Download The wbbD gene of E. coli strain VW187

Glycobiology vol. 15 no. 6 pp. 605–613, 2005
doi:10.1093/glycob/cwi038
Advance Access publication on December 29, 2004
The wbbD gene of E. coli strain VW187 (O7:K1) encodes a UDP-Gal:
GlcNAc␣-pyrophosphate-R ␤1,3-galactosyltransferase involved in the
biosynthesis of O7-specific lipopolysaccharide
John G. Riley2, Mohammed Menggad2, Pedro J. MontoyaPeleaz3, Walter A. Szarek3, Cristina L. Marolda4,
Miguel A. Valvano4, John S. Schutzbach2,
and Inka Brockhausen1,2
2
Department of Medicine, Department of Biochemistry, The Arthritis
Centre and Human Mobility Research Centre, Queen’s University,
Kingston General Hospital, Kingston, Ontario K7L 2V7, Canada;
3
Department of Chemistry, Queen’s University, Kingston, Ontario K7L
3N6, Canada; 4Department of Microbiology and Immunology, University
of Western Ontario, London, Ontario N6A 5C1, Canada
Received on September 16, 2004; revised on November 18, 2004;
accepted on December 23, 2004
In this work, we demonstrate that the wbbD gene of the O7
lipopolysaccharide (LPS) biosynthesis cluster in Escherichia coli
strain VW187 (O7:K1) encodes a galactosyltransferase involved
in the synthesis of the O7-polysaccharide repeating unit. The
galactosyltransferase catalyzed the transfer of Gal from UDPGal to the GlcNAc residue of a GlcNAc-pyrophosphate-lipid
acceptor. A mutant strain with a defective wbbD gene was unable
to form O7 LPS and lacked this specific galactosyltransferase
activity. The normal phenotype was restored by complementing
the mutant with the cloned wbbD gene. To characterize the WbbD
galactosyltransferase, we used a novel acceptor substrate containing GlcNAc␣-pyrophosphate covalently bound to a hydrophobic
phenoxyundecyl moiety (GlcNAc ␣-O-PO3-PO3-(CH2)11-Ophenyl). The WbbD galactosyltransferase had optimal activity at
pH 7 in the presence of 2.5 mM MnCl2. Detergents in the assay
did not increase glycosyl transfer. Digestion of enzyme product by
highly purified bovine testicular ␤-galactosidase demonstrated a
␤-linkage. Cleavage of product by pyrophosphatase and phosphatase, followed by HPLC and NMR analyses, revealed a disaccharide with the structure Gal ␤1-3GlcNAc. Our results
conclusively demonstrate that WbbD is a UDP-Gal: GlcNAc␣pyrophosphate-R ␤1,3-galactosyltransferase and suggest that the
novel synthetic glycolipid acceptor may be generally applicable to
characterize other bacterial glycosyltransferases.
Key words: enzyme assay/galactosyltransferase/O antigen
synthesis/undecaprenol-P
Introduction
The O-specific polysaccharide (O antigen) is the outermost
component of the lipopolysaccharide (LPS), a major con1
To whom correspondence should be addressed; email: brockhau@
post.queensu.com
Glycobiology vol. 15 no. 6 © Oxford University Press 2004; all rights reserved.
stituent of the outer membrane in Gram-negative bacteria.
The genetics and biochemistry of the pathways for the
biosynthesis and assembly of O antigens have been well
defined over the past few decades using model systems
(Raetz and Whitfield, 2002; Samuel and Reeves, 2003;
Valvano, 2003). One of these models is the O7 polysaccharide of the Escherichia coli strain VW187 (O7:K1), which
arises from the polymerization of a pentasaccharide O
repeating unit consisting of 3-VioNAc β1-2 [Rha α1-3]Man
α1-4Gal β1-3 GlcNAc α1- (L’vov et al., 1984). The synthesis of the O7 polysaccharide involves the assembly of the
repeating unit onto undecaprenol-phosphate (Und-P)
(Alexander and Valvano, 1994). The initiation reaction is
catalyzed by the integral membrane protein WecA, a
UDP-GlcNAc:Und-P GlcNAc-1-phosphate transferase that
belongs to the family of polyisoprenyl-phosphate N-acetylhexosamine-1-phosphate transferases (Valvano, 2003). This
family comprises proteins that are present both in prokaryotes and in eukaryotes and share conserved residues involved
in catalysis (Amer and Valvano, 2001, 2002).
As with other O antigens, the assembly of GlcNAcpyrophosphate-Und (GlcNAc-PP-Und)-linked O antigen
subunits is thought to occur at the cytosolic face of the plasma
membrane (Whitfield and Valvano, 1993). Four additional
sugars are subsequently added to the GlcNAc-PP-Und
intermediate to complete the formation of the O7 subunits.
These reactions are catalyzed by specific glycosyltransferases, which are either soluble enzymes or associated with
the plasma membrane. The candidate enzymes for the completion of the O7 subunit are predicted to be encoded by the
genes wbbABCD, which map within the O7 LPS biosynthesis gene cluster (Marolda et al., 1999). The lipid-linked pentasaccharide is subsequently translocated across the inner
membrane and then polymerized to form a lipid-linked
polysaccharide by transfer of repeating units to the reducing end of the growing polymer, a process that differs from
biosynthetic mechanisms of oligosaccharides in mammalian systems where the growth occurs at the nonreducing
end of the glycan chains (Brockhausen et al., 1998). This is
followed by the transfer of the completed O7-specific
polysaccharide to the lipid A-core moiety of the LPS molecule (Feldman et al., 1999; Marolda et al., 1999).
From previous studies, the WbbD protein was implicated
in the glycosyl transfer of the second sugar, galactose, to the
nascent O7 antigen repeat (Marolda et al., 1999). In the study
described in this article, we used a synthetic exogenous
acceptor substrate containing GlcNAcα-pyrophosphate
bound to a phenoxyundecyl moiety in [GlcNAc α1-O-PO2-OPO2-O-(CH2)11-O-phenyl]2− (GlcNAc-PP-PhU) to characterize
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J.G. Riley et al.
the galactosyltransferase from the E. coli strain VW187.
Using a combination of genetic and biochemical approaches,
we conclusively demonstrated that the wbbD gene encodes
a UDP-Gal: GlcNAc-R β1,3-galactosyltransferase (Galtransferase) activity involved in O7 LPS synthesis.
Results
Construction and characterization of a wbbD mutant in the
strain VW187
In a previous work, we reported the genetic organization of
the wbEcO7 biosynthesis cluster (Figure 1) and tentatively
assigned wbbD as a gene encoding a galactosyltransferase
involved in the extension of the O7 antigen subunit by adding Gal to GlcNAc-PP-Und (Marolda et al., 1999). The
gene wbbD encodes a polypeptide that belongs to the glycosyltransferase family 2 (http://afmb.cnrs-mrs. fr/~cazy/cazy/
index.html). This family includes a large group of inverting
glycosyltransferases, such as cellulose synthase, chitin synthase, dolichyl-phosphate β-D-mannosyltransferase, dolichyl-phosphate β-glucosyltransferase, GlcNAc transferase,
N-acetylgalactosaminyltransferase, hyaluronan synthase,
β1,3-glucan synthase, and others acting on unknown substrates (Coutinho et al., 2003).
To determine whether wbbD is required for O7 LPS synthesis, we constructed a mutant derivative of strain VW187
using a homologous recombination strategy that resulted in
the replacement of the wild-type wbbD gene by wbbD::aph.
The aph gene cassette encodes resistance to the antibiotic
kanamycin, and it is oriented in such manner that the transcription of the wbEcO7 genes located downstream of wbbD
is driven by the aph promoter (Figure 1). The LPS from the
resulting mutant strain, MB1, was analyzed by silver staining of LPS preparations after electrophoresis in sodium
dodecyl sulfate (SDS)/N-[tris(hydroxymethyl)methyl]glycine gels. Figure 2 shows that the LPS prepared from the
parental strain VW187 displays a typical ladder-like banding
pattern with a band distribution that is characteristic of the
O7-specific polysaccharide (Marolda et al., 1999). In contrast,
the wbbD::aph mutant MB1 only shows a rapidly migrating
band corresponding to lipid A-core oligosaccharide (Figure 2).
This band is also present in LPS prepared from strain MV501
(Figure 2), which carries a wecA::Tn10 insertion that precludes the initiation of the synthesis of the O7 repeat
(Alexander and Valvano, 1994). Neither MV501 nor MB1
bacteria gave positive slide agglutination in the presence of
O7-specific rabbit antiserum. The ability of MB1 to form O7
polysaccharide was restored by the introduction of pCM227,
which encodes a glutathione transferase (GST)-WbbD chimeric protein (Figure 1), confirming that the insertion of the
aph cassette has no effect on the expression of the downstream
genes of the wbEcO7 cluster (Figure 1). Also, this experiment
BglII
galF
lB lD lA lC z x
rm rm rm rm w
v
A B A B
io io bb bb z y
v w w w
C
B
an
an gnd'
m
m
C
bb
w
wbbD
pCM227
gst
pMB1
1 Kb
pMB4
aph
Fig. 1. Genetic organization of the O7 LPS biosynthesis cluster. The
wbEcO7 gene cluster is flanked by galF and gnd genes (Marolda et al.,
1999). Genes represented by gray shading are those encoding metabolic
enzymes for the synthesis of some of the nucleotide sugar precursors for
the O7 repeating unit: rmlBDAB, dTDP-rhamnose (Marolda and Valvano
1995); vioAB, dTDP-viosamine; and manBC, GDP-mannose (Marolda
and Valvano, 1993). The genes encoding enzymes for the synthesis of
UDP-Gal and UDP-GlcNAc are encoded elsewhere in the chromosome.
Genes represented in black fill correspond to those involved in the assembly
of the O7 repeat, and they are wzx, O antigen flippase; wbbA, predicted
rhamnosyltransferase; wbbB, predicted viosaminyltransferase; wzy,
O antigen polymerase; and wbbC, predicted mannosyltransferase. The
wbbD gene studied in this work is represented by hatch fill, and the BglII
site within the wbbD coding sequence that was used for the construction
of the wbbD::aph mutant is shown. The boundaries of the DNA inserts
from plasmids pCM227, pMB1, and pMB4 are also indicated. The arrow
upstream from rmlB denotes the location of the wbEcO7 promoter and the
direction of transcription (Marolda and Valvano, 1998).
606
Fig. 2. Analysis of LPS from wild-type and mutant strains lacking
O-chain. LPS was extracted from different strains as described under
Materials and methods and separated by SDS–PAGE followed by silver
staining. VW187, wild-type E. coli O7 strain; MB1, VW187 wbbD::aph
mutant; MB1(pCM227), MB1 mutant complemented with pCM227,
expressing the WbbD protein fused with GST; MV501, wecA::Tn10
mutant of VW187 with a defect in the GlcNAc-phosphotransferase
enzyme WecA.
E. coli O7:K1 galactosyltransferase
confirms that the fusion protein encoded by pCM227 is
functional as an enzyme involved in O-chain synthesis.
MB1(pCM227) and VW187 gave a positive slide agglutination with the O7-specific antibody, demonstrating that the
O polysaccharide produced in the complemented MB1 mutant
has the same immunoreactivity as the parental O7 LPS.
Additional experiments with the Ffm bacteriophage
showed that mutants MB1 and MV501 were sensitive to
lysis by Ffm, whereas the parental strain VW187 and the
complemented MB1(pCM227) strain were resistant, suggesting that there is sufficient surface-exposed O7 LPS in
the complemented strains to mask the Ffm receptor in the
core oligosaccharide and prevent the entry of the phage
DNA into the bacterial cells. Altogether, we conclude from
these experiments that WbbD carries an essential function
for the synthesis of O7 LPS, which is consistent with its predicted Gal-transferase activity.
The MB1 strain lacks a UDP-GlcNAc-dependent
galactosyltransferase
Gal is the second sugar transferred during synthesis of the
O7 subunit and is transferred to the GlcNAc-PP-Und
endogenous acceptor. Thus the incorporation of radioactive Gal to endogenous acceptor should be dependent on
the presence of UDP-GlcNAc in the assay. We first determined that the UDP-GlcNAc:Und-P GlcNAc-1-phosphotransferase in the homogenates prepared from the wild-type
strain VW187 was active toward endogenous substrate by
demonstrating that up to 0.13 nmol [3H]GlcNAc/assay
were incorporated into a chloroform/methanol-extractable
product. Gal-transferase assays were then carried out using
crude bacterial homogenates as the source of the enzyme
and endogenous acceptor in the presence of an excess of
UDP-GlcNAc and radiolabeled UDP-Gal. The synthesis of
the Gal-transferase product was followed by quantifying
the incorporation of [3H]Gal into a chloroform/methanolextractable fraction.
Optimal assay conditions were determined with respect
to metal ion cofactors and buffer requirements. Optimal
activity of homogenates from the parental strain VW187
(75 pmol of Gal transferred per assay, 0.36 nmol/h/mg protein) was found with 0.1 M Tris/acetate buffer, pH 8.5, and
20 mM Mg2+. Using these conditions, the incorporation of
Gal was linear with time up to 30 min and proportional to
enzyme concentration (data not shown). Table I shows that
the homogenate prepared from the wecA::Tn10 mutant
MV501 had a low endogenous Gal-transferase activity
(0.07 nmol/h/mg), relative to the activity from the parental
strain VW187 (0.36 nmol/h/mg). This is consistent with the
lack of GlcNAc-PP-Und synthesis in the absence of a functional WecA protein (Amer and Valvano, 2001, 2002). The
homogenate prepared from the wbbD::aph mutant MB1
had 25% endogenous Gal-transferase activity relative to the
activity in homogenates from the parental strain. However,
introduction of pCM227 into MB1 increased the activity in
the homogenate 2.9-fold (Table I). Homogenates from
E. coli DH5α carrying the plasmid pCM227 also showed a
relatively high activity (2.6-fold higher than the activity in
the parental strain homogenate). These results demonstrate
that the presence of an intact wbbD gene is associated with
Table I. Gal-transferase activities in homogenates from different
strains of E. coli
Relative activity (%)a
Extracts prepared from
Endogenous
substrate
GlcNAc-PP-PhU
substrate
VW187
100
100
MB1
25
4
MB1 (pCM227)
72
173
MV501
19
107
MV501 (pCM227)
ND
759
DH5a (pCM227)
261
136
a
Gal-transferase activity was assayed as described in Materials and methods,
using endogenous substrate or exogenous substrate GlcNAc-PP-PhU.
The endogenous substrate is of unknown structure and concentration,
and the enzyme product(s) or the specific Gal-transferase activity
measured is undefined. The use of exogenous substrate allows assaying
a defined activity, UDP-Gal: GlcNAc-PP-PhU β3-Gal-transferase. The
activity in homogenates of the wild–type bacteria was set to 100% (0.13
nmol/h/mg for the endogenous activity and 758 nmol/h/mg [0.0126 µmol/
min/mg] for the activity using GlcNAc-PP-PhU substrate). Assays were
carried out in at least duplicate determinations; assay results varied by
< 10%. ND, not done.
a Gal-transferase activity that is dependent on the availability of endogenous GlcNAc-PP-Und glycolipid acceptor.
Homogenates of the wild-type strain VW187 were centrifuged for 10 min at 10,000 × g. Essentially all of the activity
stayed in the pellet fraction, suggesting that the native
enzyme was associated with membranes. The recombinant
WbbD enzyme protein, expressed as a GST-fusion protein
encoded by pCM227 in DH5α, was solubilized in 0.1% TritonX100, and purified on glutathione-Sepharose. Coomassie blue
stains of the fusion protein separated by SDS–polyacrylamide gel electrophoresis (PAGE) showed a major band at
57 kDa, suggesting a high level of expression. Surprisingly,
however, the purified enzyme protein had very little activity
(less than 1% of activity of the homogenate from DH5α
carrying pCM227).
Characterization of the galactosyltransferase activity using a
novel synthetic acceptor substrate, GlcNAc-PP-PhU
In previous experiments, the rate and extent of galactosyl
transfer to endogenous acceptor was relatively low, and the
reaction could not be characterized because of the dependence on the activity of the prior enzyme in the pathway.
Also, because endogenous acceptor was limiting, it was difficult to isolate sufficient quantities of the resulting product
for a detailed characterization. Therefore, we used a new
acceptor substrate, GlcNAc-PP-PhU (Montoya-Peleaz
et al., 2005), to directly assay for the presence of the Galtransferase and to characterize both the enzymatic reaction
and the enzyme product.
The Gal-transferase activity of strain VW187 catalyzed
galactosyl transfer from UDP-Gal to GlcNAc-PP-PhU as
the acceptor in standard assays (Table I). At 30°C incubation temperature, Gal-transferase activity was reduced by
28%. The enzyme had a broad pH optimum between 6.0
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J.G. Riley et al.
and 8.0. Galactosyl transfer was dependent on the presence
of divalent cations, and 2.5 mM Mn2+ supported maximal
activity. MgCl2, CoCl2, and ZnCl2 at 5 mM concentration
yielded 11%, 38%, and 9% activity, respectively, compared
with 5 mM MnCl2. In the presence of NiCl2, ethylenediamine tetraacetic acid (EDTA) or with no addition, enzyme
activity was < 4.5%. The Km for GlcNAc-PP-PhU was
0.08 mM (Vmax 0.027 µmol/min/mg) and the Km for UDP-Gal
was 1.2 mM (Vmax 0.042 µmol/min/mg).
The activities of membrane-bound glycosyltransferases
are often stimulated by detergents. However, octylglucoside
at concentrations of 0.125% and 0.25% in the assay
decreased Gal-transferase activity by 30% and 77%, respectively, and the activity was diminished in the presence of
CHAPS at concentrations of 0.125–0.6%. Gal-transferase
retained up to 50% activity in the presence of Triton X-100
at concentrations between 0.05% and 1%.
Crude homogenates prepared from the E. coli MB1 wbbD
mutant and the parental strain VW187, as well as from
E. coli MV501 mutant and DH5α containing pCM227
(wbbD+) were assayed with exogenously added GlcNAcPP-PhU. The results were similar to those obtained from
assays using endogenous substrate (Table I). The extracts
from the MB1 mutant, which was incapable of synthesizing
O-chain due to a disruption of the wbbD gene, had very low
(4%) activity relative to that of the extracts from the parental strain VW187 (Table I). On transformation of MB1 with
pCM227, the activity was increased to 173%, most likely
due to overexpression of the WbbD protein. Similarly,
extracts from E. coli DH5α(pCM227) that do not synthesize O7 LPS but expressed the GST-WbbD construct
showed a high Gal-transferase activity (136%). Extracts
from mutant MV501, which lacks the GlcNAc-P-transferase activity provided by the WecA protein, had similar
values of Gal-transferase activity relative to those of
VW187, although this mutant was incapable of synthesizing O-chain. Complementation of MV501 with pCM227, as
expected, resulted in extracts with high Gal-transferase
activity (759%) which was due to both endogenous and
plasmid-derived Gal-transferase. These results clearly show
an association of Gal-transferase activity with the presence
of a functional wbbD gene.
separated on HPLC using an amine column and acetonitrile/water (90/10) as the mobile phase. More than 95% of
the radioactivity eluted later than free [3H]Gal but earlier
than Gal β1-4GlcNAc (Figure 3) and coeluted with Galβ13GlcNAc, showing that the linkage between Gal and
GlcNAc was not β1-4.
Galactosidases were used as a tool to determine the anomeric linkage between Gal and GlcNAc in the GalGlcNAc-PP-PhU enzyme product. The released free Gal
was monitored by measuring the radioactivity of the water
fractions of Sep-Pak eluates, whereas the intact product
was measured similarly in the methanol eluates. The
enzyme product was resistant to jack bean (β1-4-specific)
β-galactosidase (>96%) and coffee bean α-galactosidase
(>92%). However, treatment with bovine testicular β-galactosidase, which cleaves Galβ1-3,-4, and -6 linkages, resulted
in 90% of the radioactivity from enzyme product eluting
as [3H]Gal on HPLC. Thus, we concluded that Gal was
β-linked in the enzyme product.
The 600 MHz proton–nuclear magnetic resonance
(NMR) spectra of the Gal′β1-3GlcNAc standard (in
CD3OD) showed characteristic signals of the Gal H-1′ residue at 4.34 ppm and the H-1 of GlcNAcα at 3.85 ppm
(Table II). Because the H-3 signal was obscured by other
signals, it was further determined by 2D NMR, that is, correlation spectroscopy (COSY) (proton connectivity), heteronuclear
multiple-quantum
coherence
spectroscopy
(HMQC) (carbon connectivity), and nuclear Overhauser
effect spectroscopy (NOESY) (through space interaction
with H-1′). Thus, the proton and carbon chemical shifts of
H-1, H-2, H-3, and H-1′, as well as C-1, C-2, C-3, and C-1′,
Product identification
Enzyme product from assays using VW187 homogenates
containing UDP-[3H]Gal as the donor substrate and
GlcNAc-PP-PhU as the acceptor substrate was isolated by
high-performance liquid chromatography (HPLC) for
structural analysis. We previously showed by mass
spectrometry that the radioactive product was a disaccharide-PP-PhU (Montoya-Peleaz et al., 2005). When the
radioactive product was incubated with both nucleotide
pyrophosphatase and alkaline phosphatase, the radioactivity was no longer retained by Sep-Pak C18, and >95% of
the radioactivity eluted with the water fractions. This indicated cleavage of the pyrophosphate bond between the
GlcNAc residue and the lipid tail. The resulting radioactive
disaccharide did not bind to AG1x8, indicating that >95%
of the phosphate residues were cleaved, and free disaccharide
was produced. The radioactive compounds were further
608
Fig. 3. HPLC separation of disaccharide released from enzyme product.
Enzyme reaction product from GlcNAc α1-O-PO2-O-PO2-O-(CH2)11O-phenyl substrate was purified on Sep-Pak columns, incubated with
pyrophosphatase and phosphatase, and passed through an AG1x8
column. The eluate was analyzed by HPLC using an amine column and
acetonitrile/water 90:10 as the mobile phase. The elution times of
standards are shown as arrows: radioactive Gal, Gal β1-3GlcNAc, and
Gal β1-4GlcNAc. The absorbance at 195 nm and the radioactivity (cpm)
of the disaccharide released from the enzyme product are shown.
E. coli O7:K1 galactosyltransferase
Table II. Select proton and carbon NMR chemical shifts (ppm) of Gal β(1-3) GlcNAc (α) and galactosyltransferase product
Standard Gal′ β(1-3) GlcNAc (α)
D2O
Nuclei position
a
Product
CD3OD
CD3OD
H-1
4.58 (d, J = 3.0 Hz)
5.08 (d, J = 3.6 Hz)
H-2
5.80
3.99(dd, J = 3.2, 10.6 Hz)
4.14 (br d, J = 9.7 Hz)
3.85b
3.84b
H-3
NA
H-1′
5.28 (d, J = 7.0 Hz)
4.34 (d, J = 7.6 Hz);
5.58 (br s)
4.35 (d, J = 7.5 Hz)
C-1
91.7
91.2c
94.8c
C-2
53.5
53.1c
52.4c
C-3
80.9
80.5
c
80.3c
C-1′
104.0
103.7c
104.0c
a
Literature data (Lemieux and Driguez, 1975) of standard Gal β(1-3) GlcNAc (α) in deuterium oxide. The standard was also
run in deuterated methanol for better correlation with the product.
b
Determined by COSY.
c
Determined by HSQC.
OH
Thus, 1D NMR fingerprint as well as the 2D NMR analysis of
the disaccharide and intact enzyme product conclusively show
that the sugar linkage between Gal and GlcNAc is β1-3.
OH
OH
O HO
HO
OH
2
3
O
NH
O
1´
O
1
Discussion
OR
CD3OD
CD3OD
H-3
H-1’
H-2
H-1
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
Fig. 4. 600 MHz proton NMR spectrum of enzyme product
Galβ1-3GlcNAcα1-O-PO2-O-PO2-O-(CH2)11-O-phenyl. Spectra
were recorded of Gal-transferase product in deuterated methanol.
are shown in Table II. More than 1 µmol of enzyme product was produced using the highly active homogenate from
the MV501(pCM227) strain. Enzyme product was purified
by C18 Sep-Pak and HPLC and analyzed by NMR. An aliquot of the enzyme product was cleaved with pyrophosphatase and phosphatase, and the resulting disaccharide
was purified by anion exchange and HPLC.
The 600 MHz proton NMR spectrum of 200 nmol of the
purified disaccharide released from the enzyme product
showed signals identical to those for Galβ1-3GlcNAc standard. The 1D NMR spectrum of the intact enzyme product
Gal-GlcNAc-PP-PhU measured in deuterated methanol is
shown in Figure 4. The signals were assigned using the same
2D NMR procedures as used for the standard (Table II).
The shifts of GlcNAc-H-1 and H-2 show minor differences
due to the linkage of GlcNAc to pyrophosphate. However
the characteristic shifts for Gal H-1′ and GlcNAc H-3 are
similar to those of the disaccharide (Table II; Figure 4).
The O7 polysaccharide of the E. coli strain VW187 has been
used as a model system to understand the biosynthesis of
the O antigen repeating subunit structure (Valvano, 2003;
Marolda et al., 1990, 1999; Marolda and Valvano, 1993,
1995, 1998). The O7 antigen consists of a repeating pentasaccharide with the structure VioNAc α1-2 [Rha α1-3]
Man α1-4Gal β1-3 GlcNAc α1-3, which is assembled on a
C55 polyisoprenoid alcohol with subsequent polymerization to the final polysaccharide. The initial reaction in the
synthesis pathway involves the transfer of GlcNAc-P from
UDP-GlcNAc to Und-P, resulting in the formation of
GlcNAc-PP-Und, a reaction mediated by the WecA protein
(Alexander and Valvano, 1994; Amer and Valvano, 2002;
Marolda et al.,1999). The addition of the subsequent sugars
presumably requires the products of additional glycosyltransferase genes. Initial studies of Gal-transferase activities using endogenous acceptor substrate that is present in
crude homogenates from the wbbD mutant MB1 demonstrated a low enzyme activity in the presence of UDPGlcNAc, despite the fact that the GlcNAc-P-transferase
reaction occurred at normal levels. This was consistent with
the lack of O-chain formation and the high sensitivity of the
mutant to the Ffm phage. This phenotype was complemented by transforming MB1 with a functional wbbD gene
provided by plasmid pCM227, indicating that the Galtransferase activity was encoded by wbbD.
The inclusion of the synthetic GlcNAc-PP-PhU in the
crude cell homogenates obviated the need to add UDPGlcNAc to the assay mixtures, as demonstrated by high
Gal-transferase activities toward GlcNAc-PP-PhU found
in extracts of the parental strain VW187 and the GlcNAcP-transferase mutant MV501. Therefore we confirmed
that GlcNAc-PP-PhU salts are excellent substrates for the
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J.G. Riley et al.
WbbD enzyme and that GlcNAc linked through pyrophosphate to either Und or to a smaller hydrophobic chain can
serve as substrate for the Gal-transferase. Our results also
indicate that both the naturally occurring Gal-transferase
as well as the GST-Gal-transferase construct are both
active toward the new acceptor substrate GlcNAc-PP-PhU.
Enzyme product analysis unequivocally demonstrated
that the newly assayed Gal-transferase activity transfers
Gal from UDP-Gal to GlcNAc-PP-PhU in β1-3 linkage.
The Gal-transferase activity was low in the presence of
EDTA and was stimulated primarily by divalent metal ions
Mn2+, Mg2+, and Co2+. This property of Gal-transferase is
similar to that of mammalian β3- and β4-Gal-transferases.
These metal ions may be involved in binding and stabilizing
UDP-Gal in the donor binding site of the enzyme (Boeggeman
and Qasba, 2002).
Pyrophosphate-containing lipids with a phenoxyundecyl
group may also be acceptor substrates for other glycosyltransferases that catalyze the subsequent steps of O7 chain
synthesis and for glycosyltransferases of other strains of
Gram-negative bacteria. GlcNAc-PP-PhU is an excellent
substrate, possibly because, in contrast to the natural Und-P
substrate, its relatively short hydrophobic tail may not be
inserted in the membrane, thus rendering the GlcNAc residue accessible to Gal-transferase. The Gal-transferase activity was lost on purification of the protein in the absence of
membranes. The detergents used in this study appeared to
be unfavorable for mimicking the membrane environment or
producing appropriate micelle structures to support enzyme
activity. These combined results suggest that the topology
and membrane association not only of the enzymes themselves but also of substrates are important factors for the
sugar transfer reaction in bacteria. Alternatively, it is possible that additional proteins are required for full enzymatic
activity as a part of a putative protein complex.
We have devised a synthetic acceptor molecule that will
help in studying the biochemical glycosyl transfer reactions
in bacterial systems. The utility of the new compound was
(1) to allow the development of a sensitive and accurate
assay system for a specific O-chain glycosyltransferase; (2) to
characterize the crude endogenous enzyme, and to show
that the enzyme was active as a fusion protein in the presence
of bacterial membranes; (3) to follow enzyme purification;
and (4) to produce substrate for other enzymes subsequently
acting in the O-chain synthesis pathway. Current experiments are under way in our laboratories to characterize
these additional reactions for the biosynthesis of the complete O7 subunit and to determine the appropriate conditions
for the characterization of purified glycosyltransferases
that will enable us to perform structure–function studies.
kits were from Qiagen (Mississauga, Ontario). Oligonucleotide primers were purchased from Life Technologies and
Cortec (Kingston, Ontario). pGEM-T vector was from
Promega (Madison, WI). Tris-glycine SDS–PAGE gels
were purchased from Novex (San Diego, CA). Protein concentrations were determined with the BioRad (Hercules,
CA; Bradford) protein assay using bovine serum albumin
as a standard. GlcNAc-PP-PhU was synthesized as described (Montoya-Peleaz et al., 2005)
Bacterial strains and growth conditions
E. coli DH5α was used as the initial host for the cloning
experiments. Strains VW187 (E. coli O7:K1) and its wecA::
Tn10 mutant, MV501, have been previously described
(Alexander and Valvano, 1994; Valvano and Crosa, 1989).
Strain MB1 is a wbbD mutant of VW187 constructed as
described shortly. Bacteria were grown at 37°C in Luria Bertani (LB) medium consisting of 10 g NaCl, 5 g yeast extract,
and 10 g tryptone per liter, which was supplemented with
ampicillin (50 mg/ml), tetracycline (20 mg/ml), kanamycin
(50 µg/ml), and chloramphenicol (20 µg/ml), as appropriate. For some experiments, cultures were plated on LB agar
plates supplemented with 0.2% (w/v) X-Gal and 0.4 mM
IPTG. For selection against sacB, LB agar plates were supplemented with sucrose to a final concentration of 5% (w/
v). Spot assays with the rough-specific bacteriophage Ffm
(Wilkinson et al., 1972), which lyses bacteria lacking the Ochain, were carried out as described (Expert and Toussaint,
1985). O antigen reactivity was determined as described
(Valvano and Crosa, 1989).
DNA methods
Restriction enzymes, T4 DNA ligase, and Klenow DNA
polymerase were used according to the conditions recommended by the supplier. Recombinant plasmids were introduced into E. coli strains by CaCl2 transformation or
electroporation (2500 V, 5 ms) using an Eppendorf Multiporator. DNA fragments for cloning were isolated from
agarose gels using the Qiaquick kit (Qiagen, Valencia, CA).
Polymerase chain reaction (PCR) amplifications were carried out with PwoI or Taq DNA polymerase as recommended by the manufacturer. Amplification was done in an
Eppendorf thermocycler after denaturation at 94°C for 1
min, followed by 35 cycles consisting of 30 s at 94°C (denaturation), 30 s at 55°C (annealing), and 2 min at 72°C for
(extension), and a final extension cycle of 10 min at 72°C.
PCR amplification products were visualized on 1% (w/v)
agarose gels. PCR products were recovered by ethanol precipitation and ligated into the appropriate cloning vectors
as described next.
Materials and methods
Cloning of wbbD and construction of a wbbD::aph mutant
of VW187 (strain MB1)
Reagents
A 2.7-kb fragment containing the wbbD gene and the
surrounding region (Figure 1) was amplified by PCR, using
the PCR conditions just described, and the primers
5′-GGATCCTATTCGATGGGATTGATTGC-3′ and 5′GGATCCATTCCCAAAGCGAAGACCAT-3′ (underscoring indicated BamHI recognition sites not present in the
original DNA sequence that were introduced into the
All reagents were of the highest grade available and were
from Sigma Chemical (St. Louis, MO) unless otherwise
indicated. 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-Gal), isopropyl β-D-thiogalactopyranoside (IPTG),
T4 DNA ligase, and DNA polymerases were purchased from
Roche Diagnostics (Laval, Quebec). Plasmid preparation
610
E. coli O7:K1 galactosyltransferase
primers to facilitate future cloning steps). Chromosomal
DNA from the strain VW187 served as a template. The PCR
product was ligated into the thymidylated EcoRV site of
pGEM-T vector to yield plasmid pMB1 (Figure 1). The
cloned DNA fragment in pMB1 carries a BglII site located
in the middle of wbbD that was used to insert the aminoglycoside 3′-phosphotransferase gene (aph) from Tn903, which
encodes kanamycin resistance. To facilitate this construction, we first cloned aph from pUC4K into the unique
BamHI site of pKO3, resulting in the recombinant pMB2,
which encodes both chloramphenicol and kanamycin resistance. After this step, the ligation mixture of pMB1 linearized with BglII and pMB2 linearized with BamHI was
transformed into E. coli DH5α and transformants selected
on plates with kanamycin and ampicillin. One of these
recombinants contained the plasmid pMB3, which corresponded to pMB1 with the aph gene inserted into wbbD.
The plasmid pMB3 was digested with BamHI and ligated
into the temperature-sensitive suicide plasmid pKO3 that
carries the sacB gene for counterselection of double crossover recombinants. This experiment resulted in the isolation
of the mutagenic plasmid pMB4, which was transformed by
electroporation into E. coli O7 strain VW187. One of the
selected transformants was incubated at 42°C to isolate
derivatives where the pMB4 plasmid was integrated by
homologous recombination into the VW187 chromosome.
To isolate mutants with a second crossover that resulted in
the replacement of the parental wbbD by the mutated
wbbD::aph gene, purified colonies were streaked on plates
supplemented with 5% (w/v) sucrose. The loss of chloramphenicol resistance in sucrose-resistant, kanamycin-resistant colonies confirmed the loss of pMB4. One of these isolates,
designated MB1, was used for additional experiments to
confirm by PCR analysis that the correct gene replacement
took place (data not shown).
The coding region of the wbbD gene was amplified by PCR
with primers as described and cloned into the unique BamHI
site of the vector pGEX-2T. The forward primers were
designed in a way such that wbbD was translationally fused to
the distal portion of the GST gene that is present in pGEX2T. The resulting plasmid pCM227 encoded a chimeric protein of ~ 57 kDa in mass (with a 27-kDa N-terminal region
contributed by the GST, followed by a thrombin cleavage
site, and a 30-kDa C-terminal region contributed by WbbD).
LPS analysis
LPS was extracted as previously described, and analyzed by
SDS–PAGE (14% gels) and silver staining (Marolda et al.,
1990).
Enzyme preparation
Bacterial homogenates were prepared as the enzyme source
as described (Montoya-Peleaz et al., 2005). Briefly, bacteria
were grown in Luria broth containing 100 µg/ml ampicillin
and sedimented by centrifugation. Bacterial cells were sonically ruptured using a Sonic Dismembrator Model 100
(Fisher Scientific, Silver Spring, MD) for two pulses of 15 s
with 2-min intervals to allow for cooling on ice. Protein
concentrations ranged between 2 and 12 mg/ml. For purification of the fusion protein GST–WbbD, encoded by
plasmid pCM227, bacterial homogenates in phosphate
buffered saline containing 0.1% Triton X-100 were incubated with glutathione Sepharose 4B. Bound proteins were
eluted with 10 mM reduced glutathione/50 mM Tris–Cl,
pH 8.0 and stored in phosphate buffered saline. The fusion
protein was analyzed by SDS–PAGE (8% gels) and western
blots using anti-GST antibody.
Enzyme assays for GlcNAc-phospho transfer and Gal transfer
to endogenous acceptors
The GlcNAc-phospho transfer from UDP-GlcNAc to endogenous Und-P was assayed in a total volume of 0.08 ml of
50 mM 2-N-morpholinosulfonate (MES)/acetate, pH 7.0,
containing 1.5 mM UDP-[3H]GlcNAc (1270 cpm/nmol),
25 mM MgCl2, 0.8 mM EDTA, and 0.04 ml of freshly prepared enzyme homogenate (400 µg protein). After 30 min
at 37°C the reactions were quenched by the addition of 0.7
ml CHCl3/CH3OH (2:1) and thorough mixing on a vortex
mixer. After standing 15 min at room temperature, 0.6 ml
of pure solvent upper phase (a mixture of 15 ml CHCl3, 240
ml methanol, 1.83 g KCl in 235 ml water), were added to
the solution, the mixtures were vigorously stirred on a vortex mixer and subjected to centrifugation at 8000 rpm for
1.5 min to separate the two phases; the aqueous upper phase
and interface were aspirated and discarded. The organic
phase was washed two times with 0.5 ml pure solvent upper
phase and then transferred to scintillation vials, taken to
dryness, and counted in scintillation fluid. All assays were
carried out at least in duplicate.
Galactosyl transfer to endogenous acceptor was carried
out in reaction mixtures containing 2 mM UDP-GlcNAc
(for the synthesis of GlcNAc-PP-Und as a galactosyl acceptor), 75 mM MES, pH 7.0, 10 mM MgCl2, and 0.9 mM
[3H]-UDP-Gal (10,650 cpm/nmol). Reactions were incubated for 30 min at 37°C and quenched by the addition of
0.7 ml CHCl3/CH3OH (2:1). The formation of radioactive
product was measured as already described. Enzyme homogenate that was heated for 10 min at 100°C was used as a
negative control.
Enzyme assays for Gal transfer to exogenous acceptor
Standard assays for galactosyl transfer to exogenously
added substrate were carried out as described (MontoyaPeleaz et al., 2005). Briefly, 20 µl enzyme homogenate (3–12 µg
protein) were incubated in reaction mixtures of 40 µl total
volume, containing 0.5 mM GlcNAc-PP-PhU, 5 mM MnCl2,
75 mM MES buffer, pH 7, and 0.5 mM UDP-[3H]Gal (800–
4400 cpm/nmol). Enzyme product was isolated using a C18
Sep-Pak column. For further analysis, enzyme product was
isolated by reverse-phase HPLC (Montoya-Peleaz et al.,
2005). Enzyme kinetics were determined with acceptor substrate concentrations ranging from 0.05 to 1 mM (with 2
mM UDP-Gal), and 0.1 to 6 mM UDP-Gal (with 1 mM
acceptor). Km and Vmax values were established with double-reciprocal Lineweaver-Burk plots.
Galactosidase digestion
The anomeric configuration of the linkage formed in the
product was determined by digestion with specific galactosidases. Aliquots of pooled radioactive enzyme product
611
J.G. Riley et al.
from VW187 bacterial homogenate (750 cpm ) were treated
in a total volume of 100 µl with 25 µl MacIlvaine buffer (0.1
M citric acid/0.2 M Na-phosphate), pH 4.3, 10 µl of 0.1%
bovine serum albumin, and either 4 µl (0.04 U) jack bean
β-galactosidase or 10 µl (0.01 U) of bovine testicular
β-galactosidase, or 10 µl (0.54 U) green coffee bean
α-galactosidase. Mixtures were incubated for 1 h at 37°C,
diluted with 800 µl water, and applied to C18 SepPak columns. Released [3H]Gal was eluted with 5 ml water, whereas
unreacted enzyme product was eluted with 5 ml methanol.
The 1-ml fractions were counted in 5 ml scintillation fluid.
Abbreviations
COSY, correlation spectroscopy; EDTA, ethylenediamine
tetraacetic acid; GST, glutathione transferase; HMQC,
heteronuclear multiple-quantum coherence spectroscopy;
HPLC, high-performance liquid chromatography; IPTG,
isopropylβ-D-thiogalactopyranoside; LB, Luria Bertani;
LPS, lipopolysaccharide; MES, 2-N-morpholinosulfonate;
NMR, nuclear magnetic resonance; NOESY, nuclear
Overhauser effect spectroscopy; PAGE, polyacrylamide gel
electrophoresis; PCR, polymerase chain reaction; SDS,
sodium dodecyl sulfate.
Analysis of enzyme product by NMR
To prepare large amounts of enzyme product for NMR, Galtransferase assays were carried out as follows. The incubation
mixtures (a total of 8 ml) contained: 4 ml bacterial homogenate (MV501 strain complemented with plasmid pCM227),
4 µmol GlcNAc-PP-PhU, 8.08 µmol UDP-[3H]Gal (60 cpm/
nmol), 600 µmol HEPES buffer, pH 7, and 400 µmol MnCl2.
After incubation for 30 min at 37°C, 8 ml of cold water was
added and the mixtures were applied to 10 C18 Sep-Pak columns. Each column was washed with 4 ml water, and the
product was eluted with 4 ml methanol. The methanol fractions were pooled, flash evaporated, and redissolved in 500 µl
methanol. Aliquots of the first methanol fraction obtained
from the Sep-Pak column were purified by HPLC, using a
C18 column and acetonitrile/water (6:94) as the mobile phase,
as described (Brockhausen et al., 2002). Enzyme product was
dried, exchanged three times with 99.96% D2O and CH3OD,
and analyzed by 600 MHz proton NMR spectroscopy.
For digestion with phosphatases, 20 standard assays were
carried out, and the product was isolated by Sep-Pak, dried,
and redissolved in 500 µl methanol, 2 ml water, and 2 ml 0.5
M HEPES, pH 7.5. Nucleotide pyrophosphatase from Crotalus adamenteus venom (200 µl of 20 U) in 35% Tris(hydroxymethyl)aminomethane and alkaline phosphatase from intestinal
mucosa (50 µl of 1700 U) in 3 M NaCl, 1 mM MgCl2, 0.1
mM ZnCl2, 30 mM triethanolamine, pH 7.6, were added.
Mixtures were incubated for 1 h at 37°C and applied to a
1-ml AG1x8 column (Cl– form). The column was washed with
10 ml water. Eluate was lyophilized. Released disaccharide
product was separated by HPLC using an amine column and
acetonitrile/water (90:10). Fractions containing radioactivity
were combined, flash evaporated, lyophilized, exchanged
three times with 99.96% D2O, and analyzed in CD3OD with
a 600 MHz Bruker spectrometer, using 1D proton NMR
methods without water suppression. COSY, NOESY, and
HMQC methods were also applied to analyze both the intact
enzyme product as well as the released disaccharide. Control
substances were Galβ1-3GlcNAc (Gefco-Chemicals, Israel)
and Galβ1-4GlcNAc.
Acknowledgments
We thank Yi Li for growing bacteria and enzyme assays. This
work was supported by grants from the Natural Science and
Engineering Research Council of Canada (to I.B., W.A.S.,
and M.A.V.) and a Research Scientist Award from the Arthritis
Society of Canada; M.A.V. holds a Canada Research Chair in
Infectious Diseases and Microbial Pathogenesis.
612
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