From the Department of Biochemistry and Molecular
Biology, Uniformed Services University of the Health Sciences,
Bethesda, Maryland 20814-4799 and the
Department of Molecular
and Cellular Biochemistry, University of Kentucky College of Medicine,
Lexington, Kentucky 40536
Received for publication, February 19, 2003
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ABSTRACT |
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The
assembly of many bacterial cell surface polysaccharides requires the
transbilayer movement of polyisoprenoid-linked saccharide intermediates
across the cytoplasmic membrane. It is generally believed that
transverse diffusion of glycolipid intermediates is mediated by
integral membrane proteins called translocases or "flippases." The
bacterial genes proposed to encode these translocases have been
collectively designated wzx genes. The wzxE
gene of Escherichia coli K-12 has been implicated in the
transbilayer movement of Fuc4NAc-ManNAcA-GlcNAc-P-P-undecaprenol (lipid
III), the donor of the trisaccharide repeat unit in the biosynthesis of
enterobacterial common antigen (ECA). Previous studies (Feldman, M. F., Marolda, C. L., Monteiro, M. A., Perry, M. B., Parodi, A. J., and Valvano, M. (1999) J. Biol.
Chem. 274, 35129-35138) provided indirect evidence that the
wzx016 gene product of E. coli K-12
encoded a translocase capable of mediating the transbilayer movement of
N-acetylglucosaminylpyrophosphorylundecaprenol
(GlcNAc-P-P-Und), an early intermediate in the synthesis of ECA and
many lipopolysaccharide O antigens. Therefore, genetic and biochemical
studies were conducted to determine if the putative WzxO16
translocase was capable of mediating the transport of
N-acetylglucosaminylpyrophosphorylnerol (GlcNAc-P-P-Ner), a
water-soluble analogue of GlcNAc-P-P-Und. [3H]GlcNAc-P-P-Ner was transported into sealed, everted
cytoplasmic membrane vesicles of E. coli K-12 as well as a
deletion mutant lacking both the wzx016 and
wzxC genes. In contrast, [3H]GlcNAc-P-P-Ner
was not transported into membrane vesicles prepared from a
wzxE-null mutant, and metabolic radiolabeling experiments revealed the accumulation of lipid III in this mutant. The WzxE transport system exhibited substrate specificity by recognizing both a
pyrophosphoryl-linked saccharide and an unsaturated The biosynthesis of a wide variety of complex glycoconjugates in
both eucaryotic and procaryotic cells occurs by a process whereby
membrane-bound glycolipids or glycolipid precursors are synthesized on
the cytosolic face of a membrane and subsequently translocated to the
opposite side of the membrane where they serve as substrates for
additional processing reactions. In eucaryotic cells the synthesis of
N-linked oligosaccharides of glycoproteins involves the
translocation of dolichyl-linked mono- and pentasaccharide intermediates from the cytosolic leaflet to the lumenal monolayer of
the endoplasmic reticulum
(ER)1 (1-5). Similarly, the
synthesis of glycophosphatidylinositol (GPI) anchors in eucaryotic
cells is initiated on the cytoplasmic face of the ER, but the completed
anchor structure is linked to protein on the lumenal face (6, 7). The
transbilayer movement of phospholipids also occurs during the assembly
of the phospholipid bilayers of biogenic membranes in procaryotic and
eucaryotic cells (8-11). Studies using synthetic lipid bilayers
indicate that the transbilayer migration of
di(N-acetylglucosaminyl)pyrophosphoryldolichol (GlcNAc2-P-P-Dol) and spin-labeled analogues of
polyisoprenyl compounds does not occur spontaneously (12, 13). Rather,
it has been proposed that the transbilayer diffusion of polar-lipid head groups is mediated by specific integral membrane proteins called
"flippases" or "translocases" (14-17), and a substantial amount of evidence has been obtained that supports this conclusion (17-19).
Flippase proteins are also believed to be involved in the assembly of a
large group of bacterial lipopolysaccharide (LPS) O antigens
collectively referred to as Wzy-dependent O antigens (20).
Although these O antigens are structurally distinct, they are all
heteropolysaccharides comprised of oligosaccharide repeat units and all
are assembled by the same general mechanism (20). The repeat units of
these O antigens are synthesized as undecaprenyl pyrophosphate
(Und-P-P)-linked oligosaccharides on the inner face of the cytoplasmic
membrane and subsequently translocated en bloc to the
periplasmic face where they are utilized as substrates for chain
elongation by a polymerase enzyme (Wzy) (20, 21). Polymerization occurs
by the transfer of nascent polysaccharide chains from the carrier lipid
to the non-reducing termini of newly synthesized carrier lipid-linked
repeat units (22, 23). The assembly of Wzy-dependent O
antigens by this mechanism has been called "block-polymerization."
The O antigen chains are then transferred to the core-lipid A region of
LPS, and the completed LPS molecules are translocated to the exterior
leaflet of the outer membrane by an unknown mechanism.
All of the gene clusters involved in the synthesis of
Wzy-dependent O antigens contain a gene, designated
wzx, that is believed to encode the flippase that mediates
the transbilayer movement of the Und-P-P-linked repeat unit (24).
Analyses of the predicted structures of the putative O antigen
flippases have revealed that all are hydrophobic proteins with twelve
putative transmembrane domains (24). Although these proteins share very
little structural homology at the primary amino acid sequence level,
all have strikingly similar hydropathy profiles (25). Indirect evidence
in support of the proposed functions of the O antigen flippases of
Salmonella enterica serovar Typhimurium and Shigella
dysenteriae was obtained by Liu et al. (26), who
demonstrated the accumulation of single lipid-linked O antigen repeat
units on the inner face of the cytoplasmic membrane in wzx
mutants. However, the mechanism involved in flippase-mediated transbilayer movement of lipid-linked O antigen repeat units, as well
as definitive identification of the proteins involved, remains to be established.
The assembly of phosphoglyceride-linked enterobacterial common antigen
(ECAPG) of Gram-negative enteric bacteria is also believed to occur by a Wzy-dependent mechanism (27).
ECAPG is a glycolipid component of the outer membrane of
all Gram-negative enteric bacteria (27, 28). The carbohydrate portion
of ECA consists of a linear heteropolysaccharide chain comprised of
N-acetyl-D-glucosamine (GlcNAc),
N-acetyl-D-mannosaminuronic acid (ManNAcA), and
4-acetamido-4,6-dideoxy-D-galactose (Fuc4NAc) (29). These
amino sugars are linked to one another to form the trisaccharide repeat
unit
The synthesis of GlcNAc-P-P-Und is the initial step in the assembly of
the ECA trisaccharide repeat unit (30) and the repeat units of many
Wzy-dependent O antigens (20, 35, 36). It is also the first
lipid-linked intermediate in the synthesis of several Wzy-independent O
antigens (20). This reaction involves the transfer of GlcNAc 1-P from
UDP-GlcNAc to Und-P catalyzed by the enzyme UDP-GlcNAc:undecaprenyl
phosphate GlcNAc 1-P transferase (WecA) (31, 37). The wecA
gene (formerly rfe) is located in the wec gene
cluster (formerly rfe-rff), which includes many of the genes
involved in the biosynthesis of ECA (27, 38, 39). The wec
gene cluster also includes a gene designated wzxE (formerly rfbX) that is believed to encode the flippase that mediates
the transbilayer movement of the Und-P-P-linked trisaccharide repeat unit (25). Indeed, the hydropathy profile of the predicted product of
the wzxE gene is almost identical to the hydropathy profiles of the putative flippases involved in the assembly of many
Wzy-dependent O antigens (25).
The O16 O antigen repeat unit of Escherichia coli K-12/O16
is a branched pentasaccharide whose assembly is initiated by the synthesis of GlcNAc-P-P-Und catalyzed by WecA, and the assembly of this
O antigen is believed to occur by a Wzy-dependent mechanism (36, 40, 41). Recent studies reported that a complete Und-P-P-linked O16 repeat unit was not required for translocation by the
WzxO16 translocase (42). Indeed, the data presented in
these studies suggested that the E. coli WzxO16
translocase was able to translocate GlcNAc-P-P-Und.
The studies presented here were conducted to investigate a role for
wzx genes in the transbilayer movement of
Fuc4NAc-ManNAcA-GlcNAc-P-P-Und in E. coli. Attempts to
measure the flippase-mediated transbilayer movement of naturally
occurring undecaprenyl- or dolichyl-linked substrates are complicated
by the extremely hydrophobic nature of these compounds. To circumvent
this technical difficulty, we employed a variation of the experimental
strategy of Rush et al. (18, 19) who used water-soluble
citronellyl-based analogues of mannosylphosphoryldolichol (Man-P-Dol)
and glucosylphosphoryldolichol (Glc-P-Dol) to assay flippase-mediated
transport activities into intact ER vesicles. Bishop and Bell (14)
originally used a water-soluble analogue of phosphatidylcholine to
investigate one or more phosphatidylcholine flippases in sealed rat
liver microsomes. Subsequently, phospholipid analogues with short fatty
acid chains have been utilized by several other research groups to
partially purify and characterize membrane proteins involved in the
transbilayer diffusion of phospholipids (43-46). Furthermore,
water-soluble analogues of glucosylceramide have been used to study the
transbilayer movement of glucosylceramide in the Golgi apparatus (47).
The results presented here demonstrate the translocase-mediated
transport of [3H]GlcNAc-P-P-Ner into everted membrane
vesicles of E. coli. The data support the conclusion that
the observed transport was not mediated by the WzxO16
translocase, but rather by WzxE, the putative flippase involved in the
transbilayer movement of lipid III across the cytoplasmic membrane
during the assembly of ECAPG.
Materials--
(s)-Citronellol, nerol
(cis-3,7-dimethyl-2,6-octadien-1-ol), tetramethylammonium
phosphate, trichloroacetonitrile, and phosphorous oxytrichloride were
obtained from Sigma-Aldrich (St. Louis, MO). UDP-N-acetyl-D-[6-3H]glucosamine
(40-60 Ci/mmol) was purchased from American Radiolabeled Chemicals,
Inc. (St. Louis, MO). [14C]Glucose (360 mCi/mmol) was
obtained from American Radiolabeled Chemicals (St. Louis, MO), and it
was adjusted to a specific activity of 94 dpm/pmol by the addition of
non-radioactive glucose. [3H]GDP-mannose was prepared as
described previously (48). Selecto silica gel was obtained from Fisher
Scientific (Pittsburgh, PA). All other chemicals were reagent grade and
were purchased from standard commercial sources.
Bacterial Strains and Plasmids--
E. coli K-12
strains used in this study are listed in Table
I. Transductions were carried out using
phage P1 vir as described by Silhavy et al. (49).
Cultures were routinely grown at 37 °C with vigorous aeration in
Luria-Bertani (LB) broth (50) supplemented with glucose to give a final
concentration of 0.2% or on LB agar containing 0.2% glucose.
Tetracycline, ampicillin, and chloramphenicol were added to media to
give final concentrations of 10, 50, and 30 µg/ml, respectively.
Plasmid pRL160 was constructed by digestion of pCA32 (39) with
HindIII yielding an 8.4-kb fragment containing wzxE as well as additional upstream genes of the
wec gene cluster. This fragment was digested with
XmaI to yield a 7.65-kb fragment that was subsequently
ligated to the HindIII and AvaI sites of pBR322.
Plasmid pRL162 was constructed by ligation of the 8.4-kb HindIII fragment of pCA32, described above, to the
corresponding site of the low copy number vector, pWSK29 (51). Plasmid
pRL147 contained the wecA gene under the control of the
PBAD promoter, and it was constructed as follows. The
wecA gene was obtained by PCR amplification of the DNA
sequence from bp 9984 to 11141 (GenBankTM accession number
AE000454) using genomic DNA from E. coli strain AB1133 as template. The polynucleotides
5'-CTCTGAGAGCATGC-3' and 5'-GCGTCGACGTTTCCCAGGCATTGGT-3'
were used as forward and reverse primers, respectively, and an
SalI restriction site was incorporated into the reverse
primer (underlined sequence). PCR amplifications were carried out using
Taq polymerase (Sigma-Aldrich Chemicals). The amplified
sequence contained 17 bp immediately upstream of the translational
start site and 36 bp immediately downstream of the translational
termination site, and it was cloned into TA cloning site of the pCR2.1
vector (Invitrogen). The resulting construct was digested with
EcoRI and SalI, and the 1.1-kb fragment was
subcloned into the expression vector, pBAD18 (52), that was restricted
with the same enzymes to yield plasmid pRL147.
Synthesis and Purification of Radiolabeled GlcNAc-P-P-Ner and
Structurally Related Compounds--
Citronellol was phosphorylated
using phosphorous oxytrichloride as described by Danilov and Chojnacki
(53). Nerol was phosphorylated with tetrabutylammonium phosphate and
trichloroacetonitrile in anhydrous acetonitrile as described by Danilov
et al. (54). The isoprenyl phosphates were purified by
ion-exchange chromatography on DEAE-cellulose as described previously
(48).
[3H]GlcNAc-P-P-Ner was synthesized enzymatically using
the UDP-GlcNAc:undecaprenyl phosphate
N-acetylglucosaminyl-1-phosphate transferase (WecA) present
in E. coli membranes. Membrane fractions were prepared as
previously described (37). Reaction mixtures contained membranes (2.7 mg of membrane protein), 0.1 M Tris-HCl (pH 8.0), 10 mM neryl phosphate (Ner-P), 40 mM
MgCl2, 5 mM dithiothreitol, 1 mM
sodium orthovanadate, and 0.1 mM
UDP-[3H]GlcNAc (18-1800 dpm/pmol) in a total volume of 3 ml. Reaction mixtures were incubated at 21 °C for 16 h and then
subjected to centrifugation at 100,000 × g for 10 min
using a Beckman TL100.3 micro-ultracentrifuge. The supernatant solution
was removed and layered on a 15-ml column of benzyl-DEAE-cellulose
(Sigma Chemical Co., St. Louis, MO) equilibrated with 10 mM
NH4HCO3. The column was developed with two
column volumes of 10 mM NH4HCO3
followed by a 60-ml gradient of NH4HCO3 (0-1
M). Fractions containing [3H]GlcNAc-P-P-Ner
were pooled and dried by rotary evaporation under reduced pressure at
30 °C. The radiolabeled analogue was then dissolved in
H2O and desalted by gel-filtration chromatography on a
Sephadex G-10 column (1 × 30 cm) equilibrated with
H2O. Fractions containing [3H]GlcNAc-P-P-Ner
were pooled, dried by rotary evaporation, dissolved in a small volume
of CHCl3/CH3OH (2:1, v/v) and layered onto a 15-ml Selecto silica gel column equilibrated with CHCl3.
The column was then eluted with
CHCl3/CH3OH/H2O/concentrated
NH4OH (65:35:6:1, v/v), and fractions of 3.5 ml were
collected. Radiolabeled GlcNAc-P-P-Ner eluted in fractions 8-15.
Fractions containing [3H]GlcNAc-P-P-Ner were pooled,
dried by rotary evaporation, dissolved in 2 ml of CH3OH,
and stored at
Reactions mixtures for the enzymatic synthesis of
[3H]GlcNAc-P-Ner contained Bacillus cereus
membranes (6.3 mg of membrane protein), 0.04 M Tris-HCl (pH
8.0), 4 mM Ner-P, 20 mM MgCl2, 2 mM dithiothreitol, 2 mM sodium orthovanadate, 1 mM CDP-choline, and 0.1 mM
UDP-[3H]GlcNAc (18 dpm/pmol) in a total volume of 5 ml.
Reaction mixtures were incubated at 21 °C for 2 h and then
stopped by the addition of 5 ml of 95% ethanol. The particulate
fraction was removed by centrifugation in a clinical centrifuge, and
the radiolabeled GlcNAc-P-Ner was purified by chromatography on
benzyl-DEAE-cellulose and Selecto silica gel columns and subsequently
characterized as described above for [3H]GlcNAc-P-P-Ner.
[3H]GlcNAc-P-Ner was stored in CH3OH at
Enzymatic reactions for the synthesis of
[3H]GlcNAc-P-P-citronellol
([3H]GlcNAc-P-P-Cit) and
[3H]GlcNAc2-P-P-Cit contained microsomes from
Chinese hamster ovary Tn-10 cells (57) (1 mg of membrane protein), 0.1 M Tris-HCl (pH 8.5), 4 mM citronellyl phosphate
(Cit-P), 40 mM MgCl2, 2 mM sodium
orthovanadate, and 0.5 mM UDP-[3H]GlcNAc (68 dpm/pmol) in total volume of 1 ml. Reaction mixtures were incubated at
30 °C for 4 h and then subjected to centrifugation at
100,000 × g for 10 min in a Beckman TL100.3
micro-ultracentrifuge. Radiolabeled GlcNAc1-2-P-P-Cit was
purified as described above for GlcNAc-P-P-Ner. After desalting, the
fractions containing GlcNAc1-2-P-P-Cit were pooled, dried
by rotary evaporation at 30 °C under reduced pressure, and dissolved
in a minimal volume of CH3OH. The sample was then spotted
on a sheet of Whatman 3MM paper, and the paper was developed in a
descending manner for 14 h using butanol/pyridine/H2O
(6:4:3, v/v). Regions of the chromatogram containing
[3H]GlcNAc-P-P-Cit and
[3H]GlcNAc2-P-P-Cit were located using a
Bioscan System 200 Imaging Scanner. These regions were cut out, and
each compound was eluted with H2O. The H2O
eluates were dried by rotary evaporation at 30 °C under reduced
pressure and desalted by gel-filtration on Sephadex G-10 as described
above. Fractions containing the products were pooled and dried by
rotary evaporation at 30 °C under reduced pressure. The compounds
were then dissolved in 2 ml of CH3OH and stored at
Preparation of Sealed Everted Membrane Vesicles--
Everted
membrane vesicles were prepared by a modification of the procedure
described by Ambudkar et al. (58). Cells were grown with
vigorous aeration to an absorbance (600 nm) of 1.2 to 1.3 in LB broth
supplemented with glucose to give a final concentration of 0.2%. The
cells from two 500-ml cultures were harvested by centrifugation at
10,000 × g at 4 °C and washed with cold buffer consisting of 10 mM Tris-HCl (pH 7.5), 0.25 M
sucrose, and 0.14 M choline chloride. The washed cells were
resuspended in 20 ml of 10 mM Tris-HCl (pH 7.5), 0.25 M sucrose, 0.14 M choline chloride, and 0.1 mM phenylmethylsulfonyl fluoride and then disrupted by two
passages through a cooled French pressure cell at 4000 p.s.i. Pancreatic DNase was added to the lysate immediately to give a final
concentration of 0.1 mg/ml, and the lysate was incubated in an ice bath
for 60 min. Intact cells were removed by low speed centrifugation, and
membrane vesicles were harvested by centrifugation of the supernatant
solution at 150,000 × g for 60 min at 4 °C. The
pelleted vesicles were washed with cold buffer consisting of 10 mM Tris-HCl (pH 7.5), 0.25 M sucrose, and 0.14 M choline chloride and subsequently resuspended in 1.0 ml
of the same buffer. Vesicle preparations were stored at
The orientation of membrane vesicles was determined using the procedure
described by Futai (59). This procedure revealed that essentially all
of the vesicles were in the everted or "inside-out" orientation. In
addition, the integrity of vesicle membranes was verified by assaying
the ability of vesicles to maintain an NADH-generated Assay of Transport Activity by Membrane Vesicles or Human
Erythrocytes--
The standard assay mixture contained 10 mM Tris-HCl (pH 7.5), 0.14 M choline chloride,
0.125 M sucrose, 2.5 mM MgCl2,
[3H]GlcNAc-P-P-Ner (35 µM, 18 dpm/pmol),
and vesicles (120-150 µg) in a total volume of 20 µl. Reaction
mixtures were incubated at 37 °C for various periods of time and
then terminated by the addition of 0.5 ml of cold 10 mM
Tris-HCl (pH 7.4), 0.25 M sucrose, 0.14 M
choline chloride, and 5 mM CaCl2. The diluted
mixtures were immediately suction-filtered through 0.45-µm HA
filter discs, and the discs were washed with 0.5 ml of 10 mM Tris-HCl (pH 7.4), 0.25 M sucrose, 0.14 M choline chloride, and 5 mM CaCl2.
The filters were then added to counting vials and incubated with 0.5 ml
of 2% SDS for 1 h at room temperature prior to the addition of 5 ml of Ready Safe liquid scintillation fluid (Beckman). The closed vials
were incubated overnight at room temperature, and the radioactivity was
quantified using a Beckman LS 6500 liquid scintillation spectrometer.
Transport of either [14C]glucose or
[3H]GlcNAc-P-P-Ner by human erythrocytes was assayed
using the same procedure described above for membrane vesicles except
that the assays were carried out at 21 °C, and the cells were
harvested by filtration using Whatman GF/C glass-fiber filter discs
(Whatman Laboratory Products, Inc., Clifton, NJ). Human erythrocytes
were obtained from a donor (J. S. R.) as previously described (18,
19).
Assay for Lipid III Accumulation--
Strain PR4180 was grown in
120 ml of proteose peptone beef extract (PPBE) broth (60) containing
0.2% glucose and 50 µg/ml ampicillin at 37 °C with vigorous
aeration to an optical density (600 nm) = 0.5. The culture was
divided into two equal portions, and the cells in each portion were
harvested by centrifugation. The cells in each pellet were resuspended
in 6 ml of fresh PPBE broth containing either glucose or arabinose at
final concentrations of 0.2% and 0.1%, respectively.
[3H]GlcNAc (75 µCi, 4.1 Ci/mmol) was then added to both
cultures, and the cultures were incubated at 37 °C with vigorous
aeration for 20 min. Each of the cultures (~4.5 × 1010 cells/culture) was then poured into a separate beaker
containing 5 g of ice, and the cells were harvested by
centrifugation at 4 °C. The cells were then washed with 6 ml of cold
0.9% saline followed by successive extraction with 6 ml of cold 95%
ethanol and 6 ml of cold acetone. The extracted cells were dried under vacuum, and they were then either further processed for the isolation of radiolabeled lipid III or stored at
Radiolabeled lipid III was extracted from the dried cells by the
addition of 2.5 ml of chloroform:methanol:water (10:10:3, v/v) followed
by constant stirring for 15 min at room temperature. Particulate matter
was removed by centrifugation at room temperature, and the amount of
radiolabeled lipid III in each of the extracts was determined by
ascending paper chromatography on EDTA-treated SG-81 paper (Whatman) as
previously described (32).
Determination of Cell Viability--
Strain PR4180 was grown in
PPBE broth containing 0.2% glucose and 50 µg/ml ampicillin at
37 °C with vigorous aeration to an optical density (600 nm) = 0.20. The cells were then harvested by centrifugation and resuspended
in 100 ml of fresh PPBE broth containing 50 µg/ml ampicillin. The
resulting culture was then divided into two equal portions; glucose was
added to one portion to give a final concentration of 0.2%, whereas
arabinose was added to the other portion to give a final concentration
of 0.1%. Both cultures were incubated at 37 °C with vigorous
aeration, and the number of viable cells per milliliter of each culture
was determined at various intervals by plating dilutions of the
cultures onto PPBE agar plates and then counting the number of colonies
after overnight incubation of the plates at 37 °C. In addition, the optical densities of the cultures (600 nm) were monitored throughout the course of the experiment.
Uptake of [3H]GlcNAc-P-P-Ner by Everted Membrane
Vesicles--
GlcNAc-P-P-Und is the first intermediate in the
synthesis of the ECA trisaccharide repeat unit (27, 30) as well as the repeat units of many Wzy-dependent O antigens, including
the O16 antigen (20, 36, 40, 41). Recent studies provided indirect evidence that the wzxO16 gene product of
E. coli K-12/O16 functions as a translocase (42). These
studies also suggested that WzxO16 was capable of
translocating incomplete Und-P-P-linked O16 repeat units, including
GlcNAc-P-P-Und. Therefore, we conducted experiments in an attempt to
demonstrate translocase activity directly by assaying the transport of
[3H]GlcNAc-P-P-Ner, a water-soluble analogue of
GlcNAc-P-P-Und, by everted membrane vesicles prepared from E. coli K-12/O16. As illustrated in Fig.
1, the isoprenoid moieties of
GlcNAc-P-P-Ner and GlcNAc-P-P-Und are fully unsaturated. However, the
polyisoprenoid chain of GlcNAc-P-P-Und contains eleven isoprene units
and is extremely hydrophobic. In contrast, the short isoprenoid chain of GlcNAc-P-P-Ner contains only two isoprene units thus rendering this
compound water-soluble. Incubation of sealed everted membrane vesicles
prepared from E. coli S
Extraction of preloaded vesicles with methanol resulted in the recovery
of greater than 90% of the vesicle-associated radiolabel. Approximately 50% of this material was intact
[3H]GlcNAc-P-P-Ner, and the remainder was identified as
[3H]GlcNAc 1-P. However, it is important to note that
[3H]GlcNAc 1-P was not detected in reaction mixtures
following the incubation of [3H]GlcNAc-P-P-Ner with
membrane vesicles prepared from strains PR4150 or PR4156 (data not
shown). These observations support the conclusion that enzymatic
hydrolysis of the pyrophosphate linkage of
[3H]GlcNAc-P-P-Ner occurred following translocation of
the analogue into the vesicle lumen; however, the enzyme responsible
for this hydrolysis has not been identified.
The transport of GlcNAc-P-P-Ner was strictly dependent on the integrity
of membrane vesicles, and uptake of the analogue was abolished when
assay mixtures contained 0.1% Triton X-100 (data not shown). The
requirement for an intact permeability barrier was also demonstrated by
determining the rate of efflux of [3H]GlcNAc-P-P-Ner from
sealed preloaded vesicles following their incubation in isotonic and
hypotonic solutions. Dilution of preloaded vesicles into 100 volumes of
isotonic buffer (10 mM Tris-HCl, pH 8.0, 0.14 M
choline chloride, 0.125 M sucrose, 2.5 mM
MgCl2) resulted in the time-dependent efflux of
[3H]GlcNAc-P-P-Ner, and ~80% of the analogue was
released within 20 min (Fig. 3). In
contrast, the release of almost 90% of the [3H]GlcNAc-P-P-Ner from preloaded vesicles occurred
within 30 s when the vesicles were ruptured by dilution into 100 volumes of H2O. The transport of GlcNAc-P-P-Ner into
vesicles did not appear to require a proton-motive force, because it
was not affected by the addition of ATP, NADH, D-lactate,
carbonyl cyanide m-chlorophenylhydrazone or
carbonylcyanide 4-trifluoromethoxyphenylhydrazone to assay mixtures (data not shown). These observations suggest that uptake and
efflux of [3H]GlcNAc-P-P-Ner by everted membrane vesicles
occurs by an equilibrium process. Assuming this to be the case, net
transport of the radiolabeled analogue into vesicles was observed until
its distribution between the extravesicular solution and the lumen of
the vesicles reached equilibrium at 25 min (Fig. 2). Approximately 5%
of the [3H]GlcNAc-P-P-Ner was internalized at
equilibrium, and from these data an intravesicular volume of 6.7 µl
per mg of vesicle protein was calculated. This value is within the
range previously estimated for the intravesicular volume of everted
bacterial membrane vesicles (61-64).
Transport of [3H]GlcNAc-P-P-Ner into Everted Membrane
Vesicles Is Mediated by the wzxE Gene Product--
The results
described above suggest that the uptake of
[3H]GlcNAc-P-P-Ner by everted membrane vesicles occurs by
a protein-mediated process. Thus, experiments were conducted to
determine the possible role of the WzxO16 translocase in
this process. E. coli S
These results clearly indicate that the association of
[3H]GlcNAc-P-P-Ner with vesicles was the result of
transport into the vesicle lumen and was not due to nonspecific binding
of the analogue to the outer surface of the sealed vesicles. Additional
support for this conclusion was obtained by the observation that
[3H]GlcNAc-P-P-Ner was not transported by human red blood
cells under conditions whereby significant facilitated transport of glucose was observed (Fig. 4). All of
these results indicate that the ability of vesicles to transport
[3H]GlcNAc-P-P-Ner requires a functional wzxE
gene product.
The role of WzxE in the transport of [3H]GlcNAc-P-P-Ner
into membrane vesicles was further demonstrated by complementation studies. As mentioned previously, no transport of
[3H]GlcNAc-P-P-Ner into everted vesicles was observed
when the vesicles were prepared from strain PR4150
(wzxE::cm) (Table II). However, wild-type levels
of transport were observed using membrane vesicles prepared from strain
PR4184, a transformant of strain PR4150 containing plasmid pRL162.
Plasmid pRL162 contains several genes of the wec cluster,
including the wzxE gene, on an 8.4-kb HindIII
insert fragment (Fig. 5). In contrast,
transport of [3H]GlcNAc-P-P-Ner into vesicles was not
observed using vesicles prepared from strain PR4179, a transformant of
strain PR4150 containing plasmid pRL160 (Table II). Plasmid pRL160
contains an insert fragment that was generated by removal of a 745-bp
XmaI-HindIII fragment from the same 8.4-kb
HindIII fragment contained in pRL162 (Fig. 5). This
truncation resulted in a deletion of 378 bp from the 3' terminus of the
wzxE gene. All of the genes on the original 8.4-kb
HindIII insert fragment are transcribed in the same
direction. In addition, the wzxE gene is the last complete
open reading frame in this sequence of genes. Thus, the inability of
pRL160 to complement the observed transport defect of membrane vesicles
derived from strain PR4150 was attributed to a lack of a functional
wzxE gene and it was not due to a polar effect of the
truncated wzxE gene on downstream genes. Taken together, the
above findings support the conclusion that
[3H]GlcNAc-P-P-Ner is transported into everted membrane
vesicles and that transport is mediated by the wzxE gene
product.
Null Mutations in wzxE Result in the Accumulation of Lipid III and
Cell Death--
The available data indicate that synthesis of carrier
lipid-linked ECA trisaccharide repeat units (lipid III) occurs on the inner leaflet of the cytoplasmic membrane. This is followed by the
WzxE-mediated transbilayer movement of lipid III molecules to the
periplasmic face of the membrane where they are utilized for the
assembly of ECA polysaccharide chains by the block-polymerization mechanism. Accordingly, the inability of cells to synthesize a functional wzxE gene product would be expected to result in
the accumulation of lipid III. Indeed, a pronounced accumulation of radiolabeled lipid III was observed in cells when strain PR4180 (wecA::Tn10
wzxE::cm/pRL147, wecA under
control of the PBAD promoter) was incubated with
[3H]GlcNAc during the initial 20 min following the
addition of arabinose to the growth medium. In contrast, essentially no
radiolabeled lipid III was detected when cells were incubated with
[3H]GlcNAc following the addition of glucose to cultures
(Table III).
The arabinose induced overexpression of wecA in strain
PR4180 was accompanied by a loss of cell viability (Fig.
6). In contrast, cell growth and
viability was essentially unaffected following the overexpression of
wecA in wecA::Tn10 mutants
possessing a wild-type wzxE allele. These observations,
together with those described above, suggest that the lack of a
functional WzxE results in the accumulation of lipid III and that the
accumulation of this intermediate is toxic to cells. Thus, the
transduction of the wzxE::cm insertion into
recipient strains is essentially precluded unless the ability of these
strains to synthesize lipid III is first abolished. Therefore, strains
containing the wecA::Tn10 insertion
were routinely used as recipients for this purpose. However, the
wecA::Tn10 insertion mutation had no
apparent effect on the transport of [3H]GlcNAc-P-P-Ner
into membrane vesicles as indicated by the results obtained with
vesicles derived from strain 21548 (Table II).
Properties of the WzxE-mediated Transport System--
The
WzxE-mediated transport of GlcNAc-P-P-Ner into everted membrane
vesicles exhibited saturation kinetics (Fig.
7A), and the apparent
Km for GlcNAc-P-P-Ner was calculated to be ~55
µM (Fig. 7B). To examine the
specificity of the Wzx translocase, its ability to mediate the
transport of several compounds structurally related to GlcNAc-P-P-Ner
was determined. These studies clearly revealed a marked preference for
GlcNAc-P-P-Ner as a substrate (Table IV).
Indeed, the rate of transport
N-acetylglucosaminylpyrophosphorylcitronellol (GlcNAc-P-P-Cit) was markedly less than that determined for
GlcNAc-P-P-Ner despite the close structural similarity of these
compounds. Thus, the WzxE translocase is able to distinguish between
intermediates possessing fully unsaturated polyprenyl moieties and
dolichols that contain a saturated Despite the crucial role of "flippases" in membrane biology,
very little is known about their structures and the mechanism by which
they facilitate the transbilayer movement of polar lipids (17). A
critical step in discovering new information concerning this novel
class of membrane transporters is the identification of putative
proteins that function as flippases. However, the identification of
flippase proteins has been hampered by the lack of convenient
biochemical assays for their activity.
Water-soluble analogues of a variety of lipids have proven
to be useful model compounds for the study of several aspects of glycolipid biochemistry. In most cases, the increased hydrophilic properties of these analogues are due to the presence of shortened hydrocarbon chains relative to those that are present in the naturally occurring compounds. Thus, water-soluble short-chain analogues of
Man-P-Dol (18) and Glc-P-Dol (19) containing (C10)
isoprenyl chains have been used to investigate the properties of
protein(s) that may be involved in the transbilayer movement of
dolichol-linked intermediates in the ER of rat liver and pig brain
cells. In addition, Cit-P, a 10-carbon analogue of Dol-P, is recognized
as a substrate by a wide variety of enzymes involved in the eucaryotic
protein N-glycosylation pathway. These enzymes include
Man-P-Dol synthase from pig liver and brain (18), Glc-P-Dol synthase
from pig brain (19),
Man-P-Dol:Man5-8GlcNAc2-P-P-Dol
mannosyltransferase(s) from pig brain (48),
Glc-P-Dol:Glc0-3Man9GlcNAc2-P-P-Dol glucosyltransferase from pig brain (66), UDP-GlcNAc:Dol-P
GlcNAc-phosphotransferases from pig brain, CHO cell membranes and hen
oviduct (this study), and UDP-GlcNAc:GlcNAc-P-P-Dol GlcNAc-transferase
from CHO cell membranes.2
Furthermore, Man-P-Cit was utilized as a mannose donor for the in
vivo synthesis of Man9GlcNAc2-P-P-Dol by
permeabilized CHO cells (67). Several bacterial enzymes have also been
demonstrated to glycosylate Ner-P, a short-chain analogue of Und-P.
These include the Man-P-Und synthase of Micrococcus luteus
(18), the UDP-GlcNAc:undecaprenol phosphate GlcNAc 1-P transferase
(WecA) of E. coli (this study) and the
UDP-GlcNAc:undecaprenol phosphate GlcNAc transferase of Bacillus
cereus (this study). In addition, ceramide and glucosyl-ceramide analogues with 8-carbon acyl chains are substrates for all of the
enzymes involved in higher glycosphingolipid biosynthesis (47).
Analogues of phosphatidylcholine (PC) containing short fatty acyl
chains (C4 to C6) have also been used to
partially purify and characterize potential proteins involved in the
transbilayer movement of PC (14, 43-46). In this regard, it is
significant to note that the properties of the phospholipid flippase(s)
present in biogenic membrane bilayers that have been determined using water-soluble dibutyroyl-PC are entirely consistent with
properties determined using alternative methods for measuring
phospholipid "flip-flop" (reviewed in Ref. 11).
In this study, the flippase-mediated transport of GlcNAc-P-P-Ner, a
water-soluble analogue of GlcNAc-P-P-Und, into sealed everted membrane
vesicles of E. coli K-12 was investigated to assess the role
of wzx genes in the translocation of the trisaccharide-lipid intermediate involved in ECA assembly (Fig.
8). Transport of GlcNAc-P-P-Ner into
sealed and everted membrane vesicles of E. coli was found to
be time-dependent and saturable. In addition, the transport process did not require a proton-motive force, and the data presented here are consistent with the conclusion that entry of GlcNAc-P-P-Ner into the lumen of everted vesicles occurs by the process of facilitated diffusion. Facilitated diffusion is also believed to be the mechanism involved in the flippase-mediated transport of water-soluble analogues of phospholipids (14, 43-46) and polyisoprenyl-linked saccharides and
oligosaccharides into microsomal vesicles (18, 19). Indeed, the
in vivo movement of lipid III from the inner to the outer leaflet of the cytoplasmic membrane could plausibly be driven by its
utilization for the process of ECA polysaccharide chain elongation
catalyzed by WzyE.
-isoprene unit
in the carrier lipid. These results support the conclusion that the
wzxE gene encodes a membrane protein involved in the transbilayer movement of lipid III in E. coli.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3)-
-D-Fuc4NAc-(1
4)-
-D-ManNAcA-(1
4)-
-D-GlcNAc-(1
(29). The ECA trisaccharide repeat unit is synthesized as the Und-P-P-linked intermediate, Fuc4NAc-ManNAcA-GlcNAc-P-P-Und (lipid III)
(27, 30-32). The available data are consistent with the synthesis of
lipid III on the inner leaflet of the cytoplasmic membrane followed by
its transbilayer movement to the periplasmic face of the membrane where
assembly of the polysaccharide chains occurs by a block-polymerization
mechanism. The polysaccharide chains are subsequently transferred from
the carrier lipid to an as yet unidentified glyceride acceptor to yield
ECAPG molecules in which the potential reducing terminal
GlcNAc residue is linked to diacylglycerol through phosphodiester
linkage (33, 34). Completed ECAPG polymers are then
incorporated into the exterior leaflet of the outer membrane.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
E. coli K-12 strains
20 °C until used for transport assays. Formation of
[3H]GlcNAc-P-P-Ner was strictly dependent upon the
addition of exogenous Ner-P to reaction mixtures. The product was
detected as a single radioactive compound when analyzed by thin-layer
chromatography on silica gel plates developed with three different
solvent systems, and it was detected by a phospholipid-specific spray
reagent (55) and by an anisaldehyde-based spray reagent for the
detection of isoprenoid compounds (56).
N-Acetyl[3H]glucosamine was the only
radioactive product released by mild acid hydrolysis (0.01 N HCl, 100 °C, 10 min) as determined by descending paper
chromatography on Whatman 3MM paper using
butanol/pyridine/H2O (6:4:3, v/v) as the developing
solvent system.
20 °C until used for transport assays.
20 °C until used for transport assays.
-[3H]Man-P-Cit and
-[3H]Man-P-Ner
were synthesized enzymatically using a partially purified preparation
of Man-P-Und synthase from Micrococcus luteus,
GDP-[3H]mannose, and either Cit-P or Ner-P as described
previously (48).
80 °C
until used for transport assays.
pH as
determined by monitoring the quenching of acridine orange fluorescence
(58).
20 °C for later use.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
864 with
[3H]GlcNAc-P-P-Ner resulted in the
time-dependent uptake of the radiolabeled analogue (Fig.
2). In addition, the amount of
[3H]GlcNAc-P-P-Ner transported into the lumen of the
vesicles increased in linear proportion to the concentration of
vesicles in reaction mixtures (Fig. 2, inset).
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Fig. 1.
Structural relationship between
GlcNAc-P-P-undecaprenol (GlcNAc-P-P-Und) and GlcNAc-P-P-nerol
(GlcNAc-P-P-Ner).
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Fig. 2.
Uptake of [3H]GlcNAc-P-P-Ner by
everted membrane vesicles. Incubation mixtures contained 10 mM Tris-HCl (pH 7.4), 0.25 M sucrose, 0.14 M choline chloride, 2.5 mM MgCl2,
35 µM [3H]GlcNAc-PP-Ner (18 dpm/pmol), and
membrane vesicles (120-150 µg of protein) in a total volume of 20 µl. After incubation at 37 °C for the indicated periods of time,
the amount of radiolabel transported into the vesicles was determined
as described under "Experimental Procedures." Data were obtained
using membrane vesicles prepared from the following strains: S 864
(wild-type) (
); S
874 (
[wzxO16
wzxC]) (
); 14.5 (wzxE::cm) (
).
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Fig. 3.
Efflux of [3H]GlcNAc-P-P-Ner
from preloaded membrane vesicles. Incubation mixtures contained 10 mM Tris-HCl (pH 7.4), 0.25 M sucrose, 0.14 M choline chloride, 2.5 mM MgCl2,
1.6 µM [3H]GlcNAc-P-P-Ner (1800 dpm/pmol),
and 600 µg of membrane protein in a total volume of 65 µl. The
reaction mixture was incubated at 37 °C for 5 min, and a 10-µl
aliquot (zero time sample) was removed and assayed for the amount of
radiolabel transported into the vesicles as described under
"Experimental Procedures." Aliquots of 10 µl were then
removed and added to either 1 ml of assay buffer ( ) or water
(
), and they were further incubated at 37 °C for the indicated
periods of time. The amount of radiolabel retained by the vesicles was
then determined using the filtration assay as described under
"Experimental Procedures."
874 is a derivative of strain
S
864 that contains an extended deletion in the region of the
chromosome that includes the putative flippase genes involved in the
synthesis of the O16 O antigen (wzxO16) and
colanic acid (wzxC) (65). Quite unexpectedly, the kinetics of [3H]GlcNAc-P-P-Ner uptake by membrane vesicles
prepared from strain S
874 (
[wzx016
wzxC]) were the same as those observed using membrane
vesicles prepared from strain S
864 (wild-type) (Fig. 2). These
results suggested that a membrane protein other than WzxO16
or WzxC was responsible for mediating the transport of the analogue
into membrane vesicles. In this regard, it has been suggested that the
wzxE gene of the wec gene cluster encodes a putative translocase that facilitates the transbilayer movement of
Und-P-P-linked ECA trisaccharide repeat units (25). Thus, experiments
were conducted to determine if the observed transport of
[3H]GlcNAc-P-P-Ner was mediated by the wzxE
gene product. As shown in Fig. 2, uptake of
[3H]GlcNAc-P-P-Ner was not detected using everted
membrane vesicles prepared from E. coli 14.5, a
wzxE::cm insertion mutant derived from E. coli HCB33. The same results were obtained using everted membrane
vesicles prepared from strains PR4150 and PR4156, two strains that are
derivatives of strains S
874 and S
864, respectively, into which
the wzxE::Tn10cam insertion was
introduced by transduction (Table
II).
Transport of GlcNAc-P-P-Ner into everted membrane vesicles obtained
from wild-type and mutant strains of E. coli K-12
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Fig. 4.
Uptake of [14C]glucose and
[3H]GlcNAc-P-P-Ner by human red blood cells.
Incubation mixtures contained 10 mM Tris-HCl (pH 7.4), 0.25 M sucrose, 0.14 M choline chloride, 2.5 mM MgCl2, freshly obtained human red blood
cells (containing the indicated amount of phospholipid), and either
0.08 µM [14C]glucose (94 dpm/pmol) or 0.08 µM [3H]GlcNAc-P-P-Ner (18 dpm/pmol) in a
total volume of 50 µl. The mixtures were incubated for 1 min at
21 °C, and the amount of radiolabel transported into the red blood
cells was determined by filtration on Whatman GF/C glass fiber filter
discs as described under "Experimental Procedures."
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Fig. 5.
Physical map of the region of the
wec gene cluster between genes wzzE
and wecF. The regions of the gene cluster
contained in plasmids pRL160 and pRL162 are indicated by the open
rectangles.
Accumulation of [3H]GlcNAc-lipid III in E. coli 4180 (wzxE::cm) in the presence of wecA expression
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Fig. 6.
Effect of a wzxE
null-mutation on cell viability. Strain PR4180
( [wzx016 wzxC]
wecA::Tn10
wzxE::cm/pRL147, wild-type wecA
under control of the PBAD promoter) was grown at 37 °C
to an optical density (600 nm) of 0.2 in PPBE broth containing 0.2%
glucose. The cells were then harvested, resuspended in fresh broth
lacking glucose, and then divided into two equal portions. Glucose was
added to one portion to give a final concentration of 0.2% (
),
whereas arabinose was added to the other portion to give a final
concentration of 0.1% (
). Both cultures were incubated with shaking
at 37 °C, and the number of viable cells in each culture was
determined at the indicated times. As a control, the effect of
arabinose-induced expression of wecA on the cell viability
of strain PR4189 (
[wzx016 wzxC]
wecA::Tn10/pRL147, wild-type
wecA under control of the PBAD promoter) was
determined using the same conditions as described above (
).
Additional details are provided under "Experimental
Procedures."
-isoprene unit. A requirement for
a pyrophosphoryl-linked saccharide is also indicated by the inability of the vesicles to transport GlcNAc-P-Ner.
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Fig. 7.
Uptake of [3H]GlcNAc-P-P-Ner by
everted membrane vesicles is saturable. Incubation mixtures
contained 10 mM Tris-HCl (pH 7.4), 0.25 M
sucrose, 0.14 M choline chloride, 2.5 mM
MgCl2, and the indicated concentration of
[3H]GlcNAc-P-P-Ner (18 dpm/pmol), and 60 µg of membrane
protein in a total volume of 20 µl. Reaction mixtures were incubated
at 37 °C for 10 min, and the amount of radiolabel transported into
the vesicles was determined as described under "Experimental
Procedures."
Transport of GlcNAc-P-P-Ner and related compounds into everted membrane
vesicles prepared from E. coli S864
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (28K):
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Fig. 8.
Proposed roles of WzxE in the translocation
of polyisoprenyl-linked saccharides across the cytoplasmic
membrane. It is proposed that WzxE participates in the
transbilayer movement of Fuc4NAc-ManNAcA-GlcNAc-P-P-Und
(translocation 1) and GlcNAc-P-P-Und (translocation
2) in vivo. The WzxE-mediated transbilayer movement of
GlcNAc-P-P-Ner across the cytoplasmic membrane (dashed
arrow) was demonstrated in this study using sealed everted
cytoplasmic membrane vesicles.
Several experimental observations provide strong support for the conclusion that transport of GlcNAc-P-P-Ner into vesicles was mediated by WzxE. Thus, transport of GlcNAc-P-P-Ner was not observed using vesicles prepared from mutant strains possessing a null-mutation in wzxE. However, transport was fully restored when vesicles were prepared from transformants of these mutants that expressed the wild-type wzxE allele. Furthermore, no transport of [3H]GlcNAc-P-P-Ner into human red blood cells was observed under the same conditions employed for the transport of the analogue into everted membrane vesicles of E. coli. Thus, the association of [3H]GlcNAc-P-P-Ner with vesicles was not due to nonspecific binding of the radiolabeled analogue to the vesicles. Indeed, the efflux of ~80-90% of the [3H]GlcNAc-P-P-Ner contained in preloaded vesicles was observed following the incubation of these vesicles in isotonic buffer or as a result of their rupture by incubation in water. Finally, [3H]GlcNAc-labeled lipid III was found to accumulate in cells of mutant strains possessing a null-mutation in wzxE. All of these data support the conclusion that transport of GlcNAc-P-P-Ner into vesicles was facilitated by WzxE, the putative flippase involved in the assembly of the linear polysaccharide chains of phosphoglyceride-linked ECA (Fig. 8). To our knowledge, this is the first direct demonstration of a bacterial flippase activity involved in the transmembrane translocation of a polyisoprenyl-linked saccharide. It may be functionally significant that the hydropathy profile of WzxE is very similar to that of the Rft1 gene product, which is proposed to be a Man5GlcNAc2-P-P-Dol flippase in yeast (68).
A determination of the ability of several water-soluble compounds
related in structure to GlcNAc-P-P-Ner to serve as substrates of the
WzxE-mediated transport system demonstrated that a
pyrophosphoryl-linked GlcNAc substituent and an unsaturated
-isoprene unit are critical structural features required for
transport. It seems likely that the rate of WzxE-mediated transport of
Fuc4NAc-ManNAcA-GlcNAc-P-P-Ner into vesicles would be considerably
faster than that observed for GlcNAc-P-P-Ner. However, these rates have
not been determined due to the inability to synthesize the
water-soluble analogue of lipid III.
The Wzy-dependent O16 O antigen of E. coli K-12/O16 is a branched pentasaccharide, and synthesis of this O antigen is initiated by formation of GlcNAc-P-P-Und catalyzed by WecA (40, 41). Feldman et al. (42) reported that the assembly of the O16 O antigen involves translocation of Und-P-P-linked O16 repeat units across the cytoplasmic membrane mediated by the putative flippase, WzxO16. However, these studies revealed that the WzxO16 translocase does not appear to require a completed Und-P-P-linked O16 repeat unit, and data were presented that suggested that the WzxO16 translocase was able to mediate the in vivo translocation of GlcNAc-P-P-Und. It has also been suggested that GlcNAc-P-P-Und is the donor of the terminal GlcNAc residue of the outer core region of the K-12 lipopolysaccharide. Therefore, it is somewhat surprising that we failed to detect any WzxO16-mediated uptake of radiolabeled GlcNAc-P-P-Ner into vesicles obtained from E. coli strain 14.5, a wzxE::cm insertion mutant possessing wild-type wzxO16 and wzxC genes (Table II). Indeed, the data presented in the current study indicate that transport of GlcNAc-P-P-Ner into the lumen of everted vesicles was mediated exclusively by WzxE.
The flippases involved in the translocation of Und-P-P-linked repeat units of Wzy-dependent O antigens appear to exhibit broad substrate specificity. For example, the flippases involved in the translocation of the repeat unit of colanic acid (WzxC), the O antigen repeat unit of S. enterica serovar Typhimurium LT2 (WzxSeLT2), and the O antigen repeat unit of E. coli K-12/O16 (WzxO16) are also able to facilitate the in vivo transbilayer movement of the Und-P-P-linked O antigen repeat unit E. coli O7 (42). Indeed, the relaxed specificities of these flippases are quite apparent when one considers the pronounced differences in the structures of their respective natural substrates as well as the differences of these structures to that of the O7 repeat unit. In contrast, it appears that WzxE is unable to mediate the in vivo translocation of the Und-P-P-linked O7 O antigen repeat unit, because synthesis of an O7 lipopolysaccharide was not detected in a mutant strain that possessed a wild-type wzxE allele but that had an extended deletion that included the wzxO16 gene (42). Thus, the substrate specificity of WzxE appears to be rather stringent, and the available information suggests that WzxE may only function in the translocation of the Und-P-P-linked trisaccharide repeat unit of ECA. However, as suggested above, WzxE may also function in vivo for the translocation of GlcNAc-P-P-Und. The possible roles of WzxE in the translocation of various polyisoprenyl-linked saccharides are summarized in Fig. 8.
Null-mutations in wzxE result in the accumulation of lipid III and cell death. Accordingly, transduction of the wzxE::cm insertion mutation from E. coli strain 14.5 into recipient strains resulted in cell death unless the recipient strains were first rendered unable to synthesize lipid III by prior introduction of the wecA::Tn10 insertion mutation. However, strain 14.5 is not defective in the synthesis of lipid III, and indeed it is able to synthesize ECAPG. We believe that strain 14.5 possesses an unlinked suppressor mutation that compensates for the loss of a functional WzxE; however, the nature of this suppressor mutation has not yet been determined. In this regard, the results of preliminary experiments did not reveal an alteration in the structural gene for WzxO16 in strain 14.5. This finding, in conjunction with the inability of everted vesicles prepared from this strain to function in the transport of GlcNAc-P-P-Ner, further supports the conclusion that WzxO16 is unable to mediate the transport of this analogue. These observations also indicate that the putative suppressor mutation is unable to confer the ability to transport the analogue into membrane vesicles prepared from strain 14.5.
No differences in the specific activities of WzxE-mediated transport of
GlcNAc-P-P-Ner were found in the current study using membrane vesicles
prepared from either wecA::Tn10
insertion mutants or wecA+ strains. These
findings suggest that transbilayer translocation of GlcNAc-P-P-Ner is
not dependent on the formation of a complex between WecA and WzxE.
However, an interaction between WecA, WzxE, and perhaps other proteins
may be important for the translocation of a complete Und-P-P-linked ECA
trisaccharide in vivo, and future experiments will be
directed at investigating this possibility.
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ACKNOWLEDGEMENT |
---|
We acknowledge the generous gift of E. coli strain 14.5 (wzxE::cm) from Paul N. Danese.
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FOOTNOTES |
---|
* This work was supported by NIGMS, National Institutes of Health Grants GM52882 (to P. D. R.) and GM36365 (to C. J. W.).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: Dept. of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd., Bethesda, MD 20814-4799. Tel.: 301-295-3418; Fax: 301-295-3512; E-mail: rickp@usuhs.mil.
¶ Current address: the Centre for Biotechnology, Anna University, Madras 600 025, India.
Published, JBC Papers in Press, March 5, 2003, DOI 10.1074/jbc.M301750200
2 C. J. Waechter, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: ER, endoplasmic reticulum; GPI, glycosylphosphatidylinositol; GlcNAc, N-acetyl-D-glucosamine; GlcNAc2-P-P-Dol, di(N-acetylglucosaminyl)pyrophosphoryldolichol; LPS, lipopolysaccharide; Und, undecaprenol; Man, mannose; PPBE, proteose peptone beef extract; Dol, dolichol; Cit-P, citronellyl phosphate; Ner, nerol; ECAPG, phosphoglyceride-linked enterobacterial common antigen; PC, phosphatidylcholine; CHO, Chinese hamster ovary; ManNAcA, N-acetyl-D-mannosaminuronic acid; Fuc4NAc, 4-acetamido-4, 6-dideoxy-D-galactose.
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REFERENCES |
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