Chronic Intracellular Infection of Alfalfa Nodules by Sinorhizobium Meliloti Requires Correct Lipopolysaccharide Core
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Chronic intracellular infection of alfalfa nodules by Sinorhizobium meliloti requires correct lipopolysaccharide core Gordon R. O. Campbell*, Bradley L. Reuhs†, and Graham C. Walker*‡ *Department of Biology, 68-633, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139; and †Food Science Department, Purdue University, 1160 Food Science Building, West Lafayette, IN 47907 Edited by Sharon R. Long, Stanford University, Stanford, CA, and approved January 25, 2002 (received for review August 13, 2001) Our analyses of lipopolysaccharide mutants of Sinorhizobium me- infection thread release. In addition, rhizobia appear to alter liloti offer insights into how this bacterium establishes the chronic their LPS molecules during development into nitrogen-fixing intracellular infection of plant cells that is necessary for its nitro- bacteriods (5). gen-fixing symbiosis with alfalfa. Derivatives of S. meliloti strain There is evidence that the increase in hydrophobicity observed Rm1021 carrying an lpsB mutation are capable of colonizing curled for differentiated Rhizobium leguminosarum bacteroids is due to root hairs and forming infection threads in alfalfa in a manner increased amounts of long chain fatty acids attached to the lipid similar to a wild-type strain. However, developmental abnormal- A moiety and alterations in the O antigen that include a reduced ities occur in the bacterium and the plant at the stage when the proportion of charged sugar residues and an increase in acety- bacteria invade the plant nodule cells. Loss-of-function lpsB mu- lation and methylation (7). Interestingly, these changes are tations, which eliminate a protein of the glycosyltransferase I similar to those exhibited by some pathogenic bacteria on family, cause striking changes in the carbohydrate core of the infection of their hosts. For Salmonella, changes in the lipid A lipopolysaccharide, including the absence of uronic acids and a during infection, which include an increased acylation, have been 40-fold relative increase in xylose. We also found that lpsB mutants found to correlate with an increase in resistance to certain were sensitive to the cationic peptides melittin, polymyxin B, and cationic antimicrobial peptides (8). These peptides are an inte- poly-L-lysine, in a manner that paralleled that of Brucella abortus gral part of the innate immune system of many organisms, lipopolysaccharide mutants. Sensitivity to components of the including plants, and display an intrinsic affinity to the negative plant’s innate immune system may be part of the reason that this charge of bacterial outer membranes (9, 10). ϩ mutant is unable to properly sustain a chronic infection within the S. meliloti lpsB mutants have been previously described as Fix cells of its host-plant alfalfa. on alfalfa, but as affecting the timing of nodule emergence, the progress of nitrogen fixation, and the strain competitiveness for he nitrogen-fixing bacterium Sinorhizobium meliloti estab- nodulation (11). In this paper, we show that in the widely used Rm1021 strain background (12–15), the lpsB389 mutant, origi- Tlishes a symbiosis with alfalfa in which the nitrogen-fixing Ϫ bacteroid form of the bacteria lives intracellularly within organs nally isolated in a TnphoA-based screen for Fix mutants (16), called nodules that form on the root of the plant (1, 2). and other lpsB null mutants invade nodules normally but are Free-living rhizobia colonize curls at the tips of alfalfa root hairs unable to establish the normal chronic intracellular infection and invade the developing nodule through a tube of plant origin required for symbiosis; that the composition of their LPS core is called an infection thread. The infection thread grows through drastically altered; and that they are strikingly sensitive to certain the root hair and into the nodule, where it branches and extends cationic peptides. This cationic peptide sensitivity may in part into plant cells. Bacteria from the infection thread enter plant explain why the lpsB mutants are severely deficient in establish- cells through a process similar to endocytosis. This process ing a normal symbiosis. envelops the bacteria in a membrane of plant origin forming the Materials and Methods peribacteroid compartment, where the bacteria develop into nitrogen-fixing bacteroids. In the case of the S. meliloti-alfalfa Strains, Growth Conditions, and Microscopy. S. meliloti Rm1021 and symbiosis, a peribacteroid membrane fits tightly around each Escherichia coli strains were grown under standard conditions individual bacteroid (3, 4). (17). Plant assays were performed by using Medicago sativa cv. Lipopolysaccharides (LPS), which are a major constituent of GT-13R plus and M. sativa cv. Iroquois as described (18). the bacterial outer envelope, play a crucial, albeit poorly under- Microscopy was performed by using standard methods (3, 19). stood, role in nodule invasion in the symbiotic process (5). Complementing and Sequencing the fix389 Mutant. Cultured cells of Sinorhizobium spp. typically produce two forms S. meliloti col- of LPS: rough LPS (R-LPS), consisting of the lipid A membrane onies containing cosmid clones were selected on LB plates anchor and core oligosaccharide; and smooth (S-LPS), which containing streptomycin and tetracycline and replica plated onto LB plates spread with 109 plaque-forming units each of phages also includes an O antigen polysaccharide attached to the core oligosaccharide and extending into the environment. Relatively M9, M10, and M14, which will lyse the wild-type strain but lpsB389. little S-LPS is released from these rhizobia on extraction, and the not This cosmid library was a gift from Fred Ausubel O antigens show a uniform degree of polymerization and appear (Massachusetts General Hospital, Boston). Recombinant cos- mids were obtained by plating 108 bacteria onto plates prepared to lack structural variation. Previous work shows that the pri- mary O antigen of Sinorhizobium spp. consists of a simple glucan monomer repeating unit (6). This paper was submitted directly (Track II) to the PNAS office. At present, it is not known what structural attributes of LPS Abbreviations: LPS, lipopolysaccharide; R-LPS, rough LPS; S-LPS, smooth LPS; HPAEC, high- are required to initiate an effective symbiosis by S. meliloti. performance anion exchange chromatography; Kdo, 2-keto-3-deoxyoctulosonic acid. However, in the case of the plant symbiont Rhizobium etli, ‡To whom reprint requests should be addressed. E-mail: [email protected]. mutations that affect the presence, abundance, or chain length The publication costs of this article were defrayed in part by page charge payment. This of the O antigen result in microsymbionts, with defects ranging article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. from early blocks in infection thread formation to defects in §1734 solely to indicate this fact. 3938–3943 ͉ PNAS ͉ March 19, 2002 ͉ vol. 99 ͉ no. 6 www.pnas.org͞cgi͞doi͞10.1073͞pnas.062425699 Downloaded by guest on September 28, 2021 with phage as done for complementing cosmids, and colonies emergence, the progress of nitrogen fixation, and the strain that had regained the phage-resistant phenotype were picked competitiveness for nodulation (11). Because our lpsB mutant and the gene carrying the insertion was sequenced. had been generated by a TnphoA insertion, we decided to examine additional lpsB alleles to test whether the phenotypes LPS Analysis. PAGE and immunoblot analysis were performed as we observed were due to loss of lpsB function. Four additional previously described (20). Anti-S. meliloti Rm41, provided by lpsB mutations were generated via Tn5 insertional mutagenesis, Dale Noel (Marquette University, Milwaukee, WI), was used as each containing insertions at different sites along the length of S. meliloti Rm41 and S. meliloti Rm1021 share the same LPS core the lpsB gene at nucleotides positions 45, 438, 581, and 693 bp, serogroup (21). LPS extraction and initial purification protocols respectively. These were then transduced into strain Rm1021. are described in previous reports (6, 22). The extracted poly- We found these lpsB mutants to be indistinguishable both from saccharides were first fractionated by size-exclusion chromatog- each other and from the SmGC1 mutant with respect to every raphy (SEC) over Sephadex G-150 superfine (Pharmacia), elut- phenotype tested including a sensitivity to deoxycholic acid and ͞ ͞ ͞ ing with 0.2 M NaCl 1 mM EDTA 10 mM Tris base 0.25% SDS typical of LPS mutants, a rough colony morphology, and a deoxycholic acid, pH 9.25. The LPS was further purified by SEC resistance to 6 of 11 phage to which the parent strain Rm1021 by using a Superose 12 column (Pharmacia Biotech) and a was sensitive. In addition, all of these mutants displayed a Dionex metal-free BioLC. The column was eluted at 0.45 symbiotic deficiency on alfalfa indistinguishable from that of the ⅐ Ϫ1 ml min with 50 mm ammonium formate, pH 5, and the eluent SmGC1 mutant described below, including ineffective nodule was monitored with a refractive index detector (RID-10A, formation, and stunted plants on nitrogen-free media. The Shimadzu). The lack of detergent in the second SEC step allows similarity of the phenotypes of these independent insertion the LPS to aggregate and elute in the column void. mutants suggests that they all result from the loss of lpsB function The core oligosaccharides were released from the LPS by mild in S. meliloti strain Rm1021. acid hydrolysis (2% acetic acid, 100°C, 180 min), and the lipid A was removed by centrifugation. High-performance anion- lpsB Mutants Are Compromised in Inducing Proper Nodule Develop- exchange chromatography (HPAEC) of the LPS core oligosac- ment but Exhibit Normal Invasion Through Infection Threads. Plant charides used a Dionex Metal-free BioLC with a Dionex Car- ϫ assays were performed to compare symbiotic proficiency of lpsB boPac PA1 anion-exchange column (4 250 mm) and a pulsed mutants with that of the wild-type strain and of an exoY mutant amperometric detector, as described (6, 22). (18).