**2. Structural characteristics of exopolysaccharides**

Structures of known *R. leguminosarum* and *R. etli* (*Re*) EPSs are presented in Table 1. It should be noted that EPSs secreted by the most of the *Rlv* and *Rlt* strains were shown to have repeating units of identical structure [25-35]. These EPSs are branched heteropolysaccharides of the octasaccharide repeating unit consisting of a backbone of two glucose and two glucuronic acid residues, a side chain of three glucose and one galactose residues. The backbone contains 1-4 and 1-4 linkages only while the branch point is linked 1-6. The side chain contains 1-4 and 1-3 linkages. In general, the octasaccharide is modified by two pyruvyl, one or two non-stoichiometric O-acetyl, and one nonstoichiometric hydroxybutanoyl groups. The distribution pattern of O-acetyl and 3 hydroxybutanoyl groups may vary for some *R*. *leguminosarum* strains and was shown to be dependent on the growth phase of bacteria and culture medium [27,32].

100 The Complex World of Polysaccharides

association [9-11].

(reviewed by [9]). EPS refers to extracellular polysaccharides with slight or no cell

Among the above mentioned polysaccharides, acidic EPSs attract attention due to their diverse functions both in free-living rhizobia and during the establishment of nitrogenfixing symbiosis with host legume plants. EPS forms a biofilm layer on the cell surface which is thought to contribute to the following processes: cellular protection against environmental stresses, attachment to surfaces, nutrient gathering, and the preferential absorption of flavonoids secreted by plants along the membrane surface [9]. In addition, EPS biosynthesis is required for the effective nodulation of legumes such as *Medicago*, *Pisum*, *Trifolium*, *Leucaena*, and *Vicia* spp., which form indeterminate-type nodules (otherwise called meristematic or cylindrical) [12]. EPS-deficient mutants of *Rhizobium leguminosarum* bv. *viciae*  (hereafter *Rlv*), *R. leguminosarum* bv. *trifolii* (*Rlt*), and *Sinorhizobium meliloti* induce symbiotically defective phenotypes which include delayed root hair curling, nodules devoid of bacteria due to infection threads that abort within peripheral cells of the developing nodule, and small, partially infected, non-nitrogen-fixing nodules [12]. The precise function(s) of the EPS molecules in these associations is still unclear; however, recent studies suggest that these extracellular polymers may function as symbiotic signaling molecules

The current knowledge of EPS biosynthesis in rhizobia was based almost exclusively on the research with succinoglycan (EPS I) produced by *S. meliloti* [18]. More than 26 genes needed for the synthesis, modification, polymerization, export, and processing of *S. meliloti* EPS I were identified, and a pathway for the polysaccharide biosynthesis was proposed [19-21]. Recent discoveries on succinoglycan biosynthesis and its functioning in symbiosis are

The EPSs produced by *R. leguminosarum* strains investigated to date structurally differ from those of *S. meliloti.* At present there is fragmentary information about the *R*. *leguminosarum* EPS assembly, modification, and processing*,* as well as the role of the individual gene products in these processes. These data were reviewed in [9-11,24]. Here we report some recent and complementary data obtained in our group. On the basis of these data as well as the analysis of the sequenced *R*. *leguminosarum* and *R*. *etli* genomes we tried to outline main

Structures of known *R. leguminosarum* and *R. etli* (*Re*) EPSs are presented in Table 1. It should be noted that EPSs secreted by the most of the *Rlv* and *Rlt* strains were shown to have repeating units of identical structure [25-35]. These EPSs are branched heteropolysaccharides of the octasaccharide repeating unit consisting of a backbone of two glucose and two glucuronic acid residues, a side chain of three glucose and one galactose residues. The backbone contains 1-4 and 1-4 linkages only while the branch point is linked 1-6. The side chain contains 1-4 and 1-3 linkages. In general, the octasaccharide is modified by two pyruvyl, one or two non-stoichiometric O-acetyl, and one non-

which regulate plant responses in the infection process [13-17].

**2. Structural characteristics of exopolysaccharides** 

summarized in the exhaustive reviews [22,23].

regularities in the EPS synthesis.


**Table 1.** Structure of *R. leguminosarum* and *R. etli* EPS repeating units. Abbreviations: Glc, glucose; GlcA, glucuronic acid; Gal, galactose and Pyr, ketal pyruvate group.

However, several *R*. *leguminosarum* strains produce EPS with divergent side chains though with identical backbones and the same 1-6-linked glucosyl residue branching the side chain. Side chains of these EPS may consist of three to seven sugar residues. Up to three Gal residues can be present as in the *R. leguminosarum* bv. *phaseoli* (*Rlp*) 127K87 EPS, or terminal Gal residue can be absent as in the *Rlt* 4S EPS. In addition, in some cases side chains of EPS contain GlcA residues (*Rlp* 127K44, *Rlp* 127K38, *Rlp* 127K87, *Rlv* 248). Besides 1-4 and 1-3 linkages, sugar residues can be attached by 1-6 or 1-6 glycosidic bonds (*Rlp* 127K38, *Rlp* 127K87).

The acidic nature of EPS is explained by the presence of uronic acids and negatively charged pyruvyl groups. Similar to other representatives of *Rhizobiaceae*, the *R*. *leguminosarum* strains synthesize EPS in high-molecular-weight (HMW) and low-molecular-weight (LMW) forms [13,42]. The latter were proposed to act as signaling factors during the development of symbiosis [14,16-17,42].
