Functional and evolutionary relatedness of genes for exopolysaccharide synthesis in Rhizobium meliloti and Rhizobium sp. strain NGR234.

Rhizobium meliloti SU47 and Rhizobium sp. strain NGR234 produce distinct exopolysaccharides that have some similarities in structure. R. meliloti has a narrow host range, whereas Rhizobium strain NGR234 has a very broad host range. In cross-species complementation and hybridization experiments, we found that several of the genes required for the production of the two polysaccharides were functionally interchangeable and similar in evolutionary origin. NGR234 exoC and exoY corresponded to R. meliloti exoB and exoF, respectively. NGR234 exoD was found to be an operon that included genes equivalent to exoM, exoA, and exoL in R. meliloti. Complementation of R. meliloti exoP, -N, and -G by NGR234 R'3222 indicated that additional equivalent genes remain to be found on the R-prime. We were not able to complement NGR234 exoB with R. meliloti DNA. In addition to functional and evolutionary equivalence of individual genes, the general organization of the exo regions was similar between the two species. It is likely that the same ancestral genes were used in the evolution of both exopolysaccharide biosynthetic pathways and probably of pathways in other species as well.     

Rhizobium sp. strain NGR234 NodZ protein is a fucosyltransferase.

Rhizobium sp. strain NGR234 produces a large family of lipochitooligosaccharide Nod factors carrying specific substituents. Among them are 3-O- (or 4-O-) and 6-O-carbamoyl groups, an N-methyl group, and a 2-O-methylfucose residue which may bear either 3-O-sulfate or 4-O-acetyl substitutions. Investigations on the genetic control of host specificity revealed a number of loci which directly affect Nod factor structure. Here we show that insertion and frameshift mutations in the nodZ gene abolish fucosylation of Nod factors. In vitro assays using GDP-L-fucose as the fucose donor show that fucosyltransferase activity is associated with the nodZ gene product (NodZ). NodZ is located in the soluble protein fraction of NGR234 cells. Together with extra copies of the nodD1 gene, the nodZ gene and its associated nod box were introduced into ANU265, which is NGR234 cured of the symbiotic plasmid. Crude extracts of this transconjugant possess fucosyltransferase activity. Fusion of a His6 tag to the NodZ protein expressed in Escherichia coli yielded a protein able to fucosylate both nonfucosylated NodNGR factors and oligomers of chitin. NodZ is inactive on monomeric N-acetyl-D-glucosamine and on desulfated Rhizobium meliloti Nod factors. Kinetic analyses showed that the NodZ protein is more active on oligomers of chitin than on nonfucosylated NodNGR factors. Pentameric chitin is the preferred substrate. These data suggest that fucosylation occurs before acylation of the Nod factors.     

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.     

Two genes that regulate exopolysaccharide production in Rhizobium sp. strain NGR234: DNA sequences and resultant phenotypes.

Two closely linked genes involved in the regulation of exopolysaccharide (EPS) production in Rhizobium sp. strain NGR234, exoX and exoY, were sequenced, and their corresponding phenotypes were investigated. Inhibition of EPS synthesis occurred in wild-type strains when extra copies of exoX were introduced, but only when exoY had been deleted or mutated or was present at a lower copy number. Normal EPS synthesis occurred in Rhizobium sp. when both exoX and exoY were introduced on the same replicon. Surprisingly, the presence of multiple copies of exoY in exoY:: Tn5 mutants of NGR234 adversely affected cellular growth. This was apparent when exoY was introduced into exoY mutants on IncP1 vectors, where the copy number was approximately 10, but was not apparent when present on much larger R-prime plasmids with lower copy numbers (approximately 3 per cell). Multiple copies of exoX did not adversely affect cellular growth of any strain. The exoX gene appeared analogous, in size and phenotype, to a previously described Rhizobium leguminosarum biovar phaseoli EPS gene, psi (D. Borthakur and A.W.B. Johnston, Mol. Gen. Genet. 207:149-154, 1987), and the deduced ExoX and Psi shared strikingly similar secondary structures. Despite this, ExoX and Psi showed little homology at the primary amino acid level, except for a central region of 18 amino acids. The interaction of ExoX and ExoY could form the basis of a sensitive regulatory system for EPS acids. The interaction of ExoX and ExoY could form the basis of a sensitive regulatory system for EPS biosynthesis. The presence of a multicopy exoX in Rhizobium meliloti and R. fredii similarly abolished EPS biosynthesis in these species.     

Role of BacA in lipopolysaccharide synthesis, peptide transport, and nodulation by Rhizobium sp. strain NGR234

BacA of Sinorhizobium meliloti plays an essential role in the establishment of nitrogen-fixing symbioses with Medicago plants, where it is involved in peptide import and in the addition of very-long-chain fatty acids (VLCFA) to lipid A of lipopolysaccharide (LPS). We investigated the role of BacA in Rhizobium species strain NGR234 by mutating the bacA gene. In the NGR234 bacA mutant, peptide import was impaired, but no effect on VLCFA addition was observed. More importantly, the symbiotic ability of the mutant was comparable to that of the wild type for a variety of legume species. Concurrently, an acpXL mutant of NGR234 was created and assayed. In rhizobia, AcpXL is a dedicated acyl carrier protein necessary for the addition of VLCFA to lipid A. LPS extracted from the NGR234 mutant lacked VLCFA, and this mutant was severely impaired in the ability to form functional nodules with the majority of legumes tested. Our work demonstrates the importance of VLCFA in the NGR234-legume symbiosis and also shows that the necessity of BacA for bacteroid differentiation is restricted to specific legume-Rhizobium interactions.     

An evolutionary hot spot: the pNGR234b replicon of Rhizobium sp. strain NGR234

Rhizobium sp. strain NGR234 has an exceptionally broad host range and is able to nodulate more than 112 genera of legumes. Since the overall organization of the NGR234 genome is strikingly similar to that of the narrow-host-range symbiont Rhizobium meliloti strain 1021 (also known as Sinorhizobium meliloti), the obvious question is why are the spectra of hosts so different? Study of the early symbiotic genes of both bacteria (carried by the SymA plasmids) did not provide obvious answers. Yet, both rhizobia also possess second megaplasmids that bear, among many other genes, those that are involved in the synthesis of extracellular polysaccharides (EPSs). EPSs are involved in fine-tuning symbiotic interactions and thus may help answer the broad- versus narrow-host-range question. Accordingly, we sequenced two fragments (total, 594 kb) that encode 575 open reading frames (ORFs). Comparisons revealed 19 conserved gene clusters with high similarity to R. meliloti, suggesting that a minimum of 28% (158 ORFs) of the genetic information may have been acquired from a common ancestor. The largest conserved cluster carried the exo and exs genes and contained 31 ORFs. In addition, nine highly conserved regions with high similarity to Agrobacterium tumefaciens C58, Bradyrhizobium japonicum USDA110, and Mesorhizobium loti strain MAFF303099, as well as two conserved clusters that are highly homologous to similar regions in the plant pathogen Erwinia carotovora, were identified. Altogether, these findings suggest that >/==" BORDER="0">40% of the pNGR234b genes are not strain specific and were probably acquired from a wide variety of other microbes. The presence of 26 ORFs coding for transposases and site-specific integrases supports this contention. Surprisingly, several genes involved in the degradation of aromatic carbon sources and genes coding for a type IV pilus were also found.     

Lotus japonicus Nodulates and Fixes Nitrogen with the Broad Host Range Rhizobium sp. NGR234

Lotus japonicus possesses major advantages as a model legumefor the study of plant-microbe interactions. The relative absenceof genetic information on its normal microbial partner (i.e.,Mesorhizobium loti) could limit its utility in research. Herewe show for the first time that the broad host range Rhizobiumstrain NGR234 nodulates and fixes nitrogen in symbiosis withLotus japonicus ecotypes "Gifu" and "Funakura". We demonstratethat bacterial mutants deficient in nodulation or nitrogen fixationpossess the expected phenotype with L.japonicus. Nodulationof L.japonicus was sensitive to nitrate. Vermiculite was anefficient synthetic growth substrate, allowing axenic growthin Magenta jars. The genetic analysis of the Lotus japonicus-Mesorhizobiuminteraction should be accelerated through the use of this well-definedmicrosymbiont. (Received December 5, 1998; Accepted April 26, 1999)     

Protein-protein interactions within type III secretion system-dependent pili of Rhizobium sp. strain NGR234

Pili synthesized by the type III secretion system of Rhizobium species strain NGR234 are essential for protein secretion and thus for efficient symbiosis with many legumes. Isolation and partial purification of these pili showed that they are composed of at least three proteins, NopA, NopB, and NopX. Using biochemical assays, we show here that these proteins interact directly with one another.     

NopA is associated with cell surface appendages produced by the type III secretion system of Rhizobium sp. strain NGR234

Rhizobium sp. strain NGR234, which is capable of interacting with a large number of legumes, utilizes a variety of signaling molecules to establish nitrogen-fixing symbioses. Among these are nodulation outer proteins (Nops) that transit through a type III secretion system (TTSS). Abolition of Nop secretion affects nodulation of certain legumes. Under free-living conditions, the secretion of Nops can be induced by the addition of flavonoids. Here, we show that an in-frame deletion of nopA abolishes secretion of all other Nops and has the same impact on nodule formation as mutations that lead to a nonfunctional TTSS. This secretion-minus phenotype of the nopA mutant, as well as bioinformatics analysis of NopA itself, suggests that NopA could be an external component of the TTSS. Electron microscopy showed that NGR234 synthesizes fibrillar structures on the cell surface in a flavonoid-inducible and NopA-dependent manner. Purification of the macromolecular surface appendages revealed that NopA is a major component of these structures.     

Exo-oligosaccharides of Rhizobium sp. strain NGR234 are required for symbiosis with various legumes

Rhizobia are nitrogen-fixing bacteria that establish endosymbiotic associations with legumes. Nodule formation depends on various bacterial carbohydrates, including lipopolysaccharides, K-antigens, and exopolysaccharides (EPS). An acidic EPS from Rhizobium sp. strain NGR234 consists of glucosyl (Glc), galactosyl (Gal), glucuronosyl (GlcA), and 4,6-pyruvylated galactosyl (PvGal) residues with beta-1,3, beta-1,4, beta-1,6, alpha-1,3, and alpha-1,4 glycoside linkages. Here we examined the role of NGR234 genes in the synthesis of EPS. Deletions within the exoF, exoL, exoP, exoQ, and exoY genes suppressed accumulation of EPS in bacterial supernatants, a finding that was confirmed by chemical analyses. The data suggest that the repeating subunits of EPS are assembled by an ExoQ/ExoP/ExoF-dependent mechanism, which is related to the Wzy polymerization system of group 1 capsular polysaccharides in Escherichia coli. Mutation of exoK (NGROmegaexoK), which encodes a putative glycanase, resulted in the absence of low-molecular-weight forms of EPS. Analysis of the extracellular carbohydrates revealed that NGROmegaexoK is unable to accumulate exo-oligosaccharides (EOSs), which are O-acetylated nonasaccharide subunits of EPS having the formula Gal(Glc)5(GlcA)2PvGal. When used as inoculants, both the exo-deficient mutants and NGROmegaexoK were unable to form nitrogen-fixing nodules on some hosts (e.g., Albizia lebbeck and Leucaena leucocephala), but they were able to form nitrogen-fixing nodules on other hosts (e.g., Vigna unguiculata). EOSs of the parent strain were biologically active at very low levels (yield in culture supernatants, approximately 50 microg per liter). Thus, NGR234 produces symbiotically active EOSs by enzymatic degradation of EPS, using the extracellular endo-beta-1,4-glycanase encoded by exoK (glycoside hydrolase family 16). We propose that the derived EOSs (and not EPS) are bacterial components that play a crucial role in nodule formation in various legumes.     

Characterization of NopP, a type III secreted effector of Rhizobium sp. strain NGR234

The type three secretion system (TTSS) encoded by pNGR234a, the symbiotic plasmid of Rhizobium sp. strain NGR234, is responsible for the flavonoid- and NodD1-dependent secretion of nodulation outer proteins (Nops). Abolition of secretion of all or specific Nops significantly alters the nodulation ability of NGR234 on many of its hosts. In the closely related strain Rhizobium fredii USDA257, inactivation of the TTSS modifies the host range of the mutant so that it includes the improved Glycine max variety McCall. To assess the impact of individual TTSS-secreted proteins on symbioses with legumes, various attempts were made to identify nop genes. Amino-terminal sequencing of peptides purified from gels was used to characterize NopA, NopL, and NopX, but it failed to identify SR3, a TTSS-dependent product of USDA257. By using phage display and antibodies that recognize SR3, the corresponding protein of NGR234 was identified as NopP. NopP, like NopL, is an effector secreted by the TTSS of NGR234, and depending on the legume host, it may have a deleterious or beneficial effect on nodulation or it may have little effect.     

Purification and phosphorylation of the effector protein NopL from Rhizobium sp. NGR234

Bacterial pathogens use type III secretion systems (TTSSs) to deliver virulence factors into eukaryotic cells. These effectors perturb host-defence responses, especially signal transduction pathways. A functional TTSS was identified in the symbiotic, nitrogen-fixing bacterium Rhizobium sp. NGR234. NopL (formerly y4xL) of NGR234 is a putative symbiotic effector that modulates nodulation in legumes. To test whether NopL could interact with plant proteins, in vitro phosphorylation experiments were performed using recombinant nopL protein purified from Escherichia coli as well as protein extracts from Lotus japonicus and tobacco plants. NopL serves as a substrate for plant protein kinases as well as purified protein kinase A. Phosphorylation of NopL was inhibited by the Ser/Thr kinase inhibitor K252a as well as by PD98059, a mitogen-activated protein (MAP) kinase kinase inhibitor. It thus seems likely that, after delivery into the plant cell, NopL modulates MAP kinase pathways.     

NopB, a type III secreted protein of Rhizobium sp. strain NGR234, is associated with pilus-like surface appendages

Rhizobium sp. strain NGR234 possesses a functional type three secretion system (TTSS), through which a number of proteins, called nodulation outer proteins (Nops), are delivered to the outside of the cell. A major constraint to the identification of Nops is their low abundance in the supernatants of NGR234 strains grown in culture. To overcome this limitation, a more sensitive proteomics-based strategy was developed. Secreted proteins from wild-type NGR234 were separated by two-dimensional gel electrophoresis, and the gel was compared to similar gels containing the proteins from a TTSS mutant (NGROmegarhcN). To identify the proteins, spots unique to the NGR234 gels were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and the data were compared to the sequence of the symbiotic plasmid of NGR234. A nonpolar mutant of one of these proteins was generated called NopB. NopB is required for Nop secretion but inhibits the interaction with Pachyrhizus tuberosus and augments nodulation of Tephrosia vogelii. Flavonoids and a functional TTSS are required for the formation of some surface appendages on NGR234. In situ immunogold labeling and isolation of these pili showed that they contain NopB.     

Flavonoid-inducible modifications to rhamnan O antigens are necessary for Rhizobium sp. strain NGR234-legume symbioses

Rhizobium sp. strain NGR234 produces a flavonoid-inducible rhamnose-rich lipopolysaccharide (LPS) that is important for the nodulation of legumes. Many of the genes encoding the rhamnan part of the molecule lie between 87 degrees and 110 degrees of pNGR234a, the symbiotic plasmid of NGR234. Computational methods suggest that 5 of the 12 open reading frames (ORFs) within this arc are involved in synthesis (and subsequent polymerization) of L-rhamnose. Two others probably play roles in the transport of carbohydrates. To evaluate the function of these ORFs, we mutated a number of them and tested the ability of the mutants to nodulate a variety of legumes. At the same time, changes in the production of surface polysaccharides (particularly the rhamnan O antigen) were examined. Deletion of rmlB to wbgA and mutation in fixF abolished rhamnan synthesis. Mutation of y4gM (a member of the ATP-binding cassette transporter family) did not abolish production of the rhamnose-rich LPS but, unexpectedly, the mutant displayed a symbiotic phenotype very similar to that of strains unable to produce the rhamnan O antigen (NGRDeltarmlB-wbgA and NGROmegafixF). At least two flavonoid-inducible regulatory pathways are involved in synthesis of the rhamnan O antigen. Mutation of either pathway reduces rhamnan production. Coordination of rhamnan synthesis with rhizobial release from infection threads is thus part of the symbiotic interaction.