The exoD gene of Rhizobium meliloti encodes a novel function needed for alfalfa nodule invasion

During the symbiotic interaction between alfalfa and the nitrogen-fixing bacterium Rhizobium meliloti, the bacterium induces the formation of nodules on the plant roots and then invades these nodules. Among the bacterial genes required for nodule invasion are the exo genes, involved in production of an extracellular polysaccharide, and the ndv genes, needed for production of a periplasmic cyclic glucan. Mutations in the exoD gene result in altered exopolysaccharide production and in a nodule invasion defect. In this work we show that the stage of symbiotic arrest of exoD mutants is similar to that of other exo and ndv mutants. However, the effects of exoD mutations on exopolysaccharide production and growth on various media are different from the effects of other exo and ndv mutations. Finally, exoD mutations behave differently from other exo mutations in their ability to be suppressed or complemented extracellularly. The results suggest that exoD represents a new class of Rhizobium genes required for nodule invasion, distinct from the other exo genes and the ndv genes. We discuss models for the function of exoD.     

Exogenous suppression of the symbiotic deficiencies of Rhizobium meliloti exo mutants.

The acidic exopolysaccharide (EPS I) produced by Rhizobium meliloti during symbiosis with Medicago sativa has been shown to be required for the proper development of nitrogen-fixing nodules. Cloned DNA from the exo region of R. meliloti is shown to stimulate production of the low-molecular-weight form of this exopolysaccharide, and in this report we show that the symbiotic deficiencies of two exo mutants of R. meliloti, the exoA and exoH mutants, can be rescued by the addition of this low-molecular-weight material at the time of inoculation. For exoA and exoH mutants, rescue with a preparation containing low-molecular-weight exopolysaccharide induces the formation of nitrogen-fixing nodules which appear somewhat later and at a reduced efficiency compared with wild-type-induced nodules; however, microscopic analysis of these nodules reveals similar nodule morphology and the presence of large numbers of bacteroids in each.     

Suppression of the ndv mutant phenotype of Rhizobium meliloti by cloned exo genes

The ndvA and ndvB genes of Rhizobium meliloti are involved in the export and synthesis, respectively, of the small cyclic polysaccharide beta(1,2)glucan. We have previously shown that spontaneous symbiotic pseudorevertants of ndv mutants do not produce periplasmic beta(1,2)glucan. Here we show that the pseudorevertants also do not produce extracellular beta(1,2)glucan, but do show alterations in the amount of the major acidic exopolysaccharide produced. This exopolysaccharide is not detectably different from that produced by the wild type or by the ndv mutants. A cosmid which suppresses the symbiotic defect of both ndvA and ndvB mutants was isolated from a gene bank prepared from DNA of an ndvA pseudorevertant. This cosmid contains a number of exo genes, including exoH and exoF. Subcloning and Tn5 mutagenesis were used to show that the widely separated exoH and exoF genes are both involved in suppression of the ndv mutant phenotype and that the 3.5 kb DNA fragment which contains the exoH gene does not carry the mutation responsible for second site suppression.     

Heterologous exopolysaccharide production in Rhizobium sp. strain NGR234 and consequences for nodule development.

Rhizobium sp. strain NGR234 produces large amounts of acidic exopolysaccharide. Mutants that fail to synthesize this exopolysaccharide are also unable to nodulate the host plant Leucaena leucocephala. A hybrid strain of Rhizobium sp. strain NGR234 containing exo genes from Rhizobium meliloti was constructed. The background genetics and nod genes of Rhizobium sp. strain NGR234 are retained, but the cluster of genes involved in exopolysaccharide biosynthesis was deleted. These exo genes were replaced with genes required for the synthesis of succinoglycan exopolysaccharide from R. meliloti. As a result of the genetic manipulation, the ability of these hybrids to synthesize exopolysaccharide was restored, but the structure was that of succinoglycan and not that of Rhizobium sp. strain NGR234. The replacement genes were contained on a cosmid which encoded the entire known R. meliloti exo gene cluster, with the exception of exoB. Cosmids containing smaller portions of this exo gene cluster did not restore exopolysaccharide production. The presence of succinoglycan was indicated by staining with the fluorescent dye Calcofluor, proton nuclear magnetic resonance spectroscopy, and monosaccharide analysis. Although an NGR234 exoY mutant containing the R. meliloti exo genes produced multimers of the succinoglycan repeat unit, as does the wild-type R. meliloti, the deletion mutant of Rhizobium sp. strain NGR234 containing the R. meliloti exo genes produced only the monomer. The deletion mutant therefore appeared to lack a function that affects the multiplicity of succinoglycan produced in the Rhizobium sp. strain NGR234 background. Although these hybrid strains produced succinoglycan, they were still able to induce the development of an organized nodule structure on L. leucocephala. The resulting nodules did not fix nitrogen, but they did contain infection threads and bacteroids within plant cells. This clearly demonstrated that a heterologous acidic exopolysaccharide structure was sufficient to enable nodule development to proceed beyond the developmental barrier imposed on mutants of Rhizobium sp. strain NGR234 that are unable to synthesize any acidic exopolysaccharide.     

The ExpR/Sin quorum-sensing system controls succinoglycan production in Sinorhizobium meliloti

Sinorhizobium meliloti is a gram-negative soil bacterium capable of forming a symbiotic nitrogen-fixing relationship with its plant host, Medicago sativa. Various bacterially produced factors are essential for successful nodulation. For example, at least one of two exopolysaccharides produced by S. meliloti (succinoglycan or EPS II) is required for nodule invasion. Both of these polymers are produced in high- and low-molecular-weight (HMW and LMW, respectively) fractions; however, only the LMW forms of either succinoglycan or EPS II are active in nodule invasion. The production of LMW succinoglycan can be generated by direct synthesis or through the depolymerization of HMW products by the action of two specific endoglycanases, ExsH and ExoK. Here, we show that the ExpR/Sin quorum-sensing system in S. meliloti is involved in the regulation of genes responsible for succinoglycan biosynthesis as well as in the production of LMW succinoglycan. Therefore, quorum sensing, which has been shown to regulate the production of EPS II, also plays an important role in succinoglycan biosynthesis.     

Biochemical characterization of avirulent exoC mutants of Agrobacterium tumefaciens.

The synthesis of periplasmic beta(1-2)glucan is required for crown gall tumor formation by Agrobacterium tumefaciens and for effective nodulation of alfalfa by Rhizobium meliloti. The exoC (pscA) gene is required for this synthesis by both bacteria as well as for the synthesis of capsular polysaccharide and normal lipopolysaccharide. We tested the possibility that the pleiotropic ExoC phenotype is due to a defect in the synthesis of an intermediate common to several polysaccharide biosynthetic pathways. Cytoplasmic extracts from wild-type A. tumefaciens and from exoC mutants of A. tumefaciens containing a cloned wild-type exoC gene synthesized in vitro UDP-glucose from glucose, glucose 1-phosphate, and glucose 6-phosphate. Extracts from exoC mutants synthesized UDP-glucose from glucose 1-phosphate but not from glucose or glucose 6-phosphate. Membranes from exoC mutant cells synthesized beta(1-2)glucan in vitro when exogenous UDP-glucose was added and contained the 235-kilodalton protein, which has been shown to carry out this synthesis in wild-type cells. We conclude that the inability of exoC mutants to synthesize beta(1-2)glucan is due to a deficiency in the activity of the enzyme phosphoglucomutase (EC 2.7.5.1), which in wild-type bacteria converts glucose 6-phosphate to glucose 1-phosphate, an intermediate in the synthesis of UDP-glucose. This interpretation can account for all of the deficiencies in polysaccharide synthesis which have been observed in these mutants.     

Two genes that regulate exopolysaccharide production in Rhizobium meliloti.

We describe a new Rhizobium meliloti gene, exoX, that regulates the synthesis of the exopolysaccharide, succinoglycan, exoX resembled the psi gene of R. leguminosarum bv. phaseoli and the exoX gene of Rhizobium sp. strain NGR234 in its ability to inhibit exopolysaccharide synthesis when present in multiple copies, exoX did not appear to regulate the expression of exoP. The effect of exoX was counterbalanced by another R. meliloti gene, exoF. exoF is equivalent to Rhizobium sp. strain NGR234 exoY and resembles R. leguminosarum bv. phaseoli pss2 in its mutant phenotype and in portions of its deduced amino acid sequence. The effect of exoF on the succinoglycan-inhibiting activity of exoX depended on the relative copy numbers of the two genes. exoX-lacZ fusions manifested threefold-higher beta-galactosidase activities in exoF backgrounds than in the wild-type background. exoX mutants produced increased levels of succinoglycan. However, the exoF gene was required for succinoglycan synthesis even in an exoX mutant background. exoF did not affect the expression of exoP. Strains containing multicopy exoX formed non-nitrogen-fixing nodules on alfalfa that resembled nodules formed by exo mutants defective in succinoglycan synthesis. exoX mutants formed nitrogen-fixing nodules, indicating that, if the inhibition of succinoglycan synthesis within the nodule is necessary for nitrogen fixation, then exoX is not required for this inhibition. We present indirect evidence that succinoglycan synthesis within the nodule is not necessary for bacteroid function.     

CbrA is a stationary-phase regulator of cell surface physiology and legume symbiosis in Sinorhizobium meliloti

Sinorhizobium meliloti produces an exopolysaccharide called succinoglycan that plays a critical role in promoting symbiosis with its host legume, alfalfa (Medicago sativa). We performed a transposon mutagenesis and screened for mutants with altered succinoglycan production and a defect in symbiosis. In this way, we identified a putative two-component histidine kinase associated with a PAS sensory domain, now designated CbrA (calcofluor-bright regulator A). The cbrA::Tn5 mutation causes overproduction of succinoglycan and results in increased accumulation of low-molecular-weight forms of this exopolysaccharide. Our results suggest the cbrA::Tn5 allele leads to this succinoglycan phenotype through increased expression of exo genes required for succinoglycan biosynthesis and modification. Interestingly, CbrA-dependent regulation of exo and exs genes is observed almost exclusively during stationary-phase growth. The cbrA::Tn5 mutant also has an apparent cell envelope defect, based on increased sensitivity to a number of toxic compounds, including the bile salt deoxycholate and the hydrophobic dye crystal violet. Growth of the cbrA mutant is also slowed under oxidative-stress conditions. The CbrA-regulated genes exsA and exsE encode putative inner membrane ABC transporters with a high degree of similarity to lipid exporters. ExsA is homologous to the Escherichia coli MsbA protein, which is required for lipopolysaccharide transport, while ExsE is a member of the eukaryotic family of ABCD/hALD peroxisomal membrane proteins involved in transport of very long-chain fatty acids, which are a unique component of the lipopolysaccharides of alphaproteobacteria. Thus, CbrA could play a role in regulating the lipopolysaccharide or lipoprotein components of the cell envelope.     

Rhizobium meliloti mutants that fail to succinylate their calcofluor-binding exopolysaccharide are defective in nodule invasion

We have identified a set of Tn5-generated mutants of Rhizobium meliloti on the basis of their failure to form a fluorescent halo under UV light when grown on agar medium containing Calcofluor. These mutations define a new genetic locus we have termed exoH. Alfalfa seedlings inoculated with exoH mutants form ineffective nodules that do not contain intracellular bacteria or bacteroids. Root hair curling is significantly delayed and infection threads abort in the nodule cortex. Analyses of exopolysaccharide secreted by exoH mutants have shown that it is identical to the Calcofluor-binding exopolysaccharide secreted by the exoH+ parental strain except for the fact that it completely lacks the succinyl modification. In vitro translation of total RNA isolated from nodules induced by an exoH mutant has shown that only one of the plant-encoded nodulins is induced, as compared with the 17 nodulins induced by wild-type strains. These observations suggest that succinylation of the bacterial polysaccharide is important for its role(s) in nodule invasion and possibly nodule development.     

A symbiotic mutant of Sinorhizobium meliloti reveals a novel genetic pathway involving succinoglycan biosynthetic functions

A large-scale screen for symbiotic mutants was carried out using the model root nodulating bacterium Sinorhizobium meliloti . Several mutations in the previously uncharacterized gene msbA2 were isolated. msbA2 encodes a member of the ATP-binding cassette exporter family. This protein family is known to export a wide variety of compounds from bacterial cells. S. meliloti MsbA2 is required for the invasion of nodule tissue, with msbA2 mutant cells stimulating nodule primordium morphogenesis, but failing to invade plant tissue beyond the epidermal cell layer. msbA2 mutants do not exhibit any of the free-living traits often found to correlate with symbiotic defects, suggesting that MsbA2 may take part in a specifically symbiotic function. In strains that overproduce the symbiotic signalling polysaccharide succinoglycan, loss of MsbA2 function is extremely deleterious. This synthetic lethal phenotype can be suppressed by disrupting the succinoglycan biosynthetic genes exoY or exoA . It can also be suppressed by disrupting putative glycosyltransferase-encoding genes found upstream of msbA2 . Finally, the symbiotic phenotype of a msbA2 null mutant is suppressed by secondary mutations in these upstream transferase genes, indicating that the msbA2 mutant phenotype may be caused by an inhibitory accumulation of a novel polysaccharide that is synthesized from succinoglycan precursors.     

Genetic analyses of Rhizobium meliloti exopolysaccharides

We have recently obtained strong genetic evidence that the acidic Calcofluor-binding exopolysaccharide (EPS I) of Rhizobium meliloti Rm1021 is required for nodule invasion and possibly for later events in nodule development. Thirteen loci on the second megaplasmid have been identified that are required for, or affect, the synthesis of EPS I. Mutations in certain of these loci completely abolish the production of EPS I and result in mutants that form empty Fix- nodules. exoH mutants fail to succinylate their EPS I and form empty Fix- nodules. We have identified two unlinked regulatory loci, exoR and exoS, whose products play negative roles in the regulation of expression of the exo genes. We have recently discovered that R. meliloti has a latent capacity to synthesize a second exopolysaccharide (EPS II) that can substitute for the role(s) of EPS I in nodulation of alfalfa but not of other hosts. Possible roles for Rhizobium exopolysaccharides in nodulation are discussed.     

Induction of the second exopolysaccharide (EPSb) in Rhizobium meliloti SU47 by low phosphate concentrations.

In previous work, Rhizobium meliloti SU47 produced its alternative exopolysaccharide (EPSb [also called EPS II]) only in strains that were genetically altered to activate EPSb synthesis. Here we report that EPSb synthesis is not entirely cryptic but occurred under conditions of limiting phosphate. This was shown in several different exo mutants that are blocked in the synthesis of the normal exopolysaccharide, succinoglycan. In addition, EPSb biosynthetic gene expression was markedly increased by limiting phosphate. An apparent regulatory mutant that does not express alkaline phosphatase activity was unable to produce EPSb under these conditions. A mucR mutant that was previously shown to produce EPSb instead of the normal exopolysaccharide, succinoglycan, was not sensitive to phosphate inhibition of EPSb synthesis. No evidence was found to indicate that exoX, which affects succinoglycan synthesis, had any influence on EPSb synthesis. In contrast to limiting phosphate, limiting nitrogen or sulfur did not stimulate EPSb synthesis as it does succinoglycan.     

Genes needed for the modification, polymerization, export, and processing of succinoglycan by Rhizobium meliloti: a model for succinoglycan biosynthesis.

The major acidic exopolysaccharide of Rhizobium meliloti, termed succinoglycan, is required for nodule invasion and possibly nodule development. Succinoglycan is a polymer of octasaccharide subunits composed of one galactose residue, seven glucose residues, and acetyl, succinyl, and pyruvyl modifications, which is synthesized on an isoprenoid lipid carrier. A cluster of exo genes in R. meliloti are required for succinoglycan production, and the biosynthetic roles of their gene products have recently been determined (T.L. Reuber and G. C. Walker, Cell 74:269-280, 1993). Our sequencing of 16 kb of this cluster of exo genes and further genetic analysis of this region resulted in the discovery of several new exo genes and has allowed a correlation of the genetic map with the DNA sequence. In this paper we present the sequences of genes that are required for the addition of the succinyl and pyruvyl modifications to the lipid-linked intermediate and genes required for the polymerization of the octasaccharide subunits or the export of succinoglycan. In addition, on the basis of homologies to known proteins, we suggest that ExoN is a uridine diphosphoglucose pyrophosphorylase and that ExoK is a beta(1,3)-beta (1,4)-glucanase. We propose a model for succinoglycan biosynthesis and processing which assigns roles to the products of nineteen exo genes.     

Isolation and characterization of mutants of Rhizobium leguminosarum bv. viciae 248 with altered lipopolysaccharides: possible role of surface charge or hydrophobicity in bacterial release from the infection thread

Effects of alterations in lipopolysaccharide (LPS) structure of Rhizobium leguminosarum bv. viciae on effective symbiosis and on a number of cell surface characteristics were studied. Tn5 mutants with altered LPSs were screened for their inability to bind monoclonal antibody 3, one of three monoclonal antibodies to the tentative O-antigenic part of the wild-type LPS of strain 248. Ten class I LPS mutants completely lacked the O-antigen-containing LPS species. The class II LPS mutant had a severely diminished amount of an antigenically altered O-antigen-containing LPS. The class III LPS mutant had normal amounts of an altered, O-antigen-containing LPS. Class I and II mutants, but not the class III mutant, showed abnormal nodule development (i.e., blocked in the stage of bacterial release from the infection thread) resulting in nodules in which very few, at the most, plant cells contained bacteroids and which were unable to fix nitrogen. Class I and II mutants were nonmotile and were more sensitive to hydrophobic compounds than the parent strain. The most striking difference between the symbiotically defective class I and II LPS mutants on one hand and the wild-type strain and the class III mutant on the other hand was that the class I and II mutants have a more hydrophobic cell surface and a higher electrophoretic mobility. A role for an O-antigen-containing LPS in bacterial release from the infection thread, through its effects on general physicochemical cell surface characteristics, is proposed.     

Alfalfa root nodule invasion efficiency is dependent on Sinorhizobium meliloti polysaccharides

The soil bacterium Sinorhizobium meliloti is capable of entering into a nitrogen-fixing symbiosis with Medicago sativa (alfalfa). Particular low-molecular-weight forms of certain polysaccharides produced by S. meliloti are crucial for establishing this symbiosis. Alfalfa nodule invasion by S. meliloti can be mediated by any one of three symbiotically important polysaccharides: succinoglycan, EPS II, or K antigen (also referred to as KPS). Using green fluorescent protein-labeled S. meliloti cells, we have shown that there are significant differences in the details and efficiencies of nodule invasion mediated by these polysaccharides. Succinoglycan is highly efficient in mediating both infection thread initiation and extension. However, EPS II is significantly less efficient than succinoglycan at mediating both invasion steps, and K antigen is significantly less efficient than succinoglycan at mediating infection thread extension. In the case of EPS II-mediated symbioses, the reduction in invasion efficiency results in stunted host plant growth relative to plants inoculated with succinoglycan or K-antigen-producing strains. Additionally, EPS II- and K-antigen-mediated infection threads are 8 to 10 times more likely to have aberrant morphologies than those mediated by succinoglycan. These data have important implications for understanding how S. meliloti polysaccharides are functioning in the plant-bacterium interaction, and models are discussed.     

Molecular dissection of structure and function in the lipopolysaccharide of Rhizobium leguminosarum strain 3841 using monoclonal antibodies and genetic analysis

Following treatment with nitrosoguanidine, mutant derivatives of Rhizobium leguminosarum strain 3841 were isolated which failed to react with AFRC MAC 203. This monoclonal antibody normally recognizes a strain-specific lipopolysaccharide epitope which is developmentally regulated during legume nodule differentiation. Structural modification of lipopolysaccharide (LPS) was analysed by examining reactivity with a range of monoclonal antibodies with different epitope specificities, and also by analysis of LPS mobility changes after electrophoresis on polyacrylamide gels. One class of these LPS-defective mutants induced normal nitrogen-fixing (Fix+) nodules on peas (Pisum sativum), while another two classes of Fix- mutants were also identified, suggesting that a component of the LPS antigen that is part of the MAC 203 epitope is essential for normal nodule development leading to symbiotic nitrogen fixation. When grown under low-oxygen or low-pH culture conditions, one class of Fix- mutants completely lacked LPS-1 (the species that carries O antigen) and a second class showed a modified and truncated form of LPS-1. Mutants with defective LPS structure were also obtained after Tn5 mutagenesis of R. leguminosarum 3841 and all nine Fix- mutants were also found to lack the MAC 203 epitope. Three of these transposon-induced mutants synthesized a truncated form of LPS-1 that was structurally similar to that of the class of the NTG-induced mutants described above. These transposon-induced mutations, and the nitrosoguanidine-induced Fix- mutations, were closely linked and could be suppressed by the same cloned fragment of chromosomal DNA. The data presented here suggest that a precondition for normal nodule development of R. leguminosarum 3841 within pea nodules is the ability to synthesize relatively long-chain LPS-1 macromolecules under the physiological conditions encountered within the nodule. All mutants that lacked the ability to elongate LPS-1 macromolecules also failed to express the MAC 203 epitope.     

Rhizobium meliloti exopolysaccharides: genetic analyses and symbiotic importance.

Genetic experiments have indicated that succinoglycan (EPS I), the acidic Calcofluor-binding exopolysaccharide, of the nitrogen-fixing bacterium Rhizobium meliloti strain Rm1021 is required for nodule invasion and possibly for later events in nodule development on alfalfa and other hosts. Fourteen exo loci on the second megaplasmid have been identified that are required for, or affect, the synthesis of EPS I. Mutations in certain of these loci completely abolish the production of EPS I and result in mutants that form empty Fix- nodules. We have identified two loci, exoR and exoS, that are involved in the regulation of EPS I synthesis in the free-living state. Certain exo mutations which completely abolish EPS I production are lethal in an exoR95 or exoS96 background. Histochemical analyses of the expression of exo genes during nodulation using exo::TnphoA fusions have indicated that the exo genes are expressed most strongly in the invasion zone. In addition, we have discovered that R. meliloti has a latent capacity to synthesize a second exopolysaccharide (EPS II) that can substitute for the role(s) of EPS I in nodulation of alfalfa but not of other hosts. Possible roles for exopolysaccharides in symbiosis are discussed.