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Journal of Bacteriology, October 2000, p. 5365-5372, Vol. 182, No. 19
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
andDepartment of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801
Received 9 March 2000/Accepted 11 July 2000
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ABSTRACT |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Bacteroides thetaiotaomicron, a gram-negative obligate anaerobe, utilizes polysaccharides by binding them to its cell surface and allowing cell-associated enzymes to hydrolyze them into digestible fragments. We use the starch utilization system as a model to analyze the initial steps involved in polysaccharide binding and breakdown. In a recent paper, we reported that one of the outer membrane proteins involved, SusG, had starch-degrading activity but was not sufficient for growth on starch. Moreover, SusG alone did not have detectable starch binding activity. Previous studies have shown that starch binding is essential for starch utilization. In this paper, we report that four other outer membrane proteins, SusC through SusF, are responsible for starch binding. Results of 14C-starch binding assays show that SusC and SusD both contribute a significant amount of starch binding. SusE also appears to contribute substantially to starch binding. Using affinity chromatography, we show in vitro that these Sus proteins interact to bind starch. Moreover, protease accessibility of either SusC or SusD greatly increased when one was expressed without the other. This finding supports the hypothesis that SusC and SusD interact in the outer membrane. Evidence from additional protease accessibility studies suggests that SusC, SusE, and SusF are exposed on the cell surface. Our results demonstrate that SusC and SusD act as the major starch binding proteins on the cell surface, with SusE enhancing binding. SusF's role in starch utilization has yet to be determined, although the fact that starch protected it from proteolytic attack suggests that it does bind starch.
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INTRODUCTION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Bacteroides thetaiotaomicron, a gram-negative obligate anaerobe, can utilize polysaccharides very efficiently as a source of carbon and energy. Early studies on polysaccharide utilization by B. thetaiotaomicron showed that the enzymes that break down these polysaccharides are cell associated, with most of the enzymatic activity located in the periplasm or cytoplasm. Subsequent studies revealed that binding of the polysaccharide to the cell surface prior to hydrolysis was an important step in polysaccharide utilization (1, 2). This strategy for polysaccharide utilization may allow the bacterium to sequester hydrolysis products more efficiently and may even allow it to affix itself to a polysaccharide-containing particle.
The process of polysaccharide utilization by Bacteroides
spp. has been best studied in the case of the starch utilization system
of B. thetaiotaomicron. A cluster of eight starch
utilization (sus) genes has been identified. One of the
genes encodes a regulatory protein (SusR). When cells are grown on
maltose or starch, SusR appears to activate the promoters of
susA and the susB-G operon. It is not known
whether maltose, the presumed inducer of sus gene expression, is bound to SusR or sensed in some other way. Three genes
in the cluster encode starch-degrading enzymes (SusA, SusB, and SusG).
SusG and SusA are neopullulanases that cleave starch into mono- and
disaccharides. SusB is an 
-glucosidase that acts on the products of
SusA and SusG. SusG has a very low activity compared to SusA. In fact,
only when susA was disrupted was it possible to detect SusG
activity (12). Despite its low activity, however, SusG is
essential for growth on starch. SusG is also the only one of these
enzymes that is exposed on the cell surface (12). SusA is a
periplasmic enzyme, and SusB has been tentatively localized to the
cytoplasm (1, 12).
SusC appears to be a porin that allows uptake of maltodextrins from maltose (G2) to maltoheptaose (G7), because a mutant producing only SusC but not SusD through SusG could grow as well as the wild type on maltodextrins. A mutant lacking SusC could grow on glucose and poorly on maltose or maltotriose but not on the higher maltodextrins. The fact that SusG in combination with SusC, but without SusD through SusF, was not sufficient to allow cells to grow on starch suggested that binding of starch and further processing of it were complex. That is, SusG was not simply degrading starch on the cell surface and releasing the products for uptake through SusC.
Previously, we found that SusG made little contribution to the binding of starch to the bacterial surface (12). Nor was SusG alone able to bind starch to the cell surface. Binding of starch appears to be mediated by one or more of the other outer membrane proteins (OMP) that have no detectable enzymatic activity (SusC, SusD, SusE, and SusF). SusC alone was not sufficient to bind labeled starch to the cell surface, but a combination of SusC and SusD allowed cells to bind about 70% of the starch bound by the wild type. Since a mutation in susC had a polar effect on susD, it was not clear whether SusD alone was sufficient for this starch binding or whether SusC was playing a significant role as well. Presumably, SusE or SusF or both were necessary to account for the full level of starch binding seen with wild-type cells. If SusC, SusD, SusE, and SusF were all involved in binding starch, one or more of them should be exposed on the cell surface. Similarly, if SusC is in fact a porin for oligosaccharides as the results of previous genetic experiments suggested, SusC should be surface exposed. In this paper, we report the analysis of surface accessibility of these starch binding proteins. Furthermore, we provide genetic and biochemical evidence that SusC, SusD, and SusE appear to interact with each other to bind starch.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Bacterial strains and growth conditions.
The bacterial
strains and plasmids used in this study are listed in Table
1. All Escherichia coli
strains used in this study were grown in Luria-Bertani broth or on
Luria-Bertani agar at 37°C. B. thetaiotaomicron 5482, transposon-generated derivatives, and single-disruption mutants used in
this study have been described previously (10). For
clarification purposes, polar disruption mutants of the Sus operon are
diagrammed in Fig. 1B.
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Chemicals.
14C-starch (Nicotiana
tabacum 1) was purchased from DuPont NEN. Amylopectin, pullulan,
proteinase K, n-octyl-
-D-glucopyranoside, and
phenylmethylsulfonyl fluoride were purchased from Sigma Corp.
DNA methods.
Isolation of plasmids was done using a Wizard
Plus DNA purification system (Promega Corp., Madison, Wis.).
Dephosphorylation reactions and restriction digests were performed in
accordance with the manufacturer's instructions (Bethesda Research
Laboratories, Bethesda, Md., or New England Biolabs, Beverly, Mass.).
Transformation of E. coli DH5MCR was done by the method
of Lederberg and Cohen (7). Constructs generated in E. coli were transferred to Bacteroides recipients as
described by Shoemaker et al. (13).
Membrane preparation. Membranes were prepared by the ultracentrifugation method of Valentine and Salyers (16). Cells were grown in a defined medium with maltose as the sole carbohydrate source (0.3%) to late log phase (optical density at 650 nm of 0.6 to 0.8). The cells were washed once with 20 mM potassium phosphate buffer (pH 7.2) and resuspended in 5 ml of the same buffer. These cells were disrupted by sonication. After the cell extract was separated from insoluble material by centrifugation, the whole membranes (both inner and outer membranes) were pelleted from the cell extract by ultracentrifugation (200,000 × g for 2.5 h at 4°C). The soluble fraction was collected, and the membrane pellet was washed once with 20 mM potassium phosphate buffer and pelleted again by ultracentrifugation under the same conditions. The membrane pellet was resuspended in 20 mM potassium phosphate buffer, and the membranes were dispersed by sonication.
Expression of susD in trans.
We wished to
express susD independently of the other starch-associated
OMPs. To achieve this, we cloned the susD gene downstream of
the susA promoter on a shuttle vector,
pNLY1::PsusA. This vector was used previously to express
susG in trans. This construct was designated
pSDC27. We introduced pSDC27 into 
susC, which does not
express any of the starch-associated OMPs, to create the strain susC(pSDC27). We also introduced this plasmid into
susD, a disruption mutant that produced SusC, but not
SusD through SusG, to determine if the resulting strain,
susD(pSDC27), acted similarly to susE in
terms of protein expression and binding characteristics. Using antisera
directed against SusC and SusD, we determined by immunoblot analysis
whether SusC and SusD were expressed in susC(pSDC27) and
susD(pSDC27). We also used immunoblot analysis to
confirm that the other Sus OMPs were not being produced in these
strains. Moreover, we determined that these mutants were not able to
grow on starch.
14C-starch binding experiments. To determine the contribution of various Sus OMPs to binding of starch to intact cells, we measured binding activities of various mutants using a modification of the procedure of Anderson and Salyers (1). Intact cells were used instead of membranes because previous studies showed that membranes isolated from cells bound much less labeled starch than intact cells (1). Cells were grown in a defined medium containing 0.3% maltose to an optical density at 650 nm of 0.5 to 0.6. The cells were pelleted by centrifugation and washed twice with phosphate-buffered saline (PBS) (pH 7.4) to dissociate any loose capsular material. The cells were resuspended in PBS to an optical density at 650 nm of 0.4. Subsequently, the cell suspensions were incubated in a mixture of 14C-starch and unlabeled amylopectin (200 µg/ml in PBS stock) for 5 min under aerobic conditions. Under aerobic conditions, the cells do not internalize or accumulate starch except for that initially bound (1). The 5-min time point is used for convenience, since we have found previously that harvesting cells at earlier or later times makes no difference in the amount of starch bound. Apparently, whatever starch is going to be bound is bound within the first minute, and the amount does not increase even with incubation times of several hours. Under aerobic conditions, there is no evidence for translocation and uptake of the starch molecules. Under anaerobic conditions, uptake can be demonstrated (1). To separate binding from uptake, we use the aerobic conditions.
The cells were harvested by centrifugation for 45 s, and the supernatant fluid was discarded. The cell pellet was washed twice with 500 µl of PBS without disrupting the cell pellet. After the washes, the cell pellet was resuspended in 100 µl of PBS buffer, transferred to 2 ml of scintillation fluid, and counted on a Beckman 600IS scintillation counter. In previous experiments, we had found that starch bound to the cells was bound tightly enough not to be dislodged by washing with buffer. Thus, the binding we are measuring is irreversible, and there is no evidence that this binding involves transport of the starch into the cell. B. thetaiotaomicron 4007 was used as a wild-type control because it contained the tetQ gene, which was used in the chromosomal insertional disruptions to generate the other mutants tested. We regard binding by the strainsusC as
nonspecific binding and subtracted these values from the binding seen
in other strains. Values are reported in micrograms of starch bound per
milligram of cell protein. These values were obtained by multiplying
the total counts per minute by a dilution factor, which was the ratio of labeled starch to total starch in each assay. That number was converted by an empirical constant (based on observed counts per minute
per given amount of starch) to disintegrations per minute, which
allowed the total micrograms of starch bound to be calculated by using
the reported values of 2.2 × 106 disintegrations per
min per µg of starch. Experimental values were standardized to the
whole-cell protein concentration, which was determined using a Bio-Rad
DC protein assay kit, using bovine serum albumin as a standard.
Proteolysis experiments. Accessibility to proteinase K was used to determine whether SusC, SusD, SusE, or SusF proteins were exposed on the cell surface according to the procedure of Shipman et al. (12). Cells were inoculated with 0.5 to 1.0 ml of an overnight culture of VPI-grown cells to 100 ml of defined media containing 0.3% maltose. These cells were grown to an optical density at 650 nm of 0.6 to 0.8 and harvested by centrifugation at room temperature. The cell pellet was washed twice at room temperature with 100 mM potassium phosphate buffer (pH 7.2) in order to dissociate any loose capsular material from the cells. Subsequently, the cells were resuspended in 9 ml of 100 mM potassium phosphate buffer. Fresh proteinase K (20-mg/ml stock) was added to a final concentration of 2 mg/ml. This high concentration of proteinase K was required to see any degradation of outer membrane proteins. The cells were incubated at 37°C with occasional mixing. Samples of 2 ml each were removed at various intervals. Phenylmethylsulfonyl fluoride was added to a final concentration of 10 mM for each sample to stop proteinase K activity. The cells were harvested by centrifugation and washed with 2 ml of a 100 mM potassium phosphate buffer-phenylmethylsulfonyl fluoride solution. The final cell pellet was resuspended in 100 mM potassium phosphate buffer.
After the cells were disrupted by sonication, the protein concentration was determined using a Bio-Rad DC protein assay kit with bovine serum albumin as a standard. Approximately 100 µg of protein from each sample was resuspended in Laemmli buffer and electrophoresed on a sodium dodecyl sulfate-8% polyacrylamide gel (SDS-8% PAGE). The gel was transferred to a Bio-Rad Trans-Blot nitrocellulose membrane, and SusC, SusD, SusE, and SusF proteins were detected using antisera directed against the corresponding protein. The secondary antibody used was a goat anti-mouse antibody conjugated with horseradish peroxidase supplied in the Bio-Rad Opti-4CN kit, which also supplied the detection substrate. As a control, we repeated the above-described procedure except for the addition of proteinase K to ascertain whether SusC, SusD, SusE, and SusF were stable during the incubation period in the absence of proteinase K. In some cases, long incubation times were used. This raises the question of whether after such long incubations the outer membrane was still intact. To confirm that the outer membrane was still intact, we routinely tested for the presence of a periplasmic marker, SusA (12). In all experiments shown here, SusA was intact and present at the same level at the end of the digestion process as at the beginning (data not shown). The SusA antibody was detected by using a Bio-Rad goat anti-rabbit-horseradish peroxidase Opti-4CN substrate kit. To determine if starch would protect the proteins from cleavage by proteinase K, we followed the procedure as outlined above but incubated wild-type cells with proteinase K and amylopectin (final concentration, 2 mg/ml).Affinity chromatography. To determine whether the Sus OMPs were acting as a complex, we tested their binding to an amylose resin mixture (New England Biolabs), which contained amylose covalently bonded to agarose beads. A volume of 10 ml of this resin was packed into a chromatography column. The resin was washed with 5 column volumes of MBP buffer (20 mM Tris, 20 mM NaCl, 20 mM EDTA) and used for the following experiments. After each experiment, the column was regenerated according to the manufacturer's instructions.
Cells were grown in 700 ml of a defined medium supplemented with 0.3% maltose. After reaching an optical density at 650 nm of 0.8 to 1.0, these cells were harvested by centrifugation. The cell pellet was washed once with 20 mM potassium phosphate buffer, and a membrane fraction was obtained according to the procedure of Valentine and Salyers (16). Membranes were resuspended in 20 mM potassium phosphate buffer and dispersed by sonication. The protein concentration was determined using a Bio-Rad DC assay kit with bovine serum albumin as a standard. This suspension was added to 0.1 M KPO4-0.15 M KCl buffer supplemented with 1.5% n-octyl--D-glucopyranoside to a concentration
of 5 mg of protein per ml of buffer. Previous work has shown that these
conditions release all the Sus proteins from the membranes
(10). After the membranes were solubilized, remaining
unsolubilized material was pelleted by ultracentrifugation, and the
solubilized proteins were collected for further purification. In one
experiment, membrane proteins from susC(pSDC27) were
incubated with membrane proteins from susD overnight at
4°C before loading onto the column.
Prior to loading of the proteins, the affinity matrix was washed with 1 column volume of MBP buffer supplemented with 0.75% n-octyl--D-glucopyranoside for detergent
equilibration with the sample to be loaded. This step was needed to
ensure that the protein remained solubilized in the column. Next, the
solubilized membrane protein (25 to 30 mg of cell protein) was loaded
onto the column, and the column was washed with 5 column volumes of MBP
buffer with 0.75% n-octyl--D-glucopyranoside
at an S/V ratio of 2 column volumes/h. This wash was collected for
further analysis. The proteins that remained on the column were eluted
with 5 column volumes of the same buffer, to which maltose had been
added (final concentration, 100 mM). This maltose eluant was collected
for further analysis.
The maltose eluant and wash were concentrated more than 50-fold by
tangential flow-filtration (Amicon Centri-prep concentrator MW 10,000).
The concentrated proteins were resuspended in Laemmli buffer and
electrophoresed on an SDS-8% polyacrylamide gel. The proteins were
transferred to a Bio-Rad Trans-Blot nitrocellulose membrane. This
nitrocellulose membrane was treated with antisera directed against the
appropriate Sus proteins using a goat anti-mouse-horseradish peroxidase Opti-4CN substrate kit.
In one experiment, solubilized membranes from a mutant that produced
SusC but not SusD and solubilized membranes from a cell that produced
SusD but not SusC were mixed and incubated prior to passage through the
column. The purpose of this experiment was to determine whether one of
these proteins was helping to fold the other one or whether their
cooperation in attaching to the starch column was more likely to be due
to interaction that allowed them to stick to the column, whereas they
were incapable of doing this alone.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Both SusC and SusD are needed for starch binding by intact
cells.
Previous 14C-starch binding assays had shown
that a mutant expressing only SusC and SusD (susE) had
approximately 70% of wild-type starch binding activity. A mutant
expressing SusC alone (susD) had very little starch
binding activity. It is important to note that this assay measures
tight binding of starch to the cell surface and does not involve
transport of starch into the cell. During our assays, the cells bound
starch almost immediately but did not continue to accumulate it over
time (1; present study). Nor do the cells appear to
lose starch over time, as would be expected if SusG, the OMP with
starch-degrading activity, were degrading and releasing starch from the
cells. That is, after several hours of incubation at 37°C, there is
no decrease in the amount of labeled starch bound to wild-type cells.
This suggests that whatever degradation of starch is carried out by
SusG under these conditions is coupled to retention of starch,
presumably by other proteins in the starch surface binding complex.
susC).
According to an immunoblot of the membrane fraction, this strain
produced SusD at a level about two times lower than the wild type (Fig.
1). In contrast, when susC was expressed from the chromosome
and susD was expressed from the plasmid, the SusD protein
was present at wild-type levels. This suggests a possible stabilizing
effect of SusC on the production or assembly of the SusD protein, which
we provide evidence for in a later section.
We used the strains expressing SusC and/or SusD to determine whether
SusD was sufficient for starch binding. As shown in Fig. 2, the strains expressing SusC only
(susD) or SusD only [susC(pSDC27)] had no
significant starch binding activity. However, strains expressing both
SusC and SusD [susE and susD(pSDC27)]
bound starch at about half of wild-type levels at saturating starch
concentrations (Fig. 2). Expression of susD in
trans from the plasmid [susD(pSDC27)] rather
than from the chromosome (susE) affected starch binding very little. The level of binding was somewhat lower when SusD was
provided from the plasmid, as expected from the lower level of SusD
produced by this strain (Fig. 2). This result confirmed that the clone
was complementing successfully the chromosomal disruption of
susD, even though the gene was present in multiple copies
and was somewhat underproduced on the plasmid. Thus, SusD is not
sufficient for starch binding, and both SusD and SusC are needed for
significant starch binding by B. thetaiotaomicron. Nevertheless, SusC and SusD are not sufficient for growth, since cells
expressing both OMPs but not SusE, SusF, and SusG did not grow at all
on starch.
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SusC and SusD interact in the outer membrane.
Since both SusC
and SusD were necessary for starch binding, it seemed likely that they
interacted with each other in the outer membrane. A first line of
evidence for such an interaction came from mutants expressing either
SusC (susD) or SusD [susC(pSDC27)] alone. The proteins in these mutants showed considerably different protease sensitivities than proteins in a wild-type cell which produced
all the starch OMPs. SusC and SusD, when expressed by themselves, were
degraded significantly after 30 min of proteolytic attack (Fig. 3B).
This was a much faster degradation than that seen in wild-type cells.
Addition of amylopectin during treatment with proteinase K did not
protect SusC and SusD when they were produced (data not shown). This
finding agrees with the 14C-starch binding data, which show
that SusC and SusD individually do not bind starch.
susD(pSDC27)], both proteins were not degraded as readily by proteinase K as was SusC or
SusD alone (Fig. 3B). This change in protease accessibility suggests
that SusE and SusF are not required for protection from protease attack
and that SusC and SusD interact to stabilize each other in the outer
membrane. This interaction also seems to be necessary for starch
binding, since cells only bind starch when both SusC and SusD are
expressed. Another explanation of the results of this experiment is
that SusC or SusD acts as a chaperonin to ensure the proper folding of
the other protein. In a later section, results are presented that argue
against this hypothesis and for an interaction that allows the two
proteins to bind starch as a multimer.
SusE and SusF contribute to starch binding.
Since SusC and
SusD accounted for only about half of wild-type binding activity, it
seemed likely that SusE or SusF or both would be responsible for the
rest. When SusE was produced along with SusC and SusD
(susF), the strain bound amounts of starch similar to
those bound by the wild type (Fig. 2). Thus, SusE was making some
contribution to starch binding. We had seen this same wild-type binding
activity in a strain expressing SusC, SusD, SusE, and SusF
(susG) (12). Thus, SusF seemed to play a
limited role, if any, in binding, according to this assay.
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SusC, SusD, and SusE interact in vitro to bind starch.
As
another approach to determine if starch OMPs interacted to bind starch,
we characterized these interactions biochemically using an
amylose-agarose column. To determine whether the starch binding OMPs
would bind to the column, solubilized membrane proteins from B. thetaiotaomicron were loaded onto the column. This experiment was
first performed on proteins from wild-type cells to determine the
selectivity of this method for the starch-associated OMPs. Proteins
that bound to the amylose resin were eluted with a high concentration
of maltose. Figure 5A shows that the
column bound mainly the starch-associated OMPs, SusC through SusG. A
few other faint bands of unknown identity were evident on SDS gels of
material eluted from the column, but SusC through SusG were the major
proteins. These maltose eluant proteins were confirmed as
starch-associated OMPs by immunoblotting, using antisera directed
against each protein (data not shown).
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susD) or SusD [susC(pSDC27)] alone were
run through the column, the corresponding protein was present in the
wash but not in the maltose elution fraction (Fig. 5B). Thus, when expressed individually, these proteins did not bind the starch column.
This finding is consistent with the results of the
14C-starch binding assay. Interestingly, when both proteins
were expressed either in the susD(pSDC27) or
susE strain, SusD was retained on the column. Most of
SusC washed through the column, although a very small amount relative
to the amount of SusD did appear in the maltose eluant (Fig. 5B). This
result suggests that SusC and SusD together interact in such a way as
to allow the protein(s) to bind to the starch column, but not as
effectively as in the wild type. This may help explain why the mutant
expressing just SusC and SusD did not bind starch at wild-type levels.
Additionally, when membrane proteins from the
susC(pSDC27) and susD strains were mixed
before loading onto the column, both SusC and SusD were retained by the
column (Fig. 5B). This provides evidence that these proteins, when
expressed individually, are folding properly and can interact with each
other in vitro but are not as effective a complex as the complete suite
of binding proteins in wild-type cells.
When SusE was expressed along with SusC and SusD (in the strain
susF), all three proteins were retained on the starch
column (Fig. 5B; SusE is not shown). In fact, SusC was now retained on the column, not eluted in the wash. These results provide additional evidence of interactions between SusC, SusD, and SusE. This overall enhancement of binding activity by SusE agrees with the results from
the 14C-starch binding assay, where SusE expression
enhanced binding by intact cells to nearly wild-type levels.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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We had shown previously that SusG is responsible for the starch-degrading activity detectable in the outer membrane fraction of B. thetaiotaomicron but did not contribute to starch binding by the cells (12). In this study we showed that SusC, SusD, and SusE together bind as much starch as wild-type cells. Evidence that these proteins form a complex comes from two sources. First, when either SusC or SusD was present in outer membranes without the other, no starch binding occurred. When both were present, cells could bind at 50% of the wild-type level. Moreover, SusC and SusD alone were each much more accessible to exogenously added proteinase K than when both were present. This finding suggests that SusC and SusD stabilize each other in the outer membrane.
A second line of evidence for complex formation was the finding that although SusC and SusD alone did not bind to a starch column, SusC and SusD together allowed SusD and some SusC to be retained on the column. The fact that SusC was not retained as efficiently as SusD could be a reflection of the fact that when these proteins are in a membrane, their interaction is stabilized, whereas in a solubilized form it is weaker. The surprising thing about this finding was that SusD interacted so strongly with the column when so little SusC was retained, whereas SusD alone did not bind the column at all. One possible interpretation is that SusC causes some conformational change in SusD or actually modifies SusD in some way that persists even when SusC is absent. We have seen no evidence for a covalent modification of SusD when SusC is present. That is, migration of SusD in SDS gels appears unchanged. An even more surprising finding was that mixing SusC and SusD from different strains before passing them over the column resulted in more binding of SusC to the column. This result shows that SusC is not acting as a chaperone for SusD or vice versa, because both proteins were properly folded enough to be stably maintained in the cell. Yet it is difficult to understand why mixing them after production and localization would lead to a more effective binding complex. What this does show, however, is that SusC and SusD can associate even in the solubilized form to form a complex that allows them to bind the starch column.
Results from the 14C-starch binding assay, the proteinase K accessibility experiments, and the in vitro column assay support the hypothesis that SusE plays a role in starch binding and may stabilize the SusC-SusD-SusE complex. With SusE present, binding of starch by intact cells was increased. Moreover, starch protected SusE from proteinase K digestion. Finally, in the in vitro column assay, SusE allowed SusC to be retained more efficiently along with SusD on the column. The role of SusF remains a mystery. Results from the starch binding assay suggest that its role in starch binding is minimal. Yet it is clearly exposed on the bacterial surface, and starch protected it from proteinase K digestion. One possible explanation for this result is that SusE, which does enhance binding, might have protected SusF simply by tethering the starch so that nearby SusF was partially protected. If SusF does play a role in starch binding, it appears to be a minor one. An unanswered question is why the cells need SusE and SusF at all, since SusC and SusD are sufficient to bind starch. These proteins do not add to the tightness of binding of starch to the cells, because binding to cells producing only SusC and SusD is just as irreversible as binding to wild-type cells. One possible explanation comes from considering what the binding assay does not measure: translocation of the starch. Under conditions used to measure binding, there is no accumulation of label by the cells after the initial binding step. Thus, the assay presumably measures binding independently of uptake and further utilization of starch. At present, there is no assay for the putative translocation step. Possibly SusE and SusF play a role in this step. Also absent from most of the mutants used in this study was SusG, one of the starch-degrading enzymes. SusE and SusF may play a role in interacting with SusG, which makes no contribution to starch binding.
Results of the protease accessibility experiments show that SusE and SusF are surface exposed. The data for SusC and SusD are less clear-cut. The fact that when SusC and SusD were produced separately in intact cells they were accessible to protease digestion suggests that they are surface exposed. When both are present, however, digestion by exogenous protease was minor in the case of SusC and not detectable in the case of SusD. SusC has many homologues in the B. thetaiotaomicron genome and in the Porphyromonas gingivalis genome (6, 11). The amino acid sequence of SusC, together with the fact that SusC is necessary and sufficient for utilization of intermediate-sized oligomers of glucose (9), suggests that SusC might be a porin. In this role, SusC would have to be surface exposed in order to admit the oligosaccharides to the periplasmic space.
Since SusD is important for binding long-chain starch, it would seem that this protein too should be exposed on the surface. It may be that the interaction of SusC and SusD, further stabilized by membrane components, produces a complex that renders SusD inaccessible to protease attack. This possibility is supported by the observation of protease accessibility in membranes isolated from disrupted cells. SusC in these membrane fragments was rapidly digested by proteinase K, while SusD was stable even after SusC had disappeared. Yet when SusC was not present at all, SusD was degraded completely, even in intact cells. As with the column data, this finding supports the possibility that SusD interacts with SusC.
The starch utilization system of B. thetaiotaomicron has unique features compared to other studied systems. The majority of components are involved in substrate attachment rather than hydrolysis. Other characterized surface-associated multiprotein complexes are composed mainly of enzymes and other proteins that help localize or assemble them to the complex. The complex that is most closely analogous to the starch utilization system is the cellulosome found in cellulolytic clostridial species. The cellulosome is a complex of cellulases and scaffolding proteins which is either secreted into the extracellular medium or embedded in the cell surface (3). For the cellulosome, the cellulose binding domains and catalytic sites are primarily on the same protein. There is one protein, CipC, which may play a role similar to that of the combination of SusC and SusD. It is thought to keep cellulose in a position favorable to enzymatic attack by other proteins (8). Nevertheless, in contrast to the Sus system, the cellulosome utilizes a majority of enzymes rather than noncatalytic binding proteins for its function.
The interactions of the Sus OMPs are very important for binding and presumably for translocation as well. A similar phenomenon can be found in the maltose transport system in the cytoplasmic membrane of E. coli. Three proteins act as a membrane-associated multiprotein complex to transport maltose into the cytoplasm: MalF, MalG, and MalK (4). Through similar protease accessibility experiments, Traxler and Beckwith found that MalF and MalG interact and assemble in the inner membrane to form a functional translocation complex with two copies of MalK (15). Our system differs in several aspects from this one. First, the complex is associated with the outer membrane. Second, it not only translocates but strongly binds and then cleaves a large substrate either before or during translocation into the periplasm. The starch utilization system of B. thetaiotaomicron appears to be the first example of an outer membrane-associated multiprotein complex that separates two functions, substrate binding and hydrolysis, using different proteins for each function.
Although the Sus proteins characterized to date mediate the early steps in starch utilization, there are clearly other proteins that should be part of the utilization process that have still not been identified. These include such proteins as the equivalents of E. coli MalF, MalG, and MalK. There may be redundant systems for maltose utilization in B. thetaiotaomicron, because to date no mutants have been found that fail to use maltose.
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ACKNOWLEDGMENTS |
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We thank Kyu Hong Cho for excellent technical advice on the affinity chromatography experiments as well as many helpful discussions. We also thank James Imlay for critical review of the manuscript and many helpful suggestions throughout this project.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, 601 South Goodwin Ave., Urbana, IL 61801. Phone: (217) 333-7378. Fax: (217) 244-8485. E-mail: abigails{at}uiuc.edu.
Present address: Department of Microbiology and Molecular Genetics,
Harvard Medical School, Boston, MA 02115.
Present address: Department of Biology, Jordan Hall, Indiana
University, Bloomington, IN 47405.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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AP, no amylopectin was added. The lanes are
labeled according to the time that had elapsed after addition of
proteinase K. In all cases, the amount of the periplasmic protein SusA
was the same at all stages of digestion, and no breakdown products were
detected (not shown).
