From the Department of Microbiology, University of Guelph, Guelph, Ontario, N1G 2W1 Canada
Received for publication, October 4, 2000, and in revised form, October 26, 2000
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ABSTRACT |
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Wzc proteins are tyrosine autokinases. They are
found in some important bacterial pathogens of humans and livestock as
well as plant-associated bacteria, and are often encoded within gene clusters determining synthesis and assembly of capsular and
extracellular polysaccharides. Autophosphorylation of
Wzccps is essential for assembly of the serotype K30
group 1 capsule in Escherichia coli O9a:K30, although a
genetically unlinked Wzccps-homologue (Etk) can also
participate with low efficiency. While autophosphorylation of
Wzccps is required for assembly of high molecular weight
K30 capsular polysaccharide, it is not essential for either the
synthesis of the K30 repeat units or for activity of the K30 polymerase enzyme. Paradoxically, the cognate phosphotyrosine protein phosphatase for Wzccps, Wzbcps, is also required for
capsule expression. The tyrosine-rich domain at the C terminus of
Wzccps was identified as the site of phosphorylation and
autophosphorylation of Wzc requires a functional Walker A motif.
Intermolecular transphosphorylation of Wzccps was detected
in strains expressing a combination of mutant Wzccps
derivatives. The N- and C-terminal domains of Wzccps were
expressed independently to mimic the situation found naturally in
Gram-positive bacteria. In this format, both domains were required for
phosphorylation of the Wzccps C terminus, and for capsule assembly. Regulation by a post-translational phosphorylation event represents a new dimension in the assembly of bacterial cell-surface polysaccharides.
Capsular and exopolysaccharides play crucial roles in the biology
of many bacteria, acting either as virulence determinants that
withstand host-cell defenses, or in establishing symbiotic relationships between bacteria and plants. More than 80 different capsular structures (known as K antigens) are produced by
Escherichia coli isolates and these are subdivided into four
different groups based on genetic and biochemical criteria (1). Surface
polysaccharides with similar features are formed by other bacterial
genera. The his-linked cps loci encode enzymes
for the assembly of group 1 capsular K-antigens in E. coli
and Klebsiella pneumoniae. The cps loci all
contain a conserved region comprising the first 4 genes
(orfX, wza, wzb, and
wzccps) (2), indicating a shared role in
CPS1 expression. Following
the conserved genes is a serotype-specific region encoding enzymes that
participate in synthesis of polysaccharide repeat units and their
polymerization via a Wzy-dependent mechanism (3). The
Wzy-mediated polymerization reaction is thought to result in formation
of an undecaprenyl pyrophosphate-linked glycan at the periplasmic face
of the plasma membrane. The nascent polymer is then translocated to the
cell surface via a process that requires outer membrane complexes
formed by multimers of Wzacps (4). These complexes resemble
the "secretins" for secretion of proteins via type II and type III systems.
A minor form of the group 1 K antigen is expressed on the cell surface
in the form of low molecular weight K-antigenic oligosaccharides (one
or a few K-repeat units) linked to LPS lipid A-core and termed KLPS (5). Surface expression of KLPS follows a
distinct LPS translocation pathway that does not require
Wzacps complexes in the outer membrane (4). The capsule
structure evident in electron micrographs is formed from high molecular
weight capsular K antigen (i.e. CPS) that, unlike
KLPS, is not linked to lipid A-core (5). Despite these
organizational differences, undecaprenyl pyrophosphoryl-linked intermediates provide the substrate for the Wzy polymerase required for
synthesis of both CPS and KLPS (3).
The Wzccps proteins in E. coli and K. pneumoniae strains that produce group 1 capsular polysaccharides
are essentially identical. Homologues are also found in
plant-associated Gram-negative bacteria that synthesize related
extracellular polysaccharides. Examples include Erwinia
amylovora, Xanthomonas campestris, Ralstonia
solanacearum, and Sinorhizobium meliloti (reviewed in
Ref. 6). In E. coli, Wzccps participates in the
surface assembly of CPS but is not required for KLPS
synthesis (3). Deletion of the wzc homologue (exoP) in S. meliloti also eliminates high
molecular polysaccharide formation without affecting the ability to
synthesize repeat units (7). The Wzc proteins therefore likely play a
role in high-level polymerization and it has been suggested that they
belong to a larger family of "polysaccharide copolymerase"
proteins that dictate chain length in surface polymers including
capsules, exopolysaccharides, and LPS O antigens (8).
Investigation of the purified Wzccps homologue (Ptk) in
Acinetobacter johnsonii by Cozzone and co-workers (9)
first showed that Wzc proteins possessed tyrosine autokinase activity.
Subsequent studies by the same group confirmed similar properties for
purified Wzcca from E. coli K-12 (10). The
wzcca gene is located in the locus responsible
for synthesis of the slime exopolysaccharide known as colanic acid,
whose production is dependent on growth conditions (e.g. temperatures
below 30 °C) (11). Phosphorylation of Ptk and Wzcca
appears to proceed by a novel phosphorylation mechanism because, unlike
eukaryotic kinases, Ptk and Wzcca contain a Walker A box
ATP-binding motif (12) that is required for phosphorylation at
multiple, but presently unidentified, tyrosine residues (13). Ptk and
Wzcca are dephosphorylated by the cognate phosphotyrosine protein phosphatases Ptp and Wzbca, respectively, and these
activities have been confirmed in vitro using purified
proteins (10, 14). Based on conserved gene products and preliminary
biosynthesis data, colanic acid appears to be synthesized by a
Wzy-dependent pathway similar to group 1 capsules (reviewed
in Ref. 1). However, despite the fact that the colanic acid
biosynthesis locus is widespread in E. coli, it is absent in
E. coli strains with group 1 capsules (1). The Wzc
homologues from the group 1 CPS and colanic acid synthesis loci are
well conserved (51.9% identity; 83.1% total similarity), as are the
corresponding Wzb proteins (51.0% identity; 76.2% similarity) (3).
Thus they likely play similar roles in both biosynthetic systems.
Interestingly, homologues of Wzc are also found in important
Gram-positive pathogens including Streptococcus pneumoniae
(15, 16), Streptococcus agalactiae (17, 18), and
Staphylococcus aureus (19, 20). However, in these bacteria
the N- and C-terminal domains of Wzc are represented in two separate
polypeptides (15, 20). The CpsC protein of S. pneumoniae is
equivalent to the membrane-associated N-terminal domain of Wzc while
the CpsD protein is homologous to the C-terminal Walker box-containing
domain of Wzc. Recent investigations have shown that CpsD is
phosphorylated at a tyrosine-rich C-terminal domain (21). The product
of the cpsB gene may be involved in dephosphorylation of
CpsC (21), although sequence analysis shows it lacks the sequence
features (and catalytic-site motifs) found in other small
phosphotyrosine protein phosphatases. Cognate phosphatases have not
(yet) been identified in all Wzc-containing systems. Intriguingly,
phosphorylation of CpsD is proposed to negatively regulate capsule
biosynthesis in S. pneumoniae (21).
While autokinase and phosphotyrosine protein phosphatase activity has
been demonstrated for some Wzc and Wzb homologues, the relationship
between Wzc phosphorylation and capsular polysaccharide assembly has
not been investigated in Gram-negative bacteria. Here, we show that two
genetically unlinked wzc homologues on the chromosome of
E. coli O9a:K30 contribute to different extents in the
assembly of the K30 capsule. The phosphorylated tyrosine residues of
Wzccps are located to the C-terminal 17 residues of Wzccps and assembly of the E. coli group 1 capsule is demonstrated to require both phosphorylation-competent
Wzccps and active Wzbcps phosphatase. The
effect of phosphorylated Wzccps on capsule assembly differs
from that of phosphorylated CpsD in S. pneumoniae.
Bacterial Strains, Plasmids, and Growth Conditions--
The
bacterial strains and plasmids used in this study are listed in Table
I. All strains were grown and
maintained in Luria-Bertani (LB) medium at 37 °C. Where appropriate,
media were supplemented with antibiotics to the following
concentrations: ampicillin (Ap, 100 µg ml Construction of Chromosomal Insertion Mutations in etk and
wzb--
The etk gene is located in a gene cluster at 22 min on the E. coli K12 chromosomal map and is downstream of
wza and wzb homologues. A chromosomal
wza22 min::aadA insertion
mutation (strain CWG258) was reported previously (4). The
spectinomycin-resistance (aadA) gene cassette is flanked by
transcriptional terminators (22) and its polarity therefore eliminates
expression of downstream genes (i.e. wzb22
min and etk). The pWQ129 suicide delivery plasmid carrying wza22 min::aadA
was used to mutate E. coli CWG315
(wzccps::aacC1) to generate
CWG285 (wzccps::aacC1
wza22 min::aadA), by allelic exchange. To generate the chromosomal wzbcps
mutation, a 1.25-kilobase fragment spanning
wzbcps was amplified by PCR and cloned. The nonpolar aph3A kanamycin-resistance cassette was inserted
into the XbaI site within wzbcps.
Plasmid pWQ146, a pMAK705-based suicide-delivery construct, was then
used to transfer the mutation onto the chromosome of CWG343
(wza22 min::aadA
wzbcps::aph3A) by allelic
exchange. Details of the approach for allelic exchange are described
elsewhere (4). In all cases, correct insertions in chromosomal genes were confirmed by analysis of products of PCR reactions and by sequencing across the insertion using primers flanking the targeted region.
PCR Amplification and Cloning of etk, wzccps, and Its
Deletion Derivatives--
Restriction and DNA-modifying enzymes were
used according to the manufacturer's instructions. Transformation was
done by electroporation using a Gene Pulser from Bio-Rad (23). PCR
amplifications were carried out using a Perkin-Elmer GeneAmp PCR System
2400 thermocycler. Oligonucleotide synthesis and automated DNA
sequencing services were provided by the Guelph Molecular Supercentre
(University of Guelph, Ontario, Canada). The sequences of
oligonucleotides are listed in Table II.
All amplification reactions were performed in 50-µl reactions using
Pwo polymerase (Roche Molecular Biochemicals) and conditions
optimized for each primer pair. PCR products and plasmid DNA fragments
were purified using a QIAquick PCR purification kit (Qiagen) and/or
Geneclean II (Bio101). To clone wzccps, the 2.5-kilobase open reading frame was amplified by PCR using primers TW6
and JD102. The PCR product was digested with EcoRI and
PstI and cloned into pBAD18-Km to give plasmid pWQ130. The
wzccps gene was also amplified as an N-terminal
His6-tagged fusion protein using primer pair TW20-TW19.
These primers introduced NdeI and EcoRI
restriction sites facilitating cloning in pET28a(+) (Novagen), resulting in plasmid pWQ141. The etk gene was amplified
using the primer pair JD141-JD142. These primers introduced flanking MfeI and PstI restriction sites, allowing the
product to be ligated to the EcoRI and PstI sites
of pBAD24, resulting in plasmid pWQ131. Derivatives of
wzccps with C-terminal deletions were generated by PCR, using primer TW6 and reverse primers that introduced novel termination codons. Primers TW7 (for Wzc1-704) and TW8
(for Wzc1-480) also provided appropriate restriction sites
to facilitate cloning of the amplified fragments in pBAD18-Cm. The
resulting plasmids were pWQ133 (expressing
Wzc1-704) and pWQ139 (Wzc1-480).
The DNA fragment encoding the C-terminal domain of Wzccps
(Wzc486-721) was amplified using the primer pairs
TW9-JD102 and the product was cloned in pBAD24, giving plasmid pWQ140.
In all cases, the sequences of PCR-amplified genes were determined to
ensure no errors were introduced during amplification.
Localization of Wzccps in Subcellular
Fractions--
Expression of Wzccps and its mutant
derivatives was achieved using the pBAD arabinose-inducible expression
vectors. Bacteria were grown to mid-exponential phase and
expression of the Wzccps derivative was induced by adding
0.02% L-arabinose. After an induction period of 15 min to
2 h, the cells were harvested and resuspended in 100 mM Tris-HCl, pH 7.5, containing 100 mM
MgCl2 and 1 mM phenylmethylsulfonyl fluoride
(buffer A). Subcellular fractionation was performed at 4 °C. The
cell suspension was lysed by ultrasonication, after which unbroken
bacteria and large debris were removed by centrifugation at 4,000 × g for 8 min. The cell-free lysate was centrifuged at 40,000 × g for 30 min, resulting in a
cytosol-periplasm fraction (supernatant) and a pellet containing cell
envelopes. The cell envelopes were resuspended in buffer A and further
separated into inner and outer membranes by solubilization with 2%
(w/v) Sarkosyl in buffer A for 30 min (24). The outer membrane is
insoluble under these conditions, and was collected as a pellet at
40,000 × g for 30 min. Wzccps was
identified in the cellular fractions by SDS-PAGE and Western immunoblotting.
Expression and Partial Purification of GST-Wzccps
Fusion Protein--
To generate an N-terminal glutathione
S-transferase (GST)-Wzccps fusion derivative,
the wzccps open reading frame was amplified by
PCR using primers AP05 and AP06 (Table II). After digestion of the
fragment with EcoRI and SalI at sites introduced
in the primers, the fragment was cloned into pGEX-4T3 (Amersham
Pharmacia Biotech) giving plasmid pWQ144. E. coli BL21
( Expression and Purification of
His6-Wzbcps--
The
wzbcps gene was amplified as a 506-bp fragment
using primers JD152 and JD153 and cloned into pBAD24 to give pWQ147.
For purification, a His6-Wzbcps derivative was
used. The wzbcps open reading frame was
amplified by PCR using primers AP03 and AP04 (Table II). After
digestion of the product at the NdeI and BamHI sites introduced in the primers, the fragment was cloned in pET28a(+) giving plasmid pWQ145 and generating an N-terminal His6-tag
on Wzbcps. The same approach was used to generate pWQ149
expressing the His6-tagged version of the mutated
derivative WzbC13S. The protein was purified from cell-free
lysates of E. coli BL21 ( Site-directed Mutagenesis--
Specific mutations were
constructed in vitro using a modified procedure of the
QuikChangeTM site-directed mutagenesis kit from Stratagene.
Briefly, complementary oligonucleotides were designed to contain the
desired codon change (Table II). To generate
WzccpsK540R, the template consisted of plasmid
pWQ130 (wzccps cloned in pBAD18-Km). The
Wzbcps catalytic site mutant, WzbC13S, was
generated from plasmid pWQ147 by site-directed mutagenesis using
primers AP01 and AP02, to give pWQ148. Following PCR amplification, the
reaction products were purified using the QIAquick PCR purification kit. The DNA was digested twice with DpnI to eliminate
template, and the remaining DNA was ligated and introduced into
E. coli DH5 Production of Antibodies Specific for
Wzccps--
Wzccps was overexpressed as an
N-terminal His6-tagged derivative from plasmid pWQ141 in
E. coli BL21 ( Western Immunoblot Analysis of Wzccps--
The
protein content of separated cell fractions or whole cell protein
lysates was analyzed by SDS-PAGE (25) using 10% polyacrylamide resolving gels stained with Coomassie Brilliant Blue. For Western blotting, samples were electrophoretically transferred to
WestranTM polyvinylidene difluoride membranes (Schleicher
and Schuell). The transfer buffer was 25 mM Tris, 192 mM glycine, 0.1% SDS, and 20% methanol.
Wzccps was detected in Western blots using the rabbit
polyclonal antiserum and a goat anti-rabbit secondary antibody (Caltag,
Burligame, CA). To detect proteins containing phosphotyrosine residues,
Western blots were probed using PY20 monoclonal anti-phosphotyrosine antibody (Transduction Laboratories, New York) and an anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, Inc.). Both
secondary antibodies were conjugated to alkaline phosphatase and nitro
blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate was used
for detection.
In Vitro Phosphorylation and Dephosphorylation of
GST-Wzccps--
Autophosphorylation of
GST-Wzccps was performed using a modification of the
approaches described by Vincent et al. (10). Approximately
80 µg of GST-Wzccps was incubated for up to 15 min at
37 °C with 10 µCi of [ SDS-PAGE Analysis of Cell-surface Polysaccharides--
Cell
surface polysaccharides from proteinase K-digested whole cell lysates
were isolated as described by Hitchcock and Brown (26), and analyzed by
electrophoresis on 10-20% Tricine SDS-PAGE gels from Novex (San
Diego, CA). Following electrophoresis, the LPS-containing molecules
were visualized by silver staining (27). For Western blotting, samples
were electrophoretically transferred to 0.45-µm BioTrace NT membranes
(Gelman Science) and probed with polyclonal rabbit anti-K30 serum (28),
which recognizes the identical serotype K30 repeat unit found in both
K30LPS and K30 CPS. Rabbit polyclonal antibodies specific
for the K40 antigen were described previously (29).
Bacteriophage Sensitivity Assays--
To test for assembly of
the K30 capsular layer, strains were assessed for their sensitivity to
lysis by specific bacteriophages. The presence of surface-expressed K30
capsular antigen was determined using bacteriophage K30, which requires
the K30 antigen as its receptor (30). Bacteriophage O9-1 is specific
for the LPS O9a antigen (31). In the O9a:K30 wild-type background, the
capsular layer masks the LPS O9a antigen and this strain is O9-1
resistant, but a reduction or absence of K30 capsular antigen unmasks
the bacteriophage O9-1 receptor.
Two Functional wzc Homologues on the Chromosome of E. coliO9a:K30--
The wzccps gene is located in the K30
antigen biosynthesis (cps) locus of a prototype group 1 capsule-producing strain, E. coli E69 (O9a:K30) (3). When
examined in Western blots using anti-K30 antigen antibodies, the
cell-surface polysaccharides of the wild-type strain (E. coli E69) showed reactivity in both the K30LPS
fraction (containing primarily a single K30 repeat unit) and in high
molecular weight CPS (Fig. 1).
Previously, we showed that cell lysates of E. coli CWG315
(wzccps::aacC1) still contained immunoreactive K30 capsular antigen, but the amounts were
significantly lower than those observed in wild-type lysates (Fig. 1).
In addition, E. coli CWG315 was unable to assemble a capsular structure that masked the underlying LPS O9a antigen. The
wzccps mutation in CWG315 has no discernible
effect on KLPS (3).
Subsequent studies by others have shown that many E. coli
strains contain a homologue of wzccps,
designated etk (32). The etk gene is located in
the appA (22 min) region of the sequenced E. coli
K12 genome. The predicted Etk protein is an autokinase that is only
expressed in some E. coli strains. It shares 74% similarity
(57% identity) with Wzccps (data not shown). Although the
biological role of Etk is unknown, the 22-min locus contains several
open reading frames whose predicted products are homologous to proteins
involved in polysaccharide expression (4). The products of the last
three open reading frames are part of the same transcriptional unit and
encode homologues of Wza, Wzb, and Wzc (i.e.
Etk). This entire region was amplified by PCR and shown to
have essentially the same sequence in E. coli K12 and
E. coli O9a:K30 (data not shown). Furthermore, it was
demonstrated that the wza22 min gene product
functions with low efficiency in the translocation of E. coli K30 CPS (4). To assess whether the small amount of K30 CPS
remaining in E. coli CWG315
(wzccps::aacC1) resulted
from etk expression, we constructed CWG285
(wzccps::aacC1 wza22
min::aadA) in which wzb22
min and etk expression is eliminated by the polar
aadA cassette inserted in wza22 min.
The capsule phenotypes of these strains were assessed by Western
blotting and bacteriophage-sensitivity assays.
Western blot analysis of E. coli CWG258
(wza22 min::aadA) using
anti-K30 antigen antibodies showed no significant reduction in
synthesis of the CPS compared with the wild-type (Fig. 1) and the
mutant strain showed no reaction with bacteriophage O9-1, indicating
the maintenance of a protective K30 capsule. In the double mutant,
CWG285 (wzccps::aacC1
wza22 min::aadA), formation of
CPS was completely abolished. No reaction was detected with the
capsule-specific bacteriophage K30 and the strain was sensitive to
bacteriophage O9-1. Although no CPS was synthesized, CWG285 showed a
significant increase in the extent of polymerization (8-10 repeat
units) of K30LPS reflected in the ladder of immunoreactive material.
Plasmids carrying the wzccps and etk
genes were used to complement the K30 capsule synthesis defect in
CWG285 (wzccps::aacC1 wza22 min::aadA). Expression of
plasmid pWQ130-encoded Wzccps restored synthesis of K30 CPS
and reduced the degree of polymerization of KLPS to the
typical wild-type 1-2 repeat units (Fig. 1). While K30 CPS synthesis
was restored in the complemented strain, the amount never achieved that
seen in the wild-type strain. The reason(s) for this is unclear. While
phosphorylated Wzc is certainly overexpressed in the complemented
strain (see below), the precise amount of Wzccps that is
competent in CPS synthesis is unknown. This result could also reflect
issues relating to stoichiometry of the components in a putative
multienzyme complex. Expression of plasmid pWQ131-encoded Etk resulted
in smaller amounts of CPS being formed (relative to Wzccps)
and the polymerization of K30LPS remained at the higher level. The lower activity of Etk is consistent with the absence of a
detectable CPS phenotype of CWG258 (wza22
min::aadA). From these data and the
phenotypes of CWG258 and CWG315 (above), we conclude that
etk encodes a functional homologue of Wzccps
that participates with low efficiency, relative to Wzccps,
in assembly of the K30 capsule. The influence of Etk is not apparent in
the presence of functional Wzccps. In all subsequent
experiments, strains carrying a wza22
min::aadA mutation were used to simplify the analysis and isolate the effects of Wzccps and
Wzbcps, without the complications of etk and
wzb22 min.
Subcellular Location of Phosphorylated Wzccps--
To
examine Wzccps localization in a known genetic background,
it was overexpressed in E. coli DH5 In Vitro Autophosphorylation of Wzccps and Its
Dephosphorylation by Wzbcps--
To confirm the
autophosphorylation of Wzccps without ambiguity, we
employed the strategy used to establish autophosphorylation of
Wzcca (10). The GST-Wzccps fusion protein was
purified by chromatography on glutathione-Sepharose 4B. Based on
Coomassie Blue-stained SDS-PAGE gels, this protein preparation was
>90% pure (Fig. 3 panel A).
The purified protein showed autokinase activity, incorporating
radioactivity from [ Phosphorylation of Wzccps Is Essential for Capsule
Synthesis--
Sequence alignments revealed that Wzccps
(like its homologues) contains a Walker A or kinase-1a motif, commonly
found in kinases (Fig. 4). The sequence
Ala534-Ser-Pro-Ser-Ala-Gly-Lys-Thr541 fits the
general consensus sequence motif (AG(X4GK)ST). Two putative Walker B or kinase-2 motifs (hhhhD; where h represents a hydrophobic amino acid) were detected. The optimal spatial distances between Walker
A and B motifs are approximately 61 or 145 amino acid residues (34).
While neither of the two Walker B motif candidates
(VLFID562 and LIIID642) provides such optimal
spacing, the aspartic acid residue within the sequence
VIID651 of the A. johnsonii Ptk protein (which
corresponds to the LIIID642 motif in Wzccps)
was found to be essential for ATP binding (13).
Tyrosine phosphorylation of Ptk requires a functional Walker A box for
ATP binding and hydrolysis (13). To confirm that Wzccps
autophosphorylation follows a similar mechanism, PCR-based site-directed mutagenesis was used to generate WzcK540R
(Fig. 4). Residues equivalent to lysine 540 in the Walker A
kinase-1a-motif are believed to be required for ATP hydrolysis in the
phosphotransfer reaction (34, 35). The WzcK540R derivative
was detected with anti-Wzccps antibodies (Fig.
5A) and was expressed at
levels equivalent to the wild-type Wzccps protein. When
probed with monoclonal PY20 anti-phosphotyrosine antibody, only the
wild-type protein was phosphorylated (Fig. 5B). The function
of WzcK540R in capsule assembly was also completely
abolished. No K30 capsular antigen could be detected in Western blots
using polyclonal anti-K30 serum (Fig. 5C) or in phage
sensitivity assays (data not shown) and the increased level of
KLPS polymerization was not altered by this derivative.
These data show that a functional Walker box, capable of ATP binding,
is necessary for Wzccps function in capsular assembly.
C-terminal Tyrosine Residues Provide the Sites of Phosphorylation
and Are Required for Wzccps to Function in Capsule
Formation--
Previous studies suggested that Ptk, and presumably its
homologues, are phosphorylated at multiple tyrosine residues (9) but
the site of phosphorylation was not determined. Sequence alignments including Wzccps homologues from Gram-negative and
Gram-positive bacteria revealed several highly conserved C-terminal
tyrosine residues (Fig. 4) and while this work was in progress
phosphorylation of CpsD was shown to occur in the C-terminal
tyrosine-rich domain (21). Seven of the last 17 amino acid residues of
Wzccps are tyrosine residues. To address the relevance of
this tyrosine-enriched domain, the 17 C-terminal amino acids were
removed in a truncated version of Wzccps designated
Wzc1-704 (Fig. 3). Wzc1-704 was
reactive with anti-Wzccps antibody (Fig. 5A) and
localized to the inner membrane (data not shown) but the derivative
could no longer undergo tyrosine phosphorylation (Fig. 5B).
Furthermore, the Wzc1-704 protein was no longer able to
complement the CPS-deficient phenotype in strain CWG285
(wzccps::aacC1 wza22
min::aadA) (Fig. 5C), consistent with the proposal that phosphorylation of these tyrosine residues is
required for normal Wzccps function.
To determine whether the truncated Wzc1-704 derivative
still retains the ability to bind ATP, it was coexpressed in CWG285
with the WzcK540R protein. This resulted in
transphosphorylation of WzcK540R (Fig. 5B) and
generation of Wzc activity that restored the assembly of CPS (Fig.
5C).
Activity of the Phosphotyrosine-protein Phosphatase,
Wzbcps, Is Required for Capsule Assembly--
Since Wzb
homologues are known to dephosphorylate their cognate Wzc proteins (10,
14) and the phosphotyrosine-protein phosphatase activity for
Wzbcps was confirmed as described above, we
constructed CWG343 (wza22
min::aadA
wzbcps::aph3A) to examine the
effect of Wzccps dephosphorylation on CPS assembly. The
presence or absence of Wzbcps has no significant effect on
the amount of Wzc polypeptide produced (data not shown), or on the
amount of phosphorylated Wzccps (Fig.
6). However, CWG343 retains only trace amounts of K30 CPS synthesis, detectable as immunoreactive material in
Western blots (Fig. 6) and by sensitivity to the K30 CPS-specific bacteriophage K30 (data not shown). Introduction of pWQ147
(Wzbcps+) restored synthesis of K30 CPS
indicating that the defect in CWG343 was attributable only to the
single nonpolar insertion in wzbcps. Coomassie
Blue-stained SDS-PAGE gels of CWG343(pWQ147) whole cell protein lysates
showed the presence of an overexpressed protein with the apparent
molecular weight (16,604 predicted) expected of Wzbcps
(data not shown).
One possible interpretation of these data is that the phenotype of
CWG343 is due to a loss of protein-protein interactions, rather
than a simple loss of phosphotyrosine protein phosphatase activity. To
address this question, we constructed pWQ148 and pWQ149 in which
Wzbcps has a C13S mutation. The phosphotyrosine-protein phosphatase signature motif (H/V)C(X5)R(S/T) (36) contains
a nucleophilic cysteine residue (Cys13 in
Wzbcps) that forms a phosphocysteine intermediate during
catalysis. This residue is essential for catalysis (37, 38) and has
been shown to be required for the activity of Wzbca (14).
As expected, the WzbC13S showed no activity in
vitro against phosphorylated Wzccps (Fig. 3,
panel D). Although the mutant protein was made in and
readily detectable in CWG343 cell lysates (data not shown), it was
unable to restore CPS synthesis (Fig. 6). Thus the defect in CWG343 is attributable to the loss of phosphotyrosine-protein phosphatase activity.
A Functional Wzccps Comprising Independently Expressed
N- and C-terminal Domains--
Wzccps homologues in
Gram-positive bacteria are encoded as two distinct proteins with the
corresponding genes generally found next to one another in the same
cluster. For example, in S. pneumoniae CpsC resembles the
N-terminal membrane-anchored part of Wzccps. The adjacent
gene (cpsD) encodes a predicted polypeptide equivalent to
the C-terminal hydrophilic domain containing the ATP-binding motif and
the conserved tyrosine residues (15). Both polypeptides are required
for autophosphorylation (21). To determine whether different domains of
Wzccps would also be functional as separate polypeptides,
initiation and termination codons were introduced and fragments
expressing the N and C termini of Wzccps were cloned independently. The break point (between amino acid residues 481 and
494) was dictated by sequence alignments with known Wzccps homologues in Gram-positive bacteria (data not shown). A termination codon was introduced after Lys480 generating the N-terminal
polypeptide Wzc1-480. The Gly486 codon was
replaced with an ATG start codon to express the soluble C-terminal
polypeptide Wzc486-721. The Wzc1-480 and
Wzc486-721 derivatives were cloned behind the
arabinose-inducible promoter in pBAD18-Cm and pBAD24, generating
plasmids pWQ139 and pWQ140, respectively.
The Wzccps N- and C-terminal domains (Wzc1-480
and Wzc486-721) were expressed and located in the inner
membrane and cytosol, respectively (data not shown). While the
truncated Wzc1-480 N-terminal fragment was readily
detected in Western blots with anti-Wzccps antibodies (Fig.
7A), the
Wzc486-721 C-terminal fragment was expressed at only low
levels and could only be detected in heavily overloaded SDS-PAGE gels
(data not shown). When expressed alone in strain E. coli
CWG285, neither part of Wzccps could function in capsule
formation (Fig. 7C). However, when both proteins were
expressed simultaneously in CWG285, CPS synthesis was restored. The
function in capsule formation was correlated with phosphorylation of
the Wzc486-721 C-terminal domain, as this only occurred
when both parts of Wzccps were coexpressed (Fig.
7B). The amount of CPS was lower than wild-type and a
masking K30 capsule was not formed based on the sensitivity of the
transformant to bacteriophage O9-1. The low efficiency in the two-part
construct may be related to low level expression of soluble
Wzc486-721 and its phosphorylation in a CWG285
background. It is also conceivable that the stoichiometry of the two
Wzccps domains is important and this cannot be controlled
in the two-plasmid system.
The N Terminus of Wzccps Cannot Serve as a Functional
Replacement for the LPS O-Antigen Chain-length Regulator, Wzz--
The
N-terminal domains of Gram-negative Wzc proteins contain two putative
transmembrane helices flanking a periplasmic loop. This topology
resembles that of Wzz, the LPS O antigen chain-length determinant and
the two classes of proteins share sequence similarity (6, 8, 39). Wzz
proteins lack the additional C-terminal sequences that, in Wzc, contain
the ATP-binding motif and tyrosine-rich domain. Wzz determines the
distribution of O-antigen chain lengths and generates a modal pattern
in LPS size distribution, evident as clusters of bands in SDS-PAGE. To
determine whether the N terminus of Wzccps can alter chain
length modality as does Wzz, we used the E. coli K40 antigen
as a reporter system. The K40 antigen is a group 4 capsule (1) in which
the majority of the polysaccharide is linked to lipid A-core as an LPS
O antigen. Wzz controls the modality of the K40 LPS (29) and the
wzz::aacC1 mutant (CWG290) shows a loss
of a modal cluster of K40 LPS bands in SDS-PAGE gels and accumulation
of lower molecular weight bands (Fig. 8).
This is thought to reflect a bias in the polymerization system favoring chain-length termination rather than further polymerization (reviewed in Ref. 6). A modal cluster of bands was restored in the SDS-PAGE profile when CWG290 was transformed by plasmid pWQ30 carrying the
wzz gene from E. coli O75 (28). In contrast,
expression of the N-terminal domain of Wzc (Wzc1-480) in
CWG290 did not bring about the dramatic modality resulting from
WzzO75 expression. However, it did alter the polymerization profile evident in SDS-PAGE. Furthermore, the effect was dependent on
the extent of Wzc1-480 expression (reflecting varying
levels of induction by increasing arabinose concentrations).
Interestingly, full-length Wzccps did not generate the same
altered profile of K40 LPS even at the highest concentration of
arabinose (0.2%) (Fig. 8) and, as expected, the C terminus alone had
no effect (data not shown).
In this study we investigated the structure of Wzccps
from E. coli E69 (O9a:K30) and its role in capsule
formation. Two separable domains were identified and phosphorylation of
the Wzccps tyrosine autokinase was shown to be essential
for synthesis of CPS. The conserved features in Wzc homologues suggest
a shared and widespread role in prokaryotes. Since many of the
Wzc-containing bacteria require capsular or extracellular
polysaccharides as essential virulence determinants, Wzc homologues
play a crucial role in pathogenesis of these organisms. Regulation by a
post-translational phosphorylation event represents a new dimension in
the assembly of bacterial cell-surface polysaccharides.
Previously, we showed that a nonpolar insertion mutation in
wzccps significantly lowered the amount of CPS
synthesis in E. coli CWG315, resulting in a defective
capsular layer that was unable to mask underlying LPS O9a antigen (3).
Here, we established that the residual expression of CPS on the cell
surface of strain CWG315 is due to a second functional Wzc homologue,
encoded by the unlinked etk (formerly ep85) gene.
Ilan and colleagues (32) have shown that the Etk tyrosine autokinase is
only expressed in a subset of pathogenic E. coli strains,
but its functional role in E. coli was not determined and
its contribution to virulence has not been tested. The etk
gene is located in the appA (22 min) region of the E. coli K-12 genome in a cluster encoding homologues of Wza and Wzb,
as well as other genes whose predicted products share similarities with
enzymes involved in biogenesis of cell-surface polysaccharides. The
22-min locus lacks either a Wzy-polymerase homologue or an ABC
transporter, features that define the primary pathways for bacterial
cell-surface polysaccharide biogenesis (reviewed in Refs. 1 and 40).
Despite the apparent lack of a full spectrum of biosynthetic components
necessary for its function as an independent polysaccharide expression
locus, the 22-min locus gene products can cooperate with functions
encoded by the cps locus (4). Wzacps forms a
multimeric outer membrane complex that resembles the secretins
for secretion of proteins by type II and type III secretion systems
(4). When expressed from a multicopy plasmid, the wza22
min gene product can also function with low efficiency in
assembly of the K30 capsule. This is consistent with the current
finding that Etk participates with low efficiency, relative to
Wzccps, in CPS synthesis. In light of this functional data,
Etk should be renamed Wzc22 min, joining Wzccps
and Wzcca (colanic acid biosynthesis) as the third known
Wzc homologue in E. coli. Although the colanic acid
biosynthesis locus is also widespread in E. coli and
represents an additional source of Wzc activity, this locus is absent
in E. coli isolates with group 1 capsules (reviewed in Ref.
1).
Tyrosine autokinase activity has been documented in Ptk from A. johnsonii (41), Wzcca from E. coli K-12
(10), Etk from E. coli (32), AmsA from E. amylovora (32), and now Wzccps from E. coli
isolates with group 1 capsules. However, the exact function of these
proteins and the role of their phosphorylation in the biology of their
respective microorganisms have not been established. The involvement of
a Walker A motif in autokinase activity in these enzymes suggests that
the kinase catalytic mechanism differs from that in eukaryotic protein
kinases (13). Phosphorylation of Ptk requires ATP binding and
hydrolysis (13) and a similar activity in Wzccps was
confirmed experimentally by site-directed mutation of the invariant
Lys540 of the Walker A box. This residue is believed to be
involved in ATP binding and hydrolysis (34, 35). The
WzcK540R mutation prevented phosphorylation of Wzc and
completely abolished its function in capsule assembly, providing the
first indication that ATP binding is essential, and that the
phosphorylated form of Wzccps is the one that is functional
in CPS expression. The truncated Wzccps derivative
(Wzc1-704) identified the highly conserved C-terminal
tyrosine-rich domain residues as the site of phosphorylation and
confirmed the essential requirement for Wzccps
phosphorylation in group 1 CPS synthesis. While the CPS phenotypes
arising from an ATP-binding defect and a lack of the phosphorylation
site are identical, we cannot rule out the possibility that they have
different detrimental effects on the CPS synthesis process. Although
the role of phosphorylation of Wzccps has not been directly
tested in other Gram-negative CPS synthesis systems, production of the
exopolysaccharide succinoglycan is drastically reduced in S. meliloti strains expressing a derivative of ExoP (Wzc) lacking the
C terminus (42). By analogy to Wzc, this region of ExoP contains the
phosphorylated tyrosine residues. As with the E. coli group
1 CPS system, the exoP-delete strain is still able to
synthesize lipid-linked glycan repeat units (7).
The effect of Wzc phosphorylation on group 1 CPS expression differs
from that seen in the representative Gram-positive system from S. pneumoniae (21), where domains resembling the N and C termini of
Gram-negative Wzc are found in separate polypeptides encoded by
adjacent genes. Proteins sharing sequence similarity and features with
the C-terminal domain of Wzccps include CpsD in S. pneumoniae (15) and S. agalactiae (17, 18), CapB from S. aureus (19), EpsD from Streptococcus
thermophilus (43), and EpsB from Lactococcus lactis
(44). CpsC, CapA, EpsC, and EpsA encode the corresponding
membrane-associated domains, respectively. In S. pneumoniae
CpsD, a Walker box mutation renders the protein unable to
autophosphorylate and generates bacteria that synthesize only trace
amounts of CPS (21). The size of the remaining CPS product was not
reported. In contrast, the phosphorylation of the tyrosine residues
per se was not required for CPS synthesis in S. pneumoniae. In fact, replacement of the C-terminal tyrosines with
phenylalanine leads to a mucoid phenotype, presumed to reflect an
increase in CPS expression. The differences in phenotype in the
E. coli and S. pneumoniae systems are not simply
due to unexpected effects arising from the use of a C-terminal deletion
derivative for the E. coli analyses. The ability of
WzcK540R and Wzc1-704 to undergo
intermolecular transphosphorylation and restore CPS synthesis is
consistent with the notion that the phenotype arising from
Wzc1-704 is due to a loss of phosphorylation. Preliminary
experiments using site-directed mutagenesis to change tyrosine residues
to phenylalanines demonstrate that phosphorylation occurs at multiple tyrosines in Wzccps, and work is in progress to determine
which tyrosine residues are important. However, a mutant in which all the tyrosines are mutated lacks phosphorylation and confers a CPS
phenotype identical to that of
Wzc1-704.2 Thus,
the S. pneumoniae system differs from the E. coli
group 1 CPS in two respects: CpsD mutants defective in ATP
binding/hydrolysis and tyrosine phosphorylation have different effects
on CPS phenotype, and phosphorylated tyrosine may act as a negative
effector in S. pneumoniae. The role of
phosphotyrosine-protein phosphatase activity in S. pneumoniae is currently unclear. Although the product of an
adjacent gene (cpsB) is implicated in dephosphorylation of
CpsD (21), the putative CpsB protein lacks the motif (and active site
cysteine) typical of phosphotyrosine-protein phosphatase. While CpsB
might reflect a new type of phosphatase, such an activity has not yet
been tested.
Given the overall similarity in the components for
Wzy-dependent CPS biosynthesis in Gram-positive and
Gram-negative bacteria, the differential effect of the
phosphorylation-deficient Wzccps and CpsD derivatives was
surprising. The two domains of Wzccps could be expressed as
independent polypeptides to mimic the Gram-positive situation and these
constructs were able to function in both phosphorylation of the
C-terminal domain and in capsule assembly. However, there are some
significant structural differences between CpsCD and Wzccps. The extra-cytoplasmic membrane loop of CpsC and its
homologues are usually 270-300 residues smaller than the corresponding
domain in Wzccps (6, 8). Another interesting feature
distinguishing the Gram-negative and S. pneumoniae systems
is the fact that the tyrosines in CpsD are arranged in a
(YGX)4 motif. Whether these latter features play a role in
the apparent differences in the requirement for Wzccps/CpsD
phosphorylation in CPS synthesis remains to be established.
Phosphorylated Ptk and Wzcca are substrates for the
phosphotyrosine-protein phosphatases, Ptp and Wzb, and these enzymes
were found to be functionally interchangeable (10). Wzccps
is also dephosphorylated by Wzbcps. Unexpectedly, a
Wzbcps mutant also lacks K30 CPS biosynthesis. To rule out
the possibility that the CPS phenotype in CWG343
(wzb::aacC1) reflected a loss of
important protein-protein interactions rather than the
phosphotyrosine-protein phosphatase activity per se, we used
a catalytically inactive mutant (WzbC13S). The inability of
this mutant to complement the defect in CWG343 strongly supports the
notion that phosphatase activity is essential. The most likely
explanation for the CPS phenotype of the wzbcps mutant is an inability to dephosphorylate Wzccps. This is
based on activity of Wzbcps against Wzccps, the
fact that Wzccps is the only known tyrosine-phosphorylated
protein in the CPS biosynthesis system, and the fact that these
E. coli strains have no other detectable
tyrosine-phosphorylated proteins other than Etk. The requirement for
both autokinase and phosphatase activity in CPS biosynthesis raises the
interesting possibility that cycling between phosphorylated and
dephosphorylated Wzccps is involved in CPS assembly.
However, attempts to demonstrate cycling in vitro were not
successful (data not shown). Either the activities are confined to
one-time events, or an essential component or condition needed for
cycling is absent in the in vitro system. The level of
autokinase activity is insufficient to address this question in
vivo.
Although the results presented here provide the first indication of the
requirement for phosphorylated Wzccps in assembly of the
K30 capsule, its exact role has yet to be resolved. The resemblance of
Wzc homologues from Gram-negative and Gram-positive bacteria suggests
they may act at conserved stages in the assembly process and the most
logical candidate is polymerization. Significantly, the Wzc homologues
in both Gram-positive and Gram-negative bacteria are involved in the
assembly of polysaccharides that appear to be polymerized by
Wzy-dependent polymerization systems. The
Wzccps protein is a member of the cytoplasmic
membrane-periplasmic auxiliary protein 1 family (39). More recently,
these proteins have been suggested to be part of a larger family
including Wzz, a protein associated with chain length determination of
Wzy-dependent LPS O antigens. An alternative name,
polysaccharide copolymerase was proposed (8). The membrane-periplasmic
auxiliary/polysaccharide copolymerase proteins are associated with loci
for capsule and exopolysaccharide expression in Gram-positive and
Gram-negative bacteria and they share similarities in the transmembrane
topology. Wzc proteins are distinguished from Wzz by an extended C
terminus that contains the ATP-binding motif and phosphotyrosines,
suggesting a more complex functional role for Wzc.
Wzy polymerase proteins catalyze glycosidic bond formation and are
therefore specific for a given polymer repeat-unit structure. There is
little primary sequence relatedness shared by Wzy homologues but their
hydropathy profiles are similar. A given Wzz protein can interact with
polymerization systems for different LPS O antigen repeat units (see,
for example, Refs. 28 and 45-47). This suggests few (if any) limits
are imposed on Wzz function by either Wzy primary structure or by the
repeat unit structure of the polysaccharide product. However, the
chain-length modalities are certainly sensitive to differences in Wzz
primary sequences (45-47). In contrast to the sequence variation in
Wzz within different E. coli serogroups, both E. coli and K. pneumoniae have cps gene
clusters encoding essentially identical Wzccps proteins
(2). Wzz regulation is confined to lipid A core-linked polymers and
while it can generate modality in KLPS, it does not
influence assembly of the E. coli group 1 CPS (28). The
natural lack of modality in KLPS in the wild-type strains
results from the absence of wzz on the chromosome of
E. coli isolates with group 1 capsules (28). Conversely, we
show here that Wzccps cannot restore modality in the
absence of Wzz. While Wzc1-480 does not influence O-chain
length in precisely the same manner as Wzz, it does alter the profile
of O-antigen-substituted LPS molecules in SDS-PAGE, suggesting it can
interact with and modulate an O-antigen polymerization system providing
that the C-terminal domain is absent.
It should be noted that enzymatic activity directly involving
polymerization has not been proven for either Wzz or Wzc and their role
could be indirect. In E. coli, polymerization reactions for
K30LPS and K30 CPS share the same Wzy-polymerase enzyme (3) and K30LPS molecules comprising 8-10 repeat units of K30
antigen are formed in strains devoid of Wzccps and Etk.
Thus, Wzccps is not an essential partner for Wzy activity
per se. However, Wzccps might be essential for a
specific subset of substrates, or enzyme complexes devoted to assembly
of the high molecular weight capsular K30 antigen. For example,
Wzccps may regulate the flow of substrates into high
molecular weight K30 CPS, accounting for the increased polymerization
of K30LPS evident in the absence of CPS synthesis. Wzc
proteins could also potentially influence the synthesis of CPS by
effecting formation of a capsule assembly complex. Both Wzz and Wzc
proteins have periplasmic domains that are predicted to form
coiled-coils (8), a feature important for protein-protein interactions.
In the case of Wzz, oligomerization has been demonstrated by
cross-linking methods (47). The demonstration that coexpression of
WzcK540R and Wzc1-704 allows intermolecular
transphosphorylation and restoration of capsule expression strongly
suggests that Wzccps proteins can also interact with one
another. If Wzccps plays a role in assembling a functional
complex for assembly of the K30 CPS, the presence of polymerized
KLPS can only be explained by separate biosynthetic complexes for the polymerization of K30 CPS (Wzc-dependent)
and K30LPS (Wzc-independent). The phenotype of E. coli CWG285 (wzccps::aacC1 wza22 min::aadA) would then be
explained by the absence of functional CPS polymerization complexes.
The availability of additional Wzy enzyme and its lipid-linked
substrates for KLPS complexes would be reflected in the
higher level of KLPS polymerization. This issue provides
the direction for future studies.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1),
chloramphenicol (Cm, 34 µg ml
1), gentamicin (Gm, 30 µg ml
1), kanamycin (Km, 50 µg
ml
1), and spectinomycin (Sp, 100 µg ml
1).
Expression of genes cloned in pET and pBAD derivatives was induced using 0.1-0.5 mM
isopropyl-1-thio-
-D-galactopyranoside and 0.02%
L-arabinose, respectively.
Bacterial strains and plasmids used in this study
Primers used in this study
DE3) transformed with pWQ144 was used to express and partially
purify GST-Wzccps by affinity chromatography on
glutathione-Sepharose 4B (Amersham Pharmacia Biotech), using a
modification of the approach described by Vincent et al.
(10). Briefly, cell-free lysates were prepared by sonication in
phosphate-buffered saline. After sonication, Triton X-100 was added to
give a final concentration of 1%. Cellular debris were then removed by
centrifugation at 30,000 × g for 30 min and the
cell-free lysate was mixed with chromatography matrix on ice for 30 min, before packing in a column for washing and elution. The column was
washed with 3 × 2 ml of phosphate-buffered saline containing 1%
Triton X-100. Elution was performed using buffer B (50 mM
Tris-HCl, pH 8.0) containing 10 mM glutathione and 0.1%
Triton X-100. Fractions containing the GST-Wzccps protein were pooled and dialyzed against buffer C (25 mM Tris-HCl,
pH 7.0, 1 mM dithiothreitol, 5 mM
MgCl2).
DE3) containing pWQ145, prepared
in buffer D (50 mM Na phosphate buffer, pH 8.0, 300 mM NaCl) containing 10 mM imidazole. After removing cellular debris by centrifugation at 30,000 × g for 30 min, the His6-Wzb was purified by
Ni2+ affinity chromatography. The Ni2+-NTA
matrix was washed 2 times in 2 ml of buffer D containing 20 mM imidazole, and His6-Wzbcps was
eluted with 4 × 250 µl of buffer D containing 250 mM imidazole.
by electrotransformation. The mutated
derivative was resequenced (both strands) to confirm the mutation and
verify that no other changes were introduced.
DE3). Membranes were prepared as described
above and the His6-Wzccps protein was
solubilized by incubation in buffer containing 1% Triton X-100 and 8 M urea for 20 min on ice. The solubilized protein extract
was applied to a Ni2+-NTA column (Qiagen) and
His6-Wzccps was eluted using 250 mM
imidazole. After dialysis, purified His6-Wzccps
was used to immunize a New Zealand White rabbit. Immune serum was
absorbed against E. coli BL21 (
DE3) whole cells,
essentially removing all nonspecific antibodies.
-33P]ATP (PerkinElmer Life
Sciences, 3000 Ci/mmol) in a reaction volume of 0.2 ml. Samples were
removed at intervals, transferred to 2 × SDS-PAGE sample buffer
and heated at 100 °C for 5 min. The phosphorylated
GST-Wzccps was detected by SDS-PAGE followed by
autoradiography. Dephosphorylation of Wzccps-GST was
assessed using in vitro phosphorylated substrate. After 15 min incubation in the phosphorylation assay, ~200 µg of
His6-Wzbcps (or catalytically inactive
WzbC13S) was added at 37 °C and 10 × concentrated
buffer was added to give a final concentration of 100 mM
sodium citrate, pH 6.5, and 1 mM EDTA. At intervals,
samples were removed, transferred to 2 × SDS-PAGE sample buffer and
heated at 100 °C for 5 min prior to SDS-PAGE and autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (27K):
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Fig. 1.
Two functional Wzc homologues contribute to
formation of the E. coli K30 capsule. Panel
A shows a silver-stained Tricine-PAGE analysis of cell-surface
polysaccharides and panel B, the corresponding Western blot
probed with polyclonal K30 antiserum. Lane 1, wild-type
E. coli E69 (O9a:K30); lane 2, CWG258
(wza22 min::aadA);
lane 3, CWG315
(wzccps::aacC1); lane
4, CWG315 complemented by plasmid pWQ130
(Wzccps+); lane 5, CWG285
(wzccps::aacC1 wza22
min::aadA); lane 6, CWG285
complemented by plasmid pWQ131 (Etk+); and lane
7, CWG285 complemented by plasmid pWQ130
(Wzccps+). Each construct was tested for
susceptibility to bacteriophage K30 that requires K30 CPS as its
receptor. The receptor for bacteriophage O9-1 is serotype O9a LPS; this
bacteriophage can only lyse bacteria where the receptor is unmasked by
reduction or elimination of the K30 capsule.
. Although E. coli K-12 derivatives like DH5
contain functional copies of
wzcca and etk, these are not evident
in control samples. Etk may not be expressed in E. coli
DH5
(32) and there is no significant transcription of the colanic
acid genes (including wzcca) in E. coli K-12 at 37 °C (reviewed in Ref. 33). To determine the
subcellular localization of Wzccps, cell envelopes from
arabinose-induced E. coli DH5
(pWQ130) cells were
separated into the inner membrane (Sarkosyl soluble) and outer membrane
(Sarkosyl insoluble) fractions. A Western blot of the membrane
fractions was probed with a commercial monoclonal anti-phosphotyrosine
antibody (PY20) which is known to react with homologues of
Wzccps (32). The phosphorylated Wzccps protein
(predicted molecular weight = 79,558) is localized in the inner
membrane (Fig. 2), as expected from
sequence features and the relationships shared among the Wzc family of
tyrosine autokinases. This location was further confirmed by examining membranes fractionated by isopycnic sucrose gradient centrifugation (data not shown).
View larger version (32K):
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Fig. 2.
Localization of overexpressed
Wzccps in an E. coli
DH5 -background. A,
Coomassie Blue-stained SDS-PAGE; B, corresponding Western
blot, probed with monoclonal anti-phosphotyrosine antibody (PY20).
MW, molecular weight markers; lane 1, whole cell
lysate; lane 2, cell envelope fraction; lane 3,
Sarkosyl-solubilized inner membrane fraction; and lane 4, Sarkosyl-insoluble outer membrane fraction.
-33P]ATP in a
time-dependent manner (Fig. 3, panel B). The
cognate phosphatase (Wzbcps) was purified as a
His6-tagged derivative to >90% purity (Fig. 3,
panel A). As previously shown for Wzbca (10),
Wzbcps catalyzed the dephosphorylation of
Wzccps (Fig. 3, panel C).
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Fig. 3.
In vitro autokinase activity of
GST-Wzccps and its dephosphorylation by
Wzbcps. Panel A shows soluble fusion
proteins, GST-Wzccps and
His6-Wzbcps, purified by affinity
chromatography procedures. The GST-Wzccps protein shows an
apparent molecular weight of 105,000 (predicted
Mr = 106, 267; native Wzccps
Mr = 79,558). The apparent molecular weight of
His6-Wzbcps is 18,000 (predicted
Mr = 18,796; native Wzbcps
Mr = 16,604). Panel B is an
autoradiogram showing autophosphorylation of GST-Wzccps
using [ -33P]ATP as substrate. The only phosphorylated
protein comigrated with Wzccps and only the relevant part
of the gel is shown. Panel C shows the dephosphorylation of
33P-labeled GST-Wzccps by purified
His6-Wzbcps. Panel D shows an
equivalent dephosphorylation experiment using the catalytically
inactive His6-WzbC13S.
View larger version (25K):
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Fig. 4.
Structural features of
Wzccps. A, C-terminal sequence alignment of
selected Wzccps homologues from Gram-positive and
Gram-negative bacteria. The GenBankTM accession for each
homologue is indicated. The arrows indicate the 7 tyrosine
residues in Wzccps from E. coli O9a:K30.
Identical residues are indicated by asterisks (*). Residues
with high (:) or low (.) levels of similarity are also identified.
B, schematic organization of Wzccps and its
mutant derivatives. The location and sequence of the Walker A
ATP-binding motif of wild-type Wzccps and the mutant
WzcK540R are shown. The strategy for separating the N-
(Wzc1-480) and C-terminal (Wzc486-721)
domains was based on the "two-part" Wzccps homologues
in Gram-positive bacteria. The transmembrane (TM) helices
and tyrosine-containing C-terminal domain are indicated.
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Fig. 5.
Phosphorylation of Wzccps is
essential for capsule synthesis. Panel A shows a
Western blot of whole cell protein lysates developed using
anti-Wzccps antibodies, showing expression of
Wzccps and its mutant derivatives expressed from plasmids
in E. coli CWG285
(wzccps::aacC1 wza22
min::aadA). Lane 1, CWG285 (no
plasmid); lane 2, pWQ130 (Wzccps); lane
3, pWQ132 (WzcK540R); lane 4, pWQ133
(Wzc1-704) lane 5, pWQ132 plus
pWQ133. Panel B shows a Western blot of the same samples
probed with monoclonal anti-phosphotyrosine antibody (PY20) to detect
the phosphorylation of the Wzccps derivatives. Panel
C shows a Western blot of the cell-surface polysaccharides from
the same constructs probed with polyclonal K30 antiserum.
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Fig. 6.
Assembly of CPS also requires a functional
Wzb protein. Panel A shows a Western blot of whole cell
protein lysates probed with PY20 anti-phosphotyrosine antibodies, and
panel B shows cell-surface polysaccharides from the same
constructs in a Western blot probed with K30 antiserum. Lane
1, E. coli CWG258 (wza22
min::aadA); lane 2, CWG285
(wzccps::aacC1 wza22
min::aadA); lane 3, CWG343
(wza22 min::aadA
wzbcps::aph3A); lane
4, CWG343 containing pWQ148 (WzbC13S); lane
5, CWG343 containing pWQ147 (Wzbcps).
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Fig. 7.
Functional Wzccps comprising
independently expressed N- and C-terminal domains. The various
Wzccps constructs were expressed in E. coli
CWG285 (wzccps::aacC1
wza22 min::aadA). Western blots
of whole cell protein lysates were developed using
anti-Wzccps (panel A), and PY20
anti-phosphotyrosine (panel B) antibodies. Lane
1, pWQ130 (Wzccps); lane 2, CWG285 no
plasmid (negative control); lane 3, pWQ139
(Wzc1-480); lane 4, pWQ140
(Wzc486-721); lane 5, pWQ139 plus pWQ140.
Panel C shows the cell-surface polysaccharide phenotype of
the same strains in a Western blot probed with polyclonal K30
antiserum.
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Fig. 8.
The N-terminal domain of Wzccps
cannot functionally replace Wzz. The figure shows a Western blot
of cell-surface polysaccharides of E. coli O8:K40 and its
derivatives. The blot was probed with anti-K40 antibodies. Lanes
1 and 2 show the wild-type strain (2775) and its
wzz::aacC1 derivative (CWG290).
Lane 3 shows CWG290 transformed with pWQ139 (encoding
Wzc1-480) grown in the presence of 0.2% glucose to
repress the pBAD promoter. Lanes 4 and 5 show the
same strain after induction with 0.02 and 0.2% arabinose,
respectively. Lane 6 shows CWG290 transformed with pWQ30
(Wzz075+) to illustrate restoration of
modality. The sample in lane 7 is from CWG290 transformed
with pWQ130 (Wzccps+) induced with 0.2%
arabinose.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported in part by an operating grant from the Canadian Institutes of Health Research (to C. 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.
Supported by Erwin Schrödinger Postdoctoral Fellowship
J1594-GEN from the Austrian Science Fund.
§ Supported by postgraduate scholarships from the Natural Science and Engineering Research Council of Canada.
¶ Received an undergraduate (USRA) award from the Natural Science and Engineering Research Council of Canada.
Canadian Institutes of Health Research Senior Scientist. To
whom correspondence should be addressed: Dept. of Microbiology, University of Guelph, Guelph, Ontario, N1G 2W1 Canada. Tel.:
519-824-4120 (ext. 3478); Fax: 519-837-1802; E-mail:
cwhitfie@uoguelph.ca.
Published, JBC Papers in Press, October 26, 2000, DOI 10.1074/jbc.M009092200
2 J. Hocking, A. Paiment, and C. Whitfield, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: CPS, capsular polysaccharide; LPS, lipopolysaccharide; KLPS, lipopolysaccharide lipid A-core carrying a short oligosaccharide comprised of K-antigen repeat units; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PCR, polymerase chain reaction.
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