From the 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
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ABSTRACT |
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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.
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
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,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 -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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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.
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EXPERIMENTAL PROCEDURES |
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Chemicals and Other Materials--
[-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 [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
[
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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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 pagL construct. Allelic
exchange was performed in strain CS401, as has been described (46).
Resolution of the integrant resulted in a
pagL
strain CS586, which was verified using PCR and Southern blot analysis.
To create a nonpolar 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
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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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--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
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.
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RESULTS |
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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
-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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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 -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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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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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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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.
|
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.
|
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
(phoPCpagL) 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.
|
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
-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
-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).
|
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).
|
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.
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DISCUSSION |
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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 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
-barrel conformation that is characteristic of outer membrane
proteins, consisting of only eight anti-parallel
-strands (59).
Analysis of the PagL amino acid sequence (Fig. 9) with programs for
predicting secondary structure reveals significant
-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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