Rhizobial exopolysaccharides: Genetic regulation of their synthesis and relevance in symbiosis with legumes

International Journal of Molecular Sciences Review Rhizobial Exopolysaccharides: Genetic Regulation of Their Synthesis and Relevance in Symbiosis with Legumes Sebastián Acosta-Jurado 1, Francisco Fuentes-Romero 1, Jose-Enrique Ruiz-Sainz 1, Monika Janczarek 2,* and José-María Vinardell 1,*   Citation: Acosta-Jurado, S.; Fuentes-Romero, F.; Ruiz-Sainz, J.-E.; Janczarek, M.; Vinardell, J.-M. Rhizobial Exopolysaccharides: Genetic Regulation of Their Synthesis and Relevance in Symbiosis with Legumes. Int. J. Mol. Sci. 2021,22, 6233. https://doi.org/10.3390/ ijms22126233 Academic Editor: Shawn A. Christensen Received: 5 May 2021 Accepted: 6 June 2021 Published: 9 June 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Department of Microbiology, University of Sevilla, Avda. Reina Mercedes 6, 41012 Seville, Spain; [email protected] (S.A.-J.); [email protected] (F.F.-R.); [email protected] (J.-E.R.-S.) 2Department of Industrial and Environmental Microbiology, Institute of Biological Sciences, Faculty of Biology and Biotechnology, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland *Correspondence: [email protected] (M.J.); [email protected] (J.-M.V.); Tel.: +48-81-537-5909 (M.J.); +34-954-554-330 (J.-M.V.) Abstract: Rhizobia are soil proteobacteria able to engage in a nitrogen-fixing symbiotic interaction with legumes that involves the rhizobial infection of roots and the bacterial invasion of new organs formed by the plant in response to the presence of appropriate bacterial partners. This interaction relies on a complex molecular dialogue between both symbionts. Bacterial N-acetyl-glucosamine oligomers called Nod factors are indispensable in most cases for early steps of the symbiotic interaction. In addition, different rhizobial surface polysaccharides, such as exopolysaccharides (EPS), may also be symbiotically relevant. EPS are acidic polysaccharides located out of the cell with little or no cell association that carry out important roles both in free-life and in symbiosis. EPS production is very complexly modulated and, frequently, co-regulated with Nod factors, but the type of co-regulation varies depending on the rhizobial strain. Many studies point out a signalling role for EPS-derived oligosaccharides in root infection and nodule invasion but, in certain symbiotic couples, EPS can be dispensable for a successful interaction. In summary, the complex regulation of the production of rhizobial EPS varies in different rhizobia, and the relevance of this polysaccharide in symbiosis with legumes depends on the specific interacting couple. Keywords: rhizobia; legume; rhizobium–legume symbiosis; exopolysaccharide synthesis; quorum sensing (QS); nodulation; nod regulon; flavonoids; RosR/MucR; SyrM 1. Introduction Rhizobia is the common name for a group of soil proteobacteria able to enter into symbiosis with legume plants [ 1 ]. As part of this interaction, rhizobia infect legume roots, the plant forms new organs (root nodules), and these organs are invaded by rhizobia: all together, these events are referred as the nodulation process. Inside nodules, rhizobia are hosted by symbiotic plant cells, in which they differentiate into bacteroids able to fix N 2 into ammonia that, mostly, is assimilated by the plant [ 2 , 3 ]. Thanks to this symbiosis, legumes can grow in soils poor in nitrogen, which is important from both an ecological and an agricultural point of view. The inoculation of legumes with appropriate rhizobia can reduce or even eliminate the necessity of the application of nitrogen fertilizers (expensive and highly polluting), which makes the use of rhizobial inoculants very appropriate for sustainable agricultural practices [4,5]. The rhizobia–legume symbiotic interaction is characterized by being specific: each rhizobial strain can nodulate with a definite set of legumes (known as the nodulation range), which can be narrow or broad, and each legume species can be nodulated by a set of rhizobial strains [ 6 ]. This specificity relies on a complex molecular dialogue that is established between both symbionts [ 6 – 9 ]. Although there are exceptions [ 1 , 10 ], in most of the Int. J. Mol. Sci. 2021,22, 6233. https://doi.org/10.3390/ijms22126233 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021,22, 6233 2 of 27 rhizobia–legume symbiotic interactions this dialogue can be summarized as follows [ 9 , 10 ]: plant root exudates contain different nutrients that can feed rhizobia, including flavonoids, which, when appropriate and through the activation of the bacterial LysR regulator NodD, trigger the expression of bacterial nodulation genes and thus, the production and secretion of a set of different Nod factors. These rhizobial molecules are N-acetyl-glucosamine oligosaccharides harbouring different chemical decorations that, when appropriate, are perceived by LysM receptors present in root hair membranes, eliciting different responses necessary for bacterial infection and the start of the nodule organogenesis program. The most evolved rhizobial infection pathway is through infection threads (IT): briefly, following cytoskeletal reorganization, root hairs curve and trap rhizobial cells that previously adhered to their tips. These bacteria become enclosed in a kind of pocket wherein the action of lytic enzymes produced by both symbionts leads to local plant cell-wall degradation. At that point, root hair membrane invaginates and originates a channel, the IT, that will be used by rhizobia to penetrate into the root. These channels grow towards the root cortex, where, in parallel to the infection process, nodule organogenesis had started. Several cortical cells dedifferentiated into meristematic cells and gave rise to the formation of the nodule primordium. Later, certain cells inside the nodule differentiate into high-size, symbiotic cells through a process of endoreplication (replication of the genome in the absence of cell division). IT, which have continued to grow and have also branched out, eventually enter into the forming nodule and liberate rhizobia into the nodule endoreduplicated cells by endocytosis. Inside these cells, rhizobia differentiate into bacteroids, able to fix nitrogen. In addition to Nod factors, other rhizobial molecules play important roles in symbiotic interaction with legumes [ 7 , 8 ]. On the one hand, some rhizobia, such as S. fredii or B. japonicum , deliver effector proteins into plant cells through a type 3 secretion system [11] . These effector proteins mainly alter host signalling and suppress plant defences. On the other hand, different rhizobial surface polysaccharides participate in the nodulation process, most probably acting as signals required for the progression of the interaction and/or preventing plant defence mechanisms [ 7 , 8 , 12 , 13 ]. Rhizobia are Gram-negative bacteria, so they have an inner membrane (cytoplasmic membrane) and an outer membrane, separated by the periplasmic space that also contains the peptidoglycan layer. The main rhizobial surface polysaccharides studied so far are cyclic glucans (CG), lipopolysaccharides (LPS), capsular polysaccharides (CPS), K-antigen capsular polysaccharides (KPS) and exopolysaccharides (EPS). CG are cyclic homopolymers of glucose residues that can harbour different substitutions such as 1-phosphoglycerol or succinate. CG are mainly present in the periplasmic space but can also be secreted to the extracellular milieu. LPS are very complex glycolipid molecules located in the outer leaflet of the outer membrane of Gram-negative bacteria. KPS are acidic polysaccharides analogous to the group II of K-antigens described in Escherichia coli that, among rhizobia, are present in the Sinorhizobium (=Ensifer) genus and located, as LPS, in the outer leaflet of the outer membrane. In Rhizobium leguminosarum, the term “capsular polysaccharide” (CPS) refers to different polysaccharides, either neutral or acidic, that form a matrix surrounding the bacteria [ 14 ]. Finally, EPS are acidic polysaccharides secreted to the cellular environment and located on the cell surface. The symbiotic relevance of each of these polysaccharides notably varies depending on the symbiotic couple analysed, with the only exception of CG, which have been shown to be essential in all the rhizobia–legume interactions studied so far [8,15]. There are other rhizobial surface polysaccharides, and some of them may play a role in symbiosis, reviewed by [ 7 ]. In Rhizobium leguminosarum, glucomannan (a water-soluble surface heteropolymer, consisting of 95% of glucose and mannose and small amounts of galactose and rhamnose), also known as neutral polysaccharide, is essential for the attachment of this bacterium to a lectin present in Vicia sativa and Pisum sativum root hairs. Also in R. leguminosarum, a neutral gel-forming polysaccharide containing galactose, mannose, and glucose in a molar ratio 4:1:1 is formed in in vitro cultures at the late stationary phase, but, apparently, it does not play a role in symbiosis. Finally, cellulose Int. J. Mol. Sci. 2021,22, 6233 3 of 27 fibrils produced by rhizobia may participate in bacterial attachment to host root hairs and biofilm formation. 2. A General View of Rhizobial Exopolysaccharides As mentioned above, EPS are acidic polysaccharides located out of the cell with little or no cell association [ 8 ]. These polysaccharides perform different functions, such as protection against environmental stresses, attachment to biotic and abiotic surfaces, and nutrient gathering [ 7 , 16 ]. During interactions with plants, diverse functions have been proposed for EPS from both pathogenic and symbiotic bacteria reviewed by [ 13 ], such as the sequestration of calcium ions, ROS (reactive oxygen species) scavenging, prevention of cellulose-mediated cell agglutination, tolerance to acidic pH, participation in biofilm formation, and host surface attachment. In addition, there is evidence of the role of EPS as a key signal for rhizobial infection in several rhizobia–legume couples as Rhizobium leguminosarum bv. trifolii/Trifolium spp., Sinorhizobium meliloti/Medicago sativa and Mesorhizobium loti/Lotus japonicus [7,16–19]. Rhizobial EPS are complex and high-molecular-mass heteropolymers whose structure varies at the species level. The repeating units are constituted of a variable number of hexose and uronic acid residues showing either alpha or beta glycosidic linkages. They can also be either linear or side-branched. In addition to sugars, several non-carbohydrate substituents can appear, mostly succinate, pyruvate, or acetate, which contribute to the acidic character of this polysaccharide [ 8 , 16 , 20 ]. Figure 1shows the structure of the rhizobial EPS that will be treated in detail in this review. As it will be mentioned later, rhizobial EPS can be produced as either highor low-molecular-mass forms (HMM and LMM). Figure 1. EPS structures of different rhizobia [ 7 , 8 , 16 , 21 ]. ( a )R. leguminosarum bv. trifolii; ( b ) EPS I (succinoglycan) of S. meliloti; ( c ) EPS II (galactoglucan) of S. meliloti; ( d )M. loti; ( e )S. fredii. EPS structure is normally conserved at the species level but, in R. leguminosarum, it varies at the biovar level and in biovar trifolii, it may vary at the strain level. The structure shown in this figure is that found in most R. leguminosarum bv. trifolii strains, such as Rt42.2, RBL5599, and LPR5. Ac, acetate; Gal, D-galactose; GalA, D-galacturonic acid; Glc, D-glucose; GlcA, D-glucuronic acid; Pyr, pyruvate; RibA, D-riburonic acid; Suc, succinate. Int. J. Mol. Sci. 2021,22, 6233 4 of 27 Traditionally, the symbiotic relevance of rhizobial EPS has been related to the type of nodule ontogeny [ 16 , 17 ]. Thus, this polysaccharide appeared to be essential for indeterminate nodule-forming symbiosis, such as S. meliloti/M. sativa or R. leguminosarum bv. trifolii/Trifolium spp., but dispensable for determinate nodule-forming symbiosis, such as Sinorhizobium fredii/Glycine max and M. loti/L. japonicus. According to Stacey et al. [ 22 ] this difference may be due to the fact that the IT of indeterminate nodules are narrower than those of determinate nodules, and that the EPS-mediated matrix could be more important in the former ones. In this review, we summarize the current knowledge about rhizobial EPS, focusing on the complex regulation of its production and on the symbiotic relevance of this polysaccharide. For the sake of clarity, we will restrict this review to four rhizobial species where EPS has been studied in more detail: R. leguminosarum,S. meliloti,S. fredii, and M. loti. Supplementary Material Table S1 lists all of the genes (including regulatory ones) involved in EPS production that are mentioned in this work. These studies will show that the symbiotic importance of rhizobial EPS depends more on the specific couple analysed than on the type of nodule, determinate or indeterminate, formed by the host legume. 3. Rhizobium leguminosarum Rhizobium leguminosarum is a rhizobial species in which three biovars, trifolii,viciae and phaseoli, can be distinguished based on the type of legumes infected. Each of these biovars exhibit a narrow host-range. R. leguminosarum bv. trifolii strains establish symbiosis with Trifolium, bv. viciae with Pisum,Vicia,Lens, and Lathyrus, and bv. phaseoli with Phaseolus spp. plants. The symbiotic importance of EPS in R. leguminosarum has been related to the type of nodules formed by these host plants [ 6 , 7 , 23 , 24 ]. Trifolium,Pisum,Vicia,Lathyrus,Lens, belonging to the galegoid clade of subfamily Papilionoideae, form indeterminate nodules, whereas determinate nodules are formed by phaseolid legumes such as Phaseolus. It is generally accepted that, in general, the production of EPS is required for the effective symbiosis of R. leguminosarum trifolii and viciae strains with their host plants, which form indeterminate nodules, but not for bv. phaseoli strains, which establish symbiosis with bean plants forming determinate nodules. Despite these differences in the symbiotic importance of EPS, R. leguminosarum strains produce EPS showing similar, but not identical, structures [ 16 ] In most R. leguminosarum bv. trifolii strains (such as Rt42.2, RBL5599, LPR5, etc.), this polymer consists of octasaccharide repeating units which contain D-glucose, D-glucuronic acid and D-galactose in a molar ratio 5:2:1, joined by β -1,3 and β -1,4 glycosidic bonds, and are modified by non-sugar (acetyl and pyruvyl) groups (Figure 1), but in other strains (such as 4S), the galactose residue is not present [ 16 , 25 – 28 ]. In R. leguminosarum bv. viciae, the EPS repeating unit is very similar to the R. leguminosarum bv. trifolii octasaccharide but including an additional D-glucuronic acid residue [16,28]. Similarly to other microorganisms, EPS synthesis in rhizobia is a complex process requiring the coordinated activity of many enzymatic and transporter proteins [ 29 ]. Genes involved in the synthesis and secretion of rhizobial EPS are usually grouped in large clusters located on chromosomes or megaplasmids [ 30 – 33 ]. In the case of R. leguminosarum, a great majority of genes engaged in EPS production are located on a large chromosomal region, called Pss-I [ 34 – 37 ], that encompasses nearly 30 genes. This region is highly conserved among all the so far sequenced R. leguminosarum genomes, although differences in the genetic organization, due to rearrangements, can be found at the strain level [31,34,35,38–40] . Figure 2shows the Pss-I region from R. leguminosarum bv. trifolii Rt24.2. This clustering of genes involved in EPS synthesis most probably reflects their coordinated regulation of expression, including the influence of various environmental factors (light, root exudates, nitrogen and phosphate limitation, catabolite repression, stress conditions) [ 41 – 46 ]. The Pss-I region contains the exo5 gene, responsible for the synthesis of glucuronic acid (GlcA), genes encoding glycosyl transferases responsible for EPS subunit synthesis (pssCDE, pssJIFGH,pssS), genes involved in the addition of non-sugar modifications to EPS subunits Int. J. Mol. Sci. 2021,22, 6233 5 of 27 (pssRMK), and genes involved in EPS assembly and secretion (pssTNOP,pssL) ( Figure 2 ). The exo5 gene codes for a UDP-glucose dehydrogenase which converts UDP-glucose to UDP-GlcA, which, in turns, is the source for the synthesis of galacturonic acid (GalA). A mutant in this gene displays pleiotropic effects (i.e., changes in bacterial cell envelope and ineffective symbiosis), because its inability to synthesize uronic acids results in the absence of EPS and capsular polysaccharide (CPS) production and in the synthesis of an altered LPS that lacks GalA residues [14,47]. Figure 2. Genetic organization of EPS synthesis clusters found in R. leguminosarum bv. trifolii Rt24.2, S. meliloti 1021, S. fredii HH103 and NGR234, and M. loti R7A [30,35,48–50]. Some chromosomal genes crucial for EPS production, such as exoB and pssA, are located outside the Pss-I region. The exoB gene codes for a UDP-glucose 4-epimerase that synthesizes UDP-galactose, being a donor of this sugar in the synthesis of EPS and other galactose-containing polysaccharides [ 51 , 52 ]. An exoB mutant produces EPS lacking galactose residues and is unable to properly invade host roots (abnormal nodules inefficient in nitrogen fixation were formed on clover roots). Figure 3shows a putative model for the different steps of EPS subunit assembly and export in R. leguminosarum bv. trifolii strains in which this subunit is an octasaccharide containing D-glucose, D-glucuronic acid and D-galactose in a molar ratio 5:2:1. The pssA gene is involved in the first step of the EPS subunit assembly. This gene encodes a glucosyl-isoprenylphosphate transferase that transfers glucose-1-phosphate from UDP-glucose to the lipid carrier [ 53 ]. Mutations in pssA totally abolish EPS synthesis, leading to the induction of empty (without bacteria), non-nitrogen-fixing root nodules on host plants (clover, pea and vetch) [ 44 , 54 – 58 ]. The transcription of pssA in both ex planta and in planta conditions is at a low level, indicating that the expression of this gene, which is crucial for EPS synthesis, is under very stringent regulation [44,59–61]. Int. J. Mol. Sci. 2021,22, 6233 6 of 27 Figure 3. A model for EPS synthesis and export in R. leguminosarum bv. trifolii [ 29 , 35 , 36 , 53 , 55 , 62 – 73 ]. All pss genes involved in subsequent steps of the EPS subunit synthesis belong to the Pss-I region. The addition of two glucuronosyl residues to the growing subunit is performed by a glucuronosylβ -1,4-glucosyltransferase and a glucuronosylβ -1,4glucuronosyltransferase encoded by pssDE and pssC, respectively [ 36 , 53 , 55 , 62 ]. Interestingly, a pssD mutant shows very similar phenotypic traits as the pssA mutant (lack of EPS synthesis and non-nitrogen-fixing nodules induced on clover and vetch roots). The fourth step of the unit assembly is most probably conducted by PssS, which is a unique enzyme among R. leguminosarum glycosyltransferases engaged in the EPS synthesis because it generates an exclusive α -1,4-glucosyl bound [ 35 ]. However, there is little information about glycosyl transferases involved in the subsequent steps of EPS synthesis. Based on the CAZy database (Carbohydrate Active Enzymes), some pss genes located in the Pss-I region are predicted to be engaged in this process (pssF,pssG,pssH,pssI) [ 63 , 64 ]. As recently evidenced, pssJ codes for a galactosyl transferase that is involved in the addition of the terminal sugar residue to the octasaccharide subunits [ 65 ]. A ∆ pssJ deletion mutant produces EPS lacking terminal galactose in the side chain of the subunit (Figure 1). However, the lack of addition of this galactose residue does not block EPS polymerization. Three pss genes from the Pss-I region are involved in non-sugar modifications of the EPS structure (Figure 3); pssR codes for a putative acetyl transferase, whereas pssM and pssK code for ketal pyruvate transferases [ 66 ]. Mutation of pssM resulted in the absence of pyruvyl groups in the EPS subunits (at the sub-terminal glucose) (Figure 1) and the induction of non-nitrogen-fixing nodules on the host plant: in these nodules, the normal invasion and release of bacteria into plant cells were observed, but their differentiation into bacteroids was essentially impaired, revealing the essentiality of the addition of pyruvyl residues for the symbiotic function of this polysaccharide. The polymerization and secretion of EPS in R. leguminosarum are carried out by proteins encoded by the pssL and pssTNOP genes from the Pss-I cluster (Figure 3). Detailed information about these processes can be found in recent reviews [ 29 , 64 ]. Briefly, the polymerization of EPS subunits is performed by a Wzx/Wzy system that is composed of a Wzx-type translocase (flippase), a Wzy-type polysaccharide polymerase and a copolymerase. In R. leguminosarum, PssL is most probably a Wzx translocase involved in the transfer of the EPS subunits across the inner membrane. This type of polysaccharide transporter proteins (Wzx translocases) is characterized by a specific topology containing Int. J. Mol. Sci. 2021,22, 6233 7 of 27 numerous (10–14) predicted transmembrane (TM) segments. PssT, a Wzy-type protein containing 12 TMs, is the polysaccharide polymerase responsible for the polymerization of the EPS subunits [ 67 ]. PssP is another component of this system (PCP, polysaccharide co-polymerase) that is involved in the determination of the EPS chains length [ 68 ]. This protein has been classified as a bacterial tyrosine kinase [ 69 – 71 ]. PssP displays a significant identity to S. meliloti ExoP and other membrane-periplasmic auxiliary (OMA) proteins that are involved in the synthesis of HMM surface polysaccharides. Transport of EPS outside bacterial cells is conducted through a channel in the outer membrane formed by Wza-type proteins that are lipoproteins forming octameric α -helical channels spanning both inner and outer membranes [ 72 ]. In R. leguminosarum, PssN plays the role of this outer membrane translocase that is able to form homooligomeric structures and interact with PssP [73]. Other genes related to EPS synthesis are also located in the Pss-I region, such as prsDE (coding for components of a type 1 secretion system) and plyA (coding for a glycanase that cleaves EPS, affecting its processing) [ 35 , 62 , 74 ]. This secretion system shows an atypical broad substrate specificity, exporting at least 13 protein substrates (e.g., glycanases (PlyA, PlyB, and PlyC), rhizobial adhesion proteins (RapA2, RapB, and RapC), and the nodulation protein NodO) [ 75 – 78 ]. A prsD mutant synthesizes EPS of a higher polymerization degree than that of the wild-type strain and elicits a higher number of nodules incapable of fixing nitrogen [75]. Recently, the role in EPS synthesis of another gene belonging to the Pss-I region, pssZ, has been shown (Figure 2). This gene codes for a protein belonging to the bacterial serine/threonine protein phosphatases family, and its inactivation totally abolished EPS production in R. leguminosarum bv. trifolii [ 79 ]. Comparative transcriptomic analysis of the pssZ mutant and the wild-type strain identified a large number of genes differentially expressed in these two backgrounds, including genes related to several cellular processes (signalling, transcription regulation, synthesis of cell-surface polysaccharides, cell division and motility, bacterial metabolism) [ 61 ]. Interestingly, expression of almost all pss genes located in the Pss-I region was totally inhibited in the pssZ mutant. These data confirmed the important role of the serine/threonine protein phosphatase PssZ in EPS synthesis, most probably via its participation in a regulatory network [ 71 ]. In bacteria, in addition to twocomponent signalling systems, alternative regulatory pathways controlled by Hanks-type serine/threonine kinases and serine/threonine phosphatases also play an essential role in regulation of many processes, such as growth and cell division, cell-wall biogenesis, sporulation, biofilm formation, stress response, and metabolic and developmental processes, as well as either symbiotic or pathogenic interactions with higher host organisms [ 70 , 80 ]. In fact, the pssZ mutation resulted in pleiotropic effects in rhizobial cells: a lack of EPS production, decreased growth kinetics and motility, altered cell-surface properties, and failure to infect the host plant. The global regulator RosR is involved in the positive regulation of EPS synthesis in R. leguminosarum. This protein is encoded by the chromosomal rosR gene that is not linked to the Pss-I region [ 81 , 82 ] and that is present in the genomes of all strains belonging to the three R. leguminosarum biovars and the closely related species R. etli and R. gallicum [83,84]. R. leguminosarum rosR also exhibits a significant similarity with A. tumefaciens ros [ 85 ], mucR of S. meliloti [ 86 ] and S. fredii [ 87 , 88 ], as well as with mucR of pathogenic Brucella abortus [ 89 ]. All these genes code for transcriptional regulators belonging to the α -proteobacteria Ros/MucR family of zinc-finger proteins which regulate the expression of genes required for the successful interactions of these bacteria with eukaryotic hosts. The R. leguminosarum bv. trifolii rosR mutant produces substantially less EPS (including a decreased LMM to HMM ratio) than the wild-type strain and establishes impaired symbiosis with clover plants [ 81 , 90 ]. In addition, alterations in the polysaccharide constituent of LPS, changes in membrane and secreted proteins, and decreased motility were observed in the rosR mutant [ 90 , 91 ]. On the other hand, additional rosR copies (similarly to multiple pssA copies) resulted in a nearly twofold increase of EPS synthesis and enhanced nodulation and symbiotic effectiveness [ 92 ]. Transcriptome profiling of the rosR mutant revealed the addi- Int. J. Mol. Sci. 2021,22, 6233 8 of 27 tional role of RosR in motility, synthesis of cell-surface components, carbon and nitrogen transport and metabolism, and other cellular processes [ 37 ]. Among a large group of genes (1106) differentially transcribed in the rosR mutant, a majority (63%) were up-regulated (suggesting that RosR functions mainly as a negative regulator), including prsD,rapA1 (autoaggregation protein), ndvA (CG transport protein to the periplasm), and genes of various transcriptional regulators (i.e., nitrogen regulatory protein P-II, phosphate regulatory system PhoBR, LacI, LuxR-type regulator RaiR). In contrast, several genes involved in EPS synthesis (pssA,pssC,pssI, and pssS) and cell motility (including the regulator rem and several genes coding for flagellar proteins) were down-regulated in the rosR mutant. This finding suggests the participation of RosR in a complex regulatory network. Regulation of rosR expression is very complex and involves four regulatory proteins: RosR, CRP, NodD, and PhoB, which modulate rosR transcription in response to various environmental factors (phosphate limitation, flavonoids, carbon source) [ 81 , 82 , 93 ] (Figure 4). A high rosR expression is ensured by the action of its strong promoter and two additional (upstream promoter and TGN-extended − 10) elements [ 82 , 94 ]. Moreover, other sequence motifs (RosR-box bound by RosR, a LysR motif recognized by NodD, cAMP-CRP, PHO boxes recognized by PhoB, and numerous inverted repeats important for rosR mRNA stability) are engaged in the regulation of rosR expression [ 81 , 82 , 93 , 94 ]. Regarding the effect of the C source, a slight repression of the rosR gene is observed in the presence of glucose but not in that of glycerol [81]. Some environmental factors (root exudates, phosphate limitation, and nitrogen starvation) positively affect the transcription of pssA,pssB,pssO and pssP [ 60 , 95 ]. Flavonoids and NodD enhance EPS synthesis in R. leguminosarum through the activation of rosR [ 93 ], suggesting linked positive regulation of the synthesis of both symbiotic signals (EPS and Nod factors) [96], as also happens in S. meliloti. Figure 4. Cont. Int. J. Mol. Sci. 2021,22, 6233 9 of 27 Figure 4. Regulation of EPS synthesis in different rhizobia. ( a )R. leguminosarum; (b)S. meliloti ; (c)S. fredii HH103. Details and references used are provided in the text. For each species, not all the regulatory proteins showed in the figure may be present in all strains. In R. leguminosarum, Lov-HK is present in some strains of bvs. viciae and trifolii, whereas psi and psrA are only present in bv. phaseoli. In S. meliloti ,expR is disrupted in strain 1021, avoiding the regulation of EPS production by QS. In the case of S. fredii, the positive effect of NolR and the negative effect of NodD1 and flavonoids on EPS production have only been proven for strain HH103. There are different regulatory proteins (ExoR, Psi, PsrA, ExpR, and PssB) that have a negative effect on EPS production in R. leguminosarum. ExoR is an orthologue of S. meliloti ExoR and is involved in pssA repression [ 44 , 58 ]. An exoR mutant produces 3-fold more EPS than the wild-type strain and induces the formation of both effective and ineffective nodules on the host plant [ 97 ]. The pssB gene is located upstream of pssA and negatively affects EPS production, although its precise role in this process remains to be established. pssB encodes an inositol monophosphate phosphatase, and mutants in this gene produce more EPS than the wild-type R. leguminosarum bv. trifolii and viciae strains and induce non-nitrogen-fixing nodules on their host plants [ 82 , 98 – 100 ]. The psi (a polysaccharide inhibition) and psrA Int. J. Mol. Sci. 2021,22, 6233 16 of 27 In contrast to that found in R. leguminosarum and S. meliloti,S. fredii HH103 EPS production is not influenced by QS mechanisms since this strain lacks a functional copy of the expR gene [ 165 ]. However, the two QS systems identified in strain NGR234 have a positive effect on EPS production and repress genes involved in motility and chemotaxis [166]. The S. fredii NGR234 EPS has revealed to be indispensable for the formation of nitrogenfixing nodules with several host-plants, such as Albizia lebbeck and Leucaena leucocephala, but the lack of this polysaccharide did not affect symbiosis with other hosts, such as cowpea (Vigna unguiculata). Interestingly, the inactivation of the NGR234 exoK gene, coding for a glycanase, resulted in a lack of production of EPS-derived oligosaccharides and in the same symbiotic phenotype as EPS - mutants, indicating that, as described previously for S. meliloti, these NGR234 exo-oligosaccharides are crucial for symbiosis with certain host plants [ 167 ]. The symbiotic role of HH103 EPS has been studied in different host plants, either forming determinate or indeterminate nodules, and in no case has this polysaccharide has proven to be crucial. In determinate-nodule-forming legumes, the absence of HH103 EPS has different effects in symbiosis depending on the host legume: positive with soybean (slight increase in plant-top dry weight and significant increase in competitiveness ability), neutral with Lotus burttii (no differences in plant development) or negative with cowpea (slight decrease in plant-top dry weight and significant decrease in competitiveness ability) [ 48 , 168 – 170 ]. The fact that EPS appears to be slightly detrimental for the symbiosis of HH103 with its natural host, soybean, opens the question of whether EPS repression through nod-geneinducing flavonoids is a mechanism for optimizing symbiosis with this legume. Regarding indeterminate-nodule-forming legumes, the HH103 exoA mutant induces the formation of nitrogen-fixing nodules with a very similar performance to those of the parental strain, on pigeon-pea (Cajanus cajan) and Glycyrrhiza uralensis [168,171]. The last legume belongs, as Medicago, to the IRLC clade [ 172 ]. In S. meliloti, EPS can be functionally replaced by KPS for effective symbiosis with alfalfa [ 139 ]. However, a double exoA rkpH mutant (producing no EPS nor KPS), although negatively affected when compared to HH103, is still able to induce the formation of nitrogen-fixing nodules on both pigeon pea and G. uralensis [ 168 , 171 ]. This fact is a good example of how “rules” (strict requirement of either EPS or KPS for effective symbiosis with an indeterminate nodule-forming legume) extracted for a model symbiotic system (S. meliloti/alfalfa) cannot always be applied to other symbiotic relations. In free-living HH103 cells, EPS has proven to be essential for biofilm formation on abiotic surfaces [ 48 , 159 ], but it is not implied in attachment to soybean roots. In fact, and in contrast to Bradyrhizobium diazoefficiens USDA110, HH103 EPS (as in all S. fredii strains described so far) is unable to bind to soybean lectin. Regarding other physiological traits, the absence of EPS impairs the low surface motility exhibited by S. fredii HH103 and negatively affects tolerance to osmotic stress (either 50–100 mM NaCl or 5–10% sucrose m/v) but has no effect on sensitivity to the oxidizing agents H2O2or paraquat [48]. 6. Mesorhizobium loti The Mesorhizobium loti R7A and MAFF303099 strains, recently reclassified as M. japonicum [173], were isolated from Lotus corniculatus and Lotus japonicus nodules, respectively [174,175]. Both strains have been extensively studied since they are able to induce nitrogen-fixing nodules on the model legume plant L. japonicus Gifu [ 176 , 177 ]. The M. loti MAFF303099 and R7A genomes have been sequenced [ 178 , 179 ]; in both strains, the symbiotic genes are grouped in the symbiosis island, a mobilizable 500 kb DNA region able to make a non-symbiotic mesorhizobia into a Lotus symbiont. The symbiosis islands of these strains share a conserved backbone sequence of 248 kb with about 98% DNA sequence identity [ 180 ]. Interestingly, a comparison of the genomes of these two strains has shown that they have different secretion systems to introduce protein effectors into the plant cells. While a T4SS is present in R7A, the MAFF303099 strain possesses a T3SS, albeit both secretion systems seem to be involved in the determination of the host range and would be functionally exchangeable [180,181]. Int. J. Mol. Sci. 2021,22, 6233 17 of 27 As has been described by Kelly et al. [ 50 ], M. loti R7A produces both HMM and LMM forms of EPS. The repeating subunit consists of a highly O-acetylated octosaccharide composed of D-glucose, D-galactose, D-GlcA and D-riburonic acid in a 5:1:1:1 ratio ( Figure 1 ). The presence of the riburonic acid as a terminal residue has only previously been reported in S. meliloti IFO13336, but in the case of this strain, it is α -D-Rib and not β -D-Rib as occurs in R7A [21]. The genes responsible for EPS production in M. loti R7A and MAFF303099 are clustered in the chromosome (Figure 2), similarly as in R. leguminosarum strains. The cluster exoQFYXUKTLAMONP (from mlr5249 to mlr5276) genes is present in both strains, whereas other genes that may be involved in EPS biosynthesis, such as possible orthologues of exoI (mll0560, mll8119 and mlr0479), exoZ (mlr6758 and mlr8032) and exoB (mll7878) are dispersed in the genome [ 178 ]. Although the acetylation of the M. loti R7A EPS was found in three out of six possible sites, the gene responsible for this substitution remains to be elucidated since, as mentioned, there are two different genes showing high identity to the S. meliloti 1021 exoZ gene, mlr6758 and mlr8032 [21]. To our knowledge, specific studies designed to analyse the regulation of the M. loti EPS have not been published. In contrast, different studies have been focused on the symbiotic role of this polysaccharide in different interactions, such as those of M. loti NZP2037 with Leucaena leucocephala and Lotus pedunculatus [ 182 ], M. loti MAFF303099 with L. japonicus B-129 [ 183 ], and M. loti R7A with L. corniculatus and L. japonicus Gifu [ 50 ]. In the case of M. loti NZP2037, the symbiotic phenotype of EPS-deficient mutants depended on the host plant: these mutants induced effective nodules with L. pedunculatus (a determinatenodule-forming legume) but empty nodules unable to fix nitrogen with L. leucocephala (which forms indeterminate nodules). In the case of M. loti MAFF303099, mutants unable to produce EPS showed the same symbiotic phenotype as the wild-type strain in symbiosis with L. japonicus B-129. The most recent studies regarding the symbiotic relevance of M. loti EPS have been carried out on the R7A strain. In that strain, the symbiotic response with L. japonicus Gifu and L. corniculatus plants differs depending on the EPS biosynthesis step affected by the gene mutation. While those mutants affected in the early steps of the EPS production (such as exoA and exoB) were able to form effective nodules (although in the case of the exoA mutant, the number of nitrogen-fixing nodules induced on L. japonicus Gifu was clearly reduced), the mutants affected in the middle to late steps of EPS synthesis were clearly impaired on both plants with more or less severity depending on the mutant tested (exoO,exoU,exoK, mlr5265 and mlr5266) [ 50 ]. Based on these results, the M. loti EPS would act as a positive signal to modulate the plant response and carry out a normal rhizobial invasion (IT formation and bacterial release). Bacterial mutants that are affected at the early steps of the EPS production lacked this polysaccharide but were able to avoid the defence responses and induce effective nodules although with a delay. The more severe symbiotic impairment phenotypes were shown by bacterial mutants producing a truncated EPS that may be recognised as a negative signal that would activate plant defence responses and, therefore, disrupt IT progression [50]. The molecular basis of the results commented above was supported by the recent finding of the L. japonicus Gifu transmembrane LysM receptor kinase, EPR3. This receptor is able to recognise and bind monomeric EPS molecules, thus monitoring the bacterial EPS status during the colonization and infection processes [ 18 ]. The recognition of the M. loti wild-type EPS by the EPR3 receptor acts as a positive signal allowing epidermal infection. However, the truncated EPS form (a penta-glycan) produced by a M. loti exoU mutant is recognised as a non-compatible EPS, and this negative signal results in the arrest of IT formation and the absence of the formation of nitrogen-fixing nodules. In the case of an exoB mutant that does not produce EPS, the absence of a negative signal allows infection and the development of nitrogen-fixing nodules but to a lower extent than in the case of the wild-type strain because of the lack of the positive signal (recognition of the appropriate EPS by EPR3). Int. J. Mol. Sci. 2021,22, 6233 18 of 27 Thus, the presence of this EPS receptor establishes a double checkpoint in the infection process of M. loti with L. japonicus Gifu plants. The M. loti R7A Nod factors are recognised by the NFR1 and NFR5 receptors and trigger the symbiotic signal transduction leading to the early steps of infection and nodule devolvement [ 184 , 185 ]. At the first steps of this signal cascade, the epr3 gene is expressed, giving rise to EPS monitoring on the root infection zone and controlling the epidermal infection [ 18 ]. Recent results show that EPS3 is also expressed at the root cortex and nodule primordia, suggesting that EPS recognition is reiterated during symbiotic interaction, and it is also involved in intercellular cortical infection [ 19 ]. Recently, it has been shown that EPR3-like receptors, which present a structure that is quite different from that of LysM receptors involved in chitooligosaccharide (NF) perception, are ubiquitous in plants, suggesting that they might be surveillance receptors monitoring carbohydrates from different microbes associated with plant roots [ 186 ]. However, in the indeterminate-nodule-forming-legume M. truncatula, the orthologue of eps3 (Mtlyk10), although playing a relevant role in symbiosis, appears to be uninvolved in the perception of the S. meliloti EPS I [187]. 7. Conclusions and Perspectives In this review we have tried to present the current knowledge about one of the most studied rhizobial surface polysaccharides, the EPS. Currently, we can point out the following relevant facts: (1) Rhizobial EPS production has been found to be extremely complex in all the rhizobial strains in which this issue has been addressed. Usually, it involves the participation of different regulatory systems, either positive or negative ones, including the participation of global regulatory proteins and, in some cases, that of QS systems. (2) In many rhizobia-legume symbioses, EPS is absolutely essential or very important for a successful interaction. This is especially true in symbioses forming indeterminate nodules, although the interactions between S. fredii HH103 and G. uralensis and C. cajan are a clear exception. Also, the fact that EPS plays a very important role in the success of rhizobial infection in the M. loti/L. japonicus couple, which forms determinate nodules, makes it difficult to formulate a general rule. Interestingly, EPS appears to be crucial for the symbiotic performance of several rhizobia exhibiting a narrow host range (such as S. meliloti,R. leguminosarum, and M. loti). (3) In some rhizobia, EPS production is connected with the nod regulon, but the type of connection (positive or negative) varies depending on the rhizobial strain. Thus, in S. meliloti and R. leguminosarum bv. trifolii, appropriate flavonoids induce Nod factor production and stimulates EPS biosynthesis, which is enhanced in parallel to that of Nod factors, whereas in S. fredii HH103, appropriate flavonoids induce Nod factor production but suppress EPS biosynthesis. (4) Rhizobial EPS, in addition to being crucial for biofilm formation and for passive protection against abiotic stresses, may play a crucial role as a molecular signal required for suppressing plant defence responses and, thus, allowing a successful infection process. There is clear evidence that, at least in S. meliloti,R. leguminosarum bv. trifolii and S. fredii NGR234, this signalling function is carried out by oligosaccharides derived from EPS. The recent discovery of the Lotus japonicus EPR3 receptor as a detector of the presence of the “correct” oligosaccharides for granting a successful infection process supports this idea. However, in this context, it is surprising that S. fredii HH103, a broad host-range rhizobial strain, represses the production of its EPS in the presence of any nod genes inducing flavonoids and, thus, in the early steps of its symbiotic interactions with any potential host legume. Might this repression be a strategy for nodulating potential host legumes that would stop infection when they perceive the HH103 EPS to inadequate? Clearly, more research is required to elucidate this issue. (5) Rhizobial EPS production may be interconnected with the biosynthesis of other symbiotic relevant surface polysaccharides. For example, in R. leguminosarum bv. Int. J. Mol. Sci. 2021,22, 6233 19 of 27 trifolii and S. fredii, the synthesis of EPS and LPS are interconnected through the rkp-2 region, whereas in S. meliloti, this genetic region participates in the production of KPS and LPS. At least in S. fredii, the production of EPS and extracellular CG is simultaneously controlled by the MucR1 regulator. (6) Rhizobial EPS symbiotic function can overlap with that of other surface polysaccharides, such KPS in the case of the interaction between S. meliloti and Medicago. However, this is not a general rule, as S. fredii mutants lacking EPS and KPS can still induce the formation of nitrogen-fixing nodules on G. uralensis and C. cajan. In summary, EPS is a symbiotically relevant surface polysaccharide in most of the rhizobia–legume symbiotic interactions studied so far, and its production is complexly regulated together with that of other bacterial relevant characteristics, such as motility. However, the classical belief stating that EPS are specifically essential in interactions with indeterminate-nodule-forming legumes is not fully true, as demonstrated by the fact that they can also be highly important in determinate-nodule-forming interactions (such as M. loti/L. japonicus) and dispensable in the symbiosis between S. fredii and G. uralensis, which forms indeterminate nodules. Thus, in our opinion, the symbiotic importance of EPS cannot be predicted but must be analysed in every single rhizobial–legume interaction. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/ijms22126233/s1. Author Contributions: All authors (S.A.-J., F.F.-R., J.-E.R.-S., M.J., J.-M.V.) contributed to the manuscript writing and figures design. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Polish National Science Centre, grant number 2018/31/B/NZ9/00663 and by the project PID2019-107634RB-I00 from the Spanish “Ministerio de Ciencia, Innovación y Universidades”. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest. Abbreviations AHL N-acyl-homoserine lactone(s) CG Cyclic glucans CPS Capsular polysaccharide EPS Exopolysaccharide GlcA Glucuronic acid GalA Galacturonic acid HMM High molecular mass IRLC Inverting repeat-lacking clade IT Infection thread(s) KPS K-antigen capsular polysaccharide LMM Low molecular mass LOV-domain Light, oxygen, and voltage domain LPS Lipopolysaccharide QS Quorum sensing TM Transmembrane References 1. Poole, P.; Ramachandran, V.; Terpolilli, J. Rhizobia: From saprophytes to endosymbionts. Nat. Rev. Microbiol. 2018 ,16, 291–303. [CrossRef] [PubMed] 2. Tsyganova, A.V.; Kitaeva, A.B.; Tsyganov, V.E. Cell differentiation in nitrogen-fixing nodules hosting symbiosomes. Funct. Plant Biol. 2017,45, 47–57. [CrossRef] [PubMed] 3. Coba de la Peña, T.; Fedorova, E.; Pueyo, J.J.; Lucas, M.M. The symbiosome: Legume and rhizobia co-evolution toward a nitrogen-fixing organelle? Front. Plant Sci. 2018,8, 2229. [CrossRef] [PubMed] Int. J. Mol. Sci. 2021,22, 6233 20 of 27 4. Oldroyd, G.E.D.; Dixon, R. Biotechnological solutions to the nitrogen problem. Curr. Opin. Biotechnol. 2014 ,26, 19–24. [CrossRef] 5. Santos, M.S.; Nogueira, M.A.; Hungria, M. Microbial inoculants: Reviewing the past, discussing the present and previewing an outstanding future for the use of beneficial bacteria in agriculture. AMB Express 2019,9, 205. [CrossRef] 6. Walker, L.; Lagunas, B.; Gifford, M.L. Determinants of host range specificity in legume-rhizobia symbiosis. Front. Microbiol. 2020 , 11, 3028. [CrossRef] 7. Janczarek, M.; Rachwał, K.; Marzec, A.; Grzadziel, J.; Palusi´nska-Szysz, M. Signal molecules and cell-surface components involved in early stages of the legume-rhizobium interactions. Appl. Soil Ecol. 2015,85, 94–113. [CrossRef] 8. López-Baena, F.J.; Ruiz-Sainz, J.E.; Rodríguez-Carvajal, M.A.; Vinardell, J.M. Bacterial molecular signals in the Sinorhizobium fredii-soybean symbiosis. Int. J. Mol. Sci. 2016,17, 755. [CrossRef] 9. Oldroyd, G.E. Speak, friend, and enter: Signalling systems that promote beneficial symbiotic associations in plants. Nat. Rev. Microbiol. 2013,11, 252–263. [CrossRef] 10. Roy, S.; Liu, W.; Nandety, R.S.; Crook, A.; Mysore, K.S.; Pislariu, C.I.; Frugoli, J.; Dickstein, R.; Udvardi, M.K. Celebrating 20 Years of Genetic Discoveries in Legume Nodulation and Symbiotic Nitrogen Fixation. Plant Cell 2020,32, 15–41. [CrossRef] 11. Staehelin, C.; Krishnan, H.B. Nodulation outer proteins: Double-edged swords of symbiotic rhizobia. Biochem. J. 2015 , 470, 263–274. [CrossRef] 12. Downie, J.A. The roles of extracellular proteins, polysaccharides and signals in the interactions of rhizobia with legume roots. FEMS Microbiol. Rev. 2010,34, 150–170. [CrossRef] 13. Wanke, A.; Malisic, M.; Wawra, S.; Zuccaro, A. Unraveling the sugar code: The role of microbial extracellular glycans in plant-microbe interactions. J. Exp. Bot. 2021,72, 15–35. [CrossRef] 14. Laus, M.C.; Logman, T.J.; van Brussel, A.A.N.; Carlson, R.W.; Azadi, P.; Gao, M.; Kijne, J.W. Involvement of exo5 in production of surface polysaccharides in Rhizobium leguminosarum and its role in nodulation of Vicia sativa subsp. nigra.J. Bacteriol. 2004 , 186, 6617–6625. [CrossRef] 15. Crespo-Rivas, J.C.; Margaret, I.; Hidalgo, A.; Buendía-Clavería, A.M.; Ollero, F.J.; López-Baena, F.J.; del Socorro Murdoch, P.; Rodríguez-Carvajal, M.A.; Soria-Díaz, M.E.; Reguera, M.; et al. Sinorhizobium fredii HH103 cgs mutants are unable to nodulate determinateand indeterminate nodule-forming legumes and overproduce an altered EPS. Mol. Plant Microbe Interact. 2009 , 22, 575–588. [CrossRef] 16. Fraysse, N.; Couderc, F.; Poinsot, V. Surface polysaccharide involvement in establishing the rhizobium-legume symbiosis. Eur. J. Biochem. 2003,270, 1365–1380. [CrossRef] 17. Jones, K.M.; Kobayashi, H.; Davies, B.W.; Taga, M.E.; Walker, G.C. How rhizobial symbionts invade plants: The SinorhizobiumMedicago model. Nat. Rev. Microbiol. 2007,5, 619–633. [CrossRef] 18. Kawaharada, Y.; Kelly, S.; Nielsen, M.W.; Hjuler, C.T.; Gysel, K.; Muszynski, A.; Carlson, R.W.; Thygesen, M.B.; Sandal, N.; Asmussen, M.H.; et al. Receptor-mediated exopolysaccharide perception controls bacterial infection. Nature 2015 , 523, 308–312. [CrossRef] 19. Kawaharada, Y.; Nielsen, M.; Kelly, S.; James, E.; Andersen, K.; Rasmussen, S.; Füchtbauer, W.; Madsen, L.; Heckmann, A.; Radutoiu, S.; et al. Differential regulation of the Epr3 receptor coordinates membrane-restricted rhizobial colonization of root nodule primordia. Nat. Commun. 2017,8, 14534–14545. [CrossRef] 20. Becker, A.; Pühler, A. Production of exopolysaccharides. In The Rhizobiaceae. Molecular Biology of Model Plant-Associated Bacteria; Spaink, H.P., Kondorosi, A., Hooykaas, J.J., Eds.; Springer: Dordrecht, The Netherlands, 1998; pp. 97–118. [CrossRef] 21. Muszy´nski, A.; Heiss, C.; Hjuler, C.T.; Sullivan, J.T.; Kelly, S.J.; Thygesen, M.B.; Stougaard, J.; Azadi, P.; Carlson, R.W.; Ronson, C.W. Structures of exopolysaccharides involved in receptor-mediated perception of Mesorhizobium loti by Lotus japonicus.J. Biol. Chem. 2016,291, 20946–20961. [CrossRef] 22. Stacey, G.; So, J.S.; Roth, L.E.; Lakshmi, S.K.B.; Carlson, R.W. A lipopolysaccharide mutant of Bradyrhizobium japonicum that uncouples plant from bacterial differentiation. Mol. Plant Microbe Interact. 1991,4, 332–340. [CrossRef] [PubMed] 23. Maroti, G.; Kondorosi, E. Nitrogen-fixing Rhizobium-legume symbiosis: Are polyploidy and host peptide-governed symbiont differentiation general principles of endosymbiosis? Front. Microbiol. 2014,5, 326. [CrossRef] [PubMed] 24. Ferguson, B.J.; Indrasumunar, A.; Hayashi, S.; Lin, M.H.; Lin, Y.H.; Reid, D.E.; Gresshoff, P.M. Molecular analysis of legume nodule development and autoregulation. J. Integr. Plant Biol. 2010,52, 61–76. [CrossRef] [PubMed] 25. Robertson, B.K.; Aman, P.; Darvill, A.G.; McNeil, M.; Albersheim, P. Host-symbiont interactions. The structure of acidic extracellular polysaccharides secreted by Rhizobium leguminosarum and Rhizobium trifolii.Plant Physiol. 1981,67, 389–400. [CrossRef] 26. Breedveld, M.W.; Canter Cremers, H.C.J.; Batley, M.; Posthumus, M.A.; Zevenhuizen, L.P.T.M.; Wijfelman, C.A.; Zehnder, A.J.B. Polysaccharide synthesis in relation to nodulation behaviour of Rhizobium leguminosarum.J. Bacteriol. 1993 , 175, 750–757. [CrossRef] 27. McNeil, M.; Darvill, J.; Darvill, A.; Albersheim, P.; van Veen, R.; Hooykaas, P.; Schilperoort, R.; Dell, A. The discernible structural features of the acidic exopolysaccharides secreted by different Rhizobium species are the same. Carbohydr. Res. 1986 , 146, 307–326. [CrossRef] 28. Spaink, H.P. Root nodulation and infection factors produced by rhizobial bacteria. Annu. Rev. Microbiol. 2000 , 54, 257–288. [CrossRef] 29. Schmid, J. Recent insights in microbial exopolysaccharide biosynthesis and engineering strategies. Curr. Opin. Biotechnol. 2018 , 53, 130–136. [CrossRef] Int. J. Mol. Sci. 2021,22, 6233 21 of 27 30. Finan, T.M.; Weidner, S.; Womg, K.; Buhrmester, J.; Chain, P.; Vorhölter, F.J.; Hernandez-Lucas, I.; Becker, A.; Cowie, A.; Gouzy, J.; et al. The complete sequence of the 1,683-kb pSymB megaplasmid from the N 2 -fixing endosymbiont Sinorhizobium meliloti.Proc. Natl. Acad. Sci. USA 2001,98, 9889–9894. [CrossRef] 31. Young, J.P.W.; Crossman, L.C.; Johnston, A.W.B.; Thomson, N.R.; Ghazoui, Z.F.; Hull, K.H.; Wexler, M.; Curson, A.R.; Todd, J.D.; Poole, P.S.; et al. The genome of Rhizobium leguminosarum has recognizable core and accessory components. Genome Biol. 2006 , 7, R34. [CrossRef] 32. González, V.; Santamaría, R.I.; Bustos, P.; Hernández-González, I.; Medrano-Soto, A.; Moreno-Hagelsieb, G.; Janga, S.C.; Ramírez, M.A.; Jiménez-Jacinto, V.; Collado-Vides, J.; et al. The partitioned Rhizobium etli genome: Genetic and metabolic redundancy in seven interacting replicons. Proc. Natl. Acad. Sci. USA 2006,103, 3834–3839. [CrossRef] 33. Kaneko, T.; Nakamura, Y.; Sato, S.; Minamisawa, K.; Uchiumi, T.; Sasamoto, S.; Watanabe, A.; Idesawa, K.; Iriguchi, M.; Kawashima, K.; et al. Complete genomic sequence of nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum USDA110. DNA Res. 2002,9, 225–256. [CrossRef] 34. Król, J.E.; Mazur, A.; Marczak, M.; Skorupska, A. Syntenic arrangements of the surface polysaccharide biosynthesis genes in Rhizobium leguminosarum.Genomics 2007,89, 237–247. [CrossRef] 35. Janczarek, M.; Rachwał, K.; Kopci´nska, J. Genetic characterization of the Pss region and the role of PssS in exopolysaccharide production and symbiosis of Rhizobium leguminosarum bv. trifolii with clover. Plant Soil 2015,396, 257–275. [CrossRef] 36. Janczarek, M.; Rachwal, K.; Turska-Szewczuk, A. A mutation in pssE affects exopolysaccharide synthesis by Rhizobium leguminosarum bv. trifolii, its surface properties, and symbiosis with clover. Plant Soil 2017,417, 331–347. [CrossRef] 37. Rachwał, K.; Matczy´nska, E.; Janczarek, M. Transcriptome profiling of a Rhizobium leguminosarum bv. trifolii rosR mutant reveals the role of the transcriptional regulator RosR in motility, synthesis of cell-surface components, and other cellular processes. BMC Genom. 2015,16, 1111. [CrossRef] 38. Reeve, W.; O’Hara, G.; Chain, P.; Ardley, J.; Bräu, L.; Nandesena, K.; Tiwari, R.; Copeland, A.; Nolan, M.; Han, C.; et al. Complete genome sequence of Rhizobium leguminosarum bv. trifolii strain WSM1325, an effective microsymbiont of annual Mediterranean clovers. Stand. Genom. Sci. 2010,2, 347–356. [CrossRef] 39. Reeve, W.; O’Hara, G.; Chain, P.; Ardley, J.; Bräu, L.; Nandesena, K.; Tiwari, R.; Malfatti, S.; Kiss, H.; Lapidus, A.; et al. Complete genome sequence of Rhizobium leguminosarum bv. trifolii strain WSM2304, an effective microsymbiont of the South American clover Trifolium polymorphum.Stand. Genom. Sci. 2010,2, 66–76. [CrossRef] 40. Sadykov, M.R.; Ivashina, T.V.; Kanapin, A.A.; Shlyapnikov, M.G.; Ksenzenko, V.N. Structural and functional organization of the exopolysaccharide biosynthesis genes in Rhizobium leguminosarum bv. viciae VF39. Mol. Biol. 1998,32, 665–671. 41. Bonomi, H.R.; Posadas, D.M.; Paris, G.; Carrica, M.D.; Frederickson, M.; Pietrasanta, L.I.; Bogomolni, R.A.; Zorreguieta, A.; Goldbaum, F.A. Light regulates attachment, exopolysaccharide production, and nodulation in Rhizobium leguminosarum through a LOV-histidine kinase photoreceptor. Proc. Natl. Acad. Sci. USA 2012,109, 12135–12140. [CrossRef] 42. Cie´sla, J.; Kopyci´nska, M.; Łukowska, M.; Bieganowski, A.; Janczarek, M. Surface properties of wild-type Rhizobium leguminosarum bv. trifolii strain 24.2 and its derivatives with different extracellular polysaccharide content. PLoS ONE 2016 , 11, e0165080. [CrossRef] 43. Kopyci´nska, M.; Lipa, P.; Cie´sla, J.; Kozieł, M.; Janczarek, M. Extracellular polysaccharide protects Rhizobium leguminosarum cells against zinc stress in vitro and during symbiosis with clover. Environ. Microbiol. Rep. 2018,10, 355–368. [CrossRef] [PubMed] 44. Janczarek, M.; Urbanik-Sypniewska, T. Expression of the Rhizobium leguminosarum bv. trifolii pssA gene involved in exopolysaccharide synthesis is regulated by RosR, phosphate and the carbon source. J. Bacteriol. 2013 ,195, 3412–3423. [CrossRef] [PubMed] 45. Janczarek, M.; Rachwał, K.; Cie´sla, J.; Ginalska, G.; Bieganowski, J. Production of exopolysaccharide by Rhizobium leguminosarum bv. trifolii and its role in bacterial attachment and surface properties. Plant Soil 2015,388, 211–227. [CrossRef] 46. Jaszek, M.; Janczarek, M.; Kuczy´nski, K.; Piersiak, T.; Grzywnowicz, K. The response of the Rhizobium leguminosarum bv. trifolii wild-type and exopolysaccharide-deficient mutants to oxidative stress. Plant Soil 2014,376, 75–94. [CrossRef] 47. Muszy´nski, A.; Laus, M.; Kijne, J.W.; Carlson, R.W. The structures of the lipopolysaccharides from Rhizobium leguminosarum RBL5523 and its UDP-glucose dehydrogenase mutant (exo5). Glycobiology 2011,21, 55–68. [CrossRef] 48. Rodriguez-Navarro, D.N.; Rodriguez-Carvajal, M.A.; Acosta-Jurado, S.; Soto, M.J.; Margaret, I.; Crespo-Rivas, J.C.; Sanjuan, J.; Temprano, F.; Gil-Serrano, A.; Ruiz-Sainz, J.E.; et al. Structure and biological roles of Sinorhizobium fredii HH103 exopolysaccharide. PLoS ONE 2014,9, e115391. [CrossRef] 49. Djordjevic, S.P.; Batley, M.; Redmond, J.W.; Rolfe, B.G. The structure of the exopolysaccharide from Rhizobium sp. strain ANU280 (NGR234). Carbohydr. Res. 1986,148, 87–99. [CrossRef] 50. Kelly, S.J.; Muszy´nski, A.; Kawaharada, Y.; Hubber, A.M.; Sullivan, J.T.; Sandal, N.; Carlson, R.W.; Stougaard, J.; Ronson, C.W. Conditional requirement for exopolysaccharide in the Mesorhizobium–Lotus symbiosis. Mol. Plant-Microbe Interact. 2013 , 26, 319–329. [CrossRef] 51. Canter Cremers, H.C.J.; Batley, M.; Redmond, J.W.; Eysdems, L.; Breedveld, M.W.; Zevenhuizen, L.P.T.M.; Pees, E.; Wijffelman, C.A.; Lugtenberg, B.J.J. Rhizobium leguminosarum exoB mutants are deficient in the synthesis of UDP-glucose 40-epimerase. J. Biol. Chem. 1990,265, 21122–21127. [CrossRef] 52. Sánchez-Andújar, B.; Coronado, C.; Philip-Hollingsworth, S.; Dazzo, F.B.; Palomares, A.J. Structure and role in symbiosis of the exoB gene of Rhizobium leguminosarum bv. trifolii.Mol. Gen. Genet. 1997,255, 131–140. [CrossRef] Int. J. Mol. Sci. 2021,22, 6233 22 of 27 53. Pollock, T.J.; Workum, W.A.; Thorne, L.; Mikolajczak, M.J.; Yamazaki, M.; Kijne, J.W.; Armentrout, R.W. Assignment of biochemical functions to glycosyl transferase genes which are essential for biosynthesis of exopolysaccharides in Sphingomonas strain S88 and Rhizobium leguminosarum.J. Bacteriol. 1998,180, 586–593. [CrossRef] 54. Rolfe, B.G.; Carlson, R.W.; Ridge, R.W.; Dazzo, F.B.; Mateos, P.F.; Pankhurst, C.E. Defective infection and nodulation of clovers by exopolysaccharide mutants of Rhizobium leguminosarum bv. trifolii.Aust. J. Plant Physiol. 1996,23, 285–303. [CrossRef] 55. van Workum, W.A.; Canter Cremers, H.C.J.; Wijfjes, A.H.M.; van der Kolk, C.; Wijffelman, C.A.; Kijne, J.W. Cloning and characterization of four genes of Rhizobium leguminosarum bv. trifolii involved in exopolysaccharide production and nodulation. Mol. Plant Microbe Interact. 1997,10, 290–301. [CrossRef] 56. Ivashina, T.V.; Khmelnitsky, M.I.; Shlyapnikov, M.G.; Kanapin, A.A.; Ksenzenko, V.N. The pss4 gene from Rhizobium leguminosarum biovar viciae VF39: Cloning, sequence and the possible role in polysaccharide production and nodule formation. Gene 1994 , 50, 111–116. [CrossRef] 57. Janczarek, M.; Król, J.; Skorupska, A. The pssB gene product of Rhizobium leguminosarum bv. trifolii is homologous to a family of inositol monophosphatases. FEMS Microbiol. Lett. 1999,173, 319–325. [CrossRef] 58. Janczarek, M.; Rachwał, K. Mutation in the pssA gene involved in exopolysaccharide synthesis leads to several physiological and symbiotic defects in Rhizobium leguminosarum bv. trifolii.Int. J. Mol. Sci. 2013,14, 3711–3735. [CrossRef] 59. Latchford, J.W.; Borthakur, D.; Johnston, A.W.B. The products of Rhizobium genes, psi and pss, which affect exopolysaccharide production, are associated with the bacterial cell surface. Mol. Microbiol. 1991,5, 2107–2114. [CrossRef] 60. Janczarek, M.; Skorupska, A. Regulation of pssA and pssB gene expression in R. leguminosarum bv. trifolii in response to environmental factors. Antonie Van Leeuwenhoek 2004,85, 217–227. [CrossRef] 61. Lipa, P.; Vinardell, J.M.; Janczarek, M. Transcriptomic studies reveal that the Rhizobium leguminosarum Serine/Threonine Protein Phosphatase PssZ has a role in the synthesis of cell-surface components, nutrient utilization, and other cellular processes. Int. J. Mol. Sci. 2019,20, 2905. [CrossRef] 62. Król, J.; Wielbo, J.; Mazur, A.; Kopci´nska, J.; Łotocka, B.; Golinowski, W.; Skorupska, A. Molecular characterization of pssCDE Genes of Rhizobium leguminosarum bv. trifolii strain TA1: pssD mutant is affected in exopolysaccharide synthesis and endocytosis of bacteria. Mol. Plant Microbe Interact. 1998,11, 1142–1148. [CrossRef] [PubMed] 63. Ivashina, T.V.; Ksenzenko, V.N. Exopolysaccharide biosynthesis in Rhizobium leguminosarum: From genes to functions. In The Complex World of Polysaccharides; Karunaratne, D.N., Ed.; InTech: Rijeka, Croatia, 2012; pp. 99–126. [CrossRef] 64. Marczak, M.; Mazur, A.; Koper, P.; ˙ Zebracki, K.; Skorupska, A. Synthesis of rhizobial exopolysaccharides and their importance for symbiosis with legume plants. Genes 2017,8, 360. [CrossRef] [PubMed] 65. Marczak, M.; Wójcik, M.; ˙ Zebracki, K.; Turska-Szewczuk, A.; Talarek, K.; Nowak, D.; Wawiórka, L.; Sie´nczyk, M.; Łupicka-Słowik, A.; Bobrek, K.; et al. PssJ is a terminal galactosyltransferase involved in the assembly of the exopolysaccharide subunit in Rhizobium leguminosarum bv. trifolii.Int. J. Mol. Sci. 2020,21, 7764. [CrossRef] [PubMed] 66. Ivashina, T.V.; Fedorova, E.E.; Ashina, N.P.; Kalinchuk, N.A.; Druzhinina, T.N.; Shashkov, A.S.; Shibaev, V.N.; Ksenzenko, V.N. Mutation in the pssM gene encoding ketal pyruvate transferase leads to disruption of Rhizobium leguminosarum bv. viciae-Pisum sativum symbiosis. J. Appl. Microbiol. 2010,109, 731–742. [CrossRef] 67. Mazur, A.; Król, J.E.; Marczak, M.; Skorupska, A. Membrane topology of PssT, the transmembrane protein component of type I exopolysaccharide transport system in the Rhizobium leguminosarum bv. trifolii strain TA1. J. Bacteriol. 2003 , 185, 2503–2511. [CrossRef] 68. Mazur, A.; Król, J.E.; Wielbo, J.; Urbanik-Sypniewska, T.; Skorupska, A. Rhizobium leguminosarum bv. trifolii PssP protein is required for exopolysaccharide biosynthesis and polymerization. Mol. Plant Microbe Interact. 2002,15, 388–397. [CrossRef] 69. Grangeasse, C.; Terreux, R.; Nessler, S. Bacterial tyrosine-kinases: Structure-function analysis and therapeutic potential. Biochim. Biophys. Acta 2010,1804, 628–634. [CrossRef] 70. Janczarek, M.; Vinardell, J.M.; Lipa, P.; Kara´s, M. Hanks-type serine/threonine protein kinases and phosphatases in bacteria: Roles in signaling and adaptation to various environments. Int. J. Mol. Sci. 2018,19, 2872. [CrossRef] 71. Lipa, M.; Janczarek, M. Phosphorylation systems in symbiotic nitrogen-fixing bacteria and their role in bacterial adaptation to various environmental stresses. PeerJ. 2020,8, e8466. [CrossRef] 72. Collins, R.F.; Derrick, J.P. Wza: A new structural paradigm for outer membrane secretory proteins? Trends Microbiol. 2007 , 15, 96–100. [CrossRef] 73. Marczak, M.; Mazur, A.; Król, J.E.; Gruszecki, W.I.; Skorupska, A. Lipoprotein PssN of Rhizobium leguminosarum bv. trifolii: Subcellular localization and possible involvement in exopolysaccharide export. J. Bacteriol. 2006,188, 6943–6952. [CrossRef] 74. Zorreguieta, A.; Finnie, C.; Downie, J.A. Extracellular glycanases of Rhizobium leguminosarum are activated on the cell surface by an exopolysaccharide-related component. J. Bacteriol. 2000,182, 1304–1312. [CrossRef] 75. Finnie, C.; Hartley, N.M.; Findlay, K.C.; Downie, J.A. The Rhizobium leguminosarum prsDE genes are required for secretion of several proteins, some of which influence nodulation, symbiotic nitrogen fixation and exopolysaccharide modification. Mol. Microbiol. 1997,25, 135–146. [CrossRef] 76. Finnie, C.; Zorreguieta, A.; Hartley, N.M.; Downie, J.A. Characterization of Rhizobium leguminosarum exopolysaccharide glycanases that are secreted via a type I exporter and have a novel heptapeptide repeat motif. J. Bacteriol. 1998,180, 1691–1969. [CrossRef] 77. Ausmees, N.; Jacobsson, K.; Lindberg, M. A unipolarly located, cell-surface-associated agglutinin, RapA, belongs to a family of Rhizobium-adhering proteins (Rap) in Rhizobium leguminosarum bv. trifolii.Microbiology 2001,147, 549–559. [CrossRef] Int. J. Mol. Sci. 2021,22, 6233 23 of 27 78. Krehenbrink, M.; Downie, J.A. Identification of protein secretion systems and novel secreted proteins in Rhizobium leguminosarum bv. viciae.BMC Genom. 2008,9, 55. [CrossRef] 79. Lipa, P.; Vinardell, J.M.; Kopci´nska, J.; Zdybicka-Barabas, A.; Janczarek, M. Mutation in the pssZ gene negatively impacts exopolysaccharide synthesis, surface properties, and symbiosis of Rhizobium leguminosarum bv. trifolii with clover. Genes 2018 , 9, 369. [CrossRef] 80. Mijakovic, I.; Grangeasse, C.; Turgay, K. Exploring the diversity of protein modifications: Special bacterial phosphorylation systems. FEMS Microbiol. Rev. 2016,40, 398–417. [CrossRef] 81. Janczarek, M.; Skorupska, A. The Rhizobium leguminosarum bv. trifolii RosR: Transcriptional regulator involved in exopolysaccharide production. Mol. Plant Microbe Interact. 2007,20, 867–881. [CrossRef] 82. Janczarek, M.; Skorupska, A. Rhizobium leguminosarum bv. trifolii rosR gene expression is regulated by catabolic repression. FEMS Microbiol. Lett. 2009,291, 112–119. [CrossRef] 83. Janczarek, M.; Kalita, M.; Skorupska, A. New taxonomic markers for identification of Rhizobium leguminosarum and discrimination between closely related species. Arch. Microbiol. 2009,191, 207–219. [CrossRef] [PubMed] 84. Bittinger, M.A.; Handelsman, J. Identification of genes in the RosR regulon of Rhizobium etli.J. Bacteriol. 2000 ,182, 1706–1713. [CrossRef] [PubMed] 85. Chou, A.Y.; Archdeacon, J.; Kado, C.I. Agrobacterium transcriptional regulator Ros is a prokaryotic zinc finger protein that regulates the plant oncogene ipt.Proc. Natl. Acad. Sci. USA 1998,95, 5293–5298. [CrossRef] [PubMed] 86. Keller, M.; Roxlau, A.; Wenig, W.M.; Schmidt, M.; Quandt, J.; Niehaus, K.; Jording, D.; Arnold, W.; Pühler, A. Molecular analysis of the Rhizobium meliloti mucR gene regulating the biosynthesis of the exopolysaccharides succinoglycan and galactoglucan. Mol. Plant Microbe Interact. 1995,8, 267–277. [CrossRef] 87. Acosta-Jurado, S.; Alias-Villegas, C.; Navarro-Gomez, P.; Zehner, S.; Murdoch, P.D.; Rodriguez-Carvajal, M.A.; Soto, M.J.; Ollero, F.J.; Ruiz-Sainz, J.E.; Gottfert, M.; et al. The Sinorhizobium fredii HH103 MucR1 global regulator is connected with the nod regulon and is required for efficient symbiosis with Lotus burttii and Glycine max cv. Williams. Mol. Plant Microbe Interact. 2016 , 29, 700–712. [CrossRef] 88. Jiao, J.; Wu, L.J.; Zhang, B.; Hu, Y.; Li, Y.; Zhang, X.X.; Guo, H.J.; Liu, L.X.; Chen, W.X.; Zhang, Z.; et al. MucR is required for transcriptional activation of conserved ion transporters to support nitrogen fixation of Sinorhizobium fredii in soybean nodules. Mol. Plant Microbe Interact. 2016,29, 352–361. [CrossRef] 89. Pirone, L.; Pitzer, J.E.; D’Abrosca, G.; Fattorusso, R.; Malgieri, G.; Pedone, E.M.; Pedone, P.V.; Roop, R.M., II; Baglivo, I. Identifying the region responsible for Brucella abortus MucR higher-order oligomer formation and examining its role in gene regulation. Sci. Rep. 2018,8, 17238. [CrossRef] 90. Janczarek, M.; Kutkowska, J.; Piersiak, T.; Skorupska, A. Rhizobium leguminosarum bv. trifolii rosR is required for interaction with clover, biofilm formation and adaptation to the environment. BMC Microbiol. 2010,10, 284. [CrossRef] 91. Rachwał, K.; Boguszewska, A.; Kopci´nska, J.; Kara´s, M.; Tchórzewski, M.; Janczarek, M. The regulatory protein RosR affects Rhizobium leguminosarum bv. trifolii protein profiles, cell surface properties, and symbiosis with clover. Front. Microbiol. 2016 , 7, 1302. [CrossRef] 92. Janczarek, M.; Jaroszuk-´ Sciseł, J.; Skorupska, A. Multiple copies of rosR and pssA genes enhance exopolysaccharide production, symbiotic competitiveness and clover nodulation in Rhizobium leguminosarum bv. trifolii.Antonie Van Leeuwenhoek 2009 , 96, 471–486. [CrossRef] 93. Janczarek, M.; Skorupska, A. Modulation of rosR expression and exopolysaccharide production in Rhizobium leguminosarum bv. trifolii by phosphate and clover root exudates. Int. J. Mol. Sci. 2011,12, 4132–4155. [CrossRef] 94. Rachwał, K.; Lipa, P.; Wojda, I.; Vinardell, J.-M.; Janczarek, M. Regulatory elements located in the upstream region of the Rhizobium leguminosarum rosR global regulator are essential for its transcription and mRNA stability. Genes 2017,8, 388. [CrossRef] 95. Wielbo, J.; Mazur, A.; Krol, J.E.; Marczak, M.; Skorupska, A. Environmental modulation of the pssTNOP gene expression in Rhizobium leguminosarum bv. trifolii.Can. J. Microbiol. 2004,50, 201–211. [CrossRef] 96. Mueller, K.; González, J.E. Complex regulation of symbiotic functions is coordinated by MucR and quorum sensing in Sinorhizobium meliloti.J. Bact. 2011,193, 485–496. [CrossRef] 97. Reeve, W.G.; Dilworth, M.J.; Tiwari, R.P.; Glenn, A.R. Regulation of exopolysaccharide production in Rhizobium leguminosarum biovar viciae WSM710 involves exoR.Microbiology 1997,143, 1951–1958. [CrossRef] 98. Ivashina, T.; Sadykov, M.P.; Thatujev, B.M.; Kanapin, A.A.; Shlapnikov, M.G.; Ksenzenko, W. The Rhizobium leguminosarum bv viciae VF39 pssB gene product participates in symbiotic fixation of molecular nitrogen. Dokl. Akad. Nauk. 1996,350, 712–715. 99. Kutkowska, J.; Janczarek, M.; Kopci´nska, J.; Urbanik-Sypniewska, T.; Skorupska, A. Effects of pssB mutation on surface polysaccharides and symbiotic phenotype of Rhizobium leguminosarum bv. trifolii.Acta Biol. Cracov. Bot. 2007,49, 81–89. 100. Janczarek, M.; Skorupska, A. The Rhizobium leguminosarum bv. trifolii pssB gene product is an inositol monophosphatase that influences exopolysaccharide synthesis. Arch. Microbiol. 2001,175, 143–151. [CrossRef] 101. Borthakur, D.; Johnston, A.W.B. Sequence of psi, a gene of the symbiotic plasmid of Rhizobium phaseoli which inhibits exopolysaccharide synthesis and nodulation and demonstration that its transcription is inhibited by psr, another gene on the symbiotic plasmid. Mol. Gen. Genet. 1987,207, 149–154. [CrossRef] Int. J. Mol. Sci. 2021,22, 6233 24 of 27 102. Mimmack, M.L.; Borthakur, D.; Jones, M.A.; Downie, J.A.; Johnston, A.W.B. The psi operon of Rhizobium leguminosarum biovar phaseoli: Identification of two genes whose products are located at the bacterial cell surface. Microbiology 1994 , 140, 1223–1229. [CrossRef] 103. Mimmack, M.L.; Hong, G.F.; Johnston, A.W.B. Sequence and regulation of psrA, a gene on the Sym plasmid of Rhizobium leguminosarum biovar phaseoli which inhibits transcription of the psi genes. Microbiology 1994 ,140, 455–461. [CrossRef] [PubMed] 104. Wisniewski-Dyé, F.; Downie, J.A. Quorum sensing in Rhizobium.Antonie Van Leeuwenhoek 2002 ,81, 397–407. [CrossRef] [PubMed] 105. Edwards, A.; Frederix, M.; Wisniewski-Dyé, F.; Jones, J.; Zorreguieta, A.; Downie, J.A. The cin and rai quorum-sensing regulatory systems in Rhizobium leguminosarum are coordinated by ExpR and CinS, a small regulatory protein coexpressed with CinI. J. Bacteriol. 2009,191, 3059–3067. [CrossRef] [PubMed] 106. Frederix, M.; Edwards, A.; McAnulla, C.; Downie, J.A. Co-ordination of quorum-sensing regulation in Rhizobium leguminosarum by induction of an anti-repressor. Mol. Microbiol. 2011,81, 994–1007. [CrossRef] 107. Bogino, P.C.; Oliva, M.M.; Sorroche, F.G.; Giordano, W. The role of bacterial biofilms and surface components in plant-bacterial associations. Int. J. Mol. Sci. 2013,14, 5838–5859. [CrossRef] 108. D’Haeze, W.; Glushka, J.; De Rycke, R.; Holsters, M.; Carlson, R.W. Structural characterization of extracellular polysaccharides of Azorhizobium caulinodans and importance for nodule initiation on Sesbania rostrata.Mol. Microbiol. 2004,52, 485–500. [CrossRef] 109. Jiménez-Guerrero, I.; Acosta-Jurado, S.; del Cerro, P.; Navarro-Gómez, P.; López-Baena, F.J.; Ollero, F.J.; Vinardell, J.M.; Pérez-Montaño, F. Transcriptomic studies of the effect of nod gene-inducing molecules in rhizobia: Different weapons, one purpose. Genes 2018,9, 1. [CrossRef] 110. Galibert, F.; Finan, T.M.; Long, S.R.; Puhler, A.; Abola, P.; Ampe, F.; Barloy-Hubler, F.; Barnett, M.J.; Becker, A.; Boistard, P.; et al. The composite genome of the legume symbiont Sinorhizobium meliloti.Science 2001,293, 668–672. [CrossRef] 111. Barnett, M.J.; Long, S.R. The Sinorhizobium meliloti SyrM regulon: Effects on global gene expression are mediated by syrA and nodD3.J. Bacteriol. 2015,197, 1792–1806. [CrossRef] 112. Barnett, M.J.; Swanson, J.A.; Long, S.R. Multiple genetic controls on Rhizobium meliloti syrA, a regulator of exopolysaccharide abundance. Genetics 1998,148, 19–32. [CrossRef] 113. Cren, M.; Kondorosi, A.; Kondorosi, E. NolR controls expression of the Rhizobium meliloti nodulation genes involved in the core Nod factor synthesis. Mol. Microbiol. 1995,15, 733–747. [CrossRef] 114. Becker, A.; Rüberg, S.; Baumgarth, B.; Bertram-Drogatz, P.A.; Quester, I.; Pühler, A. Regulation of succinoglycan and galactoglucan biosynthesis in Sinorhizobium meliloti.J. Mol. Microbiol. Biotechnol. 2002,4, 187–190. 115. Janczarek, M. Environmental signals and regulatory pathways that influence exopolysaccharide production in rhizobia. Int. J. Mol. Sci. 2011,12, 7898–7933. [CrossRef] 116. Glenn, S.A.; Gurich, N.; Feeney, M.A.; Gonzalez, J.E. The ExpR/Sin quorum-sensing system controls succinoglycan production in Sinorhizobium meliloti.J. Bacteriol. 2007,189, 7077–7088. [CrossRef] 117. Zhan, H.J.; Lee, C.C.; Leigh, J.A. Induction of the second exopolysaccharide (EPSb) in Rhizobium meliloti SU47 by low phosphate concentrations. J. Bacteriol. 1991,173, 7391–7394. [CrossRef] 118. Leigh, J.A.; Walker, G.C. Exopolysaccharides of Rhizobium: Synthesis, regulation and symbiotic function. Trends Genet. 1994 , 10, 63–67. [CrossRef] 119. Mendrygal, K.E.; González, J.E. Environmental regulation of exopolysaccharide production in Sinorhizobium meliloti.J. Bacteriol. 2000,182, 599–606. [CrossRef] 120. Krol, E.; Becker, A. Global transcriptional analysis of the phosphate starvation response in Sinorhizobium meliloti strains 1021 and 2011. Mol. Genet. Genom. 2004,272, 1–17. [CrossRef] 121. Yao, S.Y.; Luo, L.; Har, K.J.; Becker, A.; Ruberg, S.; Yu, G.Q.; Zhu, J.B.; Cheng, H.P. Sinorhizobium meliloti ExoR and ExoS proteins regulate both succinoglycan and flagellum production. J. Bacteriol. 2004,186, 6042–6049. [CrossRef] 122. Wells, D.H.; Chen, E.J.; Fisher, R.F.; Long, S.R. ExoR is genetically coupled to the ExoS-ChvI two-component system and located in the periplasm of Sinorhizobium meliloti.Mol. Microbiol. 2007,64, 647–664. [CrossRef] 123. Chen, E.J.; Fisher, R.F.; Perovich, V.M.; Sabio, E.A.; Long, S.R. Identification of direct transcriptional target genes of ExoS/ChvI two-component signaling in Sinorhizobium meliloti.J. Bacteriol. 2009,191, 6833–6842. [CrossRef] [PubMed] 124. Wang, C.; Kemp, J.; Da Fonseca, I.O.; Equi, R.C.; Sheng, X.; Charles, T.C.; Sobral, B.W.S. Sinorhizobium meliloti 1021 loss-of-function deletion mutation in chvI and its phenotypic characteristics. Mol. Plant Microbe Interact. 2010 ,23, 153–160. [CrossRef] [PubMed] 125. Gibson, K.E.; Barnett, M.J.; Toman, C.J.; Long, S.R.; Walker, G.C. The symbiosis regulator CbrA modulates a complex regulatory network affecting the flagellar apparatus and cell envelope proteins. J. Bacteriol. 2007,189, 3591–3602. [CrossRef] [PubMed] 126. Morris, J.; González, J.E. The novel genes emmABC are associated with exopolysaccharide production, motility, stress adaptation, and symbiosis in Sinorhizobium meliloti.J. Bacteriol. 2009,191, 5890–5900. [CrossRef] 127. Reed, J.W.; Glazebrook, J.; Walker, G.C. The exoR gene of Rhizobium meliloti affects RNA levels of other exo genes but lacks homology to known transcriptional regulators. J. Bacteriol. 1991,173, 3789–3794. [CrossRef] 128. Becker, A.; Küster, H.; Niehaus, K.; Pühler, A. Extension of the Rhizobium meliloti succinoglycan biosynthesis gene cluster: Identification of the exsA gene encoding an ABC transporter protein, and the exsB gene, which probably codes for a regulator of succinoglycan biosynthesis. Mol. Gen. Genet. 1995,249, 487–497. [CrossRef] 129. Pinedo, C.A.; Gage, D.J. HPrK regulates succinate-mediated catabolite repression in the gram-negative symbiont Sinorhizobium meliloti.J. Bacteriol. 2009,191, 298–309. [CrossRef] Int. J. Mol. Sci. 2021,22, 6233 25 of 27 130. Bahlawane, C.; Baumgarth, B.; Serrania, J.; Rüberg, S.; Becker, A. Fine-Tuning of galactoglucan biosynthesis in Sinorhizobium meliloti by differential WggR (ExpG)-, PhoB-, and MucR-dependent regulation of two promoters. J. Bacteriol. 2008 , 190, 3456–3466. [CrossRef] 131. Calatrava-Morales, N.; McIntosh, M.; Soto, M.J. Regulation mediated by N-acyl homoserine lactone quorum sensing signals in the rhizobium-legume symbiosis. Genes 2018,9, 263. [CrossRef] 132. Hoang, H.H.; Becker, A.; Gonzalez, J.E. The LuxR homolog ExpR, in combination with the Sin quorum sensing system, plays a central role in Sinorhizobium meliloti gene expression. J. Bacteriol. 2004,186, 5460–5472. [CrossRef] 133. Pellock, B.J.; Teplitski, M.; Boinay, R.P.; Bauer, W.D.; Walker, G.C. A LuxR homolog controls production of symbiotically active extracellular polysaccharide II by Sinorhizobium meliloti.J. Bacteriol. 2002,184, 5067–5076. [CrossRef] 134. McIntosh, M.; Krol, E.; Becker, A. Competitive and cooperative effects in quorum-sensing-regulated galactoglucan biosynthesis in Sinorhizobium meliloti.J. Bacteriol. 2008,190, 5308–5317. [CrossRef] 135. Buendia, A.M.; Enenkel, B.; Köplin, R.; Niehaus, K.; Arnold, W.; Pühler, A. The Rhizobium meliloti exoZ/exoB fragment of megaplasmid 2: ExoB functions as a UDP-glucose 4-epimerase and ExoZ shows homology to NodX of Rhizobium leguminosarum biovar viciae strain TOM. Mol. Microbiol. 1991,5, 1519–1530. [CrossRef] 136. Müller, P.; Hynes, M.; Kapp, D.; Niehaus, K.; Pühler, A. Two classes of Rhizobium meliloti infection mutants differ in exopolysaccharide production and in coinoculation properties with nodulation mutants. Mol. Gen. Genet. 1988,211, 17–26. [CrossRef] 137. Kapp, D.; Niehaus, K.; Quandt, J.; Muller, P.; Pühler, A. Cooperative action of Rhizobium meliloti nodulation and infection mutants during the process of forming mixed infected alfalfa nodules. Plant Cell 1990,2, 139–151. [CrossRef] 138. Becker, A.; Kleickmann, A.; Küster, H.; Keller, M.; Arnold, W.; Pühler, A. Analysis of the Rhizobium meliloti genes exoU, exoV,exoW,exoT, and exoI involved in exopolysaccharide biosynthesis and nodule invasion: exoU and exoW probably encode glucosyltransferases. Mol. Plant Microbe Interact. 1993,6, 735–744. [CrossRef] 139. Pellock, B.J.; Cheng, H.P.; Walker, G.C. Alfalfa root nodule invasion efficiency is dependent on Sinorhizobium meliloti polysaccharides. J. Bacteriol. 2000,182, 4310–4318. [CrossRef] 140. Fraysse, N.; Lindner, B.; Kaczynski, Z.; Sharypova, L.; Holst, O.; Niehaus, K.; Poinsot, V. Sinorhizobium meliloti strain 1021 produces a low-molecular-mass capsular polysaccharide that is a homopolymer of 3-deoxy-D-manno-oct-2-ulosonic acid harboring a phospholipid anchor. Glycobiology 2005,15, 101–108. [CrossRef] 141. Putnoky, P.; Petrovics, G.; Kereszt, A.; Grosskopf, E.; Ha, D.T.; Bánfalvi, Z.; Kondorosi, A. Rhizobium meliloti lipopolysaccharide and exopolysaccharide can have the same function in the plant-bacterium interaction. J. Bacteriol. 1990 ,172, 5450–5458. [CrossRef] 142. Jones, K.M.; Sharopova, N.; Lohar, D.P.; Zhang, J.Q.; VandenBosch, K.A.; Walker, G.C. Differential response of the plant Medicago truncatula to its symbiont Sinorhizobium meliloti or an exopolysaccharidedeficient mutant. Proc. Natl. Acad. Sci. USA 2008 , 105, 704–709. [CrossRef] 143. Barnett, M.J.; Long, S.R. Novel genes and regulators that influence production of cell surface exopolysaccharides in Sinorhizobium meliloti.J. Bacteriol. 2018,200, e00501-17. [CrossRef] [PubMed] 144. Santos, R.; Herouart, D.; Sigaud, S.; Touati, D.; Puppo, A. Oxidative burst in alfalfa–Sinorhizobium meliloti symbiotic interaction. Mol. Plant Microbe Interact. 2001,14, 86–89. [CrossRef] [PubMed] 145. Maroti, G.; Downie, J.A.; Kondorosi, E. Plant cysteine-rich peptides that inhibit pathogen growth and control rhizobial differentiation in legume nodules. Curr. Opin. Plant Biol. 2015,26, 57–63. [CrossRef] [PubMed] 146. Rinaudi, L.V.; Giordano, W. An integrated view of biofilm formation in rhizobia. FEMS Microbiol. Lett. 2010 ,304, 1–11. [CrossRef] [PubMed] 147. Margaret, I.; Becker, A.; Blom, J.; Bonilla, I.; Goesmann, A.; Göttfert, M.; Lloret, J.; Mittard-Runte, V.; Rückert, C.; Ruiz-Sainz, J.E.; et al. Symbiotic properties and first analyses of the genomic sequence of the fast growing model strain Sinorhizobium fredii HH103 nodulating soybean. J. Biotechnol. 2011,155, 11–19. [CrossRef] [PubMed] 148. Dowdle, S.F.; Bohlool, B.B. Predominance of fast-growing Rhizobium japonicum in a soybean field in the People’s Republic of China. Appl. Environ. Microbiol. 1985,50, 1171–1176. [CrossRef] 149. Pueppke, S.G.; Broughton, W.J. Rhizobium sp. strain NGR234 and R. fredii USDA257 share exceptionally broad: Nested host ranges. Mol. Plant Microbe Interact. 1999,12, 293–318. [CrossRef] 150. Temprano-Vera, F.; Rodríguez-Navarro, D.N.; Acosta-Jurado, S.; Perret, X.; Fossou, R.K.; Navarro-Gómez, P.; Zhen, T.; Yu, D.; An, Q.; Buendía-Clavería, A.M.; et al. Sinorhizobium fredii Strains HH103 and NGR234 form nitrogen fixing nodules with diverse wild soybeans (Glycine soja) from Central China but are ineffective on Northern China accessions. Front. Microbiol. 2018 , 9, 2843. [CrossRef] 151. Tian, C.F.; Zhou, Y.J.; Zhang, Y.M.; Li, Q.Q.; Zhang, Y.Z.; Li, D.F.; Wang, S.; Wang, J.; Gilbert, L.B.; Li, Y.R.; et al. Comparative genomics of rhizobia nodulating soybean suggests extensive recruitment of lineage-specific genes in adaptations. Proc. Natl. Acad. Sci. USA 2012,109, 8629–8634. [CrossRef] 152. Weidner, S.; Becker, A.; Bonilla, I.; Jaenicke, S.; Lloret, J.; Margaret, I.; Pühler, A.; Ruiz-Sainz, J.E.; Schneiker-Bekel, S.; Szczepanowski, R.; et al. Genome sequence of the soybean symbiont Sinorhizobium fredii HH103. J. Bacteriol. 2012 , 194, 1617–1618. [CrossRef] 153. Vinardell, J.M.; Acosta-Jurado, S.; Zehner, S.; Gottfert, M.; Becker, A.; Baena, I.; Blom, J.; Crespo-Rivas, J.C.; Goesmann, A.; Jaenicke, S.; et al. The Sinorhizobium fredii HH103 genome: A comparative analysis with S. fredii strains differing in their symbiotic behavior with soybean. Mol. Plant Microbe Interact. 2015,28, 811–824. [CrossRef]