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Specific complex interactions between soil bacteria belonging to Rhizobium, Sinorhizobium, Mesorhizobium, Phylorhizobium, Bradyrhizobium and Azorhizobium commonly known as rhizobia, and their host leguminous plants result in development of root nodules. Nodules are new organs that consist mainly of plant cells infected with bacteroids that provide the host plant with fixed nitrogen. Proper nodule development requires the synthesis and perception of signal molecules such as lipochitooligosaccharides, called Nod factors that are important for induction of nodule development. Bacterial surface polysaccharides are also crucial for establishment of successful symbiosis with legumes. Sugar polymers of rhizobia are composed of a number of different polysaccharides, such as lipopolysaccharides (LPS), capsular polysaccharides (CPS or K-antigens), neutral β-1, 2-glucans and acidic extracellular polysaccharides (EPS). Despite extensive research, the molecular function of the surface polysaccharides in symbiosis remains unclear.This review focuses on exopolysaccharides that are especially important for the invasion that leads to formation of indetermined (with persistent meristem) type of nodules on legumes such as clover, vetch, peas or alfalfa. The significance of EPS synthesis in symbiotic interactions of Rhizobium leguminosarum with clover is especially noticed. Accumulating data suggest that exopolysaccharides may be involved in invasion and nodule development, bacterial release from infection threads, bacteroid development, suppression of plant defense response and protection against plant antimicrobial compounds. Rhizobial exopolysaccharides are species-specific heteropolysaccharide polymers composed of common sugars that are substituted with non-carbohydrate residues. Synthesis of repeating units of exopolysaccharide, their modification, polymerization and export to the cell surface is controlled by clusters of genes, named exo/ exs, exp or pss that are localized on rhizobial megaplasmids or chromosome. The function of these genes was identified by isolation and characterization of several mutants disabled in exopolysaccharide synthesis. The effect of exopolysaccharide deficiency on nodule development has been extensively studied. Production of exopolysaccharides is influenced by a complex network of environmental factors such as phosphate, nitrogen or sulphur. There is a strong suggestion that production of a variety of symbiotically active polysaccharides may allow rhizobial strains to adapt to changing environmental conditions and interact efficiently with legumes.

We applied a genomic approach in the identification of genes required for the biosynthesis of different polysaccharides in Rhizobium leguminosarum bv. trifolii TA1 (RtTA1). Pulsed-field gel electrophoresis analyses of undigested genomic DNA revealed that the RtTA1 genome is partitioned into a chromosome and four large plasmids. The combination of sequencing of RtTA1 library BAC clones and PCR amplification of polysaccharide genes from the RtTA1 genome led to the identification of five large regions and clusters, as well as many separate potential polysaccharide biosynthesis genes dispersed in the genome. We observed an apparent abundance of genes possibly linked to lipopolysaccharide biosynthesis. All RtTA1 polysaccharide biosynthesis regions showed a high degree of conserved synteny between R. leguminosarum bv. viciae and/or Rhizobium etli. A majority of the genes displaying a conserved order also showed high sequence identity levels.

The pssT gene was identified as the fourth gene located upstream of the pssNOP gene cluster possibly involved in the biosynthesis, polymerization, and transport of exopolysaccharide (EPS) in Rhizobium leguminosarum bv. trifolii strain TA1. The hydropathy profile and homology searches indicated that PssT belongs to the polysaccharide-specific transport family of proteins, a component of the type I system of the polysaccharide transport. The predicted membrane topology of the PssT protein was examined with a series of PssT-PhoA fusion proteins and a complementary set of PssT-LacZ fusions. The results generally support a predicted topological model for PssT consisting of 12 transmembrane segments, with amino and carboxyl termini located in the cytoplasm. A mutant lacking the C-terminal part of PssT produced increased amounts of total EPS with an altered distribution of high-and low-molecular-weight forms in comparison to the wild-type RtTA1 strain. The PssT mutant produced an increased number of nitrogen fixing nodules on clover.The soil bacterium Rhizobium leguminosarum bv. trifolii has the ability to produce an acidic exopolysaccharide (EPS) that plays an important role in symbiotic interaction with clover plants. The EPS of R. leguminosarum is composed of octasaccharide repeating units containing one galactose, five glucoses, and two glucuronic acid residues with acetyl, pyruvyl, and hydroxybutanoyl modifications (20). Similarly to Sinorhizobium meliloti succinoglycan (EPS I), high-molecular-weight (HMW) and low-molecular-weight (LWM) fractions of EPS were identified in culture supernatants of R. leguminosarum (11,34).Polysaccharides consisting of repeating units are assembled on a polyisoprenyl-pyrophosphate lipid carrier at the cytoplasmic face of the inner membrane by a sequential transfer of monosaccharides from their nucleotide sugars by the action of specific glycosyltransferases. The oligosaccharides are subsequently translocated, polymerized to an HMW EPS, and transported to the cell surface (57). In R. leguminosarum, assembly of the repeating units is under the control of pssA, pssDE, pssC, pssGHI, and other, as-yet-unidentified, genes encoding glycosyltransferases (5,22,26,46,53).Recently, we identified the pssN, pssO, and pssP genes that might be constituents of a type I system involved in the polymerization and export of EPS in R. leguminosarum bv. trifolii TA1 (33, 34). On the basis of computational analysis and sequence similarity to the known proteins, PssP was identified as a member of the membrane-periplasmic auxiliary (MPA1) (42) or polysaccharide copolymerase (PCP2) (39) protein family that are involved in the synthesis of HMW EPS. PssP of R. leguminosarum bv. trifolii TA1 resembles ExoP from S. meliloti, which functions in the synthesis and polymerization of succinoglycan (EPS I) (2). Mutants of R. leguminosarum bv. trifolii and S. meliloti deleted of the pssP or exoP gene, respectively, did not produce the EPS (3, 34). Recently, similarly to other members of the PCP2 family, the autophosphor...

No abstract

Rhizobium leguminosarum bv. trifolii produces an acidic exopolysaccharide (EPS) that is important for the induction of nitrogen-fixing nodules on clover. Recently, three genes, pssN, pssO, and pssP, possibly involved in EPS biosynthesis and polymerization were identified. The predicted protein product of the pssP gene shows a significant sequence similarity to other proteins belonging to the PCP2a family that are involved in the synthesis of high-molecular-weight EPS. An R. leguminosarum bv. trifolii TA1 mutant with the entire coding region of pssP deleted did not produce the EPS. A pssP mutant with the 5' end of the gene disrupted produced exclusively low-molecular-weight EPS. A mutant that synthesized a functional N-terminal periplasmic domain but lacked the C-terminal part of PssP produced significantly reduced amounts of EPS with a slightly changed low to high molecular form ratio. Mutants affected in the PssP protein carrying a stable plasmid with a constitutively expressed gusA gene induced nodules on red clover that were not fully occupied by bacteria. A mutant with the entire pssP gene deleted infected only a few plant cells in the nodule. The pssP promoter-gusA reporter fusion was active in bacteroids during nodule development.

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Rhizobia dwell and multiply in the soil and represent a unique group of bacteria able to enter into a symbiotic interaction with plants from the Fabaceae family and fix atmospheric nitrogen inside de novo created plant organs, called nodules. One of the key determinants of the successful interaction between these bacteria and plants are exopolysaccharides, which represent species-specific homo- and heteropolymers of different carbohydrate units frequently decorated by non-carbohydrate substituents. Exopolysaccharides are typically built from repeat units assembled by the Wzx/Wzy-dependent pathway, where individual subunits are synthesized in conjunction with the lipid anchor undecaprenylphosphate (und-PP), due to the activity of glycosyltransferases. Complete oligosaccharide repeat units are transferred to the periplasmic space by the activity of the Wzx flippase, and, while still being anchored in the membrane, they are joined by the polymerase Wzy. Here we have focused on the genetic control over the process of exopolysaccharides (EPS) biosynthesis in rhizobia, with emphasis put on the recent advancements in understanding the mode of action of the key proteins operating in the pathway. A role played by exopolysaccharide in Rhizobium–legume symbiosis, including recent data confirming the signaling function of EPS, is also discussed.