In rhizobia, the process of EPS synthesis is very complex and is regulated at both transcriptional and post-transcriptional levels, with multiple regulatory systems, which up to now have been best studied in two species, S. meliloti and R. leguminosarum. Adaptation of EPS synthesis to various environmental conditions requires a complex regulatory network involving cross talk between the different regulatory components. Several environmental factors and stress conditions, such as media osmolarity, ammonium and phosphate availability and flavonoids influence EPS biosynthesis [106–108]. Also, other culture conditions, such as the type of carbon source and culture age can modify the amount and composition of EPS produced [109–111].
4.1. Regulation of EPS Synthesis in <italic>S. meliloti</italic>
In S. meliloti, several regulatory genes of EPS I and EPS II synthesis have been identified either on the chromosome (mucR, exoR, exoS, exoD, expR, syrM and phoB) or on the megaplasmid pSymB (exsB, exoX and wggR). The majority of the proteins encoded by these genes have been described as repressors (Figure 3). These include the exoR, exoS, exoX and exsB genes, which negatively affect EPS I synthesis, and the mucR, which negatively regulates EPS II synthesis [57,60,62,107,112]. On the other hand, the SyrM and PhoB proteins are positive regulators of EPS I and EPS II production, respectively [69,113]. Among the identified regulators, MucR seems to be a global regulatory protein playing a key role in both positive regulation of EPS I synthesis and negative regulation of EPS II synthesis, thus coupling these two biosynthetic pathways [113–116].
The production of S. meliloti EPS is affected by several nutritional and stress conditions. Limitations of some non-carbon nutrients, like nitrogen and sulfur, very high phosphate concentrations, and hyperosmotic stress stimulate the synthesis of EPS I [61,107]. On the other hand, phosphate starvation stimulates EPS II production [27,107,113], indicating that the concentration of this nutrient is an essential signal affecting which type of EPS will be produced by S. meliloti. Also, different osmotic conditions modify EPS biosynthesis in this bacterium. A low osmotic pressure results in the production of mainly LMW EPS I, whereas production of the HMW fraction of this polymer is stimulated by an increased osmotic pressure [117]. Recently, Jofre and Becker [59] have reported that polymerization of EPS I is affected by the ionic strength of the medium rather than osmolarity.
4.1.1. Regulation of EPS I Synthesis
In general, regulation of S. meliloti EPS I synthesis is almost negative, with the exception of the MucR and SyrM, which function as positive regulators of this process (Figure 3) [114,116,118]. To date, six regulatory genes have been found to negatively affect EPS I synthesis, including the exoX and exsB genes linked with the exo/exs cluster, and the chromosomal exoR, exoS, cbrA and emmC genes. Mutants in both exoR and exoS genes display an increased production of EPS I and a higher expression of several exo genes (exoYFQ, exoA and exoP) in relation to the parental strain [61,62,119]. Despite these similarities, they differ in their symbiotic properties; the exoR mutant induces non-nitrogen-fixing nodules on alfalfa, whereas the exoS mutant elicits the formation of effective nodules on this host plant [62]. The ExoS protein was found to be almost identical to A. tumefaciens ChvG, which is a sensor protein of a two-component regulatory system [120]. ExoS is located in the IM in the form of homodimers, and its sensor domain, located in the periplasmic space, is responsible for the recognition of an environmental signal. After the signal has been recognized, ExoS kinase activates a second component of this system, the ChvI response regulator, by its phosphorylation. Next, ChvI affects the transcription of exo genes, so that this consequently leads to the modulation of EPS I production (Figure 3) [112]. ExoR is a periplasmic protein, which translocates to the periplasm, where it physically interacts with the ExoS and inhibits ExoS/ChvI two-component signalling [121–123]. exoR expression was found to be upregulated in the absence of functional ExoR. This regulation is mediated by the ExoS/ChvI system, which positively affects the transcription of exoR [123–125]. Both the ExoR protein and the ExoS/ChvI system, apart from EPS I synthesis, are also involved in the regulation of flagellum biosynthesis genes [112].
Recently, novel S. meliloti exoS and chvI null mutants have been reported [126,127]. Phenotypic analysis of these two mutants demonstrated that the ExoS/ChvI regulatory system was also required for growth on over 21 different carbon sources. Additionally, the chvI mutant exhibited several other effects, such as failure in the growth on complex media, an altered LPS profile, hypermotility, lower tolerance to acidic conditions and synthesis of significantly less poly-3-hydroxybutyrate than the wild type strain. These data indicate that ChvI is a crucial protein engaged in S. meliloti regulatory networks involving both the bacterial cell envelope and carbon source utilization [127].
In conclusion, the ExoR, ExoS and ChvI proteins form a regulatory system which appear to be involved in several critical cell processes, such as EPS I production, nutrient utilization, motility and free-living viability [123,126,127].
exoX located in the exo/exs cluster, is another gene participating in the negative regulation of EPS I synthesis (Figure 3). It has been shown that the ratio of exoX copies to the copies of the exoY encoding an enzyme, catalyzing the initial step of repeating unit synthesis, is very important for the proper level of EPS I synthesis. ExoX is a small, inner membrane-attached protein showing a significant similarity to the previously identified PsiA of R. leguminosarum bv. phaseoli and ExoX of Rhizobium sp. NGR234 [99,100]. Mutants in exoX overproduced EPS I, whereas the presence of multiple copies of this gene inhibited the production of EPS I. Since the expression of exo genes was not affected by the gene dosage of exoX to exoY, it was suggested that ExoX functions as a post-transcriptional inhibitor of the ExoY [56,119]. The fact that both ExoX and ExoY are associated with the IM suggests a direct interaction between these two proteins [100].
exsB is a second gene in the exo/exs region, which encodes a regulator negatively affecting EPS I synthesis (Figure 3) [57]. Similarly to the phenotype of the exoX mutant, an exsB mutant produced 3-fold more EPS I than the wild type, but in contrast to the exoX mutant, additional exsB copies resulted in a decrease of EPS I production to 20% of the control. Since this effect was not caused by direct regulation of the transcription of exo genes in this mutant background (the only exception was the reduction of exoK expression) [60], negative regulation of EPS I production by ExsB must have occurred mainly at the post-transcriptional level [54,57].
Among environmental factors, ammonia has been found to significantly affect EPS I synthesis. The regulation of this process by nitrogen availability is directed by the NtrC and SyrM proteins, which under limitation of this nutrient act as positive regulators. Mutations in genes for these two regulatory proteins decrease EPS I production under nitrogen starvation. The SyrM protein increases the transcription of the exoF and exoP genes via induction of syrA expression, and additionally by direct activation of exoP transcription (Figure 3) [118,128]. On the other hand, SyrM inhibits the expression of exsH and exoK encoding endoglycanases. The exoK gene is of special interest among all exo genes of the exo/exs region in that it is a target for the action of at least three regulatory proteins (MucR, SyrM and ExsB), indicating that the level of its expression is very important for the proper degree of polymerization of EPS I.
The SyrM protein is also involved in the regulation of the expression of nod genes, which are responsible for the synthesis of another symbiotic signal, the Nod factor [128,129]. This protein belongs to the family of LysR-type transcriptional regulators and displays a similarity to NodD proteins engaged in the activation of nod genes [118]. SyrM is involved in the determination of the ratio of LMW to HMW EPS I, and this process is affected by nitrogen and luteolin, the plant flavonoid indispensable for the activation of nod genes in the presence of NodD [118,129]. These data indicate that SyrM influences both EPS I and Nod factor synthesis, suggesting that nitrogen starvation might be a factor affecting the concentration of these two signal molecules essential in the early stages of host plant infection.
The precise role of another chromosomal gene, exoD, in the regulation of EPS I synthesis is difficult to explain. Nevertheless, the gene seems to positively affect this process because an exoD mutant produces diminished amounts of EPS I [63]. Furthermore, this mutant was found to be sensitive to alkaline conditions and elicited effective nodules on host plants exclusively in a slightly acidic growth medium [63].
Among the regulatory proteins of S. meliloti, MucR seems to play a key role in the positive regulation of EPS I synthesis. A mutation in mucR results in a high level of EPS II production but only a very low level of LMW EPS I synthesis [60,114,115]. The MucR protein contains the C2H2 type zinc-finger motif and influences the transcription of some exo genes (exoH, exoX, exoY) by binding to a palindromic sequence located in promoter regions of the regulated genes, but the observed levels of modulation are not high. In the mucR mutant, the transcription of exoH and exoX was slightly increased, whereas the expression of exoK and the exoYFQ operon decreased 1.5-fold. This indicates that the positive function of MucR in EPS I synthesis is a result of both stimulation of the expression of EPS I synthesis genes and repression of exoX transcription [114,115]. Moreover, MucR negatively regulates its own transcription by binding to a palindromic sequence located in the mucR upstream region [60,115].
Recently, Morris and Gonzalez [125] have characterized a novel three-component system, consisting of the EmmA, EmmB and EmmC proteins, which appear to play a crucial role in the adaptability and survivability of S. meliloti. EmmA is a protein secreted to the periplasm, whereas EmmB and EmmC are sensor and response proteins, respectively. The emmABC genes are associated with EPS I production, motility and stress adaptation. Mutations in the emm locus cause motility defects, an increase in succinoglycan synthesis and a decrease in stress tolerance, which, in consequence, leads to the loss of the ability to form an optimal symbiosis with alfalfa [125]. The EmmABC system shows some functional similarity to the ExoR-ExoS-ChvI system, although no cross-regulation of these systems has been evidenced. This suggests that these two systems may act in parallel but separate fashions in the regulation of important cell processes, such as EPS I production and motility, in response to environmental cues.
Moreover, it has recently been demonstrated that the cbrA gene, which encodes a stationary phase-induced sensor kinase, is also associated with EPS I synthesis and motility [130]. This is confirmed by the phenotype of the cbrA mutant, which overproduces EPS I and shows cell envelope defects, a decrease in flagellar biosynthesis and symbiotic defects.
4.1.2. Regulation of EPS I Synthesis by Succinate-Mediated Catabolite Repression
Recently, succinate-mediated catabolite repression has been characterized in S. meliloti, and some proteins of the phosphotransferase system (PTS) engaged in this regulation appeared to affect EPS I production [131,132]. Although this bacterium can utilize a variety of compounds as sources of carbon, succinate is preferred carbon and energy source to other compounds (glucose, fructose, galactose and lactose), playing an especially important role in S. meliloti metabolism during both the free-living and symbiotic states. The hprK, EIIA (manX) and hpr genes for the PTS system form a cluster in the chromosome located immediately downstream of the exoS gene [132]. HPrK of a kinase/phosphatase activity is the main component of the PTS system and it regulates succinate-mediated catabolite repression through phosphorylation/dephosphorylation of its substrate, the protein HPr. A phosphorylated form of HPr activates EIIA, that leads to enhancing of catabolite repression in the presence of succinate. A mutation in hpr results in several effects, including altered carbon metabolism, production of EPS I, and sensitivity to cobalt limitation [131,132]. An EIIA mutant demonstrates slowed growth on diverse carbon sources and enhanced accumulation of HMW EPS I. Despite these strong phenotypes, both hpr and EIIA mutants exhibit wild-type nodulation and nitrogen fixation on alfalfa [131]. On the other hand, a hprK mutant overproduces EPS I and induces nodules that do not fix nitrogen, indicating that this gene, apart from the key role in catabolite repression, is also important for EPS I synthesis and effective symbiosis [132].
4.1.3. Regulation of EPS II Synthesis
Besides nitrogen, phosphate is another environmental factor which plays a significant role in the regulation of EPS synthesis in S. meliloti. In contrast to EPS I, whose production is induced in the presence of high phosphate concentrations (above 10 mM), low-phosphate conditions, typically found in soil (0.1 to 10 μM), stimulate EPS II production [19,97,103]. This polysaccharide probably plays a dominant role in the environmental conditions, where this nutrient is scarce. Recently, Rinaudi and Gonzalez [133] have shown that symbiotically active fraction of EPS II is crucial for both root colonization and biofilm formation.
In S. meliloti, phosphate regulates a considerable number of genes, including the genes involved in EPS II synthesis, through the PhoB response regulator forming a two-component regulatory system with the sensor kinase PhoR (Figure 3) [67,69,113,134–137]. In general, PhoB functions as a positive regulator which induces the expression of genes belonging to the Pho regulon under Pi limitation. In these conditions, a phosphorylated form of PhoB activates the transcription of target genes by binding to the PHO box sequence located in the promoters of phosphate-regulated genes [134,136]. Among genes belonging to the S. meliloti Pho regulon, Krol and Becker [135] have found the wgaA, wggR, wgdA and wgeA genes involved in EPS II synthesis, whose expression was significantly induced in Pi-limited cells. In upstream regions of these genes, two PHO box-like sequences have been identified [113,136].
EPS II synthesis is also regulated by two other regulatory proteins, WggR (ExpG) and MucR (Figure 3) [69,138]. WggR, encoded by a gene located in the galactoglucan synthesis gene cluster, positively affects the synthesis of EPS II under Pi limitation [67]. This protein, belonging to the Mar family of transcriptional regulators containing a helix-turn-helix motif, binds to conserved palindromic sequences located in promoter regions of the wgaA, wggR/wgdA and wgeA genes to activate their expression [69,113]. The deletion of wggR reduces EPS II synthesis under Pi starvation, whereas additional copies of this gene slightly increase EPS II production [69,113]. The second regulator, MucR, represses galactoglucan synthesis, whereas a mutation in mucR leads to the production of the HMW form of this polysaccharide only [60,107,139].
Transcription of the wga, wge and wgd operons is governed by two promoters that are differentially controlled by PhoB, WggR and MucR [69]. Upstream of the distal promoters of these genes, three motifs functioning as binding sites for the MucR, WggR and PhoB proteins have been identified, and the binding site for WggR ovelapped with the PHO box sequence. Under phosphate-sufficient conditions, the transcription of the wga, wgd and wge genes is repressed by MucR, which strongly inhibits the activity of both promoters by binding to the sequence located in the vicinity of the distal transcription start sites, and consequently, only traces of EPS II are produced [69]. The WggR protein also slightly inhibits the activity of the distal promoter under the same conditions. Under Pi starvation, WggR and phosphorylated PhoB bind to the promoter regions and activate the transcription of the wga, wgd and wge genes. This phosphate-dependent regulation of EPS II synthesis has been reported to involve the cooperative action of WggR and PhoB. This indicates that WggR plays a special role in the regulation of EPS II synthesis mediated by the PhoB and MucR proteins. The activities of PhoB, WggR and MucR regulators ensure fine-tuning of the expression of galactoglucan synthesis genes, which allows bacteria to adapt to changing environmental conditions [69,113,138]. Moreover, the transcription of wggR is activated by PhoB under Pi limitation, and both the WggR and PhoB proteins function cooperatively in the transcription activation of this regulatory gene [69].
4.1.4. Role of Quorum Sensing in the Regulation of EPS I and EPS II Synthesis
S. meliloti possesses at least three quorum-sensing systems, among them the Sin system, consisting of SinR, a LuxR-type transcriptional regulator, and SinI, an autoinducer synthase responsible for the synthesis of a series of long-chain N-acyl homoserine lactons (AHL) (Figure 3) [139,140]. AHL represent one of the classes of pheromone-like signals, called autoinducers, which are released exogenously and whose concentration acts as an indicator of population density for the bacteria. The Sin system regulates the expression of many S. meliloti genes, including genes involved in the production of EPS I and EPS II, motility, chemotaxis and other cellular processes, such as nitrogen fixation and metal transport. The regulation of most of these genes is dependent on the presence of another LuxR-type regulator, ExpR [141–144].
S. meliloti wild type exhibits a mucoid phenotype due to the presence of EPS II. However, both sinI and expR mutants remain dry and unable to produce EPS II, which confirms the role of the Sin system and the ExpR protein in the synthesis of this polysaccharide [139,141,142,145]. Expression of sinI is dependent on sinR and is enhanced by AHL-ExpR (Figure 3). Also, galactoglucan biosynthesis genes (particularly wgeB, wgdA, wggR and wgcA) are highly induced in the presence of AHL, and their expression depends not only on the production of this signal but also on the active ExpR protein [139,145]. Expression of genes of the wge, wga and wgd operons is required for the synthesis of HMW EPS II, whereas expression of wgcA, encoding glycosyltransferase, is critical for the production of symbiotically active LMW EPS II [145]. Recently, it has been established that ExpR binds to promoter regions of the sinI, wgaA and wgeA genes, and this binding is enhanced in the presence of AHL [142]. Activation of these genes by ExpR is almost completely dependent on the presence of WggR when another regulator, MucR, is present. But in the absence of MucR, WggR is not required for the positive effect of ExpR. This WggR-dependent effect is also seen with PhoB-dependent induction under Pi starvation and suggests that WggR functions as a general mediator of the expression of EPS II synthesis genes for at least two regulators (PhoB and MucR) under different conditions [69]. The close proximity of the binding sites for ExpR, WggR, PhoB and MucR suggests a complex regulation of EPS II synthesis genes involving protein-protein interaction and, possibly, some competition for the target sites, which allows changing the level of production of this polymer in response to different environmental factors [69,142]. MucR appeared to repress EPS II synthesis at an extremely low population density until the ExpR/Sin system abolishes this effect at levels of quorum sufficient for biofilm formation and invasion of the host plant [144].
Moreover, ExpR and the Sin quorum-sensing system regulate some exo/exs genes involved in EPS I production (Figure 3) [142,143,146]. ExpR stimulates the expression of exoI and exsH encoding a succinoglycan glucanase which is responsible for the generation of the LMW form of this polysaccharide [143]. In the promoter regions of these genes, binding sites for ExpR have been found [142]. These data confirm that the ExpR/Sin system is engaged in the regulation of not only EPS II, but also EPS I biosynthesis.
The regulation of EPS synthesis by sinRI is an important finding, which connects quorum sensing with the symbiotic process. A sinI mutant unable to produce AHL is deficient in invasion of alfalfa plants, indicating that the Sin system plays an essential role in the establishment of an effective symbiosis [143].
Recently, Mueller and Gonzalez [144] have reported that MucR plays a more global regulatory role, being engaged, together with the ExpR/Sin system, in the complex regulation of symbiotic functions. In planctonic S. meliloti, MucR properly coordinates a diverse set of bacterial behaviors by repressing a variety of genes, whose expression is activated only during symbiosis, including those involved in nitrogen fixation and respiration (fix genes) (Figure 3). In addition, MucR has been found to enhance the bacterial ability to induce nodule formation on the host plant by increasing Nod factor synthesis via increased expression of nodD [144].
4.1.5. Roles of the MucR, ExoR/ExoS/ChvI and ExpR/Sin Systems in the Regulation of Motility Genes
Bacterial motility is one of the critical factors for the establishment of symbiosis under natural soil conditions. The regulatory proteins MucR, ExoR and ExpR, which play an essential role in EPS synthesis, appear to be also involved in motility, indicating that the regulation of these two processes is coupled in S. meliloti (Figure 4) [112,116,141,144,147–149]. The 56-kb chromosomal region, named the flagellar region, encompasses a majority of genes associated with motility. These genes, categorized into three main classes, are expressed in a hierarchical manner [148,149]. At the top of the hierarchy are visN and visR genes, which encode subunits of the LuxR-type transcriptional activator of motility and chemotaxis genes [148,149]. The VisN/VisR regulator activates the transcription of rem, which codes for a protein postively regulating the expression of the motility and chemotaxis genes (Figure 4). A disruption of visN, visR or rem leads to the loss of flagella and, in consequence, the loss of swimming and swarming motility. Rem positively regulates the expression of its own gene and the expression of motility genes by direct binding to the promoters of flgB, fliF and orf38 [141,147]. Tambalo and associates have described recently that VisN/R-Rem cascade also participates in the regulation of a majority of flagellar, motility and chemotaxis genes (che1, motA, flaABCD, motB and mcpD) in R. leguminosarum [150,151]. All of these genes except mcpD are located within the main motility and chemotaxis gene cluster of R. leguminosarum. However, other chemotaxis and motility genes (che2 operon, flaH, flaG, flaE and mcpC) found outside of this cluster are not regulated by VisN/R and Rem activators, suggesting a role of additional regulatory system in this process.
Recently, other regulators of motility have been found, which are encoded by genes located outside this region, among them CbrA, initially described as a regulator of symbiosis [130], MucR, and the two systems, ExpR/Sin and ExoR/ExoS/ChvI (Figure 4) [112,116,130,144,147]. A lack of functional CbrA results in a decreased expression of several motility genes, among them visN, visR and rem [130]. Hoang and associates [147] demonstrated that the ExpR/Sin system controls motility gene expression via the VisN/VisR and Rem regulators in a population-density-dependent manner. This system suppresses motility by repression of visN and visR expression when the bacteria reach the late logarithmic growth phase [139]. Also, the second regulatory system, ExoR/ExoS/ChvI, which is involved in the regulation of EPS I synthesis, suppresses motility gene expression through VisN/VisR and Rem [147]. In both exoR and exoS mutants, the expression of all genes for flagellum biosynthesis was downregulated, and for this reason, the cells carrying these mutations lost the ability to swim and swarm [112]. Recently, MucR has also been found to participate in the regulatory network controlling motility in S. meliloti [116]. This protein, apart from activating EPS I and inhibiting EPS II synthesis, downregulates the expression of rem by direct binding to its upstream region.
In conclusion, all three regulatory pathways mediated by the MucR protein and the ExoR/ExoS/ChvI and ExpR/Sin regulatory systems suppress motility through repression of the expression of the regulatory visN, visR and rem genes [112,116,144,147].
4.2. Regulation of EPS Synthesis in <italic>R. leguminosarum</italic>
In contrast to S. meliloti, the knowledge about regulation of EPS biosynthesis in R. leguminosarum is much more fragmentary. Up to now, only a few regulatory genes involved in this process have been described. These include two genes, psiA and psrA, located on the symbiotic megaplasmid (pSym), and exoR, pssB, rosR and expR genes located on the chromosome of R. leguminosarum (Figure 5) [82,83,152–156].
psiA (a polysaccharide inhibition gene) and psrA (a polysaccharide restoration gene), the first to be identified among those genes, have been found on pSym of R. leguminosarum bv. phaseoli, near the nod-nif region involved in nodulation and nitrogen fixation [152]. Although a mutation in psiA does not affect EPS production, additional copies of this gene prevent EPS synthesis and abolish nodulation of Phaselous plants [81–83,152]. The inhibitory effect of extra psiA copies is overcome in the presence of multiple copies of psrA or pssA, encoding the glucosyl-IP-transferase engaged in the first step of EPS synthesis [81,82]. This indicates that for proper EPS production, a balanced number of psiA, pssA and psrA copies is required. psiA encodes a small, inner membrane-attached protein, which shows a similarity to the S. meliloti ExoX regulator. Also PssA, containing a hydrophobic N-terminal end and a transmembrane helix domain is located in the IM [81,157]. The same subcellular localization of both PsiA and PssA proteins suggests that PsiA most probably functions as a post-translational inhibitor of PssA, which binds to and inhibits the activity of this enzyme.
The second regulator, PsrA, which belongs to a family of transcriptional regulators containing a helix-turn-helix motif represses the transcription of psiA (Figure 5) [83,152]. A psrA mutant produces a decreased amount of EPS in comparison to the wild type strain, suggesting a positive role of this gene in EPS synthesis. Both a strain with multiple copies of psrA and a psiA mutant demonstrate similar symbiotic phenotypes—they induce the formation of non-nitrogen-fixing nodules on the host plant. These data suggest that psiA most probably inhibits EPS production inside nodules via the repression of psrA. The psiA and psrA genes have been identified exclusively on pSym plasmids of R. leguminosarum bv. phaseoli strains, indicating that this regulatory mechanism is specific only to this biovar [78,152].
EPS production in R. leguminosarum is also negatively regulated by exoR, which shows a significant similarity to the exoR of S. meliloti (Figure 5). An R. leguminosarum bv. viciae exoR mutant produces 3-fold more EPS than the wild-type strain similarly to the S. meliloti exoR mutant [153]; but in contrast to this latter mutant, it induces both effective and non-infective nodules on pea plants.
Also pssB, located upstream of the pssA gene, seems to be engaged in negative regulation of EPS synthesis; however, its precise role in this process is difficult to establish. A pssB mutant produces higher amounts of EPS than the wild type and induces non-nitrogen-fixing nodules on clover and vetch [154,158,159], whereas additional copies of this gene result in increased EPS production [160]. pssB encodes a protein belonging to a family of inositol monophosphate phosphatases (IMP-ases), encompassing enzymes from both prokaryotic and eukaryotic organisms [159,160]. In mammalian cells, IMPases are responsible for the conversion of inositol monophosphate to inositol, which is needed for the regeneration of phospholipids containing inositol. But the role of IMPase in the metabolism of rhizobia is unclear. This enzyme may generate the pool of inositol, which is a compound commonly found inside bacteroids of R. leguminosarum bv. viciae and also abundantly occurring in pea nodules. On the other hand, inositol catabolism seems to be important for survival and competition of rhizobial strains, being crucial for successful invasion of host plants [160].
4.2.1. Role of RosR in EPS Synthesis and Symbiosis
Recently, rosR, which seems to play a key role in the regulation of EPS synthesis among all the so far described regulatory genes, has been identified on the chromosome of R. leguminosarum bv. trifolii (Figure 5) [155]. This gene is very conserved and is present in the genomes of all strains belonging to three R. leguminosarum biovars and closely related species, R. etli and R. gallicum, suggesting its important regulatory role in other rhizobial species as well [78,161]. R. leguminosarum rosR also shares a significant identity with ros of A. tumefaciens [162], rosAR of A. radiobacter [163] and mucR of S. meliloti [60]. All these genes encode transcriptional regulators belonging to the family of Ros/MucR proteins, which possess C2H2 type zinc-finger motifs and are involved in the regulation of EPS synthesis. A genome-wide genetic screening has shown that R. etli RosR affects the expression of many functionally diverse genes, among them those responsible for the synthesis and modification of surface polysaccharides (exoB, prsD, pssK and plyA) [164]. A mutation in R. leguminosarum bv. trifolii rosR results in a substantial decrease of EPS production and ineffective symbiosis with clover, whereas additional copies of this gene lead to a nearly twofold increase of EPS synthesis [79,155], indicating that rosR functions as a positive regulator of this process. Moreover, a rosR mutant exhibits several other effects, including a decreased ratio of LMW to HMW fractions of EPS, quantitative alterations in the polysaccharide constituent of LPS, changes in membrane and secreted protein profiles, higher sensitivity to surface-active detergents and some osmolytes, and decreased motility and nodulation competitiveness [165]. But the most striking effect of rosR mutation is the considerably decreased attachment and colonization of root hairs, indicating that the mutation affects the first steps of the invasion process. On the other hand, multiple rosR copies significantly enhance competitiveness and clover nodulation in R. leguminosarum bv. trifolii strains, confirming the essential role of this gene in symbiosis [79].
The data obtained by our research group indicate that rosR expression is very complex and is modulated by different environmental factors (Figure 5) [108,155,166]. This gene demonstrates a very high level of expression, which is due to the action of its strong distal promoter as well as two additional regulatory elements, an upstream promoter element and TGN extended −10 element [166]. In addition, several other sequence motifs were identified in the rosR upstream region, among them a LysR motif recognized by proteins from the LysR family, motifs resembling E. coli cAMP-CRP binding site, PHO boxes as binding sites for the PhoB regulator, and the RosR box [155,166]. The complexity of rosR regulation is additionally enhanced by six inverted repeats of different lengths found in this region, which are most probably engaged in post-transcriptional regulation, affecting the stability of rosR transcripts [108]. We confirmed that RosR recognizes and binds to the 22-bp-long palindromic sequence, called the RosR box, and decreases the transcription of its own gene [155]. In the presence of glucose, rosR transcription is also significantly decreased, indicating that the expression of this gene is regulated by catabolic repression [166]. Moreover, rosR transcription increases in the presence of clover root exudates and the functional activator NodD [108]. Also phosphate affects rosR expression, and PHO boxes located in the rosR upstream region are most probably target sites for PhoB. These data show that four regulatory proteins (RosR, CRP-like protein, NodD and PhoB) are involved in the regulation of rosR expression in response to different environmental factors (Figure 5). Also, EPS production in R. leguminosarum is affected by phosphate and ammonia, plant flavonoids and the type of carbon source [108,166,167]. This indicates that the increase of EPS synthesis in the presence of flavonoids and glycerol, and under phosphate limitation, is mediated by rosR, whose transcription also increases significantly in these conditions. The positive role of RosR in EPS production seems to be a coupled effect of stimulation of pssA expression [155] and repression of the expression of the exoR and pssB genes, engaged in the negative regulation of this process (Figure 5) [168]. In addition, root exudates, phosphate and ammonia starvation affect the transcription of some pss genes (pssA, pssB, pssO and pssP) [79,169].
In general, a positive effect of flavonoids and nod genes on EPS production has been observed in R. leguminosarum and S. meliloti, suggesting connected regulation of the synthesis of both symbiotic signals, EPS and Nod factors. But, influence of flavonoids on EPS production seems to be different in different rhizobia, because the presence of genistein—an effective inducer of nod genes in S. fredii HH103, results in a non-mucoid phenotype [170,171]. On the other hand, nolR encoding a regulator of nod genes has a positive effect on EPS synthesis in this strain [170,171].
4.2.2. Role of Quorum Sensing in the Regulation of EPS Synthesis
EPS production in R. leguminosarum also seems to be influenced by quorum sensing (Figure 5) [140]. In R. leguminosarum bv. viciae, four different AHL-based quorum sensing systems, cin, rai, rhi and tra, have been discovered, which are involved in several processes, including plasmid transfer and nodulation [172]. The cinR, cinI and cinS genes of the cin system are at the top of this hierarchical regulatory network and induce the other (rai, rhi and tra) systems. CinI is responsible for the production of N-(3-hydroxy-7-cis-tetradecenoyl)-l-homoserine lactone, and CinR induces cinI in response to this AHL [140]. The third element of the cin system, CinS, is a small protein having an anti-repressor function, which induces the regulatory genes rhiR and raiR of the rhi and rai systems, and also plyB encoding an extracellular glycanase (Figure 5) [156,173]. This protein acts by attenuating the repressive activity of another regulator, PraR. A cloned cinS causes an apparent loss of colony mucoidy with colony aging, and this unusual phenotype is a result of the action of increased amounts of PlyB, which degrades EPS. Another protein that is required for the induction of both raiR and plyB is ExpR, which functions independently of CinI-made AHL [156,173]. R. leguminosarum expR is homologous to the expR of S. meliloti and the two proteins encoded by these genes are LuxR-type regulators. Nevertheless, several functional differences between them have been found. In contrast to the expR mutant of S. meliloti, the colony morphology of the R. leguminosarum expR mutant is very similar to the wild type. This indicates that expR regulates a lower number of genes in R. leguminosarum than its homolog from S. meliloti, and among them only one gene, plyB, which is associated with EPS production [156,173].
4.3. Role of EPS Synthesis Genes in Biofilm Formation in Rhizobia
Similarly to other bacterial EPS, rhizobial EPS play a significant role in biofilm formation, being the major components of its matrix, which provides a physical barrier against diffusion of toxic compounds and protection against environmental stresses [17]. A mutation in R. leguminosarum pssA, a key gene for EPS synthesis, completely abolishes the production of this polysaccharide and biofilm formation [174]. Also, the size of EPS is very important for normal biofilm development, as borne out by the fact that prsD, prsE, plyB and plyBplyA mutants, which produce EPS of significantly longer chains than the wild type, are impaired in biofilm formation [174]. Both mutants in the prsD and prsE genes encoding components of the PrsD-PrsE type I secretion system are only able to form biofilm of an immature structure. In addition, mutants in the plyA and plyB genes, which encode glycanases cleaving EPS, show delayed formation of a biofilm with an atypical structure. Also, RapC, RapA1 and RapA2 agglutinins secreted via the PrsD-PrsE system are responsible for the adhesion and aggregation of rhizobia [4,174]. Moreover, a mutation in rosR results in the formation of lower amounts of biofilm, which differs significantly in depth, architecture and viability of cells from that of the wild type, confirming the important role of this gene in biofilm formation [108,165]. Similarly in S. meliloti, EPS I is required for biofilm development, since an exoY mutant non-producing this polysaccharide forms an immature biofilm [175]. Rinaudi and Gonzalez have reported [133] that the ExpR/Sin quorum-sensing system controls biofilm formation in this bacterium through the production of EPS II. The symbiotically active LMW fraction of this polymer appeared to be a crucial factor for the development of biofilm and root colonization [133]. Also, the cin quorum system is engaged in biofilm formation by R. leguminosarum, because a cinS mutant forms higher amounts of biofilm than the wild type [173].
Moreover, several nutrients and stress conditions regulate biofilm formation by S. meliloti and R. leguminosarum. In S. meliloti, the concentrations of sucrose, calcium and phosphate positively correlate with biofilm formation, whereas extreme temperature and pH values have a negative effect on this process [176]. Also, nutrients such as phosphate, ammonium, type of carbon source and plant flavonoids affect biofilm formation by R. leguminosarum [108]. This finding could be explained, to some extent, by the increased production of EPS in these growth conditions. Besides EPS, other rhizobial polysaccharides and components are also involved in biofilm maturation, including Nod factors and flagellum, pointing to the complexity of this process [17]. Flagella-less mutants of S. meliloti exhibit reduced biofilming capabilities [175]. Also, common nodD1ABC genes engaged in the synthesis of core Nod factor are required for the establishment of a mature biofilm by S. meliloti [177]. Williams and co-workers described that, apart from EPS, other polysaccharides are important for attachment and biofilm formation by R. leguminosarum [178]. It has been indicated that cellulose (celA) and glucomannan (gmsA) mutants of R. leguminosarum do not form biofilms on root hairs, although they develop normal biofilms in vitro, confirming a significant role of these polymers in bacterium-plant interactions.