Rhizobium leguminosarum bv. trifolii PssP protein is required for exopolysaccharide biosynthesis and polymerization

Rhizobium leguminosarum bv. trifolii produces an acidic exopolysaccharide (EPS) that is important for the induction of nitrogen-fixing nodules on clover. Recently, three genes, pssN, pssO, and pssP, possibly involved in EPS biosynthesis and polymerization were identified. The predicted protein product of the pssP gene shows a significant sequence similarity to other proteins belonging to the PCP2a family that are involved in the synthesis of high-molecular-weight EPS. An R. leguminosarum bv. trifolii TA1 mutant with the entire coding region of pssP deleted did not produce the EPS. A pssP mutant with the 5' end of the gene disrupted produced exclusively low-molecular-weight EPS. A mutant that synthesized a functional N-terminal periplasmic domain but lacked the C-terminal part of PssP produced significantly reduced amounts of EPS with a slightly changed low to high molecular form ratio. Mutants affected in the PssP protein carrying a stable plasmid with a constitutively expressed gusA gene induced nodules on red clover that were not fully occupied by bacteria. A mutant with the entire pssP gene deleted infected only a few plant cells in the nodule. The pssP promoter-gusA reporter fusion was active in bacteroids during nodule development.     

Isolation and characterization of Azospirillum brasilense loci that correct Rhizobium meliloti exoB and exoC mutations.

The occurrence in Azospirillum brasilense of genes that code for exopolysaccharide (EPS) synthesis was investigated through complementation studies of Rhizobium meliloti Exo- mutants. These mutants are deficient in the synthesis of the major acidic EPS of Rhizobium species and form empty, non-nitrogen-fixing root nodules on alfalfa (J. A. Leigh, E. R. Signer, and G. C. Walker, Proc. Natl. Acad. Sci. USA 82:6231-6235, 1985). We demonstrated that the exoC mutation of R. meliloti could be corrected for EPS production by several cosmid clones of a clone bank of A. brasilense ATCC 29145. However, the EPS produced differed in structure from the wild-type R. meliloti EPS, and the symbiotic deficiency of the exoC mutation was not reversed by any of these cosmid clones. The exoB mutation could be corrected not only for EPS production but also for the ability to form nitrogen-fixing nodules on alfalfa by one particular cosmid clone of A. brasilense. Tn5 insertions in the cloned DNA were isolated and used to construct Azospirillum mutants with mutations in the corresponding loci by marker exchange. It was found that these mutants failed to produce the wild-type high-molecular-weight EPS, but instead produced EPSs of lower molecular weight.     

Membrane topology of PssT,the transmembrane protein component of the type I exopolysaccharide transport system in Rhizobium leguminosarum bv. trifolii strain TA1

The pssT gene was identified as the fourth gene located upstream of the pssNOP gene cluster possibly involved in the biosynthesis, polymerization, and transport of exopolysaccharide (EPS) in Rhizobium leguminosarum bv. trifolii strain TA1. The hydropathy profile and homology searches indicated that PssT belongs to the polysaccharide-specific transport family of proteins, a component of the type I system of the polysaccharide transport. The predicted membrane topology of the PssT protein was examined with a series of PssT-PhoA fusion proteins and a complementary set of PssT-LacZ fusions. The results generally support a predicted topological model for PssT consisting of 12 transmembrane segments, with amino and carboxyl termini located in the cytoplasm. A mutant lacking the C-terminal part of PssT produced increased amounts of total EPS with an altered distribution of high- and low-molecular-weight forms in comparison to the wild-type RtTA1 strain. The PssT mutant produced an increased number of nitrogen fixing nodules on clover.     

Nitrogen fixation ability of exopolysaccharide synthesis mutants of Rhizobium sp. strain NGR234 and Rhizobium trifolii is restored by the addition of homologous exopolysaccharides.

Several transposon Tn5-induced mutants of the broad-host-range Rhizobium sp. strain NGR234 produce little or no detectable acidic exopolysaccharide (EPS) and are unable to induce nitrogen-fixing nodules on Leucaena leucocephala var. Peru or siratro plants. The ability of these Exo- mutants to induce functioning nodules on Leucaena plants was restored by coinoculation with a Sym plasmid-cured (Nod- Exo+) derivative of parent strain NGR234, purified EPS from the parent strain, or the oligosaccharide from the EPS. Coinoculation with EPS or related oligosaccharide also resulted in formation of nitrogen-fixing nodules on siratro plants. In addition, an Exo- mutant (ANU437) of Rhizobium trifolii ANU794 was able to form nitrogen-fixing nodules on white clover in the presence of added EPS or related oligosaccharide from R. trifolii ANU843. These results demonstrate that the absence of Rhizobium EPSs can result in failure of effective symbiosis with both temperate and subtropical legumes.     

Characterization of polysaccharides of Rhizobium meliloti exo mutants that form ineffective nodules.

Mutants of Rhizobium meliloti SU47 with defects in the production of the Calcofluor-binding expolysaccharide succinoglycan failed to gain entry into alfalfa root nodules. In order to define better the polysaccharide phenotypes of these exo mutants, we analyzed the periplasmic oligosaccharide cyclic (1-2)-beta-D-glucan and lipopolysaccharide (LPS) in representative mutants. The exoC mutant lacked the glucan and had abnormal LPS which appeared to lack a substantial portion of the O side chain. The exoB mutant had a spectrum of LPS species which differed from those of both the wild-type parental strain and the exoC mutant. The presence of the glucan and normal LPS in the exoA, exoD, exoF, and exoH mutants eliminated defects in these carbohydrates as explanations for the nodule entry defects of these mutants. We also assayed for high- and low-molecular-weight succinoglycans. All of the exo mutants except exoD and exoH completely lacked both forms. For the Calcofluor-dim exoD mutant, the distribution of high- and low-molecular-weight forms depended on the growth medium. The haloless exoH mutant produced high-molecular-weight and only a trace of low-molecular-weight succinoglycan; the succinyl modification was missing, as was expected from the results of previous studies. The implications of these observations with regard to nodule entry are discussed.     

Host specificity of Rhizobium strains isolated from nitrogen-fixing trees and nitrogenase activities of strain GRH2 in symbiosis with Prosopis chilensis

Host plant specificity was examined in symbiosis between Rhizobium strains isolated from legume-tree root nodules and herbaceous or woody legumes from which they were isolated. Strain GRH2 isolated from Acacia cyanophylla formed effective nodules on Acacia, Prosopis and Medicago sativa as well. Nitrogenase activity, measured as acetylene reduction, of strain GRH2 in symbiosis with Prosopis chilensis was the highest (P 0.05) among the tropical legumes studied and was similar to those found for other associations involving herbaceous legumes. Relative efficiency of nitrogenase varied from 0.3 to near 1 during the light time of the photoperiod. However no hydrogen uptake activity was detected by the amperometric method used. Rhizobium strains GRH3, GRH5 and GRH9 isolated from A. melanoxylon, P. chilensis and Sophora microphylla, respectively, also showed a very low host-range specificity. All isolates were infective and effective on at least one of the herbaceous legumes tested. These data demonstrate the lack of specificity of Rhizobium strains isolated from nitrogen-fixing tree root nodules and that these strains can form effective nodules on herbaceous legumes.     

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.     

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.     

Exopolysaccharide mutants of Rhizobium loti are fully effective on a determinate nodulating host but are ineffective on an indeterminate nodulating host.

By Tn5 mutagenesis of Rhizobium loti PN184 (NZP2037 str-1) and selection for nonfluorescence of colonies on Calcofluor agar, eight independently generated expolysaccharide (EPS) mutants (three smooth and five rough) were isolated. The parent strain, PN184, was found to produce an acidic EPS. This EPS was produced. with reduced O acetylation, by the smooth EPS mutants but not by the rough EPS mutants. Lipopolysaccharide was isolated from all mutants and was identical to that of PN184 as defined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All mutants were resistant to lysis by R. loti bacteriophage phi 2037/1. Cosmids that complemented the mutations in the rough EPS mutants were isolated from a pLAFR1 gene library of NZP2037 by complementation of the nonfluorescent phenotype. The genes identified were shown to be unlinked and located on the chromosome. All mutants were fully effective when inoculated onto Lotus pedunculatus, a determinate nodulating host, but were ineffective, inducing the formation of very small nodules or tumorlike growths, when inoculated onto Leucaena leucocephala, an indeterminate nodulating host. These results, obtained in an isogenic Rhizobium background, support suggestions that acidic EPS is required for effective nodulation of indeterminate nodulating legumes but is not required for effective nodulation of determinate nodulating legumes.     

Relevance of Fucose-Rich Extracellular Polysaccharides Produced by Rhizobium sullae Strains Nodulating Hedysarum coronarium L. Legumes

Specific and complex interactions between soil bacteria, known as rhizobia, and their leguminous host plants result in the development of root nodules. This process implies a complex dialogue between the partners. Rhizobia synthesize different classes of polysaccharides: exopolysaccharides (EPS), Kdo-rich capsular polysaccharides, lipopolysaccharides, and cyclic β-(1,2)-glucans. These polymers are actors of a successful symbiosis with legumes. We focus here on studying the EPS produced by Rhizobium sullae bacteria that nodulate Hedysarum coronarium L., largely distributed in Algeria. We describe the influence of the carbon source on the production and on the composition of EPS produced by R. sullae A6 and RHF strains. High-molecular-weight EPS preserve the bacteria from desiccation. The structural characterization of the EPS produced by R. sullae strains has been performed through sugar analysis by gas chromatography-mass spectrometry. The low-molecular-weight EPS of one strain (RHF) has been totally elucidated using nuclear magnetic resonance and quantitative time-of-flight tandem mass spectrometry analyses. An unusual fucose-rich EPS has been characterized. The presence of this deoxy sugar seems to be related to nodulation capacity.     

Exopolysaccharides of Rhizobium leguminosarum biovar trifolii harbouring cloned exo region.

Rhizobium leguminosarum bv. trifolii produces an acidic exopolysaccharide (EPS) which plays an important role in the development of nitrogen-fixing nodules. Tn5 mutant of R. trifolii 93 defective in EPS production (Exo-) forms ineffective (Fix-) nodules on red clover. This Exo- mutation is complemented by the pARF1368 and pARF25 cosmids isolated from gene bank of Rhizobium trifolii TA1, but the complementation is not correlated with restoration of Fix+ phenotype. Furthermore, these cosmids introduced to wild-type of R. trifolii 24 repress its ability to form nitrogen-fixing nodules. These results might suggest that bacteria with cosmids carrying the exo region form EPS of altered structure. It has been shown by 1H-n.m.r. that exopolysaccharides produced by R. trifolii 93pARF-1368 and 93pARF25 contain less non-carbohydrate residues (acetyl, pyruvyl and 3-hydroxybutanoyl) than the wild type EPS. These data suggest that the biological activity of the exopolysaccharide of R. trifolii depends on the contents of the non-carbohydrate substitutions.     

Suppression of the Fix- phenotype of Rhizobium meliloti exoB mutants by lpsZ is correlated to a modified expression of the K polysaccharide.

The rhizobial production of extracellular polysaccharide (EPS) is generally required for the symbiotic infection of host plants that form nodules with an apical meristem (indeterminate nodules). One exception is Rhizobium meliloti AK631, an exoB mutant of Rm41, which is deficient in EPS production yet infects and fixes nitrogen (i.e., is Fix+) on alfalfa, an indeterminate nodule-forming plant. A mutation of lpsZ in AK631 results in a Fix- strain with altered phage sensitivity, suggesting that a cell surface factor may substitute for EPS in the alfalfa-AK631 symbiosis. Biochemical analyses of the cell-associated polysaccharides of AK631 and Rm5830 (AK631 lpsZ) demonstrated that the lpsZ mutation affected the expression of a surface polysaccharide that is analogous to the group II K polysaccharides of Escherichia coli; the polysaccharide contains 3-deoxy-D-manno-2-octulosonic acid or a derivative thereof in each repeating unit. Rm5830 produced a polysaccharide with altered chromatographic and electrophoretic properties, indicating a difference in the molecular weight range. Similar results were obtained in a study of Rm1021, a wild-type isolate that lacks the lpsZ gene: the introduction of lpsZ into Rm1021 exoB (Rm6903) both suppresses the Fix- phenotype and results in a modified expression of the K polysaccharide. Chromatography and electrophoresis analysis showed that the polysaccharide extracted from Rm6903 lpsZ+ differed from that of Rm6903 in molecular weight range. Importantly, the effect of LpsZ is not structurally specific, as the introduction lpsZ+ into Rhizobium fredii USDA257 also resulted in a molecular weight range change in the structurally distinct K polysaccharide produced by that strain. This evidence suggests that LpsZ has a general effect on the size-specific expression of rhizobial K polysaccharides.     

The exopolysaccharide of Rhizobium sp. YAS34 is not necessary for biofilm formation on Arabidopsis thaliana and Brassica napus roots but contributes to root colonization

Microbial exopolysaccharides (EPSs) play key roles in plant–microbe interactions, such as biofilm formation on plant roots and legume nodulation by rhizobia. Here, we focused on the function of an EPS produced by Rhizobium sp. YAS34 in the colonization and biofilm formation on non-legume plant roots ( Arabidopsis thaliana and Brassica napus ). Using random transposon mutagenesis, we isolated an EPS-deficient mutant of strain YAS34 impaired in a glycosyltransferase gene ( gta ). Wild type and mutant strains were tagged with a plasmid-born GFP and, for the first time, the EPS produced by the wild-type strain was seen in the rhizosphere using selective carbohydrate probing with a fluorescent lectin and confocal laser-scanning microscopy. We show for the fist time that Rhizobium forms biofilms on roots of non-legumes, independently of the EPS synthesis. When produced by strain YAS34 wild type, EPS is targeted at specific parts of the plant root system. Nutrient fluctuations, root exudates and bacterial growth phase can account for such a production pattern. The EPS synthesis in Rhizobium sp. YAS34 is not essential for biofilm formation on roots, but is critical to colonization of the basal part of the root system and increasing the stability of root-adhering soil. Thus, in Rhizobium sp. YAS34 and non-legume interactions, microbial EPS is implicated in root–soil interface, root colonization, but not in biofilm formation.     

Specific Amino Acid Substitutions in the Proline-Rich Motif of the Rhizobium meliloti ExoP Protein Result in Enhanced Production of Low-Molecular-Weight Succinoglycan at the Expense of High-Molecular-Weight Succinoglycan

The production of the acidic exopolysaccharide succinoglycan (EPS I) by Rhizobium meliloti exoP* mutants expressing an ExoP protein lacking its C-terminal cytoplasmic domain and by mutants characterized by specific amino acid substitutions in the proline-rich motif (RX₄PX₂PX₄SPKX₉IXGXMXGXG) located from positions 443 to 476 of the ExoP protein was analyzed. The absence of the C-terminal cytoplasmic ExoP domain (positions 484 to 786) and the substitution of both arginine₄₄₃ by isoleucine₄₄₃ and proline₄₅₇ by serine₄₅₇ within the proline-rich motif resulted in enhanced production of low-molecular-weight (LMW) EPS I at the expense of high-molecular-weight (HMW) EPS I. The ratios of HMW to LMW EPS I of the wild type and mutant strains increased with osmolarity.     

Altered exopolysaccharides of Bradyrhizobium japonicum mutants correlate with impaired soybean lectin binding, but not with effective nodule formation

 The exact mechanism(s) of infection and symbiotic development between rhizobia and legumes is not yet known, but changes in rhizobial exopolysaccharides (EPSs) affect both infection and nodule development of the legume host. Early events in the symbiotic process between Bradyrhizobium japonicum and soybean (Glycinemax [L.] Merr.) were studied using two mutants, defective in soybean lectin (SBL) binding, which had been generated from B. japonicum 2143 (USDA 3I-1b-143 derivative) by Tn5 mutagenesis. In addition to their SBL-binding deficiency, these mutants produced less EPS than the parental strain. The composition of EPS varied with the genotype and with the carbon source used for growth. When grown on arabinose, gluconate, or mannitol, the wild-type parental strain, B. japonicum 2143, produced EPS typical of DNA homology group I Bradyrhizobium, designated EPS I. When grown on malate, strain 2143 produced a different EPS composed only of galactose and its acetylated derivative and designated EPS II. Mutant 1252 produced EPS II when grown on arabinose or malate, but when grown on gluconate or mannitol, mutant 1252 produced a different EPS comprised of glucose, galactose, xylose and glucuronic acid (1:5:1:1) and designated EPS III. Mutant 1251, grown on any of these carbon sources, produced EPS III. The EPS of strain 2143 and mutant 1252 contained SBL-binding polysaccharide. The amount of the SBL-binding polysaccharide produced by mutant 1252 varied with the carbon source used for growth. The capsular polysaccharide (CPS) produced by strain 2143 during growth on arabinose, gluconate or mannitol, showed a high level of SBL binding, whereas CPS produced during growth of strain 2143 on malate showed a low level of SBL binding. However, the change in EPS composition and SBL binding of strain 2143 grown on malate did not affect the wild-type nodulation and nitrogen fixation phenotype of 2143. Mutant 1251, which produced EPS III, nodulated 2 d later than parental strain 2143, but formed effective, nitrogen-fixing tap root nodules. Mutant 1252, which produced either EPS II or III, however nodulated 5–6 d later and formed few and ineffective tap root nodules. Restoration of EPS I production in mutant 1252 correlated with restored SBL binding, but not with wild-type nodulation and nitrogen fixation. Received: 6 October 1999 / Accepted: 18 November 1999     

Construction of a single-transposon-insertion mutant in Rhizobium sp. strain TAL1145 from a double-insertion mutant

A method for developing a single-transposon-insertion mutant from a double-insertion mutant in Rhizobium is described. An exopolysaccharide (EPS)-defective mutant containing two Tn 5-lacZ insertions was complemented with cloned wild-type DNA for EPS synthesis. One of the Tn 5-lacZ insertions from the mutant was transferred to the complementing plasmid by homologous recombination. The plasmid containing the Tn 5-lacZ insertion in the gene involved in EPS synthesis was transferred into the wild-type strain and the Tn 5-lacZ was homogenized to obtain an EPS-defective mutant with a single Tn 5-lacZ insertion.     

Nodule formation in soybeans by exopolysaccharide mutants of Rhizobium fredii USDA 191

Production of exopolysaccharides by Rhizobium has been linked with efficient invasion and nodulation of leguminous plant roots by the bacteria. Exopolysaccharide-deficient (exo) mutants of Rhizobium fredii USDA 191 were isolated following Tn5-insertion mutagenesis. Five phenotypically unique exo mutants were investigated for exopolysaccharide synthesis and their ability to nodulate soybeans. The exopolysaccharides produced by these mutants were analysed for polysaccharide composition by column chromatography and thin-layer chromatography. Two mutants designed exo-3 and exo-5 were deficient in both neutral glucan and exopolysaccharide synthesis, but each induced some functional nodules on Glycine max (Peking). The remaining three mutants (exo-1, exo-2 and exo-4) synthesized neutral glucans at levels higher or lower than those in wild-type and exhibited partial exopolysaccharide deficiencies. The data imply that neither exopolysaccharides nor neutral glucans are essential for the induction of determinate nodules by R. fredii.     

Rhizobium meliloti exoG and exoJ mutations affect the exoX-exoY system for modulation of exopolysaccharide production.

R. meliloti Rm1021 normally produces an acidic Calcofluor-binding exopolysaccharide, called succinoglycan or EPS I, which is required for successful nodulation of alfalfa by this strain. At least 13 loci affecting production of EPS I were previously mapped to a cluster on the second of two symbiotic megaplasmids in Rm1021, pRmeSU47b. A putative regulatory region was originally defined by the exoG and exoJ mutations. exoG and exoJ mutants produced less exopolysaccharide than wild-type strains and induced nitrogen-fixing nodules on alfalfa with reduced efficiency compared with the wild type. These mutants appeared to produce only a low-molecular-weight form of EPS I. Mutations called exoX cause an increase in exopolysaccharide production and map in the same region as the exoG and exoJ mutations. The DNA sequence of this region reveals that it contains two open reading frames, called exoX and exoY, which have homologs in other Rhizobium species. Interestingly, the exoG insertion mutations fall in an intergenic region and may affect the expression of exoX or exoY. The exoJ mutation falls in the 3' portion of the exoX open reading frame and is probably an allele of exoX that results in altered function. exoG and exoJ mutations limit EPS I production in the presence of exoR95 or exoS96 mutations, which cause overproduction of EPS I. Gene regulation studies suggest that ExoX and ExoY constitute a system that modulates exopolysaccharide synthesis at a posttranslational level. The deduced sequence of ExoY is homologous to a protein required for an early step in xanthan gum biosynthesis, further suggesting that the modulatory system may affect the exopolysaccharide biosynthetic apparatus.     

Structure of a polysaccharide from a Rhizobium species containing 2-deoxy-beta-D-arabino-hexuronic acid.

The structure of the extracellular polysaccharide (EPS) produced by the Rhizobium sp. B strain isolated from atypical nodules on alfalfa has been determined using a combination of chemical and physical techniques (methylation analysis, high pH-anion exchange chromatography (HPAEC), mass spectrometry and 1-D and 2-D NMR spectroscopy). As opposed to the EPS from other strains of Rhizobium, the EPS from the sp. B strain contains D-Glc together with L-Rha and 2-deoxy-D-arabino-hexuronic acid. It is a polymer of a repeating unit having the following structure: --> 4)-beta-D-Glcp-(1 --> 4)-alpha-L-Rhap -(1 --> 3)-beta-D-Glcp-(1 --> 4)-2-deoxy-beta-D-GlcpA-(1 -->. The polysaccharide also contains 0.6 O-acetyl groups per sugar which have not been located.