Cloning and Overexpression of Glycosyltransferases That Generate the Lipopolysaccharide Core of Rhizobium leguminosarum

The lipopolysaccharide (LPS) core of the Gram-negative bacterium Rhizobium leguminosarum is more amenable to enzymatic study than that of Escherichia colibecause much of it is synthesized from readily available sugar nucleotides. The inner portion of the R. leguminosarum core contains mannose, galactose, and three 3-deoxy-d-manno-octulosonate (Kdo) residues, arranged in the order: lipid A-(Kdo)2-Man-Gal-Kdo–[O antigen]. A mannosyltransferase that uses GDP-mannose and the conserved precursor Kdo2-[4′-32P]lipid IVA (Kadrmas, J. L., Brozek, K. A., and Raetz, C. R. H. (1996) J. Biol. Chem. 271, 32119–32125) is proposed to represent a key early enzyme in R. leguminosarum core assembly. Conditions for demonstrating efficient galactosyl- and distal Kdo-transferase activities are now described using a coupled assay system that starts with GDP-mannose and Kdo2-[4′-32P]lipid IVA. As predicted, mannose incorporation precedes galactose addition, which in turn precedes distal Kdo transfer. LPS core mutants with Tn5 insertions in the genes encoding the putative galactosyltransferase (lpcA) and the distal Kdo-transferase (lpcB) are shown to be defective in the corresponding in vitroglycosylation of Kdo2-[4′-32P]lipid IVA. We have also discovered the new gene (lpcC) that encodes the mannosyltransferase. The gene is separated by several kilobase pairs from the lpcAB cluster. All three glycosyltransferases are carried on cosmid pIJ1848, which contains at least 20 kilobase pairs of R. leguminosarumDNA. Transfer of pIJ1848 into R. meliloti 1021 results in heterologous expression of all three enzymes, which are not normally present in strain 1021. Expression of the lpc genes individually behind the T7 promoter results in the production of eachR. leguminosarum glycosyltransferase in E. colimembranes in a catalytically active form, demonstrating thatlpcA, lpcB, and lpcC are structural genes. The lipopolysaccharide (LPS) core of the Gram-negative bacterium Rhizobium leguminosarum is more amenable to enzymatic study than that of Escherichia colibecause much of it is synthesized from readily available sugar nucleotides. The inner portion of the R. leguminosarum core contains mannose, galactose, and three 3-deoxy-d-manno-octulosonate (Kdo) residues, arranged in the order: lipid A-(Kdo)2-Man-Gal-Kdo–[O antigen]. A mannosyltransferase that uses GDP-mannose and the conserved precursor Kdo2-[4′-32P]lipid IVA (Kadrmas, J. L., Brozek, K. A., and Raetz, C. R. H. (1996) J. Biol. Chem. 271, 32119–32125) is proposed to represent a key early enzyme in R. leguminosarum core assembly. Conditions for demonstrating efficient galactosyl- and distal Kdo-transferase activities are now described using a coupled assay system that starts with GDP-mannose and Kdo2-[4′-32P]lipid IVA. As predicted, mannose incorporation precedes galactose addition, which in turn precedes distal Kdo transfer. LPS core mutants with Tn5 insertions in the genes encoding the putative galactosyltransferase (lpcA) and the distal Kdo-transferase (lpcB) are shown to be defective in the corresponding in vitroglycosylation of Kdo2-[4′-32P]lipid IVA. We have also discovered the new gene (lpcC) that encodes the mannosyltransferase. The gene is separated by several kilobase pairs from the lpcAB cluster. All three glycosyltransferases are carried on cosmid pIJ1848, which contains at least 20 kilobase pairs of R. leguminosarumDNA. Transfer of pIJ1848 into R. meliloti 1021 results in heterologous expression of all three enzymes, which are not normally present in strain 1021. Expression of the lpc genes individually behind the T7 promoter results in the production of eachR. leguminosarum glycosyltransferase in E. colimembranes in a catalytically active form, demonstrating thatlpcA, lpcB, and lpcC are structural genes. lipopolysaccharide kilobase pair(s) open reading frame polymerase chain reaction 3-deoxy-d-manno-octulosonate. Lipopolysaccharide (LPS)1 of Gram-negative bacteria is composed of lipid A (the hydrophobic membrane anchor), the core region (a non-repeating oligosaccharide), and O-antigen (a distal repeating oligosaccharide) (1Raetz C.R.H. J. Bacteriol. 1993; 175: 5745-5753Crossref PubMed Scopus (236) Google Scholar, 2Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. I. 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, 4Schnaitman C.A. Klena J.D. Microbiol. Rev. 1993; 57: 655-682Crossref PubMed Google Scholar). The O-antigen and much of the core are not required for growth (2Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. I. American Society for Microbiology, Washington, DC1996: 1035-1063Google Scholar, 5Morrison D.C. Ryan J.L. Bacterial Endotoxic Lipopolysaccharides: Molecular Biochemistry and Cellular Biology.Vol. I. CRC Press, Boca Raton, FL1992Google Scholar, 6Nikaido H. Vaara M. Neidhardt F.C. Escherichia coli and Salmonella typhimurium. I. ASM Publications, Washington, DC1987: 7-22Google Scholar, 7Rick P.D. Neidhardt F. Escherichia coli and Salmonella typhimurium. I. ASM Publications, Washington, DC1987: 648-662Google Scholar) under laboratory conditions, but mutants lacking portions of the core, especially the inner core, possess several interesting phenotypes. Inner core mutants often grow more slowly than wild type cells, are hypersensitive to certain antibiotics and display a compromised barrier to hydrophobic compounds (2Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. I. American Society for Microbiology, Washington, DC1996: 1035-1063Google Scholar, 5Morrison D.C. Ryan J.L. Bacterial Endotoxic Lipopolysaccharides: Molecular Biochemistry and Cellular Biology.Vol. I. CRC Press, Boca Raton, FL1992Google Scholar, 6Nikaido H. Vaara M. Neidhardt F.C. Escherichia coli and Salmonella typhimurium. I. ASM Publications, Washington, DC1987: 7-22Google Scholar, 7Rick P.D. Neidhardt F. Escherichia coli and Salmonella typhimurium. I. ASM Publications, Washington, DC1987: 648-662Google Scholar). In addition, the assembly of some outer membrane proteins, such as OmpF and OmpC, is altered in these mutants (8Ames G.-F. Spudich E.N. Nikaido H. J. Bacteriol. 1974; 117: 406-416Crossref PubMed Google Scholar, 9Koplow J. Goldfine H. J. Bacteriol. 1974; 117: 527-543Crossref PubMed Google Scholar, 10Laird M.W. Kloser A.W. Misra R. J. Bacteriol. 1994; 176: 2259-2264Crossref PubMed Scopus (55) Google Scholar). In nitrogen-fixing Gram-negative bacteria, like the Rhizobiaceae, the core region may influence plant host specificity and may function in signaling pathways leading to the formation of root nodules within the host plant (11Denarie J. Cullimore J. Cell. 1993; 74: 951-954Abstract Full Text PDF PubMed Scopus (244) Google Scholar, 12Fisher R.F. Long S.R. Nature. 1992; 357: 655-660Crossref PubMed Scopus (476) Google Scholar, 13Long S.R. Staskawicz B.J. Cell. 1993; 73: 921-935Abstract Full Text PDF PubMed Scopus (111) Google Scholar). For instance, in Rhizobium leguminosarum, core mutants are able to recognize their plant hosts and form nodules, but these nodules either do not fix nitrogen or do so at greatly reduced rates (14Cava J.R. Elias P.M. Turowski D.A. Noel K.D. J. Bacteriol. 1989; 171: 8-15Crossref PubMed Google Scholar, 15Cava J.R. Tao H. Noel K.D. Mol. Gen. Genet. 1990; 221: 125-128Crossref Scopus (23) Google Scholar, 16Priefer U.B. J. Bacteriol. 1989; 171: 6161-6168Crossref PubMed Google Scholar). There is remarkable diversity of LPS core structures in different species of Gram-negative bacteria. The structure of theEscherichia coli K-12 core region (Fig. 1) is one of the best characterized (2Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. I. American Society for Microbiology, Washington, DC1996: 1035-1063Google Scholar). Nearly all of the genes required for the biosynthesis of the E. coli core have been identified (2Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. I. American Society for Microbiology, Washington, DC1996: 1035-1063Google Scholar, 4Schnaitman C.A. Klena J.D. Microbiol. Rev. 1993; 57: 655-682Crossref PubMed Google Scholar,17Roncero C. Casabadan M. J. Bacteriol. 1992; 174: 3250-3260Crossref PubMed Google Scholar). However, because the inner E. coli core contains the unusual sugar,l-glycero-d-manno-heptose, the activated nucleotide form of which is not fully characterized, the reactions catalyzed by the enzymes of E. coli core biosynthesis have not been studied in depth (2Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. I. American Society for Microbiology, Washington, DC1996: 1035-1063Google 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.H. J. Biol. Chem. 1998; 273: 2799-2807Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The core structure of R. leguminosarum LPS, as partially displayed in Fig. 1, has been proposed by Carlson and co-workers (20Bhat U.R. Krishnaiah B.S. Carlson R.W. Carbohydr. Res. 1991; 220: 219-227Crossref PubMed Scopus (45) Google Scholar, 21Carlson 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, 22Forsberg L.S. Carlson R.W. J. Biol. Chem. 1998; 273: 2747-2757Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). It contains Kdo, mannose, galactose, and galacturonic acid, but lacks heptose. The enzymology of core assembly in R. leguminosarum is more amenable to study than in E. coli, given that all the relevant sugar nucleotides are available. In accordance with Carlson's structure, we have been able to identify three novel glycosyltransferases unique to extracts of R. leguminosarum that incorporate mannose (23Brozek 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), galactose (23Brozek 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), and the outer Kdo (present study) in the expected order (Fig. 2) to the conserved lipid A precursor, Kdo2-lipid IVA. We now describe the three structural genes of R. leguminosarum that encode these glycosyltransferases. Two of the genes, lpcA and lpcB, adjacent to each other on the chromosome, were reported previously (24Allaway D. Jeyaretnam B. Carlson R.W. Poole P.S. J. Bacteriol. 1996; 178: 6403-6406Crossref PubMed Google Scholar, 25Poole P.S. Schofield N.A. Reid C.J. Drew E.M. Walshaw D.L. Microbiology. 1994; 140: 2797-2809Crossref PubMed Scopus (88) Google Scholar), based on mutants with truncated core LPS structures. lpcA was partially sequenced and was proposed to encode the galactosyltransferase because of its homology to other sugar transferases and chemical characterization of LPS isolated from anlpcA::Tn5 insertion mutant (24Allaway D. Jeyaretnam B. Carlson R.W. Poole P.S. J. Bacteriol. 1996; 178: 6403-6406Crossref PubMed Google Scholar, 25Poole P.S. Schofield N.A. Reid C.J. Drew E.M. Walshaw D.L. Microbiology. 1994; 140: 2797-2809Crossref PubMed Scopus (88) Google Scholar, 26Zhang Y. Hollingsworth R.I. Priefer U.B. Carbohydr. Res. 1992; 231: 261-271Crossref PubMed Scopus (24) Google Scholar).lpcB was sequenced entirely, and although it showed no homology to any known gene, it was proposed to encode the distal Kdo-transferase based upon the absence of the distal Kdo in the LPS core isolated from an lpcB transposon insertion mutant (24Allaway D. Jeyaretnam B. Carlson R.W. Poole P.S. J. Bacteriol. 1996; 178: 6403-6406Crossref PubMed Google Scholar). We now demonstrate by means of our enzyme assays that lpcAdoes indeed encode the galactosyltransferase and that lpcBencodes the distal Kdo-transferase. In addition, we report a new gene, designated lpcC, encoding the mannosyltransferase, and describe a mutant lacking mannosyltransferase activity. lpcCis located several kb downstream of lpcA and lpcBon the chromosome (Fig. 3). All threeRhizobium genes have been overexpressed using an E. coli T7 promoter-driven system. The recombinant enzymes are catalytically active. The availability of the lpc genes should facilitate the re-engineering of LPS core structures in bothE. coli and Rhizobium. The biological significance of core structural diversity in pathogenesis and symbiosis might be revealed using this approach.Figure 3Order of genes in the lpc-dctregion of the chromosome of R. leguminosarum.The known open reading frames and the directions of transcription are indicated for the chromosomal DNA insert (derived from strain 8002) in cosmid pIJ1848 (25Poole P.S. Schofield N.A. Reid C.J. Drew E.M. Walshaw D.L. Microbiology. 1994; 140: 2797-2809Crossref PubMed Scopus (88) Google Scholar). Below pIJ1848 are the inserts present in a different set of overlapping cosmids derived from strain 3841 (25Poole P.S. Schofield N.A. Reid C.J. Drew E.M. Walshaw D.L. Microbiology. 1994; 140: 2797-2809Crossref PubMed Scopus (88) Google Scholar). The insert in each of these cosmids is at least 20 kb in length. Above pIJ1848 are shown the inserts in several smaller subclones derived from this region (25Poole P.S. Schofield N.A. Reid C.J. Drew E.M. Walshaw D.L. Microbiology. 1994; 140: 2797-2809Crossref PubMed Scopus (88) Google Scholar). The lengths of the inserts and the genes are not drawn exactly to scale. The gap indicated between dctD and chaA may be as long as 10 kb.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The following materials and kits were purchased: [γ-32P]ATP (NEN Life Science Products); Hepes, GDP-mannose, UDP-galactose, Kdo, and CTP (Sigma); Triton X-100 and bicinchoninic assay reagents (Pierce); silica gel 60 thin layer chromatography plates (E. Merck); yeast extract and tryptone (Difco); PCR reagents (Stratagene); restriction enzymes (New England Biolabs); shrimp alkaline phosphatase (U. S. Biochemical Corp); custom primers and T4 DNA ligase (Life Technologies); and Qiaex II gel extraction kit and Qiaprep Spin Miniprep kit (Qiagen). 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 strains and plasmids used, as well as their sources, are listed in Table I, and key plasmids are diagrammed in Fig. 4. E. coliSURE cells were purchased from Stratagene. Plasmid pET23a and E. coli strain BLR(DE3)pLysS were purchased from Novagen.Table IBacterial strains and plasmidsStrain or PlasmidDescriptionSource or Ref.Strain 1021Wild type R. melilotiSharon Long VF39Wild typeR. leguminosarum bv. viciae(16Priefer U.B. J. Bacteriol. 1989; 171: 6161-6168Crossref PubMed Google Scholar) VF39–86Strain VF39 lpcA::Tn5(16Priefer U.B. J. Bacteriol. 1989; 171: 6161-6168Crossref PubMed Google Scholar) 3841SmR derivative of wild type R. leguminosarum strain 300 bv. viciae(55Johnston A.W.B. Beringer J.E. J. Gen. Microbiol. 1975; 87: 343-350Crossref PubMed Scopus (157) Google Scholar) RU301Strain 3841 lpcB::Tn5(25Poole P.S. Schofield N.A. Reid C.J. Drew E.M. Walshaw D.L. Microbiology. 1994; 140: 2797-2809Crossref PubMed Scopus (88) Google Scholar) 3855SmR derivative of wild type R. leguminosarum strain 128C53 bv. viciae(56Brewin N.J. Wood E.A. Johnston A.W.B. Dibb N.J. Hombrecher G. J. Gen. Microbiol. 1982; 128: 1817-1827Google Scholar) RSKnHStrain 3855 lpcC::nptIIThis work 803E. coli donor strain for triparental mating(34Wood W.B. J. Mol. Biol. 1966; 16: 118-133Crossref PubMed Scopus (435) Google Scholar) DH5αE. coli donor strain for triparental mating(35Hanahan D. J. Mol. Biol. 1983; 166: 557-580Crossref PubMed Scopus (8216) Google Scholar) MT616E. coli helper strain for triparental mating; contains pRK600, a ColE1 replicon with RK2 tra genes, NmR(36Finan T.M. Kunkel B. DeVos G.F. Signer E.R. J. Bacteriol. 1986; 167: 66-72Crossref PubMed Google Scholar)Plasmid pIJ1848Cosmid from strain 8002; dctABD, lpcABC(25Poole P.S. Schofield N.A. Reid C.J. Drew E.M. Walshaw D.L. Microbiology. 1994; 140: 2797-2809Crossref PubMed Scopus (88) Google Scholar) pRK415Broad host range P-group cloning vector; TcR(57Keen N.T. Tamaki S. Kobayashi D. Trollinger D. Gene (Amst.). 1988; 70: 191-197Crossref PubMed Scopus (1275) Google Scholar) pRU68pRK415 plus 3.8-kbEcoRI/HindIII fragment from pIJ1848(25Poole P.S. Schofield N.A. Reid C.J. Drew E.M. Walshaw D.L. Microbiology. 1994; 140: 2797-2809Crossref PubMed Scopus (88) Google Scholar) pRU74pRK415 plus 2.4-kb EcoRI/PstI fragment from pIJ1848(25Poole P.S. Schofield N.A. Reid C.J. Drew E.M. Walshaw D.L. Microbiology. 1994; 140: 2797-2809Crossref PubMed Scopus (88) Google Scholar) pRU3000Cosmid from strain 3841 containing dctA, lpcC(25Poole P.S. Schofield N.A. Reid C.J. Drew E.M. Walshaw D.L. Microbiology. 1994; 140: 2797-2809Crossref PubMed Scopus (88) Google Scholar) pRU3001Cosmid from strain 3841 containing dctABD, lpcC(25Poole P.S. Schofield N.A. Reid C.J. Drew E.M. Walshaw D.L. Microbiology. 1994; 140: 2797-2809Crossref PubMed Scopus (88) Google Scholar) pRU3020Cosmid from strain 3841 containing dctABD, lpcABC(25Poole P.S. Schofield N.A. Reid C.J. Drew E.M. Walshaw D.L. Microbiology. 1994; 140: 2797-2809Crossref PubMed Scopus (88) Google Scholar) pRU3022Cosmid from strain 3841 containinglpcAB(25Poole P.S. Schofield N.A. Reid C.J. Drew E.M. Walshaw D.L. Microbiology. 1994; 140: 2797-2809Crossref PubMed Scopus (88) Google Scholar) Open table in a new tab Strains of Rhizobium were grown at 30 °C on TY medium (5 g of tryptone and 3 g of yeast extract per liter supplemented with 10 mm CaCl2) with neomycin at 100 μg/ml, kanamycin at 25 μg/ml, or tetracycline at 10 μg/ml, as appropriate. BLR(DE3)pLysS/pET23a, BLR(DE3)pLysS/pJK5, BLR(DE3)pLysS/pJK6, and BLR(DE3)pLysS/pJK7 were grown from a single colony in 1 liter of LB medium (10 g of tryptone, 5 g of yeast extract, 10 g of NaCl per liter) (27Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar) containing ampicillin (100 μg/ml) at 37 °C until the A 600 reached approximately 0.5. The culture was split into two equal portions, and one portion was induced with 100 μg/ml isopropyl-1-thio-β-d-galactopyranoside. Both cultures were incubated with shaking at 225 rpm for an additional 3 h at 37 °C, and the A 600 was recorded. Cells were harvested in the cold (0–4 °C) by centrifugation at 6,000 × g for 15 min. For each liter of late log phase culture (A 600 = 1), cell pellets were resuspended in 10 ml of 50 mm Hepes, pH 7.5. The cells were broken by passage through a French pressure cell at 18,000 p.s.i., yielding a protein concentration of approximately 10 mg/ml. Cellular debris was removed by centrifugation at 6,000 × g for 15 min. Washed membranes were prepared by a series of two ultracentrifugations at 100,000 × g for 60 min. The membrane pellet was resuspended in a minimal volume (1–2 ml) of 50 mm Hepes, pH 7.5. The protein concentrations of the extracts, membranes and cytosol were determined by the bicinchoninic acid assay (28Smith 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 (18709) Google Scholar) using bovine serum albumin as the standard. 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 BLR(DE3)pLysS/pJK2 (29Garrett T.A. Kadrmas J.L. Raetz C.R.H. J. Biol. Chem. 1997; 272: 21855-21864Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The labeled lipid IVA was converted to Kdo2-[4′-32P]lipid IVA using purified E. coli Kdo-transferase (30Belunis C.J. Raetz C.R.H. J. Biol. Chem. 1992; 267: 9988-9997Abstract Full Text PDF PubMed Google Scholar, 31Brozek K.A. Hosaka K. Robertson A.D. Raetz C.R.H. J. Biol. Chem. 1989; 264: 6956-6966Abstract Full Text PDF PubMed Google Scholar). The products were purified by preparative thin layer chromatography and stored at −20 °C as an aqueous dispersion (30Belunis C.J. Raetz C.R.H. J. Biol. Chem. 1992; 267: 9988-9997Abstract Full Text PDF PubMed Google Scholar, 31Brozek K.A. Hosaka K. Robertson A.D. Raetz C.R.H. J. Biol. Chem. 1989; 264: 6956-6966Abstract Full Text PDF PubMed Google Scholar). Prior to each use, these substrates were subjected to ultrasonic irradiation in a water bath for 60 s. For mannosyltransferase reactions, unless indicated, the standard reaction mixtures (10–40 μl) contained 50 mm Hepes, pH 7.5, 0.1% Triton X-100, 10 μmKdo2-[4′-32P]lipid IVA at 80,000 cpm/nmol, and 1.0 mm GDP-mannose. The enzyme source, added last to initiate the reaction, was generally 0.3 mg/ml washedRhizobium membranes. Reactions were incubated at 30 °C for 60 min, unless specified. Galactosyltransferase reactions were identical but also included 1.0 mm UDP-galactose in addition to the above components. Distal Kdo-transferase assays contained all the galactosyltransferase reaction components plus 2 mm Kdo, 5 mm CTP, 10 mmMgCl2, and 1.8 milliunits of partially purified CMP-Kdo synthase per 10 μl. CMP-Kdo is generated in situ because of its short half-life (minutes) (30Belunis C.J. Raetz C.R.H. J. Biol. Chem. 1992; 267: 9988-9997Abstract Full Text PDF PubMed Google Scholar). When assaying for the R. leguminosarum enzymes expressed in the E. coli T7 system, slightly different conditions were used. The mannosyltransferase assay was the same as above except that 0.2 mg/ml washed E. coli BLR(DE3)pLysS/pJK6 membranes were used as the enzyme source. To assay the E. coligalactosyltransferase construct (pJK7), mannosyl-Kdo2-IVA was first generated in a standard 60-min mannosyltransferase reaction utilizing 0.3 mg/ml washedRhizobium meliloti 1021/pIJ1848 (Table I) membranes. Residual R. meliloti 1021/pIJ1848 activity was then destroyed by a 20-min incubation at 65 °C. To this reaction mixture, 1.0 mm UDP-galactose and 0.2 mg/ml E. coliBLR(DE3)pLysS/pJK7 washed membranes were added. Reactions were then incubated for 30 min at 30 °C. To assay the E. colidistal Kdo-transferase construct (pJK5), galactosyl-mannosyl-Kdo2-IVA was first generated in a standard 60-min galactosyltransferase reaction utilizing 0.3 mg/ml washed R. meliloti 1021/pIJ1848 membranes. Residual R. meliloti 1021/pIJ1848 activity was then destroyed by a 20-min incubation at 65 °C. To this reaction mixture, 2 mm Kdo, 5 mm CTP, 10 mmMgCl2, 1.8 milliunits of CMP-Kdo synthase, and 0.2 mg/mlE. coli BLR(DE3)pLysS/pJK5 washed membranes were added. Reactions were then incubated for 30 min at 30 °C. Reactions were stopped by spotting 5-μl portions of the reaction mixtures onto a silica gel 60 thin layer chromatography plate. After drying in a stream of cold air, plates were developed in the solvent chloroform/pyridine/88% formic acid/water (30:70:16:10, v/v). The amount of product formed was calculated from the percent conversion of radioactive substrate (of known specific radioactivity) to product, quantified using a Molecular Dynamics PhosphorImager. Plasmids were prepared using the Qiagen Spin Prep 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. All other techniques involving manipulation of nucleic acids were from Ausubelet al. (32Ausubel 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 transformation by resuspension in 100 mm CaCl2, as described (32Ausubel 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). Plasmids were introduced into strains of Rhizobium via triparental mating (33Glazebrook J. Walker G.C. Methods Enzymol. 1991; 204: 398-418Crossref PubMed Scopus (113) Google Scholar). E. coli strain 803 (34Wood W.B. J. Mol. Biol. 1966; 16: 118-133Crossref PubMed Scopus (435) Google Scholar) or DH5α (35Hanahan D. J. Mol. Biol. 1983; 166: 557-580Crossref PubMed Scopus (8216) Google Scholar) served as the plasmid donor. E. coli strain MT616 (36Finan T.M. Kunkel B. DeVos G.F. Signer E.R. J. Bacteriol. 1986; 167: 66-72Crossref PubMed Google Scholar) provided the transfer functions. The appropriate strain of Rhizobium (see below) served as the recipient. Sequencing of a portion of lpcA and its homology to certain LPS core glycosyltransferases has previously been reported (24Allaway D. Jeyaretnam B. Carlson R.W. Poole P.S. J. Bacteriol. 1996; 178: 6403-6406Crossref PubMed Google Scholar) (accession no.X94963). Full-length lpcA was required to demonstrate the enzymatic activity of LpcA. The 5′ terminus of the gene was determined by cycle sequencing using custom-made Cy5-labeled primers to pRU68 (25Poole P.S. Schofield N.A. Reid C.J. Drew E.M. Walshaw D.L. Microbiology. 1994; 140: 2797-2809Crossref PubMed Scopus (88) Google Scholar), Thermo Sequenase, and the ALFexpress automated DNA sequencer. A restriction map of the 4.4-kb EcoRI fragment containing dctA has been constructed (37Ronson C.W. Astwood P.M. Downie J.A. J. Bacteriol. 1984; 160: 903-909Crossref PubMed Google Scholar), and the nucleotide sequence of dctAdetermined (EMBL accession no. Z11529). The region downstream of dctA on the 4.4-kb fragment was sequenced on both strands using a combination of sub-cloned fragments and custom primers, and an Applied Biosystems model 373A autosequencer. This sequence can be found under accession no. AF050103. To construct strain RSKnH, a kanamycin-resistance cassette was inserted into the HindIII site within the lpcC open reading frame. The kanamycin cassette was cloned from pUC4KIXX (Amersham Pharmacia Biotech) as aSmaI fragment into pIC20H (38Marsh J.L. Erfle M. Wykes E.J. Gene (Amst.). 1984; 32: 481-485Crossref PubMed Scopus (522) Google Scholar). It was then excised as aHindIII fragment and cloned into HindIII-digested pPN120 to give pRS5. pPN120 is a pLAFR1 derivative carrying the 4.4-kbEcoRI fragment that includes part of dctB, dctA, and 2000 base pairs downstream of dctA(37Ronson C.W. Astwood P.M. Downie J.A. J. Bacteriol. 1984; 160: 903-909Crossref PubMed Google Scholar). pRS5 was transferred by triparental mating to R. leguminosarum strain 3855, and recombination of the kanamycin resistance gene into the genome was forced by introduction of the incompatible plasmid pPH1. Southern hybridizations of EcoRI and KpnI digests of genomic DNA probed with pPN108 (37Ronson C.W. Astwood P.M. Downie J.A. J. Bacteriol. 1984; 160: 903-909Crossref PubMed Google Scholar) were used to confirm that the kanamycin cassette had recombined into the expected location. Pisum sativum seeds were surface-sterilized by washing with absolute alcohol, followed by soaking for 1 h in 12% sodium hypochlorite, followed by five washes with sterile water. The seeds were allowed to imbibe and then transferred to 550-ml jars containing a sterile moistened mix of fine vermiculite and pumice at a ratio of 3:1. The seeds were inoculated with 1 ml of a suspension of Rhizobium cells washed from a fresh GRDM plate (37Ronson C.W. Astwood P.M. Downie J.A. J. Bacteriol. 1984; 160: 903-909Crossref PubMed Google Scholar). The pots were watered with nitrogen-free nutrient solution (37Ronson C.W. Astwood P.M. Downie J.A. J. Bacteriol. 1984; 160: 903-909Crossref PubMed Google Scholar) and grown under controlled environmental conditions in a growth room at 20 °C day/15 °C night on a 12-h day/night cycle. The plant roots were examined for nodules after 3–6 weeks. The cloning of PCR generated lpcA, lpcB, and lpcC DNA into a vector under T7 promoter control is outlined in Fig. 4 (39Rosenberg 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, 40Studier F.W. Moffatt B.A. J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (4842) Google Scholar, 41Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6006) Google Scholar). The forward primers were synthesized with a clamp region, an NdeI restriction site, and a match to the coding strand starting at the translation initiation site. The reverse primer was synthesized with a clamp region, aBamHI restriction site, and a match to the anticoding strand that included the stop site. The PCR was performed using Pfupolymerase, as specified by the manufacturer. The plasmid pIJ1848 (25Poole P.S. Schofield N.A. Reid C.J. Drew E.M. Walshaw D.L. Microbiology. 1994; 140: 2797-2809Crossref PubMed Scopus (88) Google Scholar) was used as the template. Amplification was carried out in a 50-μl reaction mixture containing 100 ng of template, 20 mmTris-HCl, pH 8.8, 10 mm KCl, 10 mm(NH4)2SO4, 0.1% Triton X-100, 0.1% bovine serum albumin, 2 mm MgSO4, 200 μm of each of the dNTPs, 125 ng of each primer, and 1.2 units of Pfu polymerase. The re