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Expression Cloning and Characterization of the C28 Acyltransferase of Lipid A Biosynthesis in Rhizobium leguminosarum

2002; Elsevier BV; Volume: 277; Issue: 32 Linguagem: Inglês

10.1074/jbc.m204525200

ISSN

1083-351X

Autores

Shib Sankar Basu, Mark J. Karbarz, Christian R.H. Raetz,

Tópico(s)

Carbohydrate Chemistry and Synthesis

Resumo

An unusual feature of lipid A from plant endosymbionts of the Rhizobiaceae family is the presence of a 27-hydroxyoctacosanoic acid (C28) moiety. An enzyme that incorporates this acyl chain is present in extracts of Rhizobium leguminosarum, Rhizobium etli, andSinorhizobium meliloti but not Escherichia coli. The enzyme transfers 27-hydroxyoctacosanate from a specialized acyl carrier protein (AcpXL) to the precursor Kdo2((3-deoxy-d-manno-octulosonic acid)2)-lipid IVA. We now report the identification of five hybrid cosmids that direct the overexpression of this activity by screening ∼4000 lysates of individual colonies of anR. leguminosarum 3841 genomic DNA library in the host strain S. meliloti 1021. In these heterologous constructs, both the C28 acyltransferase and C28-AcpXL are overproduced. Sequencing of a 9-kb insert from cosmid pSSB-1, which is also present in the other cosmids, shows that acpXL and the lipid A acyltransferase gene (lpxXL) are close to each other but not contiguous. Nine other open reading frames around lpxXL were also sequenced. Four of them encode orthologues of fatty acid and/or polyketide biosynthetic enzymes. AcpXL purified from S. meliloti expressing pSSB-1 is fully acylated, mainly with 27-hydroxyoctacosanoate. Expression of lpxXL in E. coli behind a T7 promoter results in overproduction in vitro of the expected R. leguminosarumacyltransferase, which is C28-AcpXL-dependent and utilizes (3-deoxy-d-manno-octulosonic acid)2-lipid IVA as the acceptor. These findings confirm that lpxXL is the structural gene for the C28 acyltransferase. LpxXL is distantly related to the lauroyltransferase (LpxL) of E. coli lipid A biosynthesis, but highly significant LpxXL orthologues are present inAgrobacterium tumefaciens, Brucella melitensis, and all sequenced strains of Rhizobium, consistent with the occurrence of long secondary acyl chains in the lipid A molecules of these organisms. An unusual feature of lipid A from plant endosymbionts of the Rhizobiaceae family is the presence of a 27-hydroxyoctacosanoic acid (C28) moiety. An enzyme that incorporates this acyl chain is present in extracts of Rhizobium leguminosarum, Rhizobium etli, andSinorhizobium meliloti but not Escherichia coli. The enzyme transfers 27-hydroxyoctacosanate from a specialized acyl carrier protein (AcpXL) to the precursor Kdo2((3-deoxy-d-manno-octulosonic acid)2)-lipid IVA. We now report the identification of five hybrid cosmids that direct the overexpression of this activity by screening ∼4000 lysates of individual colonies of anR. leguminosarum 3841 genomic DNA library in the host strain S. meliloti 1021. In these heterologous constructs, both the C28 acyltransferase and C28-AcpXL are overproduced. Sequencing of a 9-kb insert from cosmid pSSB-1, which is also present in the other cosmids, shows that acpXL and the lipid A acyltransferase gene (lpxXL) are close to each other but not contiguous. Nine other open reading frames around lpxXL were also sequenced. Four of them encode orthologues of fatty acid and/or polyketide biosynthetic enzymes. AcpXL purified from S. meliloti expressing pSSB-1 is fully acylated, mainly with 27-hydroxyoctacosanoate. Expression of lpxXL in E. coli behind a T7 promoter results in overproduction in vitro of the expected R. leguminosarumacyltransferase, which is C28-AcpXL-dependent and utilizes (3-deoxy-d-manno-octulosonic acid)2-lipid IVA as the acceptor. These findings confirm that lpxXL is the structural gene for the C28 acyltransferase. LpxXL is distantly related to the lauroyltransferase (LpxL) of E. coli lipid A biosynthesis, but highly significant LpxXL orthologues are present inAgrobacterium tumefaciens, Brucella melitensis, and all sequenced strains of Rhizobium, consistent with the occurrence of long secondary acyl chains in the lipid A molecules of these organisms. (3-deoxy-d-manno-octulosonic acid)2 fast protein liquid chromatography high pressure liquid chromatography 4-morpholineethanesulfonic acid The lipid A moiety of lipopolysaccharide in the nitrogen-fixing endosymbionts, Rhizobium leguminosarum and Rhizobium etli, is very different compared with lipid A of other Gram-negative bacteria (1Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3522) Google Scholar, 2Que 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, 3Que 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). Lipid A of R. elti CE3 consists of at least four related components (Fig.1) (2Que 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, 3Que 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). The structural features that are distinct from other types of lipid A include the following: 1) the absence of phosphate groups at the 1- and 4′-positions (2Que 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, 3Que 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, 4Bhat U.R. Forsberg L.S. Carlson R.W. J. Biol. Chem. 1994; 269: 14402-14410Abstract Full Text PDF PubMed Google Scholar); 2) the presence of a galacturonic acid residue at the 4′-position (2Que 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, 3Que 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, 4Bhat U.R. Forsberg L.S. Carlson R.W. J. Biol. Chem. 1994; 269: 14402-14410Abstract Full Text PDF PubMed Google Scholar); 3) the presence of an unusual 27-hydroxyoctacosanoate chain (5Bhat U.R. Mayer H. Yokota A. Hollingsworth R.I. Carlson R. J. Bacteriol. 1991; 173: 2155-2159Crossref PubMed Google Scholar) in acyloxyacyl linkage at position 2′ (2Que 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, 3Que 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), modified with β-hydroxybutyrate; and 4) replacement in some species (2Que 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, 3Que 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) of the reducing end glucosamine unit with a 2-deoxy-2-aminogluconate residue (4Bhat U.R. Forsberg L.S. Carlson R.W. J. Biol. Chem. 1994; 269: 14402-14410Abstract Full Text PDF PubMed Google Scholar) (Fig. 1). Whether or not the remarkable lipid A species of R. leguminosarum and R. etli play a role in root cell infection and nodulation is unknown. It has been proposed that lipid A of Gram-negative plant pathogens (which is similar to that of Escherichia coli) may activate the innate immune system of plants just as E. coli lipid A activates innate immunity in animals (2Que 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, 3Que 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). The R. leguminosarum and R. etli lipid A species lack the structural features (i.e.the phosphate groups and the proper secondary acyl chains) needed for stimulation of innate immunity in animals (Fig. 1) (2Que 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, 3Que 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, 6Rietschel 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 (1354) Google Scholar). Structural modification of lipid A might allow the endosymbionts to avoid activating the plant immune system, thereby promoting bacteroid survival. To evaluate the function of lipid A during symbiosis, a structure-activity study would be informative. The construction of mutant strains with defined alterations in lipid A structure requires a detailed understanding of the enzymology and genetics of lipid A biosynthesis. In previous studies (7Price N.P.J. Kelly T.M. Raetz C.R.H. Carlson R.W. J. Bacteriol. 1994; 176: 4646-4655Crossref PubMed Google Scholar), we have shown that the first seven enzymes of the lipid A pathway (1Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3522) Google Scholar, 8Raetz C.R.H. Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Scopus (1046) Google Scholar), which generate the key intermediate Kdo21-lipid IVA (Fig. 2) in E. coli, are also present in R. etli and R. leguminosarum. However, additional enzymes are present in R. etli and R. leguminosarum (9Brozek 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, 10Brozek K.A. Carlson R.W. Raetz C.R.H. J. Biol. Chem. 1996; 271: 32126-32136Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) to catalyze alternative processing of Kdo2-lipid IVA. Unique enzymes of R. etli and R. leguminosarum reported so far include the following: 1) phosphatases to remove the 4′- and 1-phosphate moieties of Kdo2-lipid IVA (9Brozek 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, 11Price 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, 12Basu 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); 2) a deacylase to remove the ester-linked fatty acid at the 3-position (13Basu S.S. White K.A. Que N.L. Raetz C.R. J. Biol. Chem. 1999; 274: 11150-11158Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar); 3) a unique long chain acyltransferase that uses the special acyl carrier protein AcpXL to incorporate the 27-hydroxyoctacosanoic acid residue (Fig. 2) (10Brozek K.A. Carlson R.W. Raetz C.R.H. J. Biol. Chem. 1996; 271: 32126-32136Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar); and 4) a system for oxidizing the proximal glucosamine to aminogluconate (3Que 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). Enzymes for incorporating the 4′-galacturonic acid and β-hydroxybutyrate residues remain to be identified. We now describe the expression cloning of a 9-kb DNA fragment ofR. leguminosarum that encodes the C28 acyltransferase, as well as proteins involved in the production of the C28 donor, ACP-XL. By assaying lysates (10Brozek K.A. Carlson R.W. Raetz C.R.H. J. Biol. Chem. 1996; 271: 32126-32136Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) of individual members of aR. leguminosarum 3841 genomic cosmid library (14Ronson C.W. Astwood P.M. Downie J.A. J. Bacteriol. 1984; 160: 903-909Crossref PubMed Google Scholar) harbored in Sinorhizobium meliloti 1021 (15Galibert 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 (951) Google Scholar), five clones overexpressing C28-acyltransferase activity were identified. The acyltransferase, which is detected using Kdo2-[4′-32P] lipid IVA as the acceptor (12Basu 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, 16Brozek K.A. Hosaka K. Robertson A.D. Raetz C.R.H. J. Biol. Chem. 1989; 264: 6956-6966Abstract Full Text PDF PubMed Google Scholar) (Fig. 2), is membrane-bound, whereas the donor substrate C28-AcpXL is soluble (10Brozek K.A. Carlson R.W. Raetz C.R.H. J. Biol. Chem. 1996; 271: 32126-32136Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Sequencing of the relevant DNA insert, which is the same in all five isolates, shows clustering of the genes encoding AcpXL and the long chain fatty acyltransferase (LpxXL) together with four other proteins related to enzymes involved in the fatty acid elongation and/or polyketide biosynthesis. Mass spectrometry of the AcpXL, purified from one of the above clones, shows that it is fully acylated with a long chain fatty acid. The R. leguminosarum lpxXL gene can be overexpressed inE. coli behind the T7 promoter, and the product is catalytically active. The availability of acpXL,lpxXL, and the other genes required to acylate AcpXL should facilitate the re-engineering of lipid A structures present in strains of E. coli and Rhizobium. [γ-32P]ATP was obtained from PerkinElmer Life Sciences. Silica Gel 60 (0.25-mm) thin layer plates were purchased from EM Separation Technologies. Triton X-100 and bicinchoninic acid were from Pierce. Yeast extract and tryptone were purchased from Difco. All other chemicals were reagent grade and were obtained from either Sigma or Mallinckrodt. Restriction enzymes were from New England Biolabs, PCR reagents from Stratagene, shrimp alkaline phosphatase from U. S. Biochemical Corp., and custom primers and T4 DNA ligase from Invitrogen. All other reagents and enzymes were purchased from either Roche Molecular Biochemicals or Invitrogen. R. etlibiovar phaseoli CE3, R. leguminosarum 3841,R. leguminosarum 8401, and S. meliloti 1021 (15Galibert 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 (951) Google Scholar) were described in previous studies (9Brozek 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, 11Price 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, 17Cava J.R. Elias P.M. Turowski D.A. Noel K.D. J. Bacteriol. 1989; 171: 8-15Crossref PubMed Google Scholar). AllRhizobium and Sinorhizobium strains were grown at 30 °C in TY medium, which contain 5 g/liter tryptone, 3 g/liter yeast extract, 10 mm CaCl2, and 20 μg/ml nalidixic acid. For the growth of strains CE3 and 1021, streptomycin (200 μg/ml) was also added to the medium. The clones and subclones inS. meliloti 1021 were grown under additional selection with tetracycline (12.5 μg/ml). The Rhizobium cells were grown to A 600 of 0.8–1.2 before they were harvested. Plasmid pET23b and E. coli strain BLR(DE3)pLysS were purchased from Novagen. BLR(DE3)pLysS/pET23b and BLR(DE3)pLysS/pSSB-101 were grown from a single colony in 1 liter of LB medium (18Miller J.R. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1972Google Scholar) (10 g/liter tryptone, 5 g/liter yeast extract, and 10 g/liter NaCl containing 100 μg/ml ampicillin) at 37 °C untilA 600 reached ∼0.6. The cultures were then induced with 100 μg/ml isopropyl-1-thio-β-d-galatopyranoside and incubated with shaking at 225 rpm for an additional 3 h at 37 °C. Mid-logarithmic phase cells were harvested by centrifugation at 8,000 × g for 15 min. All procedures were carried out at 0–4 °C. The cell pellets were resuspended in 50 mm Hepes, pH 7.5, to give a final protein concentration of 5–10 mg/ml. To make crude cell extracts, the cells were broken by two passages through a French pressure cell at 18,000 pounds/square inch, and the debris was removed by centrifugation at 8,000 ×g for 15 min. Membranes were prepared from the crude cell extracts by centrifugation at 149,000 × g for 60 min. The membrane pellet was suspended in 50 mm Hepes, pH 7.5, at a protein concentration of ∼5–10 mg/ml, and the washed membranes were collected by another centrifugation at 149,000 ×g for 60 min. The washed membranes were again suspended in 50 mm Hepes, pH 7.5, at a protein concentration of ∼5–10 mg/ml. The soluble fraction from the initial centrifugation of the crude cell extract at 149,000 × g for 60 min was centrifuged a second time to remove residual membranes. Samples of washed membranes and membrane-free cytosol were stored in aliquots at −80 °C. Protein concentrations were determined by the bicinchoninic acid method (19Smith 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 (18922) Google Scholar) using bovine serum albumin as the standard. Kdo2-[4′-32P]-lipid IVA, Kdo-[4′-32P]-lipid IVA, and [4′-32P]-lipid IVA were prepared using theE. coli 4′-kinase and the appropriate Kdo transferase, as described previously (12Basu 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, 20Garrett T.A. Kadrmas J.L. Raetz C.R.H. J. Biol. Chem. 1997; 272: 21855-21864Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Aqueous dispersions of these lipids were stored at −20 °C, and prior to use they were subjected to sonic irradiation in a bath sonicator for 90 s. The reaction was carried out in 10 μl under optimized conditions at 30 °C for 10 min (32Vorachek-Warren M.K. Ramirez S. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 2002; 277: 14194-14205Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar,35Galloway S.M. Raetz C.R.H. J. Biol. Chem. 1990; 265: 6394-6402Abstract Full Text PDF PubMed Google Scholar) in 50 mm Hepes, pH 8.2, 0.2% Triton X-100, and 10 μm Kdo2-[4′-32P]-lipid IVA (20,000–50,000 cpm/nmol). Various protein fractions were added as indicated. Following the incubation, 4-μl samples were withdrawn and spotted directly onto thin layer chromatography plates that were developed in the solvent chloroform/pyridine/88% formic acid/water (30:70:16:10, v/v). Following overnight exposure to imaging screens, the extent of conversion of substrate to product was measured using a Amersham Biosciences model Storm PhosphorImager, equipped with ImageQuant software. The cosmid (pLAFR-1) (21Friedman A.M. Long S.R. Brown S.E. Buikema W.J. Ausubel F.M. Gene (Amst.). 1982; 18: 289-296Crossref PubMed Scopus (492) Google Scholar) library of R. leguminosarum 3841 genomic DNA (∼20–25 kb) (14Ronson C.W. Astwood P.M. Downie J.A. J. Bacteriol. 1984; 160: 903-909Crossref PubMed Google Scholar) inE. coli 803 host (22Wood W. J. Mol. Biol. 1966; 16: 118-133Crossref PubMed Scopus (437) Google Scholar) was generously provided by Dr. J. Downie of the John Innnes Institute, Norwich, UK. It was not possible to assay colony lysates of the E. coli host directly, because R. leguminosarum promoters are not recognized inE. coli. Accordingly, the entire library was first transferred into S. meliloti 1021 by tri-parental mating (23Glazebrook J. Walker G.C. Methods Enzymol. 1991; 204: 398-418Crossref PubMed Scopus (113) Google Scholar). E. coli strain 803 harboring the cosmid library served as the cosmid donor, whereas E. coli MT616 (24Finan T.M. Kunkel B. De Vos G.F. Signer E.R. J. Bacteriol. 1986; 167: 66-72Crossref PubMed Google Scholar) (the helper strain) provided the transfer functions. S. meliloti 1021 (the recipient) was grown on TY agar with nalidixic acid (20 μg/ml) and streptomycin (200 μg/ml) selection for 24 h. E. coli strain 803 harboring the cosmid library was grown on LB agar containing tetracycline (12.5 μg/ml) for 12 h, and the E. coli MT616 helper strain was grown on LB agar with chloramphenicol (30 μg/ml) for 12 h. The bacteria were then scraped off from their respective plates and resuspended in TY medium (0.5 ml per plate) with no antibiotics. The S. meliloti 1021 (the recipient), the E. coli strain 803 harboring the cosmid library (donor), and the E. coli MT616 helper strain were mixed in a test tube in the approximate ratio of 3:1:1, v/v. A portion (0.5 ml per plate) of the mixture was then spread in a circular area (∼5 cm diameter) on TY agar plate without antibiotic selection. Mating was allowed to take place for 24–36 h at 30 °C. The mixture was scraped off the plate surface in TY medium containing 20% glycerol and then stored at −80 °C. The successful transfer of the cosmid library into S. meliloti 1021 was verified by analysis of the cosmids isolated from several randomly chosen transformed recipient colonies grown on a TY agar plate containing nalidixic acid (20 μg/ml), streptomycin (200 μg/ml), and tetracycline (12.5 μg/ml). To examine a large number of S. meliloti 1021 cells harboring the individual members of the R. leguminosarumlibrary in the pLAFR-1 cosmid, the glycerol stock of the mating mixture was thawed and appropriately diluted to obtain 50–100 colonies per TY agar plate, supplemented with nalidixic acid (20 μg/ml), streptomycin (200 μg/ml), and tetracycline (12.5 μg/ml). The plates were incubated for 72 h at 30 °C to obtain individual colonies. For large scale screening, several thousand such colonies were re-grown one at a time for 24 h with constant shaking at 30 °C in separate wells of 96-well microtiter plates in 150 μl of TY medium, containing nalidixic acid (20 μg/ml), streptomycin (200 μg/ml), and tetracycline (12.5 μg/ml). A 50-μl portion of the culture from each well was transferred to another microtiter plate and stored as a 20% glycerol stock at −80 °C. The remaining cells were collected in each of the wells of the original microtiter plate by centrifugation at 3,660 × g for 15 min at 4 °C. The supernatants were decanted, and the cells were resuspended in 50 μl of 50 mm Hepes, pH 7.5. In order to generate concentrated lysates, the cells were broken by incubating with lysozyme (1 mg/ml) and EDTA (10 mm) for an hour at 4 °C (both added from concentrated stocks). Portions of the lysates (5 μl from each well) from four microtiter plates were combined to give 96 pools of four lysates in a fresh microtiter plate (20 μl per well final volume). The pooled cell lysates were assayed for their ability to acylate Kdo2-[4′-32P]-lipid IVA in the following manner. A fresh 96-well microtiter plate was prepared in which each well contained 2 μl of 250 mm Mes buffer, pH 6.5, 0.5% Triton X-100, 10 mm EDTA, 1.0 μmKdo2-[4′-32P]-lipid IVA and 8 μl of pooled cell lysate, which was added last to give final volume of 10 μl. These plates were incubated at 30 °C for 2 h, and a portion of each reaction mixture (5 μl) was then spotted onto a Silica Gel 60 thin layer chromatography plate (Fig.3 A, lanes 1–8). A negative control reaction without enzyme (Fig. 3 A, left lane) and positive controls containing either an R. leguminosarum 3841 or an S. meliloti crude extract (prepared by passage of cells through a French pressure cell) as the enzyme source were also spotted on each plate. The R. leguminosarum 3841 lysate provided standards for the various metabolites generated from Kdo2-[4′-32P]-lipid IVA in extracts of R. leguminosarum (Fig. 3,lane R.l.). The plates were developed in the solvent chloroform/pyridine/88% formic acid/water (30:70:16:10, v/v) and exposed to an imaging screen overnight. An Amersham Biosciences Storm PhosphorImager, equipped with ImageQuant software, was used to detect metabolism of the Kdo2-[4′-32P]-lipid IVA substrate to either more hydrophilic or more hydrophobic products. S. meliloti 1021/pSSB-1 was representative of several hybrid cosmids that directed overexpression of the long chain acyltransferase and was used for subcloning and DNA sequencing experiments. Plasmid and cosmid DNA were isolated using the Qiagen Spin Prep kit or a BIGGERprep kit (5 Prime → 3 Prime, Inc., Boulder, CO). Restriction endonucleases, shrimp alkaline phosphatase, and T4 DNA ligase were 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 DNA and cell transformation were done as described previously (25). Plasmids or cosmids were introduced into S. meliloti by tri-parental mating, as outlined above for the library screening (23Glazebrook J. Walker G.C. Methods Enzymol. 1991; 204: 398-418Crossref PubMed Scopus (113) Google Scholar).E. coli strain 803 (22Wood W. J. Mol. Biol. 1966; 16: 118-133Crossref PubMed Scopus (437) Google Scholar) or HB101 (Invitrogen) served as plasmid donors, and E. coli MT616 (24Finan T.M. Kunkel B. De Vos G.F. Signer E.R. J. Bacteriol. 1986; 167: 66-72Crossref PubMed Google Scholar) provided the transfer function. Different strains of S. meliloti (see below) served as recipients. The cosmid pSSB-1, isolated from the S. meliloti 1021/pSSB-1, was subjected to restriction digestion withEcoRI. This resulted in the release of several fragments (12.0, 6.0, 4.5, 0.8, 0.6, 0.4, 0.3, and 0.2 kb), including the ∼21-kb linearized cosmid vector (pLAFR-1). Two EcoRI (6.0 and 4.0 kb) fragments and two HindIII (6.6 and 5.8 kb) fragments were selected for subcloning into the multiple cloning sites of the pRK404A shuttle plasmid (26Ditta G. Stanfield S. Corbin D. Helinski D.R. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 7347-7351Crossref PubMed Scopus (1903) Google Scholar). These restriction fragments were resolved by agarose gel electrophoresis and were purified directly from the gel. Each fragment was then ligated into pRK404A (Fig.4). The ligation mixture was used to transform E. coli HB101. Plasmid-containing cells were selected by growth at 37 °C on LB agar supplemented with tetracycline (12.5 μg/ml). The plasmids isolated from several tetracycline resistance clones were also analyzed by restriction digestion to verify the presence of the right insert. The four plasmid constructs generated were designated as follows: pSSB-2, having the 5.8-kb HindIII DNA insert; pSSB-3, having the 4.0-kbHindIII DNA insert; pSSB-4, having the 6.0-kbEcoRI DNA insert; and pSSB-5, having the 4.0-kbEcoRI DNA insert. Each of these four plasmids was then transferred from E. coli HB101 into S. meliloti 1021, by tri-parental mating. S. meliloti 1021 recipient cells harboring pSSB-2, pSSB-3, pSSB-4, or pSSB-5 were selected on TY agar plates containing nalidixic acid (20 μg/ml), streptomycin (200 μg/ml), and tetracycline (12.5 μg/ml). Lysates prepared from these strains (16 colonies per construct) were screened in vitro for the overexpression of long chain acyltransferase activity. Both strands of the DNA inserts in pSSB-2 and pSSB-4 (isolated on larger scale from their E. coli host strains) were sequenced at the Duke sequencing system facility using AmpliTaq DNA polymerase in conjunction with an ABI 377 Prism DNA Sequencer. Sequencing of the downstream region of orf6 (see below) was carried out using pSSB-1 as the template. A PCR-amplified DNA fragment containing thelpxXL gene was cloned into pET23b vector behind the T7 promoter (Fig. 4) (54Golenbock D.T. Hampton R.Y. Qureshi N. Takayama K. Raetz C.R.H. J. Biol. Chem. 1991; 266: 19490-19498Abstract Full Text PDF PubMed Google Scholar, 55Neumeister B. Faigle M. Sommer M. Zähringer U. Stelter F. Menzel R. Schutt C. Northoff H. Infect. Immun. 1998; 66: 4151-4157Crossref PubMed Google Scholar, 56Rasool O. Freer E. Moreno E. Jarstrand C. Infect. Immun. 1992; 60: 1699-1702Crossref PubMed Google Scholar). The forward PCR primer (5′-GCGCGTCATATGAAACTGTTCATCACCCG-3′) was synthesized with a clamp region, an NdeI restriction site, and a match of the coding strand starting at the translation initiation site. The reverse primer (5′-AGATAGAATTCCTAAGGCGCGAGCGTCTGC-3′) was synthesized with a clamp region, an EcoRI restriction site, and a match to the anti-coding strand that included the stop codon. The PCR was performed using Pfu polymerase, as specified by the manufacturer. The plasmid pSSB-4 was used as the template. Amplification was carried out in a 50-μl reaction volume containing 100 ng of template, 20 mm Tris chloride, pH 8.8, 10 mm KCl, 10 mm (NH4)2SO4, 0.1% Triton X-100, 0.1% bovine serum albumin, 10 μm dNTPs, and 2 units of Pfu polymerase. The reactions were subjected to 25 cycles of denaturation (45 s, 94 °C), annealing (45 s, 55 °C), and extension (2 min, 72 °C) in a DNA thermal cycler. The reaction product was analyzed on a 1% agarose gel, was diges

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