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• W1999062199 abstract "The lipopolysaccharide (LPS) core domain of Gram-negative bacteria plays an important role in outer membrane stability and host interactions. Little is known about the biochemical properties of the glycosyltransferases that assemble the LPS core. We now report the purification and characterization of the Rhizobium leguminosarum mannosyl transferase LpcC, which adds a mannose unit to the inner 3-deoxy-d-manno-octulosonic acid (Kdo) moiety of the LPS precursor, Kdo2-lipid IVA. LpcC containing an N-terminal His6 tag was assayed using GDP-mannose as the donor and Kdo2-[4′-32P]lipid IVA as the acceptor and was purified to near homogeneity. Sequencing of the N terminus confirmed that the purified enzyme is the lpcCgene product. Mild acid hydrolysis of the glycolipid generated in vitro by pure LpcC showed that the mannosylation occurs on the inner Kdo residue of Kdo2-[4′-32P]lipid IVA. A lipid acceptor substrate containing two Kdo moieties is required by LpcC, since no activity is seen with lipid IVA or Kdo-lipid IVA. The purified enzyme can use GDP-mannose or, to a lesser extent, ADP-mannose (both of which have the α-anomeric configuration) for the glycosylation of Kdo2-[4′-32P]lipid IVA. Little or no activity is seen with ADP-glucose, UDP-glucose, UDP-GlcNAc, or UDP-galactose. A Salmonella typhimurium waaC mutant, which lacks the enzyme for incorporating the innerl-glycero-d-manno-heptose moiety of LPS, regains LPS with O-antigen when complemented withlpcC. An Escherichia coli heptose-lesswaaC-waaF deletion mutant expressing the R. leguminosarum lpcC gene likewise generates a hybrid LPS species consisting of Kdo2-lipid A plus a single mannose residue. Our results demonstrate that heterologous lpcC expression can be used to modify the structure of the Salmonella andE. coli LPS cores in living cells. The lipopolysaccharide (LPS) core domain of Gram-negative bacteria plays an important role in outer membrane stability and host interactions. Little is known about the biochemical properties of the glycosyltransferases that assemble the LPS core. We now report the purification and characterization of the Rhizobium leguminosarum mannosyl transferase LpcC, which adds a mannose unit to the inner 3-deoxy-d-manno-octulosonic acid (Kdo) moiety of the LPS precursor, Kdo2-lipid IVA. LpcC containing an N-terminal His6 tag was assayed using GDP-mannose as the donor and Kdo2-[4′-32P]lipid IVA as the acceptor and was purified to near homogeneity. Sequencing of the N terminus confirmed that the purified enzyme is the lpcCgene product. Mild acid hydrolysis of the glycolipid generated in vitro by pure LpcC showed that the mannosylation occurs on the inner Kdo residue of Kdo2-[4′-32P]lipid IVA. A lipid acceptor substrate containing two Kdo moieties is required by LpcC, since no activity is seen with lipid IVA or Kdo-lipid IVA. The purified enzyme can use GDP-mannose or, to a lesser extent, ADP-mannose (both of which have the α-anomeric configuration) for the glycosylation of Kdo2-[4′-32P]lipid IVA. Little or no activity is seen with ADP-glucose, UDP-glucose, UDP-GlcNAc, or UDP-galactose. A Salmonella typhimurium waaC mutant, which lacks the enzyme for incorporating the innerl-glycero-d-manno-heptose moiety of LPS, regains LPS with O-antigen when complemented withlpcC. An Escherichia coli heptose-lesswaaC-waaF deletion mutant expressing the R. leguminosarum lpcC gene likewise generates a hybrid LPS species consisting of Kdo2-lipid A plus a single mannose residue. Our results demonstrate that heterologous lpcC expression can be used to modify the structure of the Salmonella andE. coli LPS cores in living cells. lipopolysaccharide(s) matrix-assisted laser desorption-ionization/time of flight 3-deoxy-d-manno-octulosonic acid nuclear Overhauser effect 3-(cyclohexylamino)propanesulfonic acid Lipopolysaccharide (LPS)1 is a prominent structural component of the outer membranes of virtually all Gram-negative bacteria (1Brade H. Opal S.M. Vogel S.N. Morrison D.C. Endotoxin in Health and Disease. Marcel Dekker, Inc., New York1999Google Scholar, 2Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3423) Google Scholar). LPS may be viewed as a tripartite macromolecule. Its principal components include 1) the lipid A moiety (2Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3423) Google Scholar, 3Raetz C.R.H. Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Scopus (1041) Google Scholar, 4Raetz C.R.H. J. Bacteriol. 1993; 175: 5745-5753Crossref PubMed Scopus (236) Google Scholar, 5Rietschel 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), which is the hydrophobic outer membrane anchor; 2) the nonrepeating core oligosaccharide (1Brade H. Opal S.M. Vogel S.N. Morrison D.C. Endotoxin in Health and Disease. Marcel Dekker, Inc., New York1999Google Scholar, 2Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3423) Google Scholar); and 3) the distal repeating O-antigen polysaccharide (1Brade H. Opal S.M. Vogel S.N. Morrison D.C. Endotoxin in Health and Disease. Marcel Dekker, Inc., New York1999Google Scholar, 2Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3423) Google Scholar, 6Reeves P.R. Hobbs M. Valvano M.A. Skurnik M. Whitfield C. Coplin D. Kido N. Klena J. Maskell D. Raetz C.R.H. Rick P.D. Trends Microbiol. 1996; 4: 495-503Abstract Full Text PDF PubMed Scopus (418) Google Scholar). The lipid A moiety and its attached 3-deoxy-d-manno-octulosonic acid (Kdo) units represent the minimal LPS substructure required for bacterial viability (1Brade H. Opal S.M. Vogel S.N. Morrison D.C. Endotoxin in Health and Disease. Marcel Dekker, Inc., New York1999Google Scholar, 2Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3423) Google Scholar). All three domains of LPS are essential for full virulence during infection of animals and plants (1Brade H. Opal S.M. Vogel S.N. Morrison D.C. Endotoxin in Health and Disease. Marcel Dekker, Inc., New York1999Google Scholar). There are striking differences in the structures of diverse LPS molecules, such as those of the plant endosymbionts Rhizobium leguminosarum and Rhizobium etli compared with the enteric bacterium Escherichia coli (2Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3423) Google Scholar, 7Bhat U.R. Forsberg L.S. Carlson R.W. J. Biol. Chem. 1994; 269: 14402-14410Abstract Full Text PDF PubMed Google Scholar, 8Que N.L.S. Lin S. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 2000; 275: 28006-28016Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 9Que N.L.S. Ribeiro A.A. Raetz C.R.H. J. Biol. Chem. 2000; 275: 28017-28027Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). R. leguminosarum and R. etli lipid A molecules lack the phosphate groups found in E. coli lipid A, are modified with a galacturonic acid moiety at position 4′, and contain an unusual 28-carbon secondary acyl chain at position 2′ (Fig.1) (7Bhat U.R. Forsberg L.S. Carlson R.W. J. Biol. Chem. 1994; 269: 14402-14410Abstract Full Text PDF PubMed Google Scholar, 8Que N.L.S. Lin S. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 2000; 275: 28006-28016Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 9Que N.L.S. Ribeiro A.A. Raetz C.R.H. J. Biol. Chem. 2000; 275: 28017-28027Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 10Bhat U.R. Mayer H. Yokota A. Hollingsworth R.I. Carlson R. J. Bacteriol. 1991; 173: 2155-2159Crossref PubMed Google Scholar). The inner core domains ofR. leguminosarum and R. etli lack thel-glycero-d-manno-heptose residues found in E. coli, Salmonella, and most other Gram-negative bacteria and instead contain a mannose residue directly linked to position 5 of the inner Kdo sugar (Fig. 1) (11Bhat U.R. Krishnaiah B.S. Carlson R.W. Carbohydrate Res. 1991; 220: 219-227Crossref PubMed Scopus (45) Google Scholar, 12Carlson R.W. Reuhs B. Chen T.B. Bhat U.R. Noel K.D. J. Biol. Chem. 1995; 270: 11783-11788Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 13Kadrmas 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, 14Kadrmas J.L. 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). Despite these and other differences, the first seven enzymes that catalyze the formation of the key intermediate Kdo2-lipid IVA (Fig. 2) are the same in both organisms (15Price N.P.J. Kelly T.M. Raetz C.R.H. Carlson R.W. J. Bacteriol. 1994; 176: 4646-4655Crossref PubMed Google Scholar). These bacteria diverge in their subsequent processing of Kdo2-lipid IVA. For instance,R. leguminosarum membranes contain phosphatases that remove the 1- and 4′-phosphate moieties late in the pathway (16Price 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 (53) Google Scholar, 17Brozek 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). The mannose residue of the inner core of R. leguminosarum is incorporated by a GDP-mannose-dependent enzyme, which is encoded by the lpcC gene (13Kadrmas 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, 14Kadrmas J.L. 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). In E. coli andSalmonella, WaaC(RfaC) addsl-glycero-d-manno-heptose to the inner Kdo unit at the same place where LpcC adds the mannose moiety in R. leguminosarum or R. etli (Fig. 1) (18Sirisena 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, 19Kadrmas J.L. Raetz C.R. J. Biol. Chem. 1998; 273: 2799-2807Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). There is, however, no sequence homology between WaaC and LpcC (14Kadrmas J.L. 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, 18Sirisena 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, 19Kadrmas J.L. Raetz C.R. J. Biol. Chem. 1998; 273: 2799-2807Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), despite the formal structural similarity of heptose and mannose (Fig. 1). WaaC is thought to utilize ADP-l-glycero-d-manno-heptose as its physiological sugar nucleotide donor (2Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3423) Google Scholar, 18Sirisena 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, 20Gronow S. Oertelt C. Ervela E. Zamyatina A. Kosma P. Skurnik M. Holst O. J. Endotoxin Res. 2001; 7: 263-270Crossref PubMed Google Scholar), whereas LpcC employs GDP-mannose (14Kadrmas J.L. 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, 19Kadrmas J.L. Raetz C.R. J. Biol. Chem. 1998; 273: 2799-2807Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Both LpcC and WaaC can employ the analogue ADP-mannose (a natural product from corn) (21Passeron S. Recondo E. Dankert M. Biochim. Biophys. Acta. 1964; 89: 372-374PubMed Google Scholar, 22Dankert M. Passeron S. Recondo E. Leloir L.F. Biochem. Biophys. Res. Commun. 1964; 14: 358-362Crossref PubMed Scopus (10) Google Scholar) as an alternative substrate in vitro (13Kadrmas 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, 14Kadrmas J.L. 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, 19Kadrmas J.L. Raetz C.R. J. Biol. Chem. 1998; 273: 2799-2807Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). However, bacteria do not normally synthesize ADP-mannose. Significant orthologs of R. leguminosarum lpcC are present in the genomes of several other important proteobacteria, including S. meliloti (see accompanying manuscript (58Kanipes, M. I., Kalb, S. R., Cotter, R. J., Hozbor, D. F., Lagares, A., and Raetz, C. R. H. (2003)278, 16365–16371.Google Scholar)) (23Lagares A. Hozbor D.F. Niehaus K. Otero A.J. Lorenzen J. Arnold W. Puhler A. J. Bacteriol. 2001; 183: 1248-1258Crossref PubMed Scopus (28) Google Scholar, 24Galibert 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 Scopus (940) Google Scholar), Agrobacterium tumefaciens (25Goodner B. Hinkle G. Gattung S. Miller N. Blanchard M. Qurollo B. Goldman B.S. Cao Y. Askenazi M. Halling C. Mullin L. Houmiel K. Gordon J. Vaudin M. Iartchouk O. Epp A. Liu F. Wollam C. Allinger M. Doughty D. Scott C. Lappas C. Markelz B. Flanagan C. Crowell C. Gurson J. Lomo C. Sear C. Strub G. Cielo C. Slater S. Science. 2001; 294: 2323-2328Crossref PubMed Scopus (514) Google Scholar,26Wood D.W. Setubal J.C. Kaul R. Monks D.E. Kitajima J.P. Okura V.K. Zhou Y. Chen L. Wood G.E. Almeida Jr., N.F. Woo L. Chen Y. Paulsen I.T. Eisen J.A. Karp P.D. Bovee Sr., D. Chapman P. Clendenning J. Deatherage G. Gillet W. Grant C. Kutyavin T. Levy R. Li M.J. McClelland E. Palmieri A. Raymond C. Rouse G. Saenphimmachak C. Wu Z. Romero P. Gordon D. Zhang S. Yoo H. Tao Y. Biddle P. Jung M. Krespan W. Perry M. Gordon-Kamm B. Liao L. Kim S. Hendrick C. Zhao Z.Y. Dolan M. Chumley F. Tingey S.V. Tomb J.F. Gordon M.P. Olson M.V. Nester E.W. Science. 2001; 294: 2317-2323Crossref PubMed Scopus (642) Google Scholar), Brucella melitenesis (27DelVecchio V.G. Kapatral V. Redkar R.J. Patra G. Mujer C. Los T. Ivanova N. Anderson I. Bhattacharyya A. Lykidis A. Reznik G. Jablonski L. Larsen N. D'Souza M. Bernal A. Mazur M. Goltsman E. Selkov E. Elzer P.H. Hagius S. O'Callaghan D. Letesson J.J. Haselkorn R. Kyrpides N. Overbeek R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 443-448Crossref PubMed Scopus (450) Google Scholar) and Francisella tularensis, 2The F. tularensis genome is available on the World Wide Web at artedi.ebc.uu.se/Projects/Franciscella/. all of which lack WaaC. The presence of a mannose residue in α-1,5 linkage to the inner Kdo moiety of F. tularensis LPS was recently established by chemical analysis (28Vinogradov E. Perry M.B. Conlan J.W. Eur. J. Biochem. 2002; 269: 6112-6118Crossref PubMed Scopus (152) Google Scholar). A. tumefaciens and B. melitenesis also contain the 28-carbon secondary acyl chain seen in R. leguminosarum (29Bhat U.R. Carlson R.W. Busch M. Mayer H. Int. J. Syst. Bacteriol. 1991; 41: 213-217Crossref PubMed Scopus (68) Google Scholar,30Basu S.S. Karbarz M.J. Raetz C.R.H. J. Biol. Chem. 2002; 277: 28959-28971Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), whereas F. tularensis lipid A resembles that ofR. leguminosarum in lacking both the 1- and 4′-phosphate residues (28Vinogradov E. Perry M.B. Conlan J.W. Eur. J. Biochem. 2002; 269: 6112-6118Crossref PubMed Scopus (152) Google Scholar). The significance of these correlations remains to be determined, but all three organisms can live inside of eucaryotic cells. R. leguminosarum lpcC mutants, which lack most core and all O-antigen sugars, are able to infect their hosts and form small nodules, but the nodules do not fix nitrogen (14Kadrmas J.L. 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). Given the importance of LpcC in the biology of R. leguminosarum (14Kadrmas J.L. 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), its presence in certain human pathogens, and the general lack of biochemical information concerning glycosyl transferases involved in LPS core assembly (2Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3423) Google Scholar, 31Raetz C.R.H. Neidhardt F.C. 2nd Ed. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar), we have now purified R. leguminosarum LpcC to homogeneity and characterized some of its properties. Pure LpcC is highly selective for GDP-mannose, consistent with the reported structure of the R. leguminosarum LPS core (11Bhat U.R. Krishnaiah B.S. Carlson R.W. Carbohydrate Res. 1991; 220: 219-227Crossref PubMed Scopus (45) Google Scholar, 32Carlson R.W. Forsberg L.S. Price N.P. Bhat U.R. Kelly T.M. Raetz C.R.H. Prog. Clin. Biol. Res. 1995; 392: 25-31PubMed Google Scholar). Interestingly, thelpcC gene can complement waaC mutants ofSalmonella (18Sirisena 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), as judged by restoration of O-antigen attachment. GDP-mannose is available to LpcC in Salmonella(2Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3423) Google Scholar, 31Raetz C.R.H. Neidhardt F.C. 2nd Ed. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar, 33Elling L. Ritter J.E. Verseck S. Glycobiology. 1996; 6: 591-597Crossref PubMed Scopus (36) Google Scholar), and mannose is presumably similar enough tol-glycero-d-manno-heptose (Fig. 1) to permit the distal enzymes of Salmonella core assembly to function normally. When lpcC is expressed at 22 °C in a deletion mutant of E. coli lacking bothwaaC and waaF (34Brabetz W. Muller-Loennies S. Holst O. Brade H. Eur. J. Biochem. 1997; 247: 716-724Crossref PubMed Scopus (107) Google Scholar), a single hexose moiety (presumably mannose) is added to a portion of the Kdo2-lipid A made by this strain, further validating the utility of lpcC as a tool for LPS structure modification in living cells of diverse bacteria. The [γ-32P]ATP was purchased from PerkinElmer Life Sciences. ADP-mannose, GDP-mannose, Kdo, and HEPES were obtained from Sigma. Bicinchoninic assay reagents and Triton X-100 were purchased from Pierce. Yeast extract and peptone-tryptone were from Difco. Silica Gel 60 thin layer chromatography plates were obtained from Merck. DNA primers and T4 DNA ligase were from Invitrogen, and PCR reagents were from Stratagene. Reagent grade pyridine, chloroform, and methanol were purchased from Mallinckrodt. The E. coli deep rough mutant WBB06 (ΔwaaC-waaF) was kindly provided by Werner Brabetz (34Brabetz W. Muller-Loennies S. Holst O. Brade H. Eur. J. Biochem. 1997; 247: 716-724Crossref PubMed Scopus (107) Google Scholar). Wild-type strain W3110 was obtained from the E. coli Genetic Stock Center at Yale University.Salmonella typhimurium strains SL1377 (waaC630) (18Sirisena 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) and SL3770 (wild-type) were obtained from theSalmonella Genetic Stock Center (University of Calgary, Calgary, Canada). E. coli strain BLR(DE3)/pLysS was purchased from Novagen. All bacteria were grown in LB broth (10 g of NaCl, 10 g of peptone-tryptone, and 5 g of yeast extract per liter) (35Miller J.R. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar). When necessary, the cultures were supplemented with tetracycline (12 μg/ml) or kanamycin (30 μg/ml). Plasmids were prepared using the Qiagen Mini Prep Kit (Qiagen). Restriction endonucleases (New England Biolabs), shrimp alkaline phosphatase, and T4 ligase were used according to the manufacturer's instructions (Invitrogen). Competent cells were prepared for transformation using the calcium chloride method (36Bergmans H.E. van Die I.M. Hoekstra W.P. J. Bacteriol. 1981; 146: 564-570Crossref PubMed Google Scholar, 37Ausubel 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, Inc., New York1989Google Scholar). The plasmid pJK6A (13Kadrmas 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) was used to retrieve the lpcC gene by digesting it withBamHI and NdeI. The desired fragment was ligated into the pET28b+ vector (Novagen) behind the T7lac promoter that had been digested with the same enzymes, placing a His6 tag at the N terminus. This construct (pMKHN) contains a thrombin cleavage site immediately downstream of the His6tag. The plasmid pMKCA was used for complementation of thewaaC-deficient S. typhimurium mutant SL1377 (18Sirisena 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) or of the E. coli waaC-waaF deletion mutant WBB06 (34Brabetz W. Muller-Loennies S. Holst O. Brade H. Eur. J. Biochem. 1997; 247: 716-724Crossref PubMed Scopus (107) Google Scholar). pMKHN was digested with XhoI and BamHI to retrieve the lpcC gene, and the desired fragment was then cloned behind the lac promoter into the vector pBluescript KS+ (Stratagene), digested with the same enzymes, to generate pMKCA. The mannosyl transferase LpcC was assayed using the LPS precursor Kdo2-[4′-32P]lipid IVA, which was isolated and stored as a frozen aqueous dispersion as described previously (38Basu 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). Prior to each use, the radiolabeled substrate was subjected to ultrasonic irradiation in a water bath for 1 min. The mannosyl transferase was assayed as follows. The reaction mixture (10-μl final volume) contained 50 mm HEPES, pH 7.5, 0.1% Triton X-100, 1 mm GDP mannose, and 10 μmKdo2-[4′-32P]lipid IVA(3000–6000 cpm/nmol). The reaction was started by the addition of an appropriate amount of enzyme (2.5–5 μg/ml of pure enzyme or about 50 μg/ml of membranes of cells overexpressing lpcC behind the T7lac promoter) and incubated for the indicated times at 30 °C. The reactions were stopped by spotting 4-μl samples directly onto a silica gel thin layer plate. After drying the spots at room temperature, the plate was developed in the solvent chloroform, pyridine, 88% formic acid, water (30:70:16:10, v/v/v/v). Following removal of the solvent with a hot air stream, the plate was analyzed using an Amersham Biosciences PhosphorImager (STORM 840), equipped with ImageQuant software. BLR(DE3)/pLysS/pMKHN was grown from a single colony in 1 liter of LB medium containing 100 μg/ml ampicillin and 30 μg/ml kanamycin at 37 °C until the optical density of 600 reached ∼0.5. The culture was induced with 1 mmisopropyl-1-thio-β-d-galactopyranoside and incubated with shaking for an additional 3 h at 30 °C. Cells were harvested at 4 °C by centrifugation at 4,000 × g for 10 min, were resuspended in 30 ml of 50 mm HEPES, pH 7.5, and were broken by one passage through a French pressure cell at 18,000 p.s.i. Debris and inclusion bodies were removed by centrifugation at 4,000 × g for 15 min. Membranes were prepared by ultracentrifugation at 100,000 × g for 60 min at 4 °C. The membranes were resuspended in 20 ml of the same buffer, and the centrifugation was repeated a second time at 100,000 ×g to remove any remaining cytosol. Protein concentration was determined by the bicinchoninic method (Pierce) using bovine serum albumin as the standard. The membrane pellet was resuspended at 10–15 mg/ml in 50 mm HEPES, pH 7.5, and stored at −80 °C until further use. The washed membranes were solubilized by adding an appropriate volume of a 10% Triton X-100 stock solution to give a final concentration of 1% Triton X-100 and incubated at 4 °C for 1 h with intermittent inversion on a rotating apparatus (19Kadrmas J.L. Raetz C.R. J. Biol. Chem. 1998; 273: 2799-2807Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The solubilization mixture was then centrifuged at 100,000 × g for 60 min at 4 °C to remove any remaining particulate proteins and inclusion bodies. One ml of His-Bind resin (Novagen) was prepared for nickel affinity chromatography in a small disposable column by first washing the resin with 3 column volumes of water, 5 column volumes of 50 mmNiSO4, and 3 column volumes of the binding buffer (Novagen), which contains 5 mm imidazole, 0.5 mNaCl, and 20 mm Tris chloride, pH 7.9, with the further addition of 0.1% Triton X-100. A sample of the solubilized membrane proteins (11.2 mg), prepared as described above, was diluted 10-fold in binding buffer and was applied to the column at 4 °C at its natural flow rate. One-ml fractions were collected throughout. The column was washed and then eluted sequentially with 10 ml (5 mm), 6 ml (20 mm), and 6 ml (60 mm) of imidazole in 20 mm Tris-chloride buffer, pH 7.9, containing 0.5m NaCl and 0.1% Triton X-100. The protein content of each fraction was determined. The peak of enzyme activity, which eluted in the 60 mm imidazole wash, was determined by assaying each fraction in the linear range under standard conditions. The proteins were visualized by polyacrylamide gel electrophoresis in the presence of 12% SDS using the Bio-Rad Mini Protean II electrophoresis system. The thrombin cleavage capture kit (Novagen) was used to remove the N-terminal His6 tag and the adjacent thrombin cleavage site from the purified LpcC preparation. Approximately 5 μg of the cleaved purified protein was loaded onto a 12% polyacrylamide SDS gel along with two control lanes containing prestained standards to facilitate transfer to the blot and purified LpcC that had not been cleaved with thrombin. Electrophoresis was carried out at 200 V for 50 min using the Laemmli buffer system. The gel was then soaked in 10 mm CAPS buffer, pH 11, for about 5 min. 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 mmCAPS, pH 11. Protein bands were transferred to the membrane with a Bio-Rad SD electroblotter, according to the manufacturer's directions, at 4 °C. The transferred protein was visualized by Ponceau S staining, and the band of interest was excised. N-terminal sequencing was carried out by Dr. John Leszyk of the Worcester Foundation for Experimental Biology (Shrewsbury, MA). The sequence was determined as GSHMPDIRDV. The first two amino acid residues represent the residual thrombin cleavage site, and the single His residue is derived from the construction of the vector. The remaining sequence corresponds to the predicted N terminus of LpcC. Two 10-μl reaction mixtures in plastic microcentrifuge tubes were prepared containing 50 mm Hepes, pH 7.5, 0.1% Triton X-100, and either Kdo2-[4′-32P]lipid IVA (8,000 cpm/reaction) or mannosyl-Kdo2-[4′-32P]lipid IVA(8,000 cpm/reaction). The mannosyl-Kdo2-[4′-32P]lipid IVAwas first isolated from a larger scale mannosyl transferase assay system that had been allowed to go to completion. The purification of the mannosyl-Kdo2-[4′-32P]lipid IVA was achieved by thin layer chromatography, as for the preparation of Kdo2-[4′-32P]lipid IVA (38Basu 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). Next, 4 μl of 10% SDS and 26 μl of 50 mm sodium acetate, pH 4.5, were added to each microcentrifuge tube, and the tubes were incubated in a boiling water bath. At various times, 4-μl samples were withdrawn and spotted onto a silica TLC plate. The plate was developed in the solvent chloroform, pyridine, 88% formic acid, water (30:70:16:10, v/v/v/v) and analyzed as described previously to determine the site of mannose attachment (19Kadrmas J.L. Raetz C.R. J. Biol. Chem. 1998; 273: 2799-2807Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). To isolate LPS, 100-ml cultures of the E. coli heptose deficient mutant WBB06 containing either pBluescript KS+ or pMKCA (expressing lpcC in pBluescript KS+) were grown to an optical density of 600 = 1.7 at 22 °C. A relatively low temperature was used for this work in order to induce the biosynthesis of colanic acid (39Gottesman S. Stout V. Mol. Microbiol. 1991; 5: 1599-1606Crossref PubMed Scopus (176) Google Scholar), and therefore presumably also GDP-mannose, inE. coli. Ce" @default.
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• W1999062199 title "A Mannosyl Transferase Required for Lipopolysaccharide Inner Core Assembly in Rhizobium leguminosarum" @default.
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• W1999062199 cites W1570553763 @default.
• W1999062199 cites W1580454896 @default.
• W1999062199 cites W1595505074 @default.
• W1999062199 cites W1596946389 @default.
• W1999062199 cites W1604936501 @default.
• W1999062199 cites W1610653444 @default.
• W1999062199 cites W1743939834 @default.
• W1999062199 cites W1922098314 @default.
• W1999062199 cites W1979810819 @default.
• W1999062199 cites W1982308521 @default.
• W1999062199 cites W1985073212 @default.
• W1999062199 cites W1987572008 @default.
• W1999062199 cites W1991413554 @default.
• W1999062199 cites W1995671956 @default.
• W1999062199 cites W2015432102 @default.
• W1999062199 cites W2018965031 @default.
• W1999062199 cites W2021163176 @default.
• W1999062199 cites W2026900055 @default.
• W1999062199 cites W2028161352 @default.
• W1999062199 cites W2037518670 @default.
• W1999062199 cites W2040581338 @default.
• W1999062199 cites W2046171723 @default.
• W1999062199 cites W2055996709 @default.
• W1999062199 cites W2056915679 @default.
• W1999062199 cites W2057511828 @default.
• W1999062199 cites W2057944135 @default.
• W1999062199 cites W2063224703 @default.
• W1999062199 cites W2068143425 @default.
• W1999062199 cites W2072163165 @default.
• W1999062199 cites W2078639416 @default.
• W1999062199 cites W2084682726 @default.
• W1999062199 cites W2090510615 @default.
• W1999062199 cites W2092278008 @default.
• W1999062199 cites W2092290785 @default.
• W1999062199 cites W2092332122 @default.
• W1999062199 cites W2096757648 @default.
• W1999062199 cites W2108508364 @default.
• W1999062199 cites W2139889111 @default.
• W1999062199 cites W2145385227 @default.
• W1999062199 cites W2146948320 @default.
• W1999062199 cites W2149184205 @default.
• W1999062199 cites W2149731513 @default.
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• W1999062199 cites W2159688488 @default.
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