Achievements in the Life Sciences 10 (2016) 136–143

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Achievements in the Life Sciences

journal homepage: www.elsevier.com/locate/als

Structure of Extracellular Polysaccharides (EPS) Produced

by Rhizobia and their Functions in Legume–Bacteria
Symbiosis: — A Review
Pallab Kumar Ghosh, Tushar Kanti Maiti ⁎
Microbiology Laboratory, Department of Botany, Burdwan University, Burdwan -713104, West Bengal, India

a r t i c l e i n f o a b s t r a c t

Article history: The symbiotic diazotrophs comprise with a very diverse group of Gram negative soil bacteria,
Received 23 June 2016 collectively called as rhizobia found in nodule of legume plant. Rhizobia adopt themselves in
Received in revised form 7 November 2016 different environment including soil, rhizosphere and grown within legume roots, where they
Accepted 9 November 2016
fix nitrogen. The establishment of symbiosis is a very complicated process involving a coordinated
Available online 25 November 2016
exchange of signal between legume plants and the symbionts. The nodule development requires
synthesis of signal molecules such as Nod factors that are important for induction of nodule
Keywords: development. There are different types of surface polysaccharides such as lipopolysaccharides,
Rhizobium spp.
capsular polysaccharides, neutral and acidic polysaccharides found in rhizobia. The production
Extracellular polysaccharide
of symbiotically active polysaccharides may allow rhizobial strains to adapt themselves to
Production
Composition changing environmental conditions and interact efficiently with legume plants. Despite extensive
Succinoglycan research, the actual molecular function of the surface polysaccharides of rhizobia in symbiosis
Galactoglucan remains unclear. This review emphasized on the structural composition of extracellular polysac-
charide of different rhizobia isolated from different legume plants. The compositions of extracel-
lular polysaccharides are different in different rhizobia. The various compositions of extracellular
polysaccharides produced by the symbionts are considered as the signaling molecules essential
for determining host plant specificity. The present status of the biological functions of the exo-
polysaccharide in symbiosis such as host specificity, successful invasion, formation of infection
thread and induction of nodule formation in legume plants is also summarized here.
© 2016 Hosting by Elsevier B.V. on behalf of Far Eastern Federal University. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Structural Features of Rhizobial Exopolysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Production of Exopolysaccharides in Culture by Rhizobia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Functions of Rhizobial Exopolysaccharide in Symbiosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

Abbreviations: EPS, extracellular polysaccharide; CPS, capsular polysaccharide; LPS, lipopolysaccharides; CG, cyclic beta glucan; KPS, K-antigen polysaccharide;
NP, neutral polysaccharide.
⁎ Corresponding author.
E-mail address: tkmbu@yahoo.co.in (T.K. Maiti).
Peer review under responsibility of Far Eastern Federal University.

http://dx.doi.org/10.1016/j.als.2016.11.003
2078-1520/© 2016 Hosting by Elsevier B.V. on behalf of Far Eastern Federal University. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
 P.K. Ghosh, T.K. Maiti / Achievements in the Life Sciences 10 (2016) 136–143 137

Introduction

Polysaccharide is the most abundant organic macromolecules in nature. The biosyntheses of polysaccharide are found in
different organisms ranging from bacteria to eukarya, including plants. Gram negative soil bacteria belonging to (α and
β-proteobacteria) have the ability to produce root nodule by symbiosis with legume plants (Skorupska et al., 2006). This interaction
is initiated by the exchange of signal by diverse molecules between the two partners. Among them, plants liberate flavonoids into the
rhizospheric region that upregulate rhizobial genes responsible for nodule formation (Spaink, 2000; Schulze et al., 1998). Recognition
of the symbiont is made possible through the exchange of nod factor perception and Ca2+/calmodulin-dependent protein signaling
(Geurts and Bisseling, 2002; Oldroyd and Downie, 2004). The establishment of successful symbiosis in legume plant was made by the
production of nod factor signal and exposure of the correct surface and/or extracellular polysaccharide.
This signal exchange between the two partners seems to work at a distance in the rhizosphere and before binding of rhizobial
symbiont to the host root hairs, to induce morphogenetic changes in plant roots. These signal molecules, the Nod factors, are
sufficient for initiation of root hair changes, bacterial cell entry for infection, thread formation and activation of cortical cell divi-
sion Schulze et al. (1998) to generate the nodule primordium (Nod) (Kijne, 1992). Rhizobia colonize with plant root hairs, de-
velops infection, the bacteria multiply to form infection thread. In this thread the bacteria encircled by the peribacteroid
membrane of plant origin that differentiate into bacteroids (Fisher and Long, 1992).
Bacteroids were found to fix nitrogen by synthesizing the nitrogenase enzyme and other proteins. In return, plant supplies
carbohydrates to bacteria as a source of carbon and energy. The establishment of symbiosis is stringently controlled through a com-
plex network of signaling cascades Schulze et al. (1998). This process is partner specific and signifies that the rhizobial species can only
nodulate a limited but defined range of legume plants.
The key factors for the interaction are a number of rhizobial genes which are responsible for production of different types
of cell-surface polysaccharides such as capsular polysaccharide (CPS) that form as adherent cohesive layer on the cell surface.
However, the term exopolysaccharides (EPS) is used for polysaccharides with little or no cell association (Becker and Pühler,
1998). Cyclic beta-(1-2)-glucan is concentrated in periplasmic space of rhizobia, which plays an important role in osmotic adaptation
of bacteria Breedveld et al. (1993). Lipopolysaccharides (LPS) are anchored in outer membrane and consist of lipid A, a core polysac-
charide and repeating O-side antigen polysaccharides. Despite extensive research, the precise role of surface polysaccharides in sym-
biosis remains unclear. So the role of rhizobial polysaccharide has been the goal of many studies.
The present review describes the production and structure of different exopolysaccharides of rhizobia isolated from legume
root nodule. Attempts were also made to discuss the possible role of the exopolysaccharide in legume — rhizobia symbiosis
and nodule formation.

Structural Features of Rhizobial Exopolysaccharides

Rhizobial cell produces different types of surface polysaccharides into environment or retained at the cell surface. They comprise
extracellular polysaccharide (EPS), lipopolysaccharide (LPS), capsular polysaccharide (CPS), cyclic beta glucan (CG), K-antigen poly-
saccharide (KPS), neutral polysaccharide (NP), gel-forming-polysaccharide (GPS), and cellulose fibrils. They are species as well as
strain-specific heteropolymers and consisting of repeating units containing mainly common monosaccharides (D-glucose, D-mannose,
D-galactose, L-rhamnose, D-glucuronic and D-galacturonic acids) (Table 1). A large diversity in EPS chemical structures can be found
among rhizobia, concerning sugar composition, linkage of subunit, repeating unit size and degree of polymerization as well as non-
carbohydrate decoration (Table 1) (Laus et al., 2005; Skorupska et al., 2006; Downie, 2010; Janczarek, 2011). EPS are mainly
two types, succinoglycan (EPS I) and galactoglucan (EPS II) produced by several rhizobial strains (Reinhold et al., 1994) (Fig. 1).
EPS-I composed of octasaccharide repeating units containing one galactose and seven glucose residues (in molar ratio 1:7), joined
by β-1,3; β-1,4 and β-1,6 glycosidic linkages whereas EPS II 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).
Single repeating unit is decorated by different non-carbohydrate such as acetyl, pyruvyl and succinyl groups. Both EPS I and II
are secreted in two major fractions — High Molecular Weight (HMW) consisting of hundreds to thousands of repeating units and
Low Molecular Weight (LMW) that represents monomers, dimers and trimers in a case of EPS I and oligomers (15–20) in the case
of EPS II (Gonzalez et al., 1996, 1998; Wang et al., 1999). The pattern of non-carbohydrate modifications of EPS may be different
in various strains of the same species and depend on the phase of bacterial growth and culture medium. 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 sym-
biosis (Ivashina and Ksenzenko, 2012; Janczarek et al., 2014).

Production of Exopolysaccharides in Culture by Rhizobia

Rhizobium spp. are able to produce large amount of EPS in culture rather than EPS produced in symbiotic condition (Table 2).
The growth environment was very important for maximum exopolysaccharide production (Sutherland, 1972). Utilization of
different carbon sources for the growth and EPS production by Rhizobium sp. was reported earlier (Stowers, 1985; Breedveld
et al., 1993). However, carbon source in medium was a determining factor for the amount of EPS produced by R. tropici
CIAT899 (Staudt et al., 2012). Different carbon sources supplemented at 1% level promoted the bacterial growth and EPS produc-
tion by a Rhizobium sp. to different extent and mannitol was the most effective promoter. Higher concentrations of polysaccha-
rides were obtained when carbon:nitrogen ratio was higher in medium (Breedveld et al., 1993). Sulfate was less preferred
 138
Table 1
Different linkage and composition of monomers in EPS produced by Rhizobium spp.

Name of rhizobia EPS structure Monomer composition Reference

(%)

R. leguminosarum 128c53
R. leguminosarum 128c63
R. trifolii NA30
R. trifolii 0403
R. leguminosarum bv. trifolii 4S
R. trifolii 0403
→4Glc → α4GlcA → β4GlcA → β4Glc → β6Glc ← β4Glc ← βGlc ← βGlc(4–6)Pyr Galactosyl, glucosyl, glucuronosyl and pyruvyl Robertson et al. (1981)
←β3Gla(4–6)Pyr
→4Glc → α4GlcA → β4GlcA → β4Glc → β6Glc ← β4Glc ← βGlc ← βGlc(4–6)Pyr Galactosyl, glucosyl, glucuronosyl and pyruvyl
←β3Gla(4–6)Pyr
→4Glc → α4GlcA → β4GlcA → β4Glc → β6Glc ← β4Glc ← βGlc ← βGlc(4–6)Pyr Galactosyl, glucosyl, glucuronosyl and pyruvyl
←β3Gla(4–6)Pyr
→4Glc → α4GlcA → β4GlcA → β4Glc → β6Glc ← β4Glc ← βGlc ← βGlc(4–6)Pyr Galactosyl, glucosyl, glucuronosyl and pyruvyl
← β3Gla(4–6)Pyr
β-Glc-(1 → 3)-β-Glc-(1 → 4)-β-Glc(1 → 6)β → (4,6)-C-CH3-CO2H Galactosyl, glucosyl, glucuronosyl and pyruvyl Amemura et al. (1983)
ND Glucose:galactose:glucuronic acid = 5:1:0.9 Mikini et al. (1984)

P.K. Ghosh, T.K. Maiti / Achievements in the Life Sciences 10 (2016) 136–143
R. meliloti SU47 4,6-O-(1-carboxyethylidene)-α-D-Galp1 → 3(X-O-Ac)-β-D-Glcp1 → 3 1,3,5-tri-O-acetyl-2,4,6-tri-o-methylglucitol and penta-o-acetyl- Zhan et al. (1991)
2-mono-o-ethylgalactitol galactose pyruvate ketal group,
R. trifolii TA-1 Cyclic β-(1,2)-glucans Glucans Breedveld et al. (1990)
Rhizobium sp. NGR234 β-Glc-(1-6)-β-Glc(1-6)-β-(1-4)-β-Glc(1-4)-β(1-3)-βGal(1)-β(1-4)-GlcA(1-3)α- Galactosyl, glucosyl, glucuronosyl and pyruvyl
GlcA(1-4)α-(4-6) PyrGal-2,3-o-actyl
R. meliloti β-4Glc(1-4)-β-Glc(1-4)-β-Glc(1-3)-β–Gal(1-6)-β-Glc(1-6)-β-Glc(1-3)-β-Glc(1-3)-Glc-(4-6)Pyr Galactosyl, glucosyl, glucuronosyl and pyruvyl Zhan et al. (1990)
S. meliloti RM 1021 EPS II →3Gal → α(1,3)Glc-β(1,6)OAc → (4,6)-C-CH3-CO2H Galactose and glucose Her et al. (1990)
R. leguminosarum bv. viciae 248 β-Gla-(1 → 3)-β-Glc-(1 → 3)-β-Glc(1 → 3)β-GlcA-(1 → 4)-β-Glc(1 → 6)- Galactose, glucose and glucuronosyl Canter-Cremers
β → [(4,6)-C-CH3-CO2H]2 et al. (1991)
R. leguminosarum biovar trifoli TA-1 Cyclic β-(1,2)-glucans Glucans Breedveld et al. (1992)
S. fredii USDA205 [(→3)-α-D-Galp-(1 → 5)-α-D-Kdop-(2→)]n, [−2-O-MeManp → β-Kdo–]n Xylose, mannose, glucose and Kdo in a molar ratio of 1:1:1:5. Reuhs et al. (1993)
S. fredii USDA257 [(→3)-β-D-Manp-(1 → 5)-β-D-Kdop(2→)]n, [(→3)-β-D-2-O-MeManp-(1 → 5)- Xylose, mannose, glucose and Kdo in a molar ratio of 1:1:1:5.
β-D-Kdop-[2 →]]n
S. meliloti RM 1021 EPS I →4Glcβ → 6GlcOAc → β(1,4)Glc → β(1,3)Gal-β → 4Glcβ(1,6) ← βGlc(1,3) ← βGlc6- Succinoglycan with glycosidic linkage Reinhold et al. (1994)
OSucc ← β(1,3)Glc(4,6)c-CO2H-CH3
Rhizobium sp. ND Mannose:galactose:glucose 5.79:19.69:74.52 De and Basu (1996)
S. meliloti AK631 [−β-GlcA → Pse5N(β-OH-But)7NAc–]n Kdo(3-deoxy-D-manno-octulosonic acid) Campbell et al. (1998)
S. meliloti NGR185 [−β-GlcNAc → β-Kdo–]n Glucosyl and Kdo Reuhs et al. (1998)
S. fredii USDA208 [−α-Gal → β-Kdo–]n Galactosyl and Kdo
S. fredii USDA201 [−α-Gal → β-Kdo → α-2-O-MeHex → β-Kdo–]n Galactosyl and Kdo (2-metho-hexo)
Sinorhizobium sp. NGR234 [−β-Glc → α-Pse5NAc7NAc–]n Glucosyl
S. fredii HH303 [Rha, GalA]n Rhamnose with glucuronosyl
S. fredii HH103 [−7(3OH Bu)-Pse–]n 5-acetamido-3,5,7,9-tetradeoxy-7-[(R)-and (S)-3- Gil-Serrano et al. (1999)
hydroxybutyramido]-l-glycero-l-manno-nonulosonic acid = 3:1
S. meliloti −4Glc,β-1,4-Glcβ-1,4Glcβ-1,3Galβ-1Glcβ-1,3-Glcβ-1,3-Glcβ-1,6-Glcβ-1,6-(side chain) Galactose and reducing glucose Wang et al. (1999)
Rhizobium sp. ND Arabinose:xylose:rhamnose = 11.73:23.68:64.59 Datta and Basu (1999)
Rhizobium sp. ND Arabinose:xylose:rhamnose = 14.7:23.6:61.7
Rhizobium sp. ND Arabinose:xylose:rhamnose = 12.0:23.8:64.2
B. japonicum 2143 ND Man:Glu:Gal:GalA = 1: 2: 1: 1 Karr et al. (2000)
Rhizobium sp. TAL1145 ND Glucose:galactose = 2:1 Kaufusi et al. (2004)
Rhizobium sp. ND Arabinose:galactose:glucose:rhamnose:xylose Ghosh et al. (2005)
1.47:4.62:6.30:26.90:60.71
Rhizobium sp. strain KYGT207 (→4)-β-ManAp-(1 → 4)-β-Glcp-(1 → 4)-βGalp-(1 → 3 → 1) β-Glcp Glucose:galactose:mannuronic acid = 2:1:1 Kaci et al. (2005)
S. meliloti RM1021 ND Galactose:Glucose = 1:7 Luciana et al. (2010)
R. leguminosarum bv. trifolii Rt24.2 ND Glucose:glucuronic acid:galactose = 4.8:1.8:1.0 Janczarek (2011)
Rhizobium sp. strain PM25 ND Xylose:arabinose:mannose 0.1:0.8:99.1 Ghosh et al. (2011)
Rhizobium sp. ND Glucose and galactose Mukherjee et al. (2011)
R. sullae Cyclic-(1, 2)-glucans Glucose, galactose and fucose Gharzouli et al. (2013)
R. undicola strain N37 ND Galactose:mannose = 94.17:5.83 Ghosh et al. (2015)
 P.K. Ghosh, T.K. Maiti / Achievements in the Life Sciences 10 (2016) 136–143 139

Fig. 1. Chemical structure of rhizobial exopolysaccharides (EPS) (based on Skorupska et al., 2006; Downie, 2010; Janczarek, 2011 and Janczarek et al., 2014 review
works). Abbreviations: Glc = glucose, GlcA = glucuronic acid, Gal = galactose, Succ = succinate and Ac = acetyl.

nitrogen source for most of nitrogen sources for some strain of Rhizobium, whereas nitrate, glycine and ammonium conjugate
were most preferred as nitrogen source (Ghosh et al., 2005). Mandal et al. (2007) have reported L-asparagine increased both
growth and production by Rhizobium sp. VMA301 from V. mungo (L.) Hepper. Various studies have found that EPS production is
favored under conditions of nitrogen limitation (Doherty et al., 1988; Dudman, 1964). Under such nitrogen-limited conditions,
any excess sugars remaining can be used specifically for polysaccharide synthesis (Kucuk and Kivanc, 2009). However, thiamine
hydrochloride and nicotinic acid increased EPS production by different species of Rhizobium were observed Amemura et al., 1983;
Ghosh et al., 2011. Varied degrees of promotion of growth by isolates in the presence of vitamins might be due to differential require-
ment of vitamins as cofactors. Watson et al. (2001) stated that biotin might be involved as metabolic regulator in rhizobial growth.
EPS production also varies with time, as a function of growth phase, for many bacteria. For many rhizobacterial species, growth
and exopolysaccharide production occur simultaneously, because EPS biosynthesis being growth-associated (Datta and Basu,
1999; Ghosh et al., 2005). Several studies have indicated that the EPS yields vary with bacterial growth phase, while EPS compo-
sition remains constant through the batch cycle of growth (Breedveld et al., 1993; Ghosh et al., 2015).
140 P.K. Ghosh, T.K. Maiti / Achievements in the Life Sciences 10 (2016) 136–143

Table 2
EPS producing Rhizobium spp. strains reported in optimal condition by culture in different media.

Symbiont isolated Max EPS C-source EPS/carbon N-source C/N Composition of EPS Reference
from legume plants (g/L) (%) (g/g) ratio

R. leguminosarum bv. 2.7 Mannitol (1%) 1.13 Glutamic acid 5 Glucans Breedveld et al. (1993)
Trifolii TA-1
Rhizobium sp. 1.76 Mannitol (2%) 0.88 Potassium nitrate 20 Mannose, galactose and glucose De and Basu (1996)
Rhizobium sp. T1 1.5a Sucrose (1%) 1.5b Ammonium acetate 10 Glucose and glucuronic acid Guentas et al. (2000)
Rhizobium sp. D1 10 1.89 Mannitol (2%) 0.95 Potassium nitrate 20 Xylose, rhamnose, glucose, Ghosh et al. (2005)
galactose and arabinose
Rhizobium sp. KYGT207 2.5a Sucrose (2%) 1.25b ND ND Glucose, galactose and Kaci et al. (2005)
mannuronic acid
Rhizobium sp. VMA301 0.35 Mannitol (1%) + 0.27 L-asparagine 3.3 Ribose and mannose Mandal et al. (2007)
asparagine (0.3%)
Rhizobium sp. 1.182 Mannitol (1%) 1.18 Sodium nitrate 11.8 ND Kucuk and Kivanc
(2009)
Rhizobium sp. 0.966 Mannitol ND ND ND ND Kumari et al. (2009)
Rhizobium sp. PM25 0.596 Sucrose (1.5%) 0.39 Glycine 15 Xylose, arabinose and mannose Ghosh et al. (2011)
Rhizobium sp. 0.116 Glucose (2%) 0.058 Glycine 20 Glucose and galactose Mukherjee et al. (2011)
Rhizobium sp. 2.47 Sucrose (1%) 2.47 Ammonium sulfate 10 Glucose and maltose Sayyed et al. (2011)
R. tropici CIAT899 4.08 Sucrose (2%) 2.04 Ammonium nitrogen 20 Glucose, galactose, rhamnose Staudt et al. (2012)
and xylose
R. sullae 2.92 ND ND ND ND Glucose, galactose and fructose Razika et al. (2012)
R. radiobacter S10 2.834 ND ND ND ND Galactose, glucose, glucosamine, Zhou et al. (2014)
mannose
R. undicola strain N37 0.515 Mannitol (0.4%) 1.28 Potassium nitrate 2 Galacotse and mannose Ghosh et al. (2015)
R. tropici Semia 4077 7.45 ND ND ND ND Mannose, rhamnose, glucuronic Castellane et al. (2015)
acid, galacturonic acid, glucose,
galactose
Rhizobium sp. LBMP-C04 6.63 ND ND ND ND Rhamnose, glucose, galactose, Moretto et al. (2015)
mannose, glucuronic acid,
galacturonic acid
Rhizobium sp. PRIM-18 2.1 ND ND ND ND ND Priyanka et al. (2015)
a
Ethanol-precipitated material from supernatant.
b
Ethanol precipitated material/carbon, ND = not detected.

In regards to oxygen availability, the agitation rate controls both bacterial growth and EPS production. Bacterial growth
(in biomass) tends to be increase with higher agitation rates, under conditions where oxygen limitation is not imposed. However,
the impact of agitation on EPS production is unclear. Studies by Dudman (1964) and Kucuk and Kivanc (2009) indicate that
EPS production is optimal under conditions of low agitation. However, others have indicated that polysaccharide production is
maximized under conditions of high aeration, when oxygen limitation is not imposed (Duta et al., 2006).
Both bacterial growth and rates of EPS production are dependent upon the pH of the growth medium (Martinez-Romero et al.,
1991; Staudt et al., 2012). High level of pH resulted dramatic decrease in EPS production, particularly in the acidic range (Kucuk
and Kivanc, 2009; Staudt et al., 2012) whereas optimum EPS production was reported at slight alkaline pH value of 8.0 (Sayyed
et al., 2011). The role of bacterial growth temperature in EPS production is variable. EPS production is often favored by suboptimal
temperature Sutherland (1972). However, other studies found maximum EPS production occurring at optimal temperature
conditions by rhizobia (Sayyed et al., 2011; Ghosh et al., 2015).

Functions of Rhizobial Exopolysaccharide in Symbiosis

Several possible biological functions of rhizobial EPS are suggested by different workers (Fig. 2). These includes role in
protecting bacteria against environmental stresses, initial attachment of bacteria to the roots, involvement in stage of infection, struc-
tural role in the infection thread formation, release of bacteria from the infection threads, bacteroid development, suppression of plant
defense responses and protection against plant antimicrobial compounds (Becker and Pühler, 1998; Fraysse et al., 2003).
The primary attachment is established by recognition of root-hair lectins by the surface carbohydrates of rhizobial cell Dazzo et al.
(1984). EPS may enhance the chance of adhesion of bacteria to the tip of growing root hairs. However, EPS-deficient mutants are not
significantly affected in adhesion to the roots of several species such as clover (Rolfe et al., 1996), vetch (Van Workum et al., 1998) or
alfalfa (Cheng and Walker, 1998). The surface proteins rhicadhesins could also play an important role in the initial attachment (Smit
et al., 1992) and involvement of cellulose fibrils is suggested in this process (Laus et al., 2005). After initial attachment of
R. leguminosarum to root hairs, bacteria aggregate around the root hair surface in a cap or a biofilm formation which requires cellulose
fibrils (Laus et al., 2005; Williams et al., 2008). Rhizobial strain produce EPS in large amount were characterized by biofilm formation
that enhanced bacterial cell to better adaptation inside the host roots (Fujishige et al., 2006).
The low molecular mass of EPS in Sinorhizobium meliloti Rm1021 has been studied to know the structural requirement of EPS
for an effective nodulation (Gonzalez et al., 1996). Normally, this strain requires the presence of a succinoglycan to efficiently
infect Medicago sativa root hairs Reinhold et al. (1994). The importance of this polysaccharide in the symbiosis was confirmed in
 P.K. Ghosh, T.K. Maiti / Achievements in the Life Sciences 10 (2016) 136–143 141

Fig. 2. Different types of function related to rhizobial EPS production.

several non-EPS producing strains of Sinorhizobium meliloti and Rhizobium leguminosarum bvs. trifolii and viciae, which were symbi-
otically defective due to induction of empty or almost uninfected nodules on the respective host plants, being a result of aborted in-
fection thread elongation within the peripheral cells of the developing nodule (Ivashina and Ksenzenko, 2012).
The most important among the suggested EPS functions is its role as a signaling molecule. Rhizobial lipochitin oligosaccharides
(Nod factors), bacterial cell-surface components and low-molecular weight metabolites are engaged in the signaling. The EPS of
Mesorhizobium loti played a signaling role in symbiosis with Lotus plants forming determinate type of nodules Kelly et al.
(2013). Although several studies about rhizobial EPS and its precise function as a signal molecule in the symbiosis have not yet
been clearly established (Janczarek et al., 2014).
Moreover, EPS is especially important in symbioses with legumes that form indeterminate-type nodules, where long infection
threads are formed, as a compound indispensable for initiation and propagation of infection threads, bacterial release from the
infection threads and development of bacteroids (Brewin, 2004; Skorupska et al., 2006; Ivashina and Ksenzenko, 2012). The acidic
EPS essential for the development of an indeterminate type of nodules: Sinorhizobium meliloti/alfalfa, Rhizobium leguminosarum bv.
Viciae/Vicia sativa and bv. Trifoli/trifolium ssp. NGR234/Leucaena (Breedveld et al., 1993; Djordjevic et al., 1987).
Besides, there are several ways that plant pathogenic and symbiotic bacteria used to avoid plant defense system and to pro-
tect themselves (D'Haeze et al., 2004). The rhizobial surface polysaccharides and glucan play important roles in protection
against the host defense (D'Haeze et al., 2004). It is suggested that EPS might be act by suppressing bacterial antigens at the
stage of nodule cell infection (Wielbo et al., 2004). Recently Ciesla et al. (2016) reported that EPS plays an essential role in elec-
trophoretic mobility of rhizobial cells and their higher amounts depend on mobility as well as its acidic nature of the bacterial cell
surface.

Conclusion

Extracellular polysaccharides (EPS) are species-specific complex polymers of different carbohydrate unit secreted by bacterial
cell. This subject has been a great interest for a long time because of their importance in successful development of symbiosis with
legume hosts. The production of EPS by the rhizobia is considered to have important physiological implications and its critical
constituents seemed to be only for the invasion process leading to indeterminate and determinate nodule types. Several putative
roles have been considered for EPS synthesis such as specific signaling in the root invasion process, inhibition of plant defense
response structural requirement for this process and electrophoretic mobility of rhizobial cells.
On the other hand, new development of molecular methods that advances our understanding of the biosynthesis, regulation
and secretion of exopolysaccharides. The important goal for the future research should be the enlightenment of mechanisms of
EPS's action as signaling molecules in the initiation and development of symbiosis and mechanisms of control of the plant defense
systems which enable rhizobia to invade legume plants and their survival.

Acknowledgements

Authors gratefully acknowledged the financial support provided by the UGC, New Delhi, India, through CAS, Department of
Botany, and The University of Burdwan, Burdwan, West Bengal, India. The authors also gratefully acknowledged DST (SERB),
New Delhi, India (NPDF/2016/00323 dt. 05.07.2016) for providing National Post-Doctoral Fellowship to the first author.
142 P.K. Ghosh, T.K. Maiti / Achievements in the Life Sciences 10 (2016) 136–143

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