Symbiotic properties and first analyses of the genomic sequence of the fast growing model strain Sinorhizobium fredii HH103 nodulating soybean

Glycine max (soybean) plants can be nodulated by fast-growing rhizobial strains of the genus Sinorhizobium as well as by slow-growing strains clustered in the genus Bradyrhizobium. Fast-growing rhizobia strains with different soybean cultivar specificities have been isolated from Chinese soils and from other geographical regions. Most of these strains have been clustered into the species Sinorhizobium fredii. The S. fredii strain HH103 was isolated from soils of Hubei province, Central China and was first described in 1985. This strain is capable to nodulate American and Asiatic soybean cultivars and many other different legumes and is so far the best studied fast-growing soybean-nodulating strain. Additionally to the chromosome S. fredii HH103 carries five indigenous plasmids. The largest plasmid (pSfrHH103e) harbours genes for the production of diverse surface polysaccharides, such as exopolysaccharides (EPS), lipopolysaccharides (LPS), and capsular polysaccharides (KPS). The second largest plasmid (pSfrHH103d) is a typical symbiotic plasmid (pSym), carrying nodulation and nitrogen fixation genes. The present mini review focuses on symbiotic properties of S. fredii HH103, in particular on nodulation and surface polysaccharides aspects. The model strain S. fredii HH103 was chosen for genomic sequencing, which is currently in progress. First analyses of the draft genome sequence revealed an extensive synteny between the chromosomes of S. fredii HH103 and Rhizobium sp. NGR234.     

Structure and Biological Roles of Sinorhizobium fredii HH103 Exopolysaccharide

Here we report that the structure of the Sinorhizobium fredii HH103 exopolysaccharide (EPS) is composed of glucose, galactose, glucuronic acid, pyruvic acid, in the ratios 5∶2∶2∶1 and is partially acetylated. A S. fredii HH103 exoA mutant (SVQ530), unable to produce EPS, not only forms nitrogen fixing nodules with soybean but also shows increased competitive capacity for nodule occupancy. Mutant SVQ530 is, however, less competitive to nodulate Vigna unguiculata. Biofilm formation was reduced in mutant SVQ530 but increased in an EPS overproducing mutant. Mutant SVQ530 was impaired in surface motility and showed higher osmosensitivity compared to its wild type strain in media containing 50 mM NaCl or 5% (w/v) sucrose. Neither S. fredii HH103 nor 41 other S. fredii strains were recognized by soybean lectin (SBL). S. fredii HH103 mutants affected in exopolysaccharides (EPS), lipopolysaccharides (LPS), cyclic glucans (CG) or capsular polysaccharides (KPS) were not significantly impaired in their soybean-root attachment capacity, suggesting that these surface polysaccharides might not be relevant in early attachment to soybean roots. These results also indicate that the molecular mechanisms involved in S. fredii attachment to soybean roots might be different to those operating in Bradyrhizobium japonicum.     

Melanin Production by Rhizobium Strains

Different Rhizobium and Bradyrhizobium strains were screened for their ability to produce melanin. Pigment producers (Mel⁺) were found among strains of R. leguminosarum biovars viceae, trifolii, and phaseoli, R. meliloti, and R. fredii; none of 19 Bradyrhizobium strains examined gave a positive response. Melanin production and nod genes were plasmid borne in R. leguminosarum biovar trifolii RS24. In R. leguminosarum biovar phaseoli CFN42 and R. meliloti GR015, mel genes were located in the respective symbiotic plasmids. In R. fredii USDA 205, melanin production correlated with the presence of its smallest indigenous plasmid.     

Phage susceptibility and plasmid profile analysis of Sinorhizobium fredii

This is the first report identifying bacteriophages and documenting megaplasmids of Sinorhizobium fredii. Plasmid DNA content and bacteriophage typing of eighteen strains of S. fredii were determined. S. fredii strains fell into ten plasmid profile groups containing 1 to 6 plasmids, some evidently larger than 1000 MDa. Twenty-three S. fredii lytic phages were isolated from soil, and they lysed six different S. fredii strains. The host range and plaque morphology of these phages were studied. Susceptibility to S. fredii phages was examined for S. meliloti; Rhizobium leguminosarum bvs. viceae, trifolii and Phaseoli; R. loti; Bradyrhizobium japonicum; B. elkanii and Bradyrhizobium sp. (Arachis). Several phages that originally lysed S. fredii strain USDA 206 also lysed strains of all three S. fredii serogroups described originally by Sadowsky et al. Phages that infected S. fredii strains USDA 191 and USDA 257 were highly specific and lysed only serogroup 193 strains. S. meliloti strains L5-30 and USDA 1005 were lysed by three of the phages that lysed S. fredii strain USDA 217. No other Rhizobium or Bradyrhizobium strain tested was susceptible to lysis by any of the S. fredii phages. The present investigation indicates that phage susceptibility in conjunction with plasmid profile analysis may provide a rapid method for identification and characterization of strains of S. fredii.     

Citrate Synthase Mutants of Sinorhizobium fredii USDA257 Form Ineffective Nodules with Aberrant Ultrastructure

The tricarboxylic acid (TCA) cycle plays an important role in generating the energy required by bacteroids to fix atmospheric nitrogen. Citrate synthase is the first enzyme that controls the entry of carbon into the TCA cycle. We cloned and determined the nucleotide sequence of the gltA gene that encodes citrate synthase in Sinorhizobium fredii USDA257, a symbiont of soybeans (Glycine max [L.] Merr.) and several other legumes. The deduced citrate synthase protein has a molecular weight of 48,198 and exhibits sequence similarity to citrate synthases from several bacterial species, including Sinorhizobium meliloti and Rhizobium tropici. Southern blot analysis revealed that the fast-growing S. fredii strains and Rhizobium sp. strain NGR234 contained a single copy of the gene located in the bacterial chromosome. S. fredii USDA257 gltA mutant HBK-CS1, which had no detectable citrate synthase activity, had diminished nodulation capacity and produced ineffective nodules on soybean. Light and electron microscopy observations revealed that the nodules initiated by HBK-CS1 contained very few bacteroids. The infected cells contained large vacuoles and prominent starch grains. Within the vacuoles, membrane structures that appeared to be reminiscent of disintegrating bacteroids were detected. The citrate synthase mutant had altered cell surface characteristics and produced three times more exopolysaccarides than the wild type produced. A plasmid carrying the USDA257 gltA gene, when introduced into HBK-CS1, was able to restore all of the defects mentioned above. Our results demonstrate that a functional citrate synthase gene of S. fredii USDA257 is essential for efficient soybean nodulation and nitrogen fixation.     

Different species and symbiotic genotypes of field rhizobia can nodulate Phaseolus vulgaris in Tunisian soils

A collection of 160 isolates of rhizobia nodulating Phaseolus vulgaris in three geographical regions in Tunisia was characterized by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplified 16S rDNA, nifH and nodC genes. Nine groups of rhizobia were delineated: Rhizobium gallicum biovar (bv.) gallicum, Rhizobium leguminosarum bv. phaseoli and bv. viciae, Rhizobium etli bv. phaseoli, Rhizobium giardinii bv. giardinii, and four groups related to species of the genus Sinorhizobium, Sinorhizobium meliloti, Sinorhizobium medicae and Sinorhizobium fredii. The most abundant rhizobial species were R. gallicum, R. etli, and R. leguminosarum encompassing 29–20% of the isolates each. Among the isolates assigned to R. leguminosarum, two-thirds were ineffective in nitrogen fixation with P. vulgaris and harbored a symbiotic gene typical of the biovar viciae. The S. fredii-like isolates did not nodulate soybean plants but formed numerous effective nodules on P. vulgaris. Comparison of nodC gene sequences showed that their symbiotic genotype was not related to that of S. fredii, but to that of the S. fredii-like reference strain GR-06, which was isolated from a bean plant grown in a Spanish soil. An additional genotype including 16% of isolates was found to be closely related to species of the genus Agrobacterium. However, when re-examined, these isolates did not nodulate their original host.     

Serological Relatedness of Rhizobium fredii to Other Rhizobia and to the Bradyrhizobia

Several isolates of Rhizobium fredii were examined for their serological relatedness to each other, to Bradyrhizobium japonicum, and to other fast- and slow-growing rhizobia. Immunofluorescence, agglutination, and immunodiffusion analyses indicated that R. fredii contains at least three separate somatic serogroups, USDA 192, USDA 194, and USDA 205. There was no cross-reaction between any of the R. fredii isolates and 13 of the 14 B. japonicum somatic serogroups tested. Cross-reactions were obtained with antisera from R. fredii and serogroup 122 of B. japonicum, Rhizobium meliloti, and several fast-growing Rhizobium spp. for Leucaena, Sesbania, and Lablab species. The serological relationship between R. fredii and R. meliloti was examined in more detail, and of 23 R. meliloti strains examined, 8 shared somatic antigens with the type strains from all three R. fredii serogroups. The serological relatedness of R. fredii to B. japonicum and R. meliloti appears to be unique since the strains are known to be biochemically and genetically diverse.     

The Symbiotic Biofilm of Sinorhizobium fredii SMH12, Necessary for Successful Colonization and Symbiosis of Glycine max cv Osumi,Is Regulated by Quorum Sensing Systems and Inducing Flavonoids via NodD1

Bacterial surface components, especially exopolysaccharides, in combination with bacterial Quorum Sensing signals are crucial for the formation of biofilms in most species studied so far. Biofilm formation allows soil bacteria to colonize their surrounding habitat and survive common environmental stresses such as desiccation and nutrient limitation. This mode of life is often essential for survival in bacteria of the genera Mesorhizobium, Sinorhizobium, Bradyrhizobium, and Rhizobium. The role of biofilm formation in symbiosis has been investigated in detail for Sinorhizobium meliloti and Bradyrhizobium japonicum. However, for S. fredii this process has not been studied. In this work we have demonstrated that biofilm formation is crucial for an optimal root colonization and symbiosis between S. fredii SMH12 and Glycine max cv Osumi. In this bacterium, nod-gene inducing flavonoids and the NodD1 protein are required for the transition of the biofilm structure from monolayer to microcolony. Quorum Sensing systems are also required for the full development of both types of biofilms. In fact, both the nodD1 mutant and the lactonase strain (the lactonase enzyme prevents AHL accumulation) are defective in soybean root colonization. The impairment of the lactonase strain in its colonization ability leads to a decrease in the symbiotic parameters. Interestingly, NodD1 together with flavonoids activates certain quorum sensing systems implicit in the development of the symbiotic biofilm. Thus, S. fredii SMH12 by means of a unique key molecule, the flavonoid, efficiently forms biofilm, colonizes the legume roots and activates the synthesis of Nod factors, required for successfully symbiosis.     

Distribution and diversity of rhizobia associated with wild soybean (Glycine soja Sieb. & Zucc.) in Northwest China

A total of 155 nodule isolates that originated from seven sites in Northwest China were characterized by PCR-RFLP of the 16S rRNA gene and sequence analysis of multiple core genes (16S rRNA, recA, atpD, and glnII) in order to investigate the diversity and biogeography of Glycine soja-nodulating rhizobia. Among the isolates, 80 were Ensifer fredii, 19 were Ensifer morelense, 49 were Rhizobium radiobacter, and 7 were putative novel Rhizobium species. The phylogenies of E. fredii and E. morelense isolates in a concatenate tree (assembly of all housekeeping genes) were generally consistent with those in individual gene trees. However, incongruence was found in the phylogenies of the different genes of Rhizobium isolates, indicating that lateral transfer or recombination possibly occurred in these gene loci. Despite their species identity, all the isolates in this study formed a single lineage related to E. fredii in nodAand nifH gene phylogenies, which also indicated that the symbiotic genes were laterally transferred between different species. Biogeographic patterns were found at the species and strain genomic type levels, as revealed by BOXA1R fingerprinting, demonstrating that the evolution of rhizobial populations in different geographic locations was related to soil types, altitude and spatial effects. This study is the first to report that E. morelense, R. radiobacter, and Rhizobium sp. are microsymbionts of G. soja, as well as showing that the diversity of G. soja rhizobia is enhanced and new rhizobia have evolved in Northwest China.     

Effects of the <Emphasis Type="Italic">purL</Emphasis> Gene Expression Level on the Competitive Nodulation Ability of <Emphasis Type="Italic">Sinorhizobium fredii</Emphasis>

Purine pathway in Rhizobium is important during the nodulation processes. The purL gene in Sinorhizobium fredii (S. fredii) has been identified to be required for the whole establishment of a nitrogen-fixing nodule. To get a better understanding of the purL gene’s impacts on Rhizobium–plant interaction, the competitive nodulation abilities of S. fredii containing different purL expression plasmids were studied. Several kinds of coinoculations were performed, including using different bacterial concentration ratios, with or without the supplementation of purine source in the plant nutrient solution, and the delayed coinoculation tests. The results indicated that the competitive nodule occupancy of S. fredii was affected significantly by the purL expression level during the early nodulation periods. The mutant strain containing no purL expression could not elicit competitive nodules both in the presence and absence of purine source. A positive linear correlation within certain limits was observed between strain’s competitive nodule occupancy and purL gene expression level. All these results suggested that the purL gene played a role in the competitive nodulation of S. fredii.     

NopC Is a Rhizobium-Specific Type 3 Secretion System Effector Secreted by Sinorhizobium (Ensifer) fredii HH103

Sinorhizobium (Ensifer) fredii HH103 is a broad host-range nitrogen-fixing bacterium able to nodulate many legumes, including soybean. In several rhizobia, root nodulation is influenced by proteins secreted through the type 3 secretion system (T3SS). This specialized secretion apparatus is a common virulence mechanism of many plant and animal pathogenic bacteria that delivers proteins, called effectors, directly into the eukaryotic host cells where they interfere with signal transduction pathways and promote infection by suppressing host defenses. In rhizobia, secreted proteins, called nodulation outer proteins (Nops), are involved in host-range determination and symbiotic efficiency. S. fredii HH103 secretes at least eight Nops through the T3SS. Interestingly, there are Rhizobium-specific Nops, such as NopC, which do not have homologues in pathogenic bacteria. In this work we studied the S. fredii HH103 nopC gene and confirmed that its expression was regulated in a flavonoid-, NodD1- and TtsI-dependent manner. Besides, in vivo bioluminescent studies indicated that the S. fredii HH103 T3SS was expressed in young soybean nodules and adenylate cyclase assays confirmed that NopC was delivered directly into soybean root cells by means of the T3SS machinery. Finally, nodulation assays showed that NopC exerted a positive effect on symbiosis with Glycine max cv. Williams 82 and Vigna unguiculata. All these results indicate that NopC can be considered a Rhizobium-specific effector secreted by S. fredii HH103.     

Genetic Analysis Reveals the Essential Role of Nitrogen Phosphotransferase System Components in Sinorhizobium fredii CCBAU 45436 Symbioses with Soybean and Pigeonpea Plants

The nitrogen phosphotransferase system (PTS^Ntr) consists of EI^Ntr, NPr, and EIIA^Ntr. The active phosphate moiety derived from phosphoenolpyruvate is transferred through EI^Ntr and NPr to EIIA^Ntr. Sinorhizobium fredii can establish a nitrogen-fixing symbiosis with the legume crops soybean (as determinate nodules) and pigeonpea (as indeterminate nodules). In this study, S. fredii strains with mutations in ptsP and ptsO (encoding EI^Ntr and NPr, respectively) formed ineffective nodules on soybeans, while a strain with a ptsN mutation (encoding EIIA^Ntr) was not defective in symbiosis with soybeans. Notable reductions in the numbers of bacteroids within each symbiosome and of poly-β-hydroxybutyrate granules in bacteroids were observed in nodules infected by the ptsP or ptsO mutant strains but not in those infected with the ptsN mutant strain. However, these defects of the ptsP and ptsO mutant strains were recovered in ptsP ptsN and ptsO ptsN double-mutant strains, implying a negative role of unphosphorylated EIIA^Ntr in symbiosis. Moreover, the symbiotic defect of the ptsP mutant was also recovered by expressing EI^Ntr with or without the GAF domain, indicating that the putative glutamine-sensing domain GAF is dispensable in symbiotic interactions. The critical role of PTS^Ntr in symbiosis was also observed when related PTS^Ntr mutant strains of S. fredii were inoculated on pigeonpea plants. Furthermore, nodule occupancy and carbon utilization tests suggested that multiple outputs could be derived from components of PTS^Ntr in addition to the negative role of unphosphorylated EIIA^Ntr.     

Nodulation and Nitrogen Fixation Efficacy of Rhizobium fredii with Phaseolus vulgaris Genotypes

Phaseolus plant introduction (PI) genotypes (consisting of 684 P. vulgaris, 26 P. acutifolius, 39 P. lunatus, and 5 P. coccineus accessions) were evaluated for their ability to form effective symbioses with strains of six slow-growing (Bradyrhizobium) and four fast-growing (Rhizobium fredii) soybean rhizobia. Of the 684 P. vulgaris genotypes examined, three PIs were found to form effective nitrogen-fixing symbioses with the R. fredii strains. While none of the Bradyrhizobium strains nodulated any of the genotypes tested, some produced large numbers of undifferentiated root proliferations (hypertrophies). A symbiotic plasmid-cured R. fredii strain failed to nodulate the P. vulgaris PIs and cultivars, suggesting that P. vulgaris host range genes are Sym plasmid borne in the fast-growing soybean rhizobia.     

Ensifer fredii symbiovar vachelliae nodulates endemic Vachellia gummifera in semiarid Moroccan areas

The purpose of this work was to study the genetic diversity of the nodule-forming bacteria associated with native populations of Vachellia gummifera growing wild in Morocco. The nearly complete 16S rRNA gene sequences from three selected strains, following ARDRA and REP-PCR results, revealed they were members of the genus Ensifer and the sequencing of the housekeeping genes recA, gyrB, dnaK and rpoB, and their concatenated phylogenetic analysis, showed that the 3 strains belong to the species E. fredii. Based on the nodC and nodA phylogenies, the 3 strains clearly diverged from the type and other reference strains of E. fredii and formed a clearly separated cluster. The strains AGA1, AGA2 and AGB23 did not form nodules on Glycine max, Phaseolus vulgaris and Medicago truncatula, and effectively nodulated V. gummifera, Acacia cyanophylla, Prosopis chilensis and Leucaena leucocephala. Based on similarities of the nodC and nodA symbiotic genes and differences in the host range, the strains isolated from Moroccan endemic V. gummifera may form a different symbiovar within Ensifer species, for which the name “vachelliae” is proposed.     

Characterization of a class II 5-enopyruvylshikimate-3-phosphate synthase with high tolerance to glyphosate from Sinorhizobium fredii

5-Enopyruvylshikimate-3-phosphate synthase (EPSP synthase) is an important enzyme in the shikimate pathway mediating the biosynthesis of aromatic compounds in plants and microorganisms. A novel class II EPSP synthase AroA _{S. fredii} from Sinorhizobium fredii NGR234 was overexpressed in Escherichia coli BL21. It was purified to homogeneity and its catalytic properties were studied. The enzyme exhibited optimum catalytic activity at pH 8.0 and 50 °C. It was stable below 40 °C, and over a broad range of pH 5.0–9.0. The EPSP synthase was increasingly activated by 100 mM of the chlorides of NH₄ ⁺, K⁺, Na⁺ and Li⁺. Kinetic analysis of AroA _{S. fredii} suggested that the enzyme exhibited a high glyphosate tolerance and high level of affinity for phosphoenolpyruvate, which indicates the enzyme with a high potential for structural and functional studies and its potential usage for the generation of transgenic crops resistant to the herbicide.     

Exopolysaccharide-Deficient Mutants of Rhizobium fredii HH303 Which Are Symbiotically Effective

Nineteen Tn5-induced mutants of Rhizobium fredii HH303 defective in acidic exopolysaccharide synthesis were isolated by screening for lack of Calcofluor fluorescence. They were grouped by complementation analysis by using Rhizobium meliloti cosmids carrying exo genes. All of the 19 mutants were symbiotically effective or partially effective, indicating that the major bacterial acidic exopolysaccharide of this strain of R. fredii may not be required for symbiotic development in the soybean.     

Effects of inoculation with plant growth promoting rhizobacteria (PGPRs) andSinorhizobium fredii on biological nitrogen fixation, nodulation and growth ofGlycine max cv. Osumi

We investigated the effects of three plant growth promoting rhizobacteria (PGPR), on Biological Nitrogen Fixation (BNF), nodulation and growth promotion by soybean (Glycine max) var. Osumi plants. The strains, Aur 6, Aur 9 and Cell 4, belong toPsedomonas fluorescens, Chryseobacterium balustinum andSerratia fonticola, respectively. Inoculation modes for the PGPRs andSinorhizobium fredii (carried out through irrigation), were examined. In the first mode, PGPRs andS. fredii were co-inoculated. In the second mode, we first inoculatedS. fredii and after the PGPRs, which were added 5 or 10 days later (each inoculation being an independent treatment). In the third mode, the PGPRs were inoculated first, and theS. fredii was inoculated 5 days later. We also included treatments inoculated with only the PGPRs (one PGPR per treatment) and only withS. fredii. Plants were maintained in a greenhouse under controlled environmental conditions, and were sampled 3 months after sowing. The results obtained showed the effects of the inoculation sequence. The most significant effects on growth parameters (stem plus leaf weight and fresh root weight) were found when inoculations with PGPR andS. fredii were at different times or when we inoculated only with PGPR and the plants were watered with nitrogen. Co-inoculation had no positive effects on any parameter, probably due to competition between the PGPR andS. fredii. Our results indicate that the inoculation modes with PGPR and rhizobia play a very important role in the effects produced. Thus, although plant growth promoting rhizobacteria may interact synergistically with root-nodulating rhizobia, plant growth promoting rhizobacteria selected for one crop should be assessed for potentially hazardous effects on other crops before being used as inoculants.