovered a novel oxidase that acts on 1-dephosphorylated lipid A species to generate the unique 2-aminogluconate moiety found in the proximal unit of R. leguminosarum lipid A (43Que-Gewirth N.L.S. Lin S. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 2003; 278: 12109-12119Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 44Que-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) (see Fig. 1). We now report the expression cloning of the R. leguminosarum gene that encodes the lipid A 1-phosphatase (see Fig. 1). A hybrid cosmid capable of directing the overexpression of 1-phosphatase activity was identified by assaying cell lysates of individual clones of a R. leguminosarum 3841 genomic DNA library (45Ronson C.W. Astwood P.M. Downie J.A. J. Bacteriol. 1984; 160: 903-909Crossref PubMed Google Scholar) harbored in R. etli CE3 (46Cava J.R. Elias P.M. Turowski D.A. Noel K.D. J. Bacteriol. 1989; 171: 8-15Crossref PubMed Google Scholar). The 1-phosphatase, which can be detected with either E. coli Kdo2-[4′-32P]lipid IVA or its precursor [4′-32P]lipid IVA as substrates (see Fig. 1) (39Brozek 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), is associated with the inner membrane. Sequencing of a 7-kb DNA insert derived from the hybrid cosmid permitted the identification of the structural gene (designated lpxE from the German eins) encoding the 1-phosphatase (see Fig. 1). The R. leguminosarum lpxE gene, which encodes a protein of 244 amino acid residues, can be overexpressed in E. coli behind the T7lac promoter, and the enzyme is catalytically active. The expression of lpxE in Sinorhizobium meliloti confers resistance to polymyxin. The availability of the lpxE gene should facilitate the re-engineering of lipid A structures in Gram-negative bacteria and enhance our understanding of the biological functions of 1-dephosphorylated lipid A species. Chemicals and Materials—[γ-32P]ATP was obtained from Perkin-Elmer Life Sciences. Silica gel 60 thin layer plates (0.25 mm) were purchased from EM Separation Technology (Merck). Triton X-100 and bicinchoninic acid were from Pierce. Yeast extract and tryptone were purchased from Difco. All other chemicals were reagent grade and were obtained from either Sigma or Mallinckrodt. All of the restriction enzymes were from Invitrogen or New England Biolabs. PCR reagents were purchased from Stratagene. Shrimp alkaline phosphatase was purchased from U.S. Biochemical Corp. Seaplaque low temperature melting agarose was purchased from FMC Bioproducts (Rockland, ME). A "Dark Reader," utilized for DNA manipulations, was purchased from Epicenter Technologies Corporation. Custom primers and T4 DNA ligase were from Invitrogen. All other molecular biology reagents and enzymes were purchased from either Roche Applied Science or Invitrogen. Bacterial Strains and Growth Conditions—R. etli CE3 (46Cava J.R. Elias P.M. Turowski D.A. Noel K.D. J. Bacteriol. 1989; 171: 8-15Crossref PubMed Google Scholar, 47Noel K.D. Sanchez A. Fernandez L. Leemans J. Cevallos M.A. J. Bacteriol. 1984; 158: 148-155Crossref PubMed Google Scholar), R. leguminosarum 3841 (40Basu S.S. Karbarz M.J. Raetz C.R.H. J. Biol. Chem. 2002; 277: 28959-28971Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 48Johnston A.W. Beringer J.E. J. Gen. Microbiol. 1975; 87: 343-350Crossref PubMed Google Scholar) and S. meliloti 1021 (49Galibert F. Finan T.M. Long S.R. Puhler A. Abola P. Ampe F. Barloy-Hubler F. Barnett M.J. Becker A. Boistard P. Bothe G. Boutry M. Bowser L. Buhrmester J. Cadieu E. Capela D. Chain P. Cowie A. Davis R.W. Dreano S. Federspiel N.A. Fisher R.F. Gloux S. Godrie T. Goffeau A. Golding B. Gouzy J. Gurjal M. Hernandez-Lucas I. Hong A. Huizar L. Hyman R.W. Jones T. Kahn D. Kahn M.L. Kalman S. Keating D.H. Kiss E. Komp C. Lelaure V. Masuy D. Palm C. Peck M.C. Pohl T.M. Portetelle D. Purnelle B. Ramsperger U. Surzycki R. Thebault P. Vandenbol M. Vorholter F.J. Weidner S. Wells D.H. Wong K. Yeh K.C. Batut J. Science. 2001; 293: 668-672Crossref PubMed Google Scholar) were grown as described previously (37Price N.P.J. Kelly T.M. Raetz C.R.H. Carlson R.W. J. Bacteriol. 1994; 176: 4646-4655Crossref PubMed Google Scholar, 40Basu S.S. Karbarz M.J. Raetz C.R.H. J. Biol. Chem. 2002; 277: 28959-28971Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), and their properties are summarized in Table I.Table IBacterial strains and plasmids used in this studyStrain or plasmidGenotype or descriptionSource or referenceStrainsR. etli CE3CFN42 SmrRefs. 46Cava J.R. Elias P.M. Turowski D.A. Noel K.D. J. Bacteriol. 1989; 171: 8-15Crossref PubMed Google Scholar and 47Noel K.D. Sanchez A. Fernandez L. Leemans J. Cevallos M.A. J. Bacteriol. 1984; 158: 148-155Crossref PubMed Google ScholarR. leguminosarum 3841Wild type strain 300 biovar viciae SmrRefs. 40Basu S.S. Karbarz M.J. Raetz C.R.H. J. Biol. Chem. 2002; 277: 28959-28971Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar and 48Johnston A.W. Beringer J.E. J. Gen. Microbiol. 1975; 87: 343-350Crossref PubMed Google ScholarS. meliloti 1021SU47 SmrSharon Long (49Galibert F. Finan T.M. Long S.R. Puhler A. Abola P. Ampe F. Barloy-Hubler F. Barnett M.J. Becker A. Boistard P. Bothe G. Boutry M. Bowser L. Buhrmester J. Cadieu E. Capela D. Chain P. Cowie A. Davis R.W. Dreano S. Federspiel N.A. Fisher R.F. Gloux S. Godrie T. Goffeau A. Golding B. Gouzy J. Gurjal M. Hernandez-Lucas I. Hong A. Huizar L. Hyman R.W. Jones T. Kahn D. Kahn M.L. Kalman S. Keating D.H. Kiss E. Komp C. Lelaure V. Masuy D. Palm C. Peck M.C. Pohl T.M. Portetelle D. Purnelle B. Ramsperger U. Surzycki R. Thebault P. Vandenbol M. Vorholter F.J. Weidner S. Wells D.H. Wong K. Yeh K.C. Batut J. Science. 2001; 293: 668-672Crossref PubMed Google Scholar)E. coliHB101hsdS20 supE44 ara14 galK2 lacY1 proA2 rpsL20 (Smr) xyl-5 mtl-1 recA13 mcrB thi-1 leuB6InvitrogenXL1-BluerecA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac[F′proAB lacl qZDM15 Tn10 (Tetr)]StratageneMT616pRK2013 Cmr, Km::Tn9 containing strain for tri-parental matingRef. 63Finan T.M. Kunkel B. De Vos G.F. Signer E.R. J. Bacteriol. 1986; 167: 66-72Crossref PubMed Google ScholarNovablue (DE3)E. coli host strain used for expressionNovagenPlasmidspLAFR-1Broad host range P-group cloning vector, mobilizable RK2 cosmid TetrRef. 60Friedman A.M. Long S.R. Brown S.E. Buikema W.J. Ausubel F.M. Gene (Amst.). 1982; 18: 289-296Crossref PubMed Google ScholarpRK404aShuttle vector TetrRef. 65Ditta G. Stanfield S. Corbin D. Helinski D.R. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 7347-7351Crossref PubMed Google ScholarpET-28aE. coli expression vectorNovagenpMJK-1pLAFR-1 derivative carrying a 25-kb fragment of R. leguminosarum 3841 = genomic DNA, which includes lpxEThis workpLpxE-2pRK404a derivative carrying a 6.9-kb EcoRI fragment, which includes lpxE from pMJK-1This workpLpxE-3pRK404a derivative carrying a 4.9-kb HindIII fragment, which includes lpxE from pMJK-1This workpLpxE-4pET-28a derivative harboring lpxE behind the T7lac promoterThis work Open table in a new tab E. coli Novablue (DE3) strains (Novagen), harboring either the empty vector control pET-28a or the hybrid plasmid pLpxE-4 (containing the lpxE gene), were grown from a single colony in 200 ml of LB broth (10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl per liter) (50Miller J.R. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar) supplemented with kanamycin (15 μg/ml) at 37 °C until the A 600 reached ∼0.6. The culture was split into two equal portions, one of which was induced with 1 mm isopropyl-1-thio-β-d-galactopyranoside. Both cultures were further incubated with shaking at 225 rpm for an additional 4 h at 25 °C. Preparation of Cell-free Extracts and Washed Membranes—All of the enzyme preparations were carried out at 0–4 °C. Protein concentration was determined by the bicinchoninic acid method (51Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (17658) Google Scholar) using bovine serum albumin as a standard (Pierce). Cell-free extracts, cytosol, and washed membranes were prepared as described previously (40Basu S.S. Karbarz M.J. Raetz C.R.H. J. Biol. Chem. 2002; 277: 28959-28971Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) and stored in aliquots at –80 °C. Preparation of Radiolabeled Substrates—The [4′-32P]lipid IVA was generated from [γ-32P]ATP and the appropriate tetraacyl-disaccharide 1-phosphate acceptor, using the overexpressed 4′-kinase present in membranes of E. coli BLR(DE3)/pLysS/pJK2 (52Garrett T.A. Kadrmas J.L. Raetz C.R.H. J. Biol. Chem. 1997; 272: 21855-21864Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 53Basu S.S. York J.D. Raetz C.R.H. J. Biol. Chem. 1999; 274: 11139-11149Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The Kdo2-[4′-32P]lipid IVA was then prepared from the [4′-32P]lipid IVA by the action of the purified E. coli Kdo transferase (KdtA) (53Basu S.S. York J.D. Raetz C.R.H. J. Biol. Chem. 1999; 274: 11139-11149Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 54Belunis C.J. Raetz C.R.H. J. Biol. Chem. 1992; 267: 9988-9997Abstract Full Text PDF PubMed Google Scholar). The Kdo2-[4′-32P]lipid IVA and [4′-32P]lipid IVA were purified by preparative thin layer chromatography (53Basu S.S. York J.D. Raetz C.R.H. J. Biol. Chem. 1999; 274: 11139-11149Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 54Belunis C.J. Raetz C.R.H. J. Biol. Chem. 1992; 267: 9988-9997Abstract Full Text PDF PubMed Google Scholar, 55Brozek K.A. Hosaka K. Robertson A.D. Raetz C.R.H. J. Biol. Chem. 1989; 264: 6956-6966Abstract Full Text PDF PubMed Google Scholar) and were stored as an aqueous dispersion at –20 °C in 10 mm Tris-HCl, pH 7.5, and 1 mm EDTA. Prior to use, all of the lipid substrates were dispersed by sonic irradiation for 1 min in a bath sonicator. The substrate [32P]lipid X was prepared from 32Pi-labeled cells of E. coli strain MN7 (56Nishijima M. Raetz C.R.H. J. Biol. Chem. 1979; 2
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