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Legumes establish mutualistic associations with nitrogen-fixing bacteria, commonly referred to as rhizobia. Most rhizobia enter roots of host plants via root hairs, where they induce the formation of infection threads. Bacterial invasion and formation of nodules containing nitrogen-fixing bacteroids (the Fix+ phenotype) depend on a molecular dialogue between the symbiotic partners. Host plants secrete flavonoids that induce the synthesis of rhizobial lipo-chito-oligosaccharides called Nod factors (24, 26) and perceive them via a specific signal transduction pathway that culminates in expression of symbiosis-specific plant genes required for nodule development (34). In many symbiotic associations, nodule formation also depends on rhizobial macromolecules that include exopolysaccharides (EPS), lipopolysaccharides, K-antigens, and cyclic β-glucans. These extracellular polysaccharides (or the oligosaccharides derived from them) are crucial in the early stages of nodule formation that begins after rhizobia have entered the host plant (3, 8, 17, 19, 25, 33, 36). In addition to a protective role, rhizobial EPS have vital but poorly understood roles in infection of legume roots (2). The EPS structures of various rhizobia, including Rhizobium sp. strain NGR234 (also called Sinorhizobium fredii NGR234), which nodulates more than 112 genera of legumes (30), have been described. The repeating subunit of the acidic EPS of NGR234 is a nonasaccharide consisting of glucosyl (Glc), galactosyl (Gal), glucuronosyl (GlcA), and 4,6-pyruvylated galactosyl (PvGal) residues (9) (Fig. ​(Fig.1A).1A). Mutants of NGR234 deficient in EPS synthesis formed nonmucoid (“dry”) colonies on agar plates and lost the capacity to induce the formation of nitrogen-fixing nodules on Leucaena leucocephala (Fix− phenotype). Instead, small empty nodules (pseudonodules) were formed (5). A number of genes of NGR234 involved in synthesis of EPS have been identified in an exo cluster (6, 15, 45), which has been sequenced (38) (Fig. ​(Fig.1B).1B). This cluster is located on the pNGR234b megaplasmid and is similar to the exo cluster of Sinorhizobium meliloti strain 1021 that is required for synthesis of succinoglycan (also called EPS I) (13). Based on known or assumed functions of exo genes in S. meliloti (2, 32), we predict that EPS synthesis in NGR234 depends on the glycosyl transferases (ExoY, ExoA, ExoL, ExoM, ExoO, and ExoU) that form an undecaprenol diphosphate-linked repeating subunit at the cytoplasmic face of the inner membrane. Probably, the lipid-linked subunits are then flipped across the inner membrane and finally assembled by an ExoQ/ExoP/ExoF-dependent polymerization system that is similar to the Wzy polymerization pathway of group 1 capsular polysaccharides in Escherichia coli (41). In this model, ExoQ is a putative polymerase and ExoP is a copolymerase belonging to the MPA1 (cytoplasmic membrane-periplasmic auxiliary protein 1) family. ExoF is probably an outer membrane auxiliary protein required for EPS export. Related genes required for secretion and export of EPS (pssL, pssT, pssP, and pssN) have been identified in Rhizobium leguminosarum bv. trifolii (reference 22 and references therein). FIG. 1. (A) Repeating subunit of the acidic EPS from NGR234 (9). Additional acetyl groups are present at unidentified positions. (B) Genetic map of the exo cluster of pNGR234b (38). The exs genes and neighboring exoB gene are not shown. BamHI fragments were cloned ... Low-molecular-weight (LMW) forms of EPS (called exo-oligosaccharides [EOSs]) have been identified in supernatants of rhizobial cultures. Nodulation experiments with purified LMW EPS applied to roots indicated that EOSs could complement the symbiotic defects of exo mutants under certain growth conditions (1, 10, 14, 40). Similar complementation effects were observed when exo mutants were coinoculated with noninvasive strains that synthesize EPS (17, 18). Although not always conclusive, the results of such complementation experiments suggest that EOSs are symbiotically active molecules in certain plants. Synthesis of EOSs seems to be controlled either by enzymes involved in export and polymerization of the lipid-linked repeating EPS subunit or by extracellular glycanases that release EOSs from high-molecular-weight (HMW) forms (14). Glycanases that cleave EPS have been identified in S. meliloti and R. leguminosarum bv. viciae. Corresponding mutants produced lower levels of EOSs without altering the symbiotic phenotype (12, 43). The previously characterized glycanase ExoK secreted by S. meliloti (43, 44) exhibits a high level of sequence similarity [e−129] to the putative ExoK protein of NGR234. In this study, we mutated exo genes of NGR234 involved in synthesis of the repeating subunit (exoL, exoY, and exoZ), polymerization (exoF, exoP, and exoQ), and production of EOSs (exoK). We provide evidence that EOSs of NGR234 are required for symbiosis with certain plants.

Analysis of two exopolysaccharide-deficient mutants of Rhizobium leguminosarum , RBL5808 and RBL5812, revealed independent Tn 5 transposon integrations in a single gene, designated exo5 . As judged from structural and functional homology, this gene encodes a UDP-glucose dehydrogenase responsible for the oxidation of UDP-glucose to UDP-glucuronic acid. A mutation in exo5 affects all glucuronic acid-containing polysaccharides and, consequently, all galacturonic acid-containing polysaccharides. Exo5-deficient rhizobia do not produce extracellular polysaccharide (EPS) or capsular polysaccharide (CPS), both of which contain glucuronic acid. Carbohydrate composition analysis and nuclear magnetic resonance studies demonstrated that EPS and CPS from the parent strain have very similar structures. Lipopolysaccharide (LPS) molecules produced by the mutant strains are deficient in galacturonic acid, which is normally present in the core and lipid A portions of the LPS. The sensitivity of exo5 mutant rhizobia to hydrophobic compounds shows the involvement of the galacturonic acid residues in the outer membrane structure. Nodulation studies with Vicia sativa subsp. nigra showed that exo5 mutant rhizobia are impaired in successful infection thread colonization. This is caused by strong agglutination of EPS-deficient bacteria in the root hair curl. Root infection could be restored by simultaneous inoculation with a Nod factor-defective strain which retained the ability to produce EPS and CPS. However, in this case colonization of the nodule tissue was impaired.