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Journal of Bacteriology, December 1999, p. 7212-7220, Vol. 181, No. 23
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Genetic Dissection of the Outer Membrane Secretin
PulD: Are There Distinct Domains for Multimerization and
Secretion Specificity?
Ingrid
Guilvout,
Kim R.
Hardie,
Nathalie
Sauvonnet, and
Anthony P.
Pugsley*
Unité de Génétique
Moléculaire, CNRS URA 1773, Institut Pasteur, 75724 Paris, France
Received 8 July 1999/Accepted 16 September 1999
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ABSTRACT |
Linker and deletion mutagenesis and gene fusions were used to probe
the possible domain structure of the dodecameric outer membrane
secretin PulD from the pullulanase secretion pathway of
Klebsiella oxytoca. Insertions of 24 amino acids close to
or within strongly predicted and highly conserved amphipathic 
strands in the C-terminal half of the polypeptide (the domain)
abolished sodium dodecyl sulfate (SDS)-resistant multimer formation
that is characteristic of this protein, whereas insertions elsewhere generally had less dramatic effects on multimer formation. However, the
domain alone did not form SDS-resistant multimers unless part of
the N-terminal region of the protein (the N domain) was produced in
trans. All of the insertions except one, close to the C
terminus of the protein, abolished function. The N domain alone was
highly unstable and did not form SDS-resistant multimers even when the
domain was present in trans. We conclude that the domain is a major determinant of multimer stability and that the N
domain contributes to multimer formation. The entire or part of the N
domain of PulD could be replaced by the corresponding region of the
OutD secretin from the pectate lyase secretion pathway of Erwinia
chrysanthemi without abolishing pullulanase secretion. This
suggests that the N domain of PulD is not involved in substrate recognition, contrary to the role proposed for the N domain of OutD,
which binds specifically to pectate lyase secreted by E. chrysanthemi (V. E. Shevchik, J. Robert-Badouy, and G. Condemine, EMBO J. 16:3007-3016, 1997).
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INTRODUCTION |
The two-step general secretory
pathway (GSP) is used by gram-negative bacteria to secrete
extracellular proteins (exoproteins) and for pilus assembly
(28). The second step in the GSP comprises several terminal
branch pathways for exoprotein translocation from the periplasm across
the outer membrane (28). The main terminal branch of the
GSP, the secreton or type II secretion pathway, is present in many
species of gram-negative bacteria. The Klebsiella oxytoca
secreton is used exclusively for the secretion of a cell surface
lipoprotein, pullulanase (PulA), and is composed of or involves up to
14 proteins: PulC, PulD, PulE, PulF, PulG, PulH, PulI, PulJ, PulK,
PulL, PulM, PulN, PulO, and PulS (28). Only two of these
proteins, PulD and PulS, are located exclusively in the outer membrane
(4, 7, 8, 13, 14), where they form a large, tightly
associated complex that has a barrel-like structure and appears to form
ion-conducting channels in artificial lipid bilayers (24).
PulD and its close homologues from other secretons belong to the
secretin family of proteins (12), all of which form large complexes that are poorly dissociated in sodium dodecyl sulfate (SDS)
at room temperature or even at 100°C (1, 10, 13, 14, 17,
20). Secretins are apparently composed of two main domains (Fig.
1). The C-terminal half, the domain,
is predicted to contain several amphipathic transmembrane (TM) strands that are probably embedded in the outer membrane in a manner
similar to other outer membrane proteins (1). Even distantly
related secretins with distinct functions share relatively high
sequence similarity in this domain (12). The N-terminal half
of secretins, the N domain, is predicted to face the periplasm and is
conserved only in secretins from related secretion pathways. The two
domains are sometimes separated by a serine-and-glycine-rich segment
(12). PulD lacks this spacer sequence, and the junction
between the two domains is experimentally defined as the extremity of
the membrane-associated fragment that is resistant to treatment with trypsin (starting at amino acid [aa] 298 [23]) (Fig.
1). PulD has a short C-terminal domain (the S domain) that binds the
pilot protein PulS, which protects PulD from proteolysis and is
essential for its insertion into outer membrane (6, 13, 14)
(Fig. 1). Other secretins also appear to have their own, specific pilot proteins that likewise bind to the C-terminal regions (5,
35).
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FIG. 1.
Schematic representation of PulD protein showing
positions of the signal peptide, the N, , and S domains, the PulS
binding site (6, 13, 14, 35, 36), the trypsin cleavage site
(23), the hypothetical TM strands (TM1 to TM13)
(1), and the 24-amino-acid insertions numbered as in Table
2. Intervals of 100 aa and the position of the C terminus (aa 660) are
also indicated.
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The role of the N domain of secretins has been addressed in two
previous studies. One of these studies provided evidence that the N
domain of a phage assembly secretin (pIV of phage f1) interacts with
another phage-encoded protein, the cytoplasmic membrane protein pI, to
determine phage assembly/secretion specificity (6). In the
other study, deletion of a 51-amino-acid segment from the N domain of
Erwinia chrysanthemi OutD (residues 66 to 116) abolished exoprotein binding and secretion, whereas other deletions abolished secretion without affecting exoprotein recognition (36).
Thus, OutD was proposed to bind specifically to exoproteins secreted by
the Out secreton and to determine substrate specificity
(36), which is in line with other studies showing that OutD
protein from Erwinia carotovora, which secreted different
exoproteins, cannot substitute for that of E. chrysanthemi
(19).
This report describes genetic and other studies designed to test the
role of each domain of PulD in multimerization and exoprotein recognition.
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MATERIALS AND METHODS |
Plasmids and strains.
Plasmids carrying pul and
out genes used in this study are listed in Table
1. Strains used were PAP105
[
(lac-pro) F' lacIq
pro+ Tn10), PAP7447 [MC4100 (F'
lacIq pro+
Tn10) with pulS, pulA, and
pulC-O integrated into malPp and with a large
deletion in pulD (13)], and PAP7446, which is
the same as PAP7447 except that pulD is wild type and
pulS has a Tn5 insertion (13). Genes
under lacZp control were induced with 1 mM
isopropyl--D-thiogalactopyranoside (IPTG), and genes
under MalT control were induced with 0.4% maltose or by introduction
of a malT (Con) allele. Cells were grown at 30°C in
Luria-Bertani medium buffered, where appropriate, with 10% M63 medium
(22) and containing antibiotics at the following concentrations: ampicillin, 200 µg/ml; tetracycline, 16 µg/ml; kanamycin, 50 µg/ml; and chloramphenicol, 25 µg/ml.
Mutagenesis and construction of gene fusions.
Transposon
TnTAP was used as previosuly described (11) to
create pulD-phoA gene fusions in pCHAP3671 (Table 1).
Kanamycin-resistant clones were examined for production of stable
PulD-PhoA hybrids by SDS-polyacrylamide gel electrophoresis (PAGE) and
immunoblotting with antibodies against PulD and PhoA. Selected clones
were then sequenced using a primer hybridizing with the 5' end of
phoA. After verification, the transposon was deleted by
digesting with NotI, which cleaves sites within the inverted
repeats at each end of the transposon, to leave a 24-amino-acid linker
insert (11). A stop codon was introduced at the position of
the NotI sites using a NotI-compatible linker.
Deletions in pulD were created by ligating fragments of
pulD obtained by cleaving plasmids with different
NotI inserts in the gene and restriction sites flanking the gene.
The '
pulD fragment in pCHAP1349 comprises all codons
starting at number 298, corresponding to the N terminus of the
trypsin-resistant
fragment of purified PulD in detergent micelles
(
23). The fragment
was obtained by PCR amplification.
Restriction sites introduced
by the amplification primers were used to
subclone the amplified
DNA into plasmid pCHAP4201 (a pSU18 derivative
with the 5' end
of the
phoE gene in the same orientation as
lacZp) to create a
phoE'-'pulD gene fusion in
which DNA coding for the PulD-
S fragment
is fused to the PhoE signal
peptide and the first three amino
acids (AEF) of mature PhoE
(pCHAP1349). The '
pulD fragment used
to create the fatty
acylated PulD, which was also obtained by
PCR, comprised the complete
sequence of mature PulD (
9). The
amplified fragment was
cloned into a plasmid bearing DNA coding
for the PulA signal peptide
plus five amino acids starting with
PulA-derived CD
(pCHAP1300).
The
outD gene was amplified by PCR using primers hybridizing
with the 3' end of
outC and the 5' end of
outE in
pCCP2236 (
19)
and was then subcloned using restriction sites
introduced by the
amplification primers. Segments of the
outD and
pulD genes were
exchanged by using
existing restriction endonuclease sites or
by creating new sites at
identical positions in the two genes
by site-directed mutagenesis
(
18) in such a way that minimal
sequence changes were
introduced into the polypeptides. Excised
fragments were exchanged
between two plasmids, and recombinants
were verified by restriction
analysis and by immunoblotting with
PulD antibodies to detect the
chimera.
SDS-PAGE and immunoblotting.
Proteins were separated by
SDS-PAGE in 10 or 12% acrylamide gels, were transferred onto
nitrocellulose sheets, and were incubated with the antibodies indicated
and then with horseradish peroxidase-coupled antirabbit immunoglobulin
G (IgG). In the far-Western analysis, nitrocellulose sheets were first
incubated with purified alipoPulA (pCHAP1106) then with antibodies
against PulA and then with horseradish peroxidase-coupled antirabbit
IgG. Immunoblots were developed by enhanced chemiluminescence
(Amersham). Where indicated, PulD multimers were dissociated by phenol
extraction (13) and the samples were dissolved in sample
buffer (100 mM Tris [pH 8.0], 5% SDS, 12% glycerol, 1%
2-mercaptoethanol). Unless otherwise stated, all samples were heated
for 5 min at 100°C before being loaded onto the gel. Metabolic
labeling with radioactive palmitate was carried out as described
previously (29), and labeled proteins were separated in
SDS-PAGE gels that were then fixed in 10% acetic acid, soaked in
Amplify (Amersham), and examined by fluorography.
Affinity chromatography, flotation gradients, and pullulanase
assays.
For affinity chromatography, pullulanase was bound to
amylose-agarose resin (New England Biolabs) and pCHAP3072-encoded
PulAAB-CelZ, a secretable hybrid protein comprising the
putative secretion signals from PulA (33) fused to the
cellulase CelZ of E. chrysanthemi (30), was bound
to Avicel (Serva). Membranes were solubilized in 25 mM Tris (pH 7.4)
containing 2% Triton and 1 mM EDTA (approximately 25% of the total
amount of PulD was solubilized), and insoluble material was removed by
ultracentrifugation (120,000 × g for 1 h). The
extract was then incubated with the immobilized pullulanase, which was
then washed extensively with the same buffer. Bound proteins were
eluted in 25 mM Tris containing 2% SDS and were examined by SDS-PAGE
and immunoblotting. The presence of PulA and PulAAB-CelZ in
the extracts was verified by immunoblotting with anti-PulA and
anti-CelZ (a gift from F. Barras), and the presence of PulD was
examined by immunoblotting with anti-PulD.
For flotation gradient analysis, outer membranes were first prepared by
breaking the bacteria in a French press, removing
unbroken cells by
centrifugation at 3,000 ×
g for 5 min, and then
pelleting the outer membrane by centrifugation for 1 min in a
Beckman
TL100 rotor at 120,000 ×
g. The resuspended membranes
were incubated with purified pullulanase, brought to 60% (wt/wt)
with
sucrose, and loaded in the bottom Beckman SW55 centrifuge
tube. The
membranes were then overlaid with decreasing concentrations
of sucrose
(from 56 to 35%) in 25 mM Tris and were centrifuged
at 176,000 ×
g for 24 h. Fractions collected from the tops of
the
tubes were analyzed for the presence of PulD and PulA by SDS-PAGE
and
immunoblotting.
Pullulanase was assayed as previously described (
21), except
that cells were lysed with 0.5% octylpolyoxyethylene. The level
of
secretion is the percentage of the total amount of pullulanase
that was
detectable in unlysed
cells.
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RESULTS |
Linker insertions in pulD.
In previous studies of the
pulD homologues xpsD (16) and
xcpQ (1), short linker insertions often had
little or no effect on secretin function. Therefore, we reasoned that
larger inserts might also be tolerated and could provide information on
the topology of secretin in the outer membrane and on the role of
individual domains in multimerization and substrate recognition.
Therefore, we used TnTAP, which creates phoA
fusions and has NotI restriction sites at each end so that
it can be deleted, to leave a 24-amino-acid insert containing a
cleavage site for the tobacco etch virus protease (11).
Thirty eight such inserts were created at 19 positions in PulD (Fig.
1
and Table
2). The insertions were
relatively well
dispersed throughout the gene, though there were also
some transposition
hot spots (Table
2). All of the mutants contained
PulD at levels
that ranged from 20 to 100% of those of unaltered PulD,
and degradation
products were not detected when PulS was present.
However, three
categories of mutants could be distinguished with
respect to their
proportion of the total amount of PulD present as
monomers and
their ability to complement the
pulD
mutation in strain PAP7447
(
13) (Table
2 and Fig.
2).
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FIG. 2.
Formation of SDS-resistant multimers and yields of
representative PulD variants obtained by linker insertions in the
pulD gene. Three categories of mutants were obtained:
slightly reduced total yields and unaltered multimerization (B),
slightly reduced yields and substantially reduced multimerization (C),
and slightly reduced yields and abolished or drastically reduced
multimerization (D) compared with normal PulD (A). Samples from
IPTG-induced and noninduced cells of E. coli expressing
pulS together with the pulD variants under
lacZp control on pSU18 derivatives were examined by SDS-PAGE
and immunoblotting. The same amount of total cell extract was loaded in
each lane, and each panel represents the same chemiluminescence
exposure time.
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All of the insertions except number 19 (class B) affected, to at least
some extent, the levels of SDS-resistant multimers
detected. The
distinction between mutants in classes C and D was
based on two highly
reproducible criteria (Fig.
2): normal levels
of protein and reduced,
but still appreciable, levels (>30%) of
multimers in class C mutants
compared with reduced yields (20%)
and total absence of multimers in
class D mutants. Since the multimers
produced by class C mutants formed
discrete bands that entered
into the 4.5% acrylamide stacking gel and
are resistant to dissociation
in SDS at 100°C, they are presumed to
correspond to true multimers,
rather than to aggregates (Table
2 and
Fig.
2; see
Discussion).
Apart from insertion 19, all of the PulD insertion variants were
completely inactive in complementation tests in PAP7447 (Table
2).
Since all of these variants retain the complete PulS binding
site at
the C-terminal ends, they are probably sorted to and associate
with the
outer membrane, though they might not insert correctly.
When production
of the PulD variants was increased in these cells
by IPTG induction,
the bacteria grew more slowly and lysed when
they were harvested and
washed (not shown). This phenomenon could
result from defective
insertion into the outer
membrane.
The only insertion that did not abolish ability to complement
pulD or cause lysis after IPTG induction (insertion 19, aa
640) is located within the S domain (Fig.
1, Table
2). The protein
was still protected by PulS (data not shown). Therefore, its reduced
ability to complement
pulD might be due to impaired outer
membrane
insertion, which requires the region around aa 640 in addition
to the PulS binding site (
4).
Although all of the other insertions completely abolished
complementation of
pulD in strain PAP7447, some caused
variable,
partial complementation of the
pulD mutation
carried by pCHAP1226
(data not shown). This low-level complementation
(<45% secretion)
might reflect partial activity that is manifested
only when all
other secreton components (encoded by pCHAP1226) are
present at
high
levels.
Deletion of the PulD and N domains and
trans-induced multimerization.
The data presented
above suggest that the domain might be important for SDS-resistant
multimer formation. However, these data are in contrast to the results
of previous studies showing that a hybrid protein comprising the N
domain (aa 1 to 99 or 1 to 395) of PulD fused to PhoA apparently
produced low amounts of multimer (13). Therefore, we
screened all PulD-PhoA hybrids generated by the insertion of
TnTAP for the production of SDS-resistant multimers.
Immunoblotting with PulD and PhoA antisera failed to reveal the
presence of any multimers in any case except number 19 (aa 640), which
retains all of the N and domains. Presumably, the previously
detected PulD-PhoA multimers (13) represented aggregates
rather than dodecameric complexes similar to those formed by
full-length PulD (24). It is also possible that the combination of a segment of the N domain of PulD and the form of PhoA
used in the previous experiments could nucleate a low level of multimer production.
To obtain more information on the role of the N and
domains in
multimer stability, we created truncated forms of PulD. First,
we
constructed a variant in which the
and S domains of PulD
(from aa
298) were fused to the signal peptide and the first three
amino acids
of outer membrane protein PhoE (PulD-
S; pCHAP1349).
Proteolysis of
purified PulD dodecamers causes cleavage after
residue 297 to produce a
protected dodecameric PulD-
S fragment
that is almost identical to
PulD-
S encoded by pCHAP1349 (
23).
PulD-
S produced in
vivo was protected from proteolysis by plasmid-encoded
PulS (Fig.
3), but multimers were not detectable
(data not shown).
Thus, the
S alone is unable to form SDS-resistant
multimers,
though it presumably associates with the outer membrane
through
its interaction with PulS. High level (IPTG-induced) production
of PulD-
S caused the bacteria to grow poorly and to lose viability
at the end of exponential growth, though they did not exhibit
increased
sensitivity to normally nonpenetrating agents such as
novobiocin or
rifampicin. These observations suggest that PulD-
S
causes general
membrane perturbation without producing open channels
in the outer
membrane.
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FIG. 3.
Protection of PulD-S by PulS. Extracts from
IPTG-induced cells producing PulD-S alone (pCHAP1349) or PulD-S
plus PulS (pCHAP1349 plus pCHAP585) were examined by immunoblotting
with PulD antibodies and chemiluminescence. Bands corresponding to
protected and clipped PulD-S are indicated. The band just above
PulD-S that comigrates with the 47-kDa marker protein (indicated at
left) is recognized nonspecifically by the antibodies used.
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Next, a set of eight PulD variants lacking different sections of the
domain was constructed in vitro using unique
NotI sites
inserted at different positions within the
pulD gene. The
deletions
spanned the region from aa 406 (insertion number 13), aa 281 (number
10), aa 267 (number 9), or aa 196 (number 8) up to aa 533 (insertion
number 18) or aa 640 (number 19) (Fig.
4). Variants with deletions
that
terminated at aa 640 (and therefore lacking a region essential
for
outer membrane insertion [
4]) were not detectable or
were
detectable only in IPTG-induced cultures irrespective of the
presence
of PulS, and none was present as multimers (Fig.
4). In
contrast,
proteins with deletions that terminated at aa 533 were
detectable
even without induction, and two of them (PulD-
282-533 and
PulD-
197-533)
were present as multimers. Unlike full-length PulD,
the PulD-
282-533
multimers were dissociated after heating to 100°C
in SDS (Fig.
4). Surprisingly, multimers were most abundant in the case
of
the variant with the largest deletion (PulD-
197-533) (Fig.
4).
Furthermore, multimers produced by PulD-
282-533 and PulD-
197-533
were much closer in size than would be expected from the difference
in
size of the two monomeric forms (Fig.
4), suggesting that they
could
represent smaller multimers than the dodecamer formed by
full-length
PulD (
24). However, since these multimers form discrete
bands upon SDS-PAGE, they do not correspond to aggregates. From
these
results, we can conclude that the C-terminal region of the
domain
and the PulS binding site (aa 534 to 640) stabilize the
N domain,
presumably as a result of the binding of PulS (
4).
Furthermore, the combined presence of aa 1 to 196 and 533 to 640
seems
to permit at least some degree of multimerization of truncated
PulD.
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FIG. 4.
Effects of internal deletions affecting mainly the PulD
domain on the formation of SDS-resistant multimers. The proteins,
resulting from deletions between linkers inserted at the indicated
positions in the PulD sequence (Table 2), were produced in cells that
also contained PulS. The samples at the extreme right of the lower
panel were from a strain producing full-length PulD which was included
as a control. The positions of monomeric and multimeric forms of PulD
are indicated. The samples were from strains grown with or without IPTG
to induce production of the PulD variants encoded by the
lacZp-pulD operon fusions on pUC18 and one of each pair
of samples was heated to 100°C for 5 min before being loaded on the
gel, as indicated. The positions of molecular size markers (kDa) are
indicated.
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To validate these observations, a stop codon-bearing linker was
inserted into the
NotI sites at aa 406, 281, 267, and 196
to
produce PulD truncates completely or almost completely devoid
of the
and S domains (called PulD-N406, PulD-N281, PulD-N267,
and
PulD-N196, respectively). These truncates were barely detectable,
even
in induced cultures (data not shown). To determine whether
N domains
encoded by these constructs could be stabilized by the
PulD-
S in
trans, the cells were transformed with pCHAP1349 (see
above). Immunoblots of these cells were probed with antibodies
raised
against PulD or against the first 126 aa of the protein
(
8)
to distinguish between PulD-
S and the other forms of PulD.
Both
antibodies recognize multimers formed from full-length PulD
(data not
shown). Remarkably, an increase in the levels of both
the PulD-
S and
the PulD-N variants (and of degradation products
derived therefrom) was
observed when they were produced by the
same cell, indicating mutual
stabilization by the two halves of
the protein (an example is shown in
Fig.
5). Furthermore, SDS-
and
heat-resistant multimers of PulD-
S were abundant in cells
producing
PulD-N196 (Fig.
5). This result indicates that the N
domain of PulD (or
part of it) can stabilize and cause at least
partial multimerization of
the
and S domains in
trans. However,
the association
between the two segments is apparently disrupted
by SDS, since
PulD-N196 was not found in these multimers, and
the multimers were
smaller than would be expected if they were
composed of 12 copies of
the truncated monomer (Fig.
5). Surprisingly,
multimers of the
PulD-
S were much more abundant in cells coproducing
PulD-N196 than
in cells producing the longer PulD-N variants (data
not shown).
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FIG. 5.
Stabilization and induced multimer formation in PAP7447
cells producing PulD-S and PulD-N196 encoded by separate plasmids,
both encoded by pulD variants under lacZp control
from uninduced or IPTG-induced cultures. (Note that the cells produce
only low levels of chromosome-encoded PulS.) Pairs of extracts from
cells producing one or other or both of the variants were subjected to
SDS-PAGE and were blotted onto nitrocellulose sheets. One
representative of each pair of samples was heated to 100°C for 5 min
before SDS-PAGE, as indicated. Proteins were immunodetected first with
antibodies against full-length PulD (bottom panels), and then the
nitrocellulose sheet was stripped and reprobed with antiserum against
PulD-PhoA containing only the first 126 aa of PulD, which does not
react with PulD-S (upper panels). Bands recognized nonspecifically
by this antiserum (including PhoA alkaline phosphatase) are indicated
by the letters NS. Other bands derived from PulD are indicated, as are
the positions of molecular mass markers (kDa).
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Next, we replaced the signal peptide of PulD with a lipoprotein signal
peptide and the first five amino acids of a lipoprotein
that includes
the fatty acylated N-terminal cysteine residue and
a serine at position
+2 for sorting to the outer membrane (
34).
This protein was
created to test two aspects of PulD structure
and function: the
possibility that added fatty acids could replace
the requirement for
fatty acids on PulS (
14) and the ability
of PulD to function
when its N terminus is tethered in the lipids
of the outer membrane, as
is apparently the case in the related
secretins XpsD (
15)
and BfpB (
31). The PulD derivative created
was fatty
acylated, as shown by incorporation of radioactive palmitate,
formed
multimers that were the same size as those formed by normal
PulD, and
was protected from degradation by PulS (data not shown).
However, the
protein was unable to substitute for PulD. Replacement
of the
lipoprotein signal peptide by the classical signal peptide
from the
periplasmic MalE protein restored function (data not
shown). Therefore,
tethering of the N terminus of PulD to the
outer membrane causes loss
of function but not loss of multimerization.
This result is of interest
with respect to recent data (
23)
suggesting that the N
terminus of PulD is sequestered within the
barrel formed by the
domain (
24).
PulD-OutD and OutD-PulD chimeras.
PulD cannot be replaced by
OutD (14, 26), which is in line with data indicating that
OutD could be a determinant of substrate specificity for the E. chrysanthemi secreton (19, 36). To analyze the proposed
role of the N domains of OutD and PulD in substrate recognition
(36), we used naturally existing restriction endonuclease
sites or restriction sites created in pulD (pCHAP3635) and/or outD (pCHAP3608) by site-directed mutagenesis to
construct genes coding for secretin chimeras. We expected that
pulD would be complemented only by secretin chimeras in
which the N domain was derived from PulD. In fact, the data revealed
the opposite to be true. Table 3
summarizes results obtained with clones that encoded secretin chimeras
that had the expected size and did not show evidence of proteolysis in
strains producing PulS. The following points are worthy of particular
mention.
(i) PulD multimers are more resistant than those of OutD to
dissociation in SDS. Secretin chimeras that exhibited PulD-like
resistance to dissociation invariably included the entire
and
S
domains of PulD. This result underscores the critical importance
of the
domain in multimer stability in
SDS.
(ii) Yields and multimer stability were not affected by the presence of
secreton components other than PulS (data not
shown).
(iii) Only four of the constructs complemented
pulD in
PAP7447. One of them, O
273P, (see Table
3 for notations) in
which
almost the entire N domain is derived from OutD was only
partially
active in this complementation test. The other three
(O
158P, P
67O
158P,
and
P
158O
273P), including chimeras containing the
OutD region
from positions 66 to 116 that is part of the exoprotein
binding
site (
36), were fully active and were the only ones
that were
present mainly as SDS- and heat-resistant
multimers.
(iv) The gene fusions were unable to complement
pulC or
pulG mutations. Thus, complementation of
pulD is not due to nonspecific
permeabilization of the
outer
membrane.
These data suggest that the N domain of OutD is not specific to the Out
secreton or to exoproteins secreted by it, as might
be expected from
the data of Shevchik et al. (
36). Instead,
the
domain of
PulD seems to be specifically required for the
secretin chimeras to
promote pullulanase secretion. Whether the
partial activity of the
O
273P chimera and the complete absence
of activity of the
other chimeras lacking the PulD
domain is
because of lower yields,
drastically reduced multimerization (only
trace amounts of
heat-resistant multimer were detected for O
273P
and
O
417P), incorrect assembly into the outer membrane, or the
absence of PulA recognition or interaction with another secreton
component cannot be
determined.
Next, we sought direct evidence of a direct interaction between
pullulanase and PulD using procedures very similar to those
used to
demonstrate the interaction between pectate lyase and
OutD
(
36). First, far-Western blotting experiments failed to
demonstrate any interaction between multimeric or phenol-dissociated
PulD renatured on nitrocellulose membranes and PulA. Second,
cofractionation
experiments failed to reveal any specific association
of a soluble
(alipo-) form of pullulanase and outer membranes
containing PulD.
Third, PulA did not protect PulD from degradation in
the absence
of PulS (which normally prevents PulD proteolysis by
unidentified
endogenous proteases [
13]). In addition,
different forms of
pullulanase were immobilized on specific resins and
were tested
for their ability to trap PulD in detergent-solubilized
extracts
of outer membranes. Again, none of these assays provided any
indication
of a specific interaction between PulA and PulD. Therefore,
the
possible contribution of PulA recognition PulA by PulD to secretion
specificity is
questionable.
| |
DISCUSSION |
On the basis of the data presented here and comparisons with other
secretins, we propose that PulD is composed of two major domains, an
N-terminal (N) domain predicted to face the periplasm and a domain.
The domain of the PulD is defined on the basis of two features.
First, it is the only region of the protein predicted to contain
amphipathic strands that, by analogy with porins, are likely to be
embedded in the outer membrane. Second, proteolysis of the purified
protein leaves the domain intact as a stable dodecameric complex
(23). The domain is followed by a short domain (S) to
which the pilot protein PulS binds to facilitate insertion of PulD into
the outer membrane (4).
The domain of the PulD homologue XcpQ was predicted to include 13 potential amphipathic TM strands according to an algorithm based on
the known structures of outer membrane porins (1) (Fig. 1).
There are unlikely to be 13 TM segments in PulD, because the N and S
domains probably both face the periplasm. Ten of the 13 predicted TM
segments of XcpQ are relatively well conserved in PulD and its other
homologues, two (numbers 3 and 5) are moderately conserved, and one
(number 4) is poorly conserved. Furthermore, two of the 13 predicted TM
strands (5 and 12) are not predicted to be in configuration
according to most commonly used structure prediction algorithms
(3). No other strands were predicted in the domain
by these algorithms. On the basis of these analyses, there appear to be
10 TM strands in the domain (numbers 1, 2, 3, 6, 7, 8, 9, 10, 11, and 13). Importantly, all predicted TM segments are downstream from
the proteolysis cleavage site that defines the border between the N and
domains in PulD (23) and are upstream from the
less-well-conserved region that corresponds to the S domain in PulD
(Fig. 1). Surprisingly, the entire domain contains only one
well-conserved aromatic amino acid (F573 in PulD, on the C-terminal end
of predicted TM12), whereas conserved aromatic residues, which form
belts at each end of the TM barrels, are conserved in other outer
membrane proteins such as porins (2). (Interestingly, F573
is absolutely conserved in all secretins, including those that are only
distantly related to PulD.)
Do the consequences of the insertion of 24-amino-acid peptides in PulD
conform to this prediction? Most of these insertions cause one of two
quite distinct changes to PulD: increased dissociation in SDS (or
reduced multimerization) (class C mutants) or complete absence of
multimers and reduced total yield (class D mutants) detected by
SDS-PAGE and immunoblotting (Fig. 2). The total absence of multimers in
class D mutants indicates that these insertions completely destabilize
the complex. Five out of 7 class D insertions (at aa 318 [number 12],
aa 441 [number 15], aa 465 [number 16], aa 507 [number 17], and
aa 533 [number 18]) are within or immediately adjacent to strongly
predicted TM strands (numbers 1, 6, 7, 9, and 10, respectively)
(Fig. 1) and would therefore be expected to disrupt packing of the strands. The two other insertions that abolished SDS-resistant multimer
formation (numbers 10 and 11) flank a highly conserved, relatively
hydrophobic sequence (282LVEVLTGIS290) that is
not predicted to have a structure by most algorithms and which is
upstream of the experimentally determined N terminus of the domain
(aa 298 [23]).
All of the other insertions except number 19 belong to class C. Most of
these insertions are in the N domain, but two are in the domain (at
aa 406 and 410) (Fig. 1). These insertions are in the relatively long
loop between the weakly predicted TM strands 4 and 5 and,
therefore, are likely to have less dramatic effects on packing of the
strands. Taken together, these results suggest that the correct
packing of the strands is required for multimer formation or
stabilization. Insertions elsewhere probably have less dramatic effects
because they do not affect packing of the strands and, presumably,
destabilize multimers or reduce multimer formation for other reasons.
Although the domain is important for multimer formation and/or
stability, it is not the only region of the protein involved in
multimerization because, when synthesized alone, it is exported and
interacts with PulS but does not form SDS-resistant multimers. However,
as will be reported elsewhere, when this domain is excised from
purified PulD multimers by proteolysis, it retains its multimeric structure (23). Two further experiments reported here
address the role of the N and domains in multimerization. First,
certain internal deletions in the domain did not completely abolish multimerization if the region downstream from aa 533 (including putative TM segments 11, 12, and 13) was retained (Fig. 4). This
result suggests that the C-terminal 106 aa of the domain might
possess the ability to multimerize alone or when fused to (part of) the
N domain. However, the multimers detected are considerably smaller than
would be expected for a dodecameric complex (Fig. 4). Second, isolated
N and domains stabilize each other and allow multimerization of the
domain (Fig. 5). However, these multimers are also smaller than
expected for a dodecamer and actually appear to increase in size when
heated to 100°C (Fig. 5). In both cases, the multimers detected might
represent intermediate steps in assembly of a full complex, in which
case we would conclude that both the N and domains are required for multimerization.
Studies of the OutD and PulD chimeras also contributed information on
the roles of the N and domains in multimer stability, since only
those chimeras that retained the PulD domain formed multimers that
resisted heat dissociation in SDS. However, the main purpose behind the
construction of these hybrids was to test the possibility that the N
domain of secretins determines which substrate will be secreted. We
found that a chimera in which almost all of the PulD N domain (up to aa
273) was replaced by the corresponding domain of OutD was still able to
complement the pulD mutation in strain PAP7447, whereas
chimeras in which the S domain was derived from OutD did not (Table
3). This result suggests that the N domain of PulD does not contain an
essential binding site for pullulanase or determine secretion
specificity. This is in contrast to OutD, for which there is evidence
that the N domain binds specific exoproteins (36). There are
several possible explanations for this striking inconsistency. First,
the N domain of OutD could recognize pullulanase as well as the range
of substrates normally secreted by the Out secreton of E. chrysanthemi. If this is the case, then the observed failure of
E. chrysanthemi to secrete pullulanase encoded by
pulA on a plasmid could be explained by a specific
requirement for PulC and by the inability of OutD to replace PulD due
to incompatibility between OutD and PulC. Second, specificity might be
determined by the domain of PulD. However, we did not obtain any
evidence for an interaction between PulD and pullulanase by any of
several different methods. Furthermore, pullulanase has no effect on
the conductivity of the channels formed by purified PulD in artificial
lipid bilayers (24). This leads to the third explanation,
which is that PulD does not recognize PulA. If this is the case, PulD
and OutD are more dissimilar than might be assumed from their highly
related sequences, and the inability of OutD or the chimeras containing
the domain of OutD to replace PulD in pullulanase secretion is
presumably explained by incompatibility with PulC. Since all other Pul
secreton components except PulC can be replaced by the corresponding
protein from the Out secreton without loss of pullulanase secretion
(19, 26), we tentatively conclude that PulC, rather than
PulD, participates directly in pullulanase recognition.
| |
ACKNOWLEDGMENTS |
We are grateful to Dominique Vidal-Ingigliardi for sequence
analysis and structure predictions; to Olivera Francetic, Nico Nouwen,
and Guillaume Vignon for providing access to unpublished data; to
Nathalie Nadeau for technical assistance; to Armelle Lavenir for
artwork; and to all other members of the secretion lab for their
interest and encouragement. We are also grateful to Michael Ehrmann for
the TnTAP system, for guidance on its use, and for providing
access to unpublished data; to Alan Collmer for pCHAP2236; and to Fred
Barras for providing CelZ antibodies.
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 fondamentale en Microbiologie et Maladies
infectieuses et parasitaires.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Unité de
Génétique Moléculaire, CNRS URA 1773, Institut
Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France. Phone: 33 (0) 145688494. Fax: 33 (0) 145688960. E-mail: max{at}pasteur.fr.
Present address: Institute of Infections and Immunity, University
of Nottingham, University Hospital, Nottingham NG7 2UH, United Kingdom.
| |
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Top
Abstract
Introduction
