Aug 19, 1986 - Mild acid hydrolysis of all the LPSs releases two polysaccharides ... heptose, glucuronic acid, and 2-keto-3-deoxyoctonic acid (KDO). All the.
Plant Physiol. (1987) 84, 421-427 0032-0889/87/84/0421 /07/$0 1.00/0
The Isolation and Partial Characterization of the Lipopolysaccharides from Several Rhizobium trifolii Mutants Affected in Root Hair Infection1 Received for publication August 19, 1986 and in revised form December 2, 1986
RUSSELL W. CARLSON*, ROBERT SHATTERS2, JAUH-LIN DUH3, ELROY TURNBULL4, BRIAN HANLEY5, BARRY G. ROLFE, AND MICHAEL A. DJORDJEVIC
Department ofChemistry, Eastern Illinois University, Charleston, Illinois 61920 (R.W.C., R.S., J.L.D., E.T., B.H.); and Department of Genetics, Research School ofBiological Sciences, Australian National University, Canberra City, A.C. T. 2601 Australia (B.G.R., M.A.D.) ABSTRACIr The lipopolysaccharides (LPSs) from Rhizobium trifolii ANU843 and several transposon (Tn5) symbiotic mutants derived from ANU843 were isolated and partially characterized. The mutant strains are unable to induce normal root hair curling (Hac- phenotype) or nodulation (Nodphenotype) in clover plants. The LPSs from the parent and mutants are very similar in composition. Analysis by PAGE shows that the LPSs consist of higher and lower molecular weight forms. The higher molecular weight form of the LPSs exists in several aggregation states when PAGE is done in 0.1% SDS but collapses into a single band when PAGE is done in 0.5% SDS. Mild acid hydrolysis of all the LPSs releases two polysaccharides, PS1 and PS2. Immunoblots of the PAGE gels and enzyme linked immunosorbant assay inhibition assays show that the PS1 fractions contain the immunodominant sites of the LPSs and that these sites are present in the higher molecular weight form of the LPSs. All the PS1 fractions contain methylated sugars, 2-amino-2,6-dideoxyhexose, heptose, glucuronic acid, and 2-keto-3-deoxyoctonic acid (KDO). All the PS2 fractions contain galacturonic acid, mannose, galactose, and KDO. The PS2 fractions have a molecular weight of about 700. The KDO is present at the reducing end of both the PS1 and the PS2 fractions. The PSI and PS2 fractions from the mutants contain more glucose than these fractions from the parent. The LPS from a deletion mutant contains less acyl groups than the other LPSs. Immunoblots of the LPSs show that the parent and nod A mutant LPSs contain an additional antigenic band which is not observed in the other LPSs.
Rhizobia are gram-negative bacteria which form a nitrogenfixing symbiotic relationship with legume plants. As gram-negative bacteria they have the usual surface polysaccharides consisting of EPSs,6 CPSs, and LPSs. All of these molecules have been
hypothesized to play an important role in the symbiotic infection process (for review of these polysaccharides see Carlson [3]). This
report concerns the LPSs from Rhizobium trifolii ANU843 and several of its symbiotic mutants. Several reports have presented data which show that the lectin from the host plant appears to specifically bind to the LPS from the symbiont Rhizobium (12, 14, 16). One of these reports (12) suggests that the LPS from early stationary phase R. trifolii cells binds the clover lectin, trifoliin, to a greater extent than the LPS from exponentially growing cells. It has also been reported that the LPS, as well as CPS, from Rhizobium leguminosarum inhibit the binding of these bacteria to the host root (15). Differences in the composition between the LPSs from nodulating and nonnodulating mutants of R. trifolii have been reported (21). This change in LPS composition has been correlated with the elimination of a plasmid from the nodulating strain (20,21). However, since the missing plasmid in the mutant probably contains many genes in addition to the nodulation genes, it is difficult to say whether these data imply a role for LPSs in symbiosis. A recent report shows that the LPSs from several species of Rhizobium are highly heterogeneous molecules and probably exist in several forms; e.g. those consisting of the complete LPS containing the lipid-A, core oligosaccharide and o-antigen polysaccharide and those LPSs lacking the o-antigen portion (4). Furthermore, the data in that report suggest that the 0-antigen portion of some Rhizobium LPSs may consist of a complex oligosaccharide rather than a repeating oligosaccharide as is found in the LPSs from Salmonella and Escherichia coli. In this report we describe the isolation and partial characterization of the LPSs from R. trifolii ANU843 and several of its symbiotic mutants. These mutants are all defective in the early symbiotic event known as root hair curling, i.e. they are Hac-Nod-. One mutant has a Tn5 insert in the nod D gene, another has an insert in the nod A gene, and the third has a deletion in the symbiotic plasmid (pSym) which includes nod region I (nod ABCD) and region II. The properties and sources of the mutants are listed in Table I.
'Funded by a grant from the National Science Foundation (DCB8406697). 2 Present address: Department of Genetics, Washington State UniverMATERIALS AND METHODS sity, Pullman, WA 99163. 3 Present address: Department of Chemistry, University of Cincinnati, Bacterial Strains. The strains are listed in Table I. Each strain Cincinnati, OH 45221. was grown in 12 L batches at 25°C in a yeast extract-mannitol 4Present address: P. 0. Box 660 Road Town, Tortola, British Virgin medium and aerated with filter-sterilized air (4). The bacteria Islands. 5 Present address: Department of Biochemistry, University of Illinois, ysaccharide; LPS, lipopolysaccharide; KDO, 2-keto-3-deoxyoctonic acid; Urbana, IL 61801. PBS, physiological buffered saline; TBS, tris buffered saline; ELISA, 6Abbreviations: EPS, extracellular polysaccharide; CPS, capsular pol- enzyme linked immunosorbent assay. 421
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CARLSON ET AL.
Table I. Bacterial Strains Straina Relevant Characteristics ANU843 Wild-type, Hac+Nod+ ANU85 1b nod D::Tn5 Hac-Nod-. ANU2252b nod A::Tn5 Hac-Nod-. ANU87 1 ApSym 40-50 kb deletion, Hac-Nod-. b a All strains were obtained from B. G. Rolfe (23). Resistant to
kanamycin.
were harvested at early stationary phase by centrifugation at 12,000g for about 15 min. Each preparation (just prior to harvesting) was tested by gram-staining, for growth on medium containing the appropriate antibiotic markers (Table I) and for their inability to grow on nutrient agar. There was no evidence of contamination. In addition, each strain was tested for its nodulation characteristics. Only the parent was Nod+Fix+; all the mutants were Nod-. Lipopolysaccharide Isolation Procedures. The LPSs were isolated by the hot phenol-water extraction procedure which was modified as previously described (5). Prior to extraction the bacterial pellets were suspended, by swirling, in fresh medium and centrifuged. This was repeated two more times in order to remove any adsorbed EPS from the pellets. The pellets were then suspended in PBS by blending for 3 min and centrifuged. The supernatants were tested for hexose by the anthrone assay. This was repeated until the hexose in the supernatant was greatly reduced (by about 90%). The washing in PBS was done to remove any CPS. We find that small amounts of LPS are also removed during this procedure. The pellets from the PBS wash were then extracted with hot phenol-water. The water layer was dialyzed, treated with RNase and DNase, redialyzed and lyophilized. The resultant material was subjected to gel-filtration chromatography (5). The polysaccharide portions of the LPSs were separated from the lipid-A by mild-acid hydrolysis using 1% acetic acid at 100°C for 1 h (22). The precipitated lipid-A was removed by centrifugation and the supernatant lyophilized. The polysaccharide from the supernatant was applied to a Sephadex G-50 column equilibrated with deionized water. The two polysaccharide peaks eluting from this column, PS1 and PS2, were collected and lyophilized. The lower mol wt PS2 fractions were further purified by gel-filtration chromatography using Sephadex G-25. Composition Analysis. The hexose compositions were determined by acetylation analysis. This procedure consisted of acid hydrolysis of the polysaccharides using 2 N TFA, reduction of the monosaccharides to the alditols, preparation of the alditol acetate derivatives followed by gas chromatographic analysis (1) using a 4 mm i.d. by 2 m column packed with SP2330 (Suplelco, Bellefonte, PA). Identification and quantitation was done by comparing to authentic standards. Inositol was used in all samples as an internal standard. Identification of hexoses for which standards were not available was accomplished by using combined GC/MS. Uronic acids were identified by reducing the carboxyl groups prior to acetylation analysis. Increases in, or the appearance of, a particular hexose over that of the noncarboxyl reduced sample showed that the hexose was a hexuronic acid in the noncarboxyl reduced polysaccharide. Two methods were used to identify the uronic acids. In the first method uronic acids were reduced using carbodiimide by the method of Taylor and Conrad (24). In the second method the polysaccharide was treated with HC1 in methanol, reduced with NaBH4 (8), hydrolyzed, and acetylated as described above. Uronic acids were quantitated by the method of Blumenkrantz and Asboe-Hansen (2) using glucuronic acid as the standard. KDO, acyl groups, and pyruvate were also assayed by colorimetric assays (10, 17, 27) using authentic KDO, glucose pentaacetate, and pyruvate as standards, respectively.
Plant Physiol. Vol. 84, 1987
Development of Antisera to Rhizobium trifolii ANU843. Bacteria were grown as described above. A pellet of the bacteria was suspended in sterile PBS so that the o.d. at 620 nm was about 1.0. The suspension was used to make intravenous injections into the marginal ear vein of white New Zealand rabbits according the the following schedule: D 1, 0.5 ml; d 2, 1.0 ml; d 3, 1.5 ml;d 7, 1.5 ml;d 8,2.0 ml; d 9,2.0 ml;d 21,2.0 ml. The rabbits were bled on d 28. The blood was allowed to clot at room temperature for 2 h, placed in the cold room (4°C) overnight, and centrifuged. The serum was collected, divided into aliquots, and stored in the freezer at -20°C. Polyacrylamide Gel Electrophoresis. Discontinuous slab gel electrophoresis with SDS was performed by the method of Hitchcock and Brown (1 1). The gels were stained by the silver-staining technique for LPSs described by Hitchcock and Brown. Salmonella minnesota wild-type and rough LPSs were purchased from List Biological Chemicals, Inc., Campbell, CA. Other conditions are described in the legend to figure 1. Immunoblot Procedure. The LPSs from a slab gel, run as described above, were electroblotted onto nitrocellulose paper using a Hoeffer Transphor unit. The voltage was set at 80 V for 1 h. The transfer buffer was tris/glycine 25 mM/187mM) prepared in a 20% methanol solution. The nitrocellulose paper was removed, rinsed in deionized water, and allowed to dry. The nitrocellulose paper was stained for R. trifolii antigens using a modification of a previously published procedure (6). The paper was placed in 100 ml of a TBS solution (50 mM tris/200 mM NaCl/pH 7.4 with HCI) containing 0.5% gelatin. After 30 min, 1 ml of ANU843 antisera was added to the solution and this was allowed to shake slowly for 2.5 h. The antisera solution was removed, replaced with TBS, and allowed to shake for 10 min. This was repeated two more times. After the final TBS rinsing the solution was removed, 100 ml of TBS containing 0.5% gelatin and 0.1 ml of peroxidase conjugated antirabbit goat antisera (Sigma Chemical Co.) were added. This was allowed to shake slowly overnight at room temperature. The nitrocellulose paper was rinsed with TBS as described above and then developed as described (6) with peroxide and 4-chloro-1-naphtol. The paper was removed, rinsed briefly in deionized water, and allowed to dry. Enzyme Linked Immunosorbent Assay (ELISA) Procedure. This procedure is similar to that previously described for the ELISA test (9). A coating buffer solution (pH 9.5) consisting of 1.6 g/l Na2CO3, 2.9 g/l HaHCO3, and 0.2 g/l NaN3 was prepared. One hundred ,ul of 50 ,ug/ml LPS in coating buffer was added to each well of a polystyrene microtiter plate and the plate was placed in the cold room, 4C, overnight. Serial dilutions of LPS and LPS-derived oligosaccharides were prepared in test tubes in a physiological buffered saline (PBS) Tween 20 solution which contained 800-fold diluted rabbit antisera to strain ANU843. The PBS/Tween 20 solution contained 2.2 g/l Na2HPO4. H20, 0.2 g/l NaN3, 0.2 g/l KH2PO4, 8 g/L NaCl, 0.2 g/l KCI, and 0.5 ml/l Tween 20 and was adjusted to pH 7.0. A PBS/Tween/ ANU843 antisera solution without LPS or LPS oligosaccharides was used as a positive control. A solution containing antisera to a heterologous strain, R. japonicum 61 A 123, was used as a negative control. These PBS/Tween/antisera solutions were incubated overnight at 37C. The following morning the microtiter plate was removed from the cold room, washed with PBS/Tween three times, and 100 gl of the various antisera/PBS/Tween solutions were added to each well. The plate was allowed to stand at room temperture for 1 '/2 h and then was rinsed three times with PBS/Tween. One hundred ,l of a 2000-fold diluted alkaline phosphatase conjugate-goat anti-rabbit IgG (Sigma Chemical Co.) in PBS/Tween was added to each well of the microtiter plate and the plate was incubated at 370C for 3 h. The plate was rinsed three times with PBS/Tween and 100 ul of a 1 mg/ml
RHIZOBIUM POLYSACCHARIDES solution of alkaline phosphatase substrate (p-nitrophenylphosphate, Sigma Chemical Co.) in substrate buffer (9) was added to each well. The substrate buffer consisted of 0.1 M glycine, 1 mM MgC12, and 1 mM ZnC12, adjusted to pH 10.5 with NaOH. The plate was allowed to stand for about 45 min at room temperature and the reaction was stopped by adding 50 zA of 3 M NaOH to each well. The contents of each well were diluted to 1 ml using deionized water and the absorbance was read at 405 nm.
RESULTS Lipopolysaccharide Compositions. The LPS compositions are given in Table II. All of the LPSs contain two methylated sugars, 2-o-methyl-6-deoxyhexose and 3-N-methyl-3-amino-3,6-dideoxyhexose. One or both of these sugars have been found in the LPSs from several other species of Rhizobium including R. leguminosarum, R. phaseoli and other strains of R. trifolii (3, 5, 29). The LPSs from all of the strains contain quite large amounts of heptose and uronic acid. In addition, all the LPSs have 2amino-2,6-dideoxyhexose which appears as a shoulder on the leading edge of the heptose during GC analysis. Separation between heptose and this amino sugar was not sufficient to accurately quantitate the amino sugar. All of the LPSs are quite similar in composition. On exception may be the acyl content of ANU871 LPS, which is low compared to that of the other LPSs. The components shown in Table II account for between 52 and 62% of the mass of the various LPSs. The amount of lipid-A released on mild acid hydrolysis (discussed below) accounts for 20 ± 2% of the mass of all the LPSs. While we cannot be certain, the unaccounted mass may be due to incomplete hydrolysis of these LPSs due to the stability of glycoside bonds formed by aminoglycosyl and uronosyl residues. Uronic acids were identified by two methods. In the first method the carboxyl groups were reduced using carbodiimide and then sodium borohydride (24). In the second method the LPSs were methanolyzed, and then reduced with sodium borohydride (8). After reduction, the samples were hydrolyzed with trifluoroacetic acid and acetylated as described above. For all the LPSs reduction by the first method gives an increase in only galactose while reduction by the second method results in an increase in both galactose and glucose. Colorimetric assay for uronic acid shows that the carbodiimide method results in only a 50% reduction of the uronic acid, even after reducing the LPSs three times. Thus, these LPSs apparently have both galacturonic and glucuronic acid and the carbodiimide method only reduces the galacturonic acid. At the present time it is not known why the carbodiimide method fails to reduce the glucuronic acid residues. Mild-Acid Hydrolysis of the LPSs. Mild-acid hydrolysis of
423
the LPSs followed by gel-filtration chromatography of the water soluble fraction on Sephadex G-50 results in two polysaccharide peaks, PS1 and PS2. The PS1 fraction elutes in the partially included volume while the PS2 fraction elutes with the included volume. These results are similar to those obtained for other Rhizobium LPSs (4). We observe that during mild-acid hydrolysis the LPS solutions turn a light pink. The molecule producing this color is soluble and elutes with the PS2 fractions. This molecule is not stable and the color disappears. At the present time we have not identified the molecule responsible for this pink color. The PS2 fractions were rechromatographed on Sephadex G-25; calibrated with stachyose, maltotriose and lactose; and all elute at a volume which corresponds to a mol wt of about 700. We also find that reducing the LPSs by the carbodiimide method makes them resistant to hydrolysis by mild acid. The compositions of PS1 and PS2 from all the strains examined are shown in Table III. The PS 1 fractions contain the methylated sugars, 2-amino-2,6-dideoxyhexose, heptose, glucuronic acid, and KDO. The PS2 fractions contain mannose, galactose, galacturonic acid, and KDO. The uronic acids in the PSI and PS2 fractions were identified by the methanolysis/reduction procedure described above. A significant portion of the mass of all the PS2 fractions remains unaccounted for at the present time. As previously stated above, a possible explanation for this unaccounted mass is that the large amount of galacturonic acid makes these oligosaccharides resistant to hydrolysis causing the levels of mannose, galactose, and KDO to be lower than their actual values. The compositions of the PS2 fractions may also reflect substoichiometric substitutions of sugar residues or the presence of multiple oligosaccharides with mol wt of 700 D. We have recently found that both the PS1 and PS2 fractions each consist of a mixture of at least two oligosaccharides (RW Carlson, R Shatters, unpublished data). All the PS 1 and PS2 fractions contain only trace levels of pyruvate and acyl groups suggesting that these groups may have been removed during the mild-acid hydrolysis procedure. There are several quantitative differences in the various PSI and PS2 fractions. The PSls from strains ANU2252 and ANU871 are increased in glucose compared with the parent, ANU843, PSI. Differences in mannose and galactose in the PSls may not be significant since these are minor components and could be due to slight contamination by the PS2 fractions. No significant differences between the PSls from ANU843 and ANU851 are observed. The most consistant difference seems to be that the PS2 fractions from all the mutants are increased in glucose compared with that fraction from the parent. Also the PS2 fractions from ANU2252 and ANU871 show lower levels of KDO than these fractions from ANU843 and ANU85 1.
Table II. Lipopolysaccharide Compositions of Transposon Generated Symbiotic Mutants ofR. trifolii The compositions are given in percent of mass. The above components account for 62, 58, 61, and 52% of the LPS mass from strains 843, 851, 2252, and 871, respectively. The standard deviations are calculated from a minimum of three analyses of the same sample. The values in parentheses are of the uronic acid reduced (methanolysis procedure) LPSs. The components are as follows: 2OM6DH, 2-O-methyl-6-deoxyhexose; FUC, fucose; MAN, mannose; GAL, galactose; 3NM36DH, 3-N-methyl-3-amino-3,6-dideoxyhexose; HEP, heptose; AC, acetyl groups; PY, pyruvyl groups; ND, not determined. The detector response factors for RHA and GLC were used to estimate the amounts of 2OM6DH and 3NM36DH, respectively. The sugar, 2-amino-2,6-dideoxyhexose, was also found (but not quantitated) in all the LPSs. Strain 2OM6DH FUC MAN GAL 3NM36DH GLC HEP UA PY AC KDO ANU843 5.6 ± 0.8 2.1 ± 0.3 2.8 ± 0.6 2.3 ± 0.2 5.3 ± 0.3 1.3 ± 0.5 10 ± 1.5 24 ± 1.6 2.6 ± 0.1 2.0 ± 0.5 3.9 ± 0.5
(4.5) ANU851
5.7 ± 0.3
(5.2)
ANU2252 6.3± 1.2
(4.2) ANU871
5.0 ± 0.9
(4.8)
(2.1)
(3.1)
(9.3)
2.1 ± 0.3 2.8 ± 0.4 2.4 ± 0.3
(2.1)
(3.7)
(10)
2.6±0.6 3.0±0.4 2.6±0.2
(2.0)
(3.5)
(10)
1.9 ± 0.4 2.6 + 0.8 2.0 ± 0.4
(2.3)
(3.2)
(11)
(3.1)
5.0 ± 0.6
(2.9)
5.0± 1.2
(3.4)
5.0 ± 0.9
(3.7)
(6.2)
2.3 ± 0.5
(7.6)
1.7± 1.0
(7.6)
(9.9)
(ND)
(11)
(ND)
(ND)
11 ± 1.7 21 ±2.0 2.4±0.1
(9.8)
1.3 ± 0.9 8.8 ± 2.6
(7.4)
(ND)
(ND)
(ND)
(ND)
(ND)
(ND)
(ND)
(ND)
(ND)
10 ± 1.6 19 ± 0.1 2.5 ± 0.1 2.5 ± 0.5 3.3 ± 0.3
(12)
(ND)
(ND)
19 ± 5.4
1.4 ± 0.1
(ND)
(ND)
2.0±0.1 3.3±0.4 2.2 ± 1.0 3.2 ± 0.2
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Plant Physiol. Vol. 84, 1987
Table III. Relative Sugar Compositions ofthe Polysaccharides Releasedfrom the Lipopolysaccharides by Mild-Acid Hydrolysis The sugars are defined as in Table II. In addition: GLCA = glucuronic acid and GALA = galacturonic acid. The uronic acids were quantitated by the colorimetric assay and identified by the methanolysis/reduction procedures as described in the text. The relative percent of each component is calculated by dividing the weight percent of that component by the total percent of mass accounted for by all the components. The above components account for 91, 92, 96, and 75% of the mass of the respective PSI fractions and 43, 39, 60, and 49% of the mass of the respective PS2 fractions. All the PS1 fractions contain 2-amino-2,6-dideoxyhexose which chromatographs as a shoulder on the leading edge of the heptose peak. The polysaccharides released by mild-acid hydrolysis account for approximately 80% of the LPS mass from all strains. Of these polysaccharides, 65% is PSI and 35% is PS2. 2OM6DH FUC MAN GAL 3NM36DH GLC HEP GLCA GALA KDO Sample 15 5.2 1.0 7.9 31 1.3 ANU843PSI 3.1 28 0 7.0 12 3.9 2.0 1.0 8.4 34 ANU851PSI 3.6 28 0 7.3 12 4.1 1.1 1.0 8.3 ANU2252PSI 5.8 31 29 0 6.8 15 4.3 1.2 0.8 7.3 7.3 ANU871PS1 28 28 0 8.5 TR TR ANU843PS2 0 0 10 10 0 67 0 13 0 0 10 10 0 1.0 TR ANU851PS2 0 12 66 10 0 0 ANU2252PS2 10 0 5.0 TR 0 65 9.2 0 0 ANU871PS2 10 10 0 6.0 TR 0 65 8.9
Initial analyses for KDO revealed that the PSI and PS2 fractions contained barely detectable levels of KDO, less than 0.1%, and in fact the LPSs contained low KDO levels, about 0.6%. We found that if we modified the KDO assay procedure by increasing the acid strength from 0.04 N H2SO4 to 2.0 N H2SO4 and increasing the time of hydrolysis from 30 to 60 min, the level of KDO increases to the amounts shown in Tables II and III. This indicates that the KDO is linked at the 4 or 5 position by a residue which is stable to hydrolysis in 0.04 N H2SO4. It is known that KDO linked at the 4 or 5 position gives either no color or very little color in the thiobarbituric acid assay procedure (26). In an effort to determine whether or not KDO is present at the reducing end of the PSI and PS2, these fractions were assayed for KDO after reducing with NaBH4. When KDO is reduced it will not give a color in the thiobarbituric assay procedure (26). We find that prereducing the PS1 fractions results in a 67% decrease in KDO and prereducing the PS2 fractions results in a 100% decrease in KDO. Thus, about two-thirds of the KDO in the PS1 fractions and all the KDO in the PS2 fractions are at the reducing end. PAGE and Immunochemical Analysis of the LPSs. Figure IA shows the results of PAGE analysis of all the LPSs. In addition the LPSs from Salmonella minnesota wild-type and rough mutant are included for comparison purposes. The polysaccharides released from the LPSs by mild-acid hydrolysis do not give bands when subjected to PAGE (data not shown). All of the R. trifolii LPSs examined have very similar banding patterns. There are two banding regions 'a' and 'b.' Region b consists of a single, somewhat broad gray band with a mobility slightly greater than that of the Salmonella rough LPS. The LPS from the rough Salmonella strain lacks the 0-antigen and consists of only the lipid A-core oligosaccharide. We have previously shown that, for one strain of R. leguminosarum, this broad band does not contain any of the PS1 sugars but contains the PS2 sugars, galacturonic acid and KDO (4). We have suggested that this lower mol wt broad band consists of the lipid A-core oligosaccharide (4). Whether or not this suggestion is true for the LPSs described in this report remains to be determined. Region a consists of multiple bands separated by regularly spaced intervals. These bands have a yellow/orange color. When we compare the R. trifolii LPSs with the Salmonella wild-type LPS we observe that the Salmonella LPS contains many bands separated by much smaller regularly spaced intervals. The Salmonella LPS banding pattern has been suggested to be due to LPS molecules differing
from one another by one o-antigen repeating unit (19). Such an interpretation does not seem possible for the R. trifolii LPSs examined in this report. It seems more likely that the multiple larger mol wt R. trifolii LPS bands are due to differing aggregation states of an LPS molecule which may contain a relatively short o-antigen polysaccharide rather than a long repeating oligosaccharide. To test this possibility PAGE was performed in 0.5% SDS rather than with the normal 0.1% SDS. Increasing the SDS concentration has been shown to dissociate the larger aggregation states of Salmonella and E. coli LPSs (19). The 0.5% SDS-PAGE is shown in Figure IC. Region a is reduced to a single yellow/ orange band. The multiple bands are only faintly present. The mobility of this band indicates that it is relatively low in mol wt when compared to the majority of the bands from the S. minnesota wild-type LPS. The mobility of the band in region b and the banding pattern for the LPSs from these Salmonella strains are not altered by increasing the SDS concentration to 0.5% (the data for the Salmonella strains are not shown). Figure 1 B and D, shows immunoblots of 0.1% and 0.5% SDS gels which were stained with antisera to strain ANU843. The results show that the larger mol wt form of the LPSs (i.e. region a), which exists in different aggregation states in 0.1% SDS and collapses to a single band in 0.5% SDS, contains the immunodominant site. The apparent absence of the higher mol wt bands shown in Figure lB for the LPSs from strains ANU851 and ANU2252 are not significant since these bands are visible on the original immunoblot. The lower mol wt band (i.e. region b) from each LPS shows only a slight interaction with the antisera. In addition, the immunoblots of the 0.5% SDS-PAGE of ANU843 and ANU2252 LPSs show another band, with a lower mobility than the major LPS bands, which is not observed in the LPSs from the other strains. This band is not visible in the silver stained gel. In order to determine which LPS oligosaccharides, PSI or PS2, contain the immunodominant sites these oligosaccharides were tested for their abilities to inhibit the interaction between LPS and antisera to strain ANU843. This was done by using an ELISA procedure as described above. Figure 2 shows that the PS1 from ANU85 1 inhibits the interaction between the antisera and LPS while the PS2 fraction, up to 250 ,ug/mL, has no effect on this interaction. The data for the PS1 and PS2 fractions from the other strains described in this report are much the same and the table insert in Figure 2 summarizes these results. Only PSI from ANU871 shows less inhibition than the other PSl fractions.
425
RHIZOBIUM POLYSACCHARIDES
2
1
3
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4
2
1
6
3
4
Bs
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16
-b
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FIG. 1. Wells I through 4 in (A) to (D) contain about 25 ug of ANU843, ANU85 1, ANU2252, and ANU871 LPSs, respectively. Wells 5 and 6 in (A) contain 25 gg of Salmonella Minnesota wild-type and rough LPSs, respectively. The gels are all 10 to 17% gradient gels run in either 0.1% SDS, (A) and (B), or 0.5% SDS, (C) and (D). (B) and (D) are immunoblots of 0.1 and 0.5% SDS gels, respectively. The arrows on the left edge (A) mark the locations, from top to bottom, of BSA, ovalbumin, and lysozyme, respectively. The arrows in (D) mark the location of a band which is present in the LPS from ANU843 and ANU2252 but missing in the other LPSs. This band in ANU843 LPS is very faint in the picture visible in the original immunoblot.
of
but is
CARLSON ET AL.
426
Plant Physiol. Vol. 84, 1987
0.30 E
FIG. 2. The inhibition of the antibody-LPS interaction by PSI and PS2 from strain ANU851. This plate was coated with LPS from strain ANU85 1. The procedure is as described in the text. The positive control (no inhibition) has an absorbance value of 0.364 ± 0.055 and the negative control a value of 0.017 ± 0.006. The circles, diamonds, and squares show the inhibition by PS2, PSI, and LPS, respectively. The table insert shows the percent inhibition ofthe antibody-LPS interaction by 30 ,ug/ml solutions of LPS, PS1 and PS2 from the indicated strains.
c Ln
0
LLJ (-I)
0.20
z c
Icco ax.
0.10
CONCENTRATION, ug/mL DISCUSSION The Complexity of the Rhizobium trifolii LPSs. Before comparing the mutant and parent LPSs it is necessary to discuss the general complexity of these molecules. As with the LPSs from other Rhizobium species (4, 5, 29) and other gram-negative bacteria (28) the LPSs in this report contain KDO, and mildacid hydrolysis produces a water-insoluble material and releases two water-soluble polysaccharide fractions (PS1 and PS2). The water-insoluble precipitates are presently being characterized. They do contain fatty acids and are presumably the lipid-A portions ofthe LPSs (RW Carlson, RL Hollingsworth, FB Dazzo, unpublished data). Other than these general characteristics, Rhizobium LPSs appear to be quite different from the Salmonella and E. coli LPSs. The PAGE and immunochemical results show that the larger mol wt form of these Rhizobium LPSs contains the immunodominant structure (PS 1) and appears as a single band rather than the multiple bands observed for the LPS from Salmonella minnesota wild type strain. In addition, the immunodominant form of Rhizobium LPS has a greater electrophoretic mobility than that of Salmonella LPS. These results suggest that the complete form of the Rhizobium LPSs; i.e. that which contains the o-antigen, core and lipid; apparently has a short oantigen polysaccharide compared to that of the Salmonella LPS. The complexity of the PS1 compositions would seem to support the idea that these o-antigens consist of a complex oligosaccharide rather than a repeating oligosaccharide unit, and may also indicate that there is considerable microheterogeneity in these PSI preparations. We have presented data on other Rhizobium LPSs which also support the idea that some Rhizobium oantigens may consist of a complex oligosaccharide and not of a repeating oligosaccharide (4). We observe that the PS1 fractions do not contain any detectable galacturonic acid which is a major component of PS2, presumably the core oligosaccharide. This indicates that the bonds which link PSI and PS2 to the remainder of the LPS are both subject to mild-acid hydrolysis. This is also suggested by the fact that reduction of carboxyl groups via the carbodiimide
method stabilizes both of these linkages towards hydrolysis by mild acid. The most likely explanation would be that both of these bonds involve KDO and that reduction of the carboxyl group of KDO stabilizes both bonds toward hydrolysis by 1% acetic acid. That both PSI and PS2 are linked to the remainder of the LPS via KDO is supported by the fact that both PS1 and PS2 from all the Rhizobium LPSs in this report contain KDO at their reducing ends. In addition, the large increase in the KDO content of the PSI and PS2 fractions on increasing the acid strength and the hydrolysis time during the KDO assay procedure suggests that the bonds between PS1 and KDO and between PS2 and KDO are relatively stable to acid and are at the 4 or 5 position ofthe KDO residues. One possibility is that the PSI and PS2 oligosaccharides are linked to the KDO residues via a uronosyl residue, glucuronosyl and galacturonosyl in the case of PSI and PS2, respectively. As stated earlier, we find that both PS1 and PS2 can each be separated into additional oligosaccharides by DEAE and gel-filtration chromatography. These fractions are being characterized by acetylation and methylation analysis, and by nuclear magnetic resonance spectroscropy. The fact that all Rhizobium LPSs examined thus far appear to have a galacturonic acid oligomer is interesting when one considers the recent reports showing that pectic oligomers possess various biological activities. It has been reported that pectic oligosaccharides released from plant cell walls by pathogen endopolygalacturonases possess potent elicitor activity, i.e. they stimulate the plant to produce phytoalexins (7, 18). In addition, molecules released from plant cells by endopolygalacturonase induce callus, bud, or embryo formation depending on the tissue incubation conditions (25). It would be interesting to test the LPS oligosaccharides for some of these various biological activities.
Comparison of the R. trifolii LPSs. The compositions of the PSls and PS2s, and the PAGE results suggest that there may be several differences among the various LPSs. These differences can be summarized as follows: (a) The glucose content of both PS 1 and PS2 from strains ANU2252 and ANU87 1 are increased
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over those fractions from the parent. (b) The glucose content of PS2 from strain ANU851 is increased over that fraction from the parent. (c) The acyl content of the LPS from strain 871 is decreased compared with the parent LPS. (d) The immunoblot of the 0.5% SDS PAGE shows that the LPSs from ANU843 and ANU2252 contain a band which is not present in the other LPSs. Determining if these differences are significant with regard to the mutant phenotype will require further structural analysis of these LPSs. Establishing the structure of the parental LPS will greatly facilitate our analysis of the structural alterations, if any, which are present in the various mutants. It has recently been shown, using Mu-lac insertion mutants, that the nod ABC genes as well as other nodulation genes require induction by a factor in the host root exudate (13). Thus, it is very important to reexamine the LPSs from strains which have been grown under conditions that are known to induce these genes. We have recently been able to induce large batches (12L) of Mu-lac mutants and are presently isolating the LPSs from induced strains which carry multiple copies of these nod genes. Analysis of these LPSs as well as the further structural characterization of the parental LPS is in progress.
10. HESTRIN S 1949 The reaction of acetylcholine and other carboxylic acid derivatives with hydroxlyamine and its analytical application. J Biol Chem 249: 249-261 11. HITCHCOCK PJ, TM BROWN 1983 Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J Bacteriol 154: 269-277 12. HRABAK EM, MR URBANO, FB DAZZO 1981 Growth-phase-dependent immunodeterminants of Rhizobium trifolii lipopolysaccharide which bind trifolii A, a white-clover lectin. J Bacteriol 148: 697-71 1 13. INNES RW, PL KUEMPEL, J PLAZINSKI, H CANTER-CREMERS, BG ROLFE, MA DJORDJEVIC 1985 Plant factors induce expression of nodulation and hostrange genes in Rhizobium trifolii. Mol Gen Genet 201: 426-432 14. KAMBERGER W 1979 An Ouchterlony double diffusion study on the interaction between legume lectins and rhizobial cell surface antigens. Arch Microbiol 121: 83-90 15. KATO G, Y MARUYAMA, M NAKAMURA 1980 Role ofbacterial polysaccharides in the adsorption process of the Rhizobium-pea symbiosis. Agric Biol Chem 44: 2843-2855 16. KATO G, Y MARUYAMA, M NAKAMURA 1979 Role of lectins and lipopolysaccharides in the recognition process of specific Rhizobium-legume symbiosis. Agric Biol Chem 43: 1085-1092 17. KATSUKI H, T YOSHIDA, C TANEGASHIMA, S TANAKA 1971 Improved direct method for the determination of keto acid by 2, 4-dinitrophenylhydrazones. Anal Biochem 24: 349-356 18. McNEIL M, AG DARVILL, SC FRY, P ALBERSHEIM 1984 Structure and function of the primary cell walls of plants. Annu Rev Biochem 53: 625-663 19. PETERSEN AA, EJ MCGROARTY 1985 High-molecular-weight components in
Acknowledgment-The authors thank the NIH GC/MS facility at Washington University (RR00954) for GC/MS analyses of the various LPS samples.
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