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A Special Acyl Carrier Protein for Transferring Long Hydroxylated Fatty Acids to Lipid A in Rhizobium

1996; Elsevier BV; Volume: 271; Issue: 50 Linguagem: Inglês

10.1074/jbc.271.50.32126

ISSN

1083-351X

Autores

Kathryn A. Brozek, Russell W. Carlson, ChristianR. H. Raetz,

Tópico(s)

Carbohydrate Chemistry and Synthesis

Resumo

Lipid A, the hydrophobic anchor of lipopolysaccharides in the outer membranes of Gram-negative bacteria, varies in structure among different Rhizobiaceae. The Rhizobium meliloti lipid A backbone, like that of Escherichia coli, is a β1′-6-linked glucosamine disaccharide that is phosphorylated at positions 1 and 4′. Rhizobium leguminosarum lipid A lacks both phosphates, but contains aminogluconate in place of the proximal glucosamine 1-phosphate, and galacturonic acid instead of the 4′-phosphate. A peculiar feature of the lipid As of all Rhizobiaceae is acylation with 27-hydroxyoctacosanoic acid, a long hydroxylated fatty acid not found in E. coli. We now describe an in vitro system, consisting of a membrane enzyme and a cytosolic acyl donor from R. leguminosarum, that transfers 27-hydroxyoctacosanoic acid to (Kdo)2-lipid IVA, a key lipid A precursor common to both E. coli and R. leguminosarum. The 27-hydroxyoctacosanoic acid moiety was detected in the lipid product by mass spectrometry. The membrane enzyme required the presence of Kdo residues in the acceptor substrate for activity. The cytosolic acyl donor was purified from wild-type R. leguminosarum using the acylation of (Kdo)2-[4′-32P]-lipid IVA as the assay. Amino-terminal sequencing of the purified acyl donor revealed an exact 19-amino acid match with a partially sequenced gene (orf*) of R. leguminosarum. Orf* contains the consensus sequence, DSLD, for attachment of 4′-phosphopantetheine. When the entire orf* gene was sequenced, it was found to encode a protein of 92 amino acids. Orf* is a new kind of acyl carrier protein because it is only ∼25% identical both to the constitutive acyl carrier protein (AcpP) and to the inducible acyl carrier protein (NodF) of R. leguminosarum. Mass spectrometry of purified active Orf* confirmed the presence of 4′-phosphopantetheine and 27-hydroxyoctacosanoic acid in the major species. Smaller mass peaks indicative of Orf* acylation with hydroxylated 20, 22, 24, and 26 carbon fatty acids were also observed. Given the specialized function of Orf* in lipid A acylation, we suggest the new designation AcpXL. Lipid A, the hydrophobic anchor of lipopolysaccharides in the outer membranes of Gram-negative bacteria, varies in structure among different Rhizobiaceae. The Rhizobium meliloti lipid A backbone, like that of Escherichia coli, is a β1′-6-linked glucosamine disaccharide that is phosphorylated at positions 1 and 4′. Rhizobium leguminosarum lipid A lacks both phosphates, but contains aminogluconate in place of the proximal glucosamine 1-phosphate, and galacturonic acid instead of the 4′-phosphate. A peculiar feature of the lipid As of all Rhizobiaceae is acylation with 27-hydroxyoctacosanoic acid, a long hydroxylated fatty acid not found in E. coli. We now describe an in vitro system, consisting of a membrane enzyme and a cytosolic acyl donor from R. leguminosarum, that transfers 27-hydroxyoctacosanoic acid to (Kdo)2-lipid IVA, a key lipid A precursor common to both E. coli and R. leguminosarum. The 27-hydroxyoctacosanoic acid moiety was detected in the lipid product by mass spectrometry. The membrane enzyme required the presence of Kdo residues in the acceptor substrate for activity. The cytosolic acyl donor was purified from wild-type R. leguminosarum using the acylation of (Kdo)2-[4′-32P]-lipid IVA as the assay. Amino-terminal sequencing of the purified acyl donor revealed an exact 19-amino acid match with a partially sequenced gene (orf*) of R. leguminosarum. Orf* contains the consensus sequence, DSLD, for attachment of 4′-phosphopantetheine. When the entire orf* gene was sequenced, it was found to encode a protein of 92 amino acids. Orf* is a new kind of acyl carrier protein because it is only ∼25% identical both to the constitutive acyl carrier protein (AcpP) and to the inducible acyl carrier protein (NodF) of R. leguminosarum. Mass spectrometry of purified active Orf* confirmed the presence of 4′-phosphopantetheine and 27-hydroxyoctacosanoic acid in the major species. Smaller mass peaks indicative of Orf* acylation with hydroxylated 20, 22, 24, and 26 carbon fatty acids were also observed. Given the specialized function of Orf* in lipid A acylation, we suggest the new designation AcpXL. INTRODUCTIONLipopolysaccharides, or endotoxins, comprise the outer leaflet of the outer membranes of Gram-negative bacteria (1Raetz C.R.H. Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Scopus (1030) Google Scholar, 2Raetz C.R.H. J. Bacteriol. 1993; 175: 5745-5753Crossref PubMed Scopus (234) Google Scholar, 3Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. Second Ed. Vol. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar, 4Rietschel E.T. Brade H. Sci. Am. 1992; 267: 54-61Crossref PubMed Scopus (452) 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 (1308) Google Scholar). Lipid A, the hydrophobic moiety that attaches lipopolysaccharide to the membrane, is of special interest because it is essential for bacterial growth (6Galloway S.M. Raetz C.R.H. J. Biol. Chem. 1990; 265: 6394-6402Abstract Full Text PDF PubMed Google Scholar), and its biosynthesis is a target for the design of new antibacterial agents. 1Onishi, H. R., Pelak, B. A., Gerckens, L. S., Silver, L. L., Kahan, F. M., Chen, M. H., Patchett, A. A., Williamson, J. M., Hyland, A., Anderson, M. S., and Raetz, C. R. H. (1996) Science, in press. In addition, lipid A is a potent stimulant of mammalian immune cells (3Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. Second Ed. Vol. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google 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 (1308) Google Scholar, 8Ulevitch R.J. Tobias P.S. Curr. Opin. Immunol. 1994; 6: 125-130Crossref PubMed Scopus (227) Google Scholar). The overproduction of cytokines and inflammatory mediators by macrophages upon stimulation by lipid A during severe Gram-negative infections is thought to cause some of the clinical complications of Gram-negative sepsis (3Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. Second Ed. Vol. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google 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 (1308) Google Scholar, 8Ulevitch R.J. Tobias P.S. Curr. Opin. Immunol. 1994; 6: 125-130Crossref PubMed Scopus (227) Google Scholar, 9Morrison D.C. Ryan J.L. Annu. Rev. Med. 1987; 38: 417-432Crossref PubMed Google Scholar, 10Levin J. Levin J. Alving C.R. Munford R.S. Stütz P.L. Endotoxin Research Series, Vol 2, Bacterial Endotoxin: Recognition and Effector Mechanisms. Excerpta Medica, Amsterdam1993Google Scholar). Escherichia coli lipid A, one of the best studied examples, consists of a glucosamine disaccharide backbone that is linked β1′-6 (Fig. 1), is acylated with R-3-hydroxymyristate at positions 2, 3, 2′, and 3′, and is phosphorylated at positions 1 and 4′ (1Raetz C.R.H. Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Scopus (1030) Google Scholar, 3Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. Second Ed. Vol. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google 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 (1308) Google Scholar, 11Rietschel E.T. Sidorczyk Z. Zähringer U. Wollenweber H.-W. Lüderitz O. Anderson L. Unger F.M. Bacterial Lipopolysaccharides. ACS Symposium Series. Vol. 231. American Chemical Society, Washington, D. C.1983: 214Google Scholar, 12Imoto M. Kusumoto S. Shiba T. Naoki H. Iwashita T. Rietschel E.T. Wollenweber H.-W. Galanos C. Lüderitz O. Tetrahedron Lett. 1983; 24: 4017-4020Crossref Scopus (108) Google Scholar, 13Qureshi N. Takayama K. Mascagni P. Honovich J. Wong R. Cotter R.J. J. Biol. Chem. 1988; 263: 11971-11976Abstract Full Text PDF PubMed Google Scholar). The two R-3-hydroxy moieties of the distal unit are further acylated with laurate and myristate (Fig. 1), forming acyloxyacyl groups (1Raetz C.R.H. Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Scopus (1030) Google Scholar, 3Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. Second Ed. Vol. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google 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 (1308) Google Scholar, 11Rietschel E.T. Sidorczyk Z. Zähringer U. Wollenweber H.-W. Lüderitz O. Anderson L. Unger F.M. Bacterial Lipopolysaccharides. ACS Symposium Series. Vol. 231. American Chemical Society, Washington, D. C.1983: 214Google Scholar, 12Imoto M. Kusumoto S. Shiba T. Naoki H. Iwashita T. Rietschel E.T. Wollenweber H.-W. Galanos C. Lüderitz O. Tetrahedron Lett. 1983; 24: 4017-4020Crossref Scopus (108) Google Scholar, 13Qureshi N. Takayama K. Mascagni P. Honovich J. Wong R. Cotter R.J. J. Biol. Chem. 1988; 263: 11971-11976Abstract Full Text PDF PubMed Google Scholar, 14Karibian D. Deprun C. Caroff M. J. Bacteriol. 1993; 175: 2988-2993Crossref PubMed Google Scholar, 15Myers K.R. Ulrich J.T. Qureshi N. Takayama K. Wang R. Chen L. Emary W.B. Cotter R.J. Bioconjugate Chem. 1992; 3: 540-548Crossref PubMed Scopus (6) Google Scholar). The latter are structural hallmarks of lipid A moieties from diverse sources, and they are critical for the immunostimulatory activity of endotoxins (3Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. Second Ed. Vol. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google 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 (1308) Google Scholar, 16Loppnow H. Brade H. Dürrbaum I. Dinarello C.A. Kusumoto S. Rietschel E.T. Flad H.D. J. Immunol. 1989; 142: 3229-3238PubMed Google Scholar, 17Golenbock 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). Lipid A analogs with a reduced number of acyloxyacyl moieties are of interest because some are potent endotoxin antagonists with possible utility for treating the complications of Gram-negative sepsis (3Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. Second Ed. Vol. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar, 17Golenbock 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, 18Takayama K. Qureshi N. Beutler B. Kirkland T.N. Infect. Immun. 1989; 57: 1336-1338Crossref PubMed Google Scholar, 19Christ W.J. McGuinness P.D. Asano O. Wang Y. Mullarkey M.A. Perez M. Hawkins L.D. Blythe T.A. Dubuc G.R. Robidoux A.L. J. Am. Chem. Soc. 1994; 116: 3637-3638Crossref Scopus (82) Google Scholar, 20Christ W.J. Asano O. Robidoux A.L. Perez M. Wang Y. Dubuc G.R. Gavin W.E. Hawkins L.D. McGuinness P.D. Mullarkey M.A. Lewis M.D. Kishi Y. Kawata T. Bristol J.R. Rose J.R. Rossignol D.P. Kobayashi S. Hishinuma I. Kimura A. Asakawa N. Katayama K. Yamatsu I. Science. 1995; 265: 80-83Crossref Scopus (309) Google Scholar).Given the importance of lipid A analogs as endotoxin antagonists, we have recently become interested in elucidating the enzymatic synthesis of lipid A in Rhizobium leguminosarum (21Price N.P.J. Kelly T.M. Raetz C.R.H. Carlson R.W. J. Bacteriol. 1994; 176: 4646-4655Crossref PubMed Google Scholar, 22Price 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). Lipid A of R. leguminosarum lacks the phosphate groups (23Bhat U.R. Forsberg L.S. Carlson R.W. J. Biol. Chem. 1994; 269: 14402-14410Abstract Full Text PDF PubMed Google Scholar) found in E. coli or Rhizobium meliloti lipid A (3Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. Second Ed. Vol. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar, 24Urbanik-Sypniewska T. Seydel U. Greck M. Weckesser J. Mayer H. Arch. Microbiol. 1989; 152: 527-532Crossref Scopus (28) Google Scholar, 25Zevenhuizen L.P.T.M. Scholten-Koerselman I. Posthumus M.A. Arch. Microbiol. 1980; 125: 1-8Crossref Scopus (38) Google Scholar). R. leguminosarum lipid A contains an acylated aminogluconate in place of the proximal glucosamine 1-phosphate, and a galacturonic acid residue in place of the 4′-phosphate (Fig. 1) (23Bhat U.R. Forsberg L.S. Carlson R.W. J. Biol. Chem. 1994; 269: 14402-14410Abstract Full Text PDF PubMed Google Scholar). Most remarkably, R. leguminosarum lipid A appears to lack acyloxyacyl residues, as judged by the absence of myristate, laurate, and other common short acyl chains (23Bhat U.R. Forsberg L.S. Carlson R.W. J. Biol. Chem. 1994; 269: 14402-14410Abstract Full Text PDF PubMed Google Scholar). However, lipid As of R. leguminosarum and most other Rhizobiaceae contain an unusual, long hydroxylated acyl moiety, 27-hydroxyoctacosanoic acid, not found in enterobacterial lipid A (26Bhat U.R. Mayer H. Yokota A. Hollingsworth R.I. Carlson R. J. Bacteriol. 1991; 173: 2155-2159Crossref PubMed Google Scholar, 27Bhat U.R. Carlson R.W. Busch M. Mayer H. Int. J. Syst. Bacteriol. 1991; 41: 213-217Crossref PubMed Scopus (68) Google Scholar). The exact location of the ester-linked 27-hydroxyoctacosanoic acid moiety in R. leguminosarum lipid A is uncertain (23Bhat U.R. Forsberg L.S. Carlson R.W. J. Biol. Chem. 1994; 269: 14402-14410Abstract Full Text PDF PubMed Google Scholar). It is apparently not attached to one of the R-3-hydroxy acyl chains (Fig. 1), assuming that acyloxyacyl moieties containing 27-hydroxyoctacosanoic acid have the same chemical reactivities as ordinary acyloxyacyl groups (23Bhat U.R. Forsberg L.S. Carlson R.W. J. Biol. Chem. 1994; 269: 14402-14410Abstract Full Text PDF PubMed Google Scholar, 28Wollenweber H.-W. Rietschel E.T. J. Microbiol. Methods. 1990; 11: 195-211Crossref Scopus (145) Google Scholar). The function of 27-hydroxyoctacosanoic acid is not known. Its length is about twice that of the normal fatty acids that are usually attached to enterobacterial lipid As. Other examples of related, long chain oxygen-containing fatty acids are 25-hydroxyhexacosanoic acid in Pseudomonas carboxydovorans (27Bhat U.R. Carlson R.W. Busch M. Mayer H. Int. J. Syst. Bacteriol. 1991; 41: 213-217Crossref PubMed Scopus (68) Google Scholar) and 27-keto-octacosanoic acid in Legionella (29Zähringer U. Knirel Y.A. Lindner B. Helbig J.H. Sonesson A. Marre R. Rietschel E.T. Prog. Clin. Biol. Res. 1995; 392: 113-139PubMed Google Scholar).We previously described enzymes in E. coli extracts that incorporate laurate and myristate into the key lipid A precursor, (Kdo)2-lipid IVA 2The abbreviations used are: Kdo2-keto-3-deoxyoctonateMES4-morpholineethanesulfonic acidCAPS3-(cyclohexylamino)propanesulfonic acidORFopen reading frameHPLChigh performance liquid chromatographyTMSOtrimethylsiloxyFAMEfatty acid methyl estersGLC-MSgas liquid chromatography-mass spectrometry; nod factor, nodulation factorACPacyl carrier protein. (Fig. 2) (30Brozek K.A. Raetz C.R.H. J. Biol. Chem. 1990; 265: 15410-15417Abstract Full Text PDF PubMed Google Scholar). The enzymes catalyzing these reactions are the products of the htrB and msbB genes, respectively (31Clementz T. Bednarski J. Raetz C.R.H. FASEB J. 1995; 9: A1311Crossref PubMed Scopus (271) Google Scholar, 32Clementz T. Bednarski J.J. Raetz C.R.H. J. Biol. Chem. 1996; 271: 12095-12102Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). They have a remarkable requirement for the Kdo domain, as they do not acylate lipid IVA (30Brozek K.A. Raetz C.R.H. J. Biol. Chem. 1990; 265: 15410-15417Abstract Full Text PDF PubMed Google Scholar, 32Clementz T. Bednarski J.J. Raetz C.R.H. J. Biol. Chem. 1996; 271: 12095-12102Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). This specificity explains why tetra-acylated lipid IVA rather than hexa-acylated lipid A accumulates in cells subjected to inhibition of Kdo biosynthesis or transfer (33Raetz C.R.H. Purcell S. Meyer M.V. Qureshi N. Takayama K. J. Biol. Chem. 1985; 260: 16080-16088Abstract Full Text PDF PubMed Google Scholar, 34Strain S.M. Armitage I.M. Anderson L. Takayama K. Qureshi N. Raetz C.R.H. J. Biol. Chem. 1985; 260: 16089-16098Abstract Full Text PDF PubMed Google Scholar, 35Rick P.D. Fung L.W.-M. Ho C. Osborn M.J. J. Biol. Chem. 1977; 252: 4904-4912Abstract Full Text PDF PubMed Google Scholar, 36Rick P.D. Osborn M.J. J. Biol. Chem. 1977; 252: 4895-4903Abstract Full Text PDF PubMed Google Scholar, 37Goldman R. Kohlbrenner W. Lartey P. Pernet A. Nature. 1987; 329: 162-164Crossref PubMed Scopus (118) Google Scholar, 38Hammond S.M. Claesson A. Jansson A.M. Larsson L.G. Pring B.G. Town C.M. Ekström B. Nature. 1987; 327: 730-732Crossref PubMed Scopus (140) Google Scholar). The E. coli Kdo dependent late acyltransferases do not function with fatty acids longer than 14 carbons, and they require acyl chain activation by acyl carrier protein (30Brozek K.A. Raetz C.R.H. J. Biol. Chem. 1990; 265: 15410-15417Abstract Full Text PDF PubMed Google Scholar, 32Clementz T. Bednarski J.J. Raetz C.R.H. J. Biol. Chem. 1996; 271: 12095-12102Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar).Fig. 2The acceptor, (Kdo)2-[4′-32P]-lipid IVA, used to detect a novel long chain acyl donor in the cytosol of R. leguminosarum. The radioactive 4′ P atom is indicated by the arrow. Evidence for the generation of Kdo2-[4′-32P]-lipid IVA in R. leguminosarum extracts has been presented (21Price N.P.J. Kelly T.M. Raetz C.R.H. Carlson R.W. J. Bacteriol. 1994; 176: 4646-4655Crossref PubMed Google Scholar, 22Price 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).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Given that R. leguminosarum extracts contain all the enzymes needed to synthesize the conserved precursor, (Kdo)2-lipid IVA (Fig. 2) (21Price N.P.J. Kelly T.M. Raetz C.R.H. Carlson R.W. J. Bacteriol. 1994; 176: 4646-4655Crossref PubMed Google Scholar), we examined the possibility that (Kdo)2-[4′-32P]-lipid IVA may function as an acceptor for the 27-hydroxyoctacosanoic acid moiety. In preliminary experiments (39Brozek K.A. Kadrmas J.L. Raetz C.R.H. FASEB J. 1995; 9: A1376Google Scholar), described in the accompanying manuscript (62Brozek 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), we observed a putative acylation reaction of (Kdo)2-[4′-32P]-lipid IVA in crude extracts of R. leguminosarum. We now demonstrate that this reaction requires both a membrane component and a cytosolic factor, and that it indeed represents the addition of 27-hydroxyoctacosanoic acid to (Kdo)2-lipid IVA. It occurs in extracts of several strains of R. leguminosarum and R. meliloti, but not E. coli. The R. leguminosarum cytosolic factor has been purified, cloned, and sequenced. It is a new member of the acyl carrier protein family, designated AcpXL, that functions in transfers of long hydroxylated fatty acids. AcpXL is distinct from both the constitutive acyl carrier protein (40Platt M.W. Miller K.J. Lane W.S. Kennedy E.P. J. Bacteriol. 1990; 172: 5440-5444Crossref PubMed Google Scholar), AcpP, involved in synthesis of 12-18 carbon acyl chains and from the inducible acyl carrier protein (41Geiger O. Spaink H.P. Kennedy E.P. J. Bacteriol. 1991; 173: 2872-2878Crossref PubMed Google Scholar, 42Denarie J. Debelle F. Prome J.-C. Annu. Rev. Biochem. 1996; 65: 503-535Crossref PubMed Scopus (670) Google Scholar), NodF, required for the generation of polyunsaturated fatty acids found in the nod factors of R. leguminosarum.EXPERIMENTAL PROCEDURESMaterials[γ-32P]ATP and 32Pi were products of DuPont NEN. HEPES, MES, Kdo, and trypsin immobilized on inert beads were obtained from Sigma. The following materials and kits purchased were: Centricon, Centriprep, and Microcon centrifugation devices from Amicon; Silica Gel-60 thin layer plates, 0.25 mm, from EM Science; bicinchoninic acid and Pronase immobilized on inert beads from Pierce Chemical Co.; Sequagel DNA Sequencing Gel Reagents and Protogel Polyacrylamide Gel Reagents from National Diagnostics; Superose-12 FPLC column from Pharmacia; Pre-stained Polypeptide Standards from Bio-Rad; BIGGER prep DNA preparation kits from 5′→ 3′, Inc.; Sequenase Version 2.0 DNA sequencing kit from U. S. Biochemical Corp.; polyvinyldifluoride membranes, Immobilon-P from Millipore; and bulk silica gel, Davisil grade 63%, 100-200 mesh, 60 A from Sigma. DNA primers for sequencing were custom-made by Life Technologies, Inc.Bacterial Strains and Growth ConditionsBacterial strains are listed in Table I. All cells were grown on TY medium, containing 5 g of tryptone and 3 g of yeast extract per liter, and supplemented with 10 mM CaCl2. Rhizobia were selected with 20 μg/ml nalidixic acid (all strains) and 200 μg/ml streptomycin sulfate (R. leguminosarum 8401, R. leguminosarum CE3, R. meliloti 1021, and R. meliloti GMI255). All strains were grown at 30°C. All strains are essentially wild-type (21Price N.P.J. Kelly T.M. Raetz C.R.H. Carlson R.W. J. Bacteriol. 1994; 176: 4646-4655Crossref PubMed Google Scholar, 22Price 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), except that 8401 is lacking a symbiotic plasmid (J. A. Downie, John Innes Institute, Norwich, United Kingdom), and GMI255 carries a deletion of 280 kilobases in its symbiotic plasmid (43Truchet G. Debelle F. Vasse J. Terzaghi B. Garnerone A.M. Rosenberg C. Batut J. Maillet F. Denarie J. J. Bacteriol. 1985; 164: 1200-1210Crossref PubMed Google Scholar). Both 8401 and GM1255 are nod−.Table I.Bacterial strains employed in this workSpeciesBiovarStrainSource (Ref.)PropertiesR. leguminosarumViciae8401J. Downie (21Price N.P.J. Kelly T.M. Raetz C.R.H. Carlson R.W. J. Bacteriol. 1994; 176: 4646-4655Crossref PubMed Google Scholar)Strep.R, Nod−Viciae11954ATCCaATCC, American Type Culture Collection, Bethesda, MD.Wild type, Nod+Viciae10004ATCCWild-type, Nod+EtliCE3D. Noel (61Cava J.R. Elias P.M. Turowski D.A. Noel K.D. J. Bacteriol. 1989; 171: 8-15Crossref PubMed Google Scholar)Strep.R, Nod+Phaseoli14482ATCCWild-type, Nod+Trifolii14480ATCCWild-type, Nod+R. Meliloti1021S. LongStrep.R, Nod+10310ATCCWild-type, Nod+GMI255S. Long (43Truchet G. Debelle F. Vasse J. Terzaghi B. Garnerone A.M. Rosenberg C. Batut J. Maillet F. Denarie J. J. Bacteriol. 1985; 164: 1200-1210Crossref PubMed Google Scholar)Nod− (deletion)E. coliR477J. Adler (7Nishijima M. Raetz C.R.H. J. Biol. Chem. 1979; 254: 7837-7844Abstract Full Text PDF PubMed Google Scholar)F−rpsL 136W3110CGSCbCGSC,E. coli Genetic Stock Center, Yale University.K12 wild-typeS17-1/pCS115U. B. Priefer (49Colonna-Romano S. Arnold W. Schluter A. Boistard P. Pühler A. Priefer U.B. Mol. Gen. Genet. 1990; 223: 138-147Crossref PubMed Scopus (54) Google Scholar)Orf* cosmida ATCC, American Type Culture Collection, Bethesda, MD.b CGSC,E. coli Genetic Stock Center, Yale University. Open table in a new tab Preparation of Cell-free ExtractsBacterial cultures were harvested in late logarithmic phase (A550 = 0.6-1.0) by centrifugation at 8000 × gav for 15 min, and the cell pellet was resuspended in 50 mM HEPES, pH 7.5, to give a final protein concentration of 5-15 mg/ml. The cells were broken by passage through a French pressure cell at 18,000 p.s.i. Unbroken cells and debris were removed by another centrifugation at 8000 × gav for 15 min. Extracts were prepared and handled at 0-4°C. Protein concentrations were determined with bicinchoninic acid (44Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18446) Google Scholar), using bovine serum albumin for the standard curve. Subcellular fractions were prepared by centrifugation of the crude extract in 25 mM HEPES, pH 7.5, at 150,000 × gav for 60 min. The membrane pellet was removed, and resuspended in the original volume of buffer. The centrifugation was repeated on both the cytosol and the resuspended membranes. These final preparations are referred to as “cytosol” and “washed membranes.”When strain 8401 was used for large scale preparations of cytosolic acyl donor, frozen cells were employed. A 150-liter culture was grown in a 200-liter New Brunswick fermenter, and cells were harvested at A550 = 1.0 using a Sharples centrifuge. The cell paste was stored at −80°C, and portions were thawed as needed in 2-3 ml of 50 mM HEPES, pH 7.5, per gram of cell paste. Cells were then broken by passage through a French pressure cell at 18,000 p.s.i., and debris was removed by centrifugation at 8000 × gav for 15 min. The crude extract and acyl donating cytosolic fractions were then prepared as above.Preparation of Radioactive Substrates(Kdo)2-[4′-32P]-lipid IVA and [4′-32P]-lipid IVA were prepared as described previously (22Price 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, 45Hampton R.Y. Raetz C.R.H. Methods Enzymol. 1992; 209: 466-475Crossref PubMed Scopus (12) Google Scholar). Aqueous dispersions of these lipids were stored at −20°C, and they were subjected to sonic irradiation in a bath sonicator for 2 min prior to use.Assay of (Kdo)2-lipid IVA AcylationThe conditions for observing the acylation of (Kdo)2-[4′-32P]-lipid IVA (formation of product a) were optimized. Reactions contained 50 mM HEPES, pH 8.2, 0.2% Triton X-100, and 10 μM (Kdo)2-[4′-32P]-lipid IVA, at approximately 20,000-50,000 cpm/nmol, in 10 μl. Crude extracts, membranes, and cytosolic fractions were present at the concentrations indicated. Acylation reactions were carried out at 30°C for 60 min, or as otherwise indicated. Following incubation, 5-μl samples were withdrawn and spotted onto thin-layer chromatography plates that were then developed in the solvent chloroform, pyridine, 88% formic acid, water (30:70:16:10, v/v). Exposure to imaging screens at room temperature was carried out overnight. Extent of conversion of substrate to product(s) was measured using a Molecular Dynamics PhosphorImager operated with ImageQuant software.Polyacrylamide Gel ElectrophoresisPolyacrylamide gels of 15% were cast in a Bio-Rad Protean II apparatus, according to the conditions of Laemmli (46Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206038) Google Scholar), but without SDS. Samples were not denatured or reduced prior to loading in glycerol-containing buffer. Gels were run at constant current of 25 mA, using a buffer consisting of 3.1 g of Tris base and 14.4 g of glycine per liter. Gels were stained with Coomassie Blue.Chemical Hydrolyses of (Kdo)2-[4′-32P]-lipid IVA and Its Acylated Derivative (Product a)Acylation reactions (10 μl) were set up as described above, except that carrier-free (Kdo)2-[4′-32P]-lipid IVA was used (1 × 107 cpm/nmol), the chemical concentration of which was about 0.2 μM. Reaction RI contained no membranes or cytosolic factor. Reaction RII contained 0.1 mg/ml 8401 membranes, and 0.3 mg/ml of DEAE-purified cytosolic factor (see below). Reaction RIII contained a system for generating (Kdo)2-[lauroyl]-[4′-32P]-lipid IVA as a control, using 45 μg/ml of an extract that overproduces HtrB but lacks MsbB (32Clementz T. Bednarski J.J. Raetz C.R.H. J. Biol. Chem. 1996; 271: 12095-12102Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar), (Kdo)2-[4′-32P]-lipid IVA, and 182 μM lauroyl-ACP. Acylation reactions were incubated at 30°C for 60 min. Three 1-μl portions were withdrawn from each reaction. Each 1-μl portion was subjected to a different treatment. (a) For mild base hydrolysis, 1 μl of reaction mixture was combined with 9 μl of chloroform/methanol (2:1, v/v), and 1 μl of 1.25 M NaOH. The mixture was incubated at room temperature for 30 min, after which time 1 μl of 1.25 M HCl was added. After mixing, a 5-μl portion was applied to a thin-layer plate. (b) For acid hydrolysis, 1 μl of reaction mixture was combined with 9 μl of 0.1 M HCl in a 0.65-ml Microfuge tube, which was sealed with a boiling clip. The tube was floated in a boiling water bath for 30 min. Next, 0.8 μl of 1.25 M NaOH and 1 μl of 10% SDS were added. The latter helped recover all the radioactivity from the tube, without affecting the hydrolysis. A 5-μl sample was then

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