From the University of Manchester, 1.800 Stopford Building, School of Biological Sciences, Oxford Road, Manchester, M13 9PT, United Kingdom
Received for publication, September 7, 2000, and in revised form, November 14, 2000
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ABSTRACT |
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The cell surface expression of group 2 capsular
polysaccharides involves the translocation of the polysaccharide from
its site of synthesis on the inner face of the cytoplasmic membrane onto the cell surface. The transport process is independent of the
repeat structure of the polysaccharide, and translocation across the
periplasm requires the cytoplasmic membrane-anchored protein KpsE and
the periplasmic protein KpsD. In this paper we establish the topology
of the KpsE protein and demonstrate that the C terminus interacts with
the periplasmic face of the cytoplasmic membrane. By chemical
cross-linking we show that KpsE is likely to exist as a dimer and that
dimerization is independent of the other Kps proteins or the synthesis
of capsular polysaccharide. No interaction between KpsD and KpsE could
be demonstrated by chemical cross-linking, although in the presence of
both KpsE and Lpp, KpsD could be cross-linked to a 7-kDa protein of
unknown identity. In addition, we demonstrate that KpsD is present not only within the periplasm but is also in both the cytoplasmic and outer
membrane fractions and that the correct membrane association of KpsD
was dependent on KpsE, Lpp, and the secreted polysaccharide molecule.
Both KpsD and KpsE showed increased proteinase K sensitivity in the
different mutant backgrounds, reflecting conformational changes in the
KpsD and KpsE proteins as a result of the disruption of the transport
process. Collectively the data suggest that the trans-periplasmic
export involves KpsD acting as the link between the cytoplasmic
membrane transporter and the outer membrane with KpsE acting to
facilitate this transport process.
The export of capsular polysaccharides in Gram-negative bacteria
from their site of synthesis on the inner face of the cytoplasmic membrane onto the bacterial surface presents a unique challenge to the
micro-organism. It requires the translocation of a high molecular
weight negatively charged macromolecule across two lipid bilayers.
Understanding this process offers potential benefits in terms of
engineering polysaccharides of biomedical importance in bacteria
and in designing new antimicrobials that inhibit this process. In
contrast to protein secretion, our understanding of how capsular
polysaccharide transport is achieved is currently scant. The expression
of capsular polysaccharides (or K antigens) in Escherichia
coli offers an experimentally tractable system in which to try to
understand the mechanisms of polysaccharide transport. E. coli can express over 80 chemically and serologically distinct
capsular polysaccharides that have been divided into four groups
according to a number of biochemical and genetic criteria (1). Group 2 capsules resemble those found on the surfaces of Neisseria
meningitidis and Hemophilus influenzae and are often expressed by pathogenic E. coli isolates causing
extraintestinal disease (2). The biochemistry and genetics of
E. coli group 2 capsules, as typified by the K1 and K5
antigens, have been studied in most detail (reviewed in Refs. 1, 3, and
4). Group 2 capsules are synthesized by a hetero-oligomeric
membrane-bound biosynthetic complex on the inner face of the
cytoplasmic membrane by the sequential action of glycosyltransferases
that elongate the polysaccharide at its nonreducing end (5). A common
export pathway, irrespective of the repeat structure of the particular polysaccharide molecule, is then used to translocate group 2 capsules from their site of synthesis onto the cell surface (3, 6). Translocation across the cytoplasmic membrane is mediated by the KpsC,
M, S, and T proteins, whereas translocation across the periplasm and
outer membrane involves the KpsD and E proteins (1, 3, 4). The KpsC and
S proteins are believed to attach
phosphatidyl-Kdo1 to the
reducing end of the nascent polysaccharide chain, and this substitution
permits the entry of the polysaccharide molecule into the export
pathway (1, 3, 7). However, the enzymology of this process still awaits
elucidation. Translocation of the polysaccharide across the cytoplasmic
membrane is achieved by the KpsM and T proteins that constitute an
ABC-2 (ATP-binding cassette type 2) transporter (8), in which KpsM is
the integral membrane protein and KpsT is the ATPase (9-12). Typically
such ABC-2 transporters involved in polysaccharide export require two accessory proteins to complete the export process, a cytoplasmic membrane-periplasmic auxiliary protein (MPA), of which KpsE is an
example, and an outer membrane auxiliary protein (8). Based on computer
analysis, a predicted topology has been suggested for MPA proteins in
which the protein is anchored to the cytoplasmic membrane via N- and
C-terminal membrane-spanning domains separated by a large periplasmic
domain (8). Typically, outer membrane auxiliary proteins are modified
by the signal peptidase II to yield a lipoprotein (8) and, in the case
of group I capsular polysaccharides, have been shown to form aggregates
in the outer membrane (13). The role of KpsD as an outer membrane
auxiliary protein is less clear as it has only limited homology to
members of this family (8). In addition, KpsD has a typical N-terminal signal sequence and is believed to be a soluble periplasmic protein (14). As such its role in the export of group 2 capsular
polysaccharides is unclear.
In this paper we demonstrate that the C terminus of KpsE interacts with
the periplasmic face of the cytoplasmic membrane probably via an
amphipathic Bacterial Strains and Plasmids--
E. coli strains
used in this study are shown in Table I. Bacteria were grown in LB
broth supplemented with 100 µg ml DNA Procedures--
Recombinant DNA procedures were performed by
standard methods (17). Restriction endonucleases and DNA-modifying
enzymes were purchased from Roche Molecular Biochemicals or Life
Technologies, Inc., and used in accordance with the manufacturers'
instructions. Pfu polymerase (cloned version) was purchased
from Stratagene and used for amplifying DNA for cloning purposes.
Splicing by Overlap Extension (18) was used to generate plasmid pTH56
in which the C-terminal 29 amino acids of KpsE are fused onto the C
terminus of K5 Bacteriophage Sensitivity Assay--
Assays were carried out
as described previously (6).
Protein Analysis Procedures--
Cell lysates for SDS-PAGE were
generated by resuspending the pellet from 1 ml of culture in 25 µl of
SDS-PAGE sample buffer (19). Total cell proteins, ~50 µg per lane,
were separated by SDS-PAGE according to the method of Laemmli (19).
Proteins were either stained with Coomassie Blue or transferred to
Immobilon-P polyvinylidene difluoride membrane (Millipore) by Western
blotting as described by High et al. (20). Antibodies to
Spheroplast Formation and Protease Digestion of Periplasmic
Proteins--
Spheroplasts were prepared using the method of Rosenow
et al. (21). Proteinase K was added at a final concentration
of 500 µg ml Cell Fractionation--
Cells were fractionated to give
cytoplasmic membrane and outer membrane fractions using a modification
of the method of Maier et al. (22). A 400-ml culture was
grown to an A600 nm of 0.8 and cooled on
ice. Cells were washed with 10 mM Tris-HCl, pH 8.0, and
resuspended in 25 ml of 10 mM Tris-HCl, pH 8.0, 0.2 mM DTT, and 20 mg of DNase I were added. Bacteria were
disrupted by passing them three times through a French pressure cell at 20,000 pounds/square inch or by sonication (5-s bursts with 10-s intervals for 3 min) on ice, and unbroken cells and debris were removed
by centrifugation (7,000 × g for 15 min). The
supernatant was subjected to centrifugation (80,000 × g for 1 h at 4 °C), and the resultant supernatant
containing the cytoplasmic and periplasmic fractions was then decanted
and saved for further analysis. The pellet containing the membrane
fraction was washed with 20 ml of 10 mM Tris-HCl, pH 8.0, 0.2 mM DTT. The membranes were resuspended in ~1 ml of 10 mM Tris-HCl, pH 8.0, 0.2 mM DTT and layered on top of a two-step sucrose gradient (7.5 ml of 54% (w/v) sucrose on a
cushion of 3 ml of 70% (w/v) sucrose in 10 mM Tris-HCl, pH 8.0). The gradient was centrifuged at 100,000 × g for
16-18 h at 6 °C, and fractions were collected from the sides of the
tube with 23-gauge needles connected to 1-ml syringes and analyzed by
SDS-PAGE followed by Western blotting. Following trichloroacetic acid
precipitation the activity of the periplasmic enzyme alkaline phosphatase and the cytoplasmic enzyme malate dehydrogenase were assayed according to the method of de Maagd and Lugtenberg (23) to
check for contamination of the membrane fractions with cytoplasmic and
periplasmic enzymes. In VivoCross-linking with DSP--
Cross-linking with was
carried out as described previously (26).
Localization of the C Terminus of KpsE--
Previous attempts
using
To establish whether the hydrophobic C terminus of KpsE interacts with
either the cytoplasmic or outer membrane, plasmid pTH56 was generated.
This plasmid encodes the C-terminal 29 amino acids of KpsE fused in
frame onto the C terminus of wild type Chemical Cross-linking of KpsE and KpsD--
To determine the
interactions between KpsE and other proteins involved in capsule
transport, whole cells of strain MS101 (Table I) were treated with the chemical
cross-linking agent DSP. Strains with specific defects either in
capsule export or in the production of several major proteins in the
bacterial envelope (see below) were also analyzed in this way. The
cross-linked proteins were separated by SDS-PAGE followed by Western
blot analysis using antisera specific to KpsE and KpsD.
In MS101 four major cross-linked complexes were detected after
treatment with DSP when KpsE-specific antiserum was used (Fig. 3, panel A). The most
predominant cross-linked product had a molecular weight consistent with
that of a dimer of KpsE. The three other cross-linked products had
molecular weights of 110, 130 and in excess of 200 kDa (Fig. 3,
panel A). Continued incubation with DSP resulted in higher
molecular weight complexes that were difficult to resolve by SDS-PAGE
(data not shown). The same pattern of cross-linking was seen in mutants
lacking either KpsD, KpsM, KpsT, KpsS, or outer membrane proteins Pal,
Lpp, OmpT, LamB, OmpF, OmpA, and OmpC (data not shown) suggesting that
these proteins were not being cross-linked to KpsE. Cross-linking using
cells expressing kpsE as the only gene from the capsular
cluster resulted in the same pattern of cross-linking to that observed
in MS101 (data not shown) indicating that the probable dimerization of
KpsE is not dependent upon the expression of the polysaccharide
molecule or on the presence of other Kps proteins. In all cases the
cross-linking could be reversed by incubation in the presence of 50 mM DTT (data not shown). The ability of the purified KpsE
protein to dimerize in vitro (Fig. 3, panel B)
would suggest that the cross-linked complex at 86 kDa is likely to be
dimeric KpsE.
When KpsD-specific antiserum was used to analyze the cross-linked
products, a complex of 67 kDa was detected in MS101 but not in MSFE101
or MSCA102 (Fig. 4). Strain MSCA102 is an
isogenic lpp mutant of MS101 that lacks the 7-kDa major
outer membrane lipoprotein Lpp. However, Western blot analysis using
Lpp-specific antisera failed to detect any cross-linking of Lpp to KpsD
(data not shown). As such the 7-kDa protein that is cross-linked to KpsD cannot be Lpp, and its identity is as yet unknown.
Proteinase K Sensitivity of KpsE and KpsD in the Presence and
Absence of Other Proteins--
To demonstrate possible interactions
between KpsE and KpsD, their proteinase K sensitivity in different
mutant backgrounds was assessed by Western blot analysis following a
time course proteolysis of E. coli spheroplasts. After 30 min of incubation with proteinase K, no proteolysis of KpsD was
observed in spheroplasts from MS101 with limited proteolysis of KpsE
after 30 min (Fig. 5, panels A
and B). However, in MSFE101, where KpsE was absent from the
bacterial envelope, KpsD was quickly degraded by proteinase K (Fig. 5,
panel A). Likewise in strain MSTH101 (Table I) that lacks
KpsD, KpsE was very sensitive to proteinase K (Fig. 5, panel B). In MSCA102, lacking Lpp, KpsD was reproducibly more sensitive to proteinase K digestion than in MS101 (Fig. 5, panel A),
although compared with MSFE101, a significant proportion of KpsD was
still resistant to proteolysis (Fig. 5, panel A). In
contrast KpsE was rapidly degraded in MSCA102 (Fig. 5, panel
B). In all cases there was no appreciable difference in the
proteinase K sensitivity of periplasmic Detection of KpsD in the Bacterial Envelope--
In the E. coli Tol-PAL system the periplasmic protein TolB has been shown to
interact with the main peptidoglycan-associated outer membrane proteins
Pal, OmpA, and Lpp (28). This interaction results in the localization
of TolB both in the periplasm and the outer membrane (29). To establish
if KpsD also interacts with the cytoplasmic and outer membranes,
strains MS101, MSFE101, MSCA102, and MSCP101 were fractionated by a
two-step sucrose gradient to give cytoplasmic and outer membrane
fractions, which together with the soluble periplasmic fraction were
subjected to Western blot analysis (Fig.
6). In MS101, KpsD was detected in all
three fractions with the majority of the protein in the periplasm (Fig. 6). In MSFE101 lacking KpsE, KpsD was detected exclusively as soluble
protein in the periplasm with no protein detectable in either membrane
fraction (Fig. 6). In MSCA102 KpsD was distributed evenly in both
membrane fractions, with no soluble KpsD in the periplasmic fraction
(Fig. 6). In strain MSCP101 (Table I), which has all of the
polysaccharide export machinery but makes no polysaccharide, KpsD was
detected exclusively in the outer membrane fraction (Fig. 6).
The sensitivity of KpsE to carboxypeptidase demonstrates that its
C terminus must be exposed in the periplasm and not be located in the
cytoplasm as suggested previously for the MPA family of proteins (8).
In addition, the observation that the C-terminal 29 amino acids of KpsE
appear to be sufficient to localize A helical wheel plot (31) of the C-terminal 20 amino acids of KpsE
demonstrates the presence of a putative amphipathic The cross-linking data are consistent with KpsE functioning as a dimer,
and the ability to dimerize was independent of the presence of other
Kps proteins or the synthesis of capsular polysaccharide. The ability
of KpsE to dimerize is likely to be mediated by a predicted coiled-coil
structure present within KpsE (26). The cross-linking of KpsD to an
unidentified 7-kDa protein is curious. The observation that this
cross-linking was abolished in the absence of KpsE and Lpp suggested
that KpsE and Lpp were required for this interaction. One must be
cautious in assigning any specific role to Lpp in mediating this
interaction, since mutations in lpp are known to have
pleiotropic effects on the integrity of the periplasm (36), and this
could explain the differences in the pattern of KpsD cross-linking in
an lpp mutant. The failure to cross-link KpsE and KpsD to
each other could reflect that these proteins interact very transiently
and/or via the exported polysaccharide molecule, making it impossible
to detect the interaction with DSP cross-linking. It has been
demonstrated in type I protein secretion in Gram-negative bacteria that
the interaction between components in the secretion pathway is mediated
by the exported protein molecule (37, 38). Likewise, in the Tol system
for the uptake of colicins, it has been suggested that the colicin itself may provide a bridge between its outer membrane receptor and
periplasmic TolA protein (39). As such it is possible that a similar
situation exists in the transport of group 2 capsular polysaccharides,
whereby the exported polysaccharide molecule plays a key role in
linking the different components of the transport pathway together. The
alteration in the proteinase K sensitivity of KpsD and KpsE in the
different mutant backgrounds probably reflects conformational changes
in the KpsD and KpsE proteins as a result of the disruption of the
transport process. This supports the notion that these proteins are
likely to interact in the transport process.
Previously, KpsD has been assigned as a soluble periplasmic protein
involved in group 2 polysaccharide transport (40). The identification
of KpsD in both the cytoplasmic and outer membrane fractions suggests
that the trans-periplasmic export of group 2 capsular polysaccharides
requires the interaction of KpsD with both membranes as well as being
present in the periplasm. One interpretation is that KpsD may cycle
between the membranes moving polymer across the periplasm in the
process. In this model KpsD would engage with the polysaccharide as it
is exported across the cytoplasmic membrane by the KpsMT transporter
before docking with the outer membrane and transporting the
polysaccharide across the periplasm. Alternatively, some form of
trans-periplasmic export complex consisting of KpsD may be formed.
Which ever model is correct it would appear that KpsE is vital to allow
the interactions between KpsD and the respective membranes, and it is
possible that KpsE functions to bring the membranes together to
facilitate this process. The lack of soluble periplasmic KpsD in
MSCA102 suggests that in the absence of Lpp the export process is
disrupted such that either a trans-periplasmic complex cannot form or
KpsD cannot cycle between the two membranes. Whether this reflects a
specific role for Lpp in the transport process or is a consequence of
pleiotropic effects of the lpp mutation on the integrity of the periplasm are as yet unresolved. The lack of detectable KpsD in
either the periplasmic or cytoplasmic membrane fractions of strain
MSCP101, which is unable to synthesize K5 polysaccharide, indicates
that there is an absolute requirement for the polysaccharide molecule
to be engaged in the translocation process for the trans-periplasmic export pathway to be operational. In the absence of polysaccharide the
KpsD protein is detected in the outer membrane fraction. These results
support the notion that the polysaccharide molecule itself may mediate
interactions between proteins involved in the different stages of the
transport process and extends our earlier observations that there is a
direct coupling between polysaccharide biosynthesis and export (5).
The translocation of polysaccharide across the outer membrane is still
an area of conjecture. Unlike group 1 capsular polysaccharides, which
have an outer membrane pore-forming protein (13), no such protein is
encoded by group 2 capsule gene clusters. The observation that strain
BL21[DE3]omp8(pPC6), which lacks OmpT, LamB, OmpF, OmpA and OmpC
(41), is still able to make a K5
capsule3 would suggest that
these major outer membrane proteins do not function in this capacity.
Taken as a whole the data presented in the paper provide the first
information on the transport of group 2 capsular polysaccharides across
the periplasm and the roles of KpsE and KpsD in this process. The
identification of homologues of these Kps proteins in other Gram-negative pathogens (3) indicate these findings will be applicable
in the transport of capsular polysaccharides in these other medically
important micro-organisms.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix. By chemical cross-linking we demonstrate that
KpsE is likely to exist as a dimer and that dimerization is independent
of the other Kps proteins or the synthesis of capsular polysaccharide.
No interaction between KpsD and KpsE could be demonstrated by chemical
cross-linking although in the presence of both KpsE and Lpp, KpsD could
be cross-linked to a 7-kDa protein of unknown identity. In contrast,
both KpsD and KpsE showed increased sensitivity to proteinase K when
either KpsE or KpsD were absent. The KpsD protein was detected not only
in the periplasm but also in both the cytoplasmic and outer membrane
fractions, and the correct membrane association of KpsD was dependent
on KpsE, Lpp, and the secreted polysaccharide molecule. Collectively,
the data suggest that the trans-periplasmic export of group 2 capsular polysaccharide involves KpsD acting as the link between the cytoplasmic membrane transporter and the outer membrane with KpsE acting to facilitate this transport process.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
ampicillin, 200 µg ml
1 streptomycin, 25 µg ml
1 chloramphenicol, or 75 µg
ml
1 kanamycin as required. Plasmid pPC6
confers resistance to chloramphenicol and encodes for the expression of
the K5 capsule (15). Plasmid pBR322 (16) was used as a source of
periplasmic
-lactamase.
-lactamase. The primers
5'-GATCCGATCGATAACCCTGATAAATGCTTC-3' (SOE1A) plus
5'-CCAGCAGGCGATCAAATACCAATGCTTAATCAGTGA-3' (SOE1B) were used in PCR A;
the primer 5'-TATTTGATCGCCTGCTGG-3' (SOE1C) was used with primer
5'-GTGCTCATCGATTTAGTCTCGGTGATCTTC-3' (SOE1D) in PCR B. The reaction
products from PCRs A and B were combined in a ratio 1:1 and subjected
to PCR C using the primers SOE1A and SOE1D. The reaction product from
PCR C was digested with ClaI and inserted into the
ClaI site in pACYC184.
-lactamase and chloramphenicol acetyltransferase were obtained from
CP Laboratories; antiserum against KpsE was previously generated (21).
Antibodies to purified KpsD were generated in the
laboratory.2
1, and spheroplasts were
incubated at 37 °C for the appropriate time before the enzyme was
inhibited by the addition of trichloroacetic acid to a final
concentration of 20%. Carboxypeptidases A and B were added to final
concentrations of 200 and 100 µg ml
1,
respectively, and samples were incubated at 37 °C for 24 h
before the enzyme was inhibited by the addition of 20% trichloroacetic acid. After trichloroacetic acid precipitation, the pellet was washed
with acetone, dissolved in the appropriate volume of SDS-PAGE sample
buffer, and analyzed by SDS-PAGE followed by Western blotting.
-NADH oxidase activity was measured in all the
fractions from the gradient as a marker for the cytoplasmic membrane
fractions (24), and the level of Kdo was used as a marker for the outer
membrane (25). Membrane fractions were typically found to exhibit less
than 2% total alkaline phosphatase and 3% total malate dehydrogenase
activities indicating that they were essentially free of periplasmic
and cytoplasmic contamination. Cytoplasmic plus periplasmic fractions
were found to contain less than 4% of the total amount of Kdo and 5%
total
-NADH oxidase activity. Cytoplasmic membrane fractions were
found to contain less than 2% of total amount of Kdo, and outer
membrane fractions contained no more than 3% total
-NADH oxidase
activity, suggesting that these fractions were also predominantly free
from contamination from other fractions of the gradient.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-lactamase fusions as topology probes had established that
KpsE was anchored to the cytoplasmic membrane via an N-terminal
membrane-spanning domain located between residues 28 and 49 (21).
However, these studies failed to assign an unequivocal location for the
C terminus of KpsE and to establish whether the predicted C-terminal
transmembrane domain located between residues 362 and 379 spanned the
cytoplasmic membrane as predicted for MPA2 proteins (8). To determine
whether the C terminus of KpsE is exposed in the periplasm,
spheroplasts of the strain JM109[DE3] (pACYC184, pTH1), which
expresses full-length KpsE protein (26), were incubated with either
proteinase K or carboxypeptidases A and B. The digestion products were
analyzed by Western blot using antisera to chloramphenicol
acetyltransferase,
-lactamase, KpsE, or EnvZ (Fig.
1). The periplasmic enzyme,
-lactamase, was fully degraded by proteinase K and extensively
degraded by carboxypeptidases A and B (Fig. 1). The extra bands of
decreasing molecular weight seen in the presence of carboxypeptidases A
and B can be explained by the slow release by these enzymes of aspartic
acid, glutamic acid, and glycine residues, all of which occur in the
C-terminal 30 amino acids of
-lactamase. KpsE was also completely
digested by proteinase K, confirming the presence of a large
periplasmic loop (Fig. 1). The addition of carboxypeptidases A and B
resulted in a limited digestion of KpsE, leading to a predominant
truncated product ~1.0 kDa smaller than wild type KpsE (Fig. 1)
confirming that the C terminus of KpsE is exposed to the periplasm. No
degradation of the cytoplasmic enzyme chloramphenicol acetyltransferase
could be detected (Fig. 1), confirming the integrity of the cytoplasmic membrane. To confirm the carboxypeptidase results, Western blot analysis using antisera against EnvZ was performed. EnvZ is an integral
cytoplasmic membrane protein with cytoplasmic N and C termini with an
extended periplasmic domain (27). No degradation of EnvZ was detectable
following carboxypeptidase treatment (Fig. 1) confirming that the
limited degradation of KpsE is due to the action of
carboxypeptidase.
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Fig. 1.
Spheroplasts of strain JM109[DE3]
(pACYC184, pTH1) were digested with either carboxypeptidases A and B
(CP) or proteinase K (PK) as
described under "Experimental Procedures" before Western blot
analysis using antisera specific for chloramphenicol acetyltransferase
(panel A), -lactamase
(panel B), KpsE (panel C), or EnvZ
(panel D). The arrows denote either
chloramphenicol acetyltransferase (panel A), BlaM
(panel B), KpsE (panel C), or EnvZ (panel
D).-ve, spheroblasts were incubated without proteinase
K and carboxypeptidases A and B.
-lactamase. Cells of strain
PA360(pTH56) were fractionated by a two-step sucrose gradient to give
cytoplasmic and outer membrane fractions and subjected to Western blot
analysis (Fig. 2). As predicted, wild type
-lactamase encoded by pBR322 was located in the soluble periplasmic fraction (Fig. 2), whereas in the case of pTH56, the presence of the C terminus of KpsE resulted in the localization of a
significant amount of the
-lactamase fusion protein to the cytoplasmic membrane (Fig. 2). The exposure of this fusion protein in
the periplasm was confirmed by proteinase K treatment of spheroplasts prior to Western blot analysis. In both cases the proteins were digested by proteinase K, whereas no degradation of cytoplasmic chloramphenicol acetyltransferase was observed indicating that the
cytoplasmic membranes of the spheroplasts were intact throughout the
experiment (data not shown). The observation that plasmid pTH56
conferred resistance to ampicillin at levels greater than 100 µg
ml
1 and that the fusion was sensitive to
carboxypeptidase digestion (data not shown) confirms that the
-lactamase fusion protein is functional and is anchored to the
periplasmic face of the cytoplasmic membrane rather than being trapped
in an unfolded state in the cytoplasmic membrane. Therefore, the
C-terminal 29 amino acids of KpsE appear to be sufficient to localize
-lactamase, the archetypal soluble periplasmic protein, to the
cytoplasmic membrane, suggesting an intimate interaction between the
C-terminal part of KpsE and the cytoplasmic membrane. The lack of other
Kps proteins in strain PA360 would indicate that this association is
not dependent on the interaction between the C terminus of KpsE and
other Kps proteins.
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Fig. 2.
Western blot analysis of periplasmic
(P) and cytoplasmic membrane (CM)
fractions of strain PA360(pBR322) (panel A) and strain
PA360(pTH56) (panel B) using antisera specific
for -lactamase. The arrows
denote either
-lactamase (panel A) or the
KpsE-
-lactamase fusion protein (panel B).
Strains used in this study
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Fig. 3.
Panel A, Western blot analysis using
KpsE-specific antisera of DSP cross-linked cells. Lane 1, PA360; lane 2, MS101. Panel B, Western blot
analysis of purified KpsE. Lane 1, a Coomassie-stained gel
of purified KpsE; lane 2, a Western blot of the same
purified protein. Molecular masses are in kDa, and the
arrows denote monomeric and dimeric KpsE.
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Fig. 4.
Western blot analysis using KpsD-specific
antisera of whole cell lysates (panel A) and those
from cells first treated with DSP (panel B).
Lane 1, MSFE101; lane 2, MSCA102; lane
3, MS101. The cross-linked KpsD complex is shown by an
arrow.
-lactamase (data not shown)
confirming that the spheroplasts of each strain were equally permeable
to proteinase K.
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Fig. 5.
Western blot analysis using KpsD-specific
antisera (panel A) or KpsE-specific antisera
(panel B) following a time course digestion with
proteinase K of spheroplasts. Samples were extracted at different
time points during the digestion, precipitated with trichloroacetic
acid, and subjected to SDS-PAGE and Western blot analysis.
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Fig. 6.
Western blot analysis using KpsD-specific
antisera of periplasmic (P), cytoplasmic membrane
(CM), and outer membrane (OM)
fractions of strains MS101, MSFE101, MSCA102, and
MSCP101.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-lactamase, the archetypal
soluble periplasmic protein, to the cytoplasmic membrane suggests an
intimate interaction between the C terminus of KpsE and the cytoplasmic
membrane. The decreased sensitivity of KpsE to carboxypeptidase
digestion when compared with
-lactamase could be a consequence of
the close association of the C terminus of KpsE with the periplasmic
face of the cytoplasmic membrane, which makes the protein less
accessible to the carboxypeptidase enzymes. A similar situation has
been observed with TolR, the C terminus of which interacts with the
periplasmic face of the cytoplasmic membrane and which has reduced
sensitivity to carboxypeptidase digestion (30).
-helix with a
segregation of hydrophobic and hydrophilic residues on either side of
the helix (data not shown). It has been demonstrated that the
interactions between PBP5 (32), PBP6 (33), and TolR (30, 34) and the
periplasmic face of the cytoplasmic membrane are mediated by C-terminal
amphipathic
-helices in which hydrophobic residues are buried in the
membrane interior and the hydrophilic residues are located on the other
side of the helix and interact with the aqueous environment (35).
Therefore, it is likely that the interaction between the C terminus of
KpsE and the periplasmic face of the cytoplasmic membrane may be also
mediated by an amphipathic
-helix. Taken as a whole these data
establish the topology of KpsE, the archetypal MPA protein, and remove
any ambiguity about the location of the C termini of this family of
proteins involved in the transport of capsular polysaccharides across
the periplasmic space in Gram-negative bacteria.
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ACKNOWLEDGEMENTS |
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We thank Dr. J.-C. Lazzaroni for antiserum against Lpp and Dr. M. Inouye for antisera against EnvZ.
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FOOTNOTES |
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* This work was supported in part by the BBSRC, the Wellcome Trust, and the Lister Institute for Preventive Medicine.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Both authors contributed equally to this work.
§ Recipient of a Wellcome Travel fellowship.
¶ Recipient of an MRC quota studentship.
Recipient of a Royal Thailand Student'ship.
** To whom correspondence should be addressed: 1.800 Stopford Bldg., School of Biological Sciences, University of Manchester, Oxford Rd., Manchester M13 9PT, UK. Tel.: 44 161 275 5601; Fax: 44 161 275 5656; E-mail: ISRobert@fs1.scg.man.ac.uk.
Published, JBC Papers in Press, November 14, 2000, DOI 10.1074/jbc.M008183200
2 C. Arrecubieta, unpublished results.
3 C. Arrecubieta and I. S. Roberts, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: Kdo, 2-keto-3-deoxyoctonate; MPA, membrane-periplasmic auxiliary protein; DSP, dithiobis(succinimidyl propionate); PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; PCR, polymerase chain reaction.
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