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.     

Inactivation of the Sinorhizobium fredii HH103 rhcJ gene abolishes nodulation outer proteins (Nops) secretion and decreases the symbiotic capacity with soybean.

It has been postulated that nodulation outer proteins (Nops) avoid effective nodulation of Sinorhizobium fredii USDA257 to nodulate with American soybeans. S. fredii HH103 naturally nodulates with both Asiatic (non-commercial) and American (commercial) soybeans. Inactivation of the S. fredii HH103 gene rhcJ, which belongs to the tts (type III secretion) cluster, abolished Nop secretion and decreased its symbiotic capacity with the two varieties of soybeans. S. fredii strains HH103 and USDA257, that only nodulates with Asian soybeans, showed different SDS-PAGE Nop profiles, indicating that these strains secrete different sets of Nops. In coinoculation experiments, the presence of strain USDA257 provoked a clear reduction of the nodulation ability of strain HH103 with the American soybean cultivar Williams. These results suggest that S. fredii Nops can act as either detrimental or beneficial symbiotic factors in a strain-cultivar-dependent manner. Differences in the flavonoid-mediated expression of rhcJ with respect to nodA were also detected. In addition, one of the Nops secreted by strain HH103 was identified as NopA.     

Distinct Cell Surface Appendages Produced by Sinorhizobium fredii USDA257 and S. fredii USDA191, Cultivar-Specific and Nonspecific Symbionts of Soybean

Sinorhizobium fredii USDA257 and S. fredii USDA191 are fast-growing rhizobia that form nitrogen-fixing nodules on soybean roots. In contrast to USDA191, USDA257 exhibits cultivar specificity and can form nodules only on primitive soybean cultivars. In response to flavonoids released from soybean roots, these two rhizobia secrete nodulation outer proteins (Nop) to the extracellular milieu through a type III secretion system. In spite of the fact that Nops are known to regulate legume nodulation in a host-specific manner, very little is known about the differences in the compositions of Nops and surface appendages elaborated by USDA191 and USDA257. In this study we compared the Nop profiles of USDA191 and USDA257 by one-dimensional (1D) and 2D gel electrophoresis and identified several of these proteins by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and liquid chromatography-tandem MS (LC-MS/MS). Examination of the surface appendages elaborated by these two strains of soybean symbionts by transmission electron microscopy revealed distinct differences in their morphologies. Even though the flagella produced by USDA191 and USDA257 were similar in their morphologies, they differed in their flagellin composition. USDA257 pili resembled long thin filaments, while USDA191 pili were short, rod shaped, and much thinner than the flagella. 2D gel electrophoresis of pilus-like appendages of USDA191 and USDA257 followed by mass spectrometry resulted in the identification of several of the Nops along with some proteins previously undetected in these strains. Some of the newly identified proteins show homology to putative zinc protease and a LabA-like protein from Bradyrhizobium sp. ORS278, fimbrial type 4 assembly proteins from Ralstonia solanacearum, and the type III effector Hrp-dependent protein from Rhizobium leguminosarum bv. trifolii.     

Genetic diversity of fast-growing rhizobia that nodulate soybean ( Glycine max L. Merr)

The fast-growing Rhizobium sp. strain NGR234, isolated from Papua New Guinea, and 13 strains of Sinorhizobium fredii, isolated from China and Vietnam, were fingerprinted by means of RAPD, REP, ERIC and ARDRA. ERIC, REP and RAPD markers revealed a considerable genetic diversity among fast-growing rhizobia. Chinese isolates showed higher levels of diversity than those strains isolated from Vietnam. ARDRA analysis revealed three different genotypes among fast-growing rhizobia that nodulate soybean, even though all belonged to a subcluster that included Sinorhizobium saheli and Sinorhizobium meliloti. Among S. fredii rhizobia, two strains, SMH13 and HH303, might be representatives of other species of nitrogen-fixing organisms. Although restriction analysis of the nifD–nifK intergenic DNA fragment confirmed the unique nature of Rhizobium sp. strain NGR234, several similarities between Rhizobium sp. strain NGR234 and S. fredii USDA257, the ARDRA analysis and the full sequence of the 16S rDNA confirmed that NGR234 is a S. fredii strain. In addition, ARDRA analysis and the full sequence of the 16S rDNA suggested that two strains of rhizobia might be representatives of other species of rhizobia.     

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.     

Molecular Cloning and Characterization of <Emphasis Type="Italic">nodD</Emphasis> Genes from <Emphasis Type="Italic">Rhizobium</Emphasis> sp. SIN-1, a Nitrogen-Fixing Symbiont of <Emphasis Type="Italic">Sesbania</Emphasis> and Other Tropical Legumes

Rhizobium sp. SIN-1, a nitrogen-fixing symbiont of Sesbania aculeata and other tropical legumes, carries two copies of nodD, both on a sym plasmid. We have isolated these two nodD genes by screening a genomic library of Rhizobium sp. SIN-1 with a nodD probe from Sinorhizobium meliloti. Nucleotide sequence and the deduced amino acid sequence analysis indicated that the nodD genes of Rhizobium sp. SIN-1 are most closely related to those of R. tropici and Azorhziobium caulinodans. Rhizobium sp. SIN-1 nodD1 complemented a S. meliloti nodD1D2D3 negative mutant for nodulation on alfalfa, but failed to complement a nodD1 mutant of S. fredii USDA191 for soybean nodulation. A hybrid nodD gene, containing the N-terminus of S. fredii USDA191 nodD1 and the C-terminus of Rhizobium sp. SIN-1 nodD1, complemented the nodD1 negative mutant of USDA191 for nodulation on soybean. Received: 17 January 2002 / Accepted: 18 February 2002     

Genetic characterization of a mutant of Sinorhizobium fredii strain USDA208 with enhanced competitive ability for nodulation of soybean, Glycine max (L.) Merr.

Sinorhizobium fredii strain USDA208 is a nitrogen-fixing bacterium that forms nodules on roots of soybean and other legume plants. We previously found that the Tn5-containing mutant 208T3, which was derived from strain USDA208, is both deficient in production of exopolysaccharides and more competitive than the wild-type strain in competing against other rhizobia for nodulation of soybean. We now demonstrate that the transposon insertion of the mutant lies in a locus that is highly homologous to a portion of the exo region, which functions in exopolysaccharide biosynthesis by Sinorhizobium meliloti. We sequenced 2906 bp surrounding the insertion site and identified three genes: exoA, exoM, and exoO. The transposon lies within exoM, a glucosyl transferase. A cosmid containing exoHKLAMONP of S. meliloti restores exopolysaccharide production by mutant 208T3 to wild-type levels. Although exo mutants of S. meliloti are defective in their abilities to form indeterminate nodules, the capacities of mutant 208T3 and its wild-type parent to form such nodules on five legume species are indistinguishable. Thus the symbiotic function of exopolysaccharide in S. fredii appears to differ fundamentally from that in S. meliloti.     

Functional Analysis of NopM, a Novel E3 Ubiquitin Ligase (NEL) Domain Effector of Rhizobium sp. Strain NGR234

Type 3 effector proteins secreted via the bacterial type 3 secretion system (T3SS) are not only virulence factors of pathogenic bacteria, but also influence symbiotic interactions between nitrogen-fixing nodule bacteria (rhizobia) and leguminous host plants. In this study, we characterized NopM (nodulation outer protein M) of Rhizobium sp. strain NGR234, which shows sequence similarities with novel E3 ubiquitin ligase (NEL) domain effectors from the human pathogens Shigella flexneri and Salomonella enterica. NopM expressed in Escherichia coli, but not the non-functional mutant protein NopM-C338A, showed E3 ubiquitin ligase activity in vitro. In vivo, NopM, but not inactive NopM-C338A, promoted nodulation of the host plant Lablab purpureus by NGR234. When NopM was expressed in yeast, it inhibited mating pheromone signaling, a mitogen-activated protein (MAP) kinase pathway. When expressed in the plant Nicotiana benthamiana, NopM inhibited one part of the plant''s defense response, as shown by a reduced production of reactive oxygen species (ROS) in response to the flagellin peptide flg22, whereas it stimulated another part, namely the induction of defense genes. In summary, our data indicate the potential for NopM as a functional NEL domain E3 ubiquitin ligase. Our findings that NopM dampened the flg22-induced ROS burst in N. benthamiana but promoted defense gene induction are consistent with the concept that pattern-triggered immunity is split in two separate signaling branches, one leading to ROS production and the other to defense gene induction.     

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.     

Symbiotic performance of common bean and soybean co-inoculated with rhizobia and <Emphasis Type="Italic">Chryseobacterium balustinum</Emphasis> Aur9 under moderate saline conditions

The effect of co-inoculating beans and soybeans with rhizobia and Chryseobacterium, a plant growth promoting bacteria (PGPR), was studied under conditions of mild saline stress. Chryseobacterium balustinum Aur9 was used with Rhizobium tropici CIAT899 or R. etli ISP42 to inoculate common bean (Phaseolus vulgaris L.), or jointly with Ensifer (Sinorhizobium) fredii SMH12 and HH103 to inoculate soybean (Glycine max (L.) Merrill). The effect of co-inoculation was studied by following nodule primordia initiation, nodulation kinetics and symbiotic performance in plants grown under moderate saline conditions (25 mM NaCl). In common bean, co-inoculation improved nodule primordia formation when compared with single inoculation (R. tropici CIAT899). However, co-inoculation did not provide benefits in the development of nodule primordia in soybean with E. fredii SMH12. The kinetic of nodulation in bean was also favored by double inocula resulting in a higher number of nodules. Long-term effects of co-inoculation on beans and soybeans depended on the rhizobial species used. In both, control and saline conditions, co-inoculation of R. tropici CIAT899 and C. balustinum Aur9 improved bean growth when compared with the single inoculation (CIAT899). However, the positive effect of double inocula on plant growth did not occur when using R. etli ISP42. Soybean plants receiving double inoculation (E. fredii SMH12 and C. balustinum Aur9) showed better symbiotic performance, mostly under saline stress, than with a single inoculation. The results indicate that co-inoculation with C. balustinum and rhizobia under mild saline conditions partially relieves the salt-stress effects, although do not always result advantageous for symbiotic N₂ fixation in legume plants.     

Growth of Fast- and Slow-Growing Rhizobia on Ethanol

Free-living soybean rhizobia and Bradyrhizobium spp. (lupine) have the ability to catabolize ethanol. Of the 30 strains of rhizobia examined, only the fast- and slow-growing soybean rhizobia and the slow-growing Bradyrhizobium sp. (lupine) were capable of using ethanol as a sole source of carbon and energy for growth. Two strains from each of the other Rhizobium species examined (R. meliloti, R. loti, and R. leguminosarum biovars phaseoli, trifolii, and viceae) failed to grow on ethanol. One Rhizobium fredii (fast-growing) strain, USDA 191, and one (slow-growing) Bradyrhizobium japonicum strain, USDA 110, grew in ethanol up to concentrations of 3.0 and 1.0%, respectively. While three of the R. fredii strains examined (USDA 192, USDA 194, and USDA 205) utilized 0.2% acetate, only USDA 192 utilized 0.1% n-propanol. None of the three strains utilized 0.1% methanol, formate, or n-butanol as the sole carbon source.     

Multiple host-specificity loci of the broad host-range Rhizobium sp. NGR234 selected using the widely compatible legume Vigna unguiculata

Specificity in legume-Rhizobium symbiosis depends on plant and rhizobial genes. As our objective was to study broad host-range determinants of rhizobia, we sought a legume and a Rhizobium with the lowest possible specificity. By inoculating 12 different legumes with a heterogenous collection of 35 fast-growing rhizobia, we found Rhizobium sp. NGR234 to be the Rhizobium and Vigna unguiculata to be the plant with the lowest specificities. Transfer of cloned fragments of the Sym-plasmid pNGR234a into heterologous rhizobia, screening for extension of host-range of the transconjugants to include V. unguiculata, and restriction mapping of the Hsn- and overlapping clones, proved that there were at least three distinct Hsn-regions (HsnI, II, and III) on pNGR234a. HsnI is located next to nodD, HsnII is linked to nifKDH and HsnIII to nodC. In addition to nodulation of Vigna, HsnI conferred upon the transconjugants the ability to nodulate Glycine max, Macroptilium atropurpureum and Psophocarpus tetragonolobus. All three Hsn-regions, when transferred to the appropriate recipients, induced root-hair-curling on M. atropurpureum. Hsn-region III was able to complement a mutation in the host-range gene nodH of R. meliloti strain 2011. Homology to nod-box-sequences could be shown only for the sub-clones containing HsnII and HsnIII, thus suggesting different regulation mechanisms for HsnI and HsnII/III.     

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.     

A nopA Deletion Mutant of Sinorhizobium fredii USDA257, a Soybean Symbiont, is Impaired in Nodulation

Sinorhizobium fredii USDA257 employs type III secretion system (T3SS) to deliver effector proteins into the host cells through pili. The nopA protein is the major component of USDA257 pili. The promoter region of USDA257 nopA possesses a well conserved tts box. Serial deletion analysis revealed that the tts box is absolutely essential for flavonoid induction of nopA. Deletion of nopA drastically lowered the number of nodules formed by USDA257 on cowpea and soybean cultivar Peking. In contrast to the parental strain, the USDA257 nopA mutant was able to form few nodules on soybean cultivars McCall and Williams 82. Light and transmission electron microscopy examination of these nodules revealed numerous starch grains both in the infected and uninfected cells.     

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.     

Molecular aspects of soybean cultivar-specific nodulation by Sinorhizobium fredii USDA257

Sinorhizobium fredii USDA257 forms nitrogen-fixing nodules in association with the primitive soybean cultivar 'Peking' but fails to initiate nodules on many advanced soybean cultivars, including 'McCall'. This distinction is controlled by a set of nodulation genes termed nolXWBTUV. Inactivation of any of these genes enables USDA257 to nodulate McCall and many other improved soybean cultivars. Mutation in the nolXWBTUV locus also alters the Nod factor structure resulting in the production of a novel molecule with glucose incorporated into the chitin backbone. Some of the genes located in the nolXWBTUV locus reveal sequence homologies to known components of the type III secretion system (TTSS) of plant and animal pathogenic bacteria. Recent studies have demonstrated the presence of a complete TTSS in USDA257 and few other symbiotic bacteria. The TTSS cluster of USDA257 contains 27 open reading frames out of which 10 code for the structural components of the TTSS. USDA257, when grown in presence of flavonoids, secrete several proteins called Nops (Nodulation Outer Proteins) into the extracellular environment. Genes located in the TTSS of USDA257 encode some of the extracellular proteins, such as NopX, NopB, and NopL. These type III secreted proteins appear to play an important role in regulating nodulation in a host-dependent manner. Failure to elaborate the Nops results in a drastic phenotypic effect on soybean nodulation, indicating that these proteins may play a pivotal role in soybean cultivar specificity. The secretion of Nops appears to be facilitated by novel filamentous appendages (pili) that are produced by USDA257 upon induction by flavonoids. Biochemical studies have demonstrated the close association of several Nops with the purified pili. However, it remains to be seen if the filamentous appendages can function as conduits for delivery of Nops into the host cell. This review examines the current state of our knowledge on the molecular aspects of soybean cultivar-specific nodulation by USDA257.