Previous Article | Next Article 
Applied and Environmental Microbiology, December 1998, p. 4930-4938, Vol. 64, No. 12
Complex Carbohydrate Research Center,
University of Georgia, Athens, Georgia
30602-4712,1 and
Department of Plant
Pathology, University of Missouri, Columbia, Missouri
652112
Received 3 June 1998/Accepted 6 October 1998
Lipopolysaccharides (LPS) and capsular polysaccharides (K antigens)
may influence the interaction of rhizobia with their specific hosts;
therefore, we conducted a comparative analysis of Sinorhizobium fredii and Sinorhizobium meliloti, which are
genetically related, yet symbiotically distinct, nitrogen-fixing
microsymbionts of legumes. We found that both species typically produce
strain-specific K antigens that consist of
3-deoxy-D-manno-2-octulosonic acid (Kdo), or
other 1-carboxy-2-keto-3-deoxy sugars (such as sialic acid), and
hexoses. The K antigens of each strain are distinguished by glycosyl
composition, anomeric configuration, acetylation, and molecular weight
distribution. One consistent difference between the K antigens of
S. fredii and those of S. meliloti is
the presence of N-acetyl groups in the polysaccharides of
the latter. In contrast to the K antigens, the LPS of
Sinorhizobium spp. are major common antigens. Rough (R) LPS
is the predominant form of LPS produced by cultured cells, and some
strains release almost no detectable smooth (S) LPS upon extraction.
Sinorhizobium spp. are delineated into two major RLPS core
serogroups, which do not correspond to species (i.e., host range). The
O antigens of the SLPS, when present, have similar degrees of
polymerization and appear to be structurally conserved throughout the
genus. Interestingly, one strain was found to be distinct from all
others: S. fredii HH303 produces a unique K antigen,
which contains galacturonic acid and rhamnose, and the RLPS did not
fall into either of the RLPS core serogroups. The results of this study
indicate that the conserved S- and RLPS of Sinorhizobium
spp. lack the structural information necessary to influence host
specificity, whereas the variable K antigens may affect strain-cultivar interactions.
The cell surface polysaccharides of
rhizobia are believed to be involved in key aspects of symbiosis, such
as infection thread initiation and development, nodule invasion, and
host specificity; however, unlike those of the Nod factor (20,
34), the specific functions of the polysaccharides have not been
determined. The uncertainty about their functions is due in part
to a lack of structural data on the cell surface components of related
rhizobia. In order to develop a general model, we examined the
lipopolysaccharides (LPS) and capsular polysaccharides (K antigens)
from representative strains of Sinorhizobium fredii and
Sinorhizobium meliloti, which are genetically related, yet
symbiotically distinct, nitrogen-fixing microsymbionts of legumes
(13).
Cultured cells of Sinorhizobium spp. typically produce two
forms of LPS: rough (R) LPS, which consists of the lipid A membrane anchor and core oligosaccharide, and smooth (S) LPS, which includes an
O antigen (3, 5). The K antigens, in contrast, lack a lipid
anchor and are structurally distinct from the LPS (28). The
two types of cell surface polysaccharides can be identified on
polyacrylamide gel electrophoresis (PAGE) gels by differential staining
(29), and they can be separated from one another by preparative PAGE, based on the presence of the hydrophobic lipid A
moiety on the LPS (16).
In this report, we show that the K antigens of S. fredii and S. meliloti typically comprise small
repeating units of hexoses and
3-deoxy-D-manno-2-octulosonic acid (Kdo) or
other 1-carboxy-2-keto-3-deoxy sugars. The polysaccharides of each
strain are distinguished by size range and structural features, such as
glycosyl residue composition and substituents. These polysaccharides,
therefore, are strain-specific antigens. In contrast, the RLPS are
common antigens that are delineated into two major serogroups, which do
not correspond to species. Little or no SLPS is released from these
rhizobia upon extraction and, when present, the O antigens show similar
degrees of polymerization and lack structural variation.
Bacterial strains and growth conditions.
The bacterial
strains used in this study are listed in Table
1. Cells were stored at
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Sinorhizobium fredii and Sinorhizobium
meliloti Produce Structurally Conserved Lipopolysaccharides and
Strain-Specific K Antigens
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
70°C in 7.5%
(vol/vol) glycerol and cultured in yeast extract-mannitol
(S. fredii) or tryptone-yeast extract broth
(Sinorhizobium sp. strain NGR234 and S. meliloti) at 28°C, as previously described (29, 30).
TABLE 1.
Bacterial strains used in this study
Analysis of crude polysaccharide preparations. For PAGE analysis, the cell-associated polysaccharides were extracted from cell pellets (3-ml cultures) by a mini-phenol-water extraction technique adapted by B. L. Reuhs and J. S. Kim: prior to extraction, the cells are resuspended in H2O and pelleted to remove exopolysaccharides (EPS). Past studies have shown that no trace of EPS is found in the subsequent extract (27). The cells were pelleted (5 min; 10,000 × g) in a microcentrifuge tube (Dot Scientific, Burton, Mich.), and the supernatant was removed. The pellet was resuspended in 0.5 ml of solution A (0.05 M Na2HPO4 · 7H2O, 0.005 M EDTA, 0.05% NaN3, pH 7) and vortexed thoroughly; 0.5 ml of solution B (90% phenol) was added, and the suspension was vortexed. The tubes were placed in a 65°C heating block for 15 min, with thorough vortexing every 5 min, and then cooled in crushed ice for 5 min. After centrifugation (5 min; 10,000 × g), the water phase was removed, dialyzed (6,000- to 8,000-molecular-weight cutoff) against deionized H2O (dH2O), and freeze-dried. This fraction typically contains both the LPS and the K antigens. The phenol phase may also be dialyzed and analyzed, if necessary. The lyophilized material was then dissolved in 10 to 20 µl of PAGE sample buffer (see below).
Aliquots of 1 or 2 µl were analyzed by deoxycholic acid-PAGE, using 18% acrylamide gels. PAGE analysis was performed with a Bio-Rad Mini-Protean II cell, and the following protocol is specific for this equipment. The resolving gel (18%) contained 6 ml of A (30% [wt/vol] acrylamide, 0.8% [wt/vol] bisacrylamide), 2 ml of B (22.71 g of Tris base/75 ml of dH2O [pH 8.8], brought to 100 ml with dH2O), 2 ml of dH2O, 17.5 µl of 10% ammonium persulfate, and 8.75 µl of N,N,N',N'-tetramethylethylenediamine (TEMED). The stacking gel (4%) contained 0.33 ml of A, 0.5 ml of C (7.69 g of Tris base/75 ml of dH2O [pH 6.8], brought to 100 ml with dH2O), 1.67 ml of dH2O, 12.5 µl of 10% ammonium persulfate, and 6.25 µl of TEMED. The running buffer consisted of 21.7 g of glycine, 4.5 g of Tris base, and 2.5 g of deoxycholic acid in 1 liter of dH2O. The sample buffer contained 4 ml of C, 5 mg of bromphenol blue, and 2 ml of glycerol, brought to 20 ml with dH2O. The gels were prerun for 10 min (15.0 mA/gel) prior to the loading of samples and were run for 40 to 60 min (15.0 mA/gel) until the buffer front, not the dye front, reached the bottom of the gel. The gels were either silver stained for LPS (35) or alcian blue-silver stained for the K antigens (6, 29). For the alcian blue-silver stain, the gel was immediately immersed in 100 ml of alcian blue solution (0.005% [wt/vol] alcian blue in ethanol-acidic acid wash [40% ethanol, 5% acidic acid in dH2O]) and gently rocked for 30 min. This was followed by a change to 100 ml of fresh solution and overnight rocking. The gel was rinsed for 1 min in dH2O, oxidized in 100 ml of 0.7% sodium metaperiodate (in dH2O) for 10 min, and washed five times in 100 ml of dH2O for 5 min (minimum) each time. One milliliter of the final wash was added to 1 ml of the silver solution (see below); if a precipitate (ppt) formed, the gel was washed two more times. The gel was then stained in 100 ml of silver solution (10% Bio-Rad silver concentrate) for 10 min and rinsed in dH2O for 1 min. The gel was rinsed again in 50 ml of Bio-Rad developer (3.0 g/200 ml of dH2O) with agitation until dark ppt formed (about 20 s) and immediately drained to remove all ppt. The color was developed with the remaining developer (2 to 5 min), and the development was stopped in 50 ml of 5% acetic acid (30 s) followed by a 2-min rinse in dH2O. The developed gels were stored in dH2O. For serotype analysis, unstained PAGE gels of the cell extracts were immediately blotted to Nytran+ (Schleicher and Schuell, Keene, N.H.) with a Trans-Blot SD apparatus (Bio-Rad). The membranes were separately incubated with polyclonal rabbit antiserum raised against S. meliloti Rm41 or S. fredii USDA205 and then with goat anti-rabbit immunoglobulin G conjugated to alkaline phosphatase (Sigma, St. Louis, Mo.). The blots were developed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate (Sigma) for 5 to 15 min. Anti-Rm41 was provided by Dale Noel (Marquette University, Milwaukee, Wis.), and anti-Rf205 was produced by B. L. Reuhs as a graduate student in the Botany Department, Eastern Illinois University. Antiserum development and immunoblotting protocols have been described previously (25).Polysaccharide purification and analysis. For large-scale preparations, the polysaccharides were extracted from cell pellets (5-liter cultures) with hot phenol-water and the water phase material was dialyzed (against H2O) and freeze-dried. The RLPS, SLPS, and K antigens were isolated by size exclusion chromatography (Sephadex G-150 superfine; Pharmacia, Uppsala, Sweden) of the water phase material, as previously described (9, 29, 30, 32). The eluted fractions were assayed colorimetrically for 1-carboxy-2-keto-3-deoxy sugars by the thiobarbituric acid assay (36) and for hexose with phenol-sulfuric acid (38); LPS-containing fractions were identified by PAGE analysis. In some cases, there was minor contamination of the K-antigen preparation by SLPS; when necessary, the intact K-antigen fractions were subjected to polymyxin-agarose affinity chromatography (Detoxi-Gel; Pierce Chemical Co.). After addition of the sample solution (in H2O), the column was washed with H2O (flowthrough fraction), which eluted the K antigens, and the LPS was then eluted with 1% deoxycholic acid in H2O.
Composition analysis of the K antigens and RLPS was performed on the intact preparations (from G-150 chromatography) by gas chromatography-mass spectrometry (GC-MS) of the trimethylsilyl (TMS) methyl glycoside derivatives (38) with a 30-m DB1 fused silica column (J&W Scientific, Folsum, Calif.) on a model 5890A gas chromatograph-mass selective detector (Hewlett-Packard, Palo Alto, Calif.). Inositol was used as an internal standard, and retention times were compared to those of authentic monosaccharide standards. The relative ratios of the glycosyl residues from the RLPS were established compared to the
-OH-fatty acids of the lipid A, which are considered
constant within the genus. This showed that the glucose (Glc) content
was also a constant; therefore, the molar ratios were normalized to Glc.
Proton nuclear resonance (1H NMR) spectroscopy was
performed with a Brüker AM 250 spectrometer. The G-150-purified K
antigens were dissolved in 2H2O, and the
spectra were obtained at 296 or 316 K. Chemical shifts (reported as
parts per million) were established relative to acetone. Solvent
suppression in the analysis of the Sinorhizobium sp. strain NGR234 K antigen was achieved by low-energy presaturation of the 2HOH signal.
Fast-atom bombardment (FAB)-MS was performed with a JEOL (Tokyo, Japan)
SX/SX 102A tandem four-sector instrument, in the negative mode, using a
xenon FAB gun (6 kV) and an accelerating voltage of 10 kV. The scan
range was 300 to 3,000 m/z. The K antigens were partially
hydrolyzed with dilute acid (1% acetic acid at 100°C for 10 min),
which hydrolyzed the acid-labile ketosidic linkages of Kdo and sialic
acid and generated oligosaccharides of 1 to 4 disaccharide repeat
units. The oligosaccharides were then freeze-dried to remove the acidic
acid. A 1-µl aliquot of a saturated sample solution (in
H2O) was added to a matrix of thioglycerol (2 µl) and
applied to the FAB probe.
High-performance anion-exchange chromatography (HPAEC) of the LPS core
oligosaccharides was performed with a metal-free BioLC (Dionex Corp.,
Sunnyvale, Calif.) with a CarboPac PA1 anion-exchange column (4 by 250 mm) and a pulsed amperometric detector (PAD), as previously described
(29, 30). The core oligosaccharides were prepared for HPAEC
by mild acid hydrolysis (1% acetic acid at 100°C for 3 to 5 h)
of the RLPS, which cleaves the Kdo linkages, followed by centrifugation
to remove the insoluble lipid A.
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
K antigens. (i) PAGE analysis. The polysaccharide extracts from cultured cells of 16 strains of Sinorhizobium spp. were analyzed by PAGE-silver staining (Fig. 1), using an alcian blue prestain to visualize the acidic K antigens (compare Fig. 3). These polysaccharides commonly migrate in a ladder pattern, which is due to a sequential degree of polymerization of structurally constant repeating units. The K antigens of S. fredii USDA205 and S. fredii USDA257 have been shown to consist of disaccharide repeats (9, 29), and the polysaccharides from 9 of the other 14 strains appear to be similar. Although the size range of each is strain specific, the ladder patterns had comparable band separations. In contrast, the polysaccharides from S. fredii USDA201, USDA192, and USDA191 (Fig. 1A, lanes 3, 8, and 9, respectively) migrated with a relatively wide band separation, indicating that they consist of larger repeating units, and the K antigens of S. fredii HH303 and S. meliloti NRG133 (Fig. 1A, lane 7, and Fig. 1B, lane 1, respectively) migrated as "smears," with no obvious ladder patterns. Importantly, the PAGE analysis demonstrated that each of the 16 strains produce acidic polysaccharides that are unrelated to the LPS. The distinction of the K antigens from the LPS was also shown by nondetergent PAGE (not shown): the K antigens migrate into the gels, whereas the LPS do not (16).
|
(ii) Structural analyses. The K antigens from six Sinorhizobium strains were selected for further analysis: these strains included S. fredii USDA201 and S. fredii HH303, which yielded atypical PAGE migration patterns; S. meliloti NRG247 and S. meliloti NRG185, which had been used in interesting immunochemical studies by P. Olsen et al. (23, 24); Sinorhizobium sp. strain NGR234, a well-studied, broad-host-range organism; and S. fredii USDA208, which was randomly chosen. The K antigens were separated from the LPS by size exclusion chromatography of the phenol-water extracts (water phase), as described in past reports (9, 29); the intact polysaccharides were analyzed for glycosyl residue composition (by GC-MS) and by 1H NMR spectroscopy, and the repeat oligosaccharides, generated by mild acid hydrolysis, were analyzed by FAB-MS.
S. meliloti NRG247.
The K antigen from
S. meliloti NRG247 was found to contain equimolar
amounts of N-acetylneuraminic acid (NeuNAc; commonly termed
sialic acid) and Glc, and a repeating unit mass ion of m/z
470 (M-1 in negative-mode FAB-MS) confirmed that it consisted of
Glc-NeuNAc disaccharides. The NeuNAc was determined to be in the
configuration by 1H NMR analysis (Fig.
2A), with methylene proton resonances at 1.7 (H3a) and 2.8 (H3c) ppm; the Glc is also
linked, with an anomeric proton resonance at 4.96 ppm
and a small J1,2 coupling. The
N-acetyl methyl resonances are found at 1.98 ppm.
|
S. meliloti NRG185.
S. meliloti
NRG185 produces a K antigen consisting of disaccharide repeats of
N-acetylglucosamine (GlcNAc) and Kdo, as determined by
equimolar amounts of the glycosyl residues and a repeating unit mass
ion of m/z 440 (M-1 for GlcNAc-Kdo). 1H NMR
analysis (Fig. 2B) showed that the Kdo is linked, with characteristic resonances at 1.8 ppm (H3a) and 2.35 ppm
(H3e), and that the GlcNAc is also linked. The chemical
shift of the anomeric proton is 4.55 ppm, and the large
J1,2 coupling is also indicative of -linked
aldoses. The polysaccharide is N acetylated (2.0 ppm) on the
GlcNAc and O acetylated (2.1 ppm) at unknown locations.
S. fredii USDA208.
The K antigen of
S. fredii USDA208 contained Gal and Kdo in a 1:1
molar ratio and was determined to consist of Gal-Kdo
disaccharides by FAB-MS, with a repeating unit mass ion of
m/z 399. The 1H NMR spectrum (Fig. 2C) contained
resonances associated with -linked Kdo (1.8 and 2.4 ppm) and
-linked Gal (5.05 ppm). The off-scale resonance at 4.0 ppm is due to contamination.
S. fredii USDA201.
The K antigen isolated
from S. fredii USDA201 contained Kdo, Gal, and an
unidentified 2-O-methylhexose (2-O-MeHex) in
a 1:1 molar ratio of Kdo to the neutral sugars. Partial acid hydrolysis released the expected disaccharides of Gal-Kdo and
2-O-MeHex-Kdo (m/z 399 and 413, respectively),
but unlike the K antigens of S. fredii USDA205 and
S. fredii USDA257 (9, 29), a mass ion of
m/z 795 showed that both the Gal-Kdo and the
2-O-MeHex-Kdo disaccharides were present in the same
polysaccharide. Thus, the unusual PAGE banding pattern of the
S. fredii USDA201 K antigen (Fig. 1) is due to a
tetrasaccharide repeating unit, although it has not been
determined how the two disaccharides are linked in the
tetrasaccharide. The hexoses were determined to be linked by
1H NMR analysis (Fig. 2D), but the complexity of the
anomeric region (5.0 to 5.2 ppm) suggests that there may be
a nonlinear arrangement of the tetrasaccharides (possibly in
branches). The resonances associated with the -linked Kdo were also
complex (1.7 to 2.4 ppm).
Sinorhizobium sp. strain NGR234.
Only Glc was
detected in the GC-MS analysis of the Sinorhizobium sp.
strain NGR234 K antigen; however, the 1H NMR spectrum (Fig.
2E) contained the methylene proton resonances, at 1.65 (H3a) and 2.6 (H3e) ppm, of a
1-carboxy-2-keto-3-deoxy sugar. Two-dimensional NMR indicated that the
acidic sugar is -linked
N-acetyl-5,7-diamino-3,5,7,9-tetradeoxynonulosonic acid (pseudaminic acid;
Pse5NAc7NAc), which is labile to the (TMS methyl glycoside) derivitization process and mild acid hydrolysis (27). The
resonance at 1.1 ppm in the 1H NMR spectrum is due to the
C-9 (methyl) protons of the Pse5NAc7NAc. Due to numerous
N-acetyl groups (1.9 and 2.0 ppm) and O-acetyl groups (2.1 ppm), the polysaccharide was much less soluble than the
others, and this resulted in poor resolution; therefore, solvent suppression was required to resolve the anomeric resonance of the
-linked Glc, at 4.65 ppm, yielding a diminished signal shown in
Figure 2E. Integration showed that the Glc and Pse5NAc7NAc residues were present in a 1:1 molar ratio (27). Thus, the
polysaccharide consists of disaccharide repeats of Glc-Pse5NAc7NAc.
S. fredii HH303. The K antigen preparation from S. fredii HH303 was found to contain rhamnose (Rha) and galacturonic acid (GalA) in a 1:1 molar ratio. 1H NMR analysis (Fig. 2F) confirmed that no labile 1-carboxy-2-keto-3-deoxy sugars, such as Pse5NAc7NAc, were present. The resonance at 1.3 ppm is due to the C-6 (methyl) protons of the Rha component, and the polysaccharide was shown to be O acetylated (2.1 ppm). Strain HH303 is the only strain of S. fredii that has been found to produce an acetylated K antigen (references 9 and 29 and this report). The polysaccharide could not be degraded into subunits by dilute acid hydrolysis, so MS was not applied.
LPS. (i) PAGE and immunoblot analyses. The same polysaccharide extracts used in the PAGE analysis of the K antigens (Fig. 1) were subjected to PAGE-silver staining, omitting the alcian blue prestain (Fig. 3), for the analysis of the LPS. This revealed a common pattern of LPS migration for Sinorhizobium spp., including both the high-mobility RLPS and the low-mobility SLPS. When present, the SLPS of most strains commonly migrates in two distinct banding regions (S. meliloti Rm41 exoB [AK631] differs because the exoB mutation affects LPS production).
|
|
|
(ii) Composition analyses. In order to determine the basis for serogroup identity, further analyses were performed on the RLPS of selected strains from each of the major serogroups. The RLPS were isolated by size exclusion chromatography of the phenol-water extracts (references 9, 29, and 32 and this report). From glycosyl residue composition analysis, the RLPS were found to consist primarily of Glc, GalA, and Kdo, with lesser amounts of Gal, Man, and glucuronic acid (GlcA) (Table 3). There was some difference in the relative abundances of Gal and Man in the RLPS from the different strains, but it did not delineate the serogroups. However, there was a striking difference between the relative amounts of GalA in the RLPS from the Rf205 serogroup (S. fredii USDA205 and Sinorhizobium sp. strain NGR234) and those in the RLPS from the Rm41 serogroup (S. fredii USDA257 and S. meliloti Rm1021): the former contain approximately one less GalA residue per core (based on a comparison to the fatty acid content [see Materials and Methods]). The excess of Gal in the RLPS preparation of S. fredii USDA205 is probably due to minor contamination from the Gal-containing K antigen produced by that strain (29).
|
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Although closely related, S. fredii, S. meliloti, and Sinorhizobium sp. strain NGR234 exhibit distinct patterns of host specificity (7). S. meliloti has a relatively narrow host range, consisting solely of indeterminate nodule-forming hosts, including Medicago spp., Melilotis spp., and Trigonella spp. In contrast, S. fredii nodulates a diversity of hosts, including both determinate and indeterminate nodule-forming plants (26), yet it does not nodulate the host plants of S. meliloti. In addition, S. fredii-soybean interactions are strain X cultivar specific (12, 14), and the genetic basis for specificity is partially understood (11, 17, 18, 21). Strain NGR234 can nodulate more than 200 different host plants, including all those that are nodulated by S. fredii, except soybeans (26). This diversity in host range among Sinorhizobium strains yields an excellent model for the study of the role of the bacterial cell surface in the determination of host specificity.
There are many possible functions for the bacterial polysaccharides in symbiosis, such as the promotion of infection thread initiation and development and the elicitation of nodulins, as well as other signal functions. Potential passive functions include the masking of negative determinants and physical protection against harmful plant products. Any such functions, active or passive, may influence compatibility in infection and, ultimately, the host range of the bacterial strain.
It has now been shown that different Sinorhizobium strains produce structurally similar, yet chemically distinct, K antigens (Table 4): 10 of the 11 Sinorhizobium K antigens that have been partially or completely characterized conform to the consensus structure, Sug-Kdx (where Sug is any hexose and Kdx is any 1-carboxy-2-keto-3-deoxy sugar). Furthermore, Agrobacterium tumefaciens, which is closely related to Sinorhizobium spp., also produces a polysaccharide that appears to conform to the consensus structure, although it has not been completely characterized (31). Despite the similarities, the K antigens of Sinorhizobium spp. are clearly strain-specific antigens. In addition to the differences in glycosyl residue composition, the polysaccharides vary in substitution patterns, linkage points, anomeric configuration, size range, and repeating unit size. One interesting feature that differs among the species is the consistent presence of N-acetyl groups in the K antigens of S. meliloti strains; these have not been found in S. fredii K antigens (Table 4). In addition, it has recently been shown that the K antigens of Sinorhizobium spp. are sulfated (27) but the level of sulfation is much lower in normally cultured cells of S. meliloti than in S. fredii. If the polysaccharides play an active role in the infection process and host specificity, any of the structural features may be important. For example, a very specific size range of EPS II has been found to promote nodulation of alfalfa by S. meliloti (10), and a genetic determinant of capsule size range (rkpZ) is required for alfalfa nodulation by EPS mutants of S. meliloti (32). In addition, specific K-antigen structure appears to be important in early recognition steps in S. meliloti-alfalfa symbiosis (1).
|
The primary function of the K antigens is probably to form a hydrated capsule that surrounds the cell and protects it against abiotic factors, such as desiccation. The advantage of the capsule, as opposed to LPS, is that it may constitute a dense matrix that extends far from the cell surface, with only a fraction of the component molecules in contact with the outer membrane. Present evidence suggests that these capsule "anchors" may be linked to the LPS via disulfide bridges (27); in contrast to studies of enteric bacteria, no evidence has been found for a lipid moiety on the K antigens of rhizobia. This was also shown in the lack of fatty acid resonances in the NMR spectra presented in this report.
The capsule may also provide protection against other microorganisms, such as phages; in fact, in a recent study it was found that although capsule mutants of S. meliloti Rm41 exoB (AK631) gained resistance to one of 12 phages tested, they lost resistance to as many as 9 of the 12 (2). This shows that there may be many important functions of the capsule in these soil bacteria and that any functions in nodulation, in relation to structural specificity, may be secondary to those that affect the survival of the cell.
Sinorhizobium spp. are characterized by conserved LPS: the RLPS of 18 of the 19 strains examined are delineated into two major core serogroups (Table 2), which are not correlated with species. Although the RLPS from each serogroup have similar glycosyl residue compositions (Table 3) and contain common core oligosaccharides (Fig. 5), it appears that a difference in the uronic acid content of the serogroup-specific core oligosaccharides is partially responsible for serogroup identity. The presence of the common oligosaccharides in both types of RLPS suggests that there should have been significant cross-reaction in the immunoblot analyses, which was not the case. Therefore, the serogroup-specific core oligosaccharides must be the immunodeterminant components of the RLPS. Present evidence indicates that the serogroup-specific components may be "outer" cores that are at the nonreducing terminus of the RLPS core (27), yielding serogroup specificity despite the presence of common "inner" cores in all Sinorhizobium LPS.
The RLPS of S. meliloti NRG23 and S. meliloti NRG53 gave marginal reactions with anti-Rm41 and probably constitute a third serogroup, and the RLPS of S. fredii HH303 was negative with both antisera, suggesting that at least four serogroups exist. Importantly, the subdivision of the LPS into these core serogroups is consistent with relationships based on immunological analyses of whole cells (8, 19, 33). This implies that the RLPS is a major common antigen in Sinorhizobium spp. whereas the K antigen is a major strain-specific antigen. This assertion is supported by monoclonal antibody (MAb) studies, performed by Olsen et al. (24), and the subsequent identification of the epitopes in this laboratory (27). The three strain-specific anti-S. meliloti MAbs used in that study recognized the K antigens of the homologous strains (and a few others), whereas two of the three common-antigen MAbs recognized the RLPS of 53 of the 60 strains tested. When the anti-RLPS MAbs were employed in immunoblotting experiments with the strains from this study, they recognized the RLPS of all strains in the anti-Rm41 serogroup (Table 2) except those of S. meliloti NRG53 and S. meliloti NRG23 (27), confirming that those strains are, in fact, in a distinct RLPS serogroup(s). This also indicated that the Rm41 serogroup is the largest in the genus Sinorhizobium.
As the core regions of both RLPS and SLPS from S. fredii USDA205 have been shown to be similar (30), the specific recognition of the Rf205 serogroup SLPS by anti-Rm41 is credible evidence that there is limited variation in O-antigen structure among Sinorhizobium strains. Also, in both the PAGE and immunoblot analyses, the SLPS, when present, migrated as two distinct bands. Characterization of the two forms of SLPS from S. fredii USDA205 has shown that they bear different O antigens. The primary O antigen (upper SLPS banding region) is a glucan, and the secondary O antigen (lower SLPS banding region) is a xylomannan (30). In this regard, Sinorhizobium spp. are unusual. The O antigens of most gram-negative bacteria are highly variable, strain-specific surface antigens (37); in this genus, that role is fulfilled by the K antigens. The structural conservation of LPS throughout the genus Sinorhizobium suggests that the LPS, in the form found in cultured cells, does not impact on host range, including strain-cultivar specificity. This does not preclude the possibility, however, that the cells may express different forms of LPS in the soil, at the point of infection.
Interestingly, the expression of the two O antigens by S. fredii USDA205 is regulated by host-specific compounds (30), and "induced" differences in K antigen expression have been demonstrated in both S. fredii and S. meliloti (30, 32). Mutations in apparent regulatory genes (nolXWBTUV) also result in a modified expression of the LPS and K antigens in S. fredii USDA257 (15). In addition, many changes in the expression of Sinorhizobium LPS and K antigens are associated with growth under varied abiotic conditions, such as pH or temperature (27). These changes imply that analysis of cultured cells represents an important first step in understanding the role of bacterial surface polysaccharides in symbiosis; however, additional analyses of bacteroides, induced cells, regulatory mutants, and cells grown under different abiotic conditions are clearly required to elucidate the structure-function relationships between the bacterial cell surface and symbiosis.
In light of the complexity of legume-rhizobium interactions, we believe that a comparative analysis of closely related strains of Sinorhizobium is a valuable asset in the study of the molecular basis for host specificity. In a similar analysis of LPS and outer membrane proteins from a panel of wild-type strains of Xanthomonas campestris, Ojanen et al. (22) illustrated a correlation between host specificity and the pathogen cell surface. Our present and future analyses may yield similar results.
| |
ACKNOWLEDGMENTS |
|---|
This work was supported by grant MCB-9728564 from the National Science Foundation and by the U.S. Department of Energy-funded Center for Plant and Microbial Complex Carbohydrate Research under grant DE-FG02-93ER-20097 (B.L.R.). S.G.P. was supported by a grant from the USDA/NRI.
We thank Malcolm O'Neill for a critical review of the manuscript.
| |
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.
Present address: College of ACES, University of Illinois, Urbana,
IL 61801.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
| 1. | Bequart-de Kozak, I., B. L. Reuhs, D. Buffard, C. Breda, J. S. Kim, R. Esnault, and A. Kondorosi. 1997. Role of the K-antigen subgroup of capsular polysaccharides in the early recognition process between Rhizobium meliloti and alfalfa leaves. Mol. Plant-Microbe Interact. 10:114-123. |
| 2. |
Campbell, G. R. O.,
B. L. Reuhs, and G. C. Walker.
1998.
Different phenotypic classes of Sinorhizobium meliloti mutants defective in synthesis of K antigen.
J. Bacteriol.
180:5432-5436 |
| 3. | Carlson, R. W., U. R. Bhat, and B. Reuhs. 1992. Rhizobium lipopolysaccharides: their structures and evidence for their importance in the nitrogen-fixing symbiotic infection of their host legumes, p. 33-44. In P. M. Gresshoff (ed.), Plant biotechnology and development. CRC Press, Boca Raton, Fla. |
| 4. | Carlson, R. W., and B. S. Krishnaiah. 1992. Structures of the oligosaccharides obtained from the core regions of the lipopolysaccharides of Bradyrhizobium japonicum 61A101c and its symbiotically defective lipopolysaccharide mutant, JS314. Carbohydr. Res. 231:205-219[Medline]. |
| 5. |
Carrion, M.,
U. R. Bhat,
B. Reuhs, and R. W. Carlson.
1990.
Isolation and characterization of the lipopolysaccharides from Bradyrhizobium japonicum.
J. Bacteriol.
172:1725-1731 |
| 6. | Corzo, J., R. Pérez-Galdona, M. León-Barrios, and A. M. Gutiérrez-Navarro. 1991. Alcian Blue fixation allows silver staining of the isolated polysaccharide component of bacterial lipopolysaccharides in polyacrylamide gels. Electrophoresis 12:439-441[Medline]. |
| 7. | deLajudie, P., A. Williams, B. Pot, D. Dewettnick, G. Maestrojuan, M. Neyra, M. D. Collins, B. Dreyfus, K. Kersters, and M. Gillis. 1995. Polyphasic taxonomy of rhizobia: emendation of the genus Sinorhizobium and description of Sinorhizobium meliloti comb. nov., Sinorhizobium shaeli sp. nov., and Sinorhizobium teranga sp. nov. Int. J. Syst. Bacteriol. 44:715-733. |
| 8. |
Dowdle, S. F., and B. B. Bohlool.
1985.
Predominance of fast-growing Rhizobium japonicum in a soybean field in the People's Republic of China.
Appl. Environ. Microbiol.
50:1171-1176 |
| 9. |
Forsberg, L. S., and B. L. Reuhs.
1997.
Structural characterization of the K antigens from Rhizobium fredii USDA257: evidence for a common structural motif, with strain-specific variation, in the capsular polysaccharides of Rhizobium spp.
J. Bacteriol.
179:5366-5371 |
| 10. |
Gonzalez, J. E.,
B. L. Reuhs, and G. C. Walker.
1996.
Low molecular weight EPS II of Rhizobium meliloti allows nodule invasion in Medicago sativa.
Proc. Natl. Acad. Sci. USA
93:8636-8641 |
| 11. | Heron, D. S., T. Ersek, H. B. Krishnan, and S. G. Pueppke. 1989. Nodulation mutants of Rhizobium fredii USDA257. Mol. Plant-Microbe Interact. 2:4-10. |
| 12. |
Heron, D. S., and S. G. Pueppke.
1984.
Mode of infection, nodulation specificity, and indigenous plasmids of 11 fast-growing Rhizobacterium japonicum strains.
J. Bacteriol.
160:1061-1066 |
| 13. |
Jarvis, B. D. W.,
H. L. Downer, and J. P. W. Young.
1992.
Phylogeny of fast-growing soybean-nodulating rhizobia supports synonymy of Sinorhizobium and Rhizobium and assignment to Rhizobium fredii.
Int. J. Syst. Bacteriol.
42:93-96 |
| 14. |
Keyser, H. H.,
T. S. Hu,
B. B. Bohlool, and D. F. Weber.
1982.
Fast-growing rhizobia isolated from root nodules of soybean.
Science
215:1631-1632 |
| 15. | Kim, J. S., and B. L. Reuhs. 1996. Extended host range mutants of Rhizobium fredii USDA257 show modified expression of the K antigens and lipopolysaccharides, abstr. L-10. In Abstracts of the 8th International Congress on Molecular Plant-Microbe Interactions. |
| 16. |
Kim, J. S.,
B. L. Reuhs,
M. M. Rahman,
B. Ridley, and R. W. Carlson.
1996.
Separation of bacterial capsular and lipopolysaccharides by preparative electrophoresis.
Glycobiology
6:433-437 |
| 17. | Kovacs, L. G., P. A. Balatti, H. B. Krishnan, and S. G. Pueppke. 1995. Transcriptional organization and expression of nolXWBTUV, a locus that regulates cultivar-specific nodulation of soybean by Rhizobium fredii USDA257. Mol. Microbiol. 17:923-933[Medline]. |
| 18. | Krishnan, H. B., and S. G. Pueppke. 1992. Inactivation of nolC conditions developmental abnormalities in nodulation of Peking soybean by Rhizobium fredii USDA257. Mol. Plant-Microbe Interact. 5:14-21. |
| 19. | Krishnan, H. B., and S. G. Pueppke. 1994. Host range, RFLP, and antigenic relationships between Rhizobium fredii strains and Rhizobium sp. NGR234. Plant Soil 161:21-29. |
| 20. | Lerouge, P., P. Roche, C. Faucher, F. Maillet, G. Truchet, J. C. Promé, and J. Dénarié. 1990. Symbiotic host-specificity of Rhizobium meliloti is determined by a sulphated and acylated glucosamine oligosaccharide signal. Nature 344:781-784[Medline]. |
| 21. | Meinhardt, L. W., H. B. Krishnan, P. A. Balatti, and S. G. Pueppke. 1993. Molecular cloning and characterization of a sym plasmid locus that regulates cultivar-specific nodulation of soybean Rhizobium fredii USDA257. Mol. Microbiol. 9:17-29[Medline]. |
| 22. |
Ojanen, T.,
I. M. Helander,
K. Haahtela,
T. K. Korhonen, and T. Laakso.
1993.
Outer membrane proteins and lipopolysaccharides in pathovars of Xanthomonas campestris.
Appl. Environ. Microbiol.
59:4143-4151 |
| 23. | Olsen, P., M. Collins, and W. Rice. 1992. Surface antigens present on vegetative Rhizobium meliloti cells may be diminished or absent when cells are in the bacteroid form. Can. J. Microbiol. 38:506-509. |
| 24. | Olsen, P., M. M. Collins, and W. A. Rice. 1994. Reactivity of MAbs raised against whole cells to a panel of Rhizobium strains. Appl. Environ. Microbiol. 60:652-661. |
| 25. | Petrovics, G., P. Putnoky, B. L. Reuhs, J. S. Kim, T. A. Thorp, D. Noel, R. W. Carlson, and A. Kondorosi. 1993. The presence of a novel type of surface polysaccharide in Rhizobium meliloti requires a new fatty acid synthase-like gene cluster involved in symbiotic nodule development. Mol. Microbiol. 8:1093-1094. |
| 26. | Pueppke, S. G., and W. J. Broughton. Symbiotic specificity in the legume-Rhizobium symbiosis: Rhizobium sp. NGR234 and R. fredii USDA257 have exceptionally broad, interrelated host ranges. Mol. Plant-Microbe Interact, in press. |
| 27. | Reuhs, B. L. Unpublished data. |
| 28. | Reuhs, B. L. 1996. Acidic capsular polysaccharides (K antigens) of Rhizobium, p. 331-336. In G. Stacey, B. Mullin, and P. M. Gresshoff (ed.), Biology of plant-microbe interactions. International Society for Molecular Plant-Microbe Interaction, St. Paul, Minn. |
| 29. |
Reuhs, B. L.,
R. W. Carlson, and J. S. Kim.
1993.
Rhizobium fredii and Rhizobium meliloti produce 3-deoxy-D-manno-2-octulosonic acid-containing polysaccharides that are structurally analogous to group II K antigens (capsular polysaccharides) found in Escherichia coli.
J. Bacteriol.
175:3570-3580 |
| 30. | Reuhs, B. L., J. S. Kim, A. Badgett, and R. W. Carlson. 1994. Production of the cell-associated polysaccharides of Rhizobium fredii USDA205 is modulated by apigenin and host root extract. Mol. Plant-Microbe Interact. 7:240-247[Medline]. |
| 31. |
Reuhs, B. L.,
J. S. Kim, and A. G. Matthysse.
1997.
Attachment of Agrobacterium tumefaciens to carrot cells and Arabidopsis wound sites is correlated with the presence of a cell-associated, acidic polysaccharide.
J. Bacteriol.
179:5372-5379 |
| 32. |
Reuhs, B. L.,
M. N. V. Williams,
J. S. Kim,
R. W. Carlson, and F. Côté.
1995.
Suppression of the Fix phenotype of Rhizobium meliloti exoB by lpsZ is correlated to a modified expression of the K polysaccharide.
J. Bacteriol.
177:4289-4296 |
| 33. |
Sadowsky, M. J.,
B. B. Bohlool, and H. H. Keyser.
1987.
Serological relatedness of Rhizobium fredii to other rhizobia and to the bradyrhizobia.
Appl. Environ. Microbiol.
53:1785-1789 |
| 34. | Truchet, G., P. Roche, P. Lerouge, J. Vasse, S. Camut, F. De Billy, J.-C. Promé, and J. Dénarié. 1991. Sulphated lipo-oligosaccharide signals of Rhizobium meliloti elicit root nodule organogenesis in alfalfa. Nature 351:670-673. |
| 35. | Tsai, C., and C. E. Frisch. 1982. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal. Biochem. 119:115-119[Medline]. |
| 36. | Weissbach, A., and J. Hurwitz. 1958. The formation of 2-keto-3-deoxyheptanoic acid in extracts of Escherichia coli B. J. Biol. Chem. 234:705-709. |
| 37. | Whitfield, C., and M. A. Valvano. 1993. Biosynthesis and expression of cell-surface polysaccharides in Gram-negative bacteria. Adv. Microb. Physiol. 35:135-246[Medline]. |
| 38. | York, W. S., A. G. Darvill, M. McNeil, T. T. Stevenson, and P. Albersheim. 1985. Isolation and characterization of plant cell walls and cell wall components. Methods Enzymol. 118:3-40. |
Applied and Environmental Microbiology, December 1998, p. 4930-4938, Vol. 64, No. 12
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Schmeisser, C., Liesegang, H., Krysciak, D., Bakkou, N., Le Quere, A., Wollherr, A., Heinemeyer, I., Morgenstern, B., Pommerening-Roser, A., Flores, M., Palacios, R., Brenner, S., Gottschalk, G., Schmitz, R. A., Broughton, W. J., Perret, X., Strittmatter, A. W., Streit, W. R.
(2009). Rhizobium sp. Strain NGR234 Possesses a Remarkable Number of Secretion Systems. Appl. Environ. Microbiol.
75: 4035-4045
[Abstract] [Full Text] -
Lerner, A., Okon, Y., Burdman, S.
(2009). The wzm gene located on the pRhico plasmid of Azospirillum brasilense Sp7 is involved in lipopolysaccharide synthesis. Microbiology
155: 791-804
[Abstract] [Full Text] -
Forsberg, L. S., Carlson, R. W.
(2008). Structural Characterization of the Primary O-antigenic Polysaccharide of the Rhizobium leguminosarum 3841 Lipopolysaccharide and Identification of a New 3-Acetimidoylamino-3-deoxyhexuronic Acid Glycosyl Component: A UNIQUE O-METHYLATED GLYCAN OF UNIFORM SIZE, CONTAINING 6-DEOXY-3-O-METHYL-D-TALOSE, N-ACETYLQUINOVOSAMINE, AND RHIZOAMINURONIC ACID (3-ACETIMIDOYLAMINO-3-DEOXY-D-GLUCO-HEXURONIC ACID). J. Biol. Chem.
283: 16037-16050
[Abstract] [Full Text] -
Simsek, S., Ojanen-Reuhs, T., Stephens, S. B., Reuhs, B. L.
(2007). Strain-Ecotype Specificity in Sinorhizobium meliloti-Medicago truncatula Symbiosis Is Correlated to Succinoglycan Oligosaccharide Structure. J. Bacteriol.
189: 7733-7740
[Abstract] [Full Text] -
Jackson, C. L, Dreaden, T. M, Theobald, L. K, Tran, N. M, Beal, T. L, Eid, M., Gao, M. Y., Shirley, R. B, Stoffel, M. T, Kumar, M V., Mohnen, D.
(2007). Pectin induces apoptosis in human prostate cancer cells: correlation of apoptotic function with pectin structure. Glycobiology
17: 805-819
[Abstract] [Full Text] -
D'Haeze, W., Leoff, C., Freshour, G., Noel, K. D., Carlson, R. W.
(2007). Rhizobium etli CE3 Bacteroid Lipopolysaccharides Are Structurally Similar but Not Identical to Those Produced by Cultured CE3 Bacteria. J. Biol. Chem.
282: 17101-17113
[Abstract] [Full Text] -
Townsend, G. E. II, Forsberg, L. S., Keating, D. H.
(2006). Mesorhizobium loti Produces nodPQ-Dependent Sulfated Cell Surface Polysaccharides. J. Bacteriol.
188: 8560-8572
[Abstract] [Full Text] -
Sharypova, L.A., Chataigne, G., Fraysse, N., Becker, A., Poinsot, V.
(2006). Overproduction and increased molecular weight account for the symbiotic activity of the rkpZ-modified K polysaccharide from Sinorhizobium meliloti Rm1021. Glycobiology
16: 1181-1193
[Abstract] [Full Text] -
Le Quere, A. J.-L., Deakin, W. J., Schmeisser, C., Carlson, R. W., Streit, W. R., Broughton, W. J., Forsberg, L. S.
(2006). Structural Characterization of a K-antigen Capsular Polysaccharide Essential for Normal Symbiotic Infection in Rhizobium sp. NGR234: DELETION OF THE rkpMNO LOCUS PREVENTS SYNTHESIS OF 5,7-DIACETAMIDO-3,5,7,9-TETRADEOXY-NON-2-ULOSONIC ACID. J. Biol. Chem.
281: 28981-28992
[Abstract] [Full Text] -
Broughton, W. J., Hanin, M., Relic, B., Kopcinska, J., Golinowski, W., Simsek, S., Ojanen-Reuhs, T., Reuhs, B., Marie, C., Kobayashi, H., Bordogna, B., Le Quere, A., Jabbouri, S., Fellay, R., Perret, X., Deakin, W. J.
(2006). Flavonoid-Inducible Modifications to Rhamnan O Antigens Are Necessary for Rhizobium sp. Strain NGR234-Legume Symbioses.. J. Bacteriol.
188: 3654-3663
[Abstract] [Full Text] -
Reuhs, B. L., Relic, B., Forsberg, L. S., Marie, C., Ojanen-Reuhs, T., Stephens, S. B., Wong, C.-H., Jabbouri, S., Broughton, W. J.
(2005). Structural Characterization of a Flavonoid-Inducible Pseudomonas aeruginosa A-Band-Like O Antigen of Rhizobium sp. Strain NGR234, Required for the Formation of Nitrogen-Fixing Nodules. J. Bacteriol.
187: 6479-6487
[Abstract] [Full Text] -
Spiers, A. J., Rainey, P. B.
(2005). The Pseudomonas fluorescens SBW25 wrinkly spreader biofilm requires attachment factor, cellulose fibre and LPS interactions to maintain strength and integrity. Microbiology
151: 2829-2839
[Abstract] [Full Text] -
Fraysse, N., Lindner, B., Kaczynski, Z., Sharypova, L., Holst, O., Niehaus, K., Poinsot, V.
(2005). Sinorhizobium meliloti strain 1021 produces a low-molecular-mass capsular polysaccharide that is a homopolymer of 3-deoxy-D-manno-oct-2-ulosonic acid harboring a phospholipid anchor. Glycobiology
15: 101-108
[Abstract] [Full Text] -
Laus, M. C., Logman, T. J., van Brussel, A. A. N., Carlson, R. W., Azadi, P., Gao, M.-Y., Kijne, J. W.
(2004). Involvement of exo5 in Production of Surface Polysaccharides in Rhizobium leguminosarum and Its Role in Nodulation of Vicia sativa subsp. nigra. J. Bacteriol.
186: 6617-6625
[Abstract] [Full Text] -
Frirdich, E., Vinogradov, E., Whitfield, C.
(2004). Biosynthesis of a Novel 3-Deoxy-D-manno-oct-2-ulosonic Acid-containing Outer Core Oligosaccharide in the Lipopolysaccharide of Klebsiella pneumoniae. J. Biol. Chem.
279: 27928-27940
[Abstract] [Full Text] -
Buendia-Claveria, A. M., Moussaid, A., Ollero, F. J., Vinardell, J. M., Torres, A., Moreno, J., Gil-Serrano, A. M., Rodriguez-Carvajal, M. A., Tejero-Mateo, P., Peart, J. L., Brewin, N. J., Ruiz-Sainz, J. E.
(2003). A purL mutant of Sinorhizobium fredii HH103 is symbiotically defective and altered in its lipopolysaccharide. Microbiology
149: 1807-1818
[Abstract] [Full Text] -
Sharypova, L. A., Niehaus, K., Scheidle, H., Holst, O., Becker, A.
(2003). Sinorhizobium meliloti acpXL Mutant Lacks the C28 Hydroxylated Fatty Acid Moiety of Lipid A and Does Not Express a Slow Migrating Form of Lipopolysaccharide. J. Biol. Chem.
278: 12946-12954
[Abstract] [Full Text] -
Gudlavalleti, S. K., Forsberg, L. S.
(2003). Structural Characterization of the Lipid A Component of Sinorhizobium sp. NGR234 Rough and Smooth Form Lipopolysaccharide. DEMONSTRATION THAT THE DISTAL AMIDE-LINKED ACYLOXYACYL RESIDUE CONTAINING THE LONG CHAIN FATTY ACID IS CONSERVED IN RHIZOBIUM AND SINORHIZOBIUM SP.. J. Biol. Chem.
278: 3957-3968
[Abstract] [Full Text] -
Fraysse, N., Jabbouri, S., Treilhou, M., Couderc, F., Poinsot, V.
(2002). Symbiotic conditions induce structural modifications of Sinorhizobium sp. NGR234 surface polysaccharides. Glycobiology
12: 741-748
[Abstract] [Full Text] -
Pellock, B. J., Teplitski, M., Boinay, R. P., Bauer, W. D., Walker, G. C.
(2002). A LuxR Homolog Controls Production of Symbiotically Active Extracellular Polysaccharide II by Sinorhizobium meliloti. J. Bacteriol.
184: 5067-5076
[Abstract] [Full Text] -
DeLoney, C. R., Bartley, T. M., Visick, K. L.
(2002). Role for Phosphoglucomutase in Vibrio fischeri-Euprymna scolopes Symbiosis. J. Bacteriol.
184: 5121-5129
[Abstract] [Full Text] -
Campbell, G. R. O., Reuhs, B. L., Walker, G. C.
(2002). Chronic intracellular infection of alfalfa nodules by Sinorhizobium meliloti requires correct lipopolysaccharide core. Proc. Natl. Acad. Sci. USA
99: 3938-3943
[Abstract] [Full Text] -
Janecka, J., Jenkins, M. B., Brackett, N. S., Lion, L. W., Ghiorse, W. C.
(2002). Characterization of a Sinorhizobium Isolate and Its Extracellular Polymer Implicated in Pollutant Transport in Soil. Appl. Environ. Microbiol.
68: 423-426
[Abstract] [Full Text] -
Lagares, A., Hozbor, D. F., Niehaus, K., Otero, A. J. L. P., Lorenzen, J., Arnold, W., Pühler, A.
(2001). Genetic Characterization of a Sinorhizobium meliloti Chromosomal Region Involved in Lipopolysaccharide Biosynthesis. J. Bacteriol.
183: 1248-1258
[Abstract] [Full Text] -
Pellock, B. J., Cheng, H.-P., Walker, G. C.
(2000). Alfalfa Root Nodule Invasion Efficiency Is Dependent on Sinorhizobium meliloti Polysaccharides. J. Bacteriol.
182: 4310-4318
[Abstract] [Full Text] -
Reuhs, B. L., Stephens, S. B., Geller, D. P., Kim, J. S., Glenn, J., Przytycki, J., Ojanen-Reuhs, T.
(1999). Epitope Identification for a Panel of Anti-Sinorhizobium meliloti Monoclonal Antibodies and Application to the Analysis of K Antigens and Lipopolysaccharides from Bacteroids. Appl. Environ. Microbiol.
65: 5186-5191
[Abstract] [Full Text] -
Vinuesa, P., Reuhs, B. L., Breton, C., Werner, D.
(1999). Identification of a Plasmid-Borne Locus in Rhizobium etli KIM5s Involved in Lipopolysaccharide O-Chain Biosynthesis and Nodulation of Phaseolus vulgaris. J. Bacteriol.
181: 5606-5614
[Abstract] [Full Text] -
van Doorn, J., Ojanen-Reuhs, T., Hollinger, T. C., Reuhs, B. L., Schots, A., Boonekamp, P. M., Oudega, B.
(1999). Development and Application of Pathovar-Specific Monoclonal Antibodies That Recognize the Lipopolysaccharide O Antigen and the Type IV Fimbriae of Xanthomonas hyacinthi. Appl. Environ. Microbiol.
65: 4171-4180
[Abstract] [Full Text] -
Forsberg, L. S., Bhat, U. R., Carlson, R. W.
(2000). Structural Characterization of the O-antigenic Polysaccharide of the Lipopolysaccharide from Rhizobium etli Strain CE3. A UNIQUE O-ACETYLATED GLYCAN OF DISCRETE SIZE, CONTAINING 3-O-METHYL-6-DEOXY-L-TALOSE AND 2,3,4-TRI-O-METHYL-L-FUCOSE. J. Biol. Chem.
275: 18851-18863
[Abstract] [Full Text] -
Lerouge, I., Laeremans, T., Verreth, C., Vanderleyden, J., Van Soom, C., Tobin, A., Carlson, R. W.
(2001). Identification of an ATP-binding Cassette Transporter for Export of the O-antigen across the Inner Membrane in Rhizobium etli Based on the Genetic, Functional, and Structural Analysis of an lps Mutant Deficient in O-antigen. J. Biol. Chem.
276: 17190-17198
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»