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J. Biol. Chem., Vol. 280, Issue 22, 21202-21211, June 3, 2005

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A Phosphoethanolamine Transferase Specific for the Outer 3-Deoxy-D-manno-octulosonic Acid Residue of Escherichia coli Lipopolysaccharide

IDENTIFICATION OF THE eptB GENE AND Ca²⁺ HYPERSENSITIVITY OF AN eptB DELETION MUTANT^*

C. Michael Reynolds, Suzanne R. Kalb, Robert J. Cotter, and Christian R. H. Raetz¶

From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 and Middle Atlantic Mass Spectrometry Laboratory, Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Received for publication, January 26, 2005 , and in revised form, March 28, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Addition of a phosphoethanolamine (pEtN) moiety to the outer 3-deoxy-D-manno-octulosonic acid (Kdo) residue of lipopolysaccharide (LPS) in WBB06, a heptose-deficient Escherichia coli mutant, occurs when cells are grown in 5–50 mM CaCl₂ (Kanipes, M. I., Lin, S., Cotter, R. J., and Raetz, C. R. H. (2001) J. Biol. Chem. 276, 1156–1163). A Ca²⁺-induced, membrane-bound enzyme was responsible for the transfer of the pEtN unit to the Kdo domain. We now report the identification of the gene encoding the pEtN transferase. E. coli yhjW was cloned and overexpressed, because it is homologous to a putative pEtN transferase implicated in the modification of the -chain heptose residue of Neisseria meningitidis lipo-oligosaccharide (Mackinnon, F. G., Cox, A. D., Plested, J. S., Tang, C. M., Makepeace, K., Coull, P. A., Wright, J. C., Chalmers, R., Hood, D. W., Richards, J. C., and Moxon, E. R. (2002) Mol. Microbiol. 43, 931–943). In vitro assays with Kdo₂-4'-[³²P]lipid A as the acceptor showed that YhjW (renamed EptB) utilizes phosphatidylethanolamine in the presence of Ca²⁺ to transfer the pEtN group. Stoichiometric amounts of diacylglycerol were generated during the EptB-catalyzed transfer of pEtN to Kdo₂-lipid A. EptB is an inner membrane protein of 574 amino acid residues with five predicted trans-membrane segments within its N-terminal region. An in-frame replacement of eptB with a kanamycin resistance cassette rendered E. coli WBB06 (but not wild-type W3110) hypersensitive to CaCl₂ at 5 mM or higher. Ca²⁺ hypersensitivity was suppressed by excess Mg²⁺ in the medium or by restoring the LPS core of WBB06. The latter was achieved by reintroducing the waaC and waaF genes, which encode LPS heptosyl transferases I and II, respectively. Our data demonstrate that pEtN modification of the outer Kdo protected cells containing heptose-deficient LPS from damage by high concentrations of Ca²⁺. Based on its sequence similarity to EptA(PmrC), we propose that the active site of EptB faces the periplasmic surface of the inner membrane.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The envelope of Gram-negative bacteria consists of an inner membrane (1, 2), a peptidoglycan cell wall (3), and an outer membrane (4). The latter is an asymmetric bilayer with glycerophospholipids on its inner surface and lipopolysaccharide (LPS)¹ on the outside surface (5, 6). LPS consists of three covalently linked portions (6, 7) as follows: 1) the lipid A moiety, a glucosamine-based saccharolipid² that serves as the hydrophobic membrane anchor of LPS; 2) the core region, a non-repeating oligosaccharide modified with phosphate-containing substituents; and 3) the O-antigen, a distal repeating oligosaccharide, which is absent in most strains of Escherichia coli K12 (6). The lipid A moiety and the 3-deoxy-D-manno-octulosonic acid (Kdo) residues of the core are essential for growth of E. coli and most other Gram-negative bacteria (6). Strains lacking all LPS sugars distal to Kdo are termed heptose-deficient or "deep rough" (6, 8). These mutants are viable under laboratory conditions (9), but are hypersensitive to serum and antibiotics (4), and often show reduced virulence in animal models (7).

Under certain conditions, strains of E. coli and Salmonella synthesize LPS molecules modified with a phosphoethanolamine (pEtN) group at position 7 of the outer Kdo residue (9) (Fig. 1). Brabetz et al. (9) have reported that E. coli WBB06, which harbors a deletion spanning the heptosyl transferase genes waaC(rfaC) and waaF(rfaF), contains heptose-deficient LPS, modified with pEtN at position 7 of the outer Kdo sugar. Kanipes et al. (10) later demonstrated that pEtN addition to LPS in WBB06 is a consequence of the presence of Ca²⁺ in the growth medium used by Brabetz et al. (9) and is unrelated to the deletion of the heptosyl transferase genes. A pEtN transferase activity is present in membranes of WBB06 grown in the presence of 5–50 mM Ca²⁺ (10). The enzyme is stimulated by exogenous phosphatidylethanolamine (PE) and is selective for the outer Kdo residue of Kdo₂-lipid A and related substrates (10). Addition of EDTA to the in vitro pEtN transferase assay was found to be inhibitory.

We have now identified the gene that encodes the Ca²⁺-induced pEtN transferase. YhjW is one of six putative pEtN transferases present in E. coli that are homologous to a Neisseria meningitidis gene required for the modification of the lipo-oligosaccharide -chain heptose with pEtN (11). We now demonstrate unambiguously that recombinant YhjW, renamed EptB, utilizes PE as its donor substrate in vitro, generates diacylglycerol as a by-product, and depends upon the presence of Ca²⁺ in the assay system for activity. Expression of EptB can be re-engineered to be dependent upon the lac promoter, in which case induction of activity no longer requires the addition of Ca²⁺ to the growth medium. Deletion of eptB in the heptose-deficient mutant WBB06 renders this strain strikingly hyper-sensitive to Ca²⁺ at concentrations 5 mM, suggesting a function for pEtN modification of the Kdo region in the maintenance of envelope stability. Deletion of eptB in wild-type W3110 does not lead to significant Ca²⁺ hypersensitivity, indicating that certain outer core sugars, when present, may provide a similar stabilizing effect.

View larger version (20K): [in this window] [in a new window]   FIG. 1. Proposed reaction catalyzed by EptB. The position of the pEtN substituent on the outer Kdo unit was determined by Brabetz et al. (9) for the pEtN-Kdo2-lipid A isolated from WBB06 grown in the presence of Ca2+. The product generated in vitro is proposed to have the same structure. The dependence of in vitro product formation on PE as the donor substrate is presented. Diacylglycerol is the proposed by-product.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bacterial Strains—The bacterial strains used in this 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 (12). When required for selection of plasmids, cells were grown in the presence of 100 µg/ml ampicillin, 12 µg/ml tetracycline, 25 µg/ml chloramphenicol, or 30 µg/ml kanamycin.

Construction of eptB Expression Vectors—The eptB (yhjW) gene of E. coli was cloned into pET28b (Novagen) behind the T7lac promoter. The predicted coding region for eptB was amplified by PCR from E. coli W3110 genomic DNA. The forward primer contained a clamp region, an NcoI site (underlined), and the eptB-coding region with its start codon (boldface). The reverse primer contained a clamp region, a BamHI site (underlined), and the coding region with its stop codon (boldface). Sequences of the forward and reverse primers were 5'-GCGCGCCCATGGTCTTATCACCTGTTTGTCCA-3' and 5'-GCGCGCGGATCCTTAGTTAGCCGCTGCCTC-3', respectively. The PCR mixture contained 300 ng of genomic DNA as template, 0.2 µg of each primer, 200 µM each of dNTP, 100 mM Tris-HCl, pH 8.8, 35 mM MgCl₂, 250 mM KCl, and 5 units of Pfu DNA polymerase (Stratagene) in a reaction volume of 0.1 ml. The reaction mixture was subjected to a 1-min denaturation at 94 °C, followed by 25 cycles of 94 °C for 1 min, 55 °C for 1 min, 72 °C for 2 min, and a final extension for 10 min at 72 °C, using the PerkinElmer Life Sciences GeneAmp PCR System 2400. The PCR product and the pET28b vector were both digested with NcoI and BamHI, ligated together, and transformed into XL-1 Blue cells (Stratagene) for propagation of the desired plasmid, designated pEptB1. In some experiments, pEptB1 was used directly for EptB protein expression in strain C41(DE3) (Table I).

The eptB gene was also moved from pEptB1 into pWSK29 (15), a lac-inducible, low copy expression vector. The XbaI/BamHI-digested fragment, consisting of the eptB gene as well as the pET28b-derived ribosome binding site (RBS), was ligated to the corresponding restriction sites of pWSK29. This plasmid, designated pEptB2, was then transformed into competent E. coli W3110 or electroporated into the heptose-deficient E. coli strain WBB06 (9).

Construction of waaC and waaC/waaF Expression Vectors—The waaC gene of E. coli (16) was cloned into pWSK29 behind the lac promoter to generate plasmid pWaaC. The coding region for waaC was amplified by PCR from E. coli W3110 genomic DNA. The forward primer contained a clamp region, an XbaI site (underlined), and an RBS (italicized) prior to the waaC start codon (boldface). The reverse primer contained a clamp region, a BamHI site (underlined), and the coding region with its stop codon (boldface). Sequences of the forward and reverse primers were 5'-GCGCGCTCTAGAAAGGAGATATAATGCGGGTTTTGATCGTTAAA-3' and 5'-GCGCGCGGATCCTTATAATGATGATAACTTTTC-3', respectively. The PCR conditions were the same as those used to amplify eptB.

A second plasmid (pWaaCF), in which both waaC (16) and waaF (6, 17) are under lac control in pWSK29, was constructed by amplifying the adjacent waaC and waaF genes from W3110 genomic DNA using a forward primer that contained a clamp region, an XbaI site (underlined), and an RBS (italicized) immediately in front of the waaF start codon (boldface). The sequence of the forward primer used is as follows: 5'-GCGCGCTCTAGAAAGGAGATATAATGAAAATACTGGTGATCGGC-3'. The reverse primer was the same as that used to amplify waaC. Again, the PCR conditions were the same as those used to amplify eptB.

Overexpression of lpxL with the T7lac System—The lpxL (htrB) structural gene of E. coli (18) was cloned into pET21a⁺ (Novagen) to generate plasmid pLpxL, as described (19). Cultures of BLR(DE3)/pLysS/pLpxL were grown in LB broth at 37 °C to A₆₀₀ of 0.6, and then 1 mM isopropyl 1-thio--D-galactopyranoside (IPTG) was added. The cultures were then further incubated at 37 °C for 3 h. Cell membranes were isolated and washed as described previously (20).

Preparation of Membranes for Assay of pEtN Transferase Activity—A single colony of WBB06 harboring either the pWSK29 vector or pEptB2 was used to inoculate 5 ml of LB broth (containing 100 µg/ml ampicillin), which was then grown overnight at 37 °C. Next, the overnight cultures were used to inoculate 200 ml of LB cultures (initial A₆₀₀ = 0.02) containing 100 µg/ml ampicillin and 1 mM IPTG. When the A₆₀₀ reached 1.0, cells were harvested by centrifugation at 4,000 x g for 20 min (0–4 °C). Cells were resuspended in 4 ml of cold 50 mM Hepes, pH 7.5, and broken by a single passage through a French pressure cell at 10,000 pounds/square inch. Cellular debris was removed by centrifugation at 7000 x g for 20 min. Membranes were prepared by ultracentrifugation at 100,000 x g for 60 min. The membranes were resuspended in 8 ml of Hepes, pH 7.5, and the ultracentrifugation was repeated a second time at 100,000 x g to remove contaminating cytosol. The final membrane pellet was stored in 750 µl of 50 mM Hepes, pH 7.5, at –80 °C. Protein concentrations were determined by the bicinchoninic acid method. In some experiments, membranes were prepared in the same manner from cultures of WBB06 grown either without CaCl₂ or in the presence of 5 or 50 mM CaCl₂.

Solubilization of EptB-expressing Membranes—Membranes were prepared from IPTG-induced C41(DE3) cells, harboring either pET28b or pEptB1, as described above. Membranes at 5 mg/ml in 50 mM Hepes, pH 7.5, were solubilized with 2% Triton X-100 in a total volume of 750 µl and rotated gently at 4 °C for 2 h. The samples were subjected to ultracentrifugation at 55,000 rpm in a Beckman TLA 110 rotor for 1 h at 4 °C in an Optima TX ultracentrifuge. The supernatants were collected, and protein concentrations were determined.

Preparation of Lipid Substrates—The substrate Kdo₂-4'-[³²P]lipid A was synthesized in three separate steps. First, 100 µCi of [-³²P]ATP, 0.125 mg/ml tetraacyldisaccharide 1-phosphate (21, 22), 1% Nonidet P-40, 5 mM MgCl₂, and 1 mg/ml beef heart cardiolipin were added to a 1.5-ml microcentrifuge tube, and the volume was adjusted to 40 µl with water. Next, 5 µl of E. coli BLR (DE3)/pLysS/pJK2 (23) membranes, which overexpress the 4'-kinase LpxK (0.05 mg/ml), were added to the tube, and the mixture was incubated at room temperature for 10 min. A second 5-µl portion of the LpxK-overexpressing membranes was then added and incubated for 10 min at room temperature to generate 4'-[³²P]lipid IV_A in 70% yield based on input [-³²P]ATP.

While the synthesis of the 4'-[³²P]lipid IV_A was in progress, the components for the second step of the reaction were assembled in a separate 1.5-ml microcentrifuge tube. The reagents for Kdo addition to the 4'-[³²P]lipid IV_A acceptor consisted of 2 µM carrier lipid IV_A, 0.1% Triton X-100, 10 mM CTP, 4 mM Kdo, 12.5 mM MgCl₂, 0.03 units of purified E. coli CMP-Kdo synthase (24, 25), and 0.65 µg/ml purified E. coli Kdo transferase (26) in total volume of 50 µl. Upon completion of the 4'-[³²P]lipid IV_A step, the reaction components needed for the addition of the Kdo residues were added to the 4'-[³²P]lipid IV_A-containing tube, and the mixture was incubated at room temperature for 30 min.

While this was in progress, the components for the third step of the synthesis of Kdo₂-4'-[³²P]lipid A were prepared. These consisted of 50 mM Hepes, pH 7.5, 0.1% Triton X-100, 50 mM NaCl, 50 mM MgCl₂, 0.1 mg/ml bovine serum albumin, and 26 µM lauroyl-acyl carrier protein (27, 28), 0.05 mg/ml E. coli BLR(DE3)/pLysS/pLpxL membranes (19), and 0.05 mg/ml E. coli BLR(DE3)/pLysS/pLpxM (27, 28) membranes in a total volume of 50 µl. Upon completion of the Kdo₂-4'-[³²P]lipid IV_A synthesis step, the above components were added, and the acylation reactions catalyzed by LpxL and LpxM were allowed to proceed at room temperature for 30 min.

The total reaction mixture was then spotted onto a 10 x 20-cm TLC plate and dried under a cold air stream. The plate was developed in the solvent system chloroform, pyridine, 88% formic acid, water (30:70:16: 10, v/v). Following chromatography, the plate was dried under a cold air stream and exposed to x-ray film for 20 s to locate the Kdo₂-4'-[³²P]lipid A. The region of the silica plate containing the product was removed by scraping, transferred to a thick-walled glass tube, and resuspended in 4 ml of an acidic single-phase Bligh/Dyer mixture (29, 30) consisting of chloroform, methanol, 0.1 M HCl (1:2:0.8, v/v). The suspension was vigorously mixed with the aid of a vortex and subjected to sonic irradiation in a bath 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 of the silica was repeated, first with 4 ml and then with 8 ml of the acidic single-phase Bligh/Dyer mixture. The extracts were pooled, passed through a 4-ml glass-wool column to remove remaining silica particles, and placed into glass tubes equipped with Teflon-lined caps. The solution was converted to a two-phase Bligh/Dyer mixture, consisting of chloroform, methanol, 0.1 M HCl (2:2:1.8, v/v). The phases were separated in a clinical centrifuge. The lower phases were transferred to new glass tubes. The upper phases was extracted a second time by the addition of fresh, pre-equilibrated acidic lower phases. The lower phases were pooled, neutralized by addition of pyridine (1 drop per 2-ml lower phase), and dried under a stream of N₂. The Kdo₂-4'-[³²P]lipid A was resuspended in 25 mM Tris-HCl, pH 7.8, containing 1 mM EDTA, 1 mM EGTA, and 0.1% Triton X-100 and then stored at –20 °C. The amount of Kdo₂-4'-[³²P]lipid A recovered was typically greater than 50 µCi. Nonradioactive carrier Kdo₂-lipid A was prepared from WBB06 in milligram quantities, as described previously (31).

Assay Conditions for Detecting pEtN Transferase Activity—The EptB transferase was assayed under optimized conditions in a 15-µl reaction mixture containing 50 mM Hepes, pH 7.5, 0.1% Triton X-100, 1 mM calcium chloride (CaCl₂), 1.25 mM dithiothreitol (DTT), 0.6 mM E. coli PE, and 10 µM Kdo₂-4'-[³²P]lipid A (50,000 cpm/nmol). Washed membranes were employed as the enzyme. Assay mixtures were incubated at 30 °C for the indicated times, and 4-µl portions were spotted onto Silica Gel 60 TLC plates to stop the reactions. Substrate and product(s) were separated using the solvent chloroform/methanol/water/acetic acid (25:15:4:4, v/v). Following chromatography, the plates were dried and analyzed using a Amersham Biosciences PhosphorImager (STORM 840), equipped with ImageQuant software.

Assay for Kdo₂-Lipid A-dependent Diacylglycerol Formation—EptB-catalyzed diacylglycerol formation was assayed in a 15-µl reaction mixture containing 50 mM Hepes, pH 7.5, 0.1% Triton X-100, 1 mM CaCl₂, 1.25 mM DTT, 200 µM Kdo₂-lipid A, and 100 µM 1-palmitoyl-2-[1-¹⁴C]linoleoylglycero-3-phosphoethanolamine ([16:0,¹⁴C-18:2]PE) at 5 x 10⁴ dpm/reaction. Triton X-100-solubilized C41(DE3)/pEptB1 membranes (0.1 mg/ml) were used as the enzyme source. Control reactions in which Kdo₂-lipid A or enzyme was omitted from the assay mixture were performed in parallel. Assay were carried out at 30 °C for the indicated times, and 5-µl portions were spotted onto Silica Gel 60 TLC plates to stop the reactions. Substrate and product(s) were separated using the solvent hexane/diethyl ether/acetic acid (30:70:1, v/v) (32). Following chromatography, the plates were dried and analyzed with a PhosphorImager, as described above. A [¹⁴C]diacylglycerol standard was generated by B. cereus phospholipase C treatment (33) of [16:0,¹⁴C-18:2]PE. Phospholipase C (0.25 units) was added to the standard 15-µl assay mixture, containing 50 mM Hepes, pH 7.5, 0.1% Triton X-100, and 100 µM ([16:0,¹⁴C-18:2]PE) (5 x 10⁴ dpm).

Separation of Inner and Outer Membranes—Membranes of W3110 harboring pEptB2 were separated by isopycnic sucrose gradient centrifugation. First, 120-ml LB cultures of W3110 harboring pEptB2 were grown with 1 mM IPTG until A₆₀₀ = 1.0. The cells were harvested as above, and membranes were prepared by the method of Osborn and Munson (34). The membranes were applied to a seven-step gradient (35) and subjected to ultracentrifugation at 35,000 rpm in a Beckman SW40.1 rotor for 18 h at 4 °C. Each fraction (0.5 ml) was assayed for NADH oxidase (inner membrane marker), phospholipase A (outer membrane marker) (36), and protein (37). Each fraction was also assayed for pEtN transferase activity.

Mild Acid Hydrolysis of pEtN-Kdo₂-4'-³²P-Lipid A Generated in Vitro—Two 10-µl reaction mixtures were prepared in 50 mM Hepes, pH 7.5, and 0.1% Triton X-100, containing either Kdo₂-4'-[³²P]lipid A (10,000 cpm) or pEtN-Kdo₂-4'-[³²P]lipid A (10,000 cpm). The pEtN-Kd-o₂-4'-[³²P]lipid A was generated under standard pEtN transferase assay conditions (described above). It was purified by TLC, as described for Kdo₂-4'-[³²P]lipid A. Next, 6.5 µl of 200 mM sodium acetate, pH 4.5, and 4 µl of 10% SDS were added to each tube, and the final volumes were adjusted to 40 µl. The reaction mixtures were placed into a heat block at 100 °C. At various times, 4-µl samples were withdrawn and spotted onto a silica TLC plate, which was developed in the solvent chloroform, pyridine, 88% formic acid, water (30:70:16:10, v/v) (10).

In-frame Replacement of eptB with a Kanamycin Resistance Cassette in E. coli WBB06—To create an in-frame replacement of eptB with the kanamycin resistance cassette (kan), WBB06 was transformed by electroporation with pKD46, an arabinose-inducible, -RED helper plasmid (38). PCR was utilized to construct a linear piece of DNA containing kan, flanked on the 5' end by an RBS plus 42 bp of chromosomal DNA upstream of eptB, and flanked 3' by 42 bp of chromosomal DNA located downstream of eptB. The forward and reverse primers are as follows: 5'-CCGCACTTTTTCCCTGCCGGGCCTGAAAAGCCACTAAGCAGGAAGGAGATATAATGAGCCATATTCAACGGGAA-3' and 5'-TAGCAAAATGCCTTTTGATCGGCGAGAAAGTCAGCAGGCCGCTTAGAAAAACTCATCGAGCAT-3'. The portion of the kanamycin cassette is underlined and the engineered RBS is in italics. The kanamycin resistance gene (Tn903) from plasmid pWKS130 (15) served as the template. PCR conditions used were the same as described above for the amplification of the eptB gene from genomic DNA. The PCR product was resolved on a 1.0% agarose gel and purified with the QIAquick gel kit. The product was electroporated into WBB06/pKD46, which had been grown at 30 °C with 1 mM L-(+)-arabinose. After growth for 2 h at 37 °C, the cells were plated on LB agar containing kanamycin (20 µg/ml) and incubated overnight at 37 °C. Kanamycin-resistant colonies were re-purified on LB kanamycin plates, which were incubated overnight at 37 °C to cure the strain of pKD46. The eptB::kan replacement on the chromosome of WBB06 was verified by PCR using external primers located 95 bp upstream (5'-GCACACTCTTTCCCCACACTTTTTCC-3') and 118 bp downstream (5'-CCTCCGACCCCTTCGTCCCGAACGAAG-3') of eptB. A single PCR product was resolved on a 1% agarose gel, purified with the QIAquick gel extraction kit, and sequenced using the same primers to confirm the replacement. The resulting strain, designated WBB06eptB::kan, was made electrocompetent by the method of Sambrook and Russell (13) and then transformed with the following pWSK29-derived plasmids: pWSK29 (vector control), pEptB2, pWaaC, or pWaaCF.

Growth of WBB060-derived Strains in the Presence of Divalent Cations—Strains were grown overnight on LB broth at 37 °C and used to inoculate 50 ml of pre-warmed LB broth to an A₆₀₀ = 0.02. Growth was allowed to continue with shaking at 250 rpm (37 °C). When A₆₀₀ reached 0.2, various divalent cations were added, as indicated below (time = 0). Growth was allowed to continue at 37 °C. Whenever the A₆₀₀ reached 0.3–0.4, the cultures were diluted 10-fold into 50 ml of pre-warmed LB, containing the relevant divalent cations, in order to keep the cells in log phase.

Extraction and Purification of LPS from E. coli WBB06—To determine whether the LPS molecules in WBB06 are covalently modified with pEtN when eptB is expressed under lac control from pWSK29 in the absence of added Ca²⁺, 1-liter cultures of WBB06 harboring either pWSK29 or pEptB2 were grown in the presence of 100 µg/ml ampicillin and 1 mM IPTG until A₆₀₀ = 1.0. The LPS was extracted and purified, as described previously (10).

In-frame Replacement of eptB with a Kanamycin Resistance Cassette in E. coli W3110—The eptB gene was also replaced with kan on the chromosome of E. coli W3110. The purified PCR product used to create the replacement of eptB on the chromosome of WBB06 (see above) was utilized to construct W3110eptb::kan. First, the PCR product was electroporated into the -RED strain, DY330 (39). After growth for 2 h at 37 °C, the cells were plated on LB agar containing kanamycin (20 µg/ml) and incubated overnight at 37 °C. Kanamycin-resistant colonies were re-purified on LB kanamycin plates. One of the kanamycin-resistant colonies was designated DY330eptB::kan. A P1_vir bacteriophage lysate of donor strain DY330eptB::kan was made as described by Miller (12) and used to tranduce stationary cells of W3110. The transduction mixture was plated onto LB agar, containing 20 µg/ml kanamycin and 5 mM sodium citrate, and then incubated at 37 °C overnight. Kanamycin-resistant colonies were re-purified with selection, and the eptB::kan replacement on the chromosome of W3110 was verified by PCR using the same primers used to confirm the eptB::kan replacement on the chromosome of WBB06 (see above). The PCR product was resolved on a 1% agarose gel, purified with the QIAquick gel extraction kit, and sequenced to confirm the replacement.

Mass Spectrometry of Kdo₂-Lipid A Samples—Spectra were acquired in the negative-ion linear mode using a Kratos (Manchester, UK) analytical matrix-assisted laser desorption ionization/time of flight (MALDI/TOF) mass spectrometer with a 337-nm nitrogen laser, a 20-kV extraction voltage, and time-delayed extraction. Each spectrum was the average of 50 shots. The lipid A samples were prepared for MALDI/TOF analysis by depositing 0.3 µl of the lipid sample, dissolved in chloroform/methanol (4:1, v/v), followed by 0.3 µl of a saturated solution of 6-aza-2-thiothymine in 50% acetonitrile and 10% tribasic ammonium citrate (9:1, v/v) as the matrix. The samples were dried at room temperature before the spectra were acquired.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
pEtN Transferase Activity in Membranes of Ca²⁺-treated WBB06 Versus EptB Overexpressing WBB06—Membranes of the heptose-deficient mutant WBB06, grown in LB broth containing 5–50 mM CaCl₂, exhibit pEtN transferase activity in vitro with the tetra-acylated LPS precursor Kdo₂-4'-[³²P]lipid IV_A as the acceptor substrate (10). The pEtN transferase activity was also observed when hexa-acylated Kdo₂-4'-[³²P]lipid A was employed as the lipid acceptor (Fig. 2, lanes 3 and 4). To determine whether the pEtN transferase activity seen in Ca²⁺-treated WBB06 is because of EptB, we examined membranes of EptB-overexpressing cells. As shown in Fig. 2, lane 6, the R_f of the product generated by membranes of WBB06/pEptB2 was the same as that produced by membranes of Ca²⁺-treated WBB06 (Fig. 2, lanes 3 and 4). However, much more product was formed with WBB06/pEptB2 membranes. No reaction was seen with the vector control WBB06/pWSK29 (Fig. 2, lane 5). The band labeled x in Fig. 2 reflects the formation of the lipid A 1-diphosphate variant (40, 41), which is generated from an as yet unidentified membrane-bound phosphate donor. Lipid A 1-diphosphate formation is partially suppressed by the presence of 1 mM Ca²⁺ in the assay system, and it is not observed when EptB-overexpressing membranes are diluted (see below).

View larger version (62K): [in this window] [in a new window]   FIG. 2. pEtN transferase activity in membranes of Ca2+-grown WBB06 versus WBB06/pEptB2. Membranes of WBB06 were assayed for pEtN transferase under standard conditions at 0.1 mg/ml for 60 min at 30 °C, and 4-µl portions were spotted onto Silica Gel 60 TLC plates to stop the reactions. Substrate and product(s) were separated using the solvent chloroform/methanol/water/acetic acid (25:15:4:4, v/v). Following chromatography, the plates were analyzed with a PhosphorImager. Lane 1, no enzyme; lane 2, WBB06, grown without added Ca2+; lane 3, WBB06, grown with 5 mM CaCl2; lane 4, WBB06, grown with 50 mM CaCl2; lane 5, WBB06/pWSK29 (vector control); and lane 6, WBB06/pEptB2. The cells used to make membranes in lanes 5 and 6 were grown on LB broth without added Ca2+. The x indicates the formation of the lipid A 1-diphosphate variant, which is generated from an endogenous, as yet unknown, phosphate donor. This activity is partially suppressed by Ca2+ in the assay mixture.

The addition of pEtN to the outer Kdo residue of LPS in living cells of WBB06 was shown previously to be dependent upon the inclusion of 5–50 mM CaCl₂ in the growth medium (10). However, it was not entirely clear if Ca²⁺ was also a necessary component of the in vitro assay system. Addition of EDTA to the assay inhibits pEtN transferase activity (10). To determine whether or not Ca²⁺ is required for pEtN transfer to Kdo₂-4'-[³²P]lipid A, diluted membranes of WBB06 overexpressing EptB were assayed in the presence or absence of 1 mM CaCl₂. Ca²⁺ greatly stimulated pEtN transferase activity in vitro when 0.025 mg/ml of membranes from EptB-overexpressing cells were employed as the enzyme source (Fig. 3B). The optimal Ca²⁺ concentration in the in vitro system was 1 mM (data not shown); higher concentrations of Ca²⁺ were inhibitory. As noted above, the formation of the lipid A 1-diphosphate (x in Fig. 3) was suppressed by Ca²⁺. All subsequent assays of EptB therefore included both 0.6 mM PE and 1 mM Ca²⁺, unless otherwise indicated. Production of pEtN-Kdo₂-lipid A by overexpressed EptB was linear for almost 60 min under the optimized conditions (Fig. 4, A and B), and it was linear with membrane protein up to 0.2 mg/ml at 30 min (data not shown).

View larger version (60K): [in this window] [in a new window]   FIG. 3. PE and Ca2+ are required for EptB activity. A, membranes from WBB06 harboring either the vector pWSK29 or pEptB2 were assayed for pEtN transferase in the absence or presence of 0.6 mM E. coli PE, as indicated. B, membranes of WBB06 harboring either the vector pWSK29 or pEptB2 were assayed for pEtN transferase in the absence or presence of 1 mM CaCl2, as indicated. The final protein concentration was of 0.025 mg/ml, and the reactions were incubated for 60 min at 30 °C in both sets of experiments.

Efficacy of PE Molecular Species as Substrates for EptB— Because E. coli PE consists of about 10 different molecular species (2, 42), we tested several commercially available PEs at 500 µM as pEtN donors for EptB. As shown in Fig. 5, PE species (16:0/18:1, 16:0/18:2, 18:0/18:1, or 18:1/18:1) with one or more double bonds were effective pEtN donors, whereas PEs with fully saturated acyl chains (16:0/16:0 and 18:0/18:0) were inactive. Similarly, saturated PEs with shorter acyl chains (10:0/10:0, 12:0/12:0, and 14:0/14:0) were not substrates (data not shown). Fully saturated PEs usually represent less than 5% of the PE molecular species present in the membranes of E. coli.

Stoichiometric Formation of Diacylglycerol and pEtN-Kdo₂-Lipid A by EptB—As shown in Fig. 6A, EptB catalyzed efficient time-dependent conversion of 200 µM Kdo₂-4'-[³²P]lipid A to pEtN-Kdo₂-4'-[³²P]lipid A with 100 µM [16:0,18:2]PE as the donor. At this ratio of concentrations, EptB-dependent diacylglycerol formation could be monitored in parallel by employing unlabeled Kdo₂-lipid A and [16:0,¹⁴C-18:2]PE as substrates (Fig. 6B). Release of [¹⁴C]diacylglycerol (Fig. 6B) followed the same time course as the formation of pEtN-Kdo₂-4'-[³²P]lipid A (Fig. 6A) with a stoichiometry of 0.9 mol of [¹⁴C]diacylglycerol/mol of pEtN-Kdo₂-4'-[³²P]lipid A at the 60-min time point. The small amount of [¹⁴C]diacylglycerol formed by EptB in the absence of added Kdo₂-lipid A (Fig. 6B, lane 13) may be due to the slow transfer of the phosphoethanolamine residue from [16:0,¹⁴C-18:2]PE to water in the absence of acceptor substrate.

View larger version (32K): [in this window] [in a new window]   FIG. 4. Time dependence of product formation by EptB. Membranes of WBB06/pEptB2 were assayed for pEtN transferase activity at 0.1 mg/ml with 10 µM Kdo2-lipid A at the indicated times. A, products were separated by TLC in the solvent chloroform/methanol/water/acetic acid (25:15:4:4, v/v) and analyzed with a PhosphorImager. B, the conversion of Kdo2-lipid A to pEtN-Kdo2-lipid A is linear with respect to the time for up to 60 min.

Inner Membrane Localization of EptB—Membranes of wild-type E. coli W3110 overexpressing EptB were subjected to isopycnic sucrose gradient centrifugation to separate inner and outer membranes. The EptB activity was found almost entirely within the inner membrane (Fig. 8). As shown in Fig. 8B, the pEtN transferase closely followed the activity of the inner membrane marker NADH oxidase (peak activity at fraction 14). As shown in Fig. 8A, we also determined the membrane protein concentration of each fraction and assayed the outer membrane marker phospholipase A (peak activity in fraction 3).

View larger version (51K): [in this window] [in a new window]   FIG. 5. Unsaturated PEs function as pEtN donor substrates for EptB. Various PE species were tested for their ability to support pEtN transferase activity in vitro. Membranes (0.025 mg/ml) from WBB06 harboring either pWSK29 or pEptB2 were assayed in the absence of added PE (lanes 2 and 3) or in the presence of E. coli PE (lanes 4 and 5), dipalmitoyl-PE (lanes 6 and 7), distearoyl-PE (lanes 8 and 9), 1-palmitoyl-2-oleoyl-PE (lanes 10 and 11), 1-palmitoyl-2-linoleoyl-PE (lanes 12 and 13), 1-stearoyl-2-oleoyl-PE (lanes 14 and 15), and dioleoyl-PE (lanes 16 and 17). Lane 1, no enzyme control; even lanes, membranes of WBB06/pWSK29; odd lanes after lane 1: membranes of WBB06/pEptB2.

Positive-mode MALDI/TOF analysis (not shown) of the LPS from WBB06/pEptB2 confirmed the presence of the pEtN substituent. The spectrum contained peaks at m/z = 2362.5, interpreted as [M + H]⁺ of pEtN-Kdo₂-lipid A, and at m/z = 2264.5 (interpreted as the B⁺₂ ion of pEtN-Kdo₂-lipid A) (44). These ions were absent in the positive-mode spectrum of the LPS purified from WBB06/pWSK29.

View larger version (45K): [in this window] [in a new window]   FIG. 6. Stoichiometric formation of diacylglycerol during pEtN transfer to Kdo2-lipid A. A, EptB-catalyzed formation of pEtN-Kdo2-4'-[32P]lipid A was assayed in a 15-µl reaction mixture containing 50 mM Hepes, pH 7.5, 0.1% Triton X-100, 1 mM CaCl2, 1.25 mM DTT, 200 µM Kdo2-4'-[32P]lipid A (5 x 104 cpm/reaction), and 100 µM [16:0,18:2]PE. Triton X-100-solubilized C41(DE3)/pEptB1 membranes (0.1 mg/ml) were used as enzyme. Assays were carried out at 30 °C, and 5-µl portions were spotted at various times onto Silica Gel 60 TLC plates to stop the reactions. Substrate and product(s) were separated using the solvent chloroform/methanol/water/acetic acid (25:15:4:4, v/v). Reactions in which PE, enzyme, or both were omitted, and the vector control, are indicated. Lanes 1–7 are samples taken at times 0, 20, 40, 60, 80, 100, and 120 min respectively. Lanes 8–11 are 120-min time points. B, EptB-catalyzed diacylglycerol formation was assayed under the same conditions as in A, but with 200 µM Kdo2-lipid A and 100 µM [16:0,14C-18:2]PE (5 x 104 dpm/reaction). Substrate and product(s) were separated using the solvent hexane/diethyl ether/acetic acid (30:70:1, v/v) (32). Following chromatography, the plates were dried and analyzed with a PhosphorImager. Reactions in which Kdo2-lipid A, enzyme, or both were omitted, and the vector control, are indicated. Lanes 1–7 are samples taken at times 0, 20, 40, 60, 80, 100, and 120 min, respectively. Lanes 8–13 are 120-min time points. A [14C]diacylglycerol standard (lane 10) was generated with B. cereus phospholipase C (PLC) (33).

View larger version (72K): [in this window] [in a new window]   FIG. 7. EptB adds pEtN to the outer Kdo residue of Kdo2-lipid A. A, Kdo2-4 '-[32P]lipid A, the substrate, was hydrolyzed at pH 4.5 in the presence of SDS at 100 °C over the indicated time course. B, the pEtN4'-[32P]Kdo2-lipid A, generated in vitro by membranes of WBB06/pEptB2, was hydrolyzed in parallel. At each time point, 4-µl portions of the hydrolysis mixtures were spotted onto a silica gel thin layer chromatography plate, which was developed in the solvent chloroform, pyridine, 88% formic acid, water (30:70:16:10, v/v). The various hydrolysis products were detected with a PhosphorImager.

View larger version (35K): [in this window] [in a new window]   FIG. 8. Inner membrane localization of EptB. Inner and outer membranes isolated from W3110/pEptB2 were separated by isopycnic sucrose density gradient centrifugation, as described previously, and 0.5-ml fractions were collected. A, the protein concentration (open squares) and the outer membrane phospholipase A activity (closed squares) were determined. B, the pEtN transferase (open circles) and the inner membrane NADH oxidase activity (closed circles) were measured.

View larger version (18K): [in this window] [in a new window]   FIG. 9. Negative ion mode MALDI/TOF mass spectrometry of LPS from WBB06/pEptB2. A, the LPS purified from E. coli strain WBB06 harboring the vector pWSK29 consists mainly of Kdo2-lipid A. B, LPS of WBB06/pEptB2 contains a significant portion of an additional compound with the mass expected for pEtN-Kdo2-lipid A, as indicated. During MALDI/TOF analysis one or both Kdo residues may be lost from the parent ion (10).

Cultures of WBB06eptB::kan harboring either pWaaC or pWaaCF were challenged with 5 mM CaCl₂ to determine whether restoration of a part (or all) of the LPS core could eliminate the Ca²⁺ sensitivity. As shown in Fig. 10C, supplying waaC in trans, which results in the incorporation of a single heptose residue (6, 8), did not alleviate the Ca²⁺ hypersensitivity. However, supplying both heptosyl transferase genes (waaC and waaF) in trans, which fully restores the LPS core (6, 8), allowed the eptB knock-out strain to grow in the presence of 5 mM Ca²⁺. These findings are consistent with the additional observation (data not shown) that deletion of eptB in wild-type E. coli W3110 does not result in Ca²⁺ sensitivity.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Phosphoethanolamine residues are commonly found as substituents of LPS (6–8) and other surface glycoconjugates in Gram-negative bacteria (45). Very little is known about the enzymes that generate the pEtN-modified molecules present in the cell envelope or about the biological function of pEtN modifications. In order to explore these issues, it is necessary to identify the genes encoding the relevant pEtN transferases and to construct mutants lacking them. In the present work we have found that the eptB gene, formerly yhjW, encodes the Ca²⁺-induced pEtN transferase known to modify the outer Kdo residue of E. coli LPS (9, 10). We have constructed a mutant in which eptB is deleted, and we found that it is extremely sensitive to added Ca²⁺ ions, provided that it is also lacking the heptose residues of the LPS core domain.

View larger version (21K): [in this window] [in a new window]   FIG. 10. Ca2+-sensitive growth of WBB06eptB::kan and its reversal by excess Mg2+ or the complete heptose domain. A, cultures of WBB06 or WBB06eptB::kan were grown to A600 = 0.2 at 37 °C, and then 5 mM CaCl2 was added to some of the cultures (time = 0), as indicated. Cells were diluted 10-fold whenever A600 reached 0.3–0.4. The plotted A600 represents the cumulative growth yield corrected for dilution. Open squares, WBB06 (no CaCl2 added); open circles, WBB06eptB::kan (no CaCl2 added); closed squares, WBB06 (+ CaCl2); closed circles, WBB06eptB::kan (+ CaCl2). B, cultures of WBB06eptB::kan were grown to A600 = 0.2 at 37 °C, and then 5 mM CaCl2 or 50 mM MgCl2 was added singly or in combination (time = 0), as indicated. Cells were diluted 10-fold whenever A600 reached 0.3–0.4. The plotted A600 represents the cumulative growth yield corrected for dilution. Open squares, WBB06eptB::kan (no added CaCl2); open circles, WBB06eptB::kan (+50 mM MgCl2); closed squares, WBB06eptB::kan (+5 mM CaCl2); closed circles, WBB06eptB::kan (+5 mM CaCl2 and 50 mM MgCl2). C, WBB06eptB::kan was transformed with the following plasmids: pWSK29 (vector), pEptB2, pWaaC, or pWaaCF. Cells were grown to A600 = 0.2 at 37 °C, and then 5 mM CaCl2 was added (time = 0), as indicated. Cells were diluted 10-fold whenever A600 reached 0.3–0.4. The plotted A600 represents the cumulative growth yield corrected for dilution. Open squares, WBB06eptB::kan/pWSK29; closed squares, WBB06eptB::kan/pEptB2; open circles, WBB06eptB::kan/pWaaC; closed circles, WBB06eptB::kan/pWaaCF.

Overexpression of the other five E. coli orthologs of NMB2010, using the lac promoter of pWSK29, led to the identification of EptA/PmrC (46), the enzyme that modifies the phosphate groups of lipid A with pEtN residues in polymyxin-resistant mutants (47, 48). The active site of PmrC/EptA has been shown by gene fusion experiments to face the periplasm (48). However, PmrC/EptA has not been purified or characterized enzymatically (48), and our EptA in vitro assay (46) was reported only in abstract form. The full characterization of EptA as an enzyme will be described elsewhere.

In addition to being induced selectively by Ca²⁺, EptB transferase activity is dependent upon the presence of 1 mM Ca²⁺ in vitro (Fig. 3). EptB joins a growing list of inner (46, 49–50) and outer (20, 51) membrane-bound enzymes that modify LPS; however, EptB is the first of these to exhibit a requirement for Ca²⁺. The exact role that Ca²⁺ plays in the transfer of pEtN from PE to the outer Kdo moiety of LPS remains to be determined. The well characterized outer membrane phospholipase A of E. coli exhibits a similar Ca²⁺ requirement, which in that case may play a role in dimer formation (52). We found that Sr²⁺ could partially stimulate the pEtN transferase activity of EptB in our in vitro system. Studies with the phospholipase A likewise revealed that Sr²⁺ could substitute for Ca²⁺ to some degree.

Our studies show that EptB utilizes several different PE molecular species (Fig. 7) provided they contain at least one double bond. EptB may have evolved to utilize the most abundant PE species normally present in the E. coli envelope (2). We have also confirmed that recombinant EptB modifies the outer Kdo residue selectively (Fig. 7) but cannot transfer pEtN to the inner Kdo unit or to the lipid A moiety at an appreciable rate. The results of the pH 4.5 hydrolysis of pEtN-Kdo₂-4'-[³²P]lipid A are definitive with regard to the outer Kdo (Fig. 7), but they do not establish the proposed localization of the pEtN unit at position 7 (Fig. 1).

The four additional EptB orthologs (besides EptA) encoded within the E. coli genome are likely to include the enzyme responsible for the addition of the pEtN moiety to the outer heptose residue of LPS (6). To date, no in vitro assays with defined acceptor substrates have been described for this heptose modification. Another one of the five EptB orthologs might be required for the transfer of pEtN from PE to a sub-set of the periplasmic membrane-derived oligosaccharides, the biosynthesis of which is induced at low osmolarity (45, 53).

EptB generates stoichiometric diacylglycerol as a by-product during the transfer of the pEtN unit from PE to Kdo₂-lipid A (Fig. 6), consistent with the scheme proposed in Fig. 1. This novel source of diacylglycerol, which is likely also generated by the other members of the EptB family, explains why the diacylglycerol that accumulates in E. coli dgk mutants is not solely due to membrane-derived oligosaccharide biosynthesis (32). In principle, the EptB reaction should be reversible and share some common mechanistic features with the better characterized eucaryotic phosphoethanolamine transferases that generate PE from CDP-ethanolamine and diacylglycerol (54). However, the EptB family of proteins displays no obvious sequence similarity to the eucaryotic phosphoethanolamine transferases, which generally contain seven predicted trans-membrane segments. Iterative analysis of certain EptB orthologs with the Psi-Blast algorithm does suggest a very distant relationship to the phosphoethanolamine transferases that participate in the assembly of the phosphatidylinositol-linked glycans in eucaryotic cells (55).

The Ca²⁺-sensitive phenotype of the heptose-deficient mutant, lacking EptB, is very intriguing, as it suggests a possible function for the pEtN modification of the outer Kdo residue. Based on the observed Ca²⁺ sensitivity of WBB06eptB::kan (Fig. 10), we propose that pEtN modification of the outer Kdo moiety of LPS is critical for tolerance of elevated levels of Ca²⁺ in heptose-deficient E. coli. It may be that modification of the outer Kdo residue with pEtN renders the outer membrane less permeable to Ca²⁺. A reduction in Ca²⁺ permeability may help maintain the very low level of intracellular Ca²⁺ (0.1 µM or less) normally present in E. coli (56). A dramatic increase in the intracellular Ca²⁺ concentration might be toxic. The fact that excess Mg²⁺ can block the growth inhibition produced by Ca²⁺ (Fig. 10B) suggests that excess Mg²⁺ may prevent Ca²⁺ from binding to a critical site that destabilizes the cell envelope, preventing the influx of Ca²⁺. The observation (Fig. 10C) that eptB mutants are not Ca²⁺-sensitive in strains with a complete core suggests that the pEtN units that are normally present on the outer heptose residue of the core may substitute for the Ca²⁺-inducible pEtN unit attached to the Kdo region. Further genetic and biochemical characterization of the Ca²⁺-sensitive phenotype of WBB06eptB::kan should provide insights into the function of this modification.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grants GM-51310 (to C. R. H. R.) and GM-64402 (to R. J. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Biochemistry, Duke University Medical Center, P. O. Box 3711, Durham, NC. Tel.: 919-684-5326; Fax: 919-684-8885; E-mail: raetz{at}biochem.duke.edu.

¹ The abbreviations used are: LPS, lipopolysaccharide; pEtN, phosphoethanolamine; PE, phosphatidylethanolamine; IPTG, isopropyl 1-thio--D-galactopyranoside; Kdo, 3-deoxy-D-manno-octulosonic acid; DTT, dithiothreitol; [16:0,18:2]PE, 1-palmitoyl-2-linoleoyl-glycero-3-phosphoethanolamine; [16:0,¹⁴C-18:2]PE, 1-palmitoyl-2-[1-¹⁴C]linoleoyl-glycero-3-phosphoethanolamine; RBS, ribosome-binding site; MALDI/TOF, matrix-assisted laser desorption ionization/time of flight.

² The term "saccharolipid" (analogous to "glycerolipid") has been introduced recently to describe molecules, such as lipid A, in which one or more fatty acyl chains are linked directly to a sugar backbone (57). Other common saccharolipids include trehalose dimycolates of mycobacteria, N-acylated nod factors of rhizobia, and O-acylated glucose derivatives of plants. LPS is classified as a saccharolipid glycan.

	ACKNOWLEDGMENTS

 
We thank M. Stephen Trent for constructing the LpxL-overexpressing strain BLR(DE3)/pLysS/pLpxL.

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