Evidence That the wzxE Gene of Escherichia coli K-12 Encodes a Protein Involved in the Transbilayer Movement of a Trisaccharide-Lipid Intermediate in the Assembly of Enterobacterial Common Antigen
2003; Elsevier BV; Volume: 278; Issue: 19 Linguagem: Inglês
10.1074/jbc.m301750200
ISSN1083-351X
AutoresP D Rick, Kathleen Barr, Krishnan Sankaran, Junko Kajimura, Jeffrey S. Rush, Charles J. Waechter,
Tópico(s)Escherichia coli research studies
ResumoThe assembly of many bacterial cell surface polysaccharides requires the transbilayer movement of polyisoprenoid-linked saccharide intermediates across the cytoplasmic membrane. It is generally believed that transverse diffusion of glycolipid intermediates is mediated by integral membrane proteins called translocases or “flippases.” The bacterial genes proposed to encode these translocases have been collectively designated wzx genes. The wzxEgene of Escherichia coli K-12 has been implicated in the transbilayer movement of Fuc4NAc-ManNAcA-GlcNAc-P-P-undecaprenol (lipid III), the donor of the trisaccharide repeat unit in the biosynthesis of enterobacterial common antigen (ECA). Previous studies (Feldman, M. F., Marolda, C. L., Monteiro, M. A., Perry, M. B., Parodi, A. J., and Valvano, M. (1999) J. Biol. Chem. 274, 35129–35138) provided indirect evidence that thewzx016 gene product of E. coli K-12 encoded a translocase capable of mediating the transbilayer movement ofN-acetylglucosaminylpyrophosphorylundecaprenol (GlcNAc-P-P-Und), an early intermediate in the synthesis of ECA and many lipopolysaccharide O antigens. Therefore, genetic and biochemical studies were conducted to determine if the putative WzxO16translocase was capable of mediating the transport ofN-acetylglucosaminylpyrophosphorylnerol (GlcNAc-P-P-Ner), a water-soluble analogue of GlcNAc-P-P-Und. [3H]GlcNAc-P-P-Ner was transported into sealed, everted cytoplasmic membrane vesicles of E. coli K-12 as well as a deletion mutant lacking both the wzx016 andwzxC genes. In contrast, [3H]GlcNAc-P-P-Ner was not transported into membrane vesicles prepared from awzxE-null mutant, and metabolic radiolabeling experiments revealed the accumulation of lipid III in this mutant. The WzxE transport system exhibited substrate specificity by recognizing both a pyrophosphoryl-linked saccharide and an unsaturated α-isoprene unit in the carrier lipid. These results support the conclusion that thewzxE gene encodes a membrane protein involved in the transbilayer movement of lipid III in E. coli. The assembly of many bacterial cell surface polysaccharides requires the transbilayer movement of polyisoprenoid-linked saccharide intermediates across the cytoplasmic membrane. It is generally believed that transverse diffusion of glycolipid intermediates is mediated by integral membrane proteins called translocases or “flippases.” The bacterial genes proposed to encode these translocases have been collectively designated wzx genes. The wzxEgene of Escherichia coli K-12 has been implicated in the transbilayer movement of Fuc4NAc-ManNAcA-GlcNAc-P-P-undecaprenol (lipid III), the donor of the trisaccharide repeat unit in the biosynthesis of enterobacterial common antigen (ECA). Previous studies (Feldman, M. F., Marolda, C. L., Monteiro, M. A., Perry, M. B., Parodi, A. J., and Valvano, M. (1999) J. Biol. Chem. 274, 35129–35138) provided indirect evidence that thewzx016 gene product of E. coli K-12 encoded a translocase capable of mediating the transbilayer movement ofN-acetylglucosaminylpyrophosphorylundecaprenol (GlcNAc-P-P-Und), an early intermediate in the synthesis of ECA and many lipopolysaccharide O antigens. Therefore, genetic and biochemical studies were conducted to determine if the putative WzxO16translocase was capable of mediating the transport ofN-acetylglucosaminylpyrophosphorylnerol (GlcNAc-P-P-Ner), a water-soluble analogue of GlcNAc-P-P-Und. [3H]GlcNAc-P-P-Ner was transported into sealed, everted cytoplasmic membrane vesicles of E. coli K-12 as well as a deletion mutant lacking both the wzx016 andwzxC genes. In contrast, [3H]GlcNAc-P-P-Ner was not transported into membrane vesicles prepared from awzxE-null mutant, and metabolic radiolabeling experiments revealed the accumulation of lipid III in this mutant. The WzxE transport system exhibited substrate specificity by recognizing both a pyrophosphoryl-linked saccharide and an unsaturated α-isoprene unit in the carrier lipid. These results support the conclusion that thewzxE gene encodes a membrane protein involved in the transbilayer movement of lipid III in E. coli. endoplasmic reticulum glycosylphosphatidylinositol N-acetyl-d-glucosamine di(N-acetylglucosaminyl)pyrophosphoryldolichol lipopolysaccharide undecaprenol mannose proteose peptone beef extract dolichol citronellyl phosphate nerol phosphoglyceride-linked enterobacterial common antigen phosphatidylcholine Chinese hamster ovary N-acetyl-d-mannosaminuronic acid 4-acetamido-4, 6-dideoxy-d-galactose The biosynthesis of a wide variety of complex glycoconjugates in both eucaryotic and procaryotic cells occurs by a process whereby membrane-bound glycolipids or glycolipid precursors are synthesized on the cytosolic face of a membrane and subsequently translocated to the opposite side of the membrane where they serve as substrates for additional processing reactions. In eucaryotic cells the synthesis ofN-linked oligosaccharides of glycoproteins involves the translocation of dolichyl-linked mono- and pentasaccharide intermediates from the cytosolic leaflet to the lumenal monolayer of the endoplasmic reticulum (ER)1 (1Hirschberg C.B. Snider M.D. Annu. Rev. Biochem. 1987; 56: 63-87Crossref PubMed Scopus (443) Google Scholar, 2Lennarz W.J. Biochemistry. 1987; 26: 7205-7210Crossref PubMed Scopus (78) Google Scholar, 3Abeijon C. Hirschberg C.B. J. Biol. Chem. 1990; 265: 14691-14695Abstract Full Text PDF PubMed Google Scholar, 4Abeijon C. Hirschberg C.B. Trends Biol. Sci. 1992; 17: 32-36Abstract Full Text PDF PubMed Scopus (195) Google Scholar, 5Schenk B. Fernandez F. Waechter C.J. Glycobiology. 2001; 11: 61R-71RCrossref PubMed Scopus (145) Google Scholar). 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Acta. 1994; 1213: 241-262Crossref PubMed Scopus (87) Google Scholar, 17Menon A.K. Trends Cell Biol. 1995; 5: 355-360Abstract Full Text PDF PubMed Scopus (76) Google Scholar), and a substantial amount of evidence has been obtained that supports this conclusion (17Menon A.K. Trends Cell Biol. 1995; 5: 355-360Abstract Full Text PDF PubMed Scopus (76) Google Scholar, 18Rush J.S. Waechter C.J. J. Cell Biol. 1995; 130: 529-536Crossref PubMed Scopus (56) Google Scholar, 19Rush J.S. van Leyen K. Overffelli O. Wolucka B. Waechter C.J. Glycobiology. 1998; 8: 1195-1205Crossref PubMed Scopus (40) Google Scholar). Flippase proteins are also believed to be involved in the assembly of a large group of bacterial lipopolysaccharide (LPS) O antigens collectively referred to as Wzy-dependent O antigens (20Keenleyside W.J. Whitfield C. Brade H. Opal S.M. Vogel S.H. Morrison D. Endotoxin in Health and Disease. Marcel Dekker, New York.1999: 331-358Google Scholar). Although these O antigens are structurally distinct, they are all heteropolysaccharides comprised of oligosaccharide repeat units and all are assembled by the same general mechanism (20Keenleyside W.J. Whitfield C. Brade H. Opal S.M. Vogel S.H. Morrison D. Endotoxin in Health and Disease. Marcel Dekker, New York.1999: 331-358Google Scholar). The repeat units of these O antigens are synthesized as undecaprenyl pyrophosphate (Und-P-P)-linked oligosaccharides on the inner face of the cytoplasmic membrane and subsequently translocated en bloc to the periplasmic face where they are utilized as substrates for chain elongation by a polymerase enzyme (Wzy) (20Keenleyside W.J. Whitfield C. Brade H. Opal S.M. Vogel S.H. Morrison D. Endotoxin in Health and Disease. Marcel Dekker, New York.1999: 331-358Google Scholar, 21McGrath B.C. Osborn M.J. J. Bacteriol. 1991; 173: 649-654Crossref PubMed Google Scholar). Polymerization occurs by the transfer of nascent polysaccharide chains from the carrier lipid to the non-reducing termini of newly synthesized carrier lipid-linked repeat units (22Bray W. Robbins P.W. Biochem. Biophys. Res. Commun. 1967; 28: 334-339Crossref PubMed Scopus (38) Google Scholar, 23Robbins P.W. Bray D. Dankert M. Wright A. Science. 1967; 158: 1536-1542Crossref PubMed Scopus (105) Google Scholar). The assembly of Wzy-dependent O antigens by this mechanism has been called “block-polymerization.” The O antigen chains are then transferred to the core-lipid A region of LPS, and the completed LPS molecules are translocated to the exterior leaflet of the outer membrane by an unknown mechanism. All of the gene clusters involved in the synthesis of Wzy-dependent O antigens contain a gene, designatedwzx, that is believed to encode the flippase that mediates the transbilayer movement of the Und-P-P-linked repeat unit (24Reeves P.R. Neuberger A. van Deenen L.L.M. Bacterial Cell Wall, New Comprehensive Biochemistry. 27. Elsevier Science Publishers, New York1994: 281-314Google Scholar). Analyses of the predicted structures of the putative O antigen flippases have revealed that all are hydrophobic proteins with twelve putative transmembrane domains (24Reeves P.R. Neuberger A. van Deenen L.L.M. Bacterial Cell Wall, New Comprehensive Biochemistry. 27. Elsevier Science Publishers, New York1994: 281-314Google Scholar). Although these proteins share very little structural homology at the primary amino acid sequence level, all have strikingly similar hydropathy profiles (25MacPherson D.F. Manning P.A. Morona R. Gene (Amst.). 1995; 155: 9-17Crossref PubMed Scopus (28) Google Scholar). Indirect evidence in support of the proposed functions of the O antigen flippases ofSalmonella enterica serovar Typhimurium and Shigella dysenteriae was obtained by Liu et al. (26Liu D. Cole R.A. Reeves P. J. Bacteriol. 1996; 178: 2102-2107Crossref PubMed Google Scholar), who demonstrated the accumulation of single lipid-linked O antigen repeat units on the inner face of the cytoplasmic membrane in wzxmutants. However, the mechanism involved in flippase-mediated transbilayer movement of lipid-linked O antigen repeat units, as well as definitive identification of the proteins involved, remains to be established. The assembly of phosphoglyceride-linked enterobacterial common antigen (ECAPG) of Gram-negative enteric bacteria is also believed to occur by a Wzy-dependent mechanism (27Rick P.D. Silver R. Neidhardt F. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd ed. ASM Press, Washington, D. C.1996: 104-122Google Scholar). ECAPG is a glycolipid component of the outer membrane of all Gram-negative enteric bacteria (27Rick P.D. Silver R. Neidhardt F. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd ed. ASM Press, Washington, D. C.1996: 104-122Google Scholar, 28Kuhn H.-M. Neter E. Mayer H. Infect. Immun. 1983; 40: 195-222Crossref Google Scholar). The carbohydrate portion of ECA consists of a linear heteropolysaccharide chain comprised ofN-acetyl-d-glucosamine (GlcNAc),N-acetyl-d-mannosaminuronic acid (ManNAcA), and 4-acetamido-4,6-dideoxy-d-galactose (Fuc4NAc) (29Lugowski C. Romanowska L. Kenne L. Lindberg B. Carbohydr. Res. 1983; 118: 173-181Crossref Scopus (75) Google Scholar). These amino sugars are linked to one another to form the trisaccharide repeat unit →3)-α-d-Fuc4NAc-(1→4)-औ-d-ManNAcA-(1→4)-α-d-GlcNAc-(1→ (29Lugowski C. Romanowska L. Kenne L. Lindberg B. Carbohydr. Res. 1983; 118: 173-181Crossref Scopus (75) Google Scholar). The ECA trisaccharide repeat unit is synthesized as the Und-P-P-linked intermediate, Fuc4NAc-ManNAcA-GlcNAc-P-P-Und (lipid III) (27Rick P.D. Silver R. Neidhardt F. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd ed. ASM Press, Washington, D. C.1996: 104-122Google Scholar, 30Rick P.D. Mayer H. Neumeyer B.A. Wolski S. Bitter-Suermann D. J. Bacteriol. 1985; 162: 494-503Crossref PubMed Google Scholar, 31Barr K. Rick P.D. J. Biol. Chem. 1987; 262: 7142-7150Abstract Full Text PDF PubMed Google Scholar, 32Barr K. Nunes-Edwards P. Rick P.D. J. Bacteriol. 1989; 171: 1326-1332Crossref PubMed Google Scholar). The available data are consistent with the synthesis of lipid III on the inner leaflet of the cytoplasmic membrane followed by its transbilayer movement to the periplasmic face of the membrane where assembly of the polysaccharide chains occurs by a block-polymerization mechanism. The polysaccharide chains are subsequently transferred from the carrier lipid to an as yet unidentified glyceride acceptor to yield ECAPG molecules in which the potential reducing terminal GlcNAc residue is linked to diacylglycerol through phosphodiester linkage (33Kuhn H.-M. Neter E. Mayer H. Infect. Immun. 1983; 40: 696-700Crossref PubMed Google Scholar, 34Rick P.D. Hubbard G.L. Kitaoka M. Nagaki H. Kinoshita H. Dowd S. Simplaceanu V. Ho C. Glycobiology. 1998; 8: 557-567Crossref PubMed Scopus (63) Google Scholar). Completed ECAPG polymers are then incorporated into the exterior leaflet of the outer membrane. The synthesis of GlcNAc-P-P-Und is the initial step in the assembly of the ECA trisaccharide repeat unit (30Rick P.D. Mayer H. Neumeyer B.A. Wolski S. Bitter-Suermann D. J. Bacteriol. 1985; 162: 494-503Crossref PubMed Google Scholar) and the repeat units of many Wzy-dependent O antigens (20Keenleyside W.J. Whitfield C. Brade H. Opal S.M. Vogel S.H. Morrison D. Endotoxin in Health and Disease. Marcel Dekker, New York.1999: 331-358Google Scholar, 35Rick P.D. Hubbard G.L. Barr K. J. Bacteriol. 1994; 176: 2877-2884Crossref PubMed Google Scholar, 36Alexander D.C. Valvano M. J. Bacteriol. 1994; 176: 7079-7084Crossref PubMed Google Scholar). It is also the first lipid-linked intermediate in the synthesis of several Wzy-independent O antigens (20Keenleyside W.J. Whitfield C. Brade H. Opal S.M. Vogel S.H. Morrison D. Endotoxin in Health and Disease. Marcel Dekker, New York.1999: 331-358Google Scholar). This reaction involves the transfer of GlcNAc 1-P from UDP-GlcNAc to Und-P catalyzed by the enzyme UDP-GlcNAc:undecaprenyl phosphate GlcNAc 1-P transferase (WecA) (31Barr K. Rick P.D. J. Biol. Chem. 1987; 262: 7142-7150Abstract Full Text PDF PubMed Google Scholar, 37Rush J.S. Rick P.D. Waechter C.J. Glycobiology. 1997; 7: 315-322Crossref PubMed Scopus (40) Google Scholar). The wecAgene (formerly rfe) is located in the wec gene cluster (formerly rfe-rff), which includes many of the genes involved in the biosynthesis of ECA (27Rick P.D. Silver R. Neidhardt F. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd ed. ASM Press, Washington, D. C.1996: 104-122Google Scholar, 38Blattner F.R. Punkett III, G. Bloch C.A. Perna N.T. Burland V. Riley M. Collado-Vides J. Glasner J.D. Rode K.K. Mayhew G.F. Gregor J. Davis N.W. Kirkpatrick H.A. Goeden M.A. Rose D.J. Maw B. Shao Y. Science. 1997; 277: 1453-1474Crossref PubMed Scopus (6056) Google Scholar, 39Meier-Dieter U. Barr K. Starman R. Hatch L. Rick P.D. J. Biol. Chem. 1992; 267: 746-753Abstract Full Text PDF PubMed Google Scholar). The wecgene cluster also includes a gene designated wzxE (formerlyrfbX) that is believed to encode the flippase that mediates the transbilayer movement of the Und-P-P-linked trisaccharide repeat unit (25MacPherson D.F. Manning P.A. Morona R. Gene (Amst.). 1995; 155: 9-17Crossref PubMed Scopus (28) Google Scholar). Indeed, the hydropathy profile of the predicted product of the wzxE gene is almost identical to the hydropathy profiles of the putative flippases involved in the assembly of many Wzy-dependent O antigens (25MacPherson D.F. Manning P.A. Morona R. Gene (Amst.). 1995; 155: 9-17Crossref PubMed Scopus (28) Google Scholar). The O16 O antigen repeat unit of Escherichia coli K-12/O16 is a branched pentasaccharide whose assembly is initiated by the synthesis of GlcNAc-P-P-Und catalyzed by WecA, and the assembly of this O antigen is believed to occur by a Wzy-dependent mechanism (36Alexander D.C. Valvano M. J. Bacteriol. 1994; 176: 7079-7084Crossref PubMed Google Scholar, 40Yao Z. Valvano M. J. Bacteriol. 1994; 176: 4133-4143Crossref PubMed Google Scholar, 41Stevenson G. Neal B. Liu D. Hobbs M. Packer N.H. Batley M. Redmond J.W. Lindquist L. Reeves P. J. Bacteriol. 1994; 176: 4144-4156Crossref PubMed Scopus (256) Google Scholar). Recent studies reported that a complete Und-P-P-linked O16 repeat unit was not required for translocation by the WzxO16 translocase (42Feldman M.F. Marolda C.L. Monteiro M.A. Perry M.B. Parodi A.J. Valvano M. J. Biol. Chem. 1999; 274: 35129-35138Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Indeed, the data presented in these studies suggested that the E. coli WzxO16translocase was able to translocate GlcNAc-P-P-Und. The studies presented here were conducted to investigate a role forwzx genes in the transbilayer movement of Fuc4NAc-ManNAcA-GlcNAc-P-P-Und in E. coli. Attempts to measure the flippase-mediated transbilayer movement of naturally occurring undecaprenyl- or dolichyl-linked substrates are complicated by the extremely hydrophobic nature of these compounds. To circumvent this technical difficulty, we employed a variation of the experimental strategy of Rush et al. (18Rush J.S. Waechter C.J. J. Cell Biol. 1995; 130: 529-536Crossref PubMed Scopus (56) Google Scholar, 19Rush J.S. van Leyen K. Overffelli O. Wolucka B. Waechter C.J. Glycobiology. 1998; 8: 1195-1205Crossref PubMed Scopus (40) Google Scholar) who used water-soluble citronellyl-based analogues of mannosylphosphoryldolichol (Man-P-Dol) and glucosylphosphoryldolichol (Glc-P-Dol) to assay flippase-mediated transport activities into intact ER vesicles. Bishop and Bell (14Bishop W.R. Bell R.M. Cell. 1985; 42: 51-60Abstract Full Text PDF PubMed Scopus (186) Google Scholar) originally used a water-soluble analogue of phosphatidylcholine to investigate one or more phosphatidylcholine flippases in sealed rat liver microsomes. Subsequently, phospholipid analogues with short fatty acid chains have been utilized by several other research groups to partially purify and characterize membrane proteins involved in the transbilayer diffusion of phospholipids (43Huijbregts R.P.H. de Kroon A.I.P.M. de Kruijff B. Biochim. Biophys. Acta. 1996; 1280: 41-50Crossref PubMed Scopus (38) Google Scholar, 44Hrafnsdóttir S. Nichols J.W. Menon A.K. 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The data support the conclusion that the observed transport was not mediated by the WzxO16translocase, but rather by WzxE, the putative flippase involved in the transbilayer movement of lipid III across the cytoplasmic membrane during the assembly of ECAPG. (s)-Citronellol, nerol (cis-3,7-dimethyl-2,6-octadien-1-ol), tetramethylammonium phosphate, trichloroacetonitrile, and phosphorous oxytrichloride were obtained from Sigma-Aldrich (St. Louis, MO). UDP-N-acetyl-d-[6-3H]glucosamine (40–60 Ci/mmol) was purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO). [14C]Glucose (360 mCi/mmol) was obtained from American Radiolabeled Chemicals (St. Louis, MO), and it was adjusted to a specific activity of 94 dpm/pmol by the addition of non-radioactive glucose. [3H]GDP-mannose was prepared as described previously (48Rush J.S. Shelling J.G. Zingg N.S. Ray P.H. Waechter C.J. J. Biol. Chem. 1993; 268: 13110-13117Abstract Full Text PDF PubMed Google Scholar). Selecto silica gel was obtained from Fisher Scientific (Pittsburgh, PA). All other chemicals were reagent grade and were purchased from standard commercial sources. E. coli K-12 strains used in this study are listed in TableI. Transductions were carried out using phage P1 vir as described by Silhavy et al. (49Silhavy T.J. Berman M.L. Enquist L.W. Experiments with Gene Fusions. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1984: 107Google Scholar). Cultures were routinely grown at 37 °C with vigorous aeration in Luria-Bertani (LB) broth (50Miller J.H. A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1992Google Scholar) supplemented with glucose to give a final concentration of 0.27 or on LB agar containing 0.27 glucose. Tetracycline, ampicillin, and chloramphenicol were added to media to give final concentrations of 10, 50, and 30 ॖg/ml, respectively. Plasmid pRL160 was constructed by digestion of pCA32 (39Meier-Dieter U. Barr K. Starman R. Hatch L. Rick P.D. J. Biol. Chem. 1992; 267: 746-753Abstract Full Text PDF PubMed Google Scholar) withHindIII yielding an 8.4-kb fragment containingwzxE as well as additional upstream genes of thewec gene cluster. This fragment was digested withXmaI to yield a 7.65-kb fragment that was subsequently ligated to the HindIII and AvaI sites of pBR322. Plasmid pRL162 was constructed by ligation of the 8.4-kbHindIII fragment of pCA32, described above, to the corresponding site of the low copy number vector, pWSK29 (51Wang F. Kushner S.R. Gene (Amst.). 1991; 100: 195-199Crossref PubMed Scopus (1013) Google Scholar). Plasmid pRL147 contained the wecA gene under the control of the PBAD promoter, and it was constructed as follows. ThewecA gene was obtained by PCR amplification of the DNA sequence from bp 9984 to 11141 (GenBankTM accession numberAE000454) using genomic DNA from E. colistrain AB1133 as template. The polynucleotides 5′-CTCTGAGAGCATGC-3′ and 5′-GCGTCGACGTTTCCCAGGCATTGGT-3′ were used as forward and reverse primers, respectively, and anSalI restriction site was incorporated into the reverse primer (underlined sequence). PCR amplifications were carried out usingTaq polymerase (Sigma-Aldrich Chemicals). The amplified sequence contained 17 bp immediately upstream of the translational start site and 36 bp immediately downstream of the translational termination site, and it was cloned into TA cloning site of the pCR2.1 vector (Invitrogen). The resulting construct was digested withEcoRI and SalI, and the 1.1-kb fragment was subcloned into the expression vector, pBAD18 (52Guzman L.-M. Belin D. Carson M.J. Beckwith J. J. Bacteriol. 1995; 177: 4121-4130Crossref PubMed Scopus (3978) Google Scholar), that was restricted with the same enzymes to yield plasmid pRL147.Table IE. coli K-12 strainsStrainDescriptionSource or Ref.SΘ864F−trp lacZ strA thi upp65Neuhard J. Thomassen E. J. Bacteriol. 1976; 126: 999-1001Crossref PubMed Google ScholarSΘ874Δ(wzx016wzxC), P2 eductant of SΘ864 with an extended deletion in the region fromudk to his65Neuhard J. Thomassen E. J. Bacteriol. 1976; 126: 999-1001Crossref PubMed Google Scholar21548thr-1 leuB6 Δ(gpt-proA)66 hisG4 argE3 thi-1 rfbd1 lacY1 ara-14 galK2 xyl-5 mtl-1 mgl-51 rpsL31 kdgK51 supE44 wecA::Tn1039Meier-Dieter U. Barr K. Starman R. Hatch L. Rick P.D. J. Biol. Chem. 1992; 267: 746-753Abstract Full Text PDF PubMed Google Scholar14.5λ−F−thr-1 leuB6 tonA31 lacY1 tsx-78 supO eda50 his-4 rfbD1 mgl-51 rpsL136 xyl-5 mtl-1 metF159 thi − 1 ara-14 wzxE::cmP. N. DanesePR4149As SΘ874, butwecA::Tn10 [21548(P1) × SΘ874]This studyPR4150As PR4149, butwzxE::cm [14.5(P1) × PR4149]This studyPR4151As SΘ864, butwecA::Tn10 [21548(P1) × SΘ864]This studyPR4156As PR4151, butwzxE::cm [14.5(P1) × PR4151]This studyPR4179PR4150/pRL160 (pBR322 containing a 3′, truncation of wzxE)This studyPR4180PR4150/pRL147 (wild-type wecA under control of the PBADpromoter)This studyPR4184PR4150/pRL162 (pWSK29 containing wild-type wzxE)This studyPR4189PR4149/pRL147 (wild-type wecA under control of the PBADpromoter)This study Open table in a new tab Citronellol was phosphorylated using phosphorous oxytrichloride as described by Danilov and Chojnacki (53Danilov L.L. Chojnacki T. FEBS Lett. 1981; 131: 310-312Crossref Scopus (75) Google Scholar). Nerol was phosphorylated with tetrabutylammonium phosphate and trichloroacetonitrile in anhydrous acetonitrile as described by Danilovet al. (54Danilov L.L. Druzhinina T.N. Kalinchak N.A. Maltev S.D. Shibaev V.N. Chem. Phys. Lipids. 1989; 51: 191-203Crossref PubMed Scopus (58) Google Scholar). The isoprenyl phosphates were purified by ion-exchange chromatography on DEAE-cellulose as described previously (48Rush J.S. Shelling J.G. Zingg N.S. Ray P.H. Waechter C.J. J. Biol. Chem. 1993; 268: 13110-13117Abstract Full Text PDF PubMed Google Scholar). [3H]GlcNAc-P-P-Ner was synthesized enzymatically using the UDP-GlcNAc:undecaprenyl phosphateN-acetylglucosaminyl-1-phosphate transferase (WecA) present in E. coli membranes. Membrane fractions were prepared as previously described (37Rush J.S. Rick P.D. Waechter C.J. Glycobiology. 1997; 7: 315-322Crossref PubMed Scopus (40) Google Scholar). Reaction mixtures contained membranes (2.7 mg of membrane protein), 0.1 m Tris-HCl (pH 8.0), 10 mm neryl phosphate (Ner-P), 40 mmMgCl2, 5 mm dithiothreitol, 1 mmsodium orthovanadate, and 0.1 mmUDP-[3H]GlcNAc (18–1800 dpm/pmol) in a total volume of 3 ml. Reaction mixtures were incubated at 21 °C for 16 h and then subjected to centrifugation at 100,000 × g for 10 min using a Beckman TL100.3 micro-ultracentrifuge. The supernatant solution was removed and layered on a 15-ml column of benzyl-DEAE-cellulose (Sigma Chemical Co., St. Louis, MO) equilibrated with 10 mmNH4HCO3. The column was developed with two column volumes of 10 mm NH4HCO3followed by a 60-ml gradient of NH4HCO3 (0–1m). Fractions containing [3H]GlcNAc-P-P-Ner were pooled and dried by rotary evaporation under reduced pressure at 30 °C. The radiolabeled analogue was then dissolved in H2O and desalted by gel-filtration chromatography on a Sephadex G-10 column (1 × 30 cm) equilibrated with H2O. Fractions containing [3H]GlcNAc-P-P-Ner were pooled, dried by rotary evaporation, dissolved in a small volume of CHCl3/CH3OH (2:1, v/v) and layered onto a 15-ml Selecto silica gel column equilibrated with CHCl3. The column was then eluted with CHCl3/CH3OH/H2O/concentrated NH4OH (65:35:6:1, v/v), and fractions of 3.5 ml were collected. Radiolabeled GlcNAc-P-P-Ner eluted in fractions 8–15. Fractions containing [3H]GlcNAc-P-P-Ner were pooled, dried by rotary evaporation, dissolved in 2 ml of CH3OH, and stored at −20 °C until used for transport assays. Formation of [3H]GlcNAc-P-P-Ner was strictly dependent upon the addition of exogenous Ner-P to reaction mixtures. The product was detected as a single radioactive compound when analyzed by thin-layer chromatography on silica gel plates developed with three different solvent systems, and it was detected by a phospholipid-specific spray reagent (55Dittmer J.C. Lester R.L. J. Lipid Res. 1964; 5: 126-127Abstract Full Text PDF PubMed Google Scholar) and by an anisaldehyde-based spray reagent for the detection of isoprenoid compounds (56Dunphy P.J. Kerr J.D. Pennock J.F. Whittle K.J. Feeney J. Biochim. Biophys. Acta. 1967; 136: 136-147Crossref PubMed Scopus (123) Google Scholar).N-Acetyl[3H]glucosamine was the only radioactive product released by mild acid hydrolysis (0.01n HCl, 100 °C, 10 min) as determined by descending paper chromatography on Whatman 3MM paper using butanol/pyridine/H2O
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