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Applied and Environmental Microbiology, November 1999, p. 5186-5191, Vol. 65, No. 11
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Epitope Identification for a Panel of
Anti-Sinorhizobium meliloti Monoclonal Antibodies and
Application to the Analysis of K Antigens and Lipopolysaccharides
from Bacteroids
Bradley L.
Reuhs,1,*
Samuel B.
Stephens,1
Daniel P.
Geller,1
John S.
Kim,1
Joshua
Glenn,1
Jessica
Przytycki,1 and
Tuula
Ojanen-Reuhs2
Complex Carbohydrate Research Center,
University of Georgia, Athens, Georgia
30602-4712,1 and Department of
Biosciences, Division of General Microbiology, SF-00014 University
of Helsinki, Finland2
Received 25 January 1999/Accepted 24 August 1999
 |
ABSTRACT |
In two published reports using monoclonal antibodies (MAbs)
generated against whole cells, Olsen et al. showed that strain-specific antigens on the surface of cultured cells of Sinorhizobium
meliloti were diminished or absent in the endophytic cells
(bacteroids) recovered from alfalfa nodules, whereas two common
antigens were not affected by bacterial differentiation (P. Olsen, M. Collins, and W. Rice, Can. J. Microbiol. 38:506-509, 1992; P. Olsen,
S. Wright, M. Collins, and W. Rice, Appl. Environ. Microbiol.
60:654-661, 1994). The nature of the antigens (i.e., the MAb
epitopes), however, were not determined in those studies. For this
report, the epitopes for five of the anti-S. meliloti MAbs
were identified by polyacrylamide gel electrophoresis-immunoblot
analyses of the polysaccharides extracted from S. meliloti
and Sinorhizobium fredii. This showed that the
strain-specific MAbs recognized K antigens, whereas the strain-cross-reactive MAbs recognized the lipopolysaccharide (LPS) core. The MAbs were then used in the analysis of the LPS and K antigens
extracted from S. meliloti bacteroids, which had been recovered from the root nodules of alfalfa, and the results supported the findings of Olsen et al. The size range of the K antigens from
bacteroids of S. meliloti NRG247 on polyacrylamide gels was altered, and the epitope was greatly diminished in abundance compared to those from the cultured cells, and no K antigens were detected in
the S. meliloti NRG185 bacteroid extract. In contrast to
the K antigens, the LPS core appeared to be similar in both cultured cells and bacteroids, although a higher proportion of the LPS fractionated into the organic phase during the phenol-water extraction of the bacteroid polysaccharides. Importantly, immunoblot analysis with
an anti-LPS MAb showed that smooth LPS production was modified in the bacteroids.
| |
TEXT |
Gram-negative bacteria of the family
Rhizobiaceae participate in a mutualistic symbiosis with
legumes. The infection process is initiated by an exchange of signal
molecules in the form of plant-derived flavonoids and bacterial Nod
factors (5). In the course of infection, the bacteria
undergo morphological changes, which result in the inclusion of highly
differentiated cells, termed bacteroids, in the root nodules of the
host plant. Although there is little information available on specific
changes in the cell surface chemistry of Sinorhizobium spp.
during infection and differentiation, Olsen et al. (10, 11)
used monoclonal antibodies (MAbs) in enzyme-linked immunosorbent assays
(ELISAs) and immunofluorescence studies of whole cells to show that
unidentified strain-specific antigens on the surface of cultured cells
of Sinorhizobium meliloti were diminished or absent in
bacteroids recovered from alfalfa nodules. In contrast, certain common
antigens were not affected by bacterial differentiation. In this study,
we determined the nature of the antigens and used the MAbs in analysis
of bacteroid extracts.
A recent report showed that capsular polysaccharide (K antigens) and
lipopolysaccharide (LPS) are important surface antigens of
Sinorhizobium spp. (16). S. meliloti
and Sinorhizobium fredii typically produce two forms of LPS:
rough LPS (R-LPS), which consists of a lipid A membrane anchor and
conserved core oligosaccharides, and smooth LPS (S-LPS), which includes
the O antigen (or O polysaccharide), and past studies have shown that
the core oligosaccharides are structurally similar in both the R-LPS
and the S-LPS of Sinorhizobium spp. (17). There
is limited variation in O-polysaccharide structure among
Sinorhizobium strains, and when present, the S-LPS migrate as two or three distinct bands in polyacrylamide electrophoresis (PAGE)
analyses. Characterization of two forms of S-LPS from S. fredii USDA205 showed that the primary O antigen is a glucan and a
secondary O antigen is a xylomannan (17). In this regard, Sinorhizobium spp. are unusual, as the O antigens of most
gram-negative bacteria are highly variable, strain-specific surface
antigens (19); in this genus, that role is fulfilled by the
K antigens.
The K antigens of Sinorhizobium spp. are major
strain-specific antigens, which commonly consist of small repeating
units of a hexose and 1-carboxy-2-keto-3-deoxy sugars, such as sialic
acid or 3-deoxy-D-manno-2-octulosonic acid
(Kdo), although they vary in glycosyl residue composition, substitution
patterns, linkage points, anomeric configuration, and size range
(6, 14-16). The K antigens of rhizobia do not possess a
lipid anchor and can be separated from the LPS by preparative PAGE,
based on the presence of the lipid A moiety on the LPS (8).
The LPS and K antigens can also be identified in PAGE analyses by
differential staining and by the use of nondetergent gels (8, 9,
15). Past studies have shown that the K antigens, as produced by
cultured cells, may be involved in an early recognition step in
S. meliloti-alfalfa interactions (1) and that
K-antigen production is affected by plant-derived signals (17,
18). This suggests that K-antigen expression is tightly regulated
by the bacterium and that it may be modified in the endophytic cell.
We employed PAGE-immunoblot analyses of extracted polysaccharides to
identify the epitopes for a panel of monoclonal antibodies (MAbs) that
were generated against whole cells of S. meliloti by Olsen
et al. (10, 11). We found that three strain-specific MAbs
recognized the K antigens of the homologous strains and that two
strain-cross-reactive MAbs recognized the LPS core. Three of the MAbs
were then used in the analysis of the polysaccharides extracted from
bacteroids of S. meliloti NRG247 and S. meliloti NRG185, which were recovered from alfalfa nodules. The results showed
that the K antigens produced by the S. meliloti NRG247 bacteroids were greatly diminished in abundance and had altered mobilities on polyacrylamide gels, and no K antigens were detected in
the polysaccharide preparation from S. meliloti NRG185
bacteroid. In contrast, the LPS core production did not appear to
be significantly modified in the endophytic bacteria, although the
S. meliloti NRG185 bacteroids were shown to produce distinct
forms of S-LPS.
Note that the terms "strain-specific" and
"strain-cross-reactive" were used in the previous reports (10,
11), so they are used in this report. However, these are relative
descriptions, as the strain-specific MAbs recognize a limited number of
other strains, and the strain-cross-reactive MAbs recognize most but not all S. meliloti strains.
Epitope identification for the anti-S. meliloti
MAbs.
The strains used in this study are described in Table
1. Cells were stored at 
70°C in 7.5%
glycerol and cultured in tryptone-yeast extract broth (S. meliloti) or yeast extract-mannitol (S. fredii) at
24°C, as previously described (15, 17). Prior to
immunoblot analysis, the polysaccharides extracted from the cultured
cells of each strain were analyzed by deoxycholic acid-PAGE, using 18% acrylamide gels, as previously described in detail (16), to determine the relative mobilities of the LPS and K antigens (data not
shown). The cell-associated polysaccharides from cell pellets of 3-ml
cultures were extracted by a mini-phenol-water method (16);
the cultures were grown to the same point (optical density at 600 nm
[OD600], 0.8 to 1.0), and the polysaccharide preparations were dissolved in identical volumes of PAGE sample buffer. The identity
of each bacterial product (R-LPS, S-LPS, and K antigens) on
polyacrylamide gels has been unequivocally established in past studies
(6, 9, 12, 14-18). The R-LPS migrates as a disperse banding
region, consisting of multiple bands (due to heterogeneity in the
core), and the S-LPS migrates as two or three low-mobility bands
(16). In contrast, the K antigens, which are polymers of
conserved repeating units, migrate in distinct ladder patterns (6,
15, 16). The PAGE migration patterns of each component were also
verified for this study by differential staining. Only the LPS appeared
on silver-stained gels, and a specific Alcian blue-silver stain
procedure that omits the oxidation step visualized only the K antigens
(3, 15). Furthermore, only the K antigens, which lack a
lipid moiety, exhibited normal migration in nondetergent polyacrylamide
gels, whereas the LPS appeared as a smear at the top of the lane (data
not shown).
Initial immunoblot analyses were performed on the extracts from a
subgroup of
S. meliloti strains used in the original work
of
Olsen et al. (
11) (Table
1). The LPS and K antigens were
separated on polyacrylamide gels and blotted to Nytran
+
membranes (Schleicher and Schuell, Keene, N.H.) with a Trans-Blot
SD
apparatus (Bio-Rad), as previously described (
16).
Individual
strips (lanes) were probed with each of five MAbs (Table
2),
which were provided by Perry Olsen
(Agriculture Canada, Beaverlodge,
Alberta, Canada). Examples of the
immunoblots are shown in Fig.
1 (not all
negative results are shown). The MAbs were delineated
into two classes: (i) the strain-specific MAbs that recognized
the K
antigens (MAbs 7 to 9) and (ii) the strain-cross-reactive
MAbs that
recognized LPS (MAbs 10 and 11). We were unable to determine
the
epitope for a third strain-cross-reactive MAb (MAb 6) described
in the
report of Olsen et al. (
11).
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FIG. 1.
Immunoblot analysis of the polysaccharide extracts from
cultured cells of S. meliloti. The immunoblots were probed
with the MAbs shown in Table 2. The MAb designations are given above
the lanes. The strains are identified in abbreviated form beneath the
lane as follows: 23, S. meliloti NRG23; 34, S. meliloti NRG34; 43, S. meliloti
NRG43; 53, S. meliloti NRG53; 133, S. meliloti
NRG133; 185, S. meliloti NRG185; 247, S. meliloti
NRG247; 282, S. meliloti NRG282; 286, S. meliloti
NRG286; 289, S. meliloti NRG289. The R-LPS, S-LPS, and
K-antigen labels identify the antigens bound by the MAbs (the S-LPS and
K antigens comigrate on polyacrylamide gels, so those regions overlap).
Only a few examples of negative results are shown.
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|
Anti-K antigen MAbs.
MAbs 7, 8, and 9 each recognized a ladder
pattern on the immunoblots, which is due to the K antigens (6,
14-18). Of the strains tested in this study, MAbs 7 and 9 were
positive only with the K antigens from the homologous strains (i.e.,
the strains used to generate the MAbs), S. meliloti NRG34
and S. meliloti NRG247, respectively (Fig. 1). Although
there was a marginal reaction of MAb 9 with S. meliloti
NRG286, this was probably not due to the primary K antigen, as the lane
was stained above the K antigen (compare MAb 9 with MAb 8 [Fig. 1]).
MAb 8 recognized the K antigens from both the homologous strain,
S. meliloti NRG185, and S. meliloti NRG286, which
is in agreement with the results of Olsen et al. (11).
In the ELISA of untreated cells performed by Olsen et al.
(
11), MAb 7 was also positive only with the homologous
strain
of the 60 strains tested, whereas MAbs 8 and 9 each recognized
19 of 60 strains; however, this included four strains that were
recognized by both MAbs 8 and 9 (
11). This observation is
interesting,
as the structures of the primary K antigens from
S. meliloti NRG185
and
S. meliloti NRG247 have been
determined, and they are quite
different (
16). The K antigen
from
S. meliloti NRG247 consists
of glucose and sialic acid
[-
-Glc
-NeuNAc-]
n and that
of
S. meliloti NRG185 contains
N-acetylglucosamine and Kdo
[-
-GlcNAc
-Kdo-]
n.
Therefore, the
cross-reactivity of MAbs 8 and 9 with some strains
must be due to
common structural features or similar secondary
K antigens, not
identical primary K
antigens.
Anti-LPS MAbs.
Strain-cross-reactive MAb 10 appeared to
recognize several forms of R-LPS, as shown by the disperse signal in
the R-LPS banding region, as well as the S-LPS from the same strains
(Fig. 1), showing that the epitope does not include the O-antigen
linkage point. Interestingly, the presence and abundance of the
highest-mobility S-LPS band (near the lower end of the region labeled
S-LPS in Fig. 1) vary from preparation to preparation, and it often
does not appear at all in Alcian blue-silver stained polyacrylamide gels (see reference 16). In this study, the band was
much fainter than the lower-mobility S-LPS bands on polyacrylamide gels
(data not shown) but yielded a much stronger signal in the immunoblot (Fig. 1).
In contrast to MAb 10, MAb 11 recognized only one major form (band) of
R-LPS from the serogroup A strains, and it did not
bind the S-LPS from
any strain. The latter phenomenon was not
due to a lower titer or
weaker signal, as overloaded blots, more-concentrated
antibody, and
longer blot development yielded much more signal
for the R-LPS and
still no binding of the S-LPS (data not shown).
In addition, MAb 11 had
negative reactions with
S. meliloti NRG23,
S. meliloti NRG53, and
S. meliloti NRG286, whereas MAb 10 yielded
a marginal reaction with the R-LPS from all three
strains.
A second series of analyses was performed with MAbs 10 and 11, using
S. meliloti strains from other sources (listed in Table
1)
as well as several
S. fredii strains and
Sinorhizobium sp.
strain NGR234 (data not shown). The
results of all immunoblot
analyses of LPS are summarized in Table
3. We found that MAbs
10 and 11 were
positive with the R-LPS from all strains previously
shown to be
positive with anti-Rm41 polyclonal antiserum (
16),
including
many strains of
S. fredii, and negative with all LPS
recognized by anti-Rf205 antiserum (
16). Thus, at least four
LPS core serogroups have been identified in the genus (
11,
16;
this report). Although Table
3 includes only those
strains that
have been tested with all MAbs and antisera, if the
results of
the ELISAs from Olsen et al. (
11) are included,
61 of 73 (84%)
strains tested fall into serogroup A, 4 strains are in
serogroup
B (5%), 3 strains are in serogroup C (4%), and the
remainder are
untyped.
Analysis of the K antigens and LPS from bacteroids.
Three of
the MAbs were then used in an analysis of polysaccharide preparations
from bacteroids of S. meliloti NRG247 and S. meliloti NRG185. For this analysis, alfalfa (Medicago
sativa cv. Apollo) seeds were sprouted in sterile 15-cm-diameter
plastic pots on 5-cm-deep coarse horticultural vermiculite (Schundler, Metuchen, N.J.) and 2-cm-deep Fafard Mix No. 3-B (Conrad Fafard, Agawam, Mass.), and the seeds were covered with 1-cm-deep vermiculite. The plants were grown in a controlled growth chamber with 14 h of
light (23°C) and 10 h of dark (18°C) and 70% humidity. The seedlings were inoculated 72 h after germination by applying 0.5 ml of bacterial culture (OD600, 0.8 to 1.0) along the base
(root pole) of the hypocotyl with a Pasteur pipette. Healthy, pink root nodules of similar size were then removed at 8 weeks postinoculation, and the bacteroids were recovered by crushing the nodules in a mortar
and pestle in 50 mM Tris-HCl, followed by filtration of the suspension
through glass wool (1 cm thick) in a 10-ml disposable syringe. The
eluted bacteroids were then pelleted, and the polysaccharides were
extracted with hot phenol-water, as described above (although convenient, collecting and freezing the nodules prior to crushing was
found to yield inconsistent results, so bacteroid recovery and
polysaccharide extraction were performed immediately after removing the
nodules from the plants). The polysaccharide preparations were then
dissolved in PAGE sample buffer to the same concentration as for the
preparations from cultured cells, and separate immunoblots were probed
with MAb 8, 9, or 10.
The results showed a significantly reduced signal with MAb 9 (anti-K
antigen) in the immunoblot analysis of the polysaccharide
preparation
from
S. meliloti NRG247 bacteroids compared to that
from
cultured cells, and the bacteroid K antigen migrated as a
low-mobility
smear, near the top of the lane (Fig.
2).
In contrast,
there was no detectable binding in the MAb 8 immunoblot
analysis
of the bacteroid polysaccharides from
S. meliloti
NRG185, suggesting
that there was no K antigen produced by the
bacteroids or that
it was structurally modified. These results fully
corroborate
those of previous studies (
10,
11), which showed
a marginal
reaction of MAb 9 with
S. meliloti NRG247
bacteroids (whole cells)
and no reaction of MAb 8 with
S. meliloti NRG185 bacteroids.
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FIG. 2.
Immunoblot analysis of the polysaccharide extracts from
alfalfa bacteroids of S. meliloti NRG185 (185b) and S. meliloti NRG247 (247b). The leftmost lane contains the extract
from cultured cells of S. meliloti NRG247 (247c) for
comparison. The immunoblots were probed with the MAbs shown in Table 2.
The two arrows indicate the migration positions of two
bacteroid-specific S-LPS banding regions not found in the analysis of
cultured cells (the fact that these bands were recognized by MAb 10 showed that it was LPS).
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The MAb 10 immunoblot of the bacteroid extracts from both
S. meliloti NRG247 and
S. meliloti NRG185 showed the
presence of
R-LPS with a mobility on polyacrylamide gels similar to
that of
R-LPS from the cultured cells (Fig.
2). This shows that the MAb
10 epitope is present in the R-LPS of bacteroids, indicating that
the
LPS core structure is similar in both cultured cells and bacteroids.
However, this did not preclude the possibility of changes in the
R-LPS
that do not affect the MAb 10 epitope; in fact, approximately
50% of
the R-LPS from the bacteroids fractionated into the phenol
phase during
the extraction compared to <5% in the extraction
of the cultured
cells, and the phenol-soluble bacteroid LPS was
also recognized by MAb
10 (data not shown). This explained the
relatively low response in the
immunoblot (compare Fig.
2 and
1) and suggested that there is some
difference in the fatty acid
content of the lipid A from the
bacteroid.
In contrast to the R-LPS, clear differences in the bacteroid-derived
S-LPS from
S. meliloti NRG185 were apparent in the
immunoblot
analysis. Interestingly, no S-LPS was detected in the
polysaccharide
preparation from the
S. meliloti NRG247
bacteroids (Fig.
2), showing
that specific bacteroid structures may
vary significantly from
strain to strain in the same host
plant.
In their analyses of endophytic sinorhizobia, Olsen et al.
(
11) found that of the 51 strains (untreated cells) that
were
positive for MAb 10 in ELISA, 50 gave a positive response with
bacteroids (98%), showing that the MAb 10 epitope in the LPS core
is
essentially unchanged in the bacteroids. Similar results were
obtained
in immunofluorescence studies (
10), in which MAb 10
showed
an equivalent affinity for cultured cells or bacteroids.
We have shown
that MAb 10 did, in fact, bind the extracted R-LPS
from bacteroids of
S. meliloti NRG185 and
S. meliloti NRG247.
These
results are in agreement with a study of "induced" cells
of
S. fredii (
17), which showed that the addition of
apigenin
or soybean root extract elicited changes in O-antigen and
K-antigen
production, but no changes were found in LPS core structure.
In
addition, the fact that much more of the R-LPS from the
S. meliloti bacteroids fractionated into the organic phase during
extraction
suggested that lipid A structure (fatty acid content) may
also
be modified during morphogenesis to the bacteroid state, without
affecting the MAb 10
epitope.
In contrast to the conserved production of the LPS epitopes in the
bacteroids, the immunofluorescence studies of Olsen et
al.
(
10) showed that the strain-specific epitopes (i.e., the
K
antigens) were diminished (
S. meliloti NRG247) or absent
(
S. meliloti NRG185) in bacteroids, and in ELISAs
(
11), the strain-specific
MAbs recognized a total of 35 strains (untreated cells), but only
2 of the 35 (6%) were positive
with the bacteroids (including
S. meliloti NRG247). We also
showed that the
S. meliloti NRG247
bacteroids produced K
antigens that were recognized by MAb 9,
although they were less
abundant and larger, whereas no K antigens
were detected with MAb 8 in
immunoblot analyses of the polysaccharide
extracts from
S. meliloti NRG185 bacteroids. Unfortunately, it
was not possible
with PAGE analyses of these preparations to determine
whether
structurally modified K antigen, which does not bind the
respective
MAbs, was produced by the bacteroids of either strain
because the
polyacrylamide gels showed an abundance of Alcian
blue-stained material
of unknown nature (data not shown). The
material may have been modified
K antigen, unrelated bacteroid-specific
polysaccharides, contaminating
plant-derived material, or some
combination of the three. We are
currently working towards improved
methods of bacteroid recovery to
overcome this
problem.
Other studies in progress have already shown that the analyses of
bacteroid structure will not be simple (
13). For example,
we
have found that changes in abiotic factors, such as temperature,
in
both the bacterial culture and plant growth conditions, also
result in
changes to the cell surface of the bacteria and bacteroids.
The use of
MAbs to study specific cell surface components from
the bacteroids of
Rhizobium leguminosarum (
2,
4,
7) have
yielded
interesting information about morphogenesis in the pea
symbiont,
including the temporal expression of specific epitopes
during nodule
development. We hope that our future studies of
bacteroids will provide
insight into the differences and similarities
in bacterial
morphogenesis in narrow-host-range symbiosis (
S. meliloti)
versus broad-host-range systems (
S. fredii), the latter
of
which includes both determinate and indeterminate host
plants.
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ACKNOWLEDGMENTS |
This work was supported in part by grant MCB-9728564 from the
National Science Foundation (to B. L. Reuhs) and by the U.S. Department of Energy-funded Center for Plant and Microbial Complex Carbohydrate Research (grant DE-FG02-93ER-20097). T. Ojanen-Reuhs was
supported by the Academy of Finland.
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FOOTNOTES |
*
Corresponding author. Mailing address: Complex
Carbohydrate Research Center, University of Georgia, 220 Riverbend Rd.,
Athens, GA 30602-4712. Phone: (706) 542-1216. Fax: (706) 542-4412. E-mail: breuhs{at}ccrc.uga.edu.
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Applied and Environmental Microbiology, November 1999, p. 5186-5191, Vol. 65, No. 11
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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