Rhizobium etli CE3 Bacteroid Lipopolysaccharides Are Structurally Similar but Not Identical to Those Produced by Cultured CE3 Bacteria
2007; Elsevier BV; Volume: 282; Issue: 23 Linguagem: Inglês
10.1074/jbc.m611669200
ISSN1083-351X
AutoresWim D’Haeze, Christine Leoff, Glenn Freshour, K. Dale Noel, Russell W. Carlson,
Tópico(s)Probiotics and Fermented Foods
ResumoRhizobium etli CE3 bacteroids were isolated from Phaseolus vulgaris root nodules. The lipopolysaccharide (LPS) from the bacteroids was purified and compared with the LPS from laboratory-cultured R. etli CE3 and from cultures grown in the presence of anthocyanin. Comparisons were made of the O-chain polysaccharide, the core oligosaccharide, and the lipid A. Although LPS from CE3 bacteria and bacteroids are structurally similar, it was found that bacteroid LPS had specific modifications to both the O-chain polysaccharide and lipid A portions of their LPS. Cultures grown with anthocyanin contained modifications only to the O-chain polysaccharide. The changes to the O-chain polysaccharide consisted of the addition of a single methyl group to the 2-position of a fucosyl residue in one of the five O-chain trisaccharide repeat units. This same change occurred for bacteria grown in the presence of anthocyanin. This methylation change correlated with the inability of bacteroid LPS and LPS from anthocyanin-containing cultures to bind the monoclonal antibody JIM28. The core oligosaccharide region of bacteroid LPS and from anthocyanin-grown cultures was identical to that of LPS from normal laboratory-cultured CE3. The lipid A from bacteroids consisted exclusively of a tetraacylated species compared with the presence of both tetra- and pentaacylated lipid A from laboratory cultures. Growth in the presence of anthocyanin did not affect the lipid A structure. Purified bacteroids that could resume growth were also found to be more sensitive to the cationic peptides, poly-l-lysine, polymyxin-B, and melittin. Rhizobium etli CE3 bacteroids were isolated from Phaseolus vulgaris root nodules. The lipopolysaccharide (LPS) from the bacteroids was purified and compared with the LPS from laboratory-cultured R. etli CE3 and from cultures grown in the presence of anthocyanin. Comparisons were made of the O-chain polysaccharide, the core oligosaccharide, and the lipid A. Although LPS from CE3 bacteria and bacteroids are structurally similar, it was found that bacteroid LPS had specific modifications to both the O-chain polysaccharide and lipid A portions of their LPS. Cultures grown with anthocyanin contained modifications only to the O-chain polysaccharide. The changes to the O-chain polysaccharide consisted of the addition of a single methyl group to the 2-position of a fucosyl residue in one of the five O-chain trisaccharide repeat units. This same change occurred for bacteria grown in the presence of anthocyanin. This methylation change correlated with the inability of bacteroid LPS and LPS from anthocyanin-containing cultures to bind the monoclonal antibody JIM28. The core oligosaccharide region of bacteroid LPS and from anthocyanin-grown cultures was identical to that of LPS from normal laboratory-cultured CE3. The lipid A from bacteroids consisted exclusively of a tetraacylated species compared with the presence of both tetra- and pentaacylated lipid A from laboratory cultures. Growth in the presence of anthocyanin did not affect the lipid A structure. Purified bacteroids that could resume growth were also found to be more sensitive to the cationic peptides, poly-l-lysine, polymyxin-B, and melittin. Root nodule development is orchestrated by a symbiotic molecular dialogue between Gram-negative Rhizobium bacteria (e.g. Azorhizobium sp., Bradyrhizobium sp., Rhizobium sp., Sinorhizobium sp.) and specific legume host plants. Nodules are newly formed organs consisting of plant cells occupied with bacteroids that provide the host plant with fixed nitrogen. In the best studied symbiotic interactions, bacteria enter the roots via susceptible curled root hairs, and intracellular infection threads guide the bacteria toward de novo nodule primordia, where internalization into plant cells takes place. Initiation of nodule development and invasion require the production of bacterial signal molecules, including fatty acylated chitin oligosaccharides known as Nod factors (1D'Haeze W. Holsters M. Glycobiology. 2002; 12: R79-R105Crossref PubMed Scopus (294) Google Scholar), and structurally complex surface polysaccharides (SPS) 3The abbreviations used are: SPS, surface polysaccharide; AGC, aeroponic growth chamber; CPS, capsular polysaccharide; DOC, deoxycholate; EPS, extracellular polysaccharide; GC-MS, gas chromatography-mass spectrometry; HPAEC, high performance anion exchange chromatography; Kdo, 3-deoxy-d-manno-oct-2-ulosonic acid; LPS, lipopolysaccharide; mAb, monoclonal antibody; MALDI-TOF, matrix-assisted laser desorption ionization-time-of-flight; NBT, nitro blue tetrazolium; PMAA, partially methylated alditol acetates; PM, peribacteroid membrane. (2Kannenberg E.L. Reuhs B.L. Forsberg L.S. Carlson R.W. Spaink H.P. Kondorosi A. Hooykaas P.J. The Rhizobiaceae. Kluwer Academic Publishers, Dordrecht, Boston, London1998: 119-154Crossref Google Scholar, 3Fraysse N. Couderc F. Poinsot V. Eur. J. Biochem. 2003; 270: 1365-1380Crossref PubMed Scopus (263) Google Scholar). The outer surface of rhizobia typically consists of SPS that include extracellular polysaccharides (EPS) that are released into the media, capsular polysaccharides that are tightly associated with the bacterial surface, and lipopolysaccharides (LPS) that are anchored in the outer membrane (4Noel K.D. Verma D.P.S. Molecular Signals in Plant-Microbe Communications. CRC Press, Inc., Boca Raton, FL1992: 341-357Google Scholar). LPS are composed of lipid A, a core oligosaccharide, and an O-antigen polysaccharide. Accumulating data demonstrate the important role that rhizobial SPS play in invasion and nodule development and their involvement in the initiation of infection and invasion, suppression of plant defense, bacterial release from infection threads, bacteroid development and senescence, induction of plant gene expression, and protection against antimicrobial compounds (2Kannenberg E.L. Reuhs B.L. Forsberg L.S. Carlson R.W. Spaink H.P. Kondorosi A. Hooykaas P.J. The Rhizobiaceae. Kluwer Academic Publishers, Dordrecht, Boston, London1998: 119-154Crossref Google Scholar, 3Fraysse N. Couderc F. Poinsot V. Eur. J. Biochem. 2003; 270: 1365-1380Crossref PubMed Scopus (263) Google Scholar). Various observations suggest that proper LPS synthesis is required for invasion and nodule development in various symbiotic interactions, including the interaction between Rhizobium etli and Phaseolus vulgaris (2Kannenberg E.L. Reuhs B.L. Forsberg L.S. Carlson R.W. Spaink H.P. Kondorosi A. Hooykaas P.J. The Rhizobiaceae. Kluwer Academic Publishers, Dordrecht, Boston, London1998: 119-154Crossref Google Scholar, 4Noel K.D. Verma D.P.S. Molecular Signals in Plant-Microbe Communications. CRC Press, Inc., Boca Raton, FL1992: 341-357Google Scholar). An R. etli mutant that lacks the O-chain polysaccharide portion of its LPS elicited the formation of infection threads on P. vulgaris; however, the bacteria ceased to develop within the root hair that formed thick walls (5Cava J.R. Elias P.M. Turowski D.A. Noel K.D. J. Bacteriol. 1989; 171: 8-15Crossref PubMed Google Scholar, 6Noel K.D. VandenBosch K.A. Kulpaca B. J. Bacteriol. 1986; 168: 1392-1401Crossref PubMed Scopus (106) Google Scholar). The formation of nodule primordia was normal, but no bacteria were released from infection threads and internalized into plant cells (6Noel K.D. VandenBosch K.A. Kulpaca B. J. Bacteriol. 1986; 168: 1392-1401Crossref PubMed Scopus (106) Google Scholar). Occasionally, some bacteria were present in intercellular spaces. It was furthermore demonstrated that not only the presence of the O-chain polysaccharide on the LPS but also the abundance of O-chain polysaccharide was important for nodulation. For example, mutant strain R. etli CE166 produced, based on PAGE analysis of the LPS, only 40% LPS containing the O-chain polysaccharide compared with the parent strain, and the symbiotic phenotype of this mutant was the same as that observed for a mutant that entirely lacks the O-chain polysaccharide (7Forsberg L.S. Noel K.D. Box J. Carlson R.W. J. Biol. Chem. 2003; 278: 51347-51359Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 8Noel K.D. Forsberg L.S. Carlson R.W. J. Bacteriol. 2000; 182: 5317-5324Crossref PubMed Scopus (53) Google Scholar). A striking feature of LPS synthesis is that it is influenced by a variety of environmental factors (9Tao H. Brewin N.J. Noel K.D. J. Bacteriol. 1992; 174: 2222-2229Crossref PubMed Google Scholar). The LPS contained in bacteria isolated from the host (bean) nodules was diminished in its ability to bind monoclonal antibodies JIM28 and JIM29. In addition, the ability to bind these mAbs was also affected by pH, O2, or phosphate concentrations and temperature. Mutants that produced O-chain polysaccharide-containing LPS that do not change in their ability to bind JIM28 or JIM29 were impaired in their nodulation frequency and development (9Tao H. Brewin N.J. Noel K.D. J. Bacteriol. 1992; 174: 2222-2229Crossref PubMed Google Scholar). In addition, it was shown that R. etli CE3, grown in the presence of P. vulgaris root or seed exudates, produced modified LPS that was no longer recognized by a particular monoclonal antibody (mAb), JIM28, specific for the O-chain polysaccharide of LPS from laboratory-cultured R. etli CE3 (10Noel K.D. Duelli D.M. Tao H. Brewin N.J. Mol. Plant-Microbe Interact. 1996; 9: 180-186Crossref Scopus (23) Google Scholar). Major compositional differences between LPS produced by CE3 cultures grown at pH 7.2 and that of pH 4.8 cultures included replacement of 2,3,4-tri-O-methylfucose by 2,3-di-O-methylfucose and an increase of 2-O-methylfucose content (11Bhat U.R. Carlson R.W. J. Bacteriol. 1992; 174: 2230-2235Crossref PubMed Google Scholar). These results showed the importance of determining the molecular/genetic basis for these subtle structural changes to R. etli LPS. Here we describe the preparation of LPS from R. etli CE3 bacteroids purified from the host root nodules, and we compare its structure to that produced by R. etli CE3 grown under normal laboratory conditions (Fig. 1). Although LPS from CE3 bacteria and bacteroids were structurally similar, we observed that bacteroid LPS was antigenically different from that of bacteria and showed a doubling in 2-O-methylfucose within the O-chain polysaccharide. Mass spectrometry analyses also demonstrated that the lipid A from bacteroid LPS lacked a β-hydroxymyristic acid acyl residue. Our results also indicated that the R. etli CE3 bacteroid population that could resume growth was significantly more sensitive to cationic peptides than R. etli laboratory-cultured bacteria. Plant Growth and Nodule Preparation—For each aeroponic growth chamber (AGC), 180 P. vulgaris seeds (black turtle; Sacajawea Organic Foods) were surface-sterilized in 50 ml of 95% ethanol for 4 min while shaking the solution manually. The ethanol was discarded, and the seeds were washed two times with sterilized deionized water. The seeds were then rinsed with 50 ml of 5% sodium hypochloride (Acros Organics) for 4 min followed by several washes with sterilized deionized water (2 liter total volume). Seeds were transferred to plastic pots containing a 0.8% agarose layer (0.8 g per 100 ml of tap water) for germination (five seeds per pot to allow enough space for the seeds to germinate) and incubated in the dark at 30 °C for 4 days. The AGC consisted of a polypropylene barrel that was not light-transparent and a lid with 150 holes through which plants could grow. A humidifier (505 Defensor from Axair AG, Pfäffikon, Switzerland) was placed on the bottom of the barrel. A tap was present in the barrel, which allowed changing of the nutrient solution in an efficient manner, and the lid-barrel contact was tight so that no nutrient solution was lost during plant growth. The entire ACG, including the humidifier, was cleaned with 98% ethanol prior to use and rinsed with 10 liters of sterilized nitrogen-free nutrient solution (12Lullien V. Barker D.G. de Lajudie P. Huguet T. Plant Mol. Biol. 1987; 9: 469-478Crossref PubMed Scopus (81) Google Scholar). Subsequently, the P. vulgaris seedlings were transferred to the AGC (one seedling per hole) and supported by some water-soaked horticultural rock wool. The latter also nicely sealed the space between the seedling and the lid material without damaging the hypocotyl. Remaining seed coats were removed manually prior to the transfer of the seedlings to the AGC. R. etli CE3 was grown as described (8Noel K.D. Forsberg L.S. Carlson R.W. J. Bacteriol. 2000; 182: 5317-5324Crossref PubMed Scopus (53) Google Scholar), and the pellets of two 300-ml overnight late exponential phase cultures were extensively washed with nutrient solution and added to the AGC after seedlings were transferred. The nutrient solution, including the CE3 inoculum, was refreshed every other day for 4 weeks. The AGCs were placed in an acclimatized plant growth room with a photoperiod of 14/10, a relative humidity of 60%, and a day and night temperature of 23 and 18 °C, respectively. Mature nodules were manually harvested 4 weeks after seedlings were transferred to the AGC. The nodules were collected in 50-ml tubes and immediately frozen until bacteroids needed to be prepared for LPS purification. R. etli CE3 Bacteroid Isolation—A slightly modified stepwise sucrose gradient-based ultracentrifugation approach as described by Ching et al. (13Ching T.M. Hedtke S. Newcomb W. Plant Physiol. 1977; 60: 771-774Crossref PubMed Google Scholar) was used for bacteroid isolation. Briefly, 5g of frozen nodules were extensively ground using a mortar and pestle until a homogeneous paste was obtained. Ten milliliters of filter-sterilized grinding buffer (13Ching T.M. Hedtke S. Newcomb W. Plant Physiol. 1977; 60: 771-774Crossref PubMed Google Scholar) were added, and the mixture was manually stirred with a glass bar for a few minutes. Six polyallomer ultracentrifugation tubes with a capacity of 12.2 ml (Beckman Coulter) were prepared by adding the stepwise sucrose gradient (i.e. from bottom to top: 2.076 ml of 57% sucrose, 2.699 ml of 52% sucrose, 2.699 ml of 50% sucrose, and 2.076 ml of 45% sucrose). Care was taken to avoid mixing of different sucrose layers. The remaining space in the tube was filled with ∼1.6 ml of the crushed nodule mixture in grinding buffer. The tubes were equilibrated, placed in an SW40Ti rotor, and ultracentrifuged at 100,000 × g for 4 h at 10 °C (Beckman Coulter). When the ultracentrifugation run was completed, tubes were carefully removed from the rotor, and the five bands were immediately transferred to a separate tube using a Pasteur pipette. The bacteroids (band 4) were washed twice (7,000 rpm for 20 min at 4 °C) with phosphate buffer, pH 7.2, and after washing, the pellet was resuspended in a final volume of 200 μl of phosphate buffer, collected in a 50-ml tube, and stored at –20 °C. When a total volume of 50 ml was obtained, the LPS was prepared using the hot phenol/water extraction as described below. Dot-blot Immunoblotting—Immunodot blot assays were prepared (14Kannenberg E.L. Brewin N.J. J. Bacteriol. 1989; 171: 4543-4548Crossref PubMed Google Scholar). Briefly, a fraction of an overnight CE3 culture or samples of the respective bands were washed with phosphate buffer, pH 7.2, and diluted to an A600 equal to 1. One microliter of the initial concentration of each sample and of 10-, 100-, and 1000-fold dilutions were spotted on a nitrocellulose membrane (Sigma) and air-dried for 1 h. The membrane was transferred to a small glass dish, which was put on a rocker set at low speed, and washed three times with TBS solution (50 mm Tris/HCl, 200 mm NaCl, pH 7.4) for 15 min. Blocking was performed by adding 20 ml of 2% (w/v) bovine serum albumin in TBS and incubation for 30 min. The membrane was incubated overnight after addition of nitrogenase antibodies (1/5000 dilution in 2% bovine serum albumin) (15Ma Y. Ludden P.W. J. Bacteriol. 2001; 183: 250-256Crossref PubMed Scopus (10) Google Scholar). The membrane was then washed with TBS solution for 2 h during which the solution was refreshed at least five times. The membrane was incubated in the presence of alkaline phosphatase anti-rat IgG (Sigma; 1/5000 dilution in 2% bovine serum albumin) and subsequently washed for 30 min in TBS solution during which the solution was refreshed at least five times. The membrane was developed in alkaline phosphatase substrate solution, containing 9 ml of Tris/HCl buffer (100 mm Tris/HCl, pH 9.6), 1 ml of nitro blue tetrazolium (NBT) solution (1 mg/ml NBT in Tris/HCl buffer plus 2% dimethyl sulfoxide), 100 μl of 5-bromo-4-chloro-indolyl phosphate (5 mg/ml in dimethylformamide), and 40 μlof1 m MgCl2. Cationic Peptide Sensitivity Assay—Overnight bacterial cultures of CE3 and CE338, the latter is affected in the synthesis of EPS (16Diebold R. Noel K.D. J. Bacteriol. 1989; 171: 4821-4830Crossref PubMed Google Scholar), and freshly isolated bacteroids were extensively washed with phosphate buffer, pH 7.2, and diluted to an A600 equal to 1. The cationic peptides tested were melittin, polymyxin B, and poly-l-lysine (Sigma). For melittin, 1 μl of a 20 μg/ml stock solution was added to 800 μl of a solution of bacteria or bacteroids and incubated for 30 min at room temperature; for polymyxin B, 3 μlofa20 μg/ml stock solution was added to 10 μl of bacteria or bacteroids and incubated for 1 h at room temperature; and for poly-l-lysine, 3 μlofa50 μg/ml stock solution was added to 10 μl of bacteria or bacteroids and incubated for 1 h at room temperature. The viability was determined as described previously (17D'Haeze W. Glushka J. De Rycke R. Holsters M. Carlson R.W. Mol. Microbiol. 2004; 52: 485-500Crossref PubMed Scopus (68) Google Scholar). This assay was repeated 10 times for each bacterial or bacteroid preparation with each cationic peptide, and a statistical analysis was performed using the Student's t test. Averages were not significantly different when p > 0.05. Microscopy Techniques—An initial microscopic examination of the various bands obtained after ultracentrifugation was done using a classical Gram staining. Material from bands 1 through 5 and cultured CE3 bacteria were stained with crystal violet followed by a safranin staining (Sigma) and thereafter immediately examined using a light microscope (Olympus, Tokyo, Japan). Transmission electron microscopy was employed to observe cultured CE3 bacteria (negative control), purified CE3 bacteroids (band 4), and sections through mature nodules (positive control). For the latter, the nodules were treated and embedded for transmission electron microscopy as described previously (18D'Haeze W. De Rycke R. Mathis R. Goormachtig S. Pagnotta S. Verplancke C. Capoen W. Holsters M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11789-11794Crossref PubMed Scopus (153) Google Scholar). The embedding of CE3 bacteria and bacteroids was done as follows (all procedures were carried out at 4 °C under rotation). Samples were extensively washed with 0.1 m cacodylate buffer (Sigma) and fixed by a gradual fixation approach. The pellets were consecutively resuspended in 0.5% formaldehyde, 0.5% glutaraldehyde in 0.1 m cacodylate buffer, 1.0% formaldehyde, 1.0% glutaraldehyde in 0.1 m cacodylate buffer, 1.5% formaldehyde, 1.5% glutaraldehyde in 0.1 m cacodylate buffer, 2.0% formaldehyde, 2.0% glutaraldehyde in 0.1 m cacodylate buffer, and finally in 2.5% formaldehyde, 2.5% glutaraldehyde in 0.1 m cacodylate buffer. Each time, the samples were incubated for 20 min. Then the pellets were washed three times with 0.1 m cacodylate buffer followed by a dehydration series, including 2 h in 30% ethanol, 2 h in 50% ethanol, overnight in 70% ethanol, 2 h in 95% ethanol, and overnight in 95% ethanol. The samples were then imbedded in LR White Hard Grade by resuspending the pellet overnight in ethanol/LR White (1/1 v/v), a step that was repeated two times. Finally, the pellets were resuspended in pure LR White and rotated overnight, which was repeated at least five times. The samples were transferred to capsules and incubated at 65 °C for 48 h to allow polymerization. Sections were made using an MT 6000-XL ultramicrotome (RMC, Inc., Tucson, AZ). Routine control sections were 1 μm thick and were stained with toluidine blue. Sections for transmission electron microscopy were 90 nm thick and collected on gilded copper slot grids (Ted Pella, Inc., Redding, CA) that were placed on Formvar bridges to dry (19Rowley J.C. Moran D.T. Ultramicroscopy. 1975; 1: 151-155Crossref PubMed Scopus (157) Google Scholar). Sections were post-stained for 2 min with 4% (w/v) aqueous uranyl acetate and for 0.5 min with lead citrate (20Reynolds E.S. J. Cell Biol. 1963; 17: 208-212Crossref PubMed Scopus (17782) Google Scholar). Sections were examined at 80 kV with a Zeiss 902A electron microscope. LPS Isolation—Crude LPS was obtained from the bacteria and bacteroids using the hot phenol/water extraction procedure (21Westphal O. Jann K. Methods Carbohydr. Chem. 1965; 5: 83-91Google Scholar), which was modified by Carlson et al. (22Carlson R.W. Sanders R.E. Napoli C. Albersheim P. Plant Physiol. 1978; 62: 912-917Crossref PubMed Google Scholar). The water phase containing the LPS was treated with RNase, DNase, and proteinase K, dialyzed, and then lyophilized (22Carlson R.W. Sanders R.E. Napoli C. Albersheim P. Plant Physiol. 1978; 62: 912-917Crossref PubMed Google Scholar). The LPS extracted into the phenol phase was treated as described by Carrion et al. (23Carrion M. Bhat U.R. Reuhs B. Carlson R.W. J. Bacteriol. 1990; 172: 1725-1731Crossref PubMed Google Scholar). The LPS was purified from these crude preparations with affinity chromatography using polymyxin B-Sepharose (Pierce) (24Forsberg L.S. Carlson R.W. J. Biol. Chem. 1998; 273: 2747-2757Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 25Ridley B.L. Jeyaretnam B.S. Carlson R.W. Glycobiology. 2000; 10: 1013-1023Crossref PubMed Scopus (28) Google Scholar). Briefly, the crude LPS was dissolved in 50 mm NH4CO3 and applied to the column. The column was then washed with 50 mm NH4CO3, followed by a solution of 300 mm triethylamine adjusted to pH 6.4 with acetic acid, and then a solution of 0.1 m NH4CO3 in 2 m urea to remove any non-LPS material from the column. The LPS was finally removed using a solution of 1% deoxycholate (DOC) in 0.1 m NH4CO3. The LPS was extensively dialyzed against a solution of 50 mm Tris base with 10% ethanol, then against deionized water, and lyophilized. For cultures grown in the presence anthocyanin, crude anthocyanin preparations were obtained by acid extraction, as described previously by Noel et al. (10Noel K.D. Duelli D.M. Tao H. Brewin N.J. Mol. Plant-Microbe Interact. 1996; 9: 180-186Crossref Scopus (23) Google Scholar), from P. vulgaris seed (cv. Midnight Black Turtle Soup supplied by Idaho Seed Bean, Twin Falls, ID). R. etli CE3 was grown in medium (8Noel K.D. Forsberg L.S. Carlson R.W. J. Bacteriol. 2000; 182: 5317-5324Crossref PubMed Scopus (53) Google Scholar) to which the crude anthocyanin extract had been added as described (10Noel K.D. Duelli D.M. Tao H. Brewin N.J. Mol. Plant-Microbe Interact. 1996; 9: 180-186Crossref Scopus (23) Google Scholar). The LPS was isolated by hot phenol/water extraction as described above and purified by Sepharose 4B chromatography after dialysis and treatment with nucleases and proteinase K (21Westphal O. Jann K. Methods Carbohydr. Chem. 1965; 5: 83-91Google Scholar, 26Carlson R.W. Kalembasa S. Turowski D. Pachori P. Noel K.D. J. Bacteriol. 1987; 169: 4923-4928Crossref PubMed Scopus (99) Google Scholar). Electrophoresis and Immunoblotting—The LPS preparations were analyzed using DOC-PAGE, and the polyacrylamide gels were stained using the Alcian blue-silver staining procedure as described previously (27Reuhs B.L. Geller D.P. Kim J.S. Fox J.E. Kolli V.S.K. Pueppke S.G. Appl. Environ. Microbiol. 1998; 64: 4930-4938Crossref PubMed Google Scholar). Immunoblotting was also performed according to the method described by Reuhs et al. (27Reuhs B.L. Geller D.P. Kim J.S. Fox J.E. Kolli V.S.K. Pueppke S.G. Appl. Environ. Microbiol. 1998; 64: 4930-4938Crossref PubMed Google Scholar). Briefly, LPS-containing gels were soaked in transfer buffer (48 mm Tris, 39 mm glycine, 20% methanol) and electrophoretically transferred to a nitrocellulose membrane using a Bio-Rad Transblot SD semi-dry transfer cell set at a current of 20 V for 20 min. The membrane was equilibrated in TBS (0.2 m NaCl, 20 mm Tris, pH 7.4) for 5 min, then blocked using 5% nonfat dry milk (Bio-Rad) in TBS, and then overlaid with a 1/100 dilution of one of the primary mAbs (JIM26, JIM27, JIM28, or JIM29) in blocking solution. The membrane was then washed (five times for 5 min in TBS) and incubated with alkaline phosphatase-conjugated secondary antibody, at 1/1000 dilution of the antibody. Finally, the membrane was equilibrated in substrate buffer (0.1 m Tris, 0.1 m NaCl, 5 mm MgCl2, pH 9.5) and developed for 5 min using a developing solution of 20 ml of substrate buffer, 128 μl of NBT stock solution (50 mg/ml NBT in 70% dimethylformamide), and 66 μl of BCIP stock solution (50 mg/ml BCIP in 100% N,N-dimethylformamide). Once the bands were visible, the reaction was stopped by washing with deionized water. LPS Analysis—Compositions were determined by the preparation and gas chromatography-mass spectrometry (GC-MS) analysis of trimethysilyl methyl glycosides (28York W.S. Darvill A.G. McNeil M. Stevenson T.T. Albersheim P. Methods Enzymol. 1985; 118: 3-40Crossref Scopus (1062) Google Scholar). This procedure was also used to determine the fatty acid composition of the LPS preparations (29Bhat U.R. Forsberg L.S. Carlson R.W. J. Biol. Chem. 1994; 269: 14402-14410Abstract Full Text PDF PubMed Google Scholar). Glycosyl composition of the LPS preparations was also determined by the preparation and GC-MS analysis of alditol acetates (28York W.S. Darvill A.G. McNeil M. Stevenson T.T. Albersheim P. Methods Enzymol. 1985; 118: 3-40Crossref Scopus (1062) Google Scholar). The location of methyl ether groups and the linkage positions of the various glycosyl residues were determined by the preparation and GC-MS analysis of partially methylated alditol acetates (PMAAs) as described by Ciucanu and Kerek (30Ciucanu I. Kerek F. Carbohydr. Res. 1984; 131: 209-217Crossref Scopus (3215) Google Scholar). Methylation was performed using tri-deuteriomethyliodide so that analysis of the partially methylated alditol acetates by GC-MS would reveal the location of the naturally occurring methyl groups on the LPS. The per-trideuteromethylated polysaccharides were hydrolyzed using 2 m trifluoroacetic acid at 121 °C for 2 h (29Bhat U.R. Forsberg L.S. Carlson R.W. J. Biol. Chem. 1994; 269: 14402-14410Abstract Full Text PDF PubMed Google Scholar). The resulting partially (trideutero) methylated glycosyl residues were reduced using sodium borodeuteride and acetylated at 80 °C with a 1/1 mixture of acetic anhydride: pyridine (29Bhat U.R. Forsberg L.S. Carlson R.W. J. Biol. Chem. 1994; 269: 14402-14410Abstract Full Text PDF PubMed Google Scholar). The partially (trideutero) methylated alditol acetates were then analyzed using GC-MS. Analysis of the core oligosaccharides was determined by subjecting the LPS preparations to 1% acetic acid for 1 h at 100°C, removing the lipid A by centrifugation, and analysis of the carbohydrates by HPAEC using a Carbo PacPA-1 (Dionex) with pulsed amperometric detection as described previously (24Forsberg L.S. Carlson R.W. J. Biol. Chem. 1998; 273: 2747-2757Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Separation was achieved using a gradient of 3–90% sodium acetate (1 m) in 100 mm NaOH at a flow rate of 1 ml/min over 50 min. The lipid A was obtained from the LPS by mild acid hydrolysis in 1% SDS in 20 mm sodium acetate, pH 4.5, as described by Caroff et al. (31Caroff M. Tacken A. Szab¢ L. Carbohydr. Res. 1988; 175: 273-282Crossref PubMed Scopus (200) Google Scholar). After hydrolysis, the SDS was removed by washing the dried hydrolysis product residue with a solution of 2/1 deionized H2O:acidified ethanol (100 μlof4 m HCl in 20 ml of ethanol). The residue was collected by centrifugation and washed again with 95% ethanol. The ethanol washing steps were repeated several times, and the final residue was suspended in deionized water and lyophilized to give a white, fluffy lipid A preparation. MALDI-TOF MS was performed in the negative ion reflectron mode with a 337 nm nitrogen laser, operating at a 20-kV extraction voltage, and with time-delayed extraction. Approximately 2 μl of a 1 mg/ml lipid A solution in chloroform:methanol (3/1, v/v) was mixed with 1 μl of trihydroxyacetophenone matrix solution (∼93.5 mg of trihydroxyacetophenone/1 ml of methanol) and applied to the probe for mass analysis. Spectra were calibrated externally using Escherichia coli lipid A (Sigma). Efficient Production of Relatively High Numbers of R. etli CE3-induced P. vulgaris Root Nodules—Thus far, the conventional system to cultivate P. vulgaris (common bean) plants for nodulation experiments is with Leonard jars, in which the roots are grown in pots filled with vermiculite. This system works well for the symbiotic interaction between P. vulgaris and R. etli CE3 but is rather labor-intensive if one needs to scale-up plant growth, which was necessary in our study because of the fact that sufficient amounts of pure LPS are required to perform proper structural analyses and additional biological experiments. Therefore, we engineered an AGC in which 150 plants can be grown at once under semi-sterile conditions (Fig. 2). To demonstrate that nodulation under the AGC conditions is at least as efficient as in the conventional Leonard jars, we determined the average number of
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