analysis of shigella flexneri wzz (rol) function by mutagenesis and cross-linking: wzz is able to...
TRANSCRIPT
Analysis of Shigella ¯exneri Wzz (Rol) functionby mutagenesis and cross-linking: Wzz is ableto oligomerize
Craig Daniels and Renato Morona*
Department of Microbiology and Immunology, The
University of Adelaide, Adelaide, South Australia,
Australia 5005.
Summary
The modal length or degree of polymerization (dp) of
the Shigella ¯exneri O-antigen is determined in an
unknown manner by the Wzz/Rol protein. The Wzz
protein is anchored into the cytoplasmic membrane
by two transmembrane domains (TM1 amino acids
32±52; TM2 amino acids 295±315) with the central
loop of the protein located in the periplasm. Plasmids
were constructed encoding hybrid Wzz proteins con-
sisting of regions of S. ¯exneri Wzz (WzzSF ) and Sal-
monella typhimurium Wzz (WzzST ). These imparted
O-antigen modal chain lengths that implied that
the carboxy-terminal region of Wzz was involved in
chain length determination. Site-directed mutagenesis
was undertaken to investigate the functional signi®-
cance of highly conserved residues in amino-/car-
boxy-terminal domains of WzzSF. Some of the WzzSF
variants resulted in O-antigen modal chain lengths
much shorter than those of wild-type WzzSF, whereas
other mutants inactivated WzzSF function entirely and
a third class had a longer O-antigen chain length dis-
tribution. The data indicate that amino acids through-
out the length of the WzzSF protein are important
in determination of O-antigen modal chain length.
In vivo cross-linking experiments were performed to
investigate the interactions between Wzz proteins.
The experiments indicated that the WzzSF protein is
able to form dimers and oligomers of at least six
WzzSF proteins. A carboxy-terminal-truncated WzzSF
protein having the amino terminal 194 amino acids
was able to oligomerize, indicating that the amino-
terminal region is suf®cient for the Wzz±Wzz inter-
action observed. Shortened WzzSF proteins having
internal deletions in the amino-terminal region were
also able to oligomerize, suggesting that residues
59±194 are not essential for oligomerization. Cross-
linking of WzzSF proteins with mutationally altered
residues showed that loss of WzzSF function may
be correlated to a reduced/altered ability to form oli-
gomers, and that mutational alteration of glycine resi-
dues in the TM2 segment affects WzzSF±WzzSF dimer
mobility in SDS polyacrylamide gels. These results
provide the ®rst evidence of protein±protein interac-
tions for proteins involved in O-antigen polysaccharide
biosynthesis.
Introduction
Complex glycolipids such as lipopolysaccharides (LPSs)
are characteristic of the outer membrane of Gram-nega-
tive bacteria. LPSs consist of three covalently linked com-
ponents: lipid A; a core sugar region; and a polymerized
chain of sugar repeat units, representing the O-antigen.
Genes required for the biosynthesis of O-antigen in Shigella
¯exneri are located in the rfb region (Macpherson et al.,
1991; 1994). The O-antigen biosynthesis process is cur-
rently believed to involve assembly of a tetrasaccharide
repeat unit on the lipid carrier bactoprenol, transfer of the
repeat unit to the periplasmic side of the membrane by
Wzx (Liu et al., 1996; Reeves et al., 1996), then polymer-
ization of the repeat units by Wzy (O-antigen polymerase),
and ®nally ligation to the lipid A-core oligosaccharide by
WaaL (O-antigen ligase). In S. ¯exneri, the number of
O-antigen repeat units attached to the lipid A-core is non-
randomly distributed (<11±16 repeats) and this modal
length [or degree of polymerization (dp)] is regulated in
an unknown manner by the wzz/rol gene product (Morona
et al., 1995). In some S. ¯exneri strains, an additional
population of LPS molecules exists, having a modal length
of $ 90 repeats; this is determined by the Cld protein
encoded on a small plasmid (pHS-2) (Stevenson et al.,
1995). Other bacteria that produce O-antigen by a Wzy-
dependent mechanism, such as Escherichia coli (Liu
and Reeves., 1994) and Salmonella typhimurium (Batche-
lor et al., 1992), also have a homologous wzz gene which
imparts a characteristic modal length to the respective
O-antigen chains.
The Wzz proteins are characterized by two conserved
transmembrane (TM) domains located in the amino-termi-
nal (TM1) and carboxy-terminal (TM2) regions, and have
Molecular Microbiology (1999) 34(1), 181±194
Q 1999 Blackwell Science Ltd
Received 19 May, 1999; revised 19 July, 1999; accepted 21 July,1999. *For correspondence. E-mail [email protected]; Tel. (�61) 8 8303 4151; Fax (�61) 8 8303 4362.
a large hydrophilic central domain located in the periplasm
(Morona et al., 1995). The Wzz homologues in members
of the Enterobacteriaceae and other bacteria share a con-
sensus proline-rich motif `PX2PX4SPKX1X10GGMXGAG8
located just before and within (underlined) TM2 (Becker
et al., 1995; Becker and Puhler, 1998). Several residues
just before and within TM1 of Wzz proteins are also highly
conserved. Wzz homologues (paralogues), including those
from members of the Enterobacteriaceae, the Wzc and
ExoP proteins, have been grouped into the MPA1 and
MPA2 (cytoplasmic membrane periplasmic auxillary pro-
teins) families of proteins involved in assembly of bacter-
ial surface polysaccharides (Paulsen et al., 1997).
The Wzz proteins show amino acid sequence similarity
with proteins associated with a diverse range of bacterial
polysaccharide biosynthesis systems. Streptococcus pneu-
moniae 19F, which produces a capsular polysaccharide
(CPS), encodes a gene cpsC, the product of which has
amino acid similarity at its carboxy-terminal end to Wzz
proteins (Guidolin et al., 1994). Acidic exopolysaccharide
succinoglycan (EPS I) produced by Sinorhizobium meliloti
requires ExoP, which also shows amino acid similarity to
Wzz proteins and also has the proline-rich motif described
above (Becker et al., 1995). These CPS/EPS systems
have an additional protein/polypeptide domain, with an
ATP-binding motif; in the case of S. pneumoniae, this is
a separate protein encoded by cpsD, whereas S. meliloti
exoP encodes a single protein containing both the Wzz
homology region and a domain with an ATP-binding motif
(Paulsen et al., 1997).
Two models have been proposed to explain the function
of Wzz proteins. Bastin et al. (1993) suggested that Wzz
interacts with Wzy and acts as a molecular timer allowing
polymerization by Wzy to continue for a set amount of
time, thereby resulting in consistent addition of repeat
units during polymerization. The alternative hypothesis
proposed by Morona et al. (1995) suggests that Wzz
acts as a molecular chaperone, facilitating the interaction
between Wzy and WaaL (the O-antigen ligase), with modal-
ity resulting from a given ratio of Wzy and WaaL. Recently,
we published data indicating the importance of the ratio
of Wzy to Wzz in determination of O-antigen chain length
distribution (Daniels et al., 1998). Additionally, the results
of Amor and Whit®eld (1997) indicate a pivotal role for
WaaL in the process of O-antigen chain length regulation,
and provide some support for the Morona et al. (1995)
model.
It has previously been reported that Wzz proteins can
in¯uence the modal chain lengths of heterologous O-anti-
gens (Batchelor et al., 1992; Bastin et al., 1993; Burrows
et al., 1997; Klee et al., 1997). A number of studies have
attempted to de®ne which region of the Wzz protein func-
tions in determining the modal length. Klee et al., 1997
compared the modal chain lengths of O-antigen from a
S. ¯exneri strain harbouring the Wzz proteins of E. coli
K-12, S. dysenteriae 1, and S. ¯exneri Y, and found them
to be quite different. The primary amino acid sequences
of these three Wzz proteins were shown to be almost iden-
tical with differences in only nine positions, ®ve of which
were conservative substitutions. Franco et al. (1998) per-
formed site-directed mutagenesis on an E. coli O2 wzz
and assessed their effect on the E. coli O111 O-antigen
modal length distribution. They reported that amino acids
distributed throughout the length of Wzz affected chain
length and concluded that modal value determination
may be an overall property of the protein. Becker and
Puhler. (1998) reported mutagenesis studies on the
ExoP protein proline-rich motif. Two of seven mutations
in this region affected EPS I production by increasing the
production of low-molecular-weight EPS I at the expense
of high-molecular-weight EPS I, highlighting the impor-
tance of this motif.
In this study, we have investigated the structure and
function of the S. ¯exneri Wzz protein. We used hybrid
proteins and heterologous complementation to attempt to
localize functional regions. Site-directed mutagenesis of
conserved and non-conserved residues was also used
to indicate which residues were essential for function and
which were directly involved in chain length determination.
A polyclonal anti-WzzSF serum was developed and used
to follow Wzz protein production and protein interactions.
We also present the ®rst biochemical evidence obtained
by in vivo cross-linking for an interaction between Wzz pro-
teins. Wzz proteins form homo-oligomers of at least six
units and the dimeric form of Wzz is highly stable. The abil-
ity of mutant and deleted Wzz proteins to form oligomers
was also investigated and correlated with their phenotypic
impact on O-antigen modal length.
Results
Comparative analysis of S. ¯exneri and S. typhimurium
Wzz proteins
To investigate the location within the S. ¯exneri Wzz
(WzzSF ) of residues functioning in O-antigen chain length
determination, we compared the WzzSF amino acid sequ-
ence with that of Salmonella enterica Typhimurium Wzz
(WzzST ). The Wzz proteins of S. ¯exneri (WzzSF) and S.
enterica Typhimurium (WzzST ) are 72% identical, differ-
ing mainly in the periplasmic domain ¯anked by the two
transmembrane domains (TM1 and TM2). Although quite
similar, the proteins impart signi®cantly different modal
chain lengths to the O-antigen of the LPSs in their wild-
type strains WzzSF (11±16 repeats) and WzzST (19±30
repeats). Franco et al. (1998) have recently classi®ed
modal lengths imparted by Wzz proteins into three cate-
gories: short (S-type; 7±16 repeats), intermediate (I-type;
Q 1999 Blackwell Science Ltd, Molecular Microbiology, 34, 181±194
182 C. Daniels and R. Morona
10±18 repeats) and long (L-type; 16±25 repeats). Under
this classi®cation, WzzSF would be S-type and WzzST an
L-type.
The wzz genes from S. ¯exneri 2457T and S. enterica
Typhimurium LT2 were cloned into the T7 overexpression
vector pRMCD77 (see Experimental procedures ; Fig. 1A),
and the plasmids introduced into S. ¯exneri wzz strain
RMA696. LPS from trans-complemented strains were com-
pared with the LPS pattern of the S. ¯exneri parent strain
2457T on an SDS 15% polyacrylamide gel (Fig. 2). As
expected, the strain containing the wzzSF (pRMCD78)
gave an identical LPS phenotype to that of the wild-type
strain (S-type; Fig. 2) and plasmid pRMCD80 (wzzST ) con-
ferred a modal chain length of (19±31 repeats; L-type),
approximately double that determined by wzzSF (11±16
repeats). To localize the residues involved in determining
the modal chain length to either the amino or carboxy
end of the protein, we constructed hybrid Wzz proteins
by fusing the two wzz genes at the common Bgl II site
(Fig. 1A) (see Experimental procedures ). The plasmid
encoding the N-WzzSF::WzzST-C protein (pRMCD106)
when used to complement RMA696 resulted in production
of LPSs with a modal length of 17±26 repeats (L-type; Fig.
2), which is close to that observed for WzzST. The strain
containing the plasmid encoding the N-WzzST::WzzSF-C
protein (pRMCD104) produced LPSs with a modal length
of 14±19 repeats (I-type; Fig. 2), which is similar to but
longer than that produced by the action of WzzSF. This
indicated that residues involved in chain length determina-
tion may be located in the carboxy-terminal region of the
Wzz protein. Klee et al. (1997) compared the amino acid
sequences of E. coli K-12, S. dysenteriae type 1 and S.
Q 1999 Blackwell Science Ltd, Molecular Microbiology, 34, 181±194
Fig. 1. Schematic representation of the Wzzconstructs.A. Structure of the WzzSF, WzzST,N-WzzST::WzzSF-C and N-WzzSF::WzzST-Cproteins. Wild-type and hybrid genes werecloned into vector pRMCD77 (seeExperimental procedures ) so they could beexpressed from the T7 RNA polymerasepromoter. Hybrid proteins were constructedusing the common Bgl II site indicated in the®gure. TM1 and TM2 representtransmembrane segment one andtransmembrane segment two respectively.B. WzzSF truncation/deletion proteins usedin formaldehyde cross-linking experiments.Deletion points are indicated by the ¯ankingresidues (Gln-161-D-Asp-194). Constructnames are indicated on the left of the ®gure.The predicted molecular weights of theproteins are indicated on the right side ofthe ®gure (in kilodaltons).C. Location of the mutated regions withinthe WzzSF protein. Aligned proteins arefrom Shigella ¯exneri (WzzSF ) (X71790),Salmonella enterica Typhimurium LT2(WzzST ) (Z17278), E. coli E4991/76(WzzE4991/76) (AF011910), Shigelladysenteriae (WzzSD) (Y07560), E. coli K12(WzzK12) (Y07559), and Sinorhizobiummeliloti (ExoPSM) (P33698). Thetransmembrane regions are overlined andresidues of the proline-rich motif are in bold.The mutated residues are indicated (Residue#) and the amino acid change is shown. Theamino acid positions of each protein areindicated on the right of the ®gure (inbrackets). Speci®c amino acid substitutionsin WzzSF were introduced by site-directedmutagenesis (see Experimental procedures ).
Wzz is an oligomer 183
¯exneri Y Wzz proteins, and reported that, although they
differed in nine positions, WzzSF differed signi®cantly at
only two sites (amino acid 267 and amino acid 270). At
position 267, the WzzSF has a basic residue (Lys) whereas
WzzST has a polar residue (Asn). We changed this residue
in WzzSF (K267 ! N) using site-directed PCR mutagen-
esis, creating plasmid pRMCD108 (see Experimental pro-
cedures ). RMA696 containing this plasmid had LPSs with
an increased modal chain length (13±20 repeats; I-type;
Fig. 2). This result showed that residue 267 is involved in
O-antigen chain length determination, but clearly other
residues are also involved in producing the modal length
(L-type) conferred by WzzST.
Mutational analysis of conserved Gly residues of TM2
The carboxy-terminal consensus region includes three
proline residues just before TM2 and four glycine residues
located within TM2 (Fig. 1C). We investigated the role in
O-antigen modal chain length determination of this highly
conserved region by using site-directed mutagenesis to
mutate the proline and glycine residues to alanine (Fig.
1C) (see Experimental procedures ). Initially, we changed
the Gly305, Gly306, Gly309 and Gly311 to alanine resi-
dues. Mutational alteration of single residues G305 ! A
or G311 ! A had no effect on the modal chain length of
LPSs when compared with the wild type (Fig. 3). The
double mutation G305 ! A/G309 ! A and the triple muta-
tion G305 ! A/G306 ! A/G309 ! A also had no effect on
the modal chain length, however the dual change of
G305 ! A/G311 ! A resulted in a marked reduction in the
modal length [3±8 repeats; very short (VS) type] (Fig. 3).
These data indicate that relatively conservative single
residue changes in the glycine-rich domain have no effect
on Wzz O-antigen chain length regulation. However, some
multiple changes (G305 ! A/G311 ! A) result in a Wzz
which confers a signi®cantly reduced O-antigen modal
chain length.
Mutational analysis of conserved Pro residues
proximal to TM2
The function of the Pro residues in the motif was investi-
gated. While the mutational alteration of P283 ! A had no
effect on O-antigen chain length pro®le (Fig. 3), the muta-
tion of P286 ! A resulted in a Wzz protein with diminished
ability to confer a modal chain length as a reduced level of
O-antigen chains with a wild-type modal length could be
seen (Fig. 3). Mutation of P292 ! A resulted in complete
loss of function of the protein as RMA696 harbouring
this plasmid (pRMCD116) had an LPS phenotype identical
to the parent wzz mutant strain (RMA696) (Fig. 3). Hence,
at least two of the three proline residues in the highly con-
served carboxy-terminal end of WzzSF are clearly essen-
tial for wild-type function of the protein in regulating modal
length.
Mutational analysis of conserved residues proximal
to and within TM1
Wzz proteins, particularly the closely related proteins from
the Enterobacteriacea, have many residues in their amino-
terminal transmembrane domains (TM1) that are highly
Q 1999 Blackwell Science Ltd, Molecular Microbiology, 34, 181±194
Fig. 2. Effect of hybrid Wzz proteins on LPS structure. This showsa silver-stained SDS 15% polyacrylamide gel withlipopolysaccharide prepared from the indicated strains. The ®rstlane contains the 2457T wild-type strain and the second lane theS. ¯exneri wzz ::KmR strain RMA696. All other tracks containRMA696 harbouring the indicated plasmid. Rough LPS (R-LPS)uncapped by O-antigen is indicated on the right side of the ®gure.The number of O-antigen repeats are shown on the left side of thegel. Samples represent 1±2 ´ 108 cells.
184 C. Daniels and R. Morona
conserved (Fig. 1C). We decided to test the importance of
the highly conserved KMTIII motif located just adjacent to,
and within, TM1. The mutational change I35 ! C had no
effect on the modal length conferred by WzzSF, however
WzzSF with a double change of I35 ! C and M32 ! T
was functional but conferred a reduced modal length on
the LPS O-antigen chain (3±8 repeats; VS-type; Fig. 3).
WzzSF with a single change of M32 ! T resulted in LPS
with modal length of 10±15 repeats; this was a slight
reduction in modal length compared with the wild type.
The change of K31 ! A resulted in loss of activity as
seen by the inability to complement the wzz defect in
RMA696 (Fig. 3). Residues in the TM1 region are clearly
essential for function and, like TM2, mutations can result
in either a dramatically reduced O-antigen modal chain
length (VS-type) or the entire loss of Wzz function.
The wzzSF plasmids described above were used in T7
polymerase overexpression experiments to ensure that
failure to complement the wzz mutant strain was not due
to an inability of the plasmids to express the various mutant
WzzSF proteins. All vectors and wzzSF plasmids were intro-
duced into E. coli DH5 containing pGP1-2 (E2096), which
encodes T7 RNA polymerase under lambda cI control
(Tabor and Richardson, 1985). T7 expression followed by
electrophoresis and Western immunoblotting using anti-
WzzSF antibodies (see Experimental procedures ) indicated
that WzzSF or mutated WzzSF could be produced from all
of the constructs (Fig. 6). The wzzST, wzzST::wzzSF and
wzzSF::wzzST plasmids were also checked for their ability
to produce Wzz-related proteins (Fig. 4A). The anti-WzzSF
serum was also able to detect WzzST and the hybrid
proteins. Interestingly, low-molecular-weight crossreac-
tive bands were visible in tracks containing WzzSF, N-
WzzST::WzzSF-C and N-WzzSF::WzzST-C but not WzzST.
The crossreactive species may be breakdown products,
and the differential detection suggests that either the
anti-WzzSF serum could not detect WzzST breakdown pro-
ducts or that WzzST is more stable than WzzSF.
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Fig. 3. Effect of Wzz point mutations onfunction. Silver-stained SDS 15%polyacrylamide gels showinglipopolysaccharide (LPS) prepared from theindicated strains. Lanes containing RMA696and its derivatives are indicated at the bottomof the gels by arrows. The plasmidsharboured within the strains are indicated atthe top of the ®gure. Rough LPS (R-LPS)uncapped by O-antigen is indicated on theright side of the ®gure. The number of O-antigen repeats are listed on the left side ofthe gel. Samples represent 1±2 ´ 108 cells.
Wzz is an oligomer 185
Detection of WzzSF oligomer formation by in vivo
cross-linking
During the assessment of Wzz production by Western
immunoblotting, we frequently detected a band with an
apparent molecular mass of 72 kDa equivalent in size to a
WzzSF dimer (Fig. 4B). The (72 kDa species was observed
in samples that had been heated to 1008C in the presence
of SDS, and no species of > 72 kDa was observed in these
samples (Fig. 4B). This suggested that WzzSF was able to
oligomerize in vivo and we undertook chemical cross-link-
ing experiments to investigate this possibility. Cross-link-
ing was performed using formaldehyde on E. coli E2096
containing pRMCD78 with the wild-type wzzSF gene (see
Experimental procedures ). Whole cell samples were
separated on SDS 10% polyacrylamide gels and analysed
by Western immunoblotting using anti-WzzSF antibodies.
As can be seen in Fig. 5, treatment with 0.5% formaldehyde
generated species migrating as <72 kDa <160 kDa and
<210 kDa in addition to the 36 kDa WzzSF protein band.
These species correspond closely to two, four or six times
the apparent molecular weight of WzzSF protein. Species
of higher molecular weight migrating more slowly than
the <210 kDa form were also detected, however these
appear to have barely entered the separating gel and
are too large to have their size extrapolated from the pro-
tein standards used. The molecular species migrating at
<72 kDa appeared to be a doublet. The doublet could
be due to the incorporation of the smaller Wzz-related pro-
tein seen in the untreated sample (Fig. 5). The reappear-
ance of the smaller product after heating to destroy the
formaldehyde cross-linking supports this suggestion.
The very large species, along with the <160 kDa and
<210 kDa species, could not be detected when the
samples were heated at 1008C for 20 min in sample buffer
containing SDS before electrophoresis (Fig. 5). The dimeric
form of <72 kDa, however, was still detected after heating
the cross-linked samples at 1008C (Fig. 5). These data
indicate that WzzSF can be cross-linked to form an oligo-
meric complex of a size corresponding to at least a hex-
amer, and the dimeric form of cross-linked WzzSF is very
stable as it is not readily dissociated. The cross-linking
data correlate well with our initial observation that the
WzzSF protein was able to form a dimer in the absence
of any cross-linking reagent, even after being heated to
1008C in the presence of SDS.
In vivo cross-linking of WzzSF with altered residues
It was possible that the altered function/lack of function
noticed for some of these proteins could be related to an
inability to form oligomers. We investigated the possible
correlation between WzzSF protein function and its oligo-
meric state by testing all of the mutant Wzz proteins for
their ability to be cross-linked. E. coli DH5 strains (E2096)
harbouring the constructs were subjected to in vivo formal-
dehyde cross-linking and whole cell samples were solubil-
ized, electrophoresed and analysed by immunoblotting
with anti-WzzSF antibodies. All of the mutant WzzSF pro-
teins generated from these constructs were able to form
oligomeric complexes similar to those of the wild-type
WzzSF (Fig. 6). The hybrid constructs producing N-
WzzSF::WzzST-C, and N-WzzST::WzzSF-C were also
able to form oligomeric complexes equivalent to that of
Q 1999 Blackwell Science Ltd, Molecular Microbiology, 34, 181±194
Fig. 4. Western immunoblot of E. coli E2096strains producing Wzz proteins.A. After electrophoresis on an SDS 15%polyacrylamide gel and transfer tonitrocellulose, proteins were detected usingaf®nity-puri®ed anti-WzzSF antibodies (seeExperimental procedures ). Lanes containwhole cell samples of bacteria (equivalentto 1 ´ 108 cells) harbouring the plasmidsindicated at the top of the ®gure. Migrationpositions of the molecular mass standards(Pharmacia) are indicated on the right side(in kilodaltons): soybean trypsin inhibitor(20.1), carbonic anhydrase (30), ovalbumin(43), bovine serum albumin (67) andphosphorylase b (94).B. Immunoblot showing WzzSF expressedin E. coli E2096 is able to dimerize in thepresence of SDS. Whole cell samples(equivalent to 1 ´ 108 cells) were heated at1008C for 5 min before electrophoresis (SDS10% polyacrylamide gel) and immunoblotting.The WzzSF (36 kDa) protein is indicated onthe right of the ®gure and the dimeric form(<72 kDa) is indicated with an asterix.Migration positions of molecular massstandards as in A are indicated in kilodaltonson the right side.
186 C. Daniels and R. Morona
the wild type WzzSF (Table 1). However, strains contain-
ing plasmid constructs pRMCD116 (WzzSF(P292A)) and
pRMCD119 (WzzSF(K31A)), both of which are unable to
impart an O-antigen modal length on RMA696 LPS, formed
less of the dimeric form and signi®cantly less of the higher
molecular weight species (Fig. 6). Strains containing plas-
mids pRMCD112, pRMCD113 and pRMCD114, encoding
wzzSF genes with G305 ! A/G309 ! A, G305 ! A/
G311 ! A and G305 ! A/G306 ! A/G309 ! A respec-
tively, show a difference in the apparent molecular mass
of the Wzz protein oligomers compared with the wild-type
Wzz (Fig. 6). This difference is particularly noticeable in
the region where the Wzz dimer migrates; the dimeric
forms of the mutated proteins have a slightly larger appar-
ent molecular mass (<80 kDa; Fig. 6; Table 1). Taken
together, these results suggest that loss of function may
be linked to a reduced/altered ability to form oligomers,
and the glycine residues in the TM2 segment in¯uence
WzzSF±WzzSF dimer conformation as the mutations affect
WzzSF±WzzSF dimer mobility in SDS polyacrylamide gels.
Formaldehyde and dithio-bis(succinimidylpropionate)
(DSP) cross-linking of Wzz in S. ¯exneri
To con®rm the cross-linking results obtained using formal-
dehyde on overexpressed WzzSF proteins in E. coli, we
performed cross-linking on wild-type S. ¯exneri. Formal-
dehyde is a small reactive molecule capable of polymeriz-
ing to a variety of lengths (Prossnitz et al., 1988), whereas
dithio-bis(succinimidylpropionate) (DSP) is a ®xed-arm-
length cross-linking reagent 12 AÊ in length. Cross-linking
was performed on wild-type S. ¯exneri Y (PE638) and E.
coli E2096 harbouring pRMCD78, using either 0.5% for-
maldehyde or 0.2 mM DSP after which cells were fractio-
nated (see Experimental procedures ). Whole cell and
cytoplasmic membrane fractions of both treated and
untreated bacteria were electrophoresed on an SDS 10%
polyacrylamide gel and Wzz was detected by immunoblot-
ting with anti-WzzSF antibodies. The results obtained for
the formaldehyde cross-linking were identical for both
the E. coli and S. ¯exneri samples (Table 1). Electrophore-
tic species corresponding to <72 kDa, <160 kDa and
<210 kDa were observed in all formaldehyde-treated
samples as expected (Table 1). The <210 kDa species
were also present in both E. coli and S. ¯exneri samples.
The DSP cross-linked samples showed a similar Wzz pro-
®le to that observed when using formaldehyde, however in
this case the Wzz dimer had two different apparent mole-
cular masses (<72 kDa, <77 kDa; Table 1). An additional
difference was noticed between the E. coli and S. ¯exneri
DSP cross-linked samples. DSP cross-linked samples
indicated only a dimeric form of the protein in S. ¯exneri,
however the high-molecular-weight species were detectable
in E. coli E2096 (pRMCD78) using this cross-linker. This
difference is likely to be a consequence of altered cross-
linking ef®ciency due to strain differences. The cross-link-
ing with DSP largely con®rmed the results obtained with
formaldehyde, and the data obtained using S. ¯exneri
correlated with what is observed in E. coli harbouring
plasmid-encoded WzzSF. We were unable to detect
WzzSF±WzzSF dimer in any S. ¯exneri samples in the
absence of cross-linking reagent.
The N-terminal domain of WzzSF is suf®cient for
oligomer formation
Plasmid pRMCD107 was initially constructed to identify
regions within WzzSF that are required for oligomer forma-
tion (Fig. 1B). Strains harbouring this construct were able
to produce a truncated WzzSF protein of <23 kDa which,
like the wild-type WzzSF, was able to dimerize in the
Q 1999 Blackwell Science Ltd, Molecular Microbiology, 34, 181±194
Fig. 5. Formaldehyde cross-linking of WzzSF in E. coli. Aftertreatment with 0.5% formaldehyde, whole cell samples wereelectrophoresed on an SDS 10% polyacrylamide gel and analysedby immunoblotting using af®nity-puri®ed anti-WzzSF antibodies (seeExperimental procedures ). Formaldehyde (0.5% form.) treated (�)and untreated (ÿ) samples are indicated at the bottom of the ®gure.Samples were heated at 608C for 10 min or at 1008C for 5 minbefore loading. The migration positions of the prestained molecularmass standards (New England Biolabs) are indicated on the rightside (in kilodaltons): triosephosphate isomerase (32.5), aldolase(47.5), glutamic dehydrogenase (62), MBP-paramyosin (83), MBP-b-galactosidase (175). Samples represent 1±2 ´ 108 cells.
Wzz is an oligomer 187
absence of cross-linking reagent (data not shown). Formal-
dehyde cross-linking of E. coli harbouring pRMCD107 con-
®rmed a dimeric species with an apparent molecular mass
of <48 kDa, and additional species were also detected
migrating as <65 kDa, <75 kDa and <210 kDa (Table 1).
These data show that the carboxy-terminal end of WzzSF
is not needed for dimerization, and that a protein±protein
interactive domain may be located in the amino-terminal
194 residues of WzzSF. To further investigate this phenom-
enon, we constructed three plasmids with sequential inter-
nal deletions within the amino-terminal residues of WzzSF.
Plasmids pRMCD138, pRMCD139 and pRMCD140 have
internal deletions of 33, 80 and 135 amino acids (Fig.
1B), resulting in Wzz proteins with apparent molecular
weights of 33, 28 and 22 kDa respectively (Table 1). Intro-
duction of pRMCD107, pRMCD138, pRMCD139 and
pRMCD140 into S. ¯exneri RMA696 did not allow restora-
tion of O-antigen modal chain length (Table 1). Initially, for-
maldehyde cross-linking of E. coli strains expressing these
proteins indicated they were able to oligomerize, however,
apart from the monomer, only very high molecular weight
species (<210 kDa) were detected. This may be a conse-
quence of reduced levels of the truncated proteins and/or
their reduced reactivity with anti-WzzSF antibodies. Sub-
sequent cross-linking experiments with strains harbouring
pRMCD138, pRMCD139 and pRMCD140, followed by
immunoblotting of over-loaded samples and increased
exposure times indicated that the internally deleted WzzSF
proteins were able to dimerize (Table 1). The apparent
molecular mass of the dimeric Wzz species generated from
cross-linking strains containing pRMCD138 (<67 kDa),
pRMCD139 (<55 kDa) and pRMCD140 (<45 kDa) were
approximately twice that of their respective monomers.
This con®rms that the <72 kDa species noted when wild-
type WzzSF is cross-linked is indeed a WzzSF dimer.
These results indicate that the TM1 region functions in
dimer formation, but do not exclude any essential roles
for the TM2 region in these interactions.
Q 1999 Blackwell Science Ltd, Molecular Microbiology, 34, 181±194
Fig. 6. Formaldehyde cross-linking of mutant WzzSF proteins in E. coli. Cells were treated as described in Experimental procedures andwhole cell samples were electrophoresed on SDS 10% polyacrylamide gels followed by immunoblotting using af®nity-puri®ed anti-WzzSF
antibodies. Formaldehyde (0.5% form.) treated (�) and untreated (ÿ) samples are indicated at the bottom of the ®gure. The migrationpositions of the prestained molecular mass standards New England Biolabs (NEB) are indicated on the right side (in kilodaltons),as describedin Fig. 5. Samples represent 1±2 ´ 108 cells.
188 C. Daniels and R. Morona
Discussion
Recent publications have emphasized the importance of
LPS O-antigen modal chain length in bacterial pathogen-
esis and immune responses to O-antigens (Attridge et
al., 1990; Hong and Payne, 1997; Klee et al., 1997; Van
Den Bosch et al., 1997). Hong and Payne (1997) showed
that the plasmid-encoded (pHS-2) Wzz/Cld protein of S.
¯exneri 2a (2457T), which imparts a modal length of
<90 repeats, is required for serum resistance. Hong and
Payne (1997) and Van Den Bosch et al. (1997) have
also shown that the chromosomally encoded Wzz protein
of S. ¯exneri 2a is required to achieve wild-type levels of
intracellular and intercellular spread in HeLa cells. Sereny
tests have also indicated that S. ¯exneri 2a wzz chromoso-
mal mutants are avirulent (Van Den Bosch et al., 1997).
The last results appear to be due to abnormal cellular loca-
lization of the normally polar located IcsA protein in the
wzz mutant. Klee et al. (1997) found that introduction of
heterologous wzz genes into a potential vaccine carrier
strain resulted in reduced masking of a heterologous
O-antigen and a concomitant increase in the immune
response of mice to the desired O-antigen. Despite their
biological importance in determination of O-antigen chain
length distribution, very little is known about the structure
and function of Wzz proteins. In this study, we attempted
to localize functional domains within the S. ¯exneri Wzz
protein. Previous attempts to localize a functional region
within E. coli Wzz proteins indicated that a range of amino
acid positions throughout the Wzz protein affected the
modal chain length (Franco et al., 1998). We found that
the Wzz protein from S. enterica Typhimurium gave an
increased O-antigen modal length in S. ¯exneri wzz strain
RMA696 (Table 1). The hybrid constructs which encode
N-WzzST::WzzSF-C and N-WzzSF::WzzST-C showed that
residues in the carboxy-terminal end of Wzz have a major
impact on the O-antigen modal chain length. Although
neither of the hybrid proteins were able to impart a modal
chain length that is identical to the two wild-type genes
used (wzzSF and wzzST ), N-WzzSF::WzzST-C (17±26
repeats) gave a modal value very close to that of the wild-
type WzzST (19±31) (Table 1). Change of the basic residue
(Lys) at position 267 in the carboxy-terminal region of
WzzSF to a polar residue (Asn) caused an increase in
the modal length from 11±16 repeat units (S-type) to
14±19 repeat units (I-type). This modest increase in length
indicates that this residue is involved in chain length deter-
mination. However, it is now obvious from our data, and that
presented by Franco et al. (1998), that residues throughout
the Wzz protein can have subtle effects on O-antigen chain
length.
To date, no experimental data for the functional sig-
ni®cance of the highly conserved glycine residues have
been reported. However, the complete conservation of
Q 1999 Blackwell Science Ltd, Molecular Microbiology, 34, 181±194
Table 1. Summary of Wzz constructs and phenotypes.
Apparent Wzz Apparent dimerPlasmid/strain Mutation/characteristics size (kDa) size (kDa)a Apparent oligomer size (kDa)a Modal lengthb
PE638 S. flexneri Y 36 72 [72, 77] 160, 210, > 210 11±16pRMCD78c Wild-type WzzSF 36 72 [72, 77] 160, 210, > 210 [160, 210, > 210] 11±16pRMCD80c Wild-type WzzST 35 ND ND 19±31pRMCD104c N-WzzST::WzzSF-C 35 70 160, 210, > 210 14±19pRMCD106c N-WzzSF::WzzST-C 36 72 160, 210, > 210 17±26pRMCD107c WzzSFD194±325 23 48 65, 75, > 210 Non-modalpRMCD108c WzzSF(K267 ! N) 36 72 160, 210, > 210 13±20pRMCD109c WzzSF(P283 ! A) 36 72 160, 210, > 210 11±16pRMCD111c WzzSF(G311 ! A) 36 74 160, 210, > 210 11±16pRMCD112c WzzSF(G305 ! A/G309) 36 80 160, 210, > 210 11±16pRMCD113c WzzSF(G305 ! A/G311 ! A) 36 80 160, 210, > 210 3±8pRMCD114c WzzSF(G305 ! A/G306 ! A/G309 ! A) 36 80d 160, 210, > 210d 11±16pRMCD116c WzzSF(P292 ! A) 36 72d 160, 210, > 210d Non-modale
pRMCD117c WzzSF(P286 ! A) 36 72 160, 210, > 210 11±16pRMCD119c WzzSF(K31 ! A) 36 72d 160, 210, > 210d Non-modalpRMCD121c WzzSF(I35 ! C) 36 72 160, 210, > 210 11±16pRMCD122c WzzSF(I35 ! C/M32 ! T) 36 72 160, 210, > 210 3±8pRMCD125c WzzSF(G305 ! A) 36 72 160, 210, > 210 11±16pRMCD127c WzzSF(M32 ! T) 36 72 160, 210, > 210 10±15pRMCD138c WzzSFDGln-161-Asp-194 33 67 > 210 Non-modalpRMCD139c WzzSFDGlu-114-Asp-194 28 55 > 210 Non-modalpRMCD140c WzzSFDThr-59-Asp-194 22 45 > 210 Non-modal
a. After cross-linking with 0.5% formaldehyde, or 0.2 mM DSP where indicated by square brackets.b. Average length of O-antigen repeat units.c. Base vector is pRMCD77.d. Reduced amounts of dimer and oligomer were observed.e. Reduced amount of O-antigen of modal length was observed.ND, not done.
Wzz is an oligomer 189
the glycine residues in the hydrophobic transmembrane
segment (Fig. 1C) led Bastin et al. (1993) to argue for
their possible involvement in protein±protein interactions.
We targeted these residues in order to determine whether
they were essential for modal chain length function in Wzz
proteins. Single-residue Wzz variants with G305 ! A or
G311 ! A did not alter the modal length from that of the
wild-type protein (Table 1). These relatively conservative
changes may not be suf®cient to signi®cantly alter this
region of the protein, and therefore no obvious effect on
function was observed. Double and triple mutant Wzz var-
iants with either G305 ! A/G309 ! A or G305 ! A/
G306 ! A/G309 ! A also did not alter the modal length.
The dual Wzz variant having G305 ! A/G311 ! A
resulted in a dramatic change in the O-antigen chain
length distribution: a modal length of 3±8 repeats was
observed (VS-type; Fig. 3; Table 1). Glycine 305 appears
to be completely conserved; it is present in Wzz-related
proteins such as the E. coli K-12 WzzE/ORF2 protein
(involved in enterobacterial common antigen biosynthesis;
Meier-Dieter et al., 1992), the Erwinia amylovora AmsA
protein (EPS biosynthesis; Bugert and Geider, 1995),
the S. pneumoniae CpsC protein (CPS biosynthesis; Gui-
dolin et al., 1994) and the S. meliloti ExoP protein (EPS
biosynthesis; Becker et al., 1995). Glycine 311 is not as
highly conserved. These residues are located at either
end of the motif in TM2 of WzzSF, and it could be that alter-
ing both of these residues in the same WzzSF protein
results in a major change in protein conformation and/or
protein±protein interaction.
Becker and Puhler (1998) have previously studied the
proline-rich motif of the S. meliloti ExoP protein. They
found that two mutations, R443 ! l and P457 ! S, resulted
in increased expression of low-molecular-weight EPS I at
the expense of high-molecular-weight EPS I. Further muta-
tions of P451 ! S and P454 ! S caused no change in EPS
I production. Amino acid sequence alignment of ExoP with
Wzz shows that the proline residues 451 and 457 from
ExoP align with the WzzSF proline residues 286 and 292
respectively (Fig. 1C). We found that altering P283 ! A
had no effect on the function of the WzzSF protein, the
P286 ! A mutation resulted in reduced Wzz activity, and
the P292 ! A mutation completely abrogated function
(Fig. 3; Table 1). These results indicate that whereas
Pro-286 is needed for ef®cient function, Pro-292, located
in the conserved SPK motif (Becker and Puhler, 1998;
Fig. 1C), is absolutely essential for Wzz function. These
observations agree with the data obtained using ExoP,
in which mutation of P457 ! S in the conserved SPK
motif had the most dramatic affect on EPS I production.
However, recent work by Gonza lez et al. (1998) has indi-
cated that the ExoP protein is involved in the production of
both high- and low-molecular-weight succinoglycan by two
alternative mechanisms, and this observation along with
the presence of an ATP-binding domain within ExoP sug-
gests that ExoP potentially has a more complex role in bio-
synthesis, making it dif®cult to correlate its function to that
of Wzz.
Alteration of residues preceding and within TM1 of WzzSF
either resulted in complete loss of function or altered func-
tion respectively. The complete loss of function resulting
from the mutation of K31 ! A was not expected. This resi-
due is highly conserved between Wzz proteins, however
it is located topologically within the cytoplasm, just before
the ®rst transmembrane region. Alteration of K31 ! A
eliminates a positively charged amino acid residue on
the cytoplasmic side of the membrane which could affect
function by destabilizing the TM1 region of the protein,
allowing either the conformation of the TM segment to be
altered or interfering with protein interactions. The single
change of I35 ! C had no effect, however changing
M32 ! T resulted in a slight reduction in O-antigen
modal length (10±15 repeats). The dual change of
I35 ! C/M32 ! T resulted in a Wzz which conferred a
dramatic reduction in modal length (3±8 repeats; Table 1).
It is interesting that dual mutations in either of the
two transmembrane regions (I35 ! C/M32 ! T or
G305 ! A G31 ! A) can result in a protein that imparts
very similar modal length. These data imply strongly that
both regions are involved either directly in modal chain
length determination and/or are required for protein±pro-
tein interaction. This could be further investigated by iden-
tifying a mutation in TM1 which suppresses the mutation in
TM2 or vice versa.
In vivo formaldehyde cross-linking has been used
extensively to study protein interactions in bacteria, includ-
ing both outer membrane proteins (Mourrain et al., 1997)
and cytoplasmic membrane proteins (Prossnitz et al.,
1988; Higgs et al., 1998). It has previously been proposed
that proteins involved in O-antigen biosynthesis may inter-
act to form a complex (Bastin et al., 1993; Morona et al.,
1995). The complex could include proteins such as Wzx,
Wzy, Wzz and WaaL, however there is currently no pub-
lished biochemical evidence to prove the existence of a
complex. In this study, we concentrated on protein inter-
actions involving the WzzSF protein. Our cross-linking
experiments using overexpressed WzzSF in E. coli DH5
indicate that WzzSF forms a homo-oligomer of at least
six units. In vivo cross-linking experiments using formalde-
hyde on wild-type S. ¯exneri also allowed detection of
WzzSF oligomers. DSP cross-linked samples indicated
only a dimeric form of the protein in S. ¯exneri, however
the high-molecular-weight species were detected in E.
coli DH5 using this cross-linker. The wild-type S. ¯exneri
strain produces S-LPS whereas the E. coli K-12 strain
used for the cross-linking experiment produces R-LPS,
furthermore DSP is hydrophobic and a larger molecule
than formaldehyde and may be hindered in its access to
Q 1999 Blackwell Science Ltd, Molecular Microbiology, 34, 181±194
190 C. Daniels and R. Morona
the WzzSF protein by the presence of the S-LPS or other
outer membrane components in S. ¯exneri. Hence, the
inability to detect complexes larger than the dimer in S.
¯exneri when cross-linked with DSP may be due to the
decreased amounts of DSP able to associate with the
WzzSF protein. However, both the formaldehyde and DSP
cross-linking data strongly support the existence of WzzSF
dimers. In the DSP cross-linked samples, the WzzSF dimer
migrated at two different apparent molecular weights: one
corresponded to the mass observed when formaldehyde
was used (<72 kDa) as seen with the WzzSF wild type
and most Wzz mutant variant proteins (Fig. 6), and the
other migrated signi®cantly more slowly (<77 kDa). The
latter is a similar size to that seen for Wzz proteins with
multiple glycine to alanine mutational alterations (Fig. 6;
Table 1). An explanation for this variation in apparent
molecular mass is that WzzSF exists in two conformations
which can only be detected in the dimeric form of the
protein.
Plasmid pRMCD107 produces a truncated protein of
<23 kDa consisting of the amino-terminal 194 residues
of WzzSF. After formaldehyde cross-linking, several new
species were generated: <48 kDa <65 kDa <75 kDa,
> 210 kDa (Table 1). The <48 kDa species observed
when this protein was cross-linked was approximately
twice that observed of the monomer and indicates that
the amino-terminal 194 residues of WzzSF are suf®cient
to allow homo-oligomer formation. The <65 kDa and
<75 kDa species, which were relatively minor in compari-
son to the other species seen, do not correlate directly with
what was observed for the wild-type protein. We would
have expected to see species of <92 kDa and <138 kDa,
equivalent to a tetramer and hexamer respectively. The
<65 kDa species could represent a trimer, however
trimeric species were not observed for wild-type WzzSF.
The lack of the carboxy-terminal 131 residues including
TM2 from the protein produced by pRMCD107 may be
contributing to the unusual cross-linking pro®le. Deletion
proteins produced by pRMCD138, pRMCD139 and
pRMCD140 had apparent molecular weights of <33 kDa,
<28 kDa and <22 kDa respectively (Table 1). Only very
large species (> 210 kDa) could be visualized when these
proteins were cross-linked and our usual amount of sam-
ple was loaded (<1±2 ´ 108 bacterial cells). Overloading
of gels with cross-linked E. coli lysates producing the pro-
teins allowed visualization of species equivalent in size to
that expected for their respective dimers (data not shown).
This indicates that residues 59±194 are not essential for
oligomerization. Taken together, these data imply that
transmembrane segment one (TM1) is involved in the
WzzSF±WzzSF interaction process, however it does not
exclude other regions of the protein from being involved.
This study has emphasized the functional signi®cance of
Wzz oligomer formation. However, the data do not prove
or disprove either of the currently suggested models for
Wzz action (Bastin et al., 1993; Morona et al., 1995).
Both models allow for possible protein±protein interactions
between enzymes involved in O-antigen assembly/trans-
location (Wzz, Wzy, WaaL), and any subsequent models
would need to take into account Wzz±Wzz interactions.
Although there appear to be no obvious interactions
between Wzz (up to the hexameric size) and any other
proteins, the ability of Wzz to associate into very large
complexes (>210 kDa) raises the possibility that Wzy
and WaaL may also be localized in these complexes. For-
mation of a large O-antigen/LPS biosynthetic complex
including Wzz would allow centralization of the LPS
assembly/transport machinery and a concomitant increase
in the ef®ciency of lipopolysaccharide expression. Investi-
gation of the presence of proteins such as Wzy and WaaL
in the large complexes will require the development and
use of speci®c antisera.
Experimental procedures
Bacterial culture conditions
All strains were grown at 378C in Luria±Bertani broth (LB;Morona et al., 1994) unless otherwise stated, except for strainscontaining pGP1-2 (308C) (Tabor and Richardson, 1985). Anti-biotics were used at the following concentrations where appro-priate: ampicillin (Ap) 150 mg mlÿ1 and kanamycin (Km)50 mg mlÿ1.
Construction of recombinant clones
All cloning/manipulations were performed using E. coli DH5a
as the recipient strain (Bethesda Research Laboratories). TheT7 polymerase expression vector pET17b (Novagen) was thebase plasmid for all wzz constructs. pET17b was ®rst digestedwith Bgl II and end-®lled with dNTPs using Klenow followed byreligation to form pRMCD77. PCR ampli®cation was performedusing standard protocols with Amplitaq DNA polymerase (Hoff-man-La Roche). wzzSF and wzzST coding regions were PCRampli®ed from S. ¯exneri 2a (2457T) (Formal et al., 1958) andS. enterica Typhimurium LT2 (EX730), respectively, using pri-mers incorporating NdeI and BamHI restriction sites (under-lined): no. 2001, 58-CAGTTAGGCATATGATGAGAGTAG-38;no. 2002, 58-TAGGATCCGAGCAGGTGTGATGTTG-38; no.2343, 58-TAGTTAGGGTACATATGACAGTG-38; no. 2344,58-CCACCATCCGGATCCGAAGC-38. The ampli®ed DNAwas then digested with NdeI and BamHI, and cloned into like-wise-digested pRMCD77, producing pRMCD78 (wzzSF ) andpRMCD80 (wzzST ) (Fig. 1A). Ampli®ed wzzSF DNA wasalso ligated into NdeI/BamHI-digested pRE1 (Reddy et al.,1989) to form pRMCD16. The sequence of the cloned PCR-ampli®ed fragments was veri®ed by DNA sequencing usingthe T7 promoter and T7 terminator primers, as recommendedby Applied Biosystems. Chimeric wzz genes were constructedby restriction enzyme digestion of pRMCD78 and pRMCD80with XbaI and Bgl II; the two fragments generated from each
Q 1999 Blackwell Science Ltd, Molecular Microbiology, 34, 181±194
Wzz is an oligomer 191
plasmid were band isolated from 1% (w/v) low-temperature-gelling agarose gels using the QIAquick Gel Extraction Kit(Qiagen). The small XbaI/Bgl II fragments (encoding theamino-terminal 194 amino acids of the Wzz proteins) fromeach plasmid were ligated with the larger fragment encodingthe carboxy-terminal end of the alternate gene. This resulted inpRMCD104 (wzzST::wzzSF ) and pRMCD106 (wzzSF::wzzST )(Fig. 1A).
Site-directed mutagenesis and deleted WzzSF
construction
Site-directed mutagenesis of codons was performed by over-lap extension using PCR and pairs of complementary primers(DNA sequences are available on request). Primer pairs wereused in combination with ¯anking primers (T7 promoter andT7 terminator) to generate mutated fusion products thatwere then fused using PCR to form the intact wzzSF gene con-taining single, double or triple codon changes (Horton, 1993).The sequence of the cloned PCR-ampli®ed fragments, includ-ing the mutations, were veri®ed by DNA sequencing using theT7 promoter and T7 terminator primers, as recommended byApplied Biosystems. Plasmid pRMCD107 was constructed bydigesting pRMCD78 with Bgl II and Not I followed by end-®llingand re-ligation (Fig. 1B). wzzSF deletion constructs were pro-duced by using the Bgl II restriction site; primers were designedincorporating a Bgl II restriction site (underlined) that boundto three different locations in the wzzSF gene: no. 2918 58-CTAGATCTTGATTCACTTTATC-38; no. 2919 58-CTAGAT-CTTCCTGATTATCCAG-38; no. 2920 58-GTGATAAGATC-TGTTGACGTCC-38. PCR using these primers and the T7promoter primer ampli®ed a DNA fragment that was thendigested with XbaI/Bgl II and ligated into band-isolatedpRMCD78 (large vector fragment containing XbaI/Bgl II)encoding the carboxy-terminal end of wzzSF. The resultingplasmids were called pRMCD138, pRMCD139 andpRMCD140 (Fig. 1B).
LPS analysis
All S. ¯exneri strains were grown for 16 h at 378C in LB con-taining appropriate antibiotics. Small-scale preparationswere made by proteinase K treatment of whole cell lysates(Hitchcock and Brown, 1983). After electrophoresis on SDS15% polyacrylamide gels, LPS was detected by silver stainingas described previously (Morona et al., 1991).
Cell fractionation, SDS±PAGE and Western
immunoblotting
Whole cell samples were prepared in sample buffer (Lugten-berg et al., 1975), except DSP cross-linked samples whichwere resuspended in sample buffer without 2-mercapto-ethanol. The remainder were fractionated to identify proteinslocated in soluble and insoluble fractions by a previously des-cribed method (Achtman et al., 1979). Wzz proteins weredetected after samples were solubilized by heating at either608C for 10 min (cross-linked) or 1008C for 5 min, then sepa-rated by SDS±PAGE and transferred to nitrocellulose mem-branes (Morona et al., 1995). Rabbit anti-WzzSF serum was
used as the primary antibody and goat anti-rabbit peroxidaseconjugate (KPL) as the secondary antibody. The blot wasdeveloped using Boehringer Mannheim chemiluminescence(POD) reagents as described by the manufacturer.
In vivo protein cross-linking
Formaldehyde cross-linking was performed by the methoddescribed by Prossnitz et al. (1988). Overnight cultures(18 h) of E2096 (E. coli DH5� pGP1-2) harbouring WzzSF
constructs were subcultured and grown with aeration at308C until they reached an OD600 of 1.0. The temperaturewas raised to 428C for 20 min to induce the production ofWzz, and cultures were then grown for an additional 60 minat 378C. Overnight cultures (18 h) of S. ¯exneri were subcul-tured 1:10 and grown with aeration for 3 h before cross-linking.Cells (E. coli or S. ¯exneri ) were harvested by centrifugationand washed once in ice-cold 10 mM K2HPO4/KH2PO4, pH 6.8,and then resuspended in the same buffer to an OD600 of 1.0.Formaldehyde (Univar 37% w/w) was added to a ®nal con-centration of 0.5%, and the samples were incubated by stand-ing for 1 h at 238C. Cross-linked samples of E. coli (1 ml) or S.¯exneri (1.5 ml) were harvested by centrifugation, washedonce in 1.5 ml of ice-cold 10 mM K2HPO4/KH2PO4, pH 6.8,and resuspended in 80 ml of sample buffer (Lugtenberg et al.,1975). Samples were either stored at ÿ208C or immediatelyfractionated. Aliquots (30±80 ml; 1±2 ´ 108 E. coli cells or 7±8 ´ 108 S. ¯exneri cells) were subjected to SDS±PAGE andWestern immunoblotting. Dithio-bis(succinimidylpropionate)(DSP) (Pierce) cross-linking was carried out as describedby Thanabalu et al. (1998). Brie¯y, cultures (50 ml) to becross-linked were washed in buffer (150 mM NaCl, 20 mMNaPO4 pH 7.2) and concentrated 10-fold in the same bufferfollowed by cross-linking with 0.2 mM DSP for 30 min at378C. DSP was quenched with 20 mM Tris, pH 7.5. Cellswere then harvested and either stored atÿ208C or immediatelyfractionated.
Preparation of Wzz speci®c antibodies
Large-scale protein preparations were produced by over-expression of WzzSF from pRMCD16 in E. coli MZ1 (Reddyet al., 1989). Brie¯y, cultures (250 ml) grown at 308C toOD600 <0.8 were induced to produce WzzSF by temperatureshift (428C/20 min) followed by a further incubation (378C/3 h). The cells were fractionated essentially as above, andthe whole membranes were solubilized in 1.67% (w/v) Sarko-syl (NL-97 Geigy), 10 mM Tris (pH 8) to separate the inner andouter membranes. WzzSF used to raise rabbit anti-WzzSF
antisera was puri®ed from the soluble component (cytoplasmicmembranes) by two cycles of SDS±PAGE. The rabbit wasimmunized (intramuscular at four sites) on day 1 with gel homo-genates in Freunds complete adjuvant. Subsequent boostswere performed on days 16, 41 and 70 using gel homo-genates in Freunds incomplete adjuvant. The rabbit wasexsanguinated by cardiac puncture under anaesthesia 20days after the last immunization and the serum was stored atÿ208C. Anti-WzzSF antibodies were immunoaf®nity puri®edfrom the anti-WzzSF sera using the method described by Sal-amitou et al. (1994), and cytoplasmic membrane extracts from
Q 1999 Blackwell Science Ltd, Molecular Microbiology, 34, 181±194
192 C. Daniels and R. Morona
E. coli (MZ1� pRMCD16) that contained WzzSF. After elutionof the antibodies, an equal volume of sterile glycerol (100%)was added, and the antibodies were stored at ÿ208C.
Acknowledgements
This study was funded by the National Health and MedicalResearch Council of Australia. C.D. is in receipt of an Austra-lian Postgraduate Research Award.
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