Rhizobial exopolysaccharides: genetic control and symbiotic functions

Anna Skorupska 0 Monika Janczarek 0 Magorzata Marczak 0 Andrzej Mazur 0 Jarosaw Krl 0 0 Address: Department of General Microbiology, University of M. Curie-Skodowska , Akademicka 19 st., 20-033 Lublin , Poland 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 -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. - Review Introduction Under nitrogen-limiting conditions, Gram-negative soil bacteria belonging to genera Rhizobium, Sinorhizobium, Mesorhizobium, Phylorhizobium, Bradyrhizobium and Azorhizobium, commonly named rhizobia, have the ability to establish root symbiosis with certain legumes. Upon stimulation by flavonoids exuded from legume roots into soil, rhizobia synthesize signaling molecules that are responsible for nodule formation [1-3]. These signaling molecules, named Nod factors, have been identified as lipochito oligosaccharides (LCOs) having diverse chemical substitutions. Nod factors are sufficient for initiation of root hair deformations (Had+, Hac+), infection thread formation (Thr+) and activation of cortical cells division [2]. Rhizobia colonize plant root hairs and infection threads develop within them. Inside the infection threads rhizobia multiply and invade developing nodules. Nodules formed on plant hosts fall into two different types: indeterminate and determinate. Temperate legumes such as clover, pea or alfalfa form indeterminate nodules, which are cylindrical in shape, with a persistent apical meristem responsible for the nodule growth. Tropical legumes such as soybean or common bean form determinate nodules, which are spherical with nonpersistent meristem. In both cases, the nodule is infected through infection thread, bacteria are released into cortical cells and surrounded by the peribacteroid membranes differentiate into bacteroids. Bacteroids synthesize the nitrogenase complex and other proteins that allow them to fix nitrogen and convert it into ammonia. In turn, plants supply bacteria with carbohydrates as a source of carbon and energy. Each step of establishment of symbiosis is tightly controlled through a complex network of signaling cascades [2-4]. Among a number of known rhizobial genes required for initiation and elongation of infection threads, the genes responsible for production of different types of cell-surface polysaccharides play a major role. Surface polysaccharides that form an adherent cohesive layer on the cell surface are designated capsular polysaccharides (CPS), whereas the term exopolysaccharides (EPS) is used for polysaccharides with little or no cell association [5,6]. Cyclic -(1,2)-glucans are generally concentrated in the periplasmic space of rhizobia, where they play an important role in osmotic adaptation of bacteria [7,8]. Lipopolysaccharides (LPS) are anchored in the outer membrane and are constituted by lipid A, a core oligosaccharide and an O-antigen polysaccharide. Despite extensive research, the precise role of the surface polysaccharides in symbiosis remains unclear. The significance of several types of surface polysaccharide has been studied extensively in model symbiosis of Sinorhizobium meliloti with alfalfa and was recently reviewed by Becker et al. [9] and Fraysse et al. [10]. This review focuses on the genetic control of exopolysaccharide synthesis, regulation and biological functions in symbiotic interactions with host legumes. Importance of EPS production in Rhizobium leguminosarum symbiotic interaction with clover is especially noticed. Structural features of rhizobial exopolysaccharides Rhizobial exopolysaccharides are species- or strain-specific heteropolysaccharides (they are composed of different kinds of monosaccharides) consisting of repeating units. They are secreted into the environment (EPS) or retained at the bacterial surface as a capsular polysaccharide (CPS). A large diversity in EPS chemical structures can be found among rhizobia, concerning sugar composition and their linkage in the single subunit, repeating unit size and degree of polymerization as well as non-carbohydrate decoration [5,11-13]. One of the best known rhizobial EPS is succinoglycan (EPS I) produced by several S. meliloti strains [14]. It is composed of octasaccharide repeating units containing one galactose and seven glucose residues (in molar ratio 1:7), joined by -1,3, -1,4 and -1,6 glycosidic linkages (Fig. 1A). Single repeating unit is decorated by acetyl, pyruvyl and succinyl groups (Fig. 1A). S. meliloti has also the ability to produce a (...truncated)