Chronic intracellular infection of nodules by 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, 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 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- 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 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 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). exo mutants are unable to synthesize succinoglycan or to initiate infection threads, resulting in empty nodules. Alfalfa Glycosyl Composition and Linkage Analyses. Glycosyl compositions plants inoculated with wild-type S. meliloti were dark green, were determined by gas chromatography-mass spectrometry averaged 16.4 cm in height, and had elongated dark pink nodules (GC-MS) analysis of the trimethylsilyl methyl glycoside deriva- after 4.5 wk of growth. All of the nodules on these plants were tives (23), by using a 30-m DB-1 fused silica column (J&W elongated and bright pink in color because of the presence of Scientific, Folsum, CA) on a 5890A GC-mass selective detector , which maintains the microaerobic conditions (Hewlett–Packard). Inositol was used as an internal standard, necessary for nitrogen fixation. In contrast, the plants inoculated and retention times were compared with authentic monosaccha- with the exoY mutant were 3.3 cm in height and had small round ride standards. Compositions are expressed as the percentage of white nodules. The plants inoculated with the SmGC1 mutant the total detected carbohydrate represented by each of the sugar moieties. had an intermediate phenotype, averaging 7.3 cm in height, having a pale green to yellow color indicating that nitrogen Cationic Peptide Assays. For all cationic peptide assays, S. meliloti fixation was much less efficient than with wild-type S. meliloti. strains were grown to an OD of Ϸ1 in LB medium. Next, 100 ␮l We observed a range of nodule phenotypes, from small, round, of cells were aliquoted to a microfuge tube, 1 ␮l of cationic and white, to elongated nodules much paler pink than wild type. peptide was added, and the tubes were incubated at room To determine the nature of the symbiotic defect of Rm1021 temperature for 1 h. The cultures were then titered by spotting lpsB mutants, we first examined infection thread formation and a series of dilutions on LB plates. The cationic peptides were development by introducing a stable encoding the green used at the following final concentrations: melittin, 20 ␮g/ml; fluorescent protein (GFP), as we have described (19). Rm1021 polymyxin B, 20 ␮g͞ml; poly(L-lysine), 50 ␮g͞ml. lpsB mutants are only slightly compromised for infection thread formation. They colonize curled root hairs as effectively as wild Results type and invade through infection threads that look normal when The fix389 Mutation Is in the lpsB Gene. We originally identified the viewed by both light and electron microscopy. Although the fix389::TnphoA mutation in a screen for symbiotically defective number of extended infection threads was approximately half MICROBIOLOGY mutants (16). Transduction of this insertion into the widely used that seen with wild-type controls, this is probably not the major Rm1021 background (12–15) resulted in the strain SmGC1, cause of their symbiotic defect because we have found that an exoZ mutant, which elongates less than 10% as many infection which exhibited a severe symbiotic deficiency similar to the ϩ original isolate (16). Through sequence analysis, we determined threads as wild type, has a Fix phenotype (19). the TnphoA mutation in the SmGC1 mutant interrupted the 1,056-bp lpsB gene at nucleotide position 716. The lpsB gene Both lpsB Rhizobia and the Host Plant Show Symbiotic Defects in encodes a protein of the glycosyl transferase 1 family that is 58% Developing Nodules at the Cellular Level. Defects in nodules elicited identical to a mannosyl transferase involved in core synthesis in by Rm1021 lpsB mutants were readily apparent on examination by Rhizobium leguminosarum, LpcC (24, 25). It has recently been light microscopy. Fig. 1A shows a cross section of the nitrogen-fixing reported that the lpsB gene product will complement the LPS zone of a nodule densely packed with wild-type bacteroids. Infected defect of an lpcC mutant (24). nodule cells are readily distinguished by their dark staining. In contrast, sections of nodules induced by an lpsB mutant had severe The SmGC1 Mutant Is Indistinguishable from Other Rm1021 lpsB defects (Fig. 1B). The plant cells from the region that should be the Mutants. Our observation that the Rm1021 mutant SmGC1 was nitrogen-fixing zone of nodules appear to be infected with fewer severely symbiotically deficient contrasted with a previous report bacteroids and to contain large vacuoles. Vacuoles are fairly that Rm6963, an lpsB derivative of strain Rm2011, is Fixϩ on common in the nitrogen-fixing zone of nodules induced by the alfalfa (26, 27), although it is affected in the timing of nodule wild-type S. meliloti; however, those present in nodules induced by

Campbell et al. PNAS ͉ March 19, 2002 ͉ vol. 99 ͉ no. 6 ͉ 3939 Downloaded by guest on September 28, 2021 Fig. 1. Light micrographs (A and B) and electron micrographs (C–E) of region corresponding to nitrogen-fixing zone in nodules induced by the Rm1021 wild-type bacteria and by an lpsB mutant. (A) Cross section through the nitrogen-fixing region [Zone III as described by Vasse et al. (4)] of an alfalfa nodule infected with the wild-type S. meliloti strain Rm1021. (Bar ϭ 18 ␮m.) (B) Cross section through region that would correspond to Zone III of an alfalfa nodule infected with an lpsB mutant. Cells contain many abnormally large vacuoles. (Bar ϭ 18 ␮m.) (C) Plant cells packed with wild-type bacteroids (for example, left arrow) from a nodule infected by the wild-type S. meliloti strain Rm1021. An infection thread (IT) and an amyloplast (right arrow) are also shown. (Bar ϭ 1.25 ␮m.) (D) Plant cells infected with an lpsB mutant. Bacteria are in various stages of degradation (arrows). (Bar ϭ 1.25 ␮m.) (E) Plant cells from a nodule containing lpsB mutant bacteria. The bacteria are in a more degraded state, frequently being within enlarged membrane compartments (arrow). Infection threads are also present (IT). A large vacuole fills most of the cytoplasm of lower plant cell (V). (Bar ϭ 1.25 ␮m.)

lpsB mutants were much larger and more numerous than in nodules in nodule development as well. The amyloplasts remain large, induced by the wild-type Rm1021 strain. even in cells that should have developed into the nitrogen-fixing Even more striking phenotypes were observed by electron zone, and many contain large vacuoles similar to ones present in microscopy. Fig. 1C shows a plant cell from the nitrogen-fixing plant cells in the senescent zone. In contrast to the striking zone of a nodule containing wild-type Rm1021 bacteria. These phenotypes of nodules induced by the Rm1021 mutants, bacteria bacteroids have heterogeneously staining cytoplasm and very in infection threads, and the infection threads themselves, ap- little extra space within the peribacteroid membrane compart- pear normal by electron microscopy (Fig. 1 C and E). ment. Fig. 1D shows the corresponding region of a nodule containing an lpsB mutant. The upper cell contains numerous Rm1021 lpsB Mutants Still Synthesize S-LPS but Are Deficient in LPS intracellular bacteria, indicating that lpsB mutants are not es- Core Biosynthesis. The cell-associated polysaccharides were ex- pecially impaired at being taken into the plant cell. However, the tracted with hot phenol-water and analyzed by deoxycholate- morphologies of these intracellular bacteria are diverse, and PAGE (Fig. 3A). The LPS from the wild-type Rm1021 strain many have refractory cytoplasm indicative of aborting bacteroids migrated as a high-mobility series of disperse bands that repre- (arrows). The defects are more extreme in some cells. Fig. 1E sent R-LPS, as well as several forms of S-LPS. This pattern is shows the mutant bacteria within dramatically enlarged mem- common for Sinorhizobium spp (20), in which R-LPS predom- brane compartments, and many of the bacteria look abnormal or inates. The LPS from the SmGC1 mutant showed a similar atrophied. Furthermore, large amyloplasts are still present. migration pattern, except that each band migrated further in the Amyloplasts are starch deposits normally present in nodule cells gel and was less reactive to LPS-specific antibodies, as has been early in development and resorbed on infection with alfalfa. Fig. observed for a different Rm2011 lpsB mutant (11, 27). These 2A shows a more enlarged view of mutant bacteria. The arrows electrophoretic patterns, along with an increase in the propor- on the right point to bacteria that have severely degraded inside tion of 2-keto-3-deoxyoctulosonic acid (Kdo), have previously bloated membrane compartments. The arrow on the left points been interpreted as being due to a truncation of the LPS core to a bacteria that has a uniformly staining cytoplasm similar to (11, 27). Furthermore, S-LPS appears to be present, indicating bacteroids seen in the senescent zone of nodules. that despite the changes to the LPS, lpsB mutants are still able Intracellular Rm1021 lpsB mutant bacteria clearly have diffi- to add O antigen. culty making the transition into bacteroids, so it is not clear what To gain further insight into the nature of these changes, we then term to use for the bacteria at this intermediate developmental applied a two-step separation method to effectively purify the LPS. stage. Occasionally, membrane compartments contain more The polysaccharides were first separated by size-exclusion chroma- than one bacterium, as in Fig. 2B, and some bacteria, such as the tography on a Sephadex G-150 column in the presence of deoxy- long one in this figure, continue to divide. These phenomena do cholate, which disaggregates the LPS. This procedure partially not normally happen in S. meliloti-alfalfa symbiosis (3, 4). separates the LPS, K antigens, cyclic ␤(1,2)-glucans, and high Furthermore, some elongated bacteroid-like bacteria appear to molecular-weight neutral polysaccharides. Interestingly, we found still contain polyhydroxybutyrate granules, which are normally that the SmGC1 lpsB mutant strain overproduced cyclic ␤(1,2)- degraded as soon as S. meliloti is released into the plant glucans, which have been implicated in osmoregulation, so their cytoplasm (Fig. 2B). The developmental defects may be in part overproduction may indicate that lpsB mutants experience some because of a fragile bacterial outer membrane as a consequence degree of osmotic stress (28). The LPS-fractions from the Sephadex of defective LPS. Fig. 2C shows an elongated bacteroid whose G-150 column were pooled and passed through a Superose 12 outer membrane is beginning to degenerate. column in the absence of detergent. This resulted in the aggregation The plant seems to have difficulty performing its normal role of the LPS, which eluted in the column void, and effectively

3940 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.062425699 Campbell et al. Downloaded by guest on September 28, 2021 Fig. 3. (A) Schematic of the LPS molecule showing the lipid A, the LPS core, and the O antigen. (B) Deoxycholic acid PAGE gel and Western blot of hot phenol-water purified LPS from wild-type and mutant LPS. R- and S-LPS are labeled accordingly. Both the smooth and rough LPS have run further in the mutant. LPS was blotted with polyclonal antibodies recognizing mostly the core region of LPS. The low intensity of the bands in the SmGC1 blot suggests that the core has been altered. (C) HPAEC-pulsed amperometric detection analysis of core oligosaccharides from R-LPS of wild-type S. meliloti and (D) the SmGC1 mutant. The mutant lacked all but one of the oligosaccharide peaks present in R-LPS core from the wild-type strain.

HPAEC-PAD pattern (Fig. 3C), which is common to the Sinorhi- zobium serogroup A (refs. 6 and 20; B.L.R., unpublished work). This complex pattern is due to heterogeneity in the LPS core oligosaccharides and the acid catalyzed conversion of some reduc- ing end Kdo residues to an anhydrous form. The LPS from the SmGC1 lpsB mutant lacked all but one of the core oligosaccharides present in the wild-type LPS. Instead, a series of oligosaccharide peaks eluted between 15 and 20 min (Fig. 3D). HPAEC separates oligosaccharides both on charge content and size, so the elution pattern suggests that the oligosaccharides released from the LPS core of the SmGC1 mutant were either smaller or lacked some acidic sugars or both. Composition analysis showed that the LPS from the SmGC1 lpsB mutant is dramatically altered from that of the wild-type strain. Glucose, glucuronic acid, galacturonic acid, and Kdo accounted for 78% of the detected carbohydrate from the wild-type R-LPS core (Table 1). In contrast, the R-LPS core Fig. 2. Intracellular lpsB mutant bacteria at a higher magnification in nodule from the lpsB mutant completely lacked uronic acid components cells from the region that would correspond to the nitrogen-fixing zone of a

but contained a larger proportion of neutral sugars and Kdo. MICROBIOLOGY wild-type nodule. (A) Degrading bacteria within enlarged membrane com- partments (right arrows). One bacterium has homogeneous staining in the ϭ cytoplasm similar to a bacteroid from the senescent zone (left arrow). (Bar Table 1. LPS core composition analysis 0.28 ␮m.) (B) An enlarged membrane compartment (right arrow) containing several partially developed bacteroids. The elongated bacterium (bottom Sugar Rm1021 RLPS SmGC1 R-LPS two arrows) appears to contain polyhydroxybutyrate granules (white circles). (Bar ϭ 0.21 ␮m.) (C) An elongated bacterium, inside an enlarged membrane Xyl 0.5 21.6 compartment, with a blebbing of bacterial outer membrane (arrow). (Bar ϭ Man 5.3 21.4 0.17 ␮m.) Gal 3.8 1.6 Glc 33.5 21 GlcA 10.8 0 separated it from the nonacylated contaminants of similar size GalA 21.2 0 (data not shown). DHA 3.6 0 The purified R-LPS was treated with mild acid hydrolysis, which Kdo 21.2 32.6 cleaves the ketosidic link of the Kdo, and the insoluble lipid A was Carbohydrate composition analysis of the R-LPS from the lpsB389 mutant removed by centrifugation. The core oligosaccharides were ana- and from the wild-type strain. Xyl, xylose; Man, mannose; Gal, galactose; Glc, lyzed by HPAEC–pulsed amperometric detection (HPAEC-PAD) glucose; GalA, galacturonic acid; GlcA, glucuronic acid; DHA, deoxyheptulo- (Fig. 3 C and D). The wild-type Rm1021 LPS yielded the expected saric acid.

Campbell et al. PNAS ͉ March 19, 2002 ͉ vol. 99 ͉ no. 6 ͉ 3941 Downloaded by guest on September 28, 2021 Table 2. Cationic peptide killing assay core with apparently the same efficiency that it adds O antigen Rm1021 % SmGC1 % to the normal LPS core. survival survival Survival ratio We have found that the LPS alteration caused by lpsB Cationic peptide after 1 h after 1 h Rm1021͞SmGC1 mutations does not impair the earlier steps of nodule formation and nodule invasion, because plants inoculated with an lpsB Melittin, 20 ␮g͞ml 2.5 1.7 ϫ 10Ϫ3 1,470 mutant have normal-looking infection threads with normal- Polymyxin B, 20 ␮g͞ml 6.1 0.043 140 looking bacteria inside them. However, the nodules themselves Poly(L-lysine), 50 ␮g͞ml 3.1 1.3 2.4 are poorly developed, and mutant bacteria within these nodules showed a wide range of phenotypes. There are a number of Cationic peptide killing assays using melittin, polymyxin B, and poly(L- lysine). Values are recorded as percent survival after a 1-h exposure to the possible explanations for this killing. The simplest is that the cationic peptide at the concentration indicated. Results shown are the aver- peribacteroid membrane compartment is potentially toxic for age of three experiments. the bacteria, and wild-type S. meliloti has mechanisms that enable it to survive in this environment. Perhaps lpsB mutations compromise the integrity of the bacterial outer envelope, how- Mannose was notably elevated in the LPS core from the mutant, ever to an extent that renders the bacterium a ‘‘conditional which may indicate that the mannose is directly linked to the lethal’’ inside the membrane compartment. This model allows a Kdo, near the reducing end of the core. Furthermore, the relatively passive contribution from both symbiotic partners to relative amount of xylose was strikingly increased. We also found the phenotypes we have seen in the nodule. that the S-LPS contains a larger percentage of glucose than An extension of this explanation would be that the alfalfa the R-LPS, which is consistent with a glucose-containing O plants are mounting an active full-scale plant innate immune antigen (6). response against the Rm1021 lpsB mutants, one that wild-type rhizobia avoid. In this view, bacteria with the wild-type LPS lpsB Mutants Show an Increased Sensitivity to Several Cationic would not trigger a plant defense response and are therefore able Peptides. LPS mutants from many pathogenic strains have been to reside within the plant unharmed. However, the plant recog- found to have altered sensitivities to cationic antimicrobial nizes the lpsB mutant as an invading pathogen and turns on its peptides. These molecules have been found to comprise an defense response to eliminate it. The altered LPS of lpsB mutants important part of the immune system of all kingdoms of life, either could be the feature that triggers the defense response, or including plants (9, 10). To explore the possibility that an it could fail to adequately mask some other feature that causes inability to properly cope with the plant’s innate immune system the plant to perceive the bacteria as a pathogen. was responsible for the symbiotic deficiencies we observed, we The inability of lpsB mutants to establish a chronic intracel- tested the sensitivity of the SmGC1 mutant to three cationic lular infection would seem to be the underlying cause of the three peptides: melittin, polymyxin B, and poly(L-lysine). These results symbiotic defects described by Lagares et al. for their Rm2011 are summarized in Table 2. Melittin proved to be the most toxic lpsB strain Rm6963, including the timing of nodule emergence, to the lpsB mutant. After a 1-h incubation, 20 ␮g͞ml of melittin the progress of nitrogen fixation, and the strain competitiveness decreased the viable count of the lpsB mutant by a factor of 5 ϫ for nodulation (11). The specific deficiencies that have been 104 versus a 40-fold reduction for the wild type. This represents reported for an S. meliloti Rm2011 lpsB mutant interacting with a 1.5 ϫ 103-fold increase in sensitivity in the mutant. Polymyxin were different in several respects from those B also showed a significant, although somewhat less substantial, we observed with alfalfa. In particular, the obvious killing of increase in toxicity for the lpsB mutant. Polymyxin B (20 ␮g͞ml) intracellular bacteria that we observed was not seen in the case killed mutant cells 144-fold more effectively than the wild-type of M. truncatula, although there were indications that plant strain. Poly(L-lysine), in contrast, only showed a 2.4-fold differ- defense responses had been induced by the lpsB mutant (27). ence in killing between the lpsB mutant and the wild-type strain. This suggests that the particular challenges that a bacterium must withstand from its host plant at various stages of the infection Discussion process may differ between particular rhizobia-plant symbioses. We have shown that in the widely used Rm1021 background To explore the possibility that an inability to properly cope with (12–15), loss of lpsB function causes a drastic alteration of the the alfalfa plant’s innate immune system was responsible for the LPS core of S. meliloti, and that this makes lpsB mutants symbiotic deficiencies we observed for Rm1021 lpsB mutants, we extremely defective in establishing the chronic intracellular examined their sensitivity to cationic peptides. Cationic antimicro- infection of plant cells that is necessary for an effective nitrogen- bial peptides are very ancient components of innate immunity, and fixing symbiosis. The altered electrophoretic migration pattern their induction pathways are highly conserved in vertebrates, in- of LPS from lpsB mutants has led to the previous suggestion that sects, and plants (9, 10). Cationic peptides derive their specificity for lpsB mutations cause a truncation of the LPS core, but that O bacteria from their affinity for the negative charge of the bacterial antigen can still be added to the truncated core (11, 27). outer envelope, and it has been found for many bacterial species However, our analysis of the core composition suggests more that sensitivity to cationic peptides is altered between smooth and complicated explanations. Although the absence of numerous rough strains (10, 29–31). Because the SmGC1 mutant is more core oligosaccharides and of uronic sugars is consistent with a sensitive to these cationic peptides, it is possible that it is also more truncation of the core, the greater than 40-fold increase in the sensitive to the cationic peptides native to alfalfa, which may be percentage of xylose cannot easily be explained by a simple present in the nodule. Interestingly, the relative sensitivity of an truncation. One possibility is that, in the absence of LpsB, other Rm1021 lpsB mutant to the three cationic peptides we tested was glycosyl transferases are able to gain access to its normal similar to the sensitivities exhibited by B. abortus LPS mutants to substrate so that other sugar moieties, in particular xylose, are these same three cationic peptides (31–33). added resulting in an altered core to which O antigen can be An alternative, although not mutually exclusive, explanation attached. Alternatively, because the lpsB mutations prevent the for the phenotypes of Rm1021 lpsB mutants is that they are synthesis of the normal LPS core, the bacteria might compensate unable to engage successfully in the cascade of signaling that by elevating the synthesis of some other minor core species queues the intracellular rhizobia to develop into nitrogen-fixing containing xylose that is not normally detected in the wild-type bacteroids. An especially interesting possibility is that the LPS strain. This second possibility is appealing because it explains serves as an active signal to alter the plant membrane compart- how lpsB mutants are still able to add O antigen to the altered ments from degradative vacuoles to symbiotic peribacteroid

3942 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.062425699 Campbell et al. Downloaded by guest on September 28, 2021 compartments that, in turn, induce the rhizobia to develop into from plant cells before undergoing any differentiation. Similarly nitrogen-fixing bacteroids. Perturbing such a signal exchange to the S. meliloti bacA mutant, a B. abortus bacA mutant was process might explain the remarkable range of developmental unable to establish a successful chronic intracellular infection defects of both an intracellular lpsB mutant and the plant vesicles (40). Our results raise the possibility that the underlying reason housing the bacteria. that the S. meliloti lpsB mutant is unable to establish a chronic This model is especially intriguing if one compares the sym- intracellular infection is related to the reason that B. abortus LPS biosis of S. meliloti with alfalfa, to the chronic bovine infection mutants cannot establish a chronic intracellular infection. caused by B. abortus. Rhizobia establish a chronic infection of the plant host in which they reside in acidic intracellular mem- We thank Ann Hirsch for useful criticism and help with the interpre- brane compartments, and brucellae establish a chronic infection tation of electron micrographs; Nicki Watson for help with the electron of their animal hosts in which they reside in acidic intracellular microscopy; and Sam Stephens for help with sample preparation and membrane compartments (34–37). We already know of one gene analysis. We also thank John Campbell, Brett Pellock, Gail Ferguson, product, BacA, required for chronic infection by both B. abortus and Kristin Levier for helpful comments and critical reading of the manuscript. This work was supported by Public Health Service Grant and S. meliloti (38–40). Similar to S. meliloti Rm1021 lpsB GM31030 from the National Institutes of Health (NIH) to G.C.W. and mutants, Rm1021 bacA mutants can invade alfalfa up to the NIH Predoctoral Training Grant T32GM07287 (to G.R.O.C.). B.L.R point of infection thread release. However, whereas lpsB mu- was supported by Grant MCB-9728564 from the National Science tants persist for a period within the membrane compartments Foundation. The electron microscopy was conducted by using the W. M. before dying, bacA mutants lyse relatively rapidly on release Keck Foundation Biological Imaging facility at the Whitehead Institute.

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