Rhizobium meliloti chromosomal loci required for suppression of exopolysaccharide mutations by lipopolysaccharide.

Mutants of alfalfa symbiont Rhizobium meliloti SU47 that fail to make extracellular polysaccharide (exo mutants) induce the formation of nodules that are devoid of bacteria and consequently do not fix nitrogen. This Fix- phenotype can be suppressed by an R. meliloti Rm41 gene that affects lipopolysaccharide structure. Here we describe mutations preventing suppression that map at two new chromosomal loci, lpsY and lpsX, present in both strains. Two other lps mutations isolated previously from SU47 also prevented suppression.     

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.     

lpsZ, a lipopolysaccharide gene involved in symbiosis of Rhizobium meliloti.

lpsZ+ is an allele that allows exo (exopolysaccharide-deficient) mutants of Rhizobium meliloti to invade nodules by modifying rhizobial lipopolysaccharide. We have cloned and sequenced the lpsZ gene. The predicted LpsZ protein has a molecular weight of 48,589 and is probably localized in the cytoplasm. A beta-glucuronidase fusion in the lpsZ gene indicates that lpsZ is not regulated by oxygen or nitrogen.     

Rhizobium meliloti lipopolysaccharide and exopolysaccharide can have the same function in the plant-bacterium interaction.

A fix region of Rhizobium meliloti 41 involved both in symbiotic nodule development and in the adsorption of bacteriophage 16-3 was delimited by directed Tn5 mutagenesis. Mutations in this DNA region were assigned to four complementation units and were mapped close to the pyr-2 and pyr-29 chromosomal markers. Phage inactivation studies with bacterial cell envelope preparations and crude lipopolysaccharides (LPS) as well as preliminary characterization of LPS in the mutants indicated that these genes are involved in the synthesis of a strain-specific LPS. Mutations in this DNA region resulted in a Fix- phenotype in AK631, an exopolysaccharide (EPS)-deficient derivative of R. meliloti 41; however, they did not influence the symbiotic efficiency of the parent strain. An exo region able to restore the EPS production of AK631 was isolated and shown to be homologous to the exoB region of R. meliloti SU47. By generating double mutants, we demonstrated that exo and lps genes determine similar functions in the course of nodule development, suggesting that EPS and LPS may provide equivalent information for the host plant.     

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.     

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.     

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.     

Regulation of Rhizobium meliloti exo genes in free-living cells and in planta examined by using TnphoA fusions.

The exo loci of Rhizobium meliloti are necessary for the production of an acidic exopolysaccharide, EPS I, that is needed for alfalfa nodule invasion by strain Rm1021. We have isolated and characterized alkaline phosphatase fusions made with TnphoA in several exo loci of R. meliloti and used these fusions to examine the subcellular localization of exo gene products and the regulation of exo genes in free-living cells and in planta. In the course of this work, we isolated a new exo locus, exoT. We have obtained evidence that several of the exo loci may encode membrane proteins. The activity of TnphoA fusions in several exo loci is increased two- to fivefold in the presence of the regulatory mutations exoR95 and exoS96. While examining the regulation of the exo gens by exoR95 and exoS96, we found that certain classes of exo mutations are lethal in an exoR95 or exoS96 background unless a plasmid complementing the exo mutation is present. This result has possible implications for the role of these exo loci in EPS I biosynthesis. We have developed a method for staining nodules specifically for the alkaline phosphatase activity present in the inducing bacteria and used this method to show that an exoF::TnphoA fusion is expressed mainly in the invasion zone of the nodule.     

Strain-ecotype specificity in Sinorhizobium meliloti-Medicago truncatula symbiosis is correlated to succinoglycan oligosaccharide structure

Molecular signals, including Nod factors and succinoglycan, are necessary for the establishment of nitrogen-fixing nodules (Fix+) in Medicago truncatula-Sinorhizobium meliloti symbiosis. This report shows that M. truncatula-S. meliloti interactions involve ecotype-strain specificity, as S. meliloti Rm41 and NRG247 are Fix+ (compatible) on M. truncatula A20 and Fix- (incompatible) on M. truncatula A17, the Fix phenotypes are reversed with S. meliloti NRG185 and NRG34, and there is a correlation between the host specificity and succinoglycan oligosaccharide structure. S. meliloti NRG185 produces oligosaccharides that are almost fully succinylated, with two succinate groups per subunit, whereas the oligosaccharides produced by S. meliloti Rm41 include many nonsuccinylated subunits, as well as subunits with a single succinate group and others with malate. The results of this study demonstrated the following: (i) incompatibility is not a consequence of an avirulence factor or lack of Nod factor activity; (ii) the Fix+ phenotypes are succinoglycan dependent; (iii) there is structural variability in the succinoglycan oligosaccharide populations between S. meliloti strains; (iv) the structural nature of the succinoglycan oligosaccharides is correlated to compatibility; most importantly, (v) an S. meliloti Rm41 derivative, carrying exo genes from an M. truncatula A17-compatible strain, produced a modified population of succinoglycan oligosaccharides (similar to the donor strain) and was Fix+ on A17.     

Interaction of nod and exo Rhizobium meliloti in alfalfa nodulation

Among the genes of Rhizobium meliloti SU47 that affect nitrogen-fixing symbiosis with alfalfa are nod genes, in which mutations block nodule induction, and exo genes, in which mutations allow nodule formation but block rhizobial exopolysaccharide production as well as nodule invasion and nitrogen fixation. To investigate whether an exo+ bacterium can "help" (that is, reverse the symbiotic defect of) an exo mutant in trans, we have coinoculated alfalfa with pairs of rhizobia of different genotypes. Coinoculant genotypes were chosen so that the exo+ helper strain was nif while the exo "indicator" strain was nif+, and thus any fixation observed was carried out by the exo coinoculant. We find that a nod exo+ coinoculant can help an exo mutant both to invade nodules and to fix nitrogen. However, a nod+ exo+ coinoculant cannot help an exo mutant: Few exo bacteria are recovered from nodules, some bacteroids differentiate into bizarre aberrant forms, and the nodules fail to fix nitrogen. In a triple coinoculation, the effect of nod+ helper supersedes that of nod helper. Implications of these results for interaction of nod and exo gene products are discussed.     

A positive correlation between bacterial autoaggregation and biofilm formation in native Sinorhizobium meliloti isolates from Argentina

Sinorhizobium meliloti is a symbiotic nitrogen-fixing bacterium that elicits nodule formation on roots of alfalfa plants. S. meliloti produces two exopolysaccharides (EPSs), termed EPS I and EPS II, that are both able to promote symbiosis. EPS I and EPS II are secreted in two major fractions that reflect differing degrees of subunit polymerization, designated high- and low-molecular-weight fractions. We reported previously that EPSs are crucial for autoaggregation and biofilm formation in S. meliloti reference strains and isogenic mutants. However, the previous observations were obtained by use of "domesticated" laboratory strains, with mutations resulting from successive passages under unnatural conditions, as has been documented for reference strain Rm1021. In the present study, we analyzed the autoaggregation and biofilm formation abilities of native S. meliloti strains isolated from root nodules of alfalfa plants grown in four regions of Argentina. 16S rRNA gene analysis of all the native isolates revealed a high degree of identity with reference S. meliloti strains. PCR analysis of the expR gene of all the isolates showed that, as in the case of reference strain Rm8530, this gene is not interrupted by an insertion sequence (IS) element. A positive correlation was found between autoaggregation and biofilm formation abilities in these rhizobia, indicating that both processes depend on the same physical adhesive forces. Extracellular complementation experiments using mutants of the native strains showed that autoaggregation was dependent on EPS II production. Our results indicate that a functional EPS II synthetic pathway and its proper regulation are essential for cell-cell interactions and surface attachment of S. meliloti.     

Rhizobium leguminosarum exoB mutants are deficient in the synthesis of UDP-glucose 4'-epimerase

Rhizobium leguminosarum bv. viciae Exo- mutant strains RBL5523,exo7::Tn5,RBL5523,exo8::Tn5 and RBL5523,exo52::Tn5 are affected in nodulation and in the syntheses of lipopolysaccharide, capsular polysaccharide, and exocellular polysaccharide. These mutants were complemented for nodulation and for the syntheses of these polysaccharides by plasmid pMP2603. The gene in which these mutants are defective is functionally homologous to the exoB gene of Rhizobium meliloti. The repeating unit of the residual amounts of EPS still made by the exoB mutants of R. leguminosarum bv. viciae lacks galactose and the substituents attached to it. The R. leguminosarum bv. viciae and R. meliloti exoB mutants fail to synthesize active UDP-glucose 4'-epimerase.     

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.     

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.     

Environmental regulation of exopolysaccharide production in Sinorhizobium meliloti

Exopolysaccharide production by Sinorhizobium meliloti is required for invasion of root nodules on alfalfa and successful establishment of a nitrogen-fixing symbiosis between the two partners. S. meliloti wild-type strain Rm1021 requires production of either succinoglycan, a polymer of repeating octasaccharide subunits, or EPS II, an exopolysaccharide of repeating dimer subunits. The reason for the production of two functional exopolysaccharides is not clear. Earlier reports suggested that low-phosphate conditions stimulate the production of EPS II in Rm1021. We found that phosphate concentrations determine which exopolysaccharide is produced by S. meliloti. The low-phosphate conditions normally found in the soil (1 to 10 microM) stimulate EPS II production, while the high-phosphate conditions inside the nodule (20 to 100 mM) block EPS II synthesis and induce the production of succinoglycan. Interestingly, the EPS II produced by S. meliloti in low-phosphate conditions does not allow the invasion of alfalfa nodules. We propose that this invasion phenotype is due to the lack of the active molecular weight fraction of EPS II required for nodule invasion. An analysis of the function of PhoB in this differential exopolysaccharide production is presented.     

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.     

Common loci for Agrobacterium tumefaciens and Rhizobium meliloti exopolysaccharide synthesis and their roles in plant interactions.

Mutants of Rhizobium meliloti have been isolated which are deficient in exopolysaccharide (EPS) production and effective nodulation of alfalfa (J. A. Leigh, E. R. Signer, and G. C. Walker, Proc. Natl. Acad. Sci. USA 82:6231-6235, 1985). We isolated approximately 100 analogous EPS-deficient (Exo) mutants of the closely related plant pathogen Agrobacterium tumefaciens, including strains whose EPS deficiencies were specifically complemented by each of five cloned R. meliloti exo loci. We also cloned A. tumefaciens genes which complemented EPS defects in three of the R. meliloti Exo mutants. In two of these cases, symbiotic defects were also complemented. All of the A. tumefaciens Exo mutants formed normal crown gall tumors on four different plant hosts, except ExoC mutants, which were nontumorigenic and unable to attach to plant cells in vitro. Like their R. meliloti counterparts, A. tumefaciens Exo mutants were deficient in production of succinoglycan, the major acidic EPS species produced by both genera. A. tumefaciens ExoC mutants also produced extremely low levels of another major EPS, cyclic 1,2-beta-D-glucan. This deficiency has been noted previously in a different set of nontumorigenic, attachment-defective A. tumefaciens mutants.     

苜蓿中华根瘤菌(Sinorhizobium meliloti)LuxR家族转录因子ExpR调节motC操纵子的表达

目前已知苜蓿中华根瘤菌(S.meliloti)Rm1021 ExpR 突变导致胞外多糖Ⅱ(EPSⅡ)的过量表达,而胞外多糖是根瘤菌成功侵染宿主植物形成有效根瘤必需的物质。软琼脂板实验发现ExpR 突变株运动能力有缺陷。但是鞭毛染色实验并没有检测到突变株的鞭毛与野生型有什么不同。通过启动子-lacZ融合子进一步研究突变株中基因表达的差异发现,ExpR以细胞密度依赖的方式调节motC操纵子的表达。由此可见,在苜蓿中华根瘤菌中,ExpR同时参与了胞外多糖Ⅱ的合成和细胞运动能力的调节。     

The acetyl substituent of succinoglycan is not necessary for alfalfa nodule invasion by Rhizobium meliloti Rm1021.

Rhizobium meliloti Rm1021 requires a Calcofluor-binding exopolysaccharide, termed succinoglycan or EPS I, to invade alfalfa nodules. We have determined that a strain carrying a mutation in the exoZ locus produces succinoglycan that lacks the acetyl substituent. The exoZ mutant nodules alfalfa normally.