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13.1 Introduction

Symbiotic nitrogen fixation (SNF) is the signature feature of legumes in which the microsymbiont collectively called as rhizobia can reduce atmospheric nitrogen (N2) into ammonia; otherwise, N2 is metabolically unavailable to higher plants. The fixed nitrogen is generally used for plant growth or the excess of fixed nitrogen is released into the rhizosphere for improving soil fertility. Hence SNF have a significant impact on sustainable agriculture. The rhizobial diversity is enormous due to their wide geographical distribution, diverse hosts, and niches they occupy all over the globe. Rhizobia are Gram-negative bacteria belonging to class alpha- beta-, and gamma-proteobacteria, including species of the Rhizobiaceae, Phyllobacteriaceae, Methylobacteriaceae, Brucellaceae, Hyphomicrobiaceae, Bradyrhizobiaceae, Burkholderiaceae, and Pseudomonadaceae families. Host specificity exists in the process of SNF. The specificity of rhizobium for a legume host plant is determined by the exchange of molecules between both symbiotic partners. Each step of establishment of symbiosis is tightly controlled through a complex network of signaling cascades. Among them, plants liberate flavonoids into the rhizospheric region that upregulate rhizobial genes responsible for nodule formation. Rhizobia produce a variety of extracellular polymeric substances (EPSs), from simple glycans to complex heteropolymers. The secretion of EPS by rhizobia is associated with the invasion process and bacteroid and nodule development, as well as being a response to environmental stresses. There are different types of surface polysaccharides such as lipopolysaccharides, capsular polysaccharides, and neutral and acidic polysaccharides found in rhizobia. The production of symbiotically active polysaccharides may also provide stress adaptability to rhizobial strains against changing environmental conditions. This chapter focuses on different kinds of polysaccharides produced by rhizobia, their genetics and biosynthesis, as well as their biological role on effective symbiosis.

13.2 Polysaccharides from Rhizobium

Rhizobia synthesize different classes of polysaccharides that are involved in establishment of functional symbiosis with host legumes (Donot et al. 2012). They include cell surface and secreted glycans like Nod factors or LCOs, extracellular polysaccharides (EPSs), lipopolysaccharides (LPSs), K-antigens , glycolipids, and cyclic glucans . Presence of surface molecules with specific structure on cell wall can be recognized by surface receptors from the plant and determine host specificity. However, the term exopolysaccharides is used for polysaccharides with little or no cell association (Becker and Pühler 1998a, b). Cyclic beta-(1-2)-glucan is localized in periplasmic space of rhizobia and reported to play an important role in osmotic adaptation (Breedveld et al. 1993). Lipopolysaccharides (LPS) containing lipid-A, a core polysaccharide, and repeating O-side antigen polysaccharides are anchored in bacterial outer membrane. This section discusses the various polysaccharides produced by rhizobia and their biological functions.

13.2.1 Nod Factors

All rhizobia produce complex mixtures of structurally diverse Nod factors . With the exception of Nod from Sinorhizobium fredii USDA191 (Bec-Ferte et al. 1996), all others consist an oligosaccharide backbone of β-1,4-linked N-acetyl-d-glucosamine and fatty acyl group attached to the nitrogen of nonreducing saccharide. Nod factors are often called lipo-chitin oligosaccharides (LCOs) due to their resemblance to oligosaccharide backbone of chitin. Structural differences in the Nod factors are a result of the following variations:

  1. 1.

    Number of N-acetyl-d-glucosamine units. LCOs of rhizobia generally contain three to six N-acetyl-d-glucosamine units. However, M. loti produces a dimeric LCO species (Olsthoorn et al. 1998).

  2. 2.

    Presence or absence of strain-specific substituents, indicated as R1–R9 (Fig. 13.1). R. leguminosarum bv. viciae strains contain only an acetyl substituent at position R4, while S. fredii NGR234 contains many modifications.

    Fig. 13.1
    figure 1

    Structure of Nod factors produced by rhizobia. R-groups stands for hydrogen (R1), hydroxy (R2, R3, R4, R5, R6, R8, and R9), and acetyl (R7)

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  3. 3.

    Structure of the fatty acyl moiety. LCOs can contain one of a broad variety of fatty acyl groups that also occur commonly as moieties of the phospholipids.

  4. 4.

    Presence or absence of special α,β-unsaturated fatty acyl moieties. LCOs of S. meliloti, R. leguminosarum biovars viciae and trifolii, R. galegae, and M. huakuii possess α,β-unsaturated fatty acyl moieties.

Nod factors act at nanomolar concentrations and induce many early molecular and physiological changes and other responses that are related to root hair infection process in the legume host plant. NFs are recognized by host plant receptors with LysM extracellular domains which are also critical in the recognition of fungi during the mutualistic mycorrhiza interaction in Parasponia andersonii. Discovery of mycorhization factor (Myc factor) as a LCO suggests that the Rhizobium NF perception system evolved from the ancient mycorrhizal symbiosis. Nod factors also play a major role in the determination of host specificity as single mutations in the NF receptors are sufficient to change specificity of the interaction at the species level. Three best examples for correlating the structure of NOD factor with host specificity are as follows: (a) presence of sulfuryl substituent in the Nod factors of S. meliloti is essential for host-specific nodulation of Medicago sativa and prevents nodulation on other host plants such as Vicia sativa (Lerouge et al. 1990); (b) O-acetyl substituent in Nod factors of R. leguminosarum bv. viciae TOM and A1 is essential for cultivar-specific nodulation of pea (Firmin et al. 1993; Ovtsyna et al. 1998); and (c) fucosyl substituent in the Nod factors of many rhizobia is essential for determining a broad host range (López-Lara et al. 1996).

Furthermore, modifications such as the carbamoyl or acetyl groups at R4 and the methyl group at R1 of nonreducing terminus are important for a broad host range (Berck et al. 1999; Corvera et al. 1999; Hanin et al. 1997; Pacios Bras et al. 2000; Pueppke and Broughton 1999). Occurrence of an α,β-unsaturated fatty acyl moiety is correlated with the capacity to nodulate the leguminous species belonging to the Galegeae tribe that form an indeterminate nodule (Hadri et al. 1998).

13.2.2 Lipopolysaccharides and K-Antigens

Rhizobial LPSs and K-antigens are tightly linked to the cell surface and often have saccharide like Kdo (3-deoxy-d-manno-2-octulosonic acid). However, rhizobial K-antigens are structurally very distinct from LPSs; especially K antigens do not always contain Kdo, and lipid anchors have not been found at all, whereas LPSs posses them. The most complete structure of LPS (Fig. 13.2) from R. etli containing lipid A, core chain, and repeat unit of the O-antigen chain has been reported (Forsberg and Carlson 1998). LPSs of various rhizobia are highly variable, especially for O-antigen, but also in their core region and the lipidA moiety (Kannenberg et al. 1998). Occurrence of the very long-chain hydroxyl fatty acids, such as 27OH-C28:0 (Hollingsworth and Carlson 1989), was exclusively reported in the LPSs and Nod factors of the β-proteobacteria (Bhat et al. 1991a, b). LPS is essential for the survival of rhizobia under all growth conditions, which makes their study very difficult through developing mutants. The fact that all non-pleiotropic LPS mutants are able to infect plant tissue to some degree indicates that LPS is not specifically involved in the initial steps of the symbiosis up to root hair infection. LPS seems to play a more apparent role in the later stages of root nodule invasion, release from the infection thread, and symbiosome development. The fact that LPSs of R. leguminosarum bv. trifolii promoted infection thread formation in clover but not with the LPS from heterologous rhizobia indicated the host-specific features of LPS. Microscopic studies of responses of plants inoculated with LPS mutants have indicated that rhizobial LPS is involved in suppressing a host-plant defense response (Perotto et al. 1994), possibly in analogy with a role for the LPS of plant pathogens (Schoonejans et al. 1987). K-antigens of S. meliloti are also involved in suppressing a host-plant defense response, and they can functionally replace EPS biosynthesis in symbiosis (Campbell et al. 1998).

Fig. 13.2
figure 2

Chemical structure of lipopolysaccharides of rhizobia

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13.2.3 Cyclic Glucans

Cyclic glucans of Rhizobium and Sinorhizobium are linked solely by α-(1,2) glycosidic bonds with degrees of polymerization ranging from 17 to 25 (R. leguminosarum) or 17–40 (S. meliloti). Species of Bradyrhizobium produce cyclic glucans containing both β-(1,3) and β-(1,6) glycosidic linkages with the degree of polymerization ranging from 10 to 13 glucose residues with branched structure (Rolin et al. 1992). Cyclic β(1,2) glucans of S. meliloti are charged by the addition of anionic substituents like phosphoglycerol (Breedveld and Miller 1998), while cyclic glucans of Bradyrhizobium spp. are uncharged but contain the zwitterionic substituent phosphocholine (Rolin et al. 1992).

NdvB protein with high molecular weight (HMW) of 319 kDa is required to form a covalent intermediate with the glucan backbone during biosynthesis cyclic β-(1,2) glucans in Sinorhizobium and Rhizobium (Zorreguieta and Ugalde 1986). A close relative of NdvB is required for the biosynthesis of β-(1,3)–β-(1,6)-linked cyclic glucan in Bradyrhizobium spp. NdvC has been identified for its involvement in the formation of the β-(1,6) linkages (Bhagwat et al. 1999). The secretion of cyclic glucans to the periplasm (during logarithmic growth) and the extracellular environment (stationary growth) is mediated by the NdvA protein, which is the ABC component of a type 1 secretion system (Figs. 13.3, 13.4 and 13.5).

Fig. 13.3
figure 3

Chemical structure of rhizobial exopolysaccharides (EPSs)

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Fig. 13.4
figure 4

Pathway for the assembly of repeating unit of EPS I in S. meliloti (REF). (i) Sugar transferases, engaged in transfer of precursors onto lipid carrier (orange and blue); (ii) modifying enzymes, decorating the unit with non-sugar moieties (green); and (iii) proteins involved in EPS assembly and export (yellow). Abbreviations used: Glc Glucose; Gal Galact; Ac Acetate; Pyr Pyruvate, Succ Succinate

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Fig. 13.5
figure 5

Biosynthesis of EPS repeating unit and polymerization in Rhizobium leguminosarum. (i) Sugar transferases, engaged in transfer of precursors onto lipid carrier (orange and blue); (ii) modifying enzymes, decorating the unit with non-sugar moieties (green); and (iii) proteins involved in EPS assembly and export (yellow). Abbreviations used: Glc Glucose; GlcA Glucuronic acid; Gal Galactose; Ac Acetate; Pyr Pyruvate; IM inner membrane

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Impaired growth of defective mutants for cyclic glucan in hypo-osmotic media and mutational studies and production of a large amount of cyclic glucans in nodules where osmotic environment is relatively high indicated their protective role against hypo-osmolarity (Gore and Miller 1993). Ability of cyclic glucan to form inclusion complexes with hydrophobic molecules suggests that cyclic glucans can serve as a means of transport for signal molecules into the plant tissues (Morris et al. 1991). It was evidenced by the increased solubility of legume-derived flavonoids and Nod factors in the presence of cyclic glucans and cyclic dextrans, respectively (Schlaman et al. 1997).

13.3 Structural Features of EPS from Rhizobia

Bacterial EPSs are generally constituted by monosaccharides and non-carbohydrate substituents (such as acetate, pyruvate, succinate, and phosphate) and classified into homo-polysaccharides and heteropolysaccharides (Donot et al. 2012). Components most commonly found in EPS are monosaccharides such as pentoses (d-arabinose and d-xylose), hexoses (d-glucose, d-galactose, d-mannose, d-allose), desoxyhexoses (l-rhamnose and l-fucose), amino sugars (d-glucosamine and d-galactosamine), or uronic acids (d-glucuronic acids and d-galacturonic acids). The linkages most commonly found between monosaccharides are 1,4-β- or 1,3-β-linkages in the backbones having strong rigidity and 1,2-α- or 1,6-α-linkages in the more flexible ones. The linkage and composition of monomers of rhizobial EPS are listed in Table 13.1. The physical properties of polysaccharides are deeply influenced by the nature and the way the monomers are arranged together and by the assemblage of single polymer chains (Silvi et al. 2013). EPSs of rhizobia are species as well as strain specific that varies in the (i) composition of monosaccharides (d-glucose, d-galactose, d-mannose, l-rhamnose, d-glucuronic, and d-galacturonic acids) and non-carbohydrate moieties (acetyl, pyruvyl, succinyl, and hydroxyl-butanoyl groups) (Skorupska et al. 2006; Downie 2010; Janczarek and Skorupska 2011), (ii) linkage of subunit, (iii) repeating unit size, and (iv) degree of polymerization.

Table 13.1 Linkage and composition of monomers in EPS produced by Rhizobium spp.
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The structural features of EPS repeating units of several rhizobial strains of R. leguminosarum, S. meliloti, B. japonicum, B. elkanii, S. fredii NGR234, R. tropici CIAT899, and R. tropici SEMIA 4080 were well established. The EPSs produced by several rhizobial strains are classified into two major types, mainly succinoglycan (EPS I) and galactoglucan (EPS II). Succinoglycan is composed of octasaccharide repeating units containing galactose and glucose residues with molar ratio of 1:7, joined by glycosidic linkages (β-1,3, β-1,4, and β-1,6), whereas galactoglucan is a polymer of disaccharide repeating unit and joined by α-1,3 and β-1,3 glycosidic bonds (Her et al. 1990; Zevenhuizen 1997). EPSs I and II are secreted as both HMW consisting of hundreds to thousands of repeating units and low molecular weight (LMW) that represents monomers, dimers, and trimers in the case of EPS I and oligomers (8–40) in the case of EPS II (Gonzalez et al. 1996, 1998; Wang et al. 1999).

13.3.1 Structural Features of EPS from Sinorhizobium

Symbiotic exopolysaccharide of S. meliloti is succinoglycan (EPS I) that contains an octasaccharide repeating unit modified with acetyl, succinyl, and pyruvyl substituents. EPS I can be polymer of high or LMW, composed of monomers, dimers, and trimers. Also, the repeating unit carries one to two succinyl groups located at the C-6 position of the seventh sugar residue and a pyruvyl group linked to the eighth sugar residue through a 4,6-ketal linkage. Galactoglucan (EPS II) of S. meliloti that can mediate infection thread formation on M. sativa at a low efficiency is a disaccharide repeating unit modified with acetyl and pyruvyl substituents.

13.3.2 Structural Features of EPS from Rhizobium

EPS of R. leguminosarum is composed of octasaccharide repeating units which contain d-glucose, d-galactose, and d-glucuronic acid residues in a molar ratio 5:1:2, additionally modified with O-acetyl and pyruvyl groups (Breedveld et al. 1993). Succinoglycan type EPS of Rhizobium sp. NGR234 is formed by repeated units containing one galactose and seven glucose molecules linked by β-1,3, β-1,4, and β-1,6 linkages, containing residues of succinyl, acetyl, and pyruvyl (Becker and Pühler 1998a, b). Acidic EPS synthesized by Rhizobium sp. NGR234 were composed of glucosyl, galactosyl, glucuronosyl, and 4,6-pyruvylated galactosyl residues with glycosidic linkages β-1,3, β-1,4, β-1,6, α-1,3, and α-1,4 (Staehelin et al. 2006). The same strain also synthesized another form of EPS, consisting alternated units of glucose and galactose with α-1,3 and β-1,3 linkages and containing residues of acetyl and pyruvyl. EPS of Rhizobium sp. strain B isolated from nodules of alfalfa contain high amounts of glucose and rhamnose (1:2) and traces of 2-deoxy-d-arabino-hexuronic acid (Guentas et al. 2001). Chemical structure of EPS repeating units produced by R. leguminosarum and R. etli is presented in Table 13.2. The pattern of non-carbohydrate modifications of EPS is strain specific as well as influenced by bacterial growth phase and culture medium. Castellane et al. (2015) classified EPS into five groups based on ester type determined from the 13C NMR spectra (Table 13.3). The first group of EPS contained acetate only (Mesorhizobium loti LMG6125 and M. huakuii LMG14107). A second group of EPS consisted of succinate only (Sinorhizobium kostiense LMG19227). The EPS of strain M. plurifarium LMG11892 contained acetate and pyruvate (group III), whereas the strain Rhizobium giardini bv. giardini H152T produced EPS which contained pyruvate and succinate (group IV). The EPS belonging to group V (Rhizobium mongolense LMG19141) contained all three esters. Non-carbohydrate modifications located in the side chain of the EPS units proved to be very important for the signaling properties of EPS in the symbiosis (Ivashina and Ksenzenko 2012; Janczarek et al. 2014).

Table 13.2 Structure of EPS repeating unit produced by R. leguminosarum and R. etli
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Table 13.3 Classification of EPS based on ester type
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13.4 Genetics of EPS Production

Complex pathway of EPS starts with the synthesis of precursors for sugar nucleotides and non-carbohydrate donors followed by sequential assembly of the repeating unit on polyprenyl lipid carriers, their modification, polymerization, and export outside of the cell. Genes required for the biosynthesis of EPS in Mesorhizobium and Sinorhizobium form large clusters on the chromosome or megaplasmids (Kaneko et al. 2000; Finan et al. 2001). The gene clusters encode the following proteins, viz., (i) enzymes involved in the biosynthesis of nucleotide sugar precursors, (ii) enzymes involved in modifying EPS with non-sugar decorations, (iii) transferases involved in the assembly of EPS repeating unit, and (iv) proteins engaged in the polymerization and export of the growing EPS chain onto the cell surface (Glucksmann et al. 1993a, b; Whitfield and Paiment 2003). Biosynthesis and regulation of succinoglycan (EPS I) as well as galactoglucan (EPS II) were extensively studied in S. meliloti, while our understanding on acidic EPS production in R. leguminosarum is limited.

Specific glycosyltransferases are involved in the sequential transfer of precursors and nucleotide diphosphosugars to a growing polysaccharide chains that are attached to undecaprenol diphosphate. The repeating unit is formed at the inner leaflet of the cytoplasmic membrane, and polymerization of individual repeating units takes place at the periplasmic face of the inner membrane (IM) after they have been flipped across the IM due to Wzx-like translocase or “flippase” activity. Polymerization is coupled to export of the growing polymer to the cell surface and engages Wzy-like polymerase and Wzc-like inner membrane-periplasmic auxiliary protein (MPA) with an ABC module that controls the chain length of the growing heteropolymers (Whitfield and Paiment 2003). Outer membrane auxiliary protein (OMA) is involved in the completion of translocation process by forming a channel in the outer membrane to facilitate the growing polysaccharide to reach the cell surface (Paulsen et al. 1997).

13.4.1 EPS Biosynthesis in Sinorhizobium

Multipartite genome of S. meliloti possesses three replicons: a chromosome (3.65 Mb) and two megaplasmids: pSymA (1.35 Mb) and pSymB (1.68 Mb). The gene cluster required for the biosynthesis of EPS I (exo/exs) is organized in several operons of megaplasmid 2 (pSymB) (Charles and Finan 1991). Analysis of S. meliloti genome sequence indicated that only 2 out of 11 regions engaged in polysaccharide biosynthesis were previously recognized on pSymB (Becker et al. 1993a, b). The genes involved in the biosynthesis of EPS I in S. meliloti are listed in Table 13.4.

Table 13.4 Genes involved in biosynthesis of EPS in Sinorhizobium meliloti
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Precursors for the biosynthesis of EPS I are produced by the proteins encoded by exoC, exoB, and exoN. exoC encodes a phosphoglucomutase that catalyzes transformation of glucose- 6-phosphate into glucose-1-phosphate. exoB encodes for the UDP-glucose-4-epimerase that converts UDP-glucose into UDP-galactose. A protein encoded by exoN gene displays UDP-glucose pirophosphorylase activity. Assembly of the repeating unit is initiated by galactosyltransferase encoded by exoY. exoF gene encodes a protein that is needed for addition of galactose to the lipid carrier. The subsequent addition of glucose residues is carried out by a complex of glucosyltransferases encoded by exoA, exoL, exoM, exoO, exoU, and exoW genes (Müller et al. 1993; Reuber and Walker 1993a, b).

Transmembrane protein with succinyl transferase activity is encoded by exoH in S. meliloti that is involved in the addition of non-sugar residues that is crucial for the polymerization and secretion of succinoglycan. A transferase encoded by exoZ gene is involved in addition of acetyl residues, while exoV is responsible for addition of pyruvyl residues. Acetyl and succinyl modifications of EPS I influence the susceptibility of the polysaccharide to cleavage by these glycanases and production of LMW EPS 1.

Polymerization of the succinoglycan repeating units and secretion of the polymer depend on the proteins encoded by the exoPQT genes (Glucksmann et al. 1993a). ExoP of S. meliloti is an autophosphorylating protein tyrosine kinase that catalyzes the formation of dimers of octasaccharide units of EPS I and forms a complex with ExoQ and ExoT proteins which participate in the secretion of succinoglycan (Niemeyer and Becker 2001). It was evidenced that ExoQ protein is indispensable for high-molecular weight (HMW) EPS I biosynthesis, while ExoT is responsible for producing low-molecular-weight (LMW) EPS (Reuber and Walker 1993a, b). ExsA protein of S. meliloti is homologous to ABC transporters and is involved in the transport of HMW EPS I. Symbiotically active, low-molecular-weight EPS I is produced in S. meliloti by a specific biosynthetic pathway, but it can also result from a cleavage of HMW succinoglycan by endoglycanases: ExoK (β-1,3-1,4-glucanase) and ExsH (succinoglycan depolymerase), the latter of which is secreted by PrsDE secretion system (York and Walker 1997).

Biosynthesis of EPS II in S. meliloti is mediated through 23 kb exp gene cluster localized on pSymB plasmid and is separated from exo/exs cluster by about 200 kb. Biosynthesis of nucleotide diphosphosugar precursors depends on the activity of ExpA7, ExpA8, ExpA9, and ExpA10 proteins, which are involved in the formation of dTDP-rhamnose. The intermediate in this synthesis dTDP-glucose serves as the donor of glucose in EPS II synthesis in contrast to UDP-glucose which is a precursor of glucose in EPS I synthesis. Other genes in the cluster were established for their involvement in polymerization of sugars (β-glucosyltransferases ExpA2 and ExpE2, galactosyl transferases ExpA3, ExpC, ExpE4, and ExpE7), export (ExpD1-ABC transporter; ExpD2-MFP-membrane fusion protein) of EPS II, and the regulation of exp gene expression (Becker et al. 1997). EPS II synthesis and secretion was blocked in defective mutants of expD1 and expD2 (Becker et al. 1997; Moreira et al. 2000).

13.4.2 EPS Biosynthesis in Rhizobium leguminosarum

Rhizobium leguminosarum comprises two biovars viciae and trifolii that differ in their host specificity and is a close relative of Rhizobium etli (formerly the third biovar—phaseoli). The genome of R. leguminosarum consists of the chromosome and 1–10 megaplasmids. R. leguminosarum bv. viciae consists of a circular chromosome of 5 Mb and six plasmids: pRL12 (870 kb), pRL11 (684 kb), pRL10 (488 kb), pRL9 (352 kb), and pRL8 (147 kb). Rhizobium leguminosarum bv. Trifolii TA1 consists of 7.3 Mb genome with five replicons via a chromosome and four plasmids: pRTA1d (800 kb), pRTA1c (650 kb), pRTA1b (600 kb), and pRTA1a (500 kb) (Król et al. 2005). Multi-cistronic operon having a core set of genes required for the assembly of the repeating units (pssEDCFGHIJS), its modification (pssKMR), polymerization (pssL), and processing (pssW) of EPS was identified in Rlv VF39 (Sadykov et al. 1998).

EPS biosynthetic clusters carry genes involved in the biosynthesis of nucleotide precursors. The genes involved in the biosynthesis of EPS in R. leguminosarum are summarized in Table 13.5. exoB gene of R. leguminosarum bv. trifolii encodes a protein showing 80% identity to the UDP-glucose 4-epimerase of S. meliloti that is involved in the biosynthesis of UDP-galactose, the donor of galactose residues for different heteropolysaccharides in rhizobia (Canter Cremers et al. 1990). exo5 encodes UDP-glucose dehydrogenase that is responsible for oxidation of UDP-glucose to UDP-glucuronic acid (Kereszt et al. 1998). pssA gene encodes for glucosyl-IP-transferase that initiates the biosynthesis of EPS in R. leguminosarum by the transferring UDP-glucose to the lipid carrier attached to the cytoplasmic membrane of R. leguminosarum bv. trifolii, viciae, and R. etli. pssD and pssE genes of both R. leguminosarum biovars possess glucuronosyl transferase activity that catalyzes the addition of a glucuronic acid residue (van Workum et al. 1997; Król et al. 1998). pssC encodes a glucuronosyl-β-1,4-glucuronosyltransferase for the addition of the second glucuronic acid residue (van Workum et al. 1997; Król et al. 1998; Sadykov et al. 1998; Pollock et al. 1998). Subsequent steps of acidic EPS synthesis were poorly studied in R. leguminosarum, although the genes pssF, pssG, pssH, pssI, pssJ, and pssS encoding putative glycosyltransferases and pssR and pssM genes predicted to encode EPS modifying enzymes were identified in R. leguminosarum bv. viciae (Sadykov et al. 1998)

Table 13.5 Genes involved in biosynthesis of EPS in Rhizobium leguminosarum
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pssT, pssN, and pssP genes are responsible for the formation of secretion system involved in assembly and export of EPS in R. leguminosarum bv. trifolii TA1. PssT is an integral inner membrane protein and pssT mutant overproduced EPS with the degree of polymerization slightly increased when compared to the wild-type strain (Mazur et al. 2003). PssN protein turned out to be similar to outer membrane auxiliary (OMA) proteins (Mazur et al. 2001). PssP is similar to membrane-periplasmic auxiliary (MPA) proteins involved in the synthesis of HMW CPS and EPS (Paulsen et al. 1997). Genetic organization of the pss gene clusters of R. leguminosarum is shown in Fig. 13.6.

Fig. 13.6
figure 6

Gene arrangement in Pss-I cluster of Rhizobium leguminosarum and R. etli

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In R. leguminosarum bv. viciae, plyA and plyB genes encode glycanases. plyA mutation did not affect EPS processing, while plyB mutant was characterized by a significant increase in culture viscosity. pssV-E operon was found in all R. leguminosarum and R. etli genomes, and 9 out of the 15 genes named as pss genes from this operon have orthologs in both biovars of R. leguminosarum and R. etli genomes. In addition, the same gene name abbreviation was assigned to six genes (pssA, pssB, pssN, pssO, pssP, and pssT) localized in other operons. The new sets of genes identified from Rlt WSM2304, Re CFN42, Re CNPAF512, and Re CIAT 652 are designated as psa (polysaccharide repeating unit assembly) and summarized in Table 13.6.

Table 13.6 Genes controlling polysaccharide repeating unit assembly for EPS biosynthesis in R. leguminosarum and R. etli
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13.5 Biological Role of Rhizobial EPS in Symbiosis

The biological roles of rhizobial EPS are postulated based on the experimental evidence observed at different stages of symbiotic interactions between host legumes with rhizobial mutants having altered gene expression. These include the involvement in early steps of legume–rhizobia symbiosis such as attachment of rhizobia on root hairs/surface, structural role in the infection thread formation, release of bacteria from infection threads, bacteroid development, suppression of plant defense responses, and protection against plant antimicrobial compounds and environmental stresses. The importance of this polysaccharide in the symbiosis was confirmed in several non-EPS producing strains of Sinorhizobium meliloti and R. leguminosarum bvs. trifolii and viciae, which were symbiotically defective due to induction of empty or almost uninfected nodules on the respective host plants, being a result of aborted infection thread elongation within the peripheral cells of the developing nodule (Ivashina and Ksenzenko 2012).

13.5.1 EPS Is Essential for Bacterial Attachment to Root Hairs

Successful recognition of appropriate rhizobial cells leads to attachment of rhizobial cell and formation of biofilm on the root surface and root hairs of host plant. Rhizobial cell attachment process has been distinguished into two as primary and secondary attachment. Primary attachment of bacterial cells to the root hair tip can be established via plant lectins, secreted at the root hair tip, which recognize specific polysaccharide structures present on the surface of the symbiotic bacteria. EPS plays an essential role in electrophoretic mobility of rhizobial cells, and their effective colonization depends on mobility as well as its acidic nature of the bacterial cell surface (Ciesla et al. 2016). EPS may also enhance the chance of adhesion of bacteria to the tip of growing root hairs. After initial attachment, rhizobial cells aggregate around the root hair surface and form biofilm-like structures. Rhizobial strains producing large amounts of EPS were characterized by biofilm formation that enhanced bacterial cell to better adaptation against stressed environment (Fujishige et al. 2006). R. leguminosarum bv. trifolii strain Rt24.2 attached with high efficiency recorded higher number of bacteria attached on root surface in comparison with its pssA and rosR defective mutants. On the other hand, EPS overproducing mutants via Rt24.2 (pBA1) and Rt24.2 (pBR1) showed more efficient attachment in comparison to the wild-type bacterial cells on clover root. The above results confirmed the crucial role of EPS in attachment and biofilm formation (Figs. 13.7 and 13.8).

Fig. 13.7
figure 7

Infection of root hair cell by Sinorhizobium meliloti. Fully extended infection thread in root hair cell infected by wild-type S. meliloti (a) and failure of infection thread extension in cells infected with exoY mutants (b)

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Fig. 13.8
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Infection of Vicia sativa subsp. nigra by wild and mutant strains of Rhizobium leguminosarum. Infection of elongated root hair by wild-type RBL5523 (a), curling of elongated root hair with no infection thread induced by EPS-deficient mutant RBL5833 (b), abortive infection threads induced by EPS- and cellulose-deficient mutant RBL5973 (c, e), infections by cellulose-deficient mutant RBL5760 originates from a curl in a short pidermal root hair (d)

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Secondary attachment of rhizobium on root surface is called “firm” attachment, since removal of attached bacteria at this stage is difficult (Ausmees et al. 1999). Cellulose fibrils on bacterial surface play a role in the firm attachment of the bacteria to the root hair. Cellulose-deficient mutant of R. leguminosarum such as RBL5760 has been reported to lack the formation of cap-like bacterial aggregates on the root hair tip. Instead, incubation of these strains with plant roots showed attachment of single bacterial cells on root hairs and the root surface. EPS-deficient mutant strains (RBL5833 and RBL5808) resulted in persistent flocculation of rhizobia due to constitutive expression of cellulose fibrils on the bacterial surface in comparison with cellulose-deficient bacteria. EPS prevents bacterial agglutination by masking the cellulose fibrils in the root hair curl. Nodule formation was arrested at the primordium stage in plants inoculated with EPS-deficient strains via RBL5833 due to cellulose-mediated agglutination of the bacterial cells in the root hair curl. Inoculation of EPS defective mutant RBL5833 has also been reported to severely reduce the number of infection sites on V. sativa roots when compared with wild-type-inoculated roots and suggested the role of EPS on enhancing primary attachment to the tip of growing root hairs as well as for infecting emerging epidermal root hairs by cellulose-deficient strains.

Cellulose deficiency of mutant RBL5760 (CF−:EPS+) does not affect nodule number or delay nodule initiation in V. sativa subsp. nigra and nodules were elongated and pink colored, an indication of nitrogen fixation. However, 95% of infection threads were originated from infection sites localized on the epidermal surface, while wild strain RBL5523 produced majority of infection threads from a curled tip of an elongated root hair. RBL5973 (CF¯:EPS¯)-inoculated root sections revealed that this EPS- and cellulose-deficient strain infects the roots via cortical root hairs. Continuous Nod factor production by these bacteria is likely to induce polarized elongation of cortical cells, since cortical root hairs are mainly found on roots after application of Nod factors (van Spronsen et al. 1994). Restoring cellulose production through complementation with celE containing plasmid pMP4698 also restored the infection of elongated root hairs to levels observed with wild-type RBL5523. The results clearly demonstrated that EPS and CF determine the site of colonization and place of origin of infection threads during nodulation process (Figs. 13.9 and 13.10).

Fig. 13.9
figure 9

Structure of biofilms formed by the R. leguminosarum bv. trifolii wild-type strain 24.2 and its mutants

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Fig. 13.10
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Attachment of R. leguminosarum bv. trifolii wild type and its mutants on sand (a) and clover roots (b)

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Roots inoculated with EPS defective mutant strains like RBL5973 (CF−:EPS−), RBL5833 (CF+:EPS−), and RBL5808 (CF+:EPS−) formed abortive infection threads originated from infection sites on the root surface (Fig. 13.10). Infection sites and infection threads were not observed in elongated root hairs. Infection threads initiated from these infection sites aborted in the first or second cortical cell layer, concomitant with an early arrest in nodule development. Nodules remained small, white, and uninfected when roots were observed for several weeks. The appearance of small uninfected nodules suggests that infection of the cortex is a prerequisite for (young) nodule formation. Abortion of the RBL5973 (CF−:EPS−)-induced infection threads before entry into the nodule primordium demonstrates the importance of EPS production for infection thread elongation. It is not clear whether infection thread extension is dependent on the presence of the succinyl substituent of succinoglycan or on the production of low-molecular-weight (trimer, dimer, and monomer) forms of succinoglycan. If succinoglycan has a role in signaling to the plant, perhaps the low-molecular-weight forms can interact more readily with the plant cell membrane.

13.5.2 EPS Is a Determinant of Host-Plant Specificity in Nodulation

Rhizobia that form indeterminate type nodules in Vicia, Medicago, Pisum, or Trifolium are reported to produce EPS for tight root hair curling, proper infection thread formation, bacteria release, bacteroid development, and the effective nodulation (van Workum et al. 1998; Rolfe et al. 1996; Pellock et al. 2000). Restoring symbiotic efficiency in exo defective mutants of S. meliloti by the addition of picomolar quantities of trimer fraction of the EPS I or EPS II fraction containing 15–20 units indicated that low-molecular-weight EPS may act as a signaling molecule during invasion process (Wang et al. 1999; Battisti et al. 1992; Gonzalez et al. 1996). In the case of R. leguminosarum bv. trifolii, purified EPS fractions restored the nodulation of exo mutants (Djordjevic et al. 1987). Fix− nodules were formed in V. sativa only on roots infected with rhizobia producing identical (R. leguminosarum bv. viciae and bv. trifolii) or similar EPS (R. leguminosarum and R. tropici). As EPS from nonhomologous rhizobial strains or structurally changed homologous EPS could not compensate for symbiotic deficiency, it was concluded that EPS structure could be one of determinants of the host specificity at early stages of root infection. These observations supported the hypothesis that host-specific infection is dependent on EPS structure (Laus et al. 2005). However, nodule initiation in V. sativa by heterologous rhizobial strains after acquisition of pSym plasmid from R. leguminosarum bv. viciae indicated that infection of root tissue can occur regardless of EPS structure, and Nod factor is the only determinant of the host specificity (Laus et al. 2004, 2005). R. leguminosarum bv. viciae producing S. meliloti Nod factor successfully infected alfalfa transgenic for pea lectin despite the different EPS structure (van Rhijn et al. 2001). The controversy on the role of EPS on host specificity is not yet resolved.

13.5.3 EPS Is Involved in the Evasion of Plant Defense Response

Plants have evolved defense mechanisms like production of antimicrobial compounds, phytoalexins, reactive oxygen species, etc., to protect themselves from biotic as well as abiotic stresses. Similarly, plant pathogens and symbiotic bacteria have also evolved mechanisms to avoid plant defense system for effective colonization. Surface polysaccharides such as EPS, CPS, LPS , and glucan produced by rhizobia play important roles to overcome host defense mechanisms (D’Haeze and Holsters 2004). Structural features of exopolysaccharides are critical for suppression of host defense responses (Pellock et al. 2000). Changes in the structure of particular surface polysaccharides generally result in an increased sensitivity to host antimicrobial compounds. EPS I defective mutants of S. meliloti elicited noninfected pseudonodules and induced plant defense response on M. sativa. Cortical cells of pseudonodules were abnormally thick and encrusted with autofluorescent phenolic compounds compared to wild-type nodules. Cell walls and wall apposition contained callose. Low-molecular-weight EPS I added to alfalfa cell cultures suppressed the alkalinization induced by yeast elicitor, while heterologous EPS or HMW EPS I failed to suppress alkalinization (Niehaus et al. 1996). These data indicated that LMW EPS I is a specific suppressor of plant defense system in the case of S. meliloti–M. sativa symbiosis. Structural changes in the EPS of exoB mutant of Bradyrhizobium japonicum can induce soybean nodules in which significant amounts of phytoalexin–glyceollin accumulated (Parniske et al. 1994). This antimicrobial compound normally accumulates during infection of soybean by pathogenic Phytophthora megasperma (Schmidt et al. 1992). In Azorhizobium caulinodans–Sesbania rostrata symbiosis, mutants deficient in EPS production were blocked at an early stage of invasion. EPS of A. caulinodans is required as a diffusion barrier protecting bacteria against toxic H2O2 generated by the host. The establishment of functional symbiosis probably depends on the suppression of plant defense mechanisms.

13.5.4 EPS Is Essential for Cell Attachment and Biofilm Formation on Abiotic Surfaces

Rhizobia have to survive long periods of time under soil conditions when the host plant is not available. Biofilm forming ability is considered as one of the survival strategy, and EPSs play a significant role in biofilm formation as an essential component of the biofilm matrix (Koo et al. 2013). The cells of pssA mutant of R. leguminosarum bv. trifolii strain Rt5819pssA completely lost its EPS production trait and produced immature/irregular pseudo-biofilm with a depth of 12.5 μm, while the wild-type strain Rt24.2 produced regular biofilm with a depth 43 μm (Janczarek et al. 2015 PS). Adhesion of rosR mutant strain Rt2472rosR was significantly impaired and an immature biofilm with the maximal depth 21.6 μm was formed. A strain Rt24.2 (pBA1) carrying multiple pssA copies overproduced EPS (156% of wild-type strain) and developed densely packed biofilm with threefold greater depth than that formed by the wild type. Survival of bacteria in the biofilms formed by the individual strains was established and presented as a ratio of live to dead cells. Majority of wild-type bacteria cells were alive (alive/dead ratio = 49), while decrease in cell viability was the highest in the pssA mutant, which does not produce any amount of EPS (a ratio of 1.68). This indicates that the lack of proper amounts of EPS significantly reduced survival of the cells. This suggests that larger amounts of EPS secreted by the bacteria into the environment enable them to form biofilm with an appropriate organization faster and survive better in this specific ecological niche. Similarly, wild-type bacteria exhibited high efficiency in attachment to sand particles, since 3.4 × 104 cells/mg of sand were attached after 1 h and 16.5 × 104 cells within 48 h post-inoculation. In contrast, the rosR mutant showed a decreased ability to adhere to this material, since the numbers of the cells attached after 1- and 48-h incubation were 33.5 and 38.4%, respectively, of those attached by the wild-type strain. While pssA mutant totally lost the ability of attachment and biofilm formation on this surface, cells adherent to sand particles accounted only for 7.6% of those detected for the control strain. EPS overproducing strains via Rt24.2 (pBA1) and Rt24.2 (pBR1) strains exhibited significantly higher efficiency in adhesion. Hence, rhizobial EPS is very important for successful and efficient adhesion of the bacteria to root or sand surfaces available in the environment.

13.6 Future Aspects for Research

Extracellular polysaccharides (EPS) are species-specific complex carbohydrate polymers of rhizobia that are involved in successful development of symbiosis with legume hosts. Several putative roles have been considered for EPS synthesis such as specific signaling in the root invasion process, inhibition of plant defense response, and electrophoretic mobility of rhizobial cells. EPS is indispensable compound for the initiation and propagation of infection threads, bacterial release from the infection threads, and development of bacteroids (Ivashina and Ksenzenko 2012) in host plants that produce indeterminate type nodules. New development of molecular methods should be explored to advance our understanding on the biosynthesis, regulation, and secretion of exopolysaccharides. Future research should enlighten the mechanisms of EPSs’ action as signaling molecules in the initiation and development of symbiosis. Despite the massive and chronic infection of nodule tissues, rhizobium–legume interactions are plant beneficial for N-requirement. Successful symbiotic development depends on the ability to actively suppress plant innate immunity that also provides an opportunity for infection by pathogenic organisms, while allowing infection by the compatible symbiont may have been a selective driving force that led to mechanisms of rhizobium host specificity. Revealing molecular network involved in the plant recognition of host-specific rhizobia and suppression of host immunity will be the key to manipulate ecological relationships in the context of agricultural systems. Studies indicated that EPS of R. tropici can be successfully utilized as alternative vehicles for inoculation as they promote symbiotic efficiency, growth, and productivity in cowpea co-inoculated with Bradyrhizobium sp., Paenibacillus graminis, and P. durus. Commercial production of EPS from rhizobia may be considered highly promising unexplored sources of microbial polysaccharides for industrial applications and soil-stabilizing agents as none of the rhizobia has been shown to be pathogenic. This may represent a potential opportunity for the bio-inoculant producing industries as best alternative activity during the non-crop seasons.