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A PhoP/PhoQ-induced Lipase (PagL) That Catalyzes 3-O-Deacylation of Lipid A Precursors in Membranes ofSalmonella typhimurium

2001; Elsevier BV; Volume: 276; Issue: 12 Linguagem: Inglês

10.1074/jbc.m010730200

ISSN

1083-351X

Autores

M. Stephen Trent, Wendy L. Pabich, Christian R.H. Raetz, Samuel I. Miller,

Tópico(s)

Vibrio bacteria research studies

Resumo

Pathogenic bacteria modify the structure of the lipid A portion of their lipopolysaccharide in response to environmental changes. Some lipid A modifications are important for virulence and resistance to cationic antimicrobial peptides. The two-component system PhoP/PhoQ plays a central role in regulating lipid A modification. We now report the discovery of a PhoP/PhoQ-activated gene (pagL) in Salmonella typhimurium, encoding a deacylase that removes the R-3-hydroxymyristate moiety attached at position 3 of certain lipid A precursors. The deacylase gene (pagL) was identified by assaying for loss of deacylase activity in extracts of 14 random TnphoA::pag insertion mutants. ThepagL gene encodes a protein of 185 amino acid residues unique to S. typhimurium and closely related organisms such as Salmonella typhi. Heterologous expression ofpagL in Escherichia coli on plasmid pWLP21 results in loss of the R-3-hydroxymyristate moiety at position 3 in ∼90% of the lipid A molecules but does not inhibit cell growth. PagL is synthesized with a 20-amino acidN-terminal signal peptide and is localized mainly in the outer membrane, as judged by assays of separated S. typhimurium membranes and by SDS-polyacrylamide gel analysis of membranes from E. coli cells that overexpress PagL. The function of PagL is unknown, given that S. typhimuriummutants lacking pagL display no obvious phenotypes, but PagL might nevertheless play a role in pathogenesis if it serves to modulate the cytokine response of an infected animal host. Pathogenic bacteria modify the structure of the lipid A portion of their lipopolysaccharide in response to environmental changes. Some lipid A modifications are important for virulence and resistance to cationic antimicrobial peptides. The two-component system PhoP/PhoQ plays a central role in regulating lipid A modification. We now report the discovery of a PhoP/PhoQ-activated gene (pagL) in Salmonella typhimurium, encoding a deacylase that removes the R-3-hydroxymyristate moiety attached at position 3 of certain lipid A precursors. The deacylase gene (pagL) was identified by assaying for loss of deacylase activity in extracts of 14 random TnphoA::pag insertion mutants. ThepagL gene encodes a protein of 185 amino acid residues unique to S. typhimurium and closely related organisms such as Salmonella typhi. Heterologous expression ofpagL in Escherichia coli on plasmid pWLP21 results in loss of the R-3-hydroxymyristate moiety at position 3 in ∼90% of the lipid A molecules but does not inhibit cell growth. PagL is synthesized with a 20-amino acidN-terminal signal peptide and is localized mainly in the outer membrane, as judged by assays of separated S. typhimurium membranes and by SDS-polyacrylamide gel analysis of membranes from E. coli cells that overexpress PagL. The function of PagL is unknown, given that S. typhimuriummutants lacking pagL display no obvious phenotypes, but PagL might nevertheless play a role in pathogenesis if it serves to modulate the cytokine response of an infected animal host. lipopolysaccharide 3-deoxy-d-manno-octulosonic acid polymerase chain reaction 3-(cyclohexylamino)propanesulfonic acid 2-[bis(2-hy-droxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol Pathogenic bacteria are capable of sensing microenvironments within the tissues of their animal hosts, leading to the expression of virulence genes necessary for bacterial survival and replication (1Jones B.D. Falkow S. Annu. Rev. 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The structure of lipid A is relatively conserved among different pathogenic Gram-negative bacteria (13Raetz C.R.H. Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Scopus (1040) Google Scholar, 14Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar, 16Zähringer U. Lindner B. Rietschel E.T. Brade H. Opal S.M. Vogel S.N. Morrison D.C. Endotoxin in Health and Disease. Marcel Dekker, Inc., New York1999: 93-114Google Scholar, 25Rietschel E.T. Brade L. Lindner B. Zähringer U. Morrison D.C. Ryan J.L. Bacterial Endotoxic Lipopolysaccharides. I. CRC Press, Inc., Boca Raton, FL1992: 3-41Google Scholar). Lipid A of E. coli and S. typhimurium is a β,1′-6-linked disaccharide of glucosamine, phosphorylated at the 1- and 4′-positions and acylated at the 2-, 3-, 2′-, and 3′-positions withR-3-hydroxymyristate (Fig. 1) (13Raetz C.R.H. Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Scopus (1040) Google Scholar, 14Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar, 16Zähringer U. Lindner B. Rietschel E.T. Brade H. Opal S.M. Vogel S.N. Morrison D.C. Endotoxin in Health and Disease. Marcel Dekker, Inc., New York1999: 93-114Google Scholar, 25Rietschel E.T. Brade L. Lindner B. Zähringer U. Morrison D.C. Ryan J.L. Bacterial Endotoxic Lipopolysaccharides. I. CRC Press, Inc., Boca Raton, FL1992: 3-41Google Scholar). The OH groups of the R-3-hydroxymyristate chains that are attached at positions 2′ and 3′ are further acylated with laurate and myristate, respectively (13Raetz C.R.H. Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Scopus (1040) Google Scholar, 14Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar, 16Zähringer U. Lindner B. Rietschel E.T. Brade H. Opal S.M. Vogel S.N. Morrison D.C. Endotoxin in Health and Disease. Marcel Dekker, Inc., New York1999: 93-114Google Scholar, 25Rietschel E.T. Brade L. Lindner B. Zähringer U. Morrison D.C. Ryan J.L. Bacterial Endotoxic Lipopolysaccharides. I. CRC Press, Inc., Boca Raton, FL1992: 3-41Google Scholar). Lipid A is glycosylated at position 6′ with two 3-deoxy-d-manno-octulosonic acid (Kdo) moieties (Fig. 1) (13Raetz C.R.H. Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Scopus (1040) Google Scholar, 14Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar, 16Zähringer U. Lindner B. Rietschel E.T. Brade H. Opal S.M. Vogel S.N. Morrison D.C. Endotoxin in Health and Disease. Marcel Dekker, Inc., New York1999: 93-114Google Scholar, 25Rietschel E.T. Brade L. Lindner B. Zähringer U. Morrison D.C. Ryan J.L. Bacterial Endotoxic Lipopolysaccharides. I. CRC Press, Inc., Boca Raton, FL1992: 3-41Google Scholar). Under certain circumstances, additional covalent modifications of lipid A are present, including 4-amino-4-deoxy-l-arabinose (4-aminoarabinose), phosphoethanolamine, palmitate, and/or 2-hydroxymyristate moieties (Fig. 1) (12Guo L. Lim K.B. Gunn J.S. Bainbridge B. Darveau R.P. Hackett M. Miller S.I. Science. 1997; 276: 250-253Crossref PubMed Scopus (480) Google Scholar, 26Helander I.M. Kilpeläinen I. Vaara M. Mol. Microbiol. 1994; 11: 481-487Crossref PubMed Scopus (146) Google Scholar, 27Nummila K. Kilpeläinen I. Zähringer U. Vaara M. Helander I.M. Mol. Microbiol. 1995; 16: 271-278Crossref PubMed Scopus (173) Google Scholar, 28Zhou Z. Lin S. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 1999; 274: 18503-18514Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). In S. typhimurium, Miller and co-workers have shown that incorporation of palmitate and 2-OH myristate moieties (12Guo L. Lim K.B. Gunn J.S. Bainbridge B. Darveau R.P. Hackett M. Miller S.I. Science. 1997; 276: 250-253Crossref PubMed Scopus (480) Google Scholar, 29Guo L. Lim K.B. Poduje C.M. Daniel M. Gunn J.S. Hackett M. Miller S.I. Cell. 1998; 95: 189-198Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar) is controlled by PhoP/PhoQ, whereas the 4-aminoarabinose and phosphoethanolamine modifications require the activation of PmrA (27Nummila K. Kilpeläinen I. Zähringer U. Vaara M. Helander I.M. Mol. Microbiol. 1995; 16: 271-278Crossref PubMed Scopus (173) Google Scholar, 30Gunn J.S. Lim K.B. Krueger J. Kim K. Guo L. Hackett M. Miller S.I. Mol. Microbiol. 1998; 27: 1171-1182Crossref PubMed Scopus (508) Google Scholar). PhoP-PhoQ mutants are more sensitive to the action of certain cationic antimicrobial peptides, in part because of the loss of palmitoylation of lipid A in the absence of the function of the PhoP-activated genepagP (29Guo L. Lim K.B. Poduje C.M. Daniel M. Gunn J.S. Hackett M. Miller S.I. Cell. 1998; 95: 189-198Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar). We have recently shown that pagP is the structural gene for a novel acyltransferase (31Bishop R.E. Gibbons H.S. Guina T. Trent M.S. Miller S.I. Raetz C.R.H. EMBO J. 2000; 19: 5071-5080Crossref PubMed Scopus (280) Google Scholar) (Fig.2) that utilizes glycerophospholipids as palmitate donors (31Bishop R.E. Gibbons H.S. Guina T. Trent M.S. Miller S.I. Raetz C.R.H. EMBO J. 2000; 19: 5071-5080Crossref PubMed Scopus (280) Google Scholar, 32Brozek K.A. Bulawa C.E. Raetz C.R.H. J. Biol. Chem. 1987; 262: 5170-5179Abstract Full Text PDF PubMed Google Scholar). PagP is the first example of a lipid A biosynthetic enzyme localized to the outer membrane (31Bishop R.E. Gibbons H.S. Guina T. Trent M.S. Miller S.I. Raetz C.R.H. EMBO J. 2000; 19: 5071-5080Crossref PubMed Scopus (280) Google Scholar). In the course of characterizing lipid A modifications in extracts of different S. typhimurium mutants, we have discovered a novel 3-O-deacylase activity that is strongly regulated by PhoP/PhoQ (Fig. 2). In the present study, we demonstrate that the 3-O-deacylase, like the PagP acyltransferase, is found mainly in the outer membrane. By assaying for 3-O-deacylase activity in extracts of PhoP-constitutive S. typhimuriumstrains harboring insertion mutations in different PhoP-activated (pag) genes (33Belden W.J. Miller S.I. Infect. Immun. 1994; 62: 5095-5101Crossref PubMed Google Scholar), the structural gene (pagL) encoding the deacylase was identified. The pagL gene was sequenced and shown to be unique to strains of Salmonella. When expressed in E. coli, PagL activity is localized in the outer membrane, and extensive lipid A 3-O-deacylation occurs without loss of cell viability. The function of pagL is unknown, since nonpolar deletions of S. typhimurium pagLdisplay no obvious phenotypes. However, partial 3-O-deacylation of Salmonella lipid A could be advantageous under certain conditions, since it might modulate the cytokine response of the host during an infection. [γ-32P]ATP was obtained from PerkinElmer Life Sciences. Silica gel 60 (0.25-mm) thin layer plates were purchased from EM Separation Technologies. Tryptone and yeast extract were from Difco. Triton X-100 and bicinchoninic acid were from Pierce. All other chemicals were reagent grade and were purchased from either Sigma or Mallinckrodt. The bacterial strains used in the present study are described in Table I. Typically, bacteria were grown at 37 °C in LB medium, which consists of 10 g of NaCl, 10 g of tryptone, and 5 g of yeast extract per liter (34Miller J.R. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar). In experiments involving Mg2+ limitation or pH changes, cells were grown in N-minimal medium (35Snavely M.D. Gravina S.A. Cheung T.T. Miller C.G. Maguire M.E. J. Biol. Chem. 1991; 266: 824-829Abstract Full Text PDF PubMed Google Scholar) with varying concentrations of Mg2+ at pH 7.7 in 100 mm Tris-HCl or at pH 5.8 in 100 mm bis-Tris buffer at 37 °C. Cells (10 ml) were first grown overnight at pH 7.7, harvested by centrifugation, washed twice with 5 ml of N-minimal medium at pH 7.7, and diluted 1:100 into N-minimal medium at pH 5.8 or 7.7, containing either low (10 μm) or high (10 mm) MgCl2. Cells were then grown into late log phase at 37 °C and harvested atA600 ranging from 0.65 to 1.0. When appropriate, cultures were supplemented with 100 μg/ml ampicillin, 12 μg/ml tetracycline, 30 μg/ml chloramphenicol, or 30 μg/ml kanamycin.Table IRelevant bacterial strains and plasmidsStrain/PlasmidDescriptionSource or referenceS. typhimurium strainsATCC 14028sWild typeATCCCS022pho-24(PhoP-constitutive)Ref. 49Miller S.I. Mekalanos J.J. J. Bacteriol. 1990; 172: 2485-2490Crossref PubMed Scopus (313) Google ScholarCS015CSO22,phoP102::Tn10d-camRef. 69Miller S.I. Kukral A.M. Mekalanos J.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5054-5058Crossref PubMed Scopus (694) Google ScholarCS400CSO22,pagA::Tn10dRef. 51Gunn J.S. Belden W.J. Miller S.I. Microb. Pathog. 1998; 25: 77-90Crossref PubMed Scopus (63) Google ScholarCS027CSO22,pagC1::TnphoARef. 51Gunn J.S. Belden W.J. Miller S.I. Microb. Pathog. 1998; 25: 77-90Crossref PubMed Scopus (63) Google ScholarCS336CSO22,pagD::TnphoARef. 51Gunn J.S. Belden W.J. Miller S.I. Microb. Pathog. 1998; 25: 77-90Crossref PubMed Scopus (63) Google ScholarCS325CSO22,pagE::TnphoARef. 51Gunn J.S. Belden W.J. Miller S.I. Microb. Pathog. 1998; 25: 77-90Crossref PubMed Scopus (63) Google ScholarCS332CSO22,pagF::TnphoARef. 51Gunn J.S. Belden W.J. Miller S.I. Microb. Pathog. 1998; 25: 77-90Crossref PubMed Scopus (63) Google ScholarCS324CSO22,pagG::TnphoARef. 51Gunn J.S. Belden W.J. Miller S.I. Microb. Pathog. 1998; 25: 77-90Crossref PubMed Scopus (63) Google ScholarCS331CSO22,pagH::TnphoARef. 51Gunn J.S. Belden W.J. Miller S.I. Microb. Pathog. 1998; 25: 77-90Crossref PubMed Scopus (63) Google ScholarCS334CSO22,pagI::TnphoARef. 51Gunn J.S. Belden W.J. Miller S.I. Microb. Pathog. 1998; 25: 77-90Crossref PubMed Scopus (63) Google ScholarCS327CSO22,pagK::TnphoARef. 51Gunn J.S. Belden W.J. Miller S.I. Microb. Pathog. 1998; 25: 77-90Crossref PubMed Scopus (63) Google ScholarCS328CSO22,pagL::TnphoARef. 51Gunn J.S. Belden W.J. Miller S.I. Microb. Pathog. 1998; 25: 77-90Crossref PubMed Scopus (63) Google ScholarCS333CSO22,pagM::TnphoARef. 51Gunn J.S. Belden W.J. Miller S.I. Microb. Pathog. 1998; 25: 77-90Crossref PubMed Scopus (63) Google ScholarCS326CSO22,pagN::TnphoARef. 51Gunn J.S. Belden W.J. Miller S.I. Microb. Pathog. 1998; 25: 77-90Crossref PubMed Scopus (63) Google ScholarCS329CSO22,pagO::TnphoARef. 51Gunn J.S. Belden W.J. Miller S.I. Microb. Pathog. 1998; 25: 77-90Crossref PubMed Scopus (63) Google ScholarCS330CSO22,pagP::TnphoARef. 51Gunn J.S. Belden W.J. Miller S.I. Microb. Pathog. 1998; 25: 77-90Crossref PubMed Scopus (63) Google ScholarCS019phoN2 zxx::6251 Tn10d-camRef. 69Miller S.I. Kukral A.M. Mekalanos J.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5054-5058Crossref PubMed Scopus (694) Google ScholarJSG435pmrA505 zjd::Tn10d-cam (PmrA-constitutive)Ref. 7Gunn J.S. Miller S.I. J. Bacteriol. 1996; 178: 6857-6864Crossref PubMed Scopus (338) Google ScholarJSG421pmrA::Tn10dRef. 7Gunn J.S. Miller S.I. J. Bacteriol. 1996; 178: 6857-6864Crossref PubMed Scopus (338) Google ScholarCS401StrepR CS019S. I. MillerCS491StrepR CS022S. I. MillerCS584CS491 ΔpagLThis workCS586CS401 ΔpagLThis workE. coli strainsW3110Wild type, F−, λ−E. coli Genetic Stock Center (Yale)MC1061araD139Δ(ara-leu)7697hsdRhdsM+E. coli Genetic Stock Center (Yale)XL-1 Blue-MRΔmcrABC, recA1,lacStratageneBLR(DE3)pLysSΔ(srl-recA)306::Tn10(DE3), Tetr/Cmr)NovagenPlasmidspET21aVector containing a T7 promoter, AmprNovagenpBB04ELPir-dependent suicide vector containing pagL::TnphoA 5′ fusion junctionRef. 51Gunn J.S. Belden W.J. Miller S.I. Microb. Pathog. 1998; 25: 77-90Crossref PubMed Scopus (63) Google ScholarpWKS30Low copy vectorRef. 70Wang R.F. Kushner S.R. Gene ( Amst. ). 1991; 100: 195-199Crossref PubMed Scopus (1011) Google ScholarpBluescript KS II+lacZ, AmprStratagenepPagLpET21a containing pagLThis workpWLP23pWKS30 containing pagLThis workpWLP21pBluescript KS II+ containing pagLThis workpWLP24ΔpagL exchange vectorThis workPKAS32Allelic exchange vectorRef. 45Skorupski K. Taylor R.K. Gene ( Amst. ). 1996; 169: 47-52Crossref PubMed Scopus (373) Google Scholar Open table in a new tab The substrate [4′-32P]lipid IVA was prepared using 100 μCi of [γ32-P]ATP, tetraacyl-disaccharide 1-phosphate acceptor and membranes from E. coli that overexpress the 4′-kinase, as previously described (36Basu S.S. York J.D. Raetz C.R. J. Biol. Chem. 1999; 274: 11139-11149Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), with the following minor changes. After the 4′-kinase reaction was completed, the assay mixture was spotted onto a 10 × 20-cm TLC plate. The plate was dried under a cold air stream and developed in the solvent system chloroform/pyridine/88% formic acid/water (50:50:16:5, v/v/v/v). Following chromatography, the plate was dried again and exposed to x-ray film for 30 s to locate the [4′-32P]lipid IVA. The region of the silica plate containing the product was removed by scraping, transferred to a thick walled glass tube, and resuspended in 3 ml of an acidic single-phase Bligh/Dyer mixture (37Bligh E.G. Dyer J.J. Can. J. Biochem. Physiol. 1959; 37: 911-918Crossref PubMed Scopus (42878) Google Scholar), consisting of chloroform/methanol/0.1 m HCl (1:2:0.8, v/v/v). The suspension was vigorously mixed with the aid of a vortex and subjected to sonic irradiation for 30 s. The silica particles were removed with a clinical centrifuge set at top speed for 10 min. The supernatant containing the 32P labeled lipid was removed, and the extraction process was repeated. The extracted materials were pooled. The solution was then converted to a two-phase Bligh/Dyer mixture (37Bligh E.G. Dyer J.J. Can. J. Biochem. Physiol. 1959; 37: 911-918Crossref PubMed Scopus (42878) Google Scholar), consisting of chloroform/methanol/0.1m HCl (2:2:1.8, v/v/v). The phases were separated in a clinical centrifuge, and the lower phase was removed to a separate tube. The resulting upper phase was extracted a second time by the addition of fresh preequilibrated lower phase. The lower phases were pooled and dried under a stream of N2. Finally, the dried lipid was resuspended in 50 mm Hepes, pH 7.5, and stored at −20 °C. To prepare Kdo2- [4′-32P]lipid IVA, the purified E. coli Kdo transferase was added to the system immediately after the 4′-kinase, as previously described (36Basu S.S. York J.D. Raetz C.R. J. Biol. Chem. 1999; 274: 11139-11149Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The Kdo2-[4′-32P]lipid IVA was isolated as described above with the exception that 50 mm ammonium acetate adjusted to pH 1.5 was used as the aqueous component instead of 0.1 m HCl in all Bligh/Dyer systems. The final yields of the desired radioactive lipid products ranged between 40 and 60 μCi from 100 μCi of [γ32-P]ATP used as the starting material. Both lipid products were stored as aqueous dispersions at −80 °C and subjected to sonic irradiation for 1 min in a bath sonicator prior to use (36Basu S.S. York J.D. Raetz C.R. J. Biol. Chem. 1999; 274: 11139-11149Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Typically, 100-ml cultures of bacteria were grown to anA600 of 1.0 at 37 °C and harvested by centrifugation at 7,000 × g for 15 min. All steps were carried out at 4 °C. Cell pellets were resuspended in 50 mm Hepes, pH 7.5, at a protein concentration of ∼3–8 mg/ml and broken by passage through a French pressure cell at 18,000 p.s.i. The crude lysate was cleared by centrifugation at 7,000 ×g for 15 min. Membranes were prepared by two centrifugation steps at 149,000 × g for 60 min with a wash of the crude membranes in 5 ml of 50 mm Hepes, pH 7.5, after the first centrifugation to ensure the removal of all cytosolic components. The final membrane pellet was resuspended in 50 mm Hepes, pH 7.5, at a protein concentration of ∼5–10 mg/ml. Cytosol from the first 149,000 × g centrifugation was subjected to a second centrifugation step for complete removal of small membrane fragments. All samples were stored in aliquots at −80 °C, and protein concentrations were determined with bicinchoninic acid (38Smith 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 (18647) Google Scholar), with bovine serum albumin as the standard. The 3-O-deacylase activity was assayed under optimized conditions in a 10-μl reaction mixture containing 50 mm Hepes, pH 8.0, 0.1% Triton X-100, 0.5m NaCl, and 10 μm [4′-32P]lipid IVA (50,000 cpm/nmol). Reaction tubes were incubated at 30 °C for the indicated times. The assays were stopped by spotting 5-μl portions of the reaction mixtures onto a silica gel 60 TLC plate. For further characterization of the 3-O-deacylase activity, the PhoPC pagP - Salmonella mutant strain (TableI) was used as the enzyme source to avoid other further acylation of the [4′-32P]lipid IVA substrate by PagP (31Bishop R.E. Gibbons H.S. Guina T. Trent M.S. Miller S.I. Raetz C.R.H. EMBO J. 2000; 19: 5071-5080Crossref PubMed Scopus (280) Google Scholar). When [4′-32P]lipid IVA was employed as the substrate, the reaction products were separated using the solvent system chloroform/pyridine/88% formic acid/water (50:50:16:5, v/v/v/v). For reactions containing Kdo2-[4′-32P]lipid IVA as the substrate, plates were developed in chloroform/pyridine/88% formic acid/water (30:70:16:10, v/v/v/v). Finally, reaction products from assays containing 32P-labeled lipid X (39Radika K. Raetz C.R.H. J. Biol. Chem. 1988; 263: 14859-14867Abstract Full Text PDF PubMed Google Scholar, 40Takayama K. Qureshi N. Mascagni P. Nashed M.A. Anderson L. Raetz C.R.H. J. Biol. Chem. 1983; 258: 7379-7385Abstract Full Text PDF PubMed Google Scholar) as the substrate were separated using the solvent chloroform/methanol/water/acetic acid (25:15:4:2, v/v/v/v). Reaction products were analyzed using a Molecular Dynamics PhosphorImager equipped with ImageQuant software. The enzyme activity was calculated by determining the percentage of the substrate converted to product, and the specific activity was expressed as nmol/min/mg. The 3-O-deacylated lipid IVA reaction product was generated in a 50-μl reaction mixture for 2 h, as described above, using membranes from the PhoPC pagP - S. typhimurium mutant strain. Mild base hydrolysis was carried out by the addition of triethylamine to a final concentration of 30%, and the reaction was incubated at 37 °C (41Basu 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). At the indicated times, 3-μl portions of the reaction mixture were removed and mixed with 3 μl of water, after which 5 μl of the resulting mixture was spotted onto a silica gel 60 TLC plate and developed in chloroform/pyidine/88% formic acid/water (50:50:16:10, v/v/v/v). As a control, the [4′-32P]lipid IVAsubstrate was also subjected to triethylamine hydrolysis under the same conditions (41Basu 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). Membranes from various strains of E. coli, S. typhimurium, orP. aeruginosa were separated by isopycnic sucrose gradient centrifugation. First, washed membranes were prepared as described above and were resuspended in 10 mm Hepes, pH 7.0, containing 0.05 mm EDTA at a protein concentration of 5 mg/ml. Membranes were applied to a seven-step gradient, prepared as described by Guy-Caffey et al. (42Guy-Caffey J.K. Rapoza M.P. Jolley K.A. Webster R.E. J. Bacteriol. 1992; 174: 2460-2465Crossref PubMed Google Scholar, 43Osborn M.J. Gander J.E. Parisi E. Carson J. J. Biol. Chem. 1972; 247: 3962-3972Abstract Full Text PDF PubMed Google Scholar), and subjected to ultracentrifugation in a Beckman SW40.1 rotor for 19 h at 3 °C. The gradient was collected in ∼0.5-ml fractions. Each fraction was then assayed for NADH oxidase as the inner membrane marker and for phospholipase A as the outer membrane marker, as previously described (44Zhou Z. White K.A. Polissi A. Georgopoulos C. Raetz C.R.H. J. Biol. Chem. 1998; 273: 12466-12467Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). The amount of protein in each fraction was determined using the bicinchoninic acid assay (38Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.

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