An Inner Membrane Enzyme in Salmonellaand Escherichia coli That Transfers 4-Amino-4-deoxy-l-arabinose to Lipid A

Attachment of the cationic sugar 4-amino-4-deoxy-l-arabinose (l-Ara4N) to lipid A is required for the maintenance of polymyxin resistance inEscherichia coli and Salmonella typhimurium. The enzymes that synthesize l-Ara4N and transfer it to lipid A have not been identified. We now report an inner membrane enzyme, expressed in polymyxin-resistant mutants, that adds one or twol-Ara4N moieties to lipid A or its immediate precursors. No soluble factors are required. A gene located near minute 51 on theS. typhimurium and E. coli chromosomes (previously termed orf5, pmrK, oryfbI) encodes the l-Ara4N transferase. The enzyme, renamed ArnT, consists of 548 amino acid residues in S. typhimurium with 12 possible membrane-spanning regions. ArnT displays distant similarity to yeast protein mannosyltransferases. ArnT adds two l-Ara4N units to lipid A precursors containing a Kdo disaccharide. However, as shown by mass spectrometry and NMR spectroscopy, it transfers only a single l-Ara4N residue to the 1-phosphate moiety of lipid IVA, a precursor lacking Kdo. Proteins with full-length sequence similarity to ArnT are present in genomes of other bacteria thought to synthesizel-Ara4N-modified lipid A, including Pseudomonas aeruginosa and Yersinia pestis. As shown in the following article (Trent, M. S., Ribeiro, A. A., Doerrler, W. T., Lin, S., Cotter, R. J., and Raetz, C. R. H. (2001) J. Biol. Chem. 276, 43132–43144), ArnT utilizes the novel lipid undecaprenyl phosphate-α-l-Ara4N as its sugar donor, suggesting that l-Ara4N transfer to lipid A occurs on the periplasmic side of the inner membrane. Attachment of the cationic sugar 4-amino-4-deoxy-l-arabinose (l-Ara4N) to lipid A is required for the maintenance of polymyxin resistance inEscherichia coli and Salmonella typhimurium. The enzymes that synthesize l-Ara4N and transfer it to lipid A have not been identified. We now report an inner membrane enzyme, expressed in polymyxin-resistant mutants, that adds one or twol-Ara4N moieties to lipid A or its immediate precursors. No soluble factors are required. A gene located near minute 51 on theS. typhimurium and E. coli chromosomes (previously termed orf5, pmrK, oryfbI) encodes the l-Ara4N transferase. The enzyme, renamed ArnT, consists of 548 amino acid residues in S. typhimurium with 12 possible membrane-spanning regions. ArnT displays distant similarity to yeast protein mannosyltransferases. ArnT adds two l-Ara4N units to lipid A precursors containing a Kdo disaccharide. However, as shown by mass spectrometry and NMR spectroscopy, it transfers only a single l-Ara4N residue to the 1-phosphate moiety of lipid IVA, a precursor lacking Kdo. Proteins with full-length sequence similarity to ArnT are present in genomes of other bacteria thought to synthesizel-Ara4N-modified lipid A, including Pseudomonas aeruginosa and Yersinia pestis. As shown in the following article (Trent, M. S., Ribeiro, A. A., Doerrler, W. T., Lin, S., Cotter, R. J., and Raetz, C. R. H. (2001) J. Biol. Chem. 276, 43132–43144), ArnT utilizes the novel lipid undecaprenyl phosphate-α-l-Ara4N as its sugar donor, suggesting that l-Ara4N transfer to lipid A occurs on the periplasmic side of the inner membrane. 4-amino-4-deoxy-l-arabinose 2-[N-morpholino]ethanesulfonic acid 3-deoxy-d-manno-octulosonic acid polymerase chain reaction phosphoethanolamine matrix assisted laser desorption-ionization/time of flight mass spectrometry Although the overall structure of the lipid A component of lipopolysaccharide is relatively conserved in diverse Gram-negative bacteria, sub-stoichiometric covalent modifications of lipid A have been identified in many organisms (Fig.1) (1Raetz C.R.H. Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Scopus (1084) Google Scholar, 2Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. 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Cotter R.J. Miller S.I. Raetz C.R.H. J. Biol. Chem. 2001; 276: 43111-43121Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), and palmitate (5Guo 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 (527) Google Scholar, 9Strain S.M. Armitage I.M. Anderson L. Takayama K. Quershi N. Raetz C.R.H. J. Biol. Chem. 1985; 260: 16089-16098Abstract Full Text PDF PubMed Google Scholar,12Bishop 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 (290) Google Scholar) (Fig. 1). S. typhimurium lipid A may, in addition, contain S-2-hydroxymyristate in place of myristate as the secondary acyl chain at position 3′ (Fig. 1) (13Bryn K. Rietschel E.T. Eur. J. Biochem. 1978; 86: 311-315Crossref PubMed Scopus (32) Google Scholar, 14Gibbons H.S. Lin S. Cotter R.J. Raetz C.R.H. J. Biol. 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An important component of the host response to infection is the production of amphipathic, cationic peptides that possess anti-microbial activity (19Ohl M.E. Miller S.I. Annu. Rev. Med. 2001; 52: 259-274Crossref PubMed Scopus (327) Google Scholar, 20Groisman E.A. J. Bacteriol. 2001; 183: 1835-1842Crossref PubMed Scopus (673) Google Scholar). Bacteria may acquire resistance to such peptides by addingl-Ara4N units to the phosphate groups of their lipid A, reducing its net negative charge, and lowering its affinity for these peptides (21Vaara M. Microbiol. Rev. 1992; 56: 395-411Crossref PubMed Google Scholar, 22Helander I.M. Kilpeläinen I. Vaara M. Mol. Microbiol. 1994; 11: 481-487Crossref PubMed Scopus (149) Google Scholar, 23Nummila K. Kilpeläinen I. Zähringer U. Vaara M. Helander I.M. Mol. Microbiol. 1995; 16: 271-278Crossref PubMed Scopus (175) Google Scholar, 24Gunn J.S. Lim K.B. Krueger J. Kim K. Guo L. Hackett M. Miller S.I. Mol. Microbiol. 1998; 27: 1171-1182Crossref PubMed Scopus (518) Google Scholar). Polymyxin is a cationic lipopeptide antibiotic produced by Gram-positive bacteria (21Vaara M. Microbiol. Rev. 1992; 56: 395-411Crossref PubMed Google Scholar). It binds to lipid A and kills Gram-negative bacteria in a manner that shares some common features with the cationic antibacterial peptides of the innate immune system (21Vaara M. Microbiol. Rev. 1992; 56: 395-411Crossref PubMed Google Scholar). Substitution of lipid A with l-Ara4N units is greatly elevated in polymyxin-resistant mutants of S. typhimurium(22Helander I.M. Kilpeläinen I. Vaara M. Mol. Microbiol. 1994; 11: 481-487Crossref PubMed Scopus (149) Google Scholar, 25Vaara M. Vaara T. Jensen M. Helander I. Nurminen M. Rietschel E.T. Makela P.H. FEBS Lett. 1981; 129: 145-149Crossref PubMed Scopus (137) Google Scholar) and E. coli K-12 (23Nummila K. Kilpeläinen I. Zähringer U. Vaara M. Helander I.M. Mol. Microbiol. 1995; 16: 271-278Crossref PubMed Scopus (175) Google Scholar). The polymyxin resistance phenotype is usually due to mutation(s) in the pmrA locus (26Roland K.L. Martin L.E. Esther C.R. Spitznagel J.K. J. Bacteriol. 1993; 175: 4154-4164Crossref PubMed Scopus (148) Google Scholar), which encodes a transcription factor that is activated during growth under mildly acidic conditions (pH < 6), in a PhoP/PhoQ-dependent manner during Mg2+limitation (10 μm), or by exposure to ferric ions (20Groisman E.A. J. Bacteriol. 2001; 183: 1835-1842Crossref PubMed Scopus (673) Google Scholar,27Gunn J.S. Miller S.I. J. Bacteriol. 1996; 178: 6857-6864Crossref PubMed Scopus (344) Google Scholar, 28Wosten M.M. Kox L.F. Chamnongpol S. Soncini F.C. Groisman E.A. Cell. 2000; 103: 113-125Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). The first two of these conditions exist within the phagolysosomes of macrophages, which engulf S. typhimuriumduring the course of an infection (19Ohl M.E. Miller S.I. Annu. Rev. Med. 2001; 52: 259-274Crossref PubMed Scopus (327) Google Scholar, 27Gunn J.S. Miller S.I. J. Bacteriol. 1996; 178: 6857-6864Crossref PubMed Scopus (344) Google Scholar, 29Soncini F.C. Garcia Vescovi E. Solomon F. Groisman E.A. J. Bacteriol. 1996; 178: 5092-5099Crossref PubMed Scopus (271) Google Scholar). PmrA activation by environmental stimuli or by appropriate point mutations within pmrA induces the expression of genes needed for polymyxin resistance and covalent modification of lipid A withl-Ara4N (24Gunn J.S. Lim K.B. Krueger J. Kim K. Guo L. Hackett M. Miller S.I. Mol. Microbiol. 1998; 27: 1171-1182Crossref PubMed Scopus (518) Google Scholar, 27Gunn J.S. Miller S.I. J. Bacteriol. 1996; 178: 6857-6864Crossref PubMed Scopus (344) Google Scholar, 30Guo L. Lim K.B. Gunn J.S. Bainbridge B. Darveau R.P. Hackett M. Miller S.I. Science. 1997; 276: 250-253Crossref PubMed Scopus (486) Google Scholar, 31Ernst R.K. Guina T. Miller S.I. J. Infect. Dis. 1999; 179 Suppl. 2: S326-S330Crossref PubMed Scopus (138) Google Scholar). A cluster of genes mapping near minute 51 in S. typhimurium and E. coli (24Gunn J.S. Lim K.B. Krueger J. Kim K. Guo L. Hackett M. Miller S.I. Mol. Microbiol. 1998; 27: 1171-1182Crossref PubMed Scopus (518) Google Scholar) have been proposed to encode a set of enzymes required for the biosynthesis of the l-Ara4N moiety and its attachment to lipid A (Fig. 2) (10Zhou Z. Lin S. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 1999; 274: 18503-18514Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). However, in vitro assays have not yet been developed to validate the functions of these putative enzymes. We now report a novel enzyme, present in inner membranes of polymyxin-resistant mutants of S. typhimurium, that can transfer one or two l-Ara4N moieties to lipid A and certain lipid A precursors. Transferase activity is dependent upon activation of the pmrA gene, either directly by mutation or secondarily by growth under appropriate conditions. The enzyme adds a singlel-Ara4N unit to the 1-phosphate moiety of the tetra-acylated lipid A precursor, lipid IVA, which lacks Kdo (Fig. 3). However, lipid A molecules containing a Kdo disaccharide are modified with two l-Ara4N units, indicating that the addition of l-Ara4N to the 4′-position is Kdo-dependent. Transferase activity is greatly elevated when the orf5(pmrK) gene ofS. typhimurium (6Gunn J.S. Ryan S.S. Van Velkinburgh J.C. Ernst R.K. Miller S.I. Infect. Immun. 2000; 68: 6139-6146Crossref PubMed Scopus (323) Google Scholar, 24Gunn J.S. Lim K.B. Krueger J. Kim K. Guo L. Hackett M. Miller S.I. Mol. Microbiol. 1998; 27: 1171-1182Crossref PubMed Scopus (518) Google Scholar), now renamed arnT (Fig.2), is expressed behind a T7lac promoter in E. coli BLR(DE3), which is itself shown here to be a polymyxin-resistant strain containing l-Ara4N modified lipid A. The l-Ara4N transferase is not dependent upon added soluble factors. As shown in the following article (32Trent M.S. Ribeiro A.A. Doerrler W.T. Lin S. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 2001; 276: 43132-43144Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), ArnT utilizes the novel lipid, undecaprenyl phosphate-α-l-Ara4N, as its donor substrate (Fig. 2). 32Pi and [γ-32P]ATP were 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. CDCl3, CD3OD, and D2O were purchased from Aldrich. All other chemicals were reagent grade and were purchased from either Sigma or Mallinckrodt. Bacterial strains are described in TableI. Typically, the bacteria were grown at 37 °C in LB broth, which contains 10 g of NaCl, 10 g of tryptone, and 5 g of yeast extract per liter (33Miller J.R. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar). For experiments requiring Mg2+ limitation or exposure to low pH, cells were grown as described previously on a defined medium (34Trent M.S. Pabich W. Raetz C.R.H. Miller S.I. J. Biol. Chem. 2001; 276: 9083-9092Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). When required for selection of plasmids, cells were grown in the presence of 100 μg/ml ampicillin, 12 μg/ml tetracycline, 30 μg/ml chloramphenicol, or 30 μg/ml kanamycin.Table IRelevant bacterial strains and plasmidsStrain or plasmidDescriptionSource or Ref.S. typhimurium ATCC 14028sWild typeATCCCS022pho-24(PhoP-constitutive)62Miller S.I. Mekalanos J.J. J. Bacteriol. 1990; 172: 2485-2490Crossref PubMed Scopus (319) Google ScholarCS015CSO22,phoP102∷Tn10d-cam47Miller S.I. Kukral A.M. Mekalanos J.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5054-5058Crossref PubMed Scopus (716) Google ScholarCS330CSO22,pagP∷TnphoA49Gunn J.S. Belden W.J. Miller S.I. Microb. Pathog. 1998; 25: 77-90Crossref PubMed Scopus (64) Google ScholarJSG435pmrA505zjd∷Tn10d-cam (PmrA-constitutive)27Gunn J.S. Miller S.I. J. Bacteriol. 1996; 178: 6857-6864Crossref PubMed Scopus (344) Google ScholarJSG421pmrA∷Tn10d(PmrA−)27Gunn J.S. Miller S.I. J. Bacteriol. 1996; 178: 6857-6864Crossref PubMed Scopus (344) Google ScholarE. coliW3110Wild type, F−, λ−E. coli Genetic Stock Center, Yale University XL-1 Blue-MRΔmcrABC, recA1, lacStratageneBLR(DE3)Δ(srl-recA)306∷Tn10(DE3), TetrNovagen NovaBlue(DE3)Δ(srl-recA)306∷Tn10(DE3), TetrNovagenPlasmids pET21a(+)Vector containing a T7lac promoter, AmprNovagen pArnTStpET21a containing S. typhimurium arnTThis work pLysSpACYC184 containing T7 lysozyme, CamrNovagen Open table in a new tab Plasmids were prepared using the Qiagen Spin Prep kit. DNA fragments were isolated from agarose gels using the Qiaex II gel extraction kit. T4 DNA ligase (Life Technologies, Inc.), restriction endonucleases (New England Biolabs), and shrimp alkaline phosphatase (U. S. Biochemical Corp.) were used according to the manufacturer's instructions. ThearnT(orf5) gene of S. typhimurium(Fig. 2) was cloned into pET21 (Novagen) behind the T7lacpromoter. The gene was amplified by PCR using S. typhimurium14028 genomic DNA as the template. The forward primer contained a clamp region, an NdeI site (underlined), and thearnT coding region with its start codon. The reverse primer contained a clamp region, a BamHI site (underlined), and the coding region with its stop codon. Sequences of the S. typhimurium forward and reverse primer were 5′-GCGCGCCATATGATGATGAAATCGATA-3′, and 5′-GCGCGCGGATCCTCATTTAGGCCGATA-3′, respectively. The PCR contained 140 ng of genomic DNA template, 0.15 μg of each primer, 200 μm each of dNTP, 100 mm Tris-HCl, pH 8.8, 35 mm MgCl2, 250 mm KCl, and 2.5 units of Pfu DNA polymerase (Stratagene) in a reaction volume of 0.05 ml. The reaction mixture was subjected to a 2-min denaturation at 95 °C followed by 25 cycles of 95 °C for 45 s, 60 °C for 45 s, 72 °C for 120 s and a final extension for 10 min at 72 °C, using the PerkinElmer Life Sciences GeneAmp PCR system 2400. The PCR product and the vector were digested with NdeI andBamHI, ligated together, and transformed into Xl-1 Blue cells (Stratagene) for propagation of the plasmid, designated pArnTSt. The plasmid was then transformed into BLR(DE3) or NovaBlue(DE3) (Table I) with or without the pLysS plasmid for overexpression of the protein. First, a single colony of E. coli containing pArnTSt was inoculated into 20 ml of LB broth and grown in a rotary shaker at 37 °C to A600 = 0.8. The culture was then used to inoculate 1 liter of fresh LB medium, and whenA600 reached ∼0.6, the culture was induced with 1 mmisopropyl-1-thio-β-d-galactopyranoside for 4 h. Crude extracts, membrane-free cytosol, and washed membranes were prepared as described below. Typically, 100-ml cultures of bacteria were grown to A600 = 1.0 at 37 °C and harvested by centrifugation at 7,000 ×g for 15 min. All steps were carried out at 0–4 °C. Cells were resuspended in 50 mm Hepes, pH 7.5, at a protein concentration of 5–10 mg/ml, and broken by one passage through a French pressure cell at 18,000 pounds/square inch. The crude lysate was centrifuged at 7,000 × g for 15 min to remove unbroken cells. Membranes were prepared by two successive centrifugations at 149,000 × g for 60 min, with a washing step inbetween to remove residual soluble 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 step was subjected to a second centrifugation to remove any remaining contaminating membranes. All membrane and cytosol preparations were stored in aliquots at −80 °C, and protein concentrations were determined with bicinchoninic acid (35Smith 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 (19429) Google Scholar), using bovine serum albumin as the standard. Cells were labeled uniformly with 5 μCi/ml 32Pi in LB broth, starting at an initial A600 of ∼0.05. Cells were then grown at 37 °C for several hours, as indicated, and harvested when A600 reached ∼1.0. The32P-labeled cells were collected using a clinical centrifuge and washed with 5 ml of phosphate-buffered saline, pH 7.4 (36Dulbecco R. Vogt M. J. Exp. Med. 1954; 99: 167-182Crossref PubMed Scopus (2046) Google Scholar). The final cell pellet was resuspended in 3 ml of a single-phase Bligh/Dyer mixture (37Bligh E.G. Dyer J.J. Can. J. Biochem. Physiol. 1959; 37: 911-918Crossref PubMed Scopus (44552) Google Scholar), consisting of chloroform/methanol/water (1:2:0.8, v/v). After 60 min, the insoluble material, which still contains the 32P-labeled lipid A covalently linked to the lipopolysaccharide core via its Kdo residues, was released by hydrolysis at 100 °C in the presence of 1% SDS at pH 4.5, as described previously (10Zhou Z. Lin S. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 1999; 274: 18503-18514Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 38Zhou Z. White K.A. Polissi A. Georgopoulos C. Raetz C.R.H. J. Biol. Chem. 1998; 273: 12466-12467.5Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). The 32P-labeled lipid A species were recovered by two-phase Bligh/Dyer extraction (10Zhou Z. Lin S. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 1999; 274: 18503-18514Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 38Zhou Z. White K.A. Polissi A. Georgopoulos C. Raetz C.R.H. J. Biol. Chem. 1998; 273: 12466-12467.5Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar) and spotted onto a Silica Gel 60 TLC plate (∼10,000 cpm/lane). The plate was developed in the solvent chloroform, pyridine, 88% formic acid, water (50:50:16:5, v/v). The plate was dried and exposed to a PhosphorImager Screen overnight to visualize the resolved32P-lipid A species. The intermediate [4′-32P]-lipid IVA was prepared using 100 μCi of [γ-32P]ATP, tetraacyldisaccharide 1-phosphate acceptor, and membranes from an E. coli strain that overexpresses the lipid 4′-kinase (39Garrett 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), as described previously (34Trent M.S. Pabich W. Raetz C.R.H. Miller S.I. J. Biol. Chem. 2001; 276: 9083-9092Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar,40Basu 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). To prepare Kdo2[4′-32P]lipid IVA, purified E. coli Kdo transferase was used in tandem with the 4′-kinase (40Basu 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, 41Belunis C.J. Raetz C.R.H. J. Biol. Chem. 1992; 267: 9988-9997Abstract Full Text PDF PubMed Google Scholar). Kdo2[32P]lipid A was prepared by labeling the heptose-deficient mutant WBB06 (42Brabetz W. Muller-Loennies S. Holst O. Brade H. Eur. J. Biochem. 1997; 247: 716-724Crossref PubMed Scopus (108) Google Scholar) with 32Piand purifying its Kdo2[32P]lipid A as reported previously (12Bishop 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 (290) Google Scholar). The l-Ara4N transferase was assayed under optimized conditions in a 10-μl reaction containing 50 mmMES, pH 6.5, 0.2% Triton X-100 and either 10 μm[4′-32P]lipid IVA or Kdo2[4′-32P]lipid IVA (each at 20,000 cpm/nmol) as the acceptor substrate. Washed membranes (0.5–1.0 mg/ml) were employed as the source of enzyme and l-Ara4N donor, as indicated. Reaction mixtures were incubated at 30 °C for various times. Enzymatic reactions were stopped by spotting 5-μl portions of the mixtures onto Silica Gel 60 thin layer chromatography plates. In most of the initial experiments, the PmrAC S. typhimurium strain JSG435 (Table I) was used for the characterization of the assay conditions, because of its apparently high levels of endogenous l-Ara4N transferase activity and the presence of the donor substrate. When [4′-32P]lipid IVA was employed as the acceptor, the substrate and reaction products were separated using the solvent chloroform, pyridine, 88% formic acid, water (50:50:16:5, v/v). For assays containing Kdo2[4′-32P]lipid IVA as the acceptor substrate, plates were developed in chloroform, pyridine, 88% formic acid, water (30:70:16:10, v/v). Following chromatography, the plates were dried and 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. The apparent specific activities were expressed in units of nmol/min/mg, recognizing that the membranes supply not only the enzyme but also the l-Ara4N donor substrate. Membranes isolated from the PmrAC S. typhimurium strain JSG435 (Table I) were separated by isopycnic sucrose gradient centrifugation as described previously (34Trent M.S. Pabich W. Raetz C.R.H. Miller S.I. J. Biol. Chem. 2001; 276: 9083-9092Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). First, washed membranes were prepared as described above and then were resuspended at a concentration of ∼5 mg/ml in 10 mm Hepes, pH 7.0, containing 0.05 mm EDTA. Membranes (2.2 ml) were applied to a 9.6-ml, seven-step sucrose gradient (43Guy-Caffey J.K. Rapoza M.P. Jolley K.A. Webster R.E. J. Bacteriol. 1992; 174: 2460-2465Crossref PubMed Google Scholar), and subjected to ultracentrifugation at 35,000 rpm in a Beckman SW40.1 rotor for 19 h at 3 °C. The gradient was collected in ∼0.5 ml fractions by piercing the bottom of the tube. Each fraction was assayed for NADH oxidase as the inner membrane marker and for phospholipase A as the outer membrane marker (38Zhou Z. White K.A. Polissi A. Georgopoulos C. Raetz C.R.H. J. Biol. Chem. 1998; 273: 12466-12467.5Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). The amount of protein in each fraction was determined using the bicinchoninic acid assay (35Smith 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 (19429) Google Scholar). Finally, each fraction was assayed forl-Ara4N transferase activity, using the optimized assay conditions described above. The lipid IVA reaction products, generated in vitro with membranes of strain JSG435, were purified by preparative thin layer chromatography. A 68-ml l-Ara4N transferase reaction mixture (see above), containing 100 μm lipid IVA and 1 mg/ml JSG435 membranes, was incubated overnight at 30 °C. The reaction mixture was then converted into a two-phase acidic Bligh/Dyer system, consisting of chloroform, methanol, 0.1m HCl (2:2:1.8, v/v), by the addition of 76 ml of chloroform, 76 ml of methanol, and 0.6 ml of 12 m HCl. After mixing, the phases were separated by centrifugation at 5,000 × g for 15 min. The lower phase was removed, and the upper phase was extracted a second time by the addition of 76 ml of a fresh lower phase derived from a two-phase Bligh/Dyer mixture of chloroform, methanol, and 0.1 m HCl. The lower phases, containing the residual substrate and the modified lipid IVA species, were pooled. Next, 4 ml of pyridine was added to neutralize remaining HCl carried over during the extraction process. The sample was dried by rotary evaporation, dissolved in 8 ml of chloroform/methanol (4:1, v/v), and spotted as lines onto the origins of 14 individual 20 × 20-cm Silica Gel 60 TLC plates (0.25-mm thickness) (∼0.57 ml per plate). The lipids were separated using the solvent system chloroform, pyridine, 88% formic acid, water (50:50:16:5, v/v). While the plates were drying at room temperature, the bands of residual lipid IVA and its modified products could be seen transiently as white zones. The latter was marked with a pencil, and the plates were dried for an additional 30 min with a cold air stream. Regions containing the putative l-Ara4N-modified lipid IVA derivatives (i.e. presumed to be lipids IIA and IIB based on their migration) (Fig. 4) were removed separately with clean razor blades and then stored in separate thick-walled glass tubes at −80 °C (10Zhou Z. Lin S. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 1999; 274: 18503-18514Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Silica chips from four separate thin layer plates containing bands with the same R f were processed together. The lipids were extracted from the chips with 6 ml of an acidic single-phase Bligh/Dyer mixture, consisting of chloroform, methanol, 0.1 m HCl (1:2:0.8, v/v). Following removal of the chips by low speed centrifugation, the supernatant was converted to a two-phase Bligh/Dyer system by adding 1.58 ml of both chloroform and water. The lower phase, which contained the desired lipid, was withdrawn, and the upper phase was re-extracted once more with an equivalent volume of fresh lower phase. The lower phases were pooled, and 6 drops of pyridine were added. The sample was dried under a stream of N2. To remove minor breakdown products and contaminating silica particles, the individual lipids recovered from the TLC plates were subjected to anion exchange chromatography on small DEAE-cellulose columns (10Zhou Z. Lin S. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 1999; 274: 18503-18514Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Each lipid sample was re-dissolved in 3 ml of chloroform/methanol/water (2:3:1, v/v) and subjected to sonic irradiation for 30 s in a bath apparatus before application to a 1-ml DEAE-cellulose column, suspended in the same solvent mixture, and equilibrated with acetate as the counter ion (10Zhou Z. Lin S. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 1999; 274: 18503-18514Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 44Raetz C.R.H. Kennedy E.P. J. Biol. Chem. 1973; 248: 1098-1105Abstract Full Text PDF PubMed Google Scholar). After application of the sample, the column was washed with 4 bed volumes of