From the Department of Biochemistry, Duke University
Medical Center, Durham, North Carolina 27710 and the
§ Department of Pharmacology and Molecular Sciences, The
Johns Hopkins University School of Medicine,
Baltimore, Maryland 21205
Received for publication, October 3, 2000, and in revised form, October 19, 2000
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ABSTRACT |
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Certain strains of Escherichia coli
and Salmonella contain lipopolysaccharide (LPS) modified
with a phosphoethanolamine (pEtN) group at position 7 of the outer
3-deoxy-D-manno-octulosonic acid (Kdo) residue.
Using the heptose-deficient E. coli mutant WBB06 (Brabetz,
W., Muller-Loennies, S., Holst, O., and Brade, H. (1997) Eur.
J. Biochem. 247, 716-724), we now demonstrate that the
critical parameter determining the presence or absence of pEtN is the
concentration of CaCl2 in the medium. As judged by mass
spectrometry, half the LPS in WBB06, grown on nutrient broth with 5 mM CaCl2, is derivatized with a pEtN group,
whereas LPS from WBB06 grown without supplemental CaCl2 is
not. Membranes from E. coli WBB06 or wild-type W3110 grown
on 5-50 mM CaCl2 contain a novel pEtN
transferase that uses the precursor
Kdo2-[4'-32P]lipid IVA as an
acceptor. Transferase is not present in membranes of E. coli grown with 5 mM MgCl2,
BaCl2, or ZnCl2. Hydrolysis of the in
vitro reaction product,
pEtN-Kdo2-[4'-32P]lipid IVA, at
pH 4.5 shows that the pEtN substituent is located on the outer Kdo
moiety. Membranes from an E. coli pss knockout mutant grown
on 50 mM CaCl2, which lack
phosphatidylethanolamine, do not contain measurable transferase
activity unless exogenous phosphatidylethanolamine is added back to the
assay system. The induction of the pEtN transferase by 5-50
mM CaCl2 suggests possible role(s) in
establishing transformation competence or resisting environmental
stress, and represents the first example of a regulated covalent
modification of the inner core of E. coli LPS.
Lipopolysaccharide
(LPS)1 is the major
constituent of the outer leaflet of the outer membranes of
Gram-negative bacteria (1-5). LPS consists of three covalently linked
domains. These are: 1) the lipid A moiety, a glucosamine-based
phospholipid that serves as the hydrophobic membrane anchor of LPS; 2)
the core region, a nonrepeating oligosaccharide decorated with several
phosphate-containing substituents; and 3) the O-antigen, a distal
repeating oligosaccharide (Fig. 1). The lipid A and the
3-deoxy-D-manno-octulosonic acid (Kdo) portions
of the LPS core are essential for the growth of Escherichia
coli and most other Gram-negative bacteria (1-5). Mutants lacking
LPS sugars distal to Kdo are usually viable, but often show reduced
virulence and sensitivity to killing by serum (5).
The inner core of E. coli K-12 LPS consists of two Kdo
residues and two (or three)
L-glycero-D-manno-heptose
moieties (Fig. 1) (2, 6). The latter are usually derivatized with
phosphate and ethanolamine pyrophosphate groups (Fig. 1) (5). Mutations in the biosynthesis or incorporation of heptose, including ones that
eliminate the phosphate-containing substituents, reduce the stability
of the outer membrane (2, 4, 7, 8). The resulting "deep rough"
phenotype includes hypersensitivity to certain antibiotics, decreased
levels of outer membrane proteins, and resistance to bacteriophages
that utilize LPS core sugars as receptors (2, 4, 7, 8).
Recently, Brabetz and co-workers (9) constructed an E. coli
deletion mutant (WBB06) that lack both heptosyltransferase I
(WaaC/RfaC) and heptosyltransferase II (WaaF/RfaF), resulting in the
production of a truncated LPS missing all sugars distal to Kdo (Fig.
1). Intriguingly, the authors noted that deletion of both
heptosyltransferases was associated with the formation of an unusual
LPS structural variant in WBB06 (Fig. 1),
characterized by a high degree of pEtN substitution at position 7 of
the outer Kdo residue (9). Modification of Kdo with pEtN had not been observed in earlier studies of similarly truncated LPS molecules (10),
isolated from heptosyltransferase-deficient point mutants of E. coli. We therefore re-examined the structure of intact LPS from
WBB06, using newer purification and MALDI-TOF mass spectrometry methods
(11). We found no evidence for a pEtN substituent on this LPS when the
cells were grown in standard LB broth (12). However, supplementation of
the medium with high concentrations of CaCl2 (5-50
mM) resulted in the appearance of a Kdo-linked pEtN moiety,
as reported by Brabetz et al. (9), who had routinely included 5 mM CaCl2 in their media to increase
the growth rate of WBB06.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic molecular structure of E. coli K-12 LPS. The heptose units of the inner core are
constitutively modified with phosphate and ethanolamine pyrophosphate
groups (5, 6, 8). In certain strains of E. coli and
Salmonella, however, a pEtN moiety has also been found on
the outer Kdo residue (5, 9). As shown in the present study, the
presence of the pEtN unit on the outer Kdo of E. coli WBB06
is dependent on 5-50 mM CaCl2 in the growth
medium.
We have now discovered a novel CaCl2-induced enzyme that
modifies the outer Kdo moiety of E. coli LPS with a pEtN
group. We have developed an assay for this enzyme using membranes from
cells grown in the presence of 5-50 mM CaCl2.
The LPS precursor, Kdo2-[4'-32P]lipid
IVA (13), functions as an acceptor of the pEtN moiety in
our in vitro system (Fig. 2).
The conditions that induce the pEtN transferase share some similarities
with the treatments that make E. coli cells competent for
DNA-mediated transformation (14, 15). The mechanism of pEtN transferase
induction by CaCl2 may provide new insights into the
functions of the LPS core. We are not aware of any previous reports
of CaCl2-induced enzymes in E. coli.
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EXPERIMENTAL PROCEDURES |
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Materials--
[-32P]ATP was purchased from
Perkin Elmer Life Science. Kdo, Hepes, CaCl2, and
phospholipids were obtained from Sigma. Triton X-100 was purchased from
Pierce. Yeast extract and tryptone were obtained from Difco. The Silica
Gel 60 thin layer chromatography plates were from Merck. Pyridine,
chloroform, and methanol were of reagent grade and were purchased from VWR.
Bacterial Strains and Growth Conditions-- The E. coli deep rough mutant strain WBB06, harboring a deletion that removes portions of both the waaC and waaF genes, was kindly provided by Dr. Werner Brabetz (9). E. coli strains W3110 and W3899 were obtained from the E. coli Genetic Stock Center, Yale University. Strains were generally grown in LB broth (10 g of NaCl, 10 g of tryptone, and 5 g of yeast extract per liter) (12). When necessary, the cultures were supplemented with tetracycline (12 µg/ml). In some experiments, CaCl2 was added to the medium at final concentrations ranging from 5 µM to 50 mM, as indicated. E. coli strain AD90 was obtained from Dr. William Dowhan at the University of Texas, Houston (16). AD90 harbors an insertion mutation in the pss gene, but is able to grow without the pss+ covering plasmid when 20-50 mM MgCl2 or CaCl2 is added to the medium. Under these conditions, the membranes of AD90 lack detectable phosphatidylethanolamine.
Preparation of E. coli Membranes--
Typically, cell-free
extracts were prepared by growing a 100-ml LB broth culture,
supplemented with or without various divalent cations, at 37 °C to
A600 = 1.7. Cells were harvested by
centrifugation at 4,000 × g for 10 min. Cells were
resuspended in 4 ml of 50 mM Hepes, pH 7.5, and broken by a
single passage through a French pressure cell at 18,000 psi. Cellular
debris was removed by centrifugation at 4000 × g for
15 min. Membranes were prepared by ultracentrifugation at 100,000 × g for 60 min. The membranes were resuspended in 8 ml of
Hepes, pH 7.5, and the ultracentrifugation was repeated a second time
at 100,000 × g to remove any contaminating cytosol. The final membrane pellet was resuspended in 1 ml 50 mM
Hepes, pH 7.5, and stored at 80 °C until further use. Protein
concentrations were determined by the bicinchoninic acid method
(Pierce) (17).
In Vitro Assay for the CaCl2-induced pEtN
Transferase--
The calcium-induced phosphoethanolamine transferase
was assayed in cell-free extracts or membrane preparations using the
LPS precursor, Kdo2-[4'-32P]lipid
IVA, which was prepared enzymatically and purified as described previously (13, 18). The radiolabeled substrate was stored at
20 °C as a frozen aqueous dispersion at ~60,000 cpm/µl in 10 mM Tris HCl, pH 7.9, with 1 mM EDTA and 1 mM EGTA. Prior to each use, the radiolabeled substrate was
subjected to ultrasonic irradiation in a water bath sonicator for 1 min. The pEtN transferase was assayed in a reaction mixture (10 µl
final volume) containing 50 mM Hepes, pH 7.5, 0.1% Triton
X-100, 1.25 mM dithiothreitol, and 2.5 µM
Kdo2-[4'-32P]lipid IVA
(~250,000 cpm/nmol). The reactions were started by addition of
enzyme, and were incubated for the indicated time periods at 30 °C.
The reactions were stopped by application of 4-µl samples directly
onto a silica gel TLC plate. After drying at room temperature, the
plate was developed in the solvent chloroform, pyridine, 88%
formic acid, water (30:70:16:10, v/v). After drying, the plate was
analyzed and the percent conversion to product quantified using a
Molecular Dynamics PhosphorImager (STORM 840), equipped with ImageQuant software.
Characterization of the in Vitro pEtN Transferase Reaction Product by Hydrolysis at pH 4.5-- Two 10-µl reaction tubes were prepared containing 50 mM Hepes, pH 7.5, 0.1% Triton X-100, 2.5 µM Kdo2-[4'-32P]lipid IVA (8,000 cpm/tube) or 2.5 µM pEtN-Kdo2-[4'-32P]lipid IVA (8,000 cpm/tube). The pEtN-Kdo2-[4'-32P]lipid IVA sample was generated using the standard pEtN transferase assay conditions, described above, and was purified by thin layer chromatography, as reported for the substrate Kdo2-[4'-32P]lipid IVA (13, 18). Next, 4 µl of 10% SDS and 26 µl of 50 mM sodium acetate, pH 4.5, were added to each tube. The tubes were then incubated in a boiling water bath. At various times, 4-µl samples were withdrawn and spotted onto a silica TLC plate. The plate was developed in the solvent chloroform, pyridine, 88% formic acid, water (30:70:16:10, v/v), and analyzed with a PhosphorImager, as described above for the assay.
Extraction and Purification of LPS from the Heptose-deficient
Mutant WBB06--
To determine what fraction of the LPS molecules in
WBB06 are covalently modified with pEtN, 100 ml of LB broth cultures of WBB06 were grown in the presence or absence of 5 mM
CaCl2 to A600 = 1.7. Cells were
harvested by centrifugation at 4,000 × g for 10 min
and washed once with 40 ml of phosphate-buffered saline (19).
The final cell pellets were resuspended in 40 ml of phosphate-buffered saline. Glycerophospholipids and LPS were extracted for 1 h at room temperature with a single phase Bligh/Dyer mixture (20), generated
by adding 100 ml of methanol and 50 ml of chloroform to the cell
suspension. This method is applicable to truncated "Re" LPS (2)
made by mutants defective in heptose biosynthesis, which is soluble in
this mixture, but not to more extensively glycosylated LPS molecules.
The precipitated proteins and nucleic acids were removed by
centrifugation at 4,000 × g for 15 min. The
supernatant was converted to a two-phase Bligh/Dyer system, which
consists of chloroform/methanol/water (2:2:1.8, v/v), by adding
appropriate volumes of chloroform and water. The phases were separated
by centrifugation at 4,000 × g for 15 min. The lower
phase was dried by rotary evaporation, re-dissolved in 5 ml of
chloroform/methanol/water (2:3:1, v/v), and applied to a 1-ml DEAE
cellulose column (acetate form) equilibrated with the same solvent
mixture (11, 21, 22). Following sample loading, the column was washed
with 10 column volumes of chloroform/methanol/water (2:3:1, v/v). The
bound phospholipids and LPS were eluted with 5-ml steps of
chloroform/methanol/aqueous ammonium acetate (2:3:1, v/v), with
ammonium acetate concentrations of 60, 120, 240, and 480 mM
successively in the aqueous component. For each step elution, 1-ml
fractions were collected. The appearance of the phospholipids and the
LPS was determined by spotting 10-µl portions of each fraction onto a
silica TLC plate. The plate was developed in the solvent chloroform,
pyridine, 88% formic acid, water (30:70:16:10, v/v). After drying, the
substances were visualized by spraying the plate with 10% sulfuric
acid in ethanol and charring on a hot plate. The Re LPS emerged at the
very end of the chloroform, methanol, 240 mM aqueous
ammonium acetate wash and at the beginning of the chloroform, methanol,
480 mM aqueous ammonium acetate step. All fractions
containing LPS were pooled and converted to a two-phase Bligh/Dyer
mixture by addition of appropriate amounts of chloroform and water. The
lower phase was dried under a stream of N2 and stored at
20 °C.
Mass Spectrometry of Intact LPS from WBB06--
Spectra were
acquired in either the positive-ion or the negative-ion mode on a
Kratos Analytical (Manchester, United Kingdom) time of flight
matrix-assisted laser desorption-ionization (MALDI) mass spectrometer,
equipped with a 337-nm nitrogen laser, a 20 kV extraction voltage, and
time-delayed extraction (11). Each spectrum was the average of 50 shots. The matrix was a mixture of saturated 6-aza-2-thiothymine in
50% aqueous acetonitrile and 10% tribasic ammonium citrate (9:1, v/v)
for the negative mode. WBB06 LPS samples were first dissolved in a
mixture of chloroform/methanol (4:1, v/v) before being mixed with the
matrix (1:1, v/v) on a slide (0.6 µl final volume). The sample/matrix
mixture was allowed to dry at room temperature prior to mass analysis.
Hexa-acylated lipid A 1,4'-bis-phosphate from E. coli, used to calibrate the spectrometer, was purchased from Sigma.
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RESULTS |
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MALDI-TOF Mass Spectrometry of LPS Isolated from E. coli
WBB06--
Re LPS from WBB06, a heptose-deficient E. coli
mutant with a deletion that inactivates the adjacent
waaC(rfaC) and the
waaF(rfaF) genes, was previously reported to
contain a pEtN substituent attached to its outer Kdo residue (9).
However, in our initial experiments, MALDI-TOF mass spectrometry of LPS
isolated from WBB06 cells grown on LB broth did not detect any
pEtN-containing species (Fig. 3, panel A). The negative mode spectrum showed major ions at
m/z 2317.2, 2237.8, 1876.2, and 1796.8 atomic mass units.
The signal at m/z 2317.2 can be attributed to
[M-H] of a hexa-acylated (Kdo)2-lipid A
variant with a 1-pyrophosphate substituent (Fig. 3, panel A,
inset) (11, 23). The signal at m/z 2237.8 atomic
mass units is interpreted as [M-H]
of the predominant
hexa-acylated (Kdo)2-lipid A 1,4'-bis-phosphate molecule (Fig. 3, panel A), characteristic of E. coli K-12. The peak at m/z 1876.2 atomic mass units
could arise by loss of the Kdo disaccharide during mass spectrometry
from the 1-pyrophosphate variant, while the peak at m/z
1796.8 atomic mass units is explained by loss of the Kdo disaccharide
from the 1,4'-bis-phosphate species. There is no evidence
for peaks indicative of LPS molecules derivatized with an additional
pEtN moiety on their outer Kdo moiety (or elsewhere), as would be
expected around 2360 or 2440 atomic mass units for the
bis-phosphate and the 1-pyrophosphate variant,
respectively.
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Effects of Supplementation with 5 mM CaCl2
in the Growth Medium on the Structure of WBB06 LPS--
The absence of
a molecular species with a pEtN substituent in the spectrum of Fig. 3,
panel A, was explained by the unanticipated effect of
CaCl2 on the structure of E. coli LPS. Brabetz
et al. (9) had noted that the growth rate of WBB06 was
increased to that of wild type E. coli by the inclusion of 5 mM CaCl2 in the medium. Therefore, their
experiments were carried out with LPS isolated from
calcium-supplemented cells. When we grew WBB06 in LB broth in the
presence of 5 mM added CaCl2, MALDI-TOF
analyses revealed that a pEtN unit was indeed present in about half of the LPS molecules (Fig. 3, panel B). The proposed structure
of this pEtN-(Kdo)2-lipid A 1,4'-bis-phosphate
species is shown in Fig. 3B, inset. The negative
mode spectrum showed major ions at m/z 2359.6, 2237.3, and
1797.4 atomic mass units. The peaks at m/z 2359.6 and 2237.3 atomic mass units are attributed to [M-H] of
pEtN-(Kdo)2-lipid A and (Kdo)2-lipid A,
respectively, both in the form of the 1,4'-bis-phosphate
species (Fig. 3, panel B). (The 1-pyrophosphate variant of
lipid A seen in Fig. 3, panel A, is apparently suppressed by
the inclusion of 5 mM CaCl2 in the growth
medium.) The peak at m/z 1797.4 could arise during mass
spectrometry by cleavage of the inner Kdo-lipid A linkage of either the
m/z 2359.6 or the m/z 2237.3 species. Whether the pEtN is attached to the outer or the inner Kdo residue cannot be
determined from the results shown in Fig. 3. However, the spectra demonstrate unequivocally that the addition of the pEtN moiety to the
Kdo region of Re LPS is absolutely dependent upon the presence of
CaCl2 in the growth medium. Addition of the pEtN
substituent to the lipid A moiety is excluded by the absence of a
significant peak at m/z 1920 atomic mass units (Fig. 3,
panel B) (11). Almost all of the minor peaks in Fig. 3 (not
labeled) are explained by the presence of a small amounts of
penta-acylated lipid A species in this particular LPS preparation
(i.e. missing the myristoyl chain that is incorporated by
MsbB) (24, 25).
Calcium-induced Expression of a Novel pEtN Transferase in E. coli
Membranes--
Washed membranes, obtained from WBB06 or wild-type
W3110 grown with or without 5 mM CaCl2, were
assayed for pEtN transferase using
Kdo2-[4'-32P]lipid IVA as the
acceptor substrate. The addition of a pEtN unit to
Kdo2-[4'-32P]lipid IVA generates
a more hydrophilic lipid product that migrates slower than
Kdo2-[4'-32P]lipid IVA. As shown
in Fig.4, membranes of E. coli
W3110 and WBB06 that had been grown in 5 mM
CaCl2 (lanes 3 and 4) supported the
conversion of Kdo2-[4'-32P]lipid
IVA to a new polar product with the expected Rf. However, membranes from the same strains grown in the absence of added CaCl2 did not (Fig. 4, lanes 1 and
2). All subsequent enzymatic experiments were done using
membranes from strain WBB06 grown in the presence of CaCl2,
since membranes of this strain displayed somewhat higher pEtN
transferase activity than membranes of W3110 (Fig. 4, lanes
4 and 3, respectively).
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A quantitative assay was developed for the calcium-induced pEtN
transferase using Kdo2-[4'-32P]lipid
IVA as substrate (Fig. 2). As expected for an enzyme that modifies the Kdo region, [4'-32P]lipid IVA
does not serve as a substrate (not shown). The reaction has a pH
optimum around 7.5 and is dependent upon the presence of the non-ionic
detergent Triton X-100 (0.05-0.1%). Dithiothreitol (1.25 mM) is slightly stimulatory (not shown). EDTA added to the assay at 1 mM is strongly inhibitory (not shown), but this
inhibition is reversed by inclusion of 1-2 mM
MgCl2 or CaCl2. In the absence of added EDTA,
however, supplementation of the assay mixture with 1-10 mM
MgCl2 or CaCl2 is not stimulatory (not shown),
suggesting that relatively low concentrations of these divalent cations
are sufficient. The final optimized reaction mixture consisted of 50 mM Hepes, pH 7.5, 0.1% Triton X-100, 1.25 mM
dithiothreitol, and 2.5 µM
Kdo2-[4'-32P]lipid IVA. Product
formation was proportional to time and protein concentration (Fig.
5, panels A and B).
The calculated specific activity of the pEtN transferase in WBB06
membranes assayed at 1.25 mg/ml was about 8 pmol/min/mg. No activity
was present in a membrane-free high-speed supernatant.
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Effect of Various Divalent Cations in the Growth Medium on the
Expression of pEtN Transferase Activity--
E. coli mutant
WBB06 was grown on LB broth in the presence of supplemental
concentrations of CaCl2 ranging from 5 µM to
50 mM. Transferase activity was the highest when the
membranes were obtained from cells grown in 50 mM
CaCl2 (Fig. 6, lane
7). No activity was observed in membranes from cells grown with
0.5 mM supplemental CaCl2 or less (Fig. 6).
WBB06 was also grown in the presence of other divalent cations to
determine whether or not the induction effect is specific for calcium.
Membranes were isolated from WBB06 cells grown with supplemental 5 mM MgCl2, BaCl2, or ZnCl2, and were assayed for transferase activity. None of
these alternative divalent cations could substitute for
CaCl2 (Fig. 7).
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Location of the pEtN Substituent in the Reaction Product as Judged by pH 4.5 Hydrolysis at 100 °C-- Hydrolysis in sodium acetate buffer, pH 4.5, at 100 °C was used to determine the location of the hydrophilic modification present in the presumed in vitro product, pEtN-Kdo2-[4'-32P]lipid IVA. Hydrolysis at pH 4.5 under these conditions cleaves only those glycosidic linkages involving the anomeric carbons of Kdo, thereby randomly splitting off the outer and the inner Kdo moieties of pEtN-Kdo2-[4'-32P]lipid IVA at comparable rates (26, 27).
Following prolonged incubation of the substrate
Kdo2-[4'-32P]lipid IVA at
100 °C, [4'-32P]lipid IVA eventually
emerges as the sole hydrolysis product (Fig.
8, panel A), but with
transient appearance of equivalent amounts of
Kdo-[4'-32P]lipid IVA and
[4'-32P]lipid IVA at early time points.
Hydrolysis of the putative
pEtN-Kdo2-[4'-32P]lipid IVA (Fig.
8, panel B) shows the same time course and pattern of
product formation. If the hydrophilic pEtN substituent were attached to
the inner Kdo residue, a different pattern of radioactive lipids would
have been observed, since release of the outer Kdo residue would have
yielded an intermediate lipid species containing a single Kdo still
derivatized with the pEtN moiety (27). The latter hydrolysis
intermediate would have migrated more slowly on silica TLC than the
intermediate Kdo-[4'-32P]lipid IVA (27). The
nearly identical time courses and patterns of fragments obtained with
Kdo2-[4'-32P]lipid IVA and
pEtN-Kdo2-[4'-32P]lipid IVA under
these hydrolysis conditions reveal that the calcium-induced pEtN
transferase modifies the outer Kdo residue.
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Demonstration that Phosphatidylethanolamine Is the Donor Substrate
for the pEtN Transferase--
E. coli cells with a null
mutation in the pss gene, which encodes phosphatidylserine
synthase, are unable to generate phosphatidylethanolamine (16). Such
mutants are able to grow slowly in the presence of 50 mM
CaCl2. Membranes of the pss null mutants AD90
grown with 50 mM CaCl2 were prepared and
assayed for pEtN transferase activity with
Kdo2-[4'-32P]lipid IVA as the
acceptor. As shown in Fig. 9 (panel
A, lane 3), the absence of phosphatidylethanolamine in E. coli membranes completely abolishes transferase activity. However,
membranes from the calcium-induced parental wild-type strain W3899 from which AD90 was derived (16) possess robust pEtN transferase activity
(Fig. 9, panel A, lane 2). The results strongly suggest that
phosphatidylethanolamine is the donor substrate for the
transferase.
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To confirm this conclusion, various commercially available phospholipid
preparations were added back to the membranes of the phosphatidylethanolamine-deficient strain AD90 in an attempt to reconstitute pEtN transferase activity. As shown in Fig. 9A,
lane 4, only phosphatidylethanolamine (1-oleoyl,2-palmitoyl)
resulted in partial restoration of enzymatic activity, whereas
phosphatidylglycerol and phosphatidylcholine with the same fatty acid
distributions (Fig. 9A, lanes 5 and 6) did not.
Phosphatidylethanolamine isolated from E. coli was the most
effective (Fig. 9B, lane 7), although dilauroyl
phosphatidylethanolamine (Fig. 9B, lane 3) and various unsaturated phosphatidylethanolamine preparations (Fig. 9B, lanes 8 and 9) were also very effective. Interestingly, both
dipalmitoyl and distearoyl phosphatidylethanolamine were completely
inactive (Fig. 9B, lanes 5 and 6). Taken
together, these findings strongly support the view that
phosphatidylethanolamine is the physiologically relevant pEtN donor in
our enzymatic system.
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DISCUSSION |
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Brabetz et al. (9) recently reported that the E. coli mutant WBB06, which harbors a deletion spanning the waaC(rfaC) and waaF(rfaF) genes, contains Re LPS that is decorated with a pEtN moiety at position 7 of the outer Kdo sugar. Previous investigators, using Re LPS isolated from E. coli strains harboring waaC point mutations (10), had not observed this modification. We have now resolved this discrepancy by demonstrating that this particular modification of LPS with pEtN in living cells is the consequence of growth in the presence of 5-50 mM CaCl2 (Fig. 3), and is unrelated to the deletion of the heptosyltransferase genes in WBB06. The earlier studies with point mutants had been carried out with cells grown in the absence of CaCl2, whereas Brabetz et al. (9) included 5 mM CaCl2 in their media to enhance the growth rate of their mutant strain.
We have found for the first time conditions for assaying an E. coli enzyme that modifies the outer Kdo sugar of the LPS core with a pEtN moiety. Membranes from wild-type W3110 and mutant WBB06 grown in the presence of 5-50 mM CaCl2 (Figs. 4 and 6) both contain a novel activity that utilizes the intermediate Kdo2-[4'-32P]lipid IVA as an acceptor substrate. The enzyme is not detected in membranes from cells grown with other divalent cations (Fig. 7). We suggest that the enzyme modifies the outer Kdo sugar with pEtN (Fig. 2) based on the following lines of evidence. First, the in vitro activity that we detect in membranes from E. coli cells grown in the presence of 5-50 mM CaCl2 parallels the in vivo modification of LPS with pEtN (Figs. 3 and 4). Second, the results from the pH 4.5 hydrolysis of the pEtN-Kdo2-[4'-32P]lipid IVA product generated in vitro show that the modification is indeed located on the outer Kdo residue (Fig. 8). Third, the calcium-induced pEtN transferase is not seen in membranes of an E. coli pss null mutant (16), which is completely deficient in the biosynthesis of phosphatidylethanolamine, strongly suggesting that phosphatidylethanolamine is the donor substrate for the pEtN moiety (Figs. 2 and 9). In fact, reconstitution of the assay system with 1 mM phosphatidylethanolamine, but not phosphatidylglycerol or phosphatidylcholine, partially restores enzymatic activity (Fig. 9). The exact location of the pEtN substituent on the outer Kdo residue of our in vitro product remains to be established.
The regulatory mechanisms that high calcium concentrations induce to bring about the modification of the outer Kdo sugar with pEtN are uncertain. In some Gram-negative bacteria, the signal transduction system, PhoP/PhoQ, is activated by low concentrations of Mg2+ or Ca2+ (10 µM) (28). The PhoP/PhoQ system regulates the expression of at least 40 genes, some of which encode enzymes that modify lipid A (29). In the presence of high concentrations of Mg2+ or Ca2+ (10 mM), the PhoP/PhoQ system is shut off (30, 31). Since pEtN transferase activity is observed only when cells are grown in 5-50 mM CaCl2 (Figs. 6 and 7), expression of the enzyme is not likely to be regulated by PhoP/PhoQ, which in contrast to our system, senses both Mg2+ and Ca2+. It will be interesting to determine the regulatory mechanism by which high CaCl2 concentrations induce the pEtN transferase. We consider a transcriptional effect the most likely possibility, but we are not aware of other E. coli enzymes or proteins that are induced by high Ca2+, perhaps only because this phenomenon has received so little attention.
Previous studies have shown that the inner heptose residue of E. coli LPS (Fig. 1) is modified in a constitutive manner with an ethanolamine pyrophosphate group (8, 32). Based on radioactive labeling studies with living cells, phosphatidylethanolamine is thought to be the precursor of the ethanolamine phosphate portion of the ethanolamine pyrophosphate moiety on heptose (33). However, in vitro systems have not been reported for ethanolamine pyrophosphate biosynthesis. It is unlikely that our calcium-induced pEtN transferase plays a role in modification of the heptose region, since the latter is independent of calcium (5, 8, 34). However, the pEtN transferases that modify the Kdo and heptose regions might display sequence or structural homology to each other. Unfortunately, the relevant genes encoding these enzymes are not yet known.
The function of our calcium-induced pEtN transferase is
uncertain. It might play a role in rendering cells competent for
DNA-mediated transformation, given that the conditions used to make
cells competent involve exposure to high levels of CaCl2
(14, 15), similar to what we use to induce the pEtN transferase.
Alternatively, the presence of the pEtN moiety on the outer Kdo sugar
might ensure resistance of the outer membrane to environmental
stresses, such as exposure to certain detergents, antibiotics, or
serum. The latter contains about 1 mM Ca2+,
which might be sufficient to trigger induction. Future studies will be
aimed at cloning the gene encoding the calcium-induced pEtN
transferase, since the characterization of a null mutant should reveal
the function of this unusual enzyme.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM-51310 (to C. R. H. R.) and GM-54882 (to R. J. C.).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.
¶ To whom correspondence should be addressed. Tel.: 919-684-5326; Fax: 919-684-8885; E-mail: raetz@biochem.duke.edu.
Published, JBC Papers in Press, October 19, 2000, DOI 10.1074/jbc.M009019200
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ABBREVIATIONS |
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The abbreviations used are: LPS, lipopolysaccharide; MALDI-TOF, matrix-assisted laser desorption-ionization time of flight; Kdo, 3-deoxy-D-manno-octulosonic acid; pEtN, phosphoethanolamine.
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