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• W2063224703 abstract "Heptosyltransferase I, encoded by the rfaC(waaC) gene of Escherichia coli, is thought to addl-glycero-d-manno-heptose to the inner 3-deoxy-d-manno-octulosonic acid (Kdo) residue of the lipopolysaccharide core. Lipopolysaccharide isolated from mutants defective in rfaC lack heptose and all other sugars distal to heptose. The putative donor, ADP-l-glycero-d-manno-heptose, has never been fully characterized and is not readily available. In cell extracts, the analog ADP-mannose can serve as an alternative donor for RfaC-catalyzed glycosylation of the acceptor, Kdo2-lipid IVA. Using a T7 promoter construct that overexpresses RfaC ∼15,000-fold, the enzyme has been purified to near homogeneity. NH2-terminal sequencing confirms that the purified enzyme is the rfaC gene product. The subunit molecular mass is 36 kDa. Enzymatic activity is dependent upon the presence of Triton X-100 and is maximal at pH 7.5. The apparentKm (determined at near saturating concentrations of the second substrate) is 1.5 mm for ADP-mannose and 4.5 μm for Kdo2-lipid IVA. Chemical hydrolysis of the RfaC reaction product at 100 °C in the presence of sodium acetate and 1% sodium dodecyl sulfate generates fragments consistent with the inner Kdo residue of Kdo2-lipid IVA as the site of mannosylation. The analog, Kdo-lipid IVA, functions as an acceptor, but is mannosylated at less than 1% the rate of Kdo2-lipid IVA. The purified enzyme displays no activity with ADP-glucose, GDP-mannose, UDP-glucose, or UDP-galactose. Mannosylation of Kdo2-lipid IVA catalyzed by RfaC proceeds in high yield and may be useful for the synthesis of lipopolysaccharide analogs. Pure RfaC can also be used together with Kdo2-[4′-32P]lipid IVA to assay for the physiological donor (presumably ADP-l-glycero-d-manno-heptose) in a crude, low molecular weight fraction isolated from wild type cells. Heptosyltransferase I, encoded by the rfaC(waaC) gene of Escherichia coli, is thought to addl-glycero-d-manno-heptose to the inner 3-deoxy-d-manno-octulosonic acid (Kdo) residue of the lipopolysaccharide core. Lipopolysaccharide isolated from mutants defective in rfaC lack heptose and all other sugars distal to heptose. The putative donor, ADP-l-glycero-d-manno-heptose, has never been fully characterized and is not readily available. In cell extracts, the analog ADP-mannose can serve as an alternative donor for RfaC-catalyzed glycosylation of the acceptor, Kdo2-lipid IVA. Using a T7 promoter construct that overexpresses RfaC ∼15,000-fold, the enzyme has been purified to near homogeneity. NH2-terminal sequencing confirms that the purified enzyme is the rfaC gene product. The subunit molecular mass is 36 kDa. Enzymatic activity is dependent upon the presence of Triton X-100 and is maximal at pH 7.5. The apparentKm (determined at near saturating concentrations of the second substrate) is 1.5 mm for ADP-mannose and 4.5 μm for Kdo2-lipid IVA. Chemical hydrolysis of the RfaC reaction product at 100 °C in the presence of sodium acetate and 1% sodium dodecyl sulfate generates fragments consistent with the inner Kdo residue of Kdo2-lipid IVA as the site of mannosylation. The analog, Kdo-lipid IVA, functions as an acceptor, but is mannosylated at less than 1% the rate of Kdo2-lipid IVA. The purified enzyme displays no activity with ADP-glucose, GDP-mannose, UDP-glucose, or UDP-galactose. Mannosylation of Kdo2-lipid IVA catalyzed by RfaC proceeds in high yield and may be useful for the synthesis of lipopolysaccharide analogs. Pure RfaC can also be used together with Kdo2-[4′-32P]lipid IVA to assay for the physiological donor (presumably ADP-l-glycero-d-manno-heptose) in a crude, low molecular weight fraction isolated from wild type cells. Lipopolysaccharide (LPS) 1The abbreviations used are: LPS, lipopolysaccharide; BSA, bovine serum albumin; PCR, polymerase chain reaction; IPTG, isopropyl-1-thio-β-d-galactopyranoside; Kdo, 3-deoxy-d-manno-octulosonic acid; CAPS, 3-(cyclohexylamino)propanesulfonic acid; Mes, 4-morpholine-ethanesulfonic acid. is a major component of the outer leaflet of the outer membranes of Gram-negative bacteria (1Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, DC1996: 1035-1063Google Scholar, 2Raetz C.R.H. Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Scopus (1041) Google Scholar, 3Rietschel E.T. Kirikae T. Schade F.U. Mamat U. Schmidt G. Loppnow H. Ulmer A.J. Zähringer U. Seydel U. Di Padova F. Schreier M. Brade H. FASEB J. 1994; 8: 217-225Crossref PubMed Scopus (1334) Google Scholar, 4Schnaitman C.A. Klena J.D. Microbiol. Rev. 1993; 57: 655-682Crossref PubMed Google Scholar). It is composed of three domains (Fig. 1): 1) a hydrophobic anchor, known as lipid A, that consists of an acylated disaccharide of glucosamine; 2) a non-repeating oligosaccharide, designated the core, that serves as a barrier to many antibiotics; and 3) the O-antigen, that extends outwards from the core and is comprised of a distinct repeating oligosaccharide. All components of LPS are required for the virulence of Gram-negative bacteria (1Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, DC1996: 1035-1063Google Scholar, 3Rietschel E.T. Kirikae T. Schade F.U. Mamat U. Schmidt G. Loppnow H. Ulmer A.J. Zähringer U. Seydel U. Di Padova F. Schreier M. Brade H. FASEB J. 1994; 8: 217-225Crossref PubMed Scopus (1334) Google Scholar, 5Hood D.W. Deadman M.E. Allen T. Masoud H. Martin A. Brisson J.R. Fleischmann R. Venter J.C. Richards J.C. Moxon E.R. Mol. Microbiol. 1996; 22: 951-965Crossref PubMed Scopus (147) Google Scholar). The O-antigen and many of the core sugars are not required for viability (1Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, DC1996: 1035-1063Google Scholar, 3Rietschel E.T. Kirikae T. Schade F.U. Mamat U. Schmidt G. Loppnow H. Ulmer A.J. Zähringer U. Seydel U. Di Padova F. Schreier M. Brade H. FASEB J. 1994; 8: 217-225Crossref PubMed Scopus (1334) Google Scholar, 6Mäkelä P.H. Stocker B.A.D. Rietschel E.T. Handbook of Endotoxin: Chemistry of Endotoxin. I. Elsevier/North-Holland Biomedical Press, Amsterdam1984: 59-137Google Scholar, 7Nikaido H. Vaara M. Neidhardt F.C. Escherichia coli and Salmonella typhimurium. 1. American Society for Microbiology, Washington, DC1987: 7-22Google Scholar, 8Reeves P. Neuberger A. van Deenen L.L.M. Bacterial Cell Wall: New Comprehensive Biochemistry. 27. Elsevier Science Publishers, New York1994: 281-314Google Scholar), but the lipid A and Kdo residues of the inner core are essential for growth of Escherichia coli and related organisms (9Galloway S.M. Raetz C.R.H. J. Biol. Chem. 1990; 265: 6394-6402Abstract Full Text PDF PubMed Google Scholar, 10Goldman R. Kohlbrenner W. Lartey P. Pernet A. Nature. 1987; 329: 162-164Crossref PubMed Scopus (120) Google Scholar, 11Hammond S.M. Claesson A. Jansson A.M. Larsson L.G. Pring B.G. Town C.M. Ekström B. Nature. 1987; 327: 730-732Crossref PubMed Scopus (140) Google Scholar, 12Onishi 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 (360) Google Scholar, 13Rick P.D. Osborn M.J. J. Biol. Chem. 1977; 252: 4895-4903Abstract Full Text PDF PubMed Google Scholar). Most of the genes of core oligosaccharide biosynthesis are contained in the rfa(waa) cluster near minute 82 on the E. coli chromosome (1Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, DC1996: 1035-1063Google Scholar, 3Rietschel E.T. Kirikae T. Schade F.U. Mamat U. Schmidt G. Loppnow H. Ulmer A.J. Zähringer U. Seydel U. Di Padova F. Schreier M. Brade H. FASEB J. 1994; 8: 217-225Crossref PubMed Scopus (1334) Google Scholar, 14Sanderson K.E. Roth J.R. Microbiol. Rev. 1988; 52: 485-532Crossref PubMed Google Scholar, 15Schnaitman C.A. Parker C.T. Pradel E. Klena J.D. Pearson N. Sanderson K.E. MacLachlan P.R. J. Bacteriol. 1991; 173: 7410-7411Crossref PubMed Google Scholar, 16Roncero C. Casabadan M. J. Bacteriol. 1992; 174: 3250-3260Crossref PubMed Google Scholar). The functions of these genes have been deduced from genetic studies, in conjunction with partial physical and chemical characterizations of isolated LPS (1Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, DC1996: 1035-1063Google Scholar, 3Rietschel E.T. Kirikae T. Schade F.U. Mamat U. Schmidt G. Loppnow H. Ulmer A.J. Zähringer U. Seydel U. Di Padova F. Schreier M. Brade H. FASEB J. 1994; 8: 217-225Crossref PubMed Scopus (1334) Google Scholar). Direct enzymatic studies of E. coli core biosynthesis beyond Kdo have been limited (1Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, DC1996: 1035-1063Google Scholar) because the structure of the core is not fully established. Consequently, the acceptor substrates of most of the enzymes involved in core glycosylation and the products generated by these enzymes are not fully characterized (1Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, DC1996: 1035-1063Google Scholar, 17Sirisena D.M. Brozek K.A. MacLachlan P.R. Sanderson K.E. Raetz C.R.H. J. Biol. Chem. 1992; 267: 18874-18884Abstract Full Text PDF PubMed Google Scholar, 18Strain S.M. Fesik S.W. Armitage I.M. J. Biol. Chem. 1983; 258: 13466-13477Abstract Full Text PDF PubMed Google Scholar, 19Strain S.M. Armitage I.M. Anderson L. Takayama K. Quershi N. Raetz C.R.H. J. Biol. Chem. 1985; 260: 16089-16098Abstract Full Text PDF PubMed Google Scholar). In vitro assays dependent upon time and protein have not generally been developed (1Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, DC1996: 1035-1063Google Scholar, 17Sirisena D.M. Brozek K.A. MacLachlan P.R. Sanderson K.E. Raetz C.R.H. J. Biol. Chem. 1992; 267: 18874-18884Abstract Full Text PDF PubMed Google Scholar, 20Rick P.D. Neidhardt F. Escherichia coli and Salmonella typhimurium. 1. American Society for Microbiology, Washington, DC1987: 648-662Google Scholar), and key synthetic donors and acceptors are not available (1Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, DC1996: 1035-1063Google Scholar). The inner core of E. coli contains 2–3 Kdo residues, 2–3 heptose residues, and several other substoichiometric substituents (Fig. 1) (1Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, DC1996: 1035-1063Google Scholar, 2Raetz C.R.H. Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Scopus (1041) Google Scholar, 3Rietschel E.T. Kirikae T. Schade F.U. Mamat U. Schmidt G. Loppnow H. Ulmer A.J. Zähringer U. Seydel U. Di Padova F. Schreier M. Brade H. FASEB J. 1994; 8: 217-225Crossref PubMed Scopus (1334) Google Scholar, 4Schnaitman C.A. Klena J.D. Microbiol. Rev. 1993; 57: 655-682Crossref PubMed Google Scholar, 20Rick P.D. Neidhardt F. Escherichia coli and Salmonella typhimurium. 1. American Society for Microbiology, Washington, DC1987: 648-662Google Scholar). Mutants that lack heptose are viable but display a deep rough phenotype (3Rietschel E.T. Kirikae T. Schade F.U. Mamat U. Schmidt G. Loppnow H. Ulmer A.J. Zähringer U. Seydel U. Di Padova F. Schreier M. Brade H. FASEB J. 1994; 8: 217-225Crossref PubMed Scopus (1334) Google Scholar). They are sensitive to detergents, hydrophobic antibiotics, and rough-specific bacteriophages (3Rietschel E.T. Kirikae T. Schade F.U. Mamat U. Schmidt G. Loppnow H. Ulmer A.J. Zähringer U. Seydel U. Di Padova F. Schreier M. Brade H. FASEB J. 1994; 8: 217-225Crossref PubMed Scopus (1334) Google Scholar, 7Nikaido H. Vaara M. Neidhardt F.C. Escherichia coli and Salmonella typhimurium. 1. American Society for Microbiology, Washington, DC1987: 7-22Google Scholar). The incorporation of the first heptose residue into LPS is thought to be catalyzed by the rfaC(waaC) gene product (1Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, DC1996: 1035-1063Google Scholar, 3Rietschel E.T. Kirikae T. Schade F.U. Mamat U. Schmidt G. Loppnow H. Ulmer A.J. Zähringer U. Seydel U. Di Padova F. Schreier M. Brade H. FASEB J. 1994; 8: 217-225Crossref PubMed Scopus (1334) Google Scholar), designated heptosyltransferase I (17Sirisena D.M. Brozek K.A. MacLachlan P.R. Sanderson K.E. Raetz C.R.H. J. Biol. Chem. 1992; 267: 18874-18884Abstract Full Text PDF PubMed Google Scholar, 21Kadrmas J.L. Brozek K.A. Raetz C.R.H. J. Biol. Chem. 1996; 271: 32119-32125Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Previous studies of E. coli and Salmonellaheptosyltransferase I suffered from some of the above mentioned limitations (17Sirisena D.M. Brozek K.A. MacLachlan P.R. Sanderson K.E. Raetz C.R.H. J. Biol. Chem. 1992; 267: 18874-18884Abstract Full Text PDF PubMed Google Scholar). Since synthetic ADP-l-glycero-d-manno-heptose was not available, partially purified preparations of ADP-heptose, isolated from cells of Shigella sonnei (22Kontrohr T. Kocsis B. J. Biol. Chem. 1981; 256: 7715-7718Abstract Full Text PDF PubMed Google Scholar), were utilized. However, the heptosyl acceptor employed, Kdo2-lipid IVA, was well characterized (23Brozek K.A. Hosaka K. Robertson A.D. Raetz C.R.H. J. Biol. Chem. 1989; 264: 6956-6966Abstract Full Text PDF PubMed Google Scholar). Kdo2-lipid IVA is thought to be capable of acquiring a complete corein vivo (24Walenga R.W. Osborn M.J. J. Biol. Chem. 1980; 255: 4257-4263Abstract Full Text PDF PubMed Google Scholar). Even so, the products generated by thisin vitro system could only be isolated in radiochemical amounts insufficient for physical analysis (17Sirisena D.M. Brozek K.A. MacLachlan P.R. Sanderson K.E. Raetz C.R.H. J. Biol. Chem. 1992; 267: 18874-18884Abstract Full Text PDF PubMed Google Scholar). Recently, we have described a new assay for heptosyltransferase I of E. coli, using commercially available ADP-mannose as an alternative donor in place of ADP-l-glycero-d-manno-heptose (21Kadrmas J.L. Brozek K.A. Raetz C.R.H. J. Biol. Chem. 1996; 271: 32119-32125Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), as shown in Fig. 2. ADP-mannose is a naturally occurring sugar nucleotide found in corn (25Dankert M. Passeron S. Recondo E. Leloir L.F. Biochem. Biophys. Res. Commun. 1964; 14: 358-362Crossref PubMed Scopus (10) Google Scholar, 26Passeron S. Recondo E. Dankert M. Biochim. Biophys. Acta. 1964; 89: 372-374PubMed Google Scholar). Here, we report the first characterization of the catalytic properties of heptosyltransferase I, using ADP-mannose as the donor and Kdo2-[4′-32P]lipid IVA as the acceptor. We have purified RfaC to near homogeneity using this optimized assay system, and we have characterized the product as mannosyl-Kdo2-lipid IVA (proposed structure shown in Fig. 2). We have also devised a new procedure for the isolation of a crude (low molecular weight) sugar nucleotide-containing fraction from various strains of E. coli and Salmonella. Assays utilizing these sugar nucleotide preparations in place of ADP-mannose demonstrate that pure RfaC is capable of glycosylating Kdo2-[4′-32P]lipid IVA with a single sugar presumed to be ADP-l-glycero-d-manno-heptose. This reaction should serve as a functional assay for the definitive isolation and structural characterization of the elusive endogenous heptosyl donor of LPS biosynthesis. Materials and kits purchased were: [γ-32P]ATP (NEN Life Science Products); Hepes, Mes, Tris, bovine serum albumin (BSA), Reactive Green 19, ADP-mannose, and all other sugar nucleotides (Sigma); Triton X-100 and bicinchoninic assay reagents (Pierce); silica gel 60 thin layer chromatography plates (E. Merck); yeast extract and tryptone (Difco); Wizard Mini-prep kit (Promega); PCR reagents (Stratagene); restriction enzymes (New England Biolabs); shrimp alkaline phosphatase (U. S. Biochemical Corp.); custom primers and T4 DNA ligase (Life Technologies, Inc.); Qiaex II gel extraction kit (Qiagen); polyacrylamide gel reagents (National Diagnostics); Centricon centrifugation devices (Amicon); and Immobilon P polyvinyldifluoride membranes (Millipore). All solvents were reagent grade. Radiochemical analysis of thin layer plates was performed with a model 425S Molecular Dynamics PhosphorImager equipped with ImageQuant software. The Clarke and Carbon E. coli strain JA200 pLC10–7 (27Clarke L. Carbon J. Cell. 1976; 9: 91-99Abstract Full Text PDF PubMed Scopus (808) Google Scholar, 28Creeger E.S. Rothfield L.I. J. Biol. Chem. 1979; 254: 804-810Abstract Full Text PDF PubMed Google Scholar, 29Nishimura A. Akiyama K. Kohara Y. Horiuchi K. Microbiol. Rev. 1992; 56: 137-151Crossref PubMed Google Scholar), containing the genes of the rfa operon on a hybrid colE1 plasmid, was obtained from the E. coli Genetic Stock Center (Yale University, New Haven, CT). E. coli SURE cells were purchased from Stratagene. Plasmid pET3a and E. colistrain BLR(DE3)pLysS were purchased from Novagen. E. colistrains D21 (wild type) (30Bowman H.G. Eriksson-Grennberg K.G. Normark S. Mattson E. Genet. Res. 1968; 12: 169-185Crossref PubMed Scopus (27) Google Scholar, 31Grundstrom T. Jaurin B. Edlund T. Normark S. J. Bacteriol. 1980; 143: 1127-1134Crossref PubMed Google Scholar) and D21f2 (rfaC−) (32Havekes L. Tommassen J. Hoekstra W. Lugtenberg B. J. Bacteriol. 1977; 129: 1-8Crossref PubMed Google Scholar) were obtained from the E. coli Genetic Stock Center. Salmonella strains SA1377 (rfaC630) (33Sanderson K.E. Wyngaarden J.V. Luderitz O. Stocker B.A.D. Can. J. Microbiol. 1974; 20: 1127-1134Crossref PubMed Scopus (14) Google Scholar), SL3600 (rfaD657) (34Lehmann V. Hammerling G. Nurminen M. Minner I. Ruschmann E. Luderitz O. Kuo T.T. Stocker B.A.D. Eur. J. Biochem. 1973; 32: 268-275Crossref PubMed Scopus (25) Google Scholar) and SL1102 (rfaE543) (35Roantree R.J. Kuo T.T. MacPhee D.G. J. Gen. Microbiol. 1977; 103: 223-234Crossref PubMed Scopus (90) Google Scholar) were obtained from the Salmonella Genetic Stock Center (University of Calgary, Calgary, Canada). The [4′-32P]lipid IVA was generated from [γ-32P]ATP and the tetra-acylated disaccharide 1-phosphate precursor, using the E. coli 4′-kinase from membranes of strain BR7 (36Hampton R.Y. Raetz C.R.H. Methods Enzymol. 1992; 209: 466-475Crossref PubMed Scopus (12) Google Scholar). The labeled lipid IVA was converted to either Kdo2-[4′-32P]lipid IVA using purified E. coli Kdo transferase (23Brozek K.A. Hosaka K. Robertson A.D. Raetz C.R.H. J. Biol. Chem. 1989; 264: 6956-6966Abstract Full Text PDF PubMed Google Scholar,37Belunis C.J. Raetz C.R.H. J. Biol. Chem. 1992; 267: 9988-9997Abstract Full Text PDF PubMed Google Scholar) or to Kdo-[4′-32P]lipid IVA using aHaemophilus influenzae extract (24Walenga R.W. Osborn M.J. J. Biol. Chem. 1980; 255: 4257-4263Abstract Full Text PDF PubMed Google Scholar, 38White K.A. Kaltashov I.A. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 1997; 272: 16555-16563Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The products were purified by preparative thin layer chromatography and stored at −20 °C as an aqueous dispersion (23Brozek K.A. Hosaka K. Robertson A.D. Raetz C.R.H. J. Biol. Chem. 1989; 264: 6956-6966Abstract Full Text PDF PubMed Google Scholar, 37Belunis C.J. Raetz C.R.H. J. Biol. Chem. 1992; 267: 9988-9997Abstract Full Text PDF PubMed Google Scholar). Prior to each use, these substrates were subjected to ultrasonic irradiation in a water bath for 60 s. Unless indicated, reaction mixtures (10–40 μl) contained 50 mm Hepes, pH 7.5, 0.1% Triton X-100, 10 μm Kdo2-[4′-32P]lipid IVA at 80,000 cpm/nmol, and 1.0 mm ADP-mannose (21Kadrmas J.L. Brozek K.A. Raetz C.R.H. J. Biol. Chem. 1996; 271: 32119-32125Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The enzyme source, added last to initiate the reaction, contained 1 mg/ml BSA when diluted to less than 1 μg/ml. The enzyme generally comprised 1/10 of the reaction volume. Reactions were incubated at 30 °C for 5–60 min. Reactions were terminated by spotting 5-μl portions of the reaction mixture onto a Silica Gel 60 thin layer plate. After drying in a stream of cold air, plates were developed in chloroform/pyridine/88% formic acid/water (30:70:16:10, v/v). The plate was dried and then exposed to a PhosphorImager screen overnight. The amount of product formed was calculated from the percent conversion of the radioactive substrate (of known specific radioactivity) to product. Plasmids were prepared using the Promega Wizard Miniprep kit. Restriction endonucleases, shrimp alkaline phosphatase, and T4 DNA ligase were all used according to the manufacturer's instructions. DNA fragments were isolated from agarose gels using a Qiaex II gel extraction kit (Qiagen). All other techniques involving manipulation of nucleic acids were from Ausubelet al. (39Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, New York1989Google Scholar). Cells were made competent for electroporation by resuspension in 10% glycerol (39Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, New York1989Google Scholar), as described. Transformation of plasmid DNA into competent cells was performed by high voltage electroporation using a Bio-Rad Gene Pulser II at 2.5 kV, 200 ohms, and 25 microfarads. The cloning of PCR-generated rfaC DNA into a vector under T7 promoter control is outlined in Fig. 3 (40Rosenberg A.H. Lade B.N. Chui D.S. Lin S., J.J., D. Studier F.W. Gene (Amst.). 1987; 56: 125-135Crossref PubMed Scopus (1044) Google Scholar, 41Studier F.W. Moffatt B.A. J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (4842) Google Scholar, 42Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6006) Google Scholar). The forward primer was synthesized with a GC clamp, an NdeI restriction site, and an rfaC coding strand starting at the translation initiation site (primer sequences are shown in legend for Fig. 3). The reverse primer was synthesized with a GC clamp, aBamHI restriction site, and an rfaC anticoding strand that includes the stop site. The PCR was performed usingPfu polymerase, as specified by the manufacturer. The plasmid pLC10–7 (28Creeger E.S. Rothfield L.I. J. Biol. Chem. 1979; 254: 804-810Abstract Full Text PDF PubMed Google Scholar, 29Nishimura A. Akiyama K. Kohara Y. Horiuchi K. Microbiol. Rev. 1992; 56: 137-151Crossref PubMed Google Scholar) was used as the template. Amplification was carried out in a 50-μl reaction mixture containing 100 ng of template, 20 mm Tris-HCl, pH 8.8, 10 mm KCl, 10 mm (NH4)2SO4, 0.1% Triton X-100, 0.1% BSA, 2 mm MgSO4, 250 μm of each of the dNTPs, 200 ng of each primer, and 1.2 units of Pfu polymerase. The reaction was subjected to 30 cycles of denaturation (1 min, 94 °C), annealing (1 min, 55 °C), and extension (1.5 min, 72 °C) in a DNA thermal cycler. The reaction product was analyzed on a 1% agarose gel, digested withNdeI and BamHI, and ligated into the expression vector pET3a that had been similarly digested. The resulting desired hybrid plasmid (designated pJK1) was transformed into E. coli SURE cells, reisolated and digested again to verify its structure, and finally transformed into cells of strain BLR(DE3)pLysS. BLR(DE3)pLysS/pET3a and BLR(DE3)pLysS/pJK1 were grown from a single colony in 1 liter of LB medium (43Miller J.R. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar) containing ampicillin (50 μg/ml) and chloramphenicol (30 μg/ml) at 37 °C until the A600 reached approximately 0.5. The culture was split into two equal portions, and one portion was induced with 100 μg/ml IPTG. Both cultures were incubated with shaking at 225 rpm for an additional 3 h at 37 °C, the A600 was recorded, and the cells were harvested by centrifugation for 10 min at 6000 × gav at 4 °C. All subsequent steps were performed either on ice or at 4 °C. The cell pellet was resuspended in a minimal volume, typically 10 ml of 50 mmHepes, pH 7.5, and broken by passage through a 5-ml French pressure cell at 18,000 p.s.i. Unbroken cells and debris were removed by centrifugation for 10 min at 6000 × g. The resulting crude extract supernatant was used to prepare membranes. The crude extract was subjected to ultracentrifugation at 100,000 ×gav for 60 min. The membrane pellet was resuspended in 1.5 ml of 50 mm Hepes, pH 7.5. The protein content of each fraction was determined by the bicinchoninic acid (BCA) assay (44Smith 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 (18713) Google Scholar) using BSA as the standard. A 1 ml portion containing 8–10 mg/ml protein of the BLR(DE3)pLysS/pJK1 membranes was mixed with an equal volume of 2% Triton X-100 and incubated on ice for 2 h with periodic gentle inversion of the tube. The solubilization mixture was then centrifuged at 100,000 × gav for 60 min to remove any unsolubilized proteins. The pellet was resuspended in 750 μl of 50 mm Hepes, pH 7.5, and the protein contents of both the solubilized and unsolubilized fractions were determined by the BCA assay (44Smith 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 (18713) Google Scholar). One gram of Reactive Green 19 resin suspended in 5 ml of water was equilibrated in a small plastic disposable column with 10 column volumes of equilibration buffer (50 mm Hepes, pH 7.5, 0.1% Triton X-100). A 4-mg sample of solubilized membrane proteins (in 1.25 ml) was diluted 10-fold with 50 mm Hepes pH 7.5, and the material was then applied to the column at a flow rate of 1 ml/min. Fractions of 2.5 ml were collected throughout. Next, the column was washed with 25 ml of equilibration buffer. Elution was carried out in three stages: 1) 25 ml of equilibration buffer plus 0.5 m NaCl, 2) 25 ml of equilibration buffer plus 1.0 m NaCl, and, finally, 3) 25 ml of equilibration buffer plus 2.5 m NaCl. The protein content of each fraction was determined using the BCA assay (44Smith 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 (18713) Google Scholar). The peak of enzyme activity was determined by assaying each fraction in the linear range under standard conditions for detection of mannose transfer to Kdo2-[4′-32P]lipid IVA. The protein in certain samples was also visualized by 10% polyacrylamide gel electrophoresis in the presence of SDS, using the Laemmli buffer system (45Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar) in conjunction with Bio-Rad Mini-Protean II electrophoresis equipment. Approximately 20 μg (400 μl) of Green 19 purified protein was concentrated 40-fold on a Microcon 10 device, according to the manufacturer's instructions. The concentrated sample was loaded onto a 10% polyacrylamide SDS gel along with a lane containing prestained standards as a control for transfer. Electrophoresis was carried out at 200 V for 50 min in a Laemmli gel buffer system. The gel was then soaked in 10 mm CAPS, pH 11, for 10 min at 4 °C. A polyvinylidene difluoride membrane was prepared while the electrophoresis was in progress by brief soaking in methanol, rinsing with water, and then soaking in 10 mm CAPS, pH 11. A Bio-Rad SD electroblotter was used according to the manufacturer's directions at 20 V for 40 min. Protein bands transferred to the membrane were visualized by Coomassie staining, and the band of interest was excised. NH2-terminal amino acid sequencing of the intact protein was carried out by Dr. John Leszyk of the Worcester Foundation for Experimental Biology, Shrewsbury, MA. Two 10-μl reaction mixtures were prepared containing 50 mm Hepes, pH 7.5, 0.1% Triton X-100, 0.4 μm Kdo2-lipid IVA (1 × 107 cpm/nmol or 40,000 cpm/reaction), and 1 mmADP-mannose. To only one tube, 0.3 μg/ml purified RfaC was added. Both tubes were incubated for 30 min at 30 °C. Next, 4 μl of 10% SDS and 26 μl of 50 mm sodium acetate pH 4.5 were added (46Cano E. Mahadevan L.C. Trends Biochem. Sci. 1995; 20: 117-122Abstract Full Text PDF PubMed Scopus (1001) Google Scholar, 47Caroff M. Tacken A. Szabó L. Carbohydr. Res. 1988; 175: 273-282Crossref PubMed Scopus (200) Google Scholar, 48Caroff M. Deprun C. Karibian D. Szabó L. J. Biol. Chem. 1991; 266: 18543-18549Abstract Full Text PDF PubMed Google Scholar) to both tubes to give a final pH of approximately 5.0, and the tubes were incubated in a boiling water bath. At 0, 1, 2, 5, 10, 20, and 30 min, 5-μl samples were withdrawn and spotted onto a silica TLC plate. The plate was developed and analyzed by PhosphorImager analysis, as described above. Five different strains (see below) were st" @default.
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