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Nico Nouwen
1,2,3
, Henning Stahlberg
2
,
Anthony P.Pugsley
1,4
and Andreas Engel
2
1
Unite
Âde Ge
Âne
Âtique Mole
Âculaire, Centre National de la Recherche
Scienti®que URA 1773, 25 rue du Dr Roux, Institut Pasteur,
75724 Paris, Cedex 15, France and
2
Maurice E.Mu
Èller-Institut,
Biozentrum, University of Basel, Klingelbergstrasse 70,
CH-4056 Basel, Switzerland
3
Present address: Department of Microbiology, University of
Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands
4
Corresponding author
e-mail: max@pasteur.fr
Secretins, a superfamily of multimeric outer
membrane proteins, mediate the transport of large
macromolecules across the outer membrane of Gram-
negative bacteria. Limited proteolysis of secretin PulD
from the Klebsiella oxytoca pullulanase secretion path-
way showed that it consists of an N-terminal domain
and a protease-resistant C-terminal domain that
remains multimeric after proteolysis. The stable
C-terminal domain starts just before the region in
PulD that is highly conserved in the secretin super-
family and apparently lacks the region at the
C-terminal end to which the secretin-speci®c pilot
protein PulS binds. Electron microscopy showed that
the stable fragment produced by proteolysis is com-
posed of two stacked rings that encircle a central
channel and that it lacks the peripheral radial spokes
that are seen in the native complex. Moreover, the
electron microscopic images suggest that the
N-terminal domain folds back into the large cavity of
the channel that is formed by the C-terminal domain
of the native complex, thereby occluding the channel,
consistent with previous electrophysiological studies
showing that the channel is normally closed.
Keywords: protein secretion/PulD/pullulanase/secretins
Introduction
Gram-negative bacteria possess several different pathways
to secrete proteins into the extracellular medium. In the
type I and type III secretion systems, secreted proteins are
transported in one step across both the inner and outer
membranes. In the secreton or type II secretion pathway,
substrates are transported ®rst across the cytoplasmic
membrane by the Sec system, and then across the outer
membrane via a specialized secretion apparatus (the
secreton) composed of or assembled by 12 or more
different proteins (Pugsley, 1993; Possot et al., 2000). One
of the most interesting features of the secreton pathway is
that it contains only one integral outer membrane protein,
protein D, which is consequently the only protein that
could conceivably form the translocation channel by
which proteins are transported across the outer membrane
(Nouwen et al., 1999).
Protein D belongs to a large superfamily of homologous
proteins, the secretins (Genin and Boucher, 1994). Besides
the D proteins, this family includes components of the
type III secretion pathway, proteins involved in DNA
uptake (natural competence) and proteins needed for the
assembly and secretion of ®lamentous phages, type IV pili
and S-layers (Genin and Boucher, 1994). Secretins show
particularly high sequence similarity in their C-terminal
halves. This region (called the bdomain in Guilvout
et al., 1999) is predicted to contain several amphipathic
b-strands similar to the transmembrane segments of porins
and, consequently, is likely to be embedded in the outer
membrane (Bitter et al., 1998; Guilvout et al., 1999). The
N-terminal half of secretins is conserved only in proteins
from related secretion pathways (Genin and Boucher,
1994) and is presumed to face the periplasm (Guilvout
et al., 1999). This domain might be involved in substrate
recognition (Shevchik et al., 1997), interaction with other
components of the secretion machinery (Feng et al., 1999)
or gating of the proposed secretin channel (Guilvout et al.,
1999). In addition, some secretins contain a small
C-terminal domain to which a secretin-speci®c pilot
protein binds (Dae¯er et al., 1997; Dae¯er and Russel,
1998). This pilot protein protects secretins from proteo-
lysis and is essential for their insertion in the outer
membrane (Hardie et al., 1996a,b; Dae¯er and Russel,
1998; Shevchik and Condemine, 1998).
To investigate whether secretins do indeed contain
different structural domains, as suggested by sequence
comparisons, we analysed the structure of secretin
biochemically. As a representative of the secretin family,
we studied the domain structure of the 633 amino acid
PulD protein, the secretin of the pullulanase-speci®c
secreton of Klebsiella oxytoca (d'Enfert et al., 1989;
Nouwen et al., 1999).
Results
Limited proteolysis of PulD
On the basis of sequence comparisons, secretins are
presumed to contain different structural domains. To
investigate whether there is biochemical evidence for this
presumed domain structure, we treated outer membranes
containing secretin PulD with limited amounts of trypsin.
PulD forms very stable high molecular weight complexes
in vivo that are not dissociated by prolonged heating at
100°C in SDS. To facilitate the analysis, samples were
treated with phenol, which converts PulD into mono-
mers, before loading onto an SDS±polyacrylamide gel.
Immunoblot analysis using an antibody raised against
almost complete PulD (His
6
-PulD) (Hardie et al., 1996a)
(Figure 1D) showed that low concentrations of trypsin
Domain structure of secretin PulD revealed by
limited proteolysis and electron microscopy
The EMBO Journal Vol. 19 No. 10 pp.2229±2236, 2000
ãEuropean Molecular Biology Organization 2229
gave rise to proteolytic fragments of ~60 and ~40 kDa
(Figure 1A). These proteolytic fragments did not react
with an antibody raised against the 99 N-terminal amino
acids of mature PulD (PulD-PhoA) (d'Enfert et al., 1987)
(Figure 1B), indicating that they do not contain the
extreme N-terminal region of PulD. Instead, a proteolytic
fragment of ~28 kDa was detected with this antibody
(Figure 1B). Interestingly, the sum of the sizes of the
40 and 28 kDa fragments is close to that of full-length
PulD. At higher trypsin concentrations, the 60 and
28 kDa fragments were completely degraded whereas
the 40 kDa fragment was trimmed to a stable fragment of
~38 kDa (Figure 1A and B). The latter proteolysis product
will be referred to hereafter as the stable C-terminal
fragment.
To determine whether the different proteolytic frag-
ments were generated by cleavage at unique trypsin
cleavage sites or at sites that are highly accessible to
proteases, the experiments were repeated with
proteinase K. At both low and high proteinase K concen-
trations, exactly the same proteolytic fragments were
generated as with trypsin (Figure 1C). Therefore, the
proteolytic fragments are likely to correspond to structural
domains in PulD.
Both N- and C-terminal fragments are integrated
into the outer membrane
Without phenol treatment, PulD migrates as a large
multimeric complex on SDS±PAGE (Hardie et al.,
1996a). However, the N-terminal 28 kDa fragment
migrated as a monomer on SDS±PAGE, indicating that,
once excised from the stable multimer, it does not retain its
multimeric state in SDS (Figure 2A). In contrast, the stable
C-terminal fragment remained multimeric in SDS
(Figure 2A, lane 3; see below). To analyse whether the
N- and stable C-terminal fragments remained associated
with the outer membrane, outer membranes were treated
with 5 M urea and then centrifuged to separate extracted
proteins from membranes. The C-terminal fragment could
not be extracted with urea, indicating that it is integrated
®rmly into the outer membrane (Figure 2D). Urea
treatment seemed to extract ~20% of the N-terminal
fragment from the membrane, whereas without this
treatment it remained entirely in the membrane fraction.
However, the urea extract also contained novel proteolytic
fragments (* and ** in Figure 2B and C) that were much
more abundant than the 28 kDa fragment. Their appear-
ance might have been due to denaturation of the protein
and degradation by endogenous proteases during sample
Fig. 1. Release of fragments from PulD by limited proteolysis with trypsin. (Aand B) Outer membranes were incubated for 15 min with the indicated
amounts of trypsin on ice. After phenol treatment to dissociate the multimers, proteins were precipitated with acetone and resuspended in SDS±PAGE
sample buffer. Samples were analysed by SDS±PAGE (10% acrylamide) and immunoblotting using anti-His
6
-PulD (almost complete PulD) or anti-
PulD-PhoA (N-terminal 99 amino acids of mature PulD. (C) Outer membranes were incubated with 5 mg/ml (+) or 100 mg/ml (++) trypsin or
proteinase K. After 15 min on ice, samples were treated and analysed as described above. (D) Schematic representation of proteins used to raise
antibodies against different regions of PulD. sp, PulD signal peptide.
N.Nouwen et al.
2230
preparation. Thus, the band at 28 kDa might not be derived
from membrane-associated N-terminal fragment. The fact
that the majority of the N-terminal fragment was not
extracted by urea indicates that this domain of PulD, like
the C-terminal domain, is integrated ®rmly into the outer
membrane.
N-terminal sequencing of the stable proteolytic
fragment
The trypsin cleavage site that generates the stable
C-terminal fragment could not be determined by proteo-
lysis of outer membranes because of the low amount of
PulD present and because the 38 kDa band was contam-
inated by other proteins that co-migrated upon SDS±
PAGE. Therefore, we generated suf®cient stable
C-terminal fragment for sequencing by treating puri®ed
PulD complex with immobilized trypsin. Proteolytic
fragments were separated on a 4±20% acrylamide gel.
Proteolysis led to the formation of a multimer of ~500 kDa
that could be dissociated with phenol to give a monomer of
~38 kDa (Figure 3A). Since this 38 kDa fragment was
multimeric, did not react with antibodies against the
N-terminal region of PulD and was exactly the same size
as the stable fragment obtained after proteolysis of outer
membranes (data not shown), we conclude that they are
identical. The determined N-terminal sequence of the
fragment was QAAKPV, indicating that cleavage had
occurred before amino acid 270 in the mature sequence of
PulD. Interestingly, this site is just before the start of the
region that is highly conserved in all secretins.
Stable C-terminal fragment lacks the PulS-binding
domain
PulS binds to the 65 C-terminal amino acids of PulD and
protects it from degradation. Proteolysis of the complex
with trypsin does not change the size of PulS (Figure 3B).
To investigate whether PulS was still associated with the
C-terminal fragment, we loaded the proteolysed complex
on an anion-exchange column. Normally, PulS co-elutes
with PulD on such a column (Figure 4A). However, after
proteolysis, PulS did not bind to the column whereas the
Fig. 3. Proteolysis of puri®ed PulD±PulS complex. (A) Puri®ed,
detergent-solubilized PulD±PulS complex was proteolysed using
trypsin-coated agarose beads. Stable proteolytic fragments were
separated by SDS±PAGE (4±20% acrylamide) and then stained with
Coomassie Brilliant Blue. (B) Samples from (A) were separated by
SDS±PAGE (12% acrylamide), electroblotted on nitrocellulose and
analysed for the presence of PulS using antibodies raised against
MalE±PulS (only that part of the blot displaying PulS is shown).
(C) Schematic representation of the mature part of PulD protein
showing the N-terminal domain (shaded), secretin homology (b) domain
(white) and PulS-binding domain (black). The trypsin cleavage site
and the calculated molecular size of the resulting fragment are also
indicated.
Fig. 2. Multimeric state and susceptibility to urea extraction of
proteolytic fragments derived from outer membrane-associated PulD.
(A) Outer membranes were treated with the indicated amount of
trypsin and analysed without phenol treatment by SDS±PAGE (11%
acrylamide) and immunoblotting with antibodies against PulD-PhoA
(lanes 1 and 2) or His
6
-PulD (lane 3). Note that in lane 3, the
multimer composed of proteolysed monomers co-migrates with
multimers composed of full-length monomer and that 40 kDa
monomers are not detected. (B±D) Outer membranes were treated with
the indicated amount of trypsin. After proteolysis, the membranes were
extracted with 5 M urea and/or pelleted. Pellet and supernatant
fractions were treated with phenol to dissociate PulD multimers and
analysed by SDS±PAGE and immunoblotting using antibodies raised
against PulD-PhoA (lanes 1 and 2, and 4±13) or His
6
-PulD (lanes 3
and 14±18).
Secretin homology domain
2231
stable C-terminal fragment did (Figure 4B). Since trypsin
does not degrade PulS (see above), this result indicates that
the C-terminal fragment lacks the PulS-binding domain
(i.e. the extreme C-terminus of PulD).
Electron microscopic analysis of the stable
C-terminal fragment
Recent electron microscopic analysis of negatively stained
samples of puri®ed PulD±PulS complex showed that it
forms ring-like structures with 12 peripheral radial spokes,
giving a total radius of 20±21 nm (Nouwen et al., 1999).
These previous analyses did not resolve structures inside
the ring. The new data presented here clearly show that
there is protein structure in the centre of the ring of the
PulD complex. Principal components analysis of 1276
images revealed two major classes of 621 and 576 images
whose averages are presented in the insets of Figure 5A.
Both average images show the ring-like structure of 14 nm
diameter surrounded by the 12 peripheral radial spokes.
The more abundant class average (left inset) reveals a plug
in the centre of the complex, while the second average
(right inset) shows a smaller, ring-like structure with a
2.6 nm diameter central hole.
Only end views of PulD±PulS complexes were clearly
distinguished when air-glow-discharged carbon ®lms were
used, preventing the analysis of side views. However, to
our surprise, when carbon ®lms were glow discharged in
the presence of pentylamine, the most abundant structures
seen were side views of single or stacked multimers
(Figure 5B). These side views suggest that the PulD
complex consists of two ring-shaped structures that are
3.5 nm apart. The lower ring is 1.5 nm thick at its
narrowest, central point in the side views, and 20 nm in
diameter, consistent with the outer diameter of the ring,
including spokes, in the end views. The upper ring is
thicker (2.3 nm) than the lower ring and has a diameter of
14 nm. This upper ring appears to form a cavity via
protrusions that fold back towards the centre of the ring,
thereby closing the cavity that it forms. Many of the
stacked multimers were doublets that resembled rugby
balls and consisted of two PulD complexes associated via
their lower rings.
Proteolysis of the complex removes PulS and >40% of
mature PulD. Thus, the structure of the remaining complex
should be drastically different from that of the original
complex. Examination of negatively stained samples of
the puri®ed stable C-terminal fragment revealed that the
peripheral spokes and the structure in the centre of the end
views were essentially absent (Figure 5C). The end views
displayed ring-shaped structures ®lled with homo-
geneously distributed material and a small central cavity
of ~1.6 nm.
The puri®ed C-terminal fragment had a strong tendency
to form long `ladder-like' structures on pentylamine-glow-
discharged carbon grids. In these structures, single com-
plexes associated not only via their lower rings but also via
their upper rings. This observation clearly indicates that
proteolysis changed the rim of the upper ring. Averages of
both single multimeric and double multimeric side views
exhibit major changes in both rings (Figure 5D, bottom
inset). The lower ring is narrower (16.7 nm compared with
20 nm in the native complex), consistent with the removal
of the spokes seen in the end views. The lateral projections
on the upper ring are longer (5.3 nm compared with 4.5 nm
in the native complex). Moreover, faint protrusions
emanating from the rim of the extensions towards the
centre appeared to be shorter and less well ordered in the
proteolysed sample, consistent with the small pore seen in
the end views.
Discussion
PulD is a member of the superfamily of secretins, outer
membrane proteins that mediate the transport of macro-
molecules across the outer membrane of many Gram-
negative bacteria. On the basis of sequence comparisons,
secretins were proposed to consist of two main domains
(Genin and Boucher, 1994; Guilvout et al., 1999). The
C-terminal domain (also called the bdomain) is highly
conserved in all members of the superfamily and is
presumably embedded in the outer membrane. The
N-terminal domain is thought to extend into the periplasm
and to interact with other components of the secreton. In
addition, some secretins contain a small C-terminal
domain to which a secretin-speci®c pilot protein binds
(in the case of PulD, this is the lipoprotein PulS). Our
biochemical analyses of secretin PulD are largely consist-
ent with such a domain organization. Limited proteolysis
of outer membranes containing PulD led to the formation
of an N-terminal 28 kDa fragment that did not retain the
SDS-resistant multimeric organization typical of native
PulD on SDS±PAGE. Under the same conditions, the
C-terminal fragments generated all retained the stable
multimeric state of PulD. This observation is in line with
those of previous studies showing that peptide insertions in
the C-terminal (b) domain of PulD had a dramatic effect
on the formation and stability of multimers. A fragment of
PulD corresponding to the stable C-terminal region of
PulD, as de®ned here, but with its PulS-binding site intact
and associated with PulS did not form multimers in vivo
(Guilvout et al., 1999). However, multimers were
Fig. 4. The stable C-terminal fragment lacks the PulS-binding domain.
Native or trypsin-proteolysed PulD±PulS complex was loaded on a
miniQÔcolumn. Flow-through fractions (lanes 2±4) and fractions
eluted with NaCl (lanes 5±7) were treated with phenol to dissociate
PulD multimers and then analysed by SDS±PAGE and immunoblotting.
(A) Native PulD±PulS complex. (B) Trypsin-proteolysed PulD±PulS
complex.
N.Nouwen et al.
2232
observed when an N-terminal fragment was produced
in trans. These multimers contained only the C-terminal
region of PulD (the N-terminal region either did not
associate with the C-terminal region or dissociated from it
in SDS) and contained <12 copies of the PulD fragment
(Guilvout et al., 1999). These results show unambiguously
that the N-terminal region does not remain oligomeric in
SDS when removed from the dodecameric C-terminal
region of PulD but is required for multimerization.
Interestingly, the 28 kDa fragment could not be
extracted with 5 M urea, indicating that it is ®rmly
integrated into the outer membrane. High amounts of
protease degraded the N-terminal 28 kDa fragment, but
multimers of an ~38 kDa C-terminal fragment appeared to
be very resistant to proteolysis, as would be expected for a
domain of PulD that is embedded in the outer membrane.
Using puri®ed, detergent-solubilized PulD±PulS complex,
we demonstrated that the fragment corresponds to a
C-terminal segment of mature PulD starting at amino acid
position 270 and lacking the PulS-binding domain as a
result of a second, undetermined proteolysis event close to
the C-terminus of PulD. Thus, the stable proteolytic
fragment corresponds almost exactly to the region (the
bdomain) that is highly conserved in the secretin
superfamily.
We recently analysed the structure of negatively stained
native PulD±PulS complex by electron microscopy
(Nouwen et al., 1999). In these studies, as in other studies
of different secretins (Koster et al., 1997; Bitter et al.,
1998; Crago and Koronakis, 1998), we only obtained clear
images of the end view of the complex. Here we present an
averaged image of the side views of the PulD±PulS
complex. It consists of a 20 nm diameter, 2.3 nm thick ring
or disc that supports a smaller cup-like structure that is
14 nm in diameter and 8.5 nm high. We hypothesize that
the broader of the two rings is integrated into the outer
membrane. This hypothesis is supported by the following:
(i) the broader ring has a strong tendency to self-associate
to form doublets, suggesting that it is very hydrophobic;
(ii) the ring is ~2.3 nm thick at its outer limits, which is
Fig. 5. Electron microscopic analysis of native and trypsin-digested PulD complex. A sample of puri®ed native or trypsin-proteolysed PulD complex
was applied to air- or pentylamine-glow-discharged thin carbon-coated grids. (A) Uranyl formate-stained images of native PulD complex on air-glow-
discharged grids; insets, averaged images of an end-on view of two major classes (n= 621 and 576). (B) Uranyl formate-stained images of native
PulD complex on pentylamine-glow-discharged grids; insets, averaged image of double multimeric (n= 150) and single multimeric (n= 217) side
views of native PulD complex. (C) Uranyl formate-stained images of proteolysed PulD complex on air-glow-discharged grids; inset, averaged image
of an end view (n= 276). (D) Uranyl formate-stained images of proteolysed PulD complex on pentylamine-glow-discharged grids; top insets,
averaged images of double complex (n= 150) and single complex (n= 217) side views of proteolysed PulD complex; bottom inset, comparison of
undigested (grey shading) and trypsin-digested (outline) secretin complexes. The number of particles (n) used for constructing the averaged image is
indicated in parentheses. The scale bar corresponds to 50 nm and the inset baseline corresponds to 25 nm.
Secretin homology domain
2233
suf®cient to span the outer membrane; and (iii) proteolysis
of the complex removes the region to which the outer
membrane-associated lipoprotein PulS binds, coincident
with a narrowing of the ring. In this model, the majority of
the complex would protrude so far into the periplasm that
the ends of the top rim of the cup would almost touch the
cytoplasmic membrane, and could therefore come into
contact with other secreton components that are located in
this membrane. In addition, folded proteins would not
have to pass the peptidoglycan layer to reach the
translocation channel since they might be captured within
the funnel of the channel close to the surface of the
cytoplasmic membrane. However, the secretin channel
must itself penetrate through the peptidoglycan layer.
Even the bulk of the bdomain might not be fully
integrated into the membrane, since less than half of the
amino acids in this domain are predicted to be in potential
transmembrane b-strands (Guilvout et al., 1999). The
loops between most of these predicted b-strands are longer
than in other outer membrane proteins that have been
analysed (Guilvout et al., 1999) and could extend into the
medium as well as into the periplasm.
In the averaged images of the side views of both the
single and double multimers, the ends of the rim of the cup
seem to fold back into the cavity of the channel. This
structure is likely to be part of the N-terminal domain of
PulD that forms the `plug' in the centre of the ring that is
seen in approximately half of the end views. In agreement
with this idea, the `plug' is totally absent in all of the end
views of the stable C-terminal fragment. Moreover, side
views of this C-terminal fragment show that, in contrast to
the native complex, the upper rims of the cup have a strong
tendency to self-associate. This indicates that drastic
changes have occurred in this region of the protein as a
result of proteolysis by trypsin. It is worth noting,
however, that almost half of the end views of the native
complex appear to have an open centre (right hand inset in
Figure 5A). This observation is dif®cult to interpret at
present but one possibility is that some of the native
secretin particles examined were in a partially open
con®guration when examined end-on.
The `plug' that could be formed by the N-terminal
domain of PulD might be analogous to a protein domain
that folds backwards into the channels formed by the outer
membrane ferrisiderophore transporters FhuA and FepA
(Ferguson et al., 1998; Locher et al., 1998; Buchanan et al.,
1999). In these transporters, the N-terminal `plug' or
`cork' domain controls channel opening and subsequent
transport of the ferrisiderophore. However, complete
displacement of the central plug would be necessary to
allow proteins to transit the secretin channel, whereas
removal of the plug is not necessary to create a channel
suf®ciently large to permit siderophore transport through
FepA and FhuA. In previous studies, we showed that the
PulD secretin complex exhibited only very low level
electrical conductance when incorporated into planar lipid
bilayers (Nouwen et al., 1999). Thus, we concluded that
these secretin complexes were in the uniformly closed
con®guration, which we now propose corresponds to the
structure with the plug in the centre of the barrel or cup-
like structure. Therefore, one would predict that the
C-terminal fragment should be in the open con®guration
and would have very high conductance when incorporated
into lipid bilayers. This was not found to be the case,
however, apparently because it is not possible to incorpor-
ate the stable C-terminal fragment into lipid bilayers
(N.Nouwen and A.Ghazi, unpublished observations). We
are currently exploring other ways to manipulate the
opening and closure of the secretin channel.
Secretins have highly conserved C-terminal regions,
thus, other secretins probably have a similar structure to
that of the C-terminal fragment of PulD. Indeed, both end
and side views of this C-terminal fragment resemble
images of secretin pIV of ®lamentous phage f1 (Linderoth
et al., 1997). The major difference between PulD and pIV
is that the side views of single complexes of pIV have only
one ring (Linderoth et al., 1997). However, pIV might
have a second ring that is not visible on the images, since
Linderoth et al. (1997) observed that the contour length of
the side views of the pIV double multimer was much
greater than twice that of the single multimer. The lower
disc and the upper cup very probably correspond to a
12-fold symmetrical structure that encircles the central
channel. Recently reported studies demonstrated that a
protease-resistant, membrane-associated C-terminal frag-
ment similar to that which we report here can be obtained
by proteolysis of the secretin XcpQ, a PulD homologue
from Pseudomonas aeruginosa (Brok et al., 1999). Like
the stable C-terminal fragment of PulD, this domain of
XcpQ retained its oligomeric structure (Brok et al., 1999).
Thus, the intrinsically multimeric C-terminal, ring-shaped
bdomain is probably a feature inherent to all secretins
Stacked rings are also seen in electron microscopic
images of the ¯agellar basal body (L- and P-ring)
(Macnab, 1996) and, most interestingly, of the complete
structure of two different type III protein secretion systems
(Kubori et al., 1998; Blocker et al., 1999). In the latter
case, it would be very interesting to see whether these
stacked rings correspond to the secretin component.
Materials and methods
Bacterial strain and growth conditions
The Escherichia coli K-12 strain NN001 (lamB ompR::Tn10) carrying
plasmid pCHAP231, encoding PulD and all other secreton proteins
needed for pullulanase secretion (d'Enfert et al., 1987), was used for
isolation of outer membranes and puri®cation of the PulD±PulS complex.
Cells were grown at 30°C in Luria±Bertani medium (Miller, 1992)
buffered with 2% M63 medium (Miller, 1992) and containing 100 mg/ml
ampicillin. The medium was supplemented with 0.4% maltose to induce
the pul operon.
SDS±PAGE and immunoblotting
Proteins were separated by SDS±PAGE in 10, 11 or 4±20% acrylamide
gels, stained with Coomassie Brilliant Blue or transferred to nitrocellulose
by semi-dry electroblotting. Immunoblots were incubated with the
antibodies indicated and then with horseradish peroxidase-coupled anti-
rabbit immunoglobulin G (IgG). Immunoblots were developed by
enhanced chemiluminescence (ECL kit; Amersham). The primary
antibodies used were a 1:4000 dilution of PulD±PhoA (d'Enfert et al.,
1989), a 1:200 dilution of an af®nity-puri®ed antibody raised against
His
6
-PulD (which lacks the ®rst 30 amino acids of mature PulD) (Hardie
et al., 1996a) and a 1:10 000 dilution of MalE±PulS (Hardie et al., 1996b).
Limited proteolysis
Outer membranes were prepared by breaking bacteria in a French press,
removing unbroken cells by centrifugation at 3000 gfor 15 min, and
pelleting the outer membrane by centrifugation for 1 min at 165 000 g.
Outer membranes were washed with 50 mM Tris pH 8.0, 1 mM EDTA,
pelleted, and resuspended in 50 mM Tris pH 8.0 at a protein concentration
of 50 mg/ml. Proteolysis was initiated by addition of the indicated amount
N.Nouwen et al.
2234
of trypsin or proteinase K to a solution containing 2 mg/ml protein. After
15 min on ice, the protease was inactivated by addition of PefablocÔ
(Interchim; ®nal concentration 100 mg/ml). Unless otherwise indicated,
PulD multimers were dissociated by phenol treatment (Hardie et al.,
1996a), precipitated overnight by addition of 2 vols of acetone at ±20°C
and dissolved in SDS±PAGE sample buffer.
To test the membrane association of the proteolytic fragments, samples
were extracted with 5 M urea and then centrifuged at 100 000 gfor 15 min.
Membrane and supernatant fractions were treated with phenol to
dissociate multimeric PulD as above and analysed for the presence of
PulD by SDS±PAGE and immunoblotting.
Protein sequence determination
Puri®ed PulD±PulS complex (1 mg/ml) was proteolysed using trypsin-
coated agarose beads (Sigma; 1:5 ratio of bead slurry to protein solution).
After 3 h on ice, the agarose beads were removed by centrifugation and
the clari®ed mixture was treated with phenol to dissociate the multimeric
complex. Proteins were precipitated by addition of 2 vols of acetone,
resuspended in SDS±PAGE sample buffer and heated to 100°C for 5 min.
After electrophoresis, proteins were electroblotted onto Immobilon P
membranes (Millipore) and the proteolytic fragment was sequenced using
an Applied Biosystems 473A protein sequencer according to the standard
procedure of the manufacturer.
Protein puri®cation and analysis
PulD±PulS complex was puri®ed as described (Nouwen et al., 1999) with
the modi®cation that PulD±PulS fractions from the size exclusion column
that contained the complex were pooled and concentrated by chroma-
tography on a miniQÔcolumn on a SmartÔ-system (Pharmacia).
Incubating puri®ed PulD±PulS complex with trypsin-coated agarose
beads (see above) generated the stable proteolytic fragment. After
proteolysis, the agarose beads were removed by centrifugation and the
clari®ed mixture was loaded on a miniQÔcolumn using a SmartÔ-
system. After loading and washing of the column, the proteolytic
fragment was eluted with 20 mM Bis-Tris-propane pH 7.0, 0.3%
Zwittergen 3±14 (Fluka), 625 mM NaCl. Fractions were analysed by
SDS±PAGE and immunoblotting for the presence of the proteolysed
fragment and PulS. Fractions containing the C-terminal fragment were
pooled and stored at ±70°C.
Electron microscopy
PulD±PulS complex and puri®ed C-terminal proteolytic fragment in
20 mM Bis-Tris-propane pH 7.0, 0.3% SB3-14, 625 mM NaCl were
adsorbed for 1 min to air- or pentylamine-glow-discharged thin carbon
®lms mounted on a thick fenestrated carbon layer on copper grids. After
three washes with distilled water, the sample was stained with uranyl
formate pH 4.2 for 10 s, blotted and air dried. Images of negatively
stained samples were recorded on a Hitachi H7000 transmission electron
microscope at 100 kV, with a nominal magni®cation of 50 0003at
500 electrons/nm
2
. Negatives were digitized by using a Leafscan 45
scanner (Leaf Systems, Westborough, MA) at 0.2 nm/pixel on the
specimen plane. The SPIDER program (Frank et al., 1996) was used to
select individual particles as 128 3128 pixel (end views) and 256 3256
pixel (side views) subframes. End views were reference-free aligned
(Penczek et al., 1992), classi®ed and the clearly visible 12-fold rotational
symmetry applied. Side views were aligned using one particle as an initial
reference and then averaged.
Acknowledgements
We are grateful to all members of the secretion laboratory of the Institut
Pasteur for their interest and encouragement and to J.D'Alayer of the
Institut Pasteur Microsequencing Laboratory for protein sequence
analysis. N.N. thanks all members of the Maurice E.Mu
Èller Institut for
their hospitality and help during his stay in Basel. This work was
supported by the European Union (Training and Mobility in Research
grant number FMRX-CT96-0004) and by a French Research Ministry
grant in the Programme fondamental en Microbiologie et Maladies
infectieuses et parasitaires and by the Maurice E.Mu
Èller foundation of
Switzerland.
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