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INTRODUCTION |
Lipopolysaccharide
(LPS)1 is a major component
of the outer membranes of Gram-negative bacteria (1-5). The lipid A
moiety of LPS makes up much of the outer monolayer of the outer
membrane (1-5). LPS acts as barrier to antibiotics (6, 7) and helps bacterial cells resist complement-mediated lysis (8). The lipid A
portion of LPS is essential for bacterial viability (9-11), and it is
also the active component of LPS responsible for many of the
pathophysiological effects associated with Gram-negative infections in
animals, including septic shock (3, 4, 11, 12).
In certain plant systems LPS is required for the establishment of
symbiosis between nitrogen-fixing strains of Rhizobium and their hosts (13, 14). Rhizobium leguminosarum mutants with truncated LPS structures lacking O-antigens are defective in generating functional nodules within root cells (15-19). Changes in LPS structure are also associated with the physiological adaptation to the symbiotic microenvironment (20, 21). For instance, developmentally regulated expression of distinct LPS epitopes can be demonstrated in
planta by immunostaining (22). Whether or not the lipid A
moiety of R. leguminosarum LPS plays a role in infection and
nodulation is unknown, since defined mutants in the lipid A pathway are
not yet available. A complete understanding of the structure,
biosynthesis, and molecular genetics of lipid A in R. leguminosarum is a prerequisite for defining its functions during symbiosis.
The structure of lipid A in R. leguminosarum is strikingly
different from that of E. coli (23-26) (Fig.
1A). R. leguminosarum lipid A lacks the 1- and
4'-phosphate groups found in the lipid A of most other Gram-negative
bacteria (23, 24). A galacturonic acid residue is incorporated in place
of the 4'-phosphate, and the proximal glucosamine 1-phosphate unit of
Escherichia coli lipid A may be replaced with an
aminogluconate moiety (23, 24) (Fig. 1A). R. leguminosarum lipid A also lacks the laurate and myristate
residues present in E. coli lipid A (3, 27) but is acylated
with an unusual 28-carbon chain (23, 24, 28). Despite these
differences, both E. coli and R. leguminosarum
employ the same seven enzymes to generate the conserved,
phosphate-containing precursor, Kdo2-lipid IVA
(Fig. 1B) (29). Several distinct enzymes must therefore
exist in R. leguminosarum that catalyze the conversion of
Kdo2-lipid IVA to R. leguminosarum
lipid A. We have recently discovered a 4'-phosphatase (30), a
1-phosphatase (31), a long chain acyltransferase (32), and a mannosyl
transferase (31, 33, 34) that are involved in the processing of
Kdo2-lipid IVA in extracts of R. leguminosarum but not of E. coli. Many additional enzymes unique to R. leguminosarum remain to be found.
We now report a novel, chemically specific and highly active
phosphotransferase reaction associated with the 4'-phosphatase of
R. leguminosarum (CE3) (Fig. 1B). In the presence
of phosphatidylinositol (PtdIns), the 4'-phosphatase can transfer the
4'-phosphate of Kdo2-[4'-32P]lipid
IVA (and related metabolites including E. coli
lipid A) to the inositol moiety of PtdIns, generating exclusively
PtdIns-4-P (Fig. 1B). The
phosphotransferase and the previously described 4'-phosphatase activity
(30) appear to be catalyzed by the same enzyme, given their identical
behavior during purification and thermal inactivation. Although the
apparent Km of the phosphotransferase for PtdIns is
about 10 times higher than that of many eucaryotic PtdIns 4-kinases
(35-37), its high specific activity in cell extracts and its absolute
specificity for the 4-position of PtdIns suggest that it could play a
significant role during symbiosis. A procaryotic pathway for the
biosynthesis of PtdIns-4-P and a direct connection between lipid A
formation and PtdIns phosphorylation have not been reported
previously.

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Fig. 1.
Structures of E. coli and
R. leguminosarum lipid A and of
Kdo2-[4'-32P]lipid IVA.
A, the 1-, 3-, and 4'-positions of each lipid A structure
are indicated. Evidence for the presence of an acyloxyacyl residue and
partially deacylated species has recently been presented by Que
et al. (25, 26). B, the same seven enzymes
catalyze the formation of Kdo2-lipid IVA in
both organisms (29). A phosphoenzyme intermediate could explain all of
the reactions catalyzed by the 4'-phosphatase/phosphotransferase of
R. leguminosarum.
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EXPERIMENTAL PROCEDURES |
Chemicals and Materials--
[
-32P]ATP was
obtained from NEN Life Science Products.
Ptd-[2-3H]Ins-4-P and [1,2-3H]Ins were
purchased from American Radiolabeled Chemicals, Inc. PtdIns-3-P,
digalactosyl diglyceride and monogalactosyl-diglyceride were obtained
from Matreya, Inc. Silica gel 60 (0.25-mm) thin layer plates were
purchased from EM Separation Technology, E. Merck. Triton X-100 and
bicinchoninic acid were from Pierce. Yeast extract and tryptone were
obtained from Difco. Other chemicals were from Sigma or Mallinckrodt.
Bacterial Strains and Growth Conditions--
R.
leguminosarum biovar phaseoli CE3 (also recently
classified as R. etli), mutant 24AR of R. leguminosarum biovar trifolii, R. leguminosarum biovar viciae 8401, Rhizobium
meliloti 1021, and E. coli W3110 were described in
previous studies (29-31, 38). All other strains of
Rhizobium were purchased from the American Type Culture
Collection. All Rhizobium strains were grown at 30 °C on
TY medium, which contains 5 g/liter tryptone, 3 g/liter yeast extract,
10 mM CaCl2, and 20 µg/ml nalidixic acid. For
the growth of strains CE3, 24AR, 1021, and 8401, streptomycin (200 µg/ml) was also added to the medium. E. coli W3110 was
grown on LB broth (39) at 30 °C.
Preparation of Radiolabeled Substrates--
The substrate
[4'-32P]lipid IVA was generated using
[
-32P]ATP, tetraacyldisaccharide 1-phosphate acceptor
(DS-1-P) (40), and membranes of E. coli BLR(DE3)/pLysS/pJK2,
which contain large amounts of the 4'-kinase (41).
Kdo2-[4'-32P]lipid IVA could then
be prepared from the [4'-32P]lipid IVA using
the purified Kdo transferase (38, 42). Alternatively, Kdo2-[4'-32P]lipid IVA was also
prepared directly from DS-1-P and [
-32P]ATP without
purification of the [4'-32P]lipid IVA
intermediate. Briefly, a 100-µCi portion of
[
-32P]ATP (3000 Ci/mmol) was dried in a 1.5-ml
polypropylene microcentrifuge tube under a stream of N2.
Next, the following components were added: (a) 25 µl of a
mixture of DS-1-P and cardiolipin, prepared by mixing 0.5 mg of DS-1-P
and 4 mg of cardiolipin in 1 ml of water, followed by ultrasonic
dispersion; (b) 5 µl of 10% Nonidet P-40, a nonionic
detergent; and (c) 10 µl of 25 mM
MgCl2. The 4'-kinase reaction was started by adding 5 µl
of membranes (500 µg/ml in 50 mM Hepes, pH 7.5) of
E. coli strain BLR(DE3)/pLysS/pJK2 and was held at room
temperature. After 10 min, a second 5-µl portion of the enzyme stock
was added, and the incubation was continued for another 10 min. The Kdo
transferase reaction was then carried out in the same tube by adding
the following: 2 µl of 5 mM lipid IVA, 25 µl of 2% Triton X-100, 50 µl of 100 mM CTP in 0.5 M Hepes, pH 7.5, 50 µl of 40 mM Kdo, 50 µl
of 100 mM MgCl2, 50 µl of purified CMP-Kdo
synthase (0.03 total units) (42, 43), and 25 µl of purified Kdo
transferase (38) (from a stock solution at 80 µg/ml). The final
reaction volume was 500 µl. After incubation at 30 °C for 30 min,
80-90% of the [
-32P]ATP was incorporated into the
Kdo2-[4'-32P]lipid IVA, as judged
by analytical thin layer chromatography and autoradiography.
Next, the reaction mixture was carefully spotted across the origin of a
20 × 20-cm silica gel 60 thin layer plate. The plate was dried
under a cold air stream and developed in the solvent chloroform/pyridine/88% formic acid/water (30:70:16:1, v/v/v/v). The
plate was again dried under a cold air stream and exposed to x-ray film
for 30-60 s to locate the Kdo2-[4'-32P]lipid
IVA band. The appropriate region was scraped and
transferred to a 30-ml sintered glass filter funnel. The silica powder
was washed with 10 ml of chloroform, and the 32P-labeled
product was then eluted with 24 ml of a single phase Bligh-Dyer mixture
(44), consisting of CHCl3/methanol/50 mM ammonium acetate in water adjusted to pH 1.5 with HCl (1:2:0.8, v/v/v).
Three 8-ml fractions were collected in glass tubes. CHCl3 (2.1 ml) and 50 mM ammonium acetate in water adjusted to pH
1.5 with HCl (2.1 ml) were added to each 8-ml fraction to form three two-phase systems. The lower phases were removed with a Pasteur pipette
and pooled into a single 15-ml Corex glass tube equipped with a Teflon
cap. Six drops of HPLC grade pyridine were added, and the solution was
dried under a stream of N2 at room temperature. While the
drying was in progress, two additional extractions of the remaining
three upper phases were carried to optimize product recovery. Each
upper phase was extracted twice with 2 ml of a fresh lower phase from a
pre-equilibrated two-phase Bligh-Dyer system, consisting of
CHCl3/methanol/50 mM ammonium acetate in water
adjusted to pH 1.5 with HCl (1:1:0.9, v/v/v). The lower phases derived
from these additional extractions were pooled into the same 15-ml Corex
tube used for the initial lower phases, and the resulting solution was
again dried under a stream of N2. The final dried
Kdo2-[4'-32P]lipid IVA
preparation was dissolved in 2 ml of CHCl3/MeOH (1:1, v/v)
with vortexing and sonic irradiation, and the material was divided
equally among four 1.5-ml polypropylene microcentrifuge tubes. These
aliquots were dried immediately under a stream of N2.
Additional residual Kdo2-[4'-32P]lipid
IVA was recovered by washing the Corex tube two more times with 2-ml portions of CHCl3/MeOH (1:1, v/v) and
distributing the solution equally among the four microcentrifuge tubes.
Following the final drying under N2, the four portions of
Kdo2-[4'-32P]lipid IVA were each
suspended by sonic irradiation in 500 µl of 10 mM
Tris-HCl, pH 7.8, containing 1 mM EDTA and 1 mM
EGTA, and they were stored at
20 °C. The entire extraction process took about 5 h. A typical final yield of
Kdo2-[4'-32P]lipid IVA (specific
activity ~8 µCi/nmol) following these extractions was ~30% of
the input [
-32P]ATP.
Kdo-[4'-32P]lipid IVA was prepared with the
monofunctional Kdo transferase from Hemophilus influenzae
(45). The substrate [4'-32P]lipid A was prepared from
compound 505 by the action of the highly overexpressed 4'-kinase (41).
The [4'-32P]lipid IVA,
Kdo-[4'-32P]lipid IVA,
Kdo2-[4'-32P]lipid IVA, and
[4'-32P]lipid A were purified by thin layer
chromatography (38, 42, 45) (or see above). These substances were
stored as aqueous dispersions at
20 °C in 10 mM
Tris-HCl, pH 7.8, containing 1 mM EDTA and 1 mM
EGTA. Prior to use, all of the substrates were dispersed again by sonic
irradiation for 1 min in a bath sonicator.
Assays of the 4'-Phosphatase and the Phosphotransferase
Reactions--
Standard assay conditions for the 4'-phosphatase were
as follows. The reaction mixture (10-20 µl) contained 50 mM MES, pH 6.5, 0.1% Triton X-100, 2 mM
dithiothreitol, 2 mM EDTA, 10 mM potassium
phosphate, and 10 µM
Kdo2-[4'-32P]lipid IVA (20,000 cpm/nmol). PtdIns (usually 1 mg/ml or at the indicated concentrations)
was added as the acceptor substrate in the phosphotransferase
reactions. When PtdIns was included as the phosphate acceptor, a 1-µl
solution of PtdIns in chloroform/methanol (4:1, v/v) was transferred to
each reaction tube, and the solvent was removed with a stream of
N2. Next, the Triton X-100 and the other reaction
components were added. The reaction mixture was subjected to sonic
irradiation to disperse the phospholipids into the detergent-containing
buffer. Reactions were initiated with enzyme. The reaction mixtures
were incubated at 30 °C for 15 min or as indicated. Reactions were
terminated by spotting 2-µl samples onto a silica gel 60 thin layer
chromatography plate, which was developed in the solvent
chloroform/pyridine/88% formic acid/water (30:70:16:10; v/v/v/v). The
remaining Kdo2-[4'-32P]lipid IVA,
the 32Pi, and the PtdIns-4-32P were
quantified on the plate using a Molecular Dynamics model 425S
PhosphorImager, equipped with ImageQuant software. The percentage conversion of substrate to product(s) was calculated for each reaction,
and the specific activities were expressed as nmol/min/mg, as indicated.
Partial Purification of the 4'-Phosphatase and Its Associated
Phosphotransferase Activity--
All enzyme preparations were carried
out at 0-4 °C. Protein was determined by the bicinchoninic acid
method (46), using bovine serum albumin as a standard. R. leguminosarum CE3 cells in late logarithmic phase
(A550 = 1.2-1.4) were harvested from 5 liters
of culture by centrifugation (8000 × g, 15 min) and
resuspended in 50 mM Hepes, pH 7.5, to give a final protein
concentration of 8-10 mg/ml. Cells were broken by two passages through
a French pressure cell at 18,000 p.s.i., and the debris was removed by centrifugation at 8000 × g for 15 min. Membranes were
prepared by ultracentrifugation at 149,000 × g for 60 min. The membrane pellet was resuspended in Buffer A (50 mM
Hepes, pH 7.5, 3% glycerol, and 10 mM potassium phosphate)
at ~4-5 mg/ml. Solubilization was carried out by the addition of
10% Triton X-100 (reduced) to yield a protein:detergent ratio of 1:2
(~0.9% final detergent concentration), followed by stirring for
2 h, and centrifugation at 149,000 × g for 60 min. The supernatant, which contained over 95% of both activities, was
collected and stored at
80 °C.
Next, a Q-Sepharose (Amersham Pharmacia Biotech) column (30 ml) was
equilibrated with Buffer B (20 mM Tris-HCl, pH 7.8, 0.15% Triton X-100 (reduced), 2 mM EDTA, 3% glycerol, and 10 mM potassium phosphate). Solubilized membranes (30 ml, 3.1 mg/ml protein) were loaded onto the column. Unbound proteins were
washed out with Buffer C (50 mM MES, pH 6.5, 0.15% reduced
Triton X-100, 2 mM EDTA, 3% glycerol, and 10 mM potassium phosphate containing 100 mM NaCl)
until A280 returned to base line. Elution was
carried out with a 400-ml linear gradient from 0.1 to 1.0 M
NaCl in Buffer C. The column fractions (8 ml) were assayed for the
4'-phosphotransferase and 4'-phosphatase. The active fractions (~80
ml total volume) were pooled, desalted, and concentrated (~3-fold) by
ultrafiltration through an Amicon (30K) filter. The concentrated
material was then diluted to the original volume with Buffer C
containing no detergent. The cycle was repeated three times. The final
concentrated active fractions (~25 ml) from the Q-Sepharose column
were then loaded onto a heparin-agarose Type 1 (Sigma) column (15 ml)
equilibrated with Buffer C. Next, the column was washed with
equilibration buffer containing 100 mM NaCl. The
4'-phosphatase/phosphotransferase activities were eluted with a 200-ml
linear gradient from 0.1 to 1.5 M NaCl in Buffer C. The
column fractions (5 ml) were assayed for both the phosphotransferase
and the 4'-phosphatase. The active fractions were pooled, concentrated
(3 fold), and desalted as above.
Cibacron Blue Column Chromatography and Substrate Elution--
A
Cibacron blue 3GA Type 300 (Sigma) column (3 ml) was equilibrated with
Buffer C (see above). Partially purified enzyme from the
heparin-agarose step (5 ml, 0.3 mg/ml protein) was loaded onto the
column and was recirculated through the column two more times. The
column was then washed successively with 5 ml of Buffer C, 15 ml of
Buffer C containing 0.5 M NaCl, 15 ml of Buffer C containing 0.5% Triton X-100 (reduced), and 10 ml of Buffer C. Finally, the elution of the 4'-phosphatase/phosphotransferase was
carried out with 35 ml of Buffer C containing 0.4 mg/ml PtdIns and 0.2 M NaCl. The column fractions (2.5 ml) were assayed for both
the phosphotransferase and the 4'-phosphatase activities.
Separation of Inner and Outer Membranes--
R.
leguminosarum (CE3) membranes were separated by isopycnic sucrose
gradient centrifugation as described previously (33). The turbidity
(A600) of each fraction was determined to
confirm the presence of membrane fragments, and each fraction was
assayed for NADH oxidase as the inner membrane marker and phospholipase A as the outer membrane marker (33). The protein content of each
fraction was determined by the bicinchoninic acid assay (46) using
bovine serum albumin as the standard. Last, each fraction was assayed
for both phosphotransferase and 4'-phosphatase activities under the
standard assay conditions described above.
Deacylation and HPLC Analysis of the 32P-Labeled
PtdIns Derivative Generated from
Kdo2-[4'-32P]lipid
IVA--
Prior to HPLC analysis, the product of the
phosphotransferase reaction and the appropriate lipid standards were
deacylated. This was accomplished by treating the compounds with
methylamine reagent (47). Briefly, a 0.5-ml portion of methylamine
reagent, prepared by bubbling methylamine (Fluka, Buchs, Switzerland)
at 0 °C through a mixture in which it is very soluble (6.2 ml of methanol, 4.6 ml of deionized water, and 1.5 ml of 1-butanol) to give a
final volume of 20 ml was added to the dried lipids (see Fig. 10
legend). The mixture was incubated at 53 °C for 40 min. Cold
1-propanol (0.2 ml) was added to the sample. After mixing, the sample
was dried and resuspended in 0.2 ml of water. This aqueous dispersion
was extracted three times with 0.5-ml portions of butanol/petroleum
ether/ethyl formate (20:4:1, v/v/v). The lower aqueous phase was dried,
and deacylation products were redissolved in 200 µl of deionized
water for HPLC analysis. Anion exchange HPLC was performed using a
Partisil SAX column (Whatman) (4.6 × 250 mm) (47). The
deacylation products were eluted (flow rate of 1 ml/min) with a linear
gradient from 10 to 510 mM
NH4H2PO4 (pH 3.8) in 50 min.
Effluent from the HPLC column flowed directly into a
BetaRAMTM in-line continuous flow scintillation detector
(INUS, Tampa, FL).
Trapping and Detection of a [32P]Phosphoenzyme
Intermediate Generated by Incubating Enzyme with
Kdo2-[4'-32P]lipid IVA--
The
enzyme (2 µg purified through the heparin-agarose step or 6 µg
purified through the Q-Sepharose step as indicated) was mixed with
(Kdo)2-[4'-32P]lipid IVA (25,000 cpm at 8 µCi/nmol) in a reaction buffer containing 50 mM
MES (pH 6.5), 0.1% Triton X-100, and 2 mM EDTA. The
reaction mixture (10 µl) was incubated at 30 °C for 5 min. The
reaction was quenched by rapidly mixing with 10 µl of water and 10 µl of 0.3 M CAPS buffer (pH 10), containing 12% SDS,
60% glycerol, and bromphenol blue dye. The sample was directly loaded,
without heating, onto a SDS-polyacrylamide gel (10%) to separate the
denatured proteins. The gels were dried and subjected to PhosphorImager analysis for visualization and quantification.
For pulse-chase experiments, three reactions (final volume of 70 µl
each) were set up in the presence of 2 µM
(Kdo)2-[4'-32P]lipid IVA (175,000 cpm/reaction) in the same buffer as described above. Phosphoenzyme
formation was initiated by adding 8.4 µg of concentrated
heparin-agarose enzyme in 3 µl to each tube. After 5 min at 30 °C,
the phosphoenzyme was chased by the addition of either 5 µl of
reaction buffer or unlabeled (Kdo)2-lipid IVA
in reaction buffer to yield final concentrations of 10 or 50 µM. The incubations were continued at 30 °C. Finally,
10-µl portions of the reaction mixtures were withdrawn at different
times after the chase (0.5, 2, 5, and 10 min), and the remaining
[32P]phosphoenzyme was trapped and detected as described above.
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RESULTS |
A Phosphotransferase in Membranes of R. leguminosarum That Uses
Kdo2-[4'-32P]lipid IVA to
Phosphorylate PtdIns--
The 4'-phosphatase found in membranes of
R. leguminosarum (biovars trifolii,
phaseoli, and viciae) but not of R. meliloti or E. coli catalyzes the release of inorganic
phosphate from the 4'-position of the lipid A disaccharide precursor
Kdo2-[4'-32P]lipid IVA (30). In
the presence of phosphatidylinositol, however, which was initially
tested as a stabilizing agent, the same membranes that dephosphorylate
Kdo2-[4'-32P]lipid IVA also
catalyze transfer of 32P from
Kdo2-[4'-32P]lipid IVA onto
PtdIns (Fig. 2, lanes
3, 5, and 6). This phosphotransferase activity is not detected in membranes of the nodulation-deficient mutant 24AR of R. leguminosarum, which lacks the
4'-phosphatase (30) and possesses a 4'-phosphate moiety on its lipid A
(48). As with the 4'-phosphatase (30), no phosphotransferase activity is seen in the cytosol.

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Fig. 2.
Presence of a PtdIns-dependent
phosphotransferase in membranes of R. leguminosarum. Membranes of different strains were
assayed for phosphotransferase activity using the standard assay. A
protein concentration of 0.2 mg/ml was used, and the incubation was
carried out for 20 min at 30 °C. The thin layer chromatographic
analysis of the reaction products generated from
Kdo2-[4'-32P]lipid IVA and PtdIns
are shown. Lane 1, no membranes; lane
2, E. coli W3110; lane 3,
R. leguminosarum biovar etli CE3; lane
4, R. leguminosarum biovar trifolii
24AR; lane 5, R. leguminosarum biovar
viciae 8401; lane 6, R. leguminosarum biovar trifolii ATCC 14479;
lane 7, R. meliloti 1021. No
PtdIns-4-P was formed in the absence of added PtdIns.
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Fractionation of R. leguminosarum CE3 membranes by sucrose
density gradient centrifugation (33) revealed that both the
4'-phosphatase and the phosphotransferase are localized in the inner
membrane (Fig. 3). The distribution of
NADH oxidase activity served as the inner membrane marker in the same
gradient fractions. Phospholipase A indicated the presence of outer
membrane fragments (Fig. 3).

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Fig. 3.
Inner membrane localization of the
4'-phosphatase and the PtdIns-dependent phosphotransferase
of R. leguminosarum. The inner and outer
membranes of strain CE3 were separated by isopycnic sucrose density
gradient centrifugation, and ~0.4-ml fractions were collected. NADH
oxidase and phospholipase A activities were assayed to locate inner and
outer membrane fragments, respectively. A, NADH oxidase and
phospholipase A activities in each fraction are expressed as a
percentage of the total activity across the entire gradient.
B, the 4'-phosphatase and the phosphotransferase activities
were assayed in each fraction.
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The Solubilized 4'-Phosphatase and Phosphotransferase Activities
Co-purify--
The possibility that the same enzyme might be
catalyzing the 4'-phosphatase and the phosphotransferase reactions was
investigated by partially purifying the 4'-phosphatase. Isolation of
membranes as the first step resulted in a 2.8-fold increase in
4'-phosphatase-specific activity (Table
I). Quantitative solubilization of both
activities was achieved with Triton X-100 (reduced) at a
detergent:protein ratio of 2:1, as described under "Experimental
Procedures." In Table I, the -fold purification and yields were
calculated based on the 4'-phosphatase activity, but the pattern was
the same if the phosphotransferase data was used (not shown).
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Table I
Solubilization and partial purification of the
4'-phosphatase/phosphotransferase
The activities were assayed with 50 µM
Kdo2-[4'-32P]lipid IVA, either with or
without 1 mg/ml PtdIns. The -fold purification and yield after each
step were calculated using the 4'-phosphatase activity data. Within the
experimental error of the assays, the ratio of the phosphotransferase
specific activity divided by the 4'-phosphatase specific activity was
the same after each step following solubilization.
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Several ion exchange resins were surveyed for their ability to bind the
solubilized 4'-phosphatase. The enzyme displayed strong, reversible
affinity for Q-Sepharose and heparin-agarose. About 80% of both
the 4'-phosphatase and the phosphotransferase bound to Q-Sepharose, and
both were quantitatively eluted from the column at ~0.3-0.4
M NaCl (Fig. 4A).
The pooled peak fractions were purified about 33-fold over the cell
extract based on the specific activity of the 4'-phosphate (Table I).
Next, column chromatography on heparin-agarose (Fig. 4B)
resulted in an additional 2.5-fold purification of the 4'-phosphatase
with an overall yield of about 75% (Table I). Importantly, the peaks
of 4'-phosphatase and the phosphotransferase activities in both of
these chromatograms (Fig. 4, A and B) overlapped each other within experimental error. Following solubilization of the
membranes, the ratio of the specific activity of the phosphotransferase to the 4'-phosphatase remained constant at each step of the
purification (2.2-2.4) (Table I). The identical chromatographic
behaviors of both activities (Fig. 4) and the constant ratios of
specific activities (Table I) indicate that a single enzyme species
probably catalyzes both the 4'-phosphatase and the phosphotransferase
reactions. However, the heparin-agarose fractions are yet not
homogeneous, as judged by SDS gel electrophoresis (not shown).

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Fig. 4.
Identical chromatographic behaviors of the
4'-phosphatase and the phosphotransferase. A,
co-elution of 4'-phosphatase and phosphotransferase activities from
Q-Sepharose. B, co-elution of 4'-phosphatase and
phosphotransferase activities from heparin-agarose.
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Following solubilization and purification, both the 4'-phosphatase and
the phosphotransferase activities are linear with time for up to 20 min
at 30 °C (Fig. 5).

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Fig. 5.
Linearity of the partially purified
4'-phosphatase and phosphotransferase reactions with time.
Partially purified enzyme (heparin-agarose step) was assayed for the
4'-phosphatase activity in the absence of PtdIns (open
circles and squares) or for the
phosphotransferase activity in the presence of PtdIns
(closed circles and squares). Protein
concentrations of 0.6 µg/ml (circles) or 1.2 µg/ml
(squares) were used, and the assays were incubated at
30 °C for the indicated times under standard conditions (30 µl
final volume). At every time point indicated, a 2-µl portion of the
reaction mixture was withdrawn and analyzed by thin layer
chromatography and PhosphorImager analysis, as described under
"Experimental Procedures." The 4'-phosphatase activity is expressed
as the amount of inorganic phosphate released per ml of reaction
mixture in absence of PtdIns, while the phosphotransferase activity is
expressed as the amount of PtdIns-4-P formed in the presence of
PtdIns.
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Thermal Inactivation and Affinity Elution Studies--
When the
heparin-agarose-purified material was preincubated at various
temperatures (30, 37, or 45 °C), the 4'-phosphatase and the
phosphotransferase activities were inactivated at the same rates (Fig.
6), further supporting the view that the
same protein may be catalyzing both reactions. Concurrent inactivation of both of the activities was also observed in the presence of higher
inorganic phosphate concentrations (40 mM) or orthovanadate (1 mM) (data not shown).

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Fig. 6.
Thermal inactivation of the 4'-phosphatase
and the phosphotransferase. Samples of the partially purified
enzyme (heparin-agarose step) were preincubated at various temperatures
(30, 37, or 45 °C) for the indicated times. A portion of the
incubation was then used to assay for remaining 4'-phosphatase and
phosphotransferase activities. The percentage of the activity remaining
is normalized to a control sample of the enzyme held on ice.
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Last, a physical association of the enzyme with PtdIns was shown by the
ability of PtdIns to elute both the 4'-phosphatase and the
phosphotransferase from a Cibacron blue-agarose column (Fig.
7) or from a heparin-agarose column (not
shown). The 4'-phosphatase/phosphotransferase bound very strongly to
Cibacron blue-agarose (type 300), since it could not be eluted with
buffers containing 0.5% Triton X-100 (reduced) and 0.5 M
NaCl. Buffer containing PtdIns (0.4 mg/ml) and 0.2 M NaCl
was effective in co-eluting both activities (Fig. 7). However, the
resulting preparation still contained several protein species, as
judged by gel electrophoresis (data not shown).

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Fig. 7.
Co-elution of the 4'-phosphatase and the
phosphotransferase activities from Cibacron blue (type 300) by
PtdIns. The chromatography was carried out as described under
"Experimental Procedures."
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Kinetic Studies and Substrate Specificity of the 4'-Phosphatase and
the Phosphotransferase--
In crude extracts, the 4'-phosphatase
functions optimally when a Kdo disaccharide is part of the substrate,
as in Kdo2-lipid IVA (30). We confirmed the Kdo
dependence of both the 4'-phosphatase and the phosphotransferase with
the 80-fold purified enzyme (heparin-agarose step). The apparent
Km for lipid IVA was about 5 times higher than that for Kdo2-lipid IVA (Table
II) with both activities, but
Kdo-[4'-32P]lipid IVA prepared with the
monofunctional Kdo transferase of H. influenzae was a good
substrate as well (Table II). Either one or two Kdo moieties also
increased the Vmax by 2-3-fold in comparison
with lipid IVA in each case (Table II). The
Vmax for the phosphotransferase, using PtdIns as
the acceptor, was 3 times higher than that of the 4'-phosphatase with
the partially purified enzyme, using Kdo2-lipid
IVA as the substrate (Table II).
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Table II
Kinetic analysis of the partially purified 4'-phosphatase and
phosphotransferase
The kinetic constants for the 4'-phosphatase and the phosphotransferase
reactions were determined from assays at varying concentrations of
Kdo2-[4'-32P]lipid IVA,
Kdo-[4'-32P]lipid IVA, or [4'-32P]lipid
IVA in the presence or absence of PtdIns (1 mg/ml). The
apparent Km and Vmax were then
estimated from double reciprocal plots. The Vmax of
the phosphotransferase reaction is higher than that of the
4'-phosphatase with each substrate.
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The ability of the phosphotransferase to tolerate structural
modifications of the donor substrate was investigated.
Kdo2-[4'-32P]1-dephospholipid IVA
(38), galactose-mannose-Kdo2-[4'-32P]lipid
IVA (34),
mannose-Kdo2-[4'-32P]lipid IVA
(34), lauroyl-Kdo2-[4'-32P]lipid
IVA (49), and [4'-32P]lipid A (41) were
tested in both assays (not shown). These substances were all utilized
efficiently. Hexa-acylated [4'-32P]lipid A was utilized
at about the same rate as tetra-acylated [4'-32P]lipid
IVA. Neither Kdo2-[1-32P]lipid
IVA nor [1-32P]lipid IVA (31)
supported the generation of PtdIns-4-32P (data not shown).
This finding indicates that the 1-phosphatase of R. leguminosarum (31) does not function as a phosphotransferase to
PtdIns. Furthermore, [32P]phosphatidic acid was inactive
as a donor for the biosynthesis of PtdIns-4-32P under these
conditions (data not shown), supporting the proposal that the
4'-phosphatase is highly selective for lipid A-like molecules as donor substrates.
The acceptor substrate specificity of the phosphotransferase was
studied with various inositol-containing substances and other membrane
lipids (Fig. 8, A and
B). The phosphotransferase exhibits a very high degree of
selectivity toward PtdIns. PtdIns from bovine liver (containing mainly
stearate and arachidonate) and PtdIns from soybean (containing mostly
palmitate and linoleate) were equally active as acceptors (Fig.
8A). No other inositol-containing compound served as an
acceptor (Fig. 8A), including PtdIns-4-P, which is actually
an inhibitor (not shown), PtdIns-3-P, lysophosphatidylinositol, or
myo-inositol. Among the other membrane lipids tested,
inefficient transfer of the 4'-phosphate of
Kdo2-[4'-32P]lipid IVA to
phosphatidylglycerol (PtdGro) was detected (Fig. 8B,
lane 4). However, the rate of transfer of
32P to PtdGro was 50 times slower than to PtdIns (Fig.
9) over a wide range of acceptor
concentrations.

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Fig. 8.
Acceptor substrate specificity of the
phosphotransferase of R. leguminosarum. Compounds
were tested as acceptors for the phosphotransferase under standard
assay conditions using partially purified (heparin-agarose) enzyme (10 µg/ml). Incubations were carried out for 40 min at 30 °C.
A, thin layer analysis of the reaction products obtained
with different inositol-containing compounds as acceptor. The lipid
acceptor substrates were used at 1 mg/ml, and inositol was used at 10 mM. Lane 1, no enzyme;
lane 2, no acceptor; lane
3, PtdIns (bovine liver); lane 4,
PtdIns (soybean); lane 5, PtdIns-4-P;
lane 6, PtdIns-3-P; lane 7,
lysophosphatidylinositol; lane 8, inositol.
B, thin layer analysis of the reaction products
obtained with different phospholipids as acceptors, each at 1 mg/ml.
Lane 1, no enzyme; lane 2,
no acceptor; lane 3, PtdIns (bovine liver);
lane 4, PtdGro; lane 5,
phosphatidylethanolamine; lane 6,
phosphatidylcholine; lane 7, phosphatidylserine;
lane 8, cardiolipin; lane
9, phosphatidic acid; lane 10,
diacylglycerol.
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Fig. 9.
Acceptor substrate specificity of the
phosphotransferase for PtdIns versus PtdGro. The
effects of PtdIns and PtdGro concentrations on phosphotransferase
specific activity were measured in the presence of excess of
Kdo2-[4'-32P]lipid IVA (50 µM). Heparin-agarose-purified enzyme (10 µg/ml) was
used, and the incubation was carried out for 10 min at 30 °C.
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Since glycosylated diacylglycerols are major components of R. leguminosarum membranes, commercially available digalactosyl diacylglycerol and monoglactosyl diacylglycerol were tested. However, neither compound was active as a phosphate acceptor (not shown).
As shown in Fig. 9, the apparent Km for PtdIns is
approximately 500 µM. This is 10-fold higher than what is
typically seen with eucaryotic PtdIns kinases (35-37), but the
specific activity of the phosphotransferase in membranes is higher than
that of many eucaryotic PtdIns kinases assayed under comparable
conditions. The relatively high apparent Km for
PtdIns therefore does not eliminate the possibility of a physiological
role for the phosphotransferase. Indeed, many key enzymes of
glycerophospholipid and lipid A biosynthesis display even higher
apparent Km values for their physiological substrates (40,
50).
Selective Transfer of the 4'-Phosphate Group of
Kdo2-Lipid IVA to the Inositol 4-Position of
PtdIns--
To determine the site of phosphorylation on PtdIns, the
32P-labeled phosphotransferase product was characterized by
chromatography and by chemical degradation. Migration of the
32P-labeled PtdIns-dependent product with
authentic PtdIns-4-P was demonstrated using silica gel 60 thin layer
chromatography plates developed with the solvents
chloroform/pyridine/88% formic acid/water (30:70:16:10, v/v/v/v) (Fig.
8) or chloroform/methanol/acetic acid (65:35:10, v/v/v) (not shown).
This procedure does not distinguish PtdIns-4-P from PtdIns-3-P.
Accordingly, the 32P-labeled PtdIns-dependent
product was deacylated by mild alkaline hydrolysis, followed by anion
exchange HPLC of the water-soluble 32P-labeled material(s)
on a Partisil SAX column (47), as described in detail in the legend to
Fig. 10. No 32P-labeled
peak was detected in the control incubated without enzyme (not shown).
The deacylated 32P-labeled PtdIns-dependent
product eluted with glycerophosphoinositol 4-phosphate (Fig. 10,
B and C), which is clearly separated from glycerophosphoinositol 3-phosphate (and other possible isomers) in this
system (47). The 32Pi produced by the
4'-phosphatase from Kdo2-[4'-32P]lipid
IVA either in the presence (Fig. 10, B and
C) or absence of PtdIns (Fig. 10A) elutes 3-4
min before glycerophosphoinositol 3-phosphate. The HPLC analysis of the
phosphotransferase reaction product therefore provides unequivocal
evidence that the 4'-phosphate of Kdo2-lipid
IVA is transferred only to the 4-position of the inositol
moiety of PtdIns. This remarkable specificity shows that the enzyme
cannot transfer the 4'-phosphate of
Kdo2-[4'-32P]lipid IVA to any
other hydroxyl group present on the surface of the Triton X-100/PtdIns
mixed micelle, since alternative phosphorylated products, like
PtdIns-3-32P or 32P-Triton X-100, are not
detected.

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Fig. 10.
Selective transfer of the 4'-phosphate group
of Kdo2-[4'-32P]lipid IVA to the
4-position of the inositol moiety of PtdIns. Three reactions (10 µl each) were carried out under standard assay conditions with 10 µM Kdo2-[4'-32P]lipid
IVA (20,000 cpm/nmol) as donor. A, with
enzyme (10 µg/ml heparin-agarose step) but without PtdIns;
B and C, with enzyme (10 µg/ml heparin-agarose
step) and with PtdIns (1 mg/ml) as the acceptor. After incubation for
40 min, internal standards consisting of PtdIns and/or its
phosphorylated derivatives in 30 µl of chloroform/methanol (1:1, v/v)
were added to each tube. A [1,2-3H]inositol-labeled
mixture (40,000 cpm) of PtdIns and phosphorylated PtdIns derivatives
isolated from yeast cells was used as the internal standard for
reactions A and B, while
Ptd-[2-3H]Ins-4-P (2000 cpm) was used as the internal
standard for tube C. Each sample was then dried by vacuum
centrifugation, followed by deacylation with methylamine and HPLC
analysis of the water-soluble deacylation products (47). The elution of
the 3H-labeled deacylated internal standards
(broken lines) and of the 32P-labeled
enzymatic reaction products (solid lines) was
monitored by in-line liquid scintillation counting. The elution
profiles for reactions A (no PtdIns, yeast standards),
B (with PtdIns, yeast standards), and C
(with PtdIns, PtdIns-4P standard) are shown in the respective
panels. The peaks for internal standards
(glycerophosphoinositol (GPI), glycerophosphoinositol
3-phosphate (GPI-3P), glycerophosphoinositol
4-phosphate (GPI-4P), and the
4'-phosphatase/phosphotransferase products
(32Pi and [32P]phosphotransferase
product) are indicated. No 32Pi or
[32P]phosphotransferase product was seen without enzyme
(not shown). The slight decrease in the retention times for the
32Pi and the GPI-4P in A
versus C probably is caused by aging of the
column following multiple cycles of chromatography.
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Evidence for a Phosphoenzyme Intermediate--
When either 6 µg
of Q-Sepharose or 2 µg of heparin-agarose
4'-phosphatase/phosphotransferase (Table I) was incubated with Kdo2-[4'-32P]lipid IVA for 5 min,
followed by the addition of buffer containing SDS at pH 10, a single
32P-labeled 68-kDa protein was detected by SDS-gel
electrophoresis and PhosphorImager analysis (Fig.
11A, lanes
2 and 3). The intensity of the radiolabeled band
increased with increasing protein concentrations (not shown). Heat
inactivation of the enzyme preparation prior to incubation with the
Kdo2-[4'-32P]lipid IVA abolished
the incorporation of 32P (Fig. 11A,
lane 1). The formation of the phosphoprotein was
transient, since maximum labeling was observed after only 2-5 min of
incubation with Kdo2-[4'-32P]lipid
IVA (not shown). Thereafter, the intensity of the band steadily decreased until it was virtually undetectable after 90 min
(not shown).

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Fig. 11.
Trapping and detection of a 68-kDa
phosphoprotein formed by incubating
Kdo2-[4'-32P]lipid IVA with
partially purified 4'-phosphatase/phosphotransferase.
A, a specific 32P-labeled phosphoprotein,
generated during a 5-min incubation of
Kdo2-[4'-32P]lipid IVA with
enzyme (as described under "Experimental Procedures") is resolved
by SDS-polyacrylamide gel electrophoresis and detected by
PhosphorImager analysis of the dried gel. Partially purified enzyme
from either the Q-Sepharose step (lane 2, 6 µg of protein)
or the heparin-agarose step (lanes 1 and
3; 2 µg of protein) was used. The enzyme for the reaction
shown in lane 1 was first heated to 65 °C for
15 min. B, PtdIns reduces the level of the phosphoprotein
generated during a 5-min incubation of
Kdo2-[4'-32P]lipid IVA with
enzyme. Reactions were performed with identical amounts (2 µg) of
heparin-agarose enzyme and Kdo2-[4'-32P]lipid
IVA in the absence (lane 1) or in the
presence (lane 2) of PtdIns (0.5 mg/ml). After 5 min, the reactions were quenched with SDS and subjected to gel
electrophoresis.
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The addition of PtdIns to the enzyme preparation together with
Kdo2-[4'-32P]lipid IVA resulted
in decreased labeling of the 68-kDa protein (Fig. 11B,
lane 2). Faster turnover of the proposed
phosphoenzyme intermediate in the presence of PtdIns (Fig. 1) may
account for this observation, since PtdIns is a better acceptor than
water. The simultaneous presence of PtdIns-4-P during the reaction of the enzyme with Kdo2-[4'-32P]lipid
IVA also resulted in decreased 32P labeling of
the protein (not shown). Dilution of the specific radioactivity of the
32P-labeled phosphoenzyme intermediate, caused by the
reverse reaction (Fig. 1) with excess unlabeled PtdIns-4-P, would
explain this effect.
A pulse-chase experiment (Fig. 12)
confirmed that the loss of 32P label from the 68-kDa
phosphoprotein was dependent both upon time and the concentration of
the unlabeled Kdo2-lipid IVA used to dilute the
specific radioactivity. The formation and turnover of the
32P-labeled phosphoprotein are consistent with the proposed
mechanism involving a phosphoenzyme intermediate (Fig. 1).

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Fig. 12.
Turnover of the labeled phosphoprotein
following a chase with unlabeled Kdo2-lipid
IVA. The enzyme preparation (heparin-agarose step) was
labeled for 5 min with Kdo2-[4'-32P]lipid
IVA as described under "Experimental Procedures."
A, B, and C are the images of the
SDS-polyacrylamide gels used to resolve the remaining
32P-labeled phosphoprotein present at different times after
being chased with 0, 10, or 50 µM nonradioactive
(Kdo)2-lipid IVA, respectively. The percentage
of the 32P-labeled phosphoprotein remaining at
various times after the start of the chase (indicated by the
arrow) is shown in D.
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The chemical stability of the 32P-labeled protein species
visualized in Figs. 11 and 12 was explored to determine the nature of the covalent linkage that might be involved. Treatment with 8 M urea or 200 mM dithiothreitol did not reduce
the intensity of the labeled band (data not shown). The
32P-labeled protein was very labile under acidic
conditions, but it was stable under mild basic or at neutral conditions
in the presence of SDS. The stability of the protein-bound
32P actually increased under mild alkaline conditions. This
pattern is consistent with a phosphohistidine residue (51, 52).
Isolation and analysis of the active site peptide labeled with
32P will be necessary to confirm this proposal.
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DISCUSSION |
The Kdo-dependent 4'-phosphatase of R. leguminosarum catalyzes a key reaction in generating a
phosphate-deficient variant of lipid A (30). Here we show that the same
enzyme preparations can also transfer the 4'-phosphate group from lipid
A and certain lipid A precursors to PtdIns. An enzymatic reaction that
directly links lipid A biosynthesis with PtdIns-4-P production has not been described previously. This unique R. leguminosarum
phosphotransferase is the first example of procaryotic enzyme capable
of generating PtdIns-4-P, a molecule that plays a central role in
eucaryotic signal transduction (53, 54).
The current studies strongly suggest that one enzyme catalyzes both the
4'-phosphatase and the phosphotransferase reactions. Like the
4'-phosphatase (30), the phosphotransferase is present in wild type
strains of R. leguminosarum but not in E. coli,
R. leguminosarum mutant 24AR, or R. meliloti. In
R. leguminosarum (CE3), both activities are localized in the
inner membrane (Fig. 3). Both activities display similar
chromatographic properties on Q-Sepharose (Fig. 4), heparin-agarose
(Fig. 4) and Cibacron blue-agarose (Fig. 7), and their thermal
inactivation profiles are identical (Fig. 6). Furthermore, both
activities co-purify (Table I) and show the same donor substrate
specificity (Table II).
The ability of one enzyme to catalyze both the 4'-phosphatase and the
phosphotransferase reaction could be explained by a mechanism involving
a covalent phosphoenzyme intermediate (Fig. 1B). The
intermediate would be generated by the initial transfer of the
4'-phosphate moiety of Kdo2-lipid IVA to the
enzyme. The intermediate could transfer the phosphate group either to
water or to the 4-OH of PtdIns. The direct demonstration (Figs. 11 and 12) of a 32P-labeled phosphoprotein, obtained by incubation
of the 80-fold purified enzyme with
Kdo2-[4'-32P]lipid IVA and chased
by nonradioactive Kdo2-lipid IVA (Fig. 12) or
PtdIns (Fig. 11B), strongly supports this scheme.
Our studies show that the phosphotransferase, like the 4'-phosphatase
(30), functions more efficiently in the presence of at least one Kdo
residue on the donor substrate (Table II). The enzyme is less sensitive
to other structural modifications of Kdo2-[4'-32P]lipid IVA, such as
removal of the 1-phosphate, the addition of extra sugars, or further
acylation. Such a dependence on Kdo may be necessary, given that the
Kdo transferase requires the presence of the 4'-phosphate residue for
catalysis (38, 42). Premature removal of the 4'-phosphate might inhibit
LPS maturation. Although the 4'-phosphatase/phosphotransferase is
associated with the inner membrane, its active site might face the
periplasm, further limiting access to LPS precursors lacking Kdo to the
4'-phosphatase (3).
A 1-phosphatase, distinct from the 4'-phosphatase and capable of
cleaving several precursors of lipid A including Kdo2-lipid IVA, is present in membranes of R. leguminosarum
(31). Using Kdo2-[1-32P]lipid IVA
(31) as the donor, however, transfer of the 1-phosphate to PtdIns was
not observed in R. leguminosarum membranes (data not shown).
Thus, phosphotransferase activity appears to be associated only with
the 4'-phosphatase of R. leguminosarum. In the presence of
[
-32P]ATP and Mg2+, no transfer of
32P to PtdIns was observed when either R. leguminosarum CE3 membranes or partially purified heparin-agarose
fractions were employed (data not shown). Hence, the R. leguminosarum phosphotransferase reaction is not attributable to a
PtdIns kinase (35-37).
Although the phosphotransferase exhibits a high degree of selectivity
toward PtdIns, the latter could not be demonstrated among the membrane
lipids of R. leguminosarum (CE3) by labeling cells with
either 32Pi or [1,2-3H]inositol.
A search for an alternative endogenous lipid acceptor among the total
membrane lipids of R. leguminosarum proved unsuccessful. Gerson and Patel (55) reported PtdIns in Rhizobium loti, but other studies (56) of different strains of R. leguminosarum failed to demonstrate PtdIns. One intriguing explanation for this anomaly might be that the synthesis of PtdIns is induced in the bacteria by root cell exudates or during symbiosis. A recent report (57) showed the presence of PtdIns in membranes of free-living Bradyrhizobium japonicum grown at low oxygen concentrations,
one of the environmental factors known to induce conversion of bacteria to bacteroids in root nodules (58).
An alternative possibility is that plant membranes supply PtdIns or a
related compound to R. leguminosarum during nodulation. PtdIns is available in the peribacteroid membranes of soybean root
nodules (58), and soybean PtdIns is an excellent acceptor substrate for
the phosphotransferase (Fig. 8). In addition, Perotto et.
al. (59) have demonstrated presence of other inositol-containing phosphoglycolipids in pea nodules. The close association of the bacteroid outer membrane with the plant-derived peribacteroid membrane
is well documented by immunostaining and electron microscopy (60-62).
Inside the symbiosome, the outer membrane of the bacteroid may actually
be fragmented (61), potentially giving PtdIns access to the
4'-phosphotransferase. PtdIns-specific lipid exchange proteins are
known to exist in plants and could serve as PtdIns carriers (63).
In heptose-deficient mutants of Salmonella typhimurium,
small quantities of exogenous glycerophospholipids can fuse with the outer membrane and move to the inner membrane (64, 65). In the case of
exogenous phosphatidylserine, the incorporated lipid is metabolized to
form phosphatidylethanolamine within the inner membrane (64, 65). While
retrograde transport of exogenous lipids is not sufficiently rapid in
Gram-negative bacteria to enable the isolation of phospholipid
auxotrophs, it is nevertheless established that such transport occurs
with a variety of lipids (64, 65). In the case of minor lipids like
PtdIns-4-P, which function at low concentrations in specific signal
transduction pathways, the possibility that the plant provides
the 4'-phosphatase/phosphotransferase with PtdIns deserves
serious consideration. The PtdIns-4-P made by R. leguminosarum from exogenous PtdIns might even be secreted again
by the bacteroids and made available to the plant.
Majerus and co-workers have recently reported a remarkable example of a
specific interaction between an enzyme made by a Gram-negative bacterium and a eucaryotic inositol phosphate signaling system (66).
Salmonella dublin secretes a virulence protein (SopB) that
hydrolyzes inositol 1,3,4,5,6-pentakis-phosphate to form inositol 1,4,5,6-tetrakis-phosphate (66). The latter
compound increases chloride secretion in animal cells, and the
catalytic activity of SopB is necessary for the induction of fluid
secretion observed in calf intestinal loops infected with S. dublin (66). SopB can also hydrolyze phosphatidylinositol
3,4,5-trisphosphate, a lipid that directly antagonizes
Ca2+-dependent chloride secretion (66). Like
R. leguminosarum, however, enteric Gram-negative bacteria
are not thought to make their own inositol phospholipids (67). If not
actually involved in making PtdIns-4-P during symbiosis, the
4'-phosphatase/phosphotransferase of R. leguminosarum might
conceivably function to remove PtdIns-4-P by catalyzing the reverse of
the proposed reactions (Fig. 1), a possibility that we have not yet examined.
Although the roles played by phosphorylated derivatives of PtdIns in
plants have not been fully established, evidence is accumulating that
signal transduction mediated by the turnover of inositol phospholipids
may indeed occur (68). For instance, Ehrhardt et al. (69)
observed calcium spiking, as is typically associated with inositol
trisphosphate signaling in animal cell systems, when alfalfa root hairs
are exposed to the R. meliloti Nod factor. In animal cells,
phosphatidylinositol kinases play additional important roles in
membrane biogenesis, secretion, vesicle trafficking, and regulation of
the actin cytoskeleton (53, 54, 70, 71). All of these processes
accompany bacterial invasion of plant root hair cells and nodule
formation (58). Since the inositol phospholipid cycle begins with the
phosphorylation of PtdIns to generate PtdIns-4-P, the R. leguminosarum phosphotransferase could play a key role in any of
the above processes during symbiosis. Cloning of the gene encoding the
4'-phosphatase/phosphotransferase and isolation of mutants specifically
defective in the enzyme should reveal the significance of the
phosphate-deficient lipid A that is found in R. leguminosarum and the biological role of the phosphotransferase activity associated with the 4'-phosphatase.