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

M. Stephen TrentDagger §, Wendy Pabich, Christian R. H. Raetz||, and Samuel I. Miller**

From the Dagger  Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 and the  Departments of Microbiology and Medicine, University of Washington, Seattle, Washington 98195

Received for publication, November 28, 2000


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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. The pagL gene encodes a protein of 185 amino acid residues unique to S. typhimurium and closely related organisms such as Salmonella typhi. Heterologous expression of pagL 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 acid N-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. typhimurium mutants 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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (1, 2). In Salmonella typhimurium and Salmonella typhi, some virulence genes are controlled by the two-component regulatory system PhoP/PhoQ (3, 4). At low levels of Mg2+, the PhoQ sensor protein phosphorylates and activates the transcriptional regulatory protein PhoP, which in turn either activates or represses over 40 different genetic loci (5, 6). A second two-component regulatory system, PmrA/PmrB, is itself PhoP/PhoQ-activated (5, 7). PmrA is also activated directly by the PmrB kinase in the presence of ferric ions or indirectly at low pH (8). Mutants altered in the PhoP/PhoQ system display greatly reduced virulence (9, 10). Homologues of both regulatory systems are present in other Gram-negative bacteria, including Escherichia coli, Pseudomonas aeruginosa, and Yersinia pestis (4, 11).

Among their many functions, the PhoP/PhoQ and the PmrA/PmrB systems regulate the expression of gene products involved in the covalent modification of lipid A (12), the glycolipid anchor of lipopolysaccharide (LPS).1 LPS is a major component of the outer leaflet of the outer membranes of Gram-negative bacteria, and the lipid A portion of LPS is the bioactive component that is also known as endotoxin (13-16). During bacterial infections of animals, lipid A activates the innate immune system through interaction with Toll-like receptors, primarily TLR-4 (17-20). The host response to lipid A includes the production of cationic antimicrobial peptides, cytokines, tissue factor, and additional immunostimulatory molecules (19-22). In limited infections, the response to lipid A helps to clear the bacteria, but in overwhelming sepsis, high levels of circulating cytokines and procoagulant activity may damage the microvasculature and precipitate the syndrome of Gram-negative septic shock with disseminated intravascular coagulation (23, 24).

The structure of lipid A is relatively conserved among different pathogenic Gram-negative bacteria (13, 14, 16, 25). Lipid A of E. coli and S. typhimurium is a beta ,1'-6-linked disaccharide of glucosamine, phosphorylated at the 1- and 4'-positions and acylated at the 2-, 3-, 2'-, and 3'-positions with R-3-hydroxymyristate (Fig. 1) (13, 14, 16, 25). 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 (13, 14, 16, 25). Lipid A is glycosylated at position 6' with two 3-deoxy-D-manno-octulosonic acid (Kdo) moieties (Fig. 1) (13, 14, 16, 25). 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) (12, 26-28). In S. typhimurium, Miller and co-workers have shown that incorporation of palmitate and 2-OH myristate moieties (12, 29) is controlled by PhoP/PhoQ, whereas the 4-aminoarabinose and phosphoethanolamine modifications require the activation of PmrA (27, 30).


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Fig. 1.   Regulated modifications of the structure of Kdo2-lipid A in S. typhimurium. The phosphate residues and acyl chains of lipid A in S. typhimurium can be derivatized in a regulated fashion (12). The phosphate moieties of lipid A can be substituted with 4-amino-4-deoxy-L-arabinose and/or phosphoethanolamine groups, both of which are under PmrA/B control (blue substituents) (26, 56). Minor species are present in which the locations of the 4-amino-4-deoxy-L-arabinose and phosphoethanolamine groups are reversed (57) (Z. Zhou and C. R. H. Raetz, manuscript in preparation) or in which both phosphates are modified with the same substituent (not shown). The addition of the palmitate chain is catalyzed by PagP, as indicated (31), and formation of the 2-hydroxymyristate group (X) requires a novel hydroxylase homologue, designated LpxO (58). The ester-linked beta -hydroxymyristoyl chain at the 3-position may be removed by the outer membrane lipase PagL, as indicated. Substituents that are incorporated or removed in a PhoP/Q-dependent manner are shown in red.

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 gene pagP (29). We have recently shown that pagP is the structural gene for a novel acyltransferase (31) (Fig. 2) that utilizes glycerophospholipids as palmitate donors (31, 32). PagP is the first example of a lipid A biosynthetic enzyme localized to the outer membrane (31).


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Fig. 2.   Reactions catalyzed by PagP and PagL. These reactions may occur in either order when lipid IVA is used as the substrate.

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. typhimurium strains harboring insertion mutations in different PhoP-activated (pag) genes (33), 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 pagL display 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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemicals and Other Materials-- [gamma -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.

Bacterial Strains-- 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 (34). In experiments involving Mg2+ limitation or pH changes, cells were grown in N-minimal medium (35) 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 at A600 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.

Preparation of Radiolabeled Substrates-- The substrate [4'-32P]lipid IVA was prepared using 100 µCi of [gamma 32-P]ATP, tetraacyl-disaccharide 1-phosphate acceptor and membranes from E. coli that overexpress the 4'-kinase, as previously described (36), 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 (37), 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 (37), consisting of chloroform/methanol/0.1 M 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 (36). 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 [gamma 32-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 (36).

Preparation of Cell-free Extracts and Membranes-- Typically, 100-ml cultures of bacteria were grown to an A600 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 (38), with bovine serum albumin as the standard.

3-O-Deacylase Assay-- 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, 0.5 M 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 (Table I) was used as the enzyme source to avoid other further acylation of the [4'-32P]lipid IVA substrate by PagP (31).

                              
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Table I
Relevant bacterial strains and plasmids

Thin Layer Chromatography-- 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 (39, 40) 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.

Mild Alkaline Base Hydrolysis Using Triethylamine-- 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 (41). 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 IVA substrate was also subjected to triethylamine hydrolysis under the same conditions (41).

Separation of Inner and Outer Membranes-- Membranes from various strains of E. coli, S. typhimurium, or P. 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. (42, 43), 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 (44). The amount of protein in each fraction was determined using the bicinchoninic acid assay (38). Each fraction was also assayed for the 3-O-deacylase activity using the standard conditions described above.

Recombinant DNA Techniques-- 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.

Sequencing of pagL Gene-- Initial sequence of the pagL region was obtained by sequencing pBB04EL, a plasmid containing the pagL::TnphoA 5' fusion junction (30). Sequence was obtained in both directions using standard techniques with a Perkin-Elmer ABI Prism 377 automated DNA sequencer equipped with Sequencher 3.0 software. The pagL sequence was subsequently verified from chromosomal DNA cloned into pBluescript (pWLP21) as described below.

Construction of a PhoPC pagL Deletion Mutant-- A nonpolar deletion of greater than 95% of the coding sequence of pagL was created using PCR amplification of flanking DNA with Pfu (Stratagene) according to the manufacturer's instructions. The flanking DNA was subsequently cloned into the allelic exchange vector, pKAS32 (45), resulting in the plasmid pWLP24 containing the Delta pagL construct. Allelic exchange was performed in strain CS401, as has been described (46). Resolution of the integrant resulted in a Delta pagL strain CS586, which was verified using PCR and Southern blot analysis.

To create a nonpolar Delta pagL in a background that constitutively expresses PhoP/PhoQ, P22HTint bacteriophage was grown on the CS401 pWLP24 integrant, and the integrated plasmid was transduced into the PhoPC streptomycin-resistant strain CS491. Resolution of the integrated plasmid resulted in a PhoPC Delta pagL strain, CS584. The successful deletion of pagL was verified by PCR and Southern blot analysis.

Construction of pWLP21 and pWLP23-- The pagL gene and its flanking sequences, including 79 base pairs upstream and 168 base pairs downstream, were amplified by PCR from S. typhimurium 14028 genomic DNA with Pfu Turbo (Stratagene) according to the manufacturer's instructions. The PCR product was cloned into both the high copy vector pBluescript KS II+ (pWLP21) and the low copy vector pWKS30 (pWLP23) (Table II).

                              
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Table II
Oligonucleotides

Overexpression of PagL behind a T7 Promoter-- The pagL gene was amplified by PCR using Pfu Turbo (Stratagene) according to the manufacturer's instructions, using S. typhimurium 14028 genomic DNA as the template. The PCR product was cloned into pET21a(+) under the control of the T7 promoter to overexpress the enzyme giving the construct, pPagL. The pagL construct was transformed into BLR(DE3)/pLysS (Novagen) for overexpression of PagL. First, a single colony of E. coli BLR(DE3)/pLysS containing pPagL was inoculated into 20 ml of LB medium and grown to an A600 = 0.8. The culture was then used to inoculate 1 liter of fresh LB medium, and at A600 of ~0.6, the cells were induced with 1 mM isopropyl-1-thio-beta -D-galactopyranoside for 4 h. Crude extracts, membrane-free cytosol, and washed membranes were prepared as described above.

Protein Gel Electrophoresis-- Protein extracts were analyzed using the Bio-Rad Protean II XI apparatus. Samples containing 40 µg of protein were solubilized in <FR><NU>1</NU><DE>3</DE></FR> volume of 2× SDS buffer and heated for 5 min. Samples were applied to a 1.5-mm-thick 15% polyacrylamide SDS gel with a 4% stacking gel (22.2 × 20 cm) and were subjected to electrophoresis at 100 V. Gels were stained with Coomassie Blue dye (2.5 mg/ml) in water/methanol/acetic acid (6:4:1, v/v/v) and were then destained with water/methanol/acetic acid (6:4:1, v/v/v). Prestained low range standards from Bio-Rad were used to estimate molecular weight.

Protein Microsequencing-- Protein from the outer membrane fraction of the E. coli expression strain BLR(DE3)pLysS containing the T7 pagL construct (pPagL) was separated by SDS-polyacrylamide gel electrophoresis as described above. The portion of the gel containing the PagL protein was excised, and the protein was transferred to an Immobilon-P polyvinylidene difluoride membrane (Millipore Corp.) in 10 mM CAPS, pH 11, in 10% methanol at 15 V for 30 min using the Bio-Rad Semi-Dry Transfer apparatus. Transferred protein was stained with 0.1% Ponceau S in 1% acetic acid for 1 min and then destained with 1% acetic acid for 10 min. The PagL protein band was excised, rinsed three times with distilled water, and subjected to high sensitivity protein microsequencing on ABI model 492A Procise Sequencer at the University of Massachusetts Medical School Core Laboratory for Protein Microsequencing and Mass Spectrometry (Worcester, MA).

Large Scale Isolation of Lipid A from E. coli Cells Expressing the Heterologous pagL Gene-- Cultures (100 ml) of the E. coli strain XL-1 Blue were grown in LB medium at 37 °C containing either pBluescript (Stratagene) or pWLP21. After A600 of ~1.0 was reached, cells were harvested, resuspended in 80 ml of phosphate-buffered saline (47), and frozen prior to lipid A isolation. Lipid A was released from cells and purified as previously described and stored frozen at -80 °C (28, 48).

Mass Spectrometry of Lipid A Species-- Spectra were obtained in the negative linear mode using a matrix-assisted laser desorption/ionization time of flight Bruker BiflexIII mass spectrometer (Bruker Daltonics, Inc., Billerica, MA). Each spectrum was the average of 100 shots. Lipid samples were dissolved in chloroform/methanol (4:1, v/v) before mixing with the matrix (lipid/matrix; 9:1, v/v). The matrix consisted of saturated 6-aza-2-thiothymine in 50% acetonitrile and 10% ammonium sulfate. The mixtures were allowed to dry at room temperature on the sample plate prior to mass analysis.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A PhoP/PhoQ-dependent Deacylase in S. typhimurium Membranes-- As shown in Fig. 3A, membranes from a strain of S. typhimurium in which the PhoP transcriptional regulatory protein is constitutively active (PhoPC) (49) are capable of converting the lipid A precursor, [4'-32P]lipid IVA, to a more hydrophilic product (lane 3) with an RF of 0.28. This product, denoted as deacyl-IVA, is the same as that produced by the 3-O-deacylases of Rhizobium etli and of P. aeruginosa, previously characterized by Basu et al. (41). The faster migrating species, lipid IVB, arises by the addition of a palmitate chain to the amide-linked beta -hydroxymyristate residue at position 2 of lipid IVA (31, 32), catalyzed by the pagP gene product (31) (Fig. 2). The additional radioactive lipid shown in lane 3 (designated deacyl-IVB) results from both the addition of a palmitate chain and 3-O-deacylation of [4'-32P]lipid IVA (Fig. 2). When membranes from a PhoPC strain, also harboring a pagP mutation (31), were used, both products containing palmitate were eliminated (lane 5).


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Fig. 3.   Identification of a 3-O-deacylase in S. typhimurium membranes and its regulation. A, membranes from the indicated strains of S. typhimurium (Table I) were assayed for 3-O-deacylase activity using assay conditions described under "Experimental Procedures." The protein concentration was 0.1 mg/ml, and assays were carried out for 30 min at 30 °C. Each reaction contained 10 µM [4'-32P]lipid IVA, and the products were separated by TLC and detected with a PhosphorImager. B, wild-type S. typhimurium membranes from cells grown at pH 7.4 in either 10 µM or 10 mM Mg2+ were assayed for deacylase activity under the conditions described above. RF values for the various metabolites were determined and are listed in ascending order: RF1 = 0.28, RF2 = 0.32, RF3 = 0.42, RRF4 = 0.49.

Membranes from wild-type S. typhimurium grown in LB broth (Fig. 3, lane 2) or in minimal medium (pH 7.4) containing 10 mM Mg2+ (Fig. 3, lane 8), conditions under which the PhoP/PhoQ system is inactive, showed little or no deacylase activity. In membranes from cells grown in minimal medium (pH 7.4) containing 10 µM Mg2+, however, the 3-O-deacylase was very active (Fig. 3, lane 9), consistent with PhoP/PhoQ regulation (5, 12). The specific activity of the deacylase in membranes of either the PhoPC strain or of wild-type S. typhimurium cells grown in limiting Mg2+ was ~0.50 nmol/min/mg. Membranes from a PhoPC strain with a pagP deletion displayed even higher specific activity (~1.35 nmol/min/mg). Since palmitoylation of lipid IVA by PagP represents a competing reaction in Salmonella membranes, a PhoPC pagP- strain was routinely used for further characterization of the deacylase.

The PmrA/PmrB two-component system was previously implicated in the covalent modification of lipid A with phosphoethanolamine and 4-aminoarabinose (26, 27, 30) and was therefore tested as a regulator of the 3-O-deacylase. Membranes from an S. typhimurium strain in which the transcriptional regulatory protein PmrA is constitutively active (PmrAC) showed no deacylase activity (Fig. 3, lane 6). Also, growth in minimal medium at a pH of 5.8, a condition known to activate the PmrA/PmrB system (5), failed to induce deacylase activity unless the Mg2+ concentration was also limiting (10 µM) (data not shown). As previously demonstrated by Basu et al. (41), E. coli membranes do not possess a deacylase when cells are grown in broth. Similarly, we found that growth in minimal medium containing 10 µM Mg2+ at pH 7.4 or 5.8 was unable to induce deacylase activity in membranes of E. coli MC1061 or W3110 (data not shown).

Mild Alkaline Hydrolysis of the S. typhimurium 3-O-Deacylase Reaction Product-- Hydrolysis of lipid A and its precursors with the mild base triethylamine at 30 °C results in the selective removal of the 3-O-linked beta -hydroxymyristoyl group, followed by gradual removal of the 3'-O-linked fatty acyl moiety (41). The triethylamine deacylation products of [4'-32P]lipid IVA are easily separated by TLC, are well characterized, and can be used as standards (41). The [4'-32P]lipid IVA substrate (Fig. 4A) and the 4'-32P hydrophilic reaction product generated by PhoPC pagP- Salmonella membranes (Fig. 4B) were treated in parallel with triethylamine. As shown in Fig. 4A, treatment of [4'-32P]lipid IVA with triethylamine results in gradual removal of both ester-linked fatty acids, giving rise to the 3- or 3'-O-deacylated materials as intermediates and the doubly O-deacylated species as the final product. The hydrophilic species generated from [4'-32P]lipid IVA by PhoPC pagP- Salmonella membranes (Fig. 4B) migrates the same as the 3-O-deacylated [4'-32P]lipid IVA standard (Fig. 4A). Further treatment of this product (Fig. 4B) with triethylamine results in formation of a compound migrating like doubly O-deacylated [4'-32P]lipid IVA. These results demonstrate that the hydrophilic material generated from [4'-32P]lipid IVA by PhoPC pagP- Salmonella membranes is probably a 3-O-deacylated species and does not contain some other hydrophilic unit that slows its migration during TLC analysis.


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Fig. 4.   Triethylamine hydrolysis of [4'-32P]lipid IVA and the hydrophilic product generated by membranes of strain CS330. As described under "Experimental Procedures," the [4'-32P]lipid IVA substrate (10 µM) was incubated either with or without membranes from the PhoPC pagP- strain (CS330) under standard assay conditions using 1 mg/ml membranes for 2 h at 30 °C. Following the reaction, the [4'-32P]lipid IVA and the hydrophilic product generated by CS330 membranes were treated with triethylamine for the indicated times, after which a portion of each sample was separated by TLC and subjected to PhosphorImager analysis.

Assay Conditions and Catalytic Properties of the PhoP/PhoQ-regulated 3-O-Deacylase-- The 3-O-deacylation of [4'-32P]lipid IVA is absolutely dependent upon the presence of the nonionic detergent, Triton X-100, with optimal activity at 0.1%. The pH optimum is 8.0, but significant activity is observed from pH 5.5 to 9.0. Divalent cations are not required. EDTA and EGTA have no effect. Increased activity is observed with higher ionic strength. Accordingly, 0.5 M NaCl is included in the assay system (data not shown). The substrate specificity of the enzyme is relatively broad (Fig. 5). The deacylase does not require the Kdo moiety for activity, showing a slightly higher activity with 10 µM [4'-32P]lipid IVA than with 10 µM Kdo2-[4'-32P]lipid IVA. The relative rate of deacylation is decreased ~10-fold when the monosaccharide precursor, lipid X (40, 50), is used as the substrate (Fig. 5) at 10 µM. Product formation by the deacylase with 10 µM [4'-32P]lipid IVA is linearly dependent upon both protein concentration (data not shown) and times less than 10 min (Fig. 5). Interestingly, we have so far been unable to demonstrate activity with hexa-acylated lipid A as the substrate (data not shown), raising an interesting paradox in light of the subcellular localization of the enzyme (see below).


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Fig. 5.   Substrate specificity and time dependence of the 3-O-deacylase. Deacylase activity was assayed under standard assay conditions with either 10 µM [4'-32P]lipid IVA, Kdo2-[4'-32P]lipid IVA, or 32P-labeled lipid X. Membranes at 1.0 mg/ml from CS330 (PhoPC, pagP- S. typhimurium) served as the enzyme source. Reaction products were separated by TLC as described under "Experimental Procedures" and subjected to PhosphorImager analysis.

The assay conditions for the Rhizobium leguminosarum/etli 3-O-deacylase (41) differ from those for the S. typhimurium enzyme with regard to pH optimum, Triton X-100 dependence, and requirement for divalent cations. Optimal deacylation conditions for P. aeruginosa membranes resemble those for the Salmonella enzyme (data not shown). However, the 3-O-deacylase of wild-type P. aeruginosa PAO1 is present in membranes prepared from cultures grown in LB broth (41). The deacylase activity of PAO1 is not increased in membranes prepared from cells grown in the presence of low Mg2+ (10 µM) (data not shown). Last, PhoP null mutants of P. aeruginosa PAO1 still make 3-O-deacylated lipid A species when grown in the presence of low Mg2+, whereas modifications with 4-aminoarabinose and palmitate are lost (11). The combined data suggest that the 3-O-deacylase of PAO1 is regulated differently than the Salmonella enzyme.

Outer Membrane Localization of the S. typhimurium 3-O-Deacylase-- The deacylase activity of S. typhimurium is localized in the particulate fraction (Fig. 6). Further separation by isopycnic density gradient centrifugation reveals that the enzyme is mainly an outer membrane protein (Fig. 7), although a small but significant fraction of the activity is also seen in the inner membrane. NADH oxidase serves as the inner membrane marker, whereas phospholipase A activity is used to locate outer membrane fragments (Fig. 7). Besides PagP (31), the 3-O-deacylase of S. typhimurium is only the second example of an outer membrane protein involved in lipid A modification. The 3-O-deacylase of P. aeruginosa is also localized mainly in the outer membrane fraction (data not shown).


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Fig. 6.   3-O-Deacylase activity is associated with the membrane fraction of S. typhimurium. The 3-O-deacylase activity of the PhoPC pagP- strain CS330 was assayed under standard assay conditions with 10 µM [4'-32P]lipid IVA. Crude extract, cytosol, or membranes served as the enzyme source. The reaction products from the indicated times were separated by TLC and subjected to PhosphorImager analysis.


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Fig. 7.   Outer membrane localization of the PhoP/PhoQ regulated 3-O-deacylase of S. typhimurium. Membranes isolated from the PhoPC pagP- strain (CS330) were separated by isopycnic sucrose density gradient centrifugation, and ~0.5-ml fractions were collected. A, phospholipase A and NADH oxidase activities were assayed as markers for outer and inner membrane fragments, respectively, and expressed as a percentage of the total activity across the gradient. B, protein concentration and 3-O-deacylase activity were assayed for each fraction.

Identification of the Structural Gene (pagL) Encoding the 3-O-Deacylase of S. typhimurium-- Previously, several S. typhimurium PhoP-activated (pag) genes were mutated using a TnphoA transposon in a PhoPC background (51). Since the deacylase is greatly induced in the PhoPC background, loss of enzymatic activity in the membranes of a particular pag mutant could reveal the structural gene encoding the deacylase or, alternatively, additional regulatory protein(s) necessary for deacylase expression. Membranes of strains containing single, distinct TnphoA mutations (Table I) were prepared and assayed for 3-O-deacylase activity. Out of 14 available pag mutants, only one (CS328) showed a loss of deacylase activity (Fig. 8). CS328 contains an insertion in a previously unreported PhoP activated gene, designated pagL. The palmitoyltransferase (PagP) activity is still present in CS328 and all other pag mutant strains except for CS330, which harbors a pagP insertion.


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Fig. 8.   Screening of extracts of S. typhimurium mutants defective in PhoP-activated genes for loss of 3-O-deacylase activity. Membranes from various PhoPC strains of S. typhimurium, each containing a separate TnphoA transposon insertion in a different pag gene, were assayed for 3-O-deacylase activity. The source of the individual pag knockouts is indicated in Table I. Membranes from wild-type S. typhimurium and a PhoPC S. typhimurium strain (CS022) served as the negative and positive controls, respectively. Assays were performed under the standard conditions for 1 h at 30 °C using 1 mg/ml membranes and 10 µM [4'-32P]lipid IVA. Products were separated by TLC and visualized by PhosphorImager analysis.

Using the DNA sequence of the transposon as the starting point, the sequence of the inactivated pag gene (pagL) was determined. A novel open reading frame was identified (Fig. 9). Comparison of the predicted PagL amino acid sequence with putative proteins in the nonredundant and incomplete microbial data bases using the BLASTp or tBLASTn programs (52, 53) revealed no obvious homologues of PagL except in other strains of Salmonella. Analysis of the amino acid sequence indicated the presence of a 15-amino acid type I signal peptide using the Signal-P Program (54), supporting the view that the protein may be localized to the outer membrane fraction. The uncleaved protein has a predicted molecular mass of ~20 kDa and a pI of ~9.0.


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Fig. 9.   Sequence of the S. typhimurium PagL protein and its secondary structure prediction. The 3-O-deacylase protein sequence was deduced from the pagL gene sequence. The cleavage site of the N-terminal signal peptide, designated by a downward arrow, occurs between amino acids 20 and 21 (AND-NVF) as evidenced by N-terminal amino acid sequencing.

A nonpolar deletion compromising greater than 95% of the pagL coding sequence was generated in the S. typhimurium strain CS401 (46), followed by transduction of the mutation into the PhoPC streptomycin-resistant strain, CS491. Membranes of CS584 (phoPCDelta pagL) contained no deacylase, as shown in Fig. 10, lane 2, when assayed under optimal conditions at 0.01 mg/ml for 10 min or even at 2 mg/ml (data not shown). To demonstrate that recovery of deacylase activity was dependent upon the pagL gene, pagL and its flanking 5' and 3' sequences were cloned into the low copy vector, pWKS30, and the resulting plasmid was named pWLP23. The 3-O-deacylase activity of CS584 was recovered upon transformation with pWLP23 (Fig. 10, lane 4). Introduction of the vector control had no effect on deacylase activity (Fig. 10, lane 3). Although these data are strongly suggestive, they do not unequivocally prove that pagL is the structural gene encoding the deacylase.


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Fig. 10.   Reconstitution of 3-O-deacylase activity in membranes of a PhoPCDelta pagL mutant by introduction of a pagL-covering plasmid. Membranes from the PhoPC Delta pagL mutant (CS584), CS584 containing the vector control (pWSK30), or CS584 containing the plasmid with the pagL gene (pWLP23) were assayed for deacylase activity. Assays were performed under standard conditions using 0.01 mg/ml membrane protein for 10 min.

Heterologous Expression of pagL in E. coli-- To obtain additional evidence that pagL is the structural gene for the 3-O-deacylase, a heterologous E. coli expression system was established. Both the control pBluescript vector and pWLP21 were transformed into E. coli strain XL-1 Blue, and the lipid A of each strain was isolated and analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry. The lipid A of XL-1 Blue, containing the control vector, consisted primarily of the hexa-acylated bis-phosphate species that is typically seen in E. coli K12 strains (28, 48), with [M - H]- at m/z 1796.9 atomic mass units in the negative mode (Fig. 11A). However, upon overexpression of pagL, the [M - H]- of the predominant lipid A species was detected at m/z 1570 atomic mass units in the negative mode, corresponding to the loss of one beta -hydroxymyristate residue from the major species seen in the vector control (Fig. 11B). The additional species at m/z 1490 atomic mass units corresponds to loss of the phosphate group from the 1-position of the 3-O-deacylated lipid A, most likely a fragment ion, since analysis of the sample by TLC followed by charring showed no such lipid. Based upon mass spectroscopy, it can be concluded that selective lipid A deacylation occurs in living cells of this heterologous construct at the 3-position. Loss of beta -hydroxymyristate at the 3'-position would also lead to the loss of the secondary myristate chain, yielding a tetra-acylated lipid A variant with a molecular weight of 1361.7, a species not seen in Fig. 11. Furthermore, heterologous expression of pagL in E. coli did not slow down cell growth (data not shown).


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Fig. 11.   Matrix-assisted laser desorption/ionization time of flight mass spectrometry of lipid A from E. coli expressing a heterologous pagL gene. The lipid A of the E. coli XL-1 Blue strain containing either pBluescript (A) or pWLP21 (pBluescript with pagL) (B) was isolated using published protocols (48). The lipid A samples were analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry in the negative linear ion mode.

There are no homologues of the pagL gene in E. coli, consistent with the observation that there is no 3-O-deacylase activity in E. coli membranes, irrespective of growth or assay conditions. The above data further support the view that pagL of S. typhimurium is the structural gene for the 3-O-deacylase.

T7 Promoter-driven Overexpression of PagL and Its Localization in the Outer Membrane-- Overproduction of PagL protein was achieved by cloning the PCR-amplified pagL gene behind a T7 promoter into the expression vector pET21a, giving the plasmid pPagL. Membranes isolated from the E. coli expression strain BLR(DE3)pLysS containing either pET21a or pPagL were assayed for 3-O-deacylase activity. The specific activity of the deacylase in membranes of PhoPC S. typhimurium was 0.50 nmol/min/mg versus 155 nmol/min/mg in the induced T7 overexpression system, a 300-fold increase in activity. Membranes from the BLR(DE3)pLysS vector control strain were inactive. As in PhoPC S. typhimurium (Fig. 7), the overexpressed PagL in E. coli was located largely in the outer membrane (Fig. 12). Comparison of the outer membranes of BLR(DE3)pLysS containing either the control vector or pPagL shows overproduction of a protein migrating with the predicted molecular mass of mature PagL (18 kDa) (Fig. 12) in the latter. To verify the presence of the signal peptide predicted by the Signal-P program and to determine the cleavage site, PagL protein from outer membranes of BLR(DE3)pLysS/pPagL was electroblotted to a polyvinylidene difluoride membrane and subjected to microsequencing. The sequence of the first 10 amino acid residues was NVFFGKGNKH, indicating that cleavage of the signal peptide occurs between amino acid residues 20 and 21 (AND-NVF) of PagL rather than between amino acids 15 and 16 as predicted by Signal-P (54) (Fig. 9).


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Fig. 12.   Overexpression and outer membrane localization of PagL in the E. coli BLR(DE3)pLysS background. Membranes from the BLR(DE3)/pLysS expression strain, containing either pET21a or pPagL plasmids, were isolated, and 40-µg samples of protein were analyzed by SDS-polyacrylamide gel electrophoresis. The inner and outer membranes of the strains were separated by isopycnic sucrose density gradient centrifugation, and 0.5-ml fractions were collected as described under "Experimental Procedures." For the fractions shown, 40-µg samples of protein were analyzed, and the gel was stained with Coomassie Blue. The positions of the molecular weight standards, PagL, and the porins are indicated.

Thermal Stability of PagL-- The outer membrane enzyme PagP resists thermal denaturation (31), since it retains ~65% of its enzymatic activity after a 10-min preincubation at 100 °C (32). PagL displays similar behavior (data not shown). The unusual thermal stability of both enzymes may be due to their relatively low molecular weights.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Over the past 15 years, nine constitutive enzymes of lipid A biosynthesis have been identified in E. coli (13, 14, 55). With few exceptions, single copies of the corresponding structural genes are present in all Gram-negative bacteria. However, the enzymes and genes responsible for the covalent modifications of lipid A (Fig. 1), which are associated with bacterial virulence and polymyxin resistance, are not yet fully characterized (4, 26, 27, 56, 57). It has been demonstrated that these modifications are controlled by the PhoP/PhoQ and PmrA/PmrB two-component regulatory systems (4, 26, 27, 56, 57) and also can be induced with metavanadate in E. coli (28). The enzymatic function of the PhoP-activated lipid A palmitoyltransferase, PagP, was recently established in our laboratories (31). Furthermore, the biosynthesis of the PhoP/PhoQ-dependent S-2-hydroxymyristate moiety found in the lipids A of S. typhimurium and certain other pathogenic bacteria was shown to depend upon a novel lipid A hydroxylase homologue, designated LpxO (58).

We now present the initial characterization, cloning, and overexpression of another lipid A-modifying enzyme, PagL, an unusual PhoP/PhoQ-activated lipase that selectively removes the ester-linked 3-O-hydroxyacyl chains of certain lipid A precursors. The S. typhimurium lipid A 3-O-deacylase activity is under the control of the PhoP/PhoQ two-component regulatory system and was discovered using an in vitro assay with the tetra-acylated lipid A precursor, [4'-32P]lipid IVA, as the substrate (Fig. 3). By assaying extracts of individual PhoPC S. typhimurium strains harboring insertion mutations in 14 separate pag loci, the gene coding for the 3-O-deacylase (pagL) was found (Fig. 8). Complementation of a PhoPC Delta pagL S. typhimurium mutant with a low copy pagL plasmid restored deacylase activity. Heterologous expression of pagL in E. coli, an organism with no deacylase activity of its own and no pagL homologue in its genome, resulted in the appearance of robust deacylase activity in extracts and in loss of the R-3-hydroxymyristate moiety at position 3 in 90% of the lipid A molecules. There was no associated impairment of cell growth. These data, taken together with the sequencing of the overproduced PagL protein, demonstrate that pagL is the structural gene for the 3-O-deacylase.

Like the palmitoyltransferase PagP (31), the 3-O-deacylase PagL is a small, thermally stable enzyme, and it is associated with outer membrane enzyme as judged by the following observations. 1) Deacylase catalytic activity was detected mostly in outer membrane fragments of a PhoPC strain of S. typhimurium by assay with the substrate lipid IVA. 2) Separation of membranes from an E. coli strain overexpressing pagL behind a T7 promoter showed that the recombinant protein was largely recovered in the outer membrane fractions, as judged by SDS-polyacrylamide gel electrophoresis (Fig. 12). 3) The overexpressed PagL protein was missing its type I signal peptide, as shown by N-terminal sequencing of the outer membrane-associated band. Because of their small sizes, both PagP (~19 kDa) and PagL (~18 kDa) may adopt the smallest possible beta -barrel conformation that is characteristic of outer membrane proteins, consisting of only eight anti-parallel beta -strands (59). Analysis of the PagL amino acid sequence (Fig. 9) with programs for predicting secondary structure reveals significant beta -sheet domains. PagL is only one of four outer membrane enzymes characterized to date (31, 60).

No homologues of the S. typhimurium 3-O-deacylase were found in the nonredundant or unfinished microbial data bases, except in S. typhi and S. paratyphi, although other Gram-negative bacteria are known to contain 3-O-deacylated lipid A species (61, 62). A related lipid A lipase activity was previously demonstrated to be present in membranes of the nitrogen-fixing bacteria R. leguminosarum and R. etli (41), which are known to contain 3-O-deacylated lipid A species (63, 64). However, the gene encoding the 3-O-deacylase activity of R. etli is unknown, and the R. etli/leguminosarum enzyme requires divalent cations for activity (41), whereas PagL does not. Other Gram-negative bacteria that contain partially 3-O-deacylated lipid A include the pathogen P. aeruginosa (11, 61), which also possesses lipid A 3-O-deacylase activity (41) localized within its outer membrane.2 Structural characterization of the lipid A from Helicobacter pylori by mass spectroscopy (65) indicates partial deacylation of the 3'-O-linked fatty acyl chain. Structural studies of lipid A from Porphyromonas gingivalis revealed that both the 3- and 3'-ester-linked fatty acids are partially removed (66). One would therefore expect additional lipid A lipases to be present in these organisms, but no obvious homologues of PagL were detected by BLASTp or PSI-BLAST searches (53) in any of these bacteria, suggesting the existence of additional, structurally distinct 3-O-deacylases.

The key remaining questions about PagL concern its biological function in Salmonella and the significance of its regulation during pathogenesis. A systematic study comparing the lipids A of diverse Salmonella strains by mass spectroscopy demonstrated partial absence of the R-3-hydroxymyristate substituent in many cases (62). Furthermore, small amounts of 3-O-deacylated lipid A species are detected among the lipid A precursors that accumulate in Kdo-deficient, temperature-sensitive mutants of S. typhimurium.2 However, PhoP/PhoQ regulation of lipid A deacylation in Salmonella was not observed in previous studies comparing the lipid A structures of wild-type and phoP mutants (12), possibly because optimal conditions for inducing the deacylase in cells were not used. Unless a low copy pagL-bearing plasmid is introduced into S. typhimurium one does not see significant 3-O-deacylation of lipid A in cells under standard PhoP/PhoQ-activating growth conditions.3 Perhaps additional as yet unknown signals are required for proper functioning of chromosomally encoded PagL.

We propose that the 3-O-deacylation of lipid A by a bacterium that is in the process of infecting an animal might result in a lower or altered immunological response, possibly aiding the bacterium in establishing a prolonged infection. It is well known that the presence of the phosphate groups at positions 1 and 4' and the number and type of fatty acyl chains play a critical role in determining the immunological activity of lipid A (67, 68). As noted above, the lipid As of H. pylori and P. gingivalis are partially deacylated, and the lipids A of both organisms display significantly lower biological activities relative to other lipid A species (65, 66). The characterization of PagL mutants with respect to their pathogenesis and the analysis of various endotoxin-related activities of 3-O-deacylated lipid A species should help to clarify the functions of PagL.

    ACKNOWLEDGEMENTS

M. S. T. thanks K. J. Babinski, S. S. Basu, and M. I. Kanipes for advice and help during the completion of this work.

    FOOTNOTES

* This research was supported by National Institutes of Health Grants GM-51310 (to C. R. H. R.) and AI-30479 (to S. I. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by National Institute of Health Grant 1 F32 AI1056-01.

|| To whom correspondence may be addressed: P. O. Box 3711, Durham, NC 27710. Tel.: 919-684-5326; Fax: 919-684-8885; E-mail: raetz@biochem.duke.edu.

** To whom correspondence may be addressed: Depts. of Microbiology and Medicine, University of Washington, K-140 Health Sciences Bldg., Box 357710, Seattle, WA 98195. Tel.: 206-616-5107; Fax: 206-616-4295; E-mail: millersi@u.washington.edu.

Published, JBC Papers in Press, December 6, 2000, DOI 10.1074/jbc.M010730200

2 S. Trent and C. R. H. Raetz, unpublished results.

3 M. S. Trent, W. Pabich, S. I. Miller, and C. R. H. Raetz, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: LPS, lipopolysaccharide; Kdo, 3-deoxy-D-manno-octulosonic acid; PCR, polymerase chain reaction; CAPS, 3-(cyclohexylamino)propanesulfonic acid; bis-Tris, 2-[bis(2-hy-droxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.

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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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