Oct 14, 1986 - column as reported by Mort and Bauer (13). LPS was isolated by phenol-water eXtraction of freeze- dried cells by the method of Johnson and ...
Vol. 169, No. 1
JOURNAL OF BACTERIOLOGY, Jan. 1987, p. 137-141
0021-9193/87/010137-05$02.00/0 Copyright © 1987, American Society for Microbiology
Cell Surface Polysaccharides from Bradyrhizobium japonicum and a Nonnodulating Mutant VELUPILLAI PUVANESARAJAH,1 FRED M. SCHELL,2 DAVID GERHOLD,1 AND GARY STACEY'* Departments of Microbiology' and Chemistry,2 The University of Tennessee, Knoxville, Tennessee 37996-0845 Received 7 July 1986/Accepted 14 October 1986
The cell surface polysaccharides of wild-type Bradyrhizobium japonicum USDA 110 and
a
nonnodulating
mutant, strain HS123, were analyzed. The capsular polysaccharide (CPS) and exopolysaccharide (EPS) of the wild type and the mutant strain do not differ in their sugar composition. CPS and EPS are composed of mannose, 4-O-methylgalactose/galactose, glucose, and galacturonic acid in a ratio of 1:1:2:1, respectively. 'H nuclear magnetic resonance spectra of the EPS and CPS of the wild type and mutant strain are very similfr, but not identical, suggesting minor structural variation in these polysaccharides. The lipopolysaccharides (LPS) of the above two strains were purified, and their compositions were determined. Gross differences in the chemical compositions of the two LPS were observed. Chemical and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses indicated that strain HS123 is a rough-type mutant lacking a complete LPS. The LPS of mutant strain HS123 is composed of mannose, glucose, glucosamine, 2-keto-3-deoxyoctulosonic acid, and lipid A. The wild-type LPS is composed of fucose, xylose, arabinose, mannose, glucose, fuvosamine, quinovosamine, glucosamine, uronic acid, 2-keto-3-deoxyoctulosonic acid, and lipid A. Preliminary sugar analysis of lipid A from B. japonicum identified mannose, while traces of glucosamine were detected. 3-Hydroxydodecanoic and 3-hydroxytetradecanoic acids formed a major portion of the fatty acids in lipid A. Lesser quantities of nonhydroxylated 16:0, 18:0, 22:0, and 24:0 acids also were detected.
unpublished data). We found strain HS123 to be a loughtype mutant differing markedly from the wild type in the composition of the LPS.
Rhizobium and Bradyrhizobium species are gram-negative bacteria that fix molecular nitrogen in symbiosis with their respective leguminous hosts. The infection process is species specific, but the factors determining this specificity are presently unknown. Investigations have focused on the initial binding of rhizobia to the host roots as an important early step at which specificity could be expressed. This step involves cell-cell contact and recognition. Host-plant lectins have been postulated to mediate this process, since these carbohydrate-binding proteins have been shown to bind specifically to various Rhizobium and Bradyrhizobium cell surface polysaccharides, exopolysaccharides (EPS), capsular polysaccharides (CPS) and lipopolysaccharides (LPS) (for a review, see reference 4). Whatever functions the bacterial cell surface polysaccharides may have in the infection process, they are ultimately dependent on the chemical structures of these molecules. In this context, the structures of EPS and CPS isolated from several Rhizobium and Bradyrhizobium strains have been established (4). In contrast, only a few reports exist in the literature that describe the partial compositions Qf LPS isolated from Rhizobium species (for a review, see reference 4). There are no published reports on the purification or the composition of LPS from slow-growing Bradyrhizobium species. To analyze the role of cell surface polysaccharides in attachment to plant root surfaces and infection by Bradyrhizobium species, we are currently studying the cell surface polysaccharides from slow-growing B. japonicum USDA 110. In addition, a nonnodulating mutant, strain HS123, that does not cause root hair curling was examined. Strain HS123 was previously described as a mutant which has reduced ability to bind to soybean roots (18). However, using a different assay developed by Pueppke (17), it was later found to attach to soybean roots at a level comparable to that of the wild type (L. Halverson, *
MATERIALS AND METHODS
Organism and culture media. The wild-type B. japonicum USDA 110 and the mutant strain HS123 were obtatined as described previously (18) and were grown on defined medium (13). Six-day-old bacterial cells were harvested by centrifugation at 16,000 x g for 20 min. The pelleted cells were washed three times with phosphate-buffered saline (0.43 g of NaH2PO4, 1.48 g of Na2HPO4, 7.2 g of NaCl per liter), rinsed with deionized water, and freeze-dried. Isolation and fractionation of polysaccharides. The supernatant fluid obtained after the removal of cells by centrifugation was concentrated by rotary evaporation at 40°C. The concentrated solution was dialyzed extensively against distilled water and freeze-dried to yield the crude EPS traction. For the isolation of CPS, the cells suspended in phosphate-buffered saline were stirred in a Waridg blender for a minute at full speed. After the removal of cells by centrifugation, the crude CPS fraction was recovered from the supernatant fluid by dialysis and lyophilization. The CPS and EPS were purified by chromatography on a DEAE-Bio-Gel column as reported by Mort and Bauer (13). LPS was isolated by phenol-water eXtraction of freezedried cells by the method of Johnson and Perry (9). After extraction, the aqueous phase was dialyzed extensively, and the polysaccharides were recovered by freeze-drying. These crude extracts were dissolved in distilled water and passed through a Dowex-1 anion (acetate form)-exchange column to remove any adhering acidic extracellular polysaccharides. After the column was washed with distilled water until no carbohydrate was detected in the effluent (as determined by the phenol-sulfuric acid method [2]), the effluent and the combined washings were concentrated by rotary evaporation and freeze-dried.
Corresponding author. 137
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PUVANESARAJAH ET AL.
Gel chromatography on agarose. Polysaccharide samples dissolved in a 0.1% aqueous solution of sodium dodecyl sulfate (SDS) and applied onto a Bio-Gel-agarose A 1.5m column (1.9 by 75 cm). Fractions (4.0 ml) were collected, and the hexose content of the various fractions was estimated by the phenol-sulfuric acid method (2). The LPS fraction eluted in the void volume and was recovered after dialysis by freeze-drying. To assess purity, the LPS fraction was dissolved in triethylamine-EDTA buffer and chromatographed on a Sepharose 4B column (5). The fractions were were
assayed for hexose and 2-keto-3-deoxyoctulosonic acid (KDO). by the phenol-sulfuric acid (2) and thiobarbituric acid (10) niethods, respectively. Lipid A. To cleave the lipid A, we treated LPS with 1% aqueous acetic acid (pH 3.1) at 100°C for 5 h. Shorter periods were less effective. Precipitated lipid A was removed by centrifugation at. 3,000 x g for 15 min, dried, and weighed. The material recovered from the supernatant fluid by lyophilization was chromatographed on a Sephadex G-50 column. The fractions were assayed for hexose content by the phenol-sulfuric acid method (2). Total fatty acids were transesterified with methanol-HCl, and the resulting fatty acid. methyl esters were treated with N,O-bis(trimethylsilyl)trifluoroacetamide. and analyzed by gas chromatography-mass spectrometry (MS) by the method of Edlund et al. (7). Analytical methods. 'H nuclear magnetic resonance spec-
tra were measured with a Nicolet NT200WB spectrometer. The spectra were run at 750C with 3-(trimethylsilyi)-1propanesulfonic acid, sodium salt, as the internal reference. Gas-liquid chromatography (GLC) was performed with a Perkin-Elmer Sigma 313 gas chromatograph with (i) a packed column of 3% SP2330 on 100/200-mesh Supelcoport (Supelco, Inc., Bellefonte, Pa.) (column A) or (ii) a packed column of 3% SP2340: on 100/120-mesh Supelcoport (column B). For the identification of neutral sugars, samples of polysaccharides were treated with 2 N trifluoroacetic acid at 1200C for 2 h (1), reduced with sodium borohydride, and analyzed by GLC or GLC-MS as alditol acetates (column A). Amino sugars were determined after hydrolysis of polysac.charides with 4 N HCl at 105°C for 16 h by: (i) autoanalysis (amino acid analyzer) and (ii) GLC-MS analyses as alditol acetates on column B. Uronic acids were determined.by the m-hydroxybiphenyl method (3), and KDO (3-deoxy-b-manno-2-octulosonic acid) was estimated by the thiobarbituric acid assay (10). LPS polyacrylamide gel electrophoresis (PAGE). LPS was electrophoresed through 4% stacking and 12% separating gels, using the buffer system of Laemmli (11) with the following modifications: 4 M urea was incorporated into the gels (20), and the SDS concentration was increased from 0.1 to 1%: in the gels and tank buffer. These modifications were reported to reduce LPS aggregation (16). Bands were made visible with LPS silver stain (20).
RESULTS EPS. The structures of the acidic EPS and CPS produced by B. japonicum have been established and are apparently identical (14). EPS and CPS consist of a repeating pentasaccharide unit in which some galactose and galacturonic acid residues carry 0-methyl and 0-acetyl substituents, respectively. In the present investigation, analyses of the EPS and CPS produced by the wild-type and mutant B. japonicum strains showed that they are composed of mannose, galactose/4-O-methylgalactose, glucose, and uronic acid in the
J. BACTERIOL. TABLE 1. Sugar analyses of the CPS and EPS of wild-type B. japonicum USDA 110 and mutant strain HS123 Sugar (molar ratio) Polysaccharide tt Mannose Galactose 4-0-methylacid galactose Glucose Uronic USDA 110 1.0 0.45 CPS 2.0 1.1 1.2 0.55 EPS 1.0 0.45 0.55 2.0 0.9 1.2
HS123 CPS EPS
1.0 1.0
0.6 0.6
0.4 0.4
2.1
2.0
1.0 1.1
1.3 1.4
molar ratio of 1:1:2:1, respectively (Table 1). This is in accord with the reported (14) pentasaccharide repeating unit. to ascertain that the EPS of both the wild type and strain HS123 possess the same structural features, the 'H nuclear magnetic resonance spectra of the two were compared. The two spectra were very similar (Fig. 1), suggesting. that the two polysaccharides are probably similar in structure. Minor variations in the degree of 0-acetyl and 0-methyl substituents (Table 1) in the two polysaccharides were observed. The wild type and mutant produce approximately equivalent amounts of EPS and CPS (150 mg of EPS and 20 mg of CPS per g of dry cells). Phenol-water extracts. Phenol-water extracts of the wild type and the mutant were first fractionated by passage through an anion-exchange column to remove any adhering acidic extracellular polysaccharides (EPS and CPS). Over 80% of the material applied to the column was unabsorbed and eluted with water. Galactose/4-O-methylgalactose, a major component of the EPS and CPS, was not detected in the polysaccharide eluted from the Dowex column, indicating the effective removal of any contaminating acidic polysaccharide. The material recovered by eluting the colunrn with 0.5 M aqueous NaCl was composed of mannose, galactose/4-O-methylgalactose, glucose, and uronic acid in the molar rattio of 1:1:2:1, which corresponds to the composition of EPS and CPS of B. japonicum. After the Dowex column, the phenol-water extracts were further fractionated by chromatography on a Bio-Gel column. Figure 2 shows the elution profile typical of the wild-type or the mutant strain extracts. Both extracts contained a higher-molecular-weight component (LPS), which elutes near the void volume, and two lower-molecularweight components, which eluted in partially included volumes. Sugar analysis of the low-molecular-weight component in fractions 40 to 43 indicated that it is composed exclusively of glucose. Such low-molecular-weight glucans have frequently been isolated from phenol-water extracts and culture filtrates of various members of the Rhizobiaceae family (4). There was no apparent diference in the -amount or composition of the glucan between the wild type and mutant. LPS. The LPS fraction, eluted near the void volume from the BioGel column, had a constant hexose/KDO ratio across the peak which indicated that it was homo.geneous in composition (Fig. 2). Compositional analyses of the LPS from the wild type and mutant strain are shown in Table 2. The carbohydrate portion of the LPS of wild-type B. japonicum consists of fucose, xylose, arabinose, mannose, glucose, glucosamine, quinovosamine, fucosamine, and uronic acid. In contrast, the glycosyl composition of LPS from strain HS123 contains only mannose, glucose, and glucosamine.
POLYSACCHARIDES FROM B. JAPONICUM AND A MUTANT
VOL. 169, 1987
23 4 5
9
8
7
6
5
139
is
1 2 0 -l PPM FIG. 1. 1H nuclear magnetic resonance spectra (200 MHz) of the exopolysaccharides of (A) wild-type USDA 110 and (B) mutant strain HS123 in D20 at 75°C. 1, H-4 of 4-0-acetyl-cL-galactopyranosiduronic acid; 2, H-1 of 4-0-acetyl-a-galactopyranosiduronic acid and a-glucopyranose; 3, H-1 of a-mannopyranose; 4, H-1 of 4-0-methyl-a-galactopyranose; 5, H-1 of f-glucopyranose; 6, 0-methyl (assignments according to Mort et al. [15]). Signal at 2.1 ppm is due to methyl hydrogens of acetyl groups. IS, 3-(Trimethylsilyl)-l-propanesulfonic acid, sodium salt, used as an internal reference. 4
The lipid A content of the LPS from strain HS123 is also much higher than that of the wild type (Table 2). Lipid A isolated from the wild-type and mutant strains contained mannose as the only detected sugar constituent. The fatty acid composition of lipid A from both the wild type and mutant strain was very similar: 3-hydroxytetradecanoic and 3-hydroxydodecanoic acids formed a major portion of the fatty acids. Smaller quantities of nonhydroxylated 16:0, 18:0, 22:0, and 24:0 acids also were detected. Treatment of wild-type LPS with 1% acetic acid for 5 h resulted in the precipitation of lipid A. Chromatography of the products (elution profile not shown) recovered from the supernatant indicated only a polymeric fraction that eluted at the void volume. This polymeric fraction was composed of all the sugar residues which were previously identified in the intact LPS. Treatment of wild-type B. japonicum LPS with 0.1 N HCI at 100°C for 45 min released a polysaccharide that also voids a Sephadex G-50 column. This polysaccharide was composed of fucosamine, xylose, and glucose, suggesting that these three sugars form part of the O-polysaccharide chain. A low-molecular-weight fraction formed during HCl treatment consisted of mannose, fucose, glucose, glucosamine, and quinovosamine and probably arises from the core region. Mild acid hydrolysis of mutant LPS resulted in a
3
low-molecular-weight polysaccharide which consisted of mannose, glucose, and glucosamine. The fact that the mutant strain HS123 LPS does not have most of the sugars found in wild-type LPS indicates that mutant HS123 is an R-type mutant defective in the synthesis of O-polysaccharide or O-polysaccharide-R-core units or both. Additional evidence for this conclusion was provided by SDSPAGE analysis of HS123 LPS. SDS-PAGE analysis. After SDS-PAGE analysis with 0.1% SDS, wild-type B. japonicum LPS resolved into seven triplets. These triplets are likely due to aggregation of the LPS units. Under the same conditions the LPS from strain HS123 comigrated with the lipid A component isolated from wild-type LPS (by 1% acetic acid hydrolysis), thus confirming the lipid A-like nature of the LPS from strain HS123. The aggregation of Escherichia coli LPS has been shown to decrease when the SDS concentration in the gel is increased (16). Increasing the SDS concentration to 1.0% resulted in the condensation of the seven triplets seen in the wild-type B. japonicum into seven major singlet bands (Fig. 3). This treatment also reduced the number of Salmonella typhimurium LPS bands from >40 to 7. Under these conditions, the LPS from mutant strain HS123 continued to migrate with lipid A.
PUVANESARAJAH ET AL.
140
J. BACTERIOL.
0.70
0.6-
0
0.5-
c"0.40
w
x
WO°.3
-
*+
0.20.1 -
0.00
T --
5
10
-
-
----T----
15
-
r
20 25 30 35 FRACTION NUMBER
40
45
50
FIG. 2. Bio-Gel A 1.5m chromatography of LPS from the wildtype USDA 110. Chromatography of the LPS from mutant strain
HS123 gives an identical profile. Symbols: A, A490 in the phenolsulfuric acid assay; *, ratios of A490 and A549 (KDO, thiobarbituric acid assay). Void volume standard (blue dextran) eluted at firaction 15.
DISCUSSION There are only a few Rhizobium LPS studies in the literature (for a review, see reference 4). In the few published reports, the structural features of LPS were not determined. Available data suggest that Rhizobium LPS are more structurally diverse than the EPS and CPS (4). There are no published reports on the purification and chemical composition of the LPS from slow-growing Bradyrhizobium species. Results of a recent study (R. W. Carlson and R. F. Koehler, Plant Physiol., 1983, vol. 72, p. 24, abstr. no. 133) suggested that slow-growing B. japonicum strains do not produce LPS. In the present investigation, we isolated, purified, and analyzed the LPS of slow-growing B. japonicum USDA 110 and a nonnodulating mutant strain, HS123. The identified constituents of the wild-type B. japonicum LPS account for approximately 60% of the dry weight. Difficulties in accounting for the total mass of Rhizobium LPS have been encountered previously in other investigations (5, 21). The reason for this discrepancy in mass, however, has not been determined. Amino sugars and uronic acid linkages in polysaccharides are resistant to complete acid hydrolysis, and therefore the quantities of these components are underestimated. It is also possible that the unaccounted for mass could be due to the presence of
other acid-labile sugar-nonsugar residues that are degraded during hydrolysis. All our attempts to detect additional acid-labile or -resistant residues have thus far been unsuccessful. The chemical structures of LPS from enterobacterial species such as Salmonella and Escherichia have been established. These LPS are generally composed of an 0specific polysaccharide chain, an R-specific core polysaccharide, and a lipid A region. Lipid A is linked to the R-specific core polysaccharide via acid-labile KDO residues. The lipid A backbone was identified as a (1-+6)-linked ,-D-glucosamine disaccharide unit in these species. GLCMS analysis of the lipid A from the wild-type and mutant B. japonicum strains showed the presence of mannose as the major sugar constituent along with traces of glucosamine. Lipid A isolated from several other Rhizobium species also contained very low percentages of glucosamine (21). Mannose and glucosamine have been identified previously in the lipid A component of Rhodomicrobium vannielii ATCC 17100 LPS (8). The hydroxyl groups of the mannose residues of R. vannielii lipid A are not substituted with fatty acids. It appears that lipid A of B. japonicum LPS possesses a different structure from the lipid A in enterobacterial LPS. In previous studies (19), a bacteriophage capable of lysing the wild-type B. japonicum USDA 110 cells was isolated. This bacteriophage appeared to interact with a phenol-water extract of the wild-type cell wall. The nonnodulating mutant strain HS123, derived from strain USDA 110, was resistant to this bacteriophage. This led to the suggestion that the mutant HS123 is somehow defective in its cell surface. In the present study, cell surface polysaccharides of mutant strain HS123 were isolated and compared with those from the wild-type strain. Results obtained show that the wild-type and mutant B. japonicum strains produce very similar EPS and CPS but dissimilar LPS. For example, LPS from strain HS123 consists only of mannose, glucose, glucosamine, KDO, and lipid A. Even though structural characterization of the LPS from the wild type and the mutant strain HS123 has not been done, chemical and SDS-PAGE analyses show 1
2
3
4
5
TABLE 2. Chemical composition of LPS from the wild-type USDA 110 and mutant strain HS123a Constituents
Fucose Xylose Arabinose Mannose Glucose Fucosamine
Quinovosamine Glucosamine Uronic acids KDO Lipid A Heptose
LPS
USDA 110
HS123
2.3 2.4 2.3 8.5 2.8 6.7 1.0 1.1 3.0 0.8 30.5
Tr Tr Tr 9.5 3.2 Tr Tr 1.0 Tr 0.7 70.0 NF
NFb
Results expressed as percentage of the dry weight of LPS. b NF, Not found.
a
FIG. 3. SDS-PAGE of LPS Lanes 1 and 5 B japonicum USDA 110 LPS; lane 2, S. typhimurium lipid A lane 3 S. typhimurium LPS; lane 4, mutant strain HS123 LPS1
VOL. 169, 1987
POLYSACCHARIDES FROM B. JAPONICUM AND A MUTANT
that the two are vastly different. Strain HS123 appears to be an R-type mutant which is incapable of producing a complete LPS. The presence of an incomplete LPS is likely responsible for the resistance of strain HS123 to the previously isolated bacteriophage (19). The establishment of an effective nitrogen-fixing rhizobium-legume symbiosis is a complex developmental process. Rhizobium cell surface polysaccharides are thought to be involved in the initial events of recognition and infection. Rhizobium trifolii LPS applied to plant roots has been shown to enhance the infection of clover by R. trifolii (6). It is not clear, however, which regions of the LPS contribute to the infection and nodulation process. Maier and Brill (12) isolated nonnodulating mutants of B. japonicum that lacked the surface antigens present on the wild type. The defect in these mutants was suggested to be in the 0-antigen portion of the LPS. Our results show that the nonnodulating mutant strain HS123 is a rough mutant lacking a complete LPS moiety. Thus, it appears that a complete LPS structure may be necessary for the ability to nodulate soybeans. ACKNOWLEDGMENTS This work was supported in part by Public Health Service grant 1-ROl GM 33494-OlAl from the National Institutes of Health and grant 04-CRCR-1-1419 from the U.S. Department of Agriculture. We acknowledge the help of the National Science Foundation Chemical Instrumentation Program in obtaining the Nicolet NT200WB spectrometer. We thank G. 0. Aspinall for helpful discussions. Janet Nickels and D. C. White are thanked for performing the fatty acid analyses. LITERATURE CITED 1. Albersheim, P., D. J. Nevins, P. D. English, and A. Karr. 1967. A method for the analysis of sugars in plant cell-wall polysaccharides by gas-liquid chromatography. Carbohydr. Res. 5:340-345. 2. Ashwell, G. 1966. New colorimetric methods of sugar analysis. Methods Enzymol. 8:85-94. 3. Blumenkrantz, N., and G. Asboe-Hansen. 1973. New method for quantitative determination of uronic acid. Anal. Biochem. 54:484-489. 4. Carlson, R. W. 1982. Surface chemistry, p. 199-234. In W. J. Broughton (ed.), Nitrogen-fixation, vol. 2: rhizobium. Clarendon Press, Oxford. 5. Carlson, R. W., R. E. Sanders, C. Napoli, and P. Albersheim. 1978. Host-symbiont interactions: purification and partial characterization of Rhizobium lipopolysaccharides. Plant Physiol. 62:912-917. 6. Dazzo, F. B., G. L. Truchet, and E. M. Hrabak. 1984. Specific enhancement of clover root hair infections by trifoliin A-binding lipopolysaccharide from Rhizobium trifolii, p. 413. In C. Veeger
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and W. E. Newton (ed.), Advances in nitrogen fixation research. Martinus Nijhoff/Dr. W. Junk Publishers, The Hague, The Netherlands. 7. Edlund, A., P. D. Nichols, R. Roffey, and D. C. White. 1985. Extractable and lipopolysaccharide fatty acid and hydroxy acid profiles from Desulfovibrio species. J. Lipid Res. 26:982-988. 8. Hoist, O., D. Borowiak, J. Wikesser, and H. Mayer. 1983. Structural studies on the phosphate-free lipid A of Rhodomicrobium vannielii ATCC 17100. Eur. J. Biochem. 137:325332. 9. Johnson, K. G., and M. B. Perry. 1975. Improved techniques for the preparation of bacterial lipopolysaccharides. Can. J. Microbiol. 22:29-34. 10. Karkhanis, Y. D., J. Y. Zeltner, J. J. Jackson, and D. J. Carlo. 1978. A new and improved microassay to determine 2-keto-3deoxyoctonate in lipopolysaccharide of Gram-negative bacteria. Anal. Biochem. 85:595-601. 11. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 12. Maier, R. J., and W. J. Brill. 1978. Involvement of Rhizobium japonicum 0-antigen in soybean nodulation. J. Bacteriol. 133:1295-1299. 13. Mort, A. J., and W. D. Bauer. 1980. Composition of the capsular and extracellular polysaccharides of Rhizobium japonicum. Changes with culture age and correlations with binding of soybean seed lectin to the bacteria. Plant Physiol. 66:158-163. 14. Mort, A. J., and W. D. Bauer. 1982. Application of two new methods for cleavage of polysaccharides into specific oligosaccharide fragments. J. Biol. Chem. 257:1870-1875. 15. Mort, A. J., J. P. Utille, G. Torri, and A. S. Perlin. 1983. High selectivity in the partial degradation of an extracellular polysaccharide of Rhizobium japonicum with liquid hydrogen fluoride: a N.M.R. spectroscopic study. Carbohydr. Res. 121:221-232. 16. Peterson, A. A., and E. J. McGroarty. 1985. High-molecularweight components in lipopolysaccharides of Salmonella typhimurium, Salmonella minnesota, and Escherichia coli. J. Bacteriol. 162:738-745. 17. Pueppke, S. G. 1984. Adsorption of slow- and fast-growing rhizobia to soybean and cowpea roots. Plant Physiol. 75:924928. 18. Stacey, G., A. S. Paau, K. D. Noel, R. J. Maier, L. J. Silver, and W. J. Brill. 1982. Mutants of Rhizobiumjaponicum defective in nodulation. Arch. Microbiol. 132:219-224. 19. Stacey, G., L. A. Pocratsky, and V. Puvanesarajah. 1984. A bacteriophage that can distinguish between wild-type Rhizobium japonicum and a nonnodulating mutant. Appl. Environ. Microbiol. 48:68-72. 20. Tsai, C. M., and C. E. Frasch. 1982. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal. Biochem. 119:115-119. 21. Zevenhuizen, L. P. T. M., H. J. Scholten-Koerselman, and M. A. Posthumus. 1980. Lipopolysaccharides of Rhizobium. Arch. Microbiol. 125:1-8.
