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.     

Cultivar-Specific Interactions of Soybean with Rhizobium fredii Are Regulated by the Genotype of the Root

Rhizobium fredii USDA257 forms nitrogen-fixing nodules on soybean cultivar Peking, but not on cultivar McCall. This pattern of nodulation persists when McCall and Peking seedlings are cultivated together in plastic growth pouches. Reciprocal grafting experiments confirm that the root genotype, and not that of the shoot, regulates such cultivar specificity. When Peking roots are grafted onto McCall seedlings, the nodulation responses of roots similarly remain unaffected. Transposon-mutant 257DH4, which is derived from USDA257, can form nitrogen-fixing nodules on McCall. Such nodulation is blocked by the presence of USDA257 in the inoculum. Grafting experiments indicate that blocking is not due to a translocatable inhibitor produced by McCall roots or triggered by their interaction with USDA257. Thus, neither freely diffusible nor graft-transmissible substances are involved in cultivar-specific interactions of soybean with R. fredii and its derivatives.     

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.     

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.     

Influence of Glycine spp. on Competitiveness of Bradyrhizobium japonicum and Rhizobium fredii

The displacement of indigenous Bradyrhizobium japonicum in soybean nodules with more effective strains offers the possibility of enhanced N₂ fixation in soybean (Glycine max (L.) Merr.). Our objective was to determine whether the wild soybean (G. soja Sieb. & Zucc.) genotype PI 468397 would cause reduced competitiveness of important indigenous B. japonicum strains USDA 31, 76, and 123 and thereby permit nodulation by Rhizobium fredii, the fast-growing microsymbiont of soybean. In an initial experiment, PI 468397 nodulated and fixed moderate amounts of N₂ with USDA 31 and 76 but, despite the formation of nodules, fixed essentially no N₂ with USDA 123. In contrast, PI 468397 formed a highly effective symbiosis with R. fredii strain USDA 193. In two subsequent experiments, Williams soybean and PI 468397 were grown in a pasteurized soil mixture or in soybean rhizobium-free soil and inoculated with both USDA 123 and USDA 193. In each experiment, more than 90% of the nodules of Williams contained USDA 123, while only a maximum of 2% were occupied with USDA 193. In contrast, in the two experiments, 16 and 11%, respectively, of the nodules produced on PI 468397 were occupied by USDA 123, while in both experiments 87% contained USDA 193. Thus, in relation to the cultivar Williams, which is commonly grown and used as a parent in soybean breeding programs in the United States, PI 468397 substantially reduced the competitive ability of B. japonicum strain USDA 123 in relation to R. fredii strain USDA 193.     

Host range,RFLP, and antigenic relationships betweenRhizobium fredii strains andRhizobium sp. NGR234

Rhizobium fredii is a nitrogen-fixing symbiont from China that combines broad host range for nodulation of legume species with cultivar specificity for nodulation of soybean. We have compared 10R. fredii strains withRhizobium sp. NGR234, a well known broad host range strain from Papua New Guinea. NGR234 nodulated 16 of 18 tested lugume species, and nodules on 14 of the 16 fixed nitrogen. TheR. fredii strains were not distinguishable from one another. They nodulated 13 of the legumes, and in only nine cases were nodules effective. All legumes nodulated byR. fredii were included within the host range of NGR234. Restriction fragment length polymorphisms (RFLPs) were detected with four DNA hybridization probes: the regulatory and commonnod genes,nodDABC; the soybean cultivar specificity gene,nolC; the nitrogenase structural genes, nifKDH; and RFRS1, a repetitive sequence fromR. fredii USDA257. A fifth locus, corresponding to a second set of soybean cultivar specificity genes,nolBTUVWX, was monomorphic. Using antisera against whole cells of threeR. fredii strains and NGR234, we separated the 11 strains into four serogroups. The anti-NGR234 sera reacted with a singleR. fredii strain, USDA191. Only one serogroup, which included USDA192, USDA201, USDA217, and USDA257, lacked cross reactivity with any of the others. Although genetic and phenotypic differences amongR. fredii strains were as great as those between NGR234 andR. fredii, our results confirm that NGR234 has a distinctly wider host range thanR. fredii.     

Regulation of nodulation in the soybean-Rhizobium symbiosis : strain and cultivar variability

Double inoculation (15 h apart) of the soybean cultivar Williams with Bradyrhizobium japonicum I-110ARS reveals a rapid regulatory plant response that inhibits nodulation of distal portions of the primary root (M Pierce, WD Bauer 1984 Plant Physiol 73: 286-290). Only living, homologous rhizobia elicit the response. We conducted similar double inoculation experiments to test the hypothesis that this is a universal phenomenon in soybean symbioses. We investigated interactions of the cultivar McCall with the slow-growing strain Bradyrhizobium sp. 3185 (=3G4b16) and strains of the fast-growing soybean symbiont, Rhizobium fredii (USDA191 [Nod⁺ on McCall] and USDA257 [Nod⁻ on McCall]). Nodulation was not detectably inhibited when USDA257 was included in various combinations with an inoculum of USDA191. Strain USDA257 cohabited nodules with strain USDA191 when plants were inoculated sequentially with both strains, but USDA257 did not nodulate McCall when a sterile culture filtrate of USDA191 was added to USDA257 inoculum. There was only a slight inhibition of nodulation of distal portions of the primary root in double inoculation experiments with McCall and strain 3185. Because these results were unexpected, we repeated the experiments with Williams and strain I-110ARS. The response was similar to that observed in the McCall × 3185 interaction. Regulation of nodulation on the primary root thus appears to be variable and depend on strain X cultivar interactions.     

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.     

Release of Flavonoids by the Soybean Cultivars McCall and Peking and Their Perception as Signals by the Nitrogen-Fixing Symbiont Sinorhizobium fredii

Sinorhizobium fredii strain USDA191 forms N-fixing nodules on the soybean (Glycine max L. Merr.) cultivars (cvs) McCall and Peking, but S. fredii strain USDA257 nodulates only cv Peking. We wondered whether specificity in this system is conditioned by the release of unique flavonoid signals from one of the cultivars or by differential perception of signals by the strains. We isolated flavonoids and used nodC and nolX, which are nod-box-dependent and -independent nod genes, respectively, to determine how signals activate genes in the microsymbionts. Seeds of cv McCall and cv Peking contain the isoflavones daidzein, genistein, and glycitein, as well as their glucosyl and malonylglucosyl glycosides. Roots exude picomolar concentrations of daidzein, genistein, glycitein, and coumestrol. Amounts are generally higher in cv Peking than in cv McCall, and the presence of rhizobia markedly influences the level of specific signals. Nanomolar concentrations of daidzein, genistein, and coumestrol induce expression of nodC and nolX in strain USDA257, but the relative nolX-inducing activities of these signals differ in strain USDA191. Glycitein and the conjugates are inactive. Strain USDA257 deglycosylates daidzin and genistin into daidzein and genistein, respectively, thereby converting inactive precursors into active inducers. Although neither soybean cultivar contains unique nod-gene-inducing flavonoids, strain- and cultivar-specific interactions are characterized by distinct patterns of signal release and response.     

Y4xP, an open reading frame located in a type III protein secretion system locus of Sinorhizobium fredii USDA257 and USDA191, encodes cysteine synthase

Sinorhizobium fredii USDA257, a soybean symbiont, exports several nodulation outer proteins (Nops) into the rhizosphere. These proteins, which are exported by a type III secretion system (TTSS), have a pivotal role in host-specific nodulation. The entire TTSS of S. fredii lies within a 31-kb region that includes conserved genes that code for secretion machinery proteins, Nops, and several open reading frames (ORF) of unknown function. Identifying the functions of these ORF is essential to understand fully the role of TTSS in nodulation. Here, we report the characterization of y4xP, an ORF of previously unknown function. Southern blot analysis revealed that USDA257 contains two copies of y4xP, while a sibling, USDA191, contains a single copy. The amino acid sequence of Y4XP is homologous to both eukaryotic and prokaryotic cysteine synthase, a key enzyme in sulfur assimilation. The coding region of USDA257 y4xP under control of T7 promoter was expressed in Escherichia coli, and the recombinant protein was purified by nickel-affinity chromatography. Antibodies generated against soybean cysteine synthase cross-reacted with the recombinant protein. A nonpolar mutant of y4xP of USDA191 showed a marked reduction in cysteine synthase activity. Enzyme activity was completely restored when the mutant was complemented with a plasmid containing the y4xP sequence. Cysteine synthase activity was confined to the cell cytosol. Extracellular protein fraction from genistein-induced USDA191 showed no cysteine synthase activity. This observation indicates that cysteine synthase, which is located in the TTSS locus, is not a type III secreted protein. A nonpolar cysteine synthase mutant was able to export all the Nops to the rhizosphere, albeit in reduced amounts compared with the wild-type USDA191. Interestingly, USDA191 cysteine synthase mutant was able to initiate nodules on 'McCall' soybean more efficiently than the wild-type. Our results demonstrate that y4xP encodes a cysteine synthase and inactivation of this gene enhances the ability of USDA191 to form nodules on 'McCall' soybean by regulating Nops production.     

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.     

Modulation of Host Gene Expression during Initiation and Early Growth of Nodules in Cowpea, Vigna unguiculata (L.) Walp

Inoculation of 2-day-old cowpea (Vigna unguiculata [L.] Walp.) seedlings with Rhizobium fredii USDA257 results in proficient nodulation of the tap root. The most abundant nodulation occurs in a region roughly corresponding to the position of the root tip at the time of inoculation. We have examined plant gene expression in this region, after inoculation with either USDA257 or a nonnodulating mutant, 257B3. After isolation of mRNA and in vitro translation, the protein products were separated by two-dimensional gel electrophoresis. Seven proteins are induced within 2.5 days after inoculation with USDA257. One additional induced protein is detectable by 3.5 days after inoculation, and three more appear by day 6. Three of the proteins that are differentially expressed at 2.5 and 3.5 days after inoculation are produced at equivalent levels after 6 days, indicating transient induction of these genes during early stages of nodule development. Several proteins were more abundant in translations of mRNA from roots that had been inoculated with the nonnodulating mutant. This was particularly true after 6 days, when nine proteins were in this class. Thus, altered plant gene expression in carefully selected, highly responsive tissue can be detected 2 days before emerging nodules are visible on the roots, and 6 to 7 days before acetylene reduction is detectable. Additionally, comparisons of ionically bound cell wall proteins isolated 6 days after inoculation revealed four that were unique to nodulating roots, suggesting that some of the nodulation-induced genes may code for structural proteins.     

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     

Expression of a Serratia marcescens chitinase gene in Sinorhizobium fredii USDA191 and Sinorhizobium meliloti RCR2011 impedes soybean and alfalfa nodulation.

A gene encoding chitinase from Serratia marcescens BJL200 was cloned into a broad-host-range vector (pRK415) and mobilized into Sinorhizobium fredii USDA191. Chitinolytic activity was detected in S. fredii USDA191 transconjugants that carried the S. marcescens chiB gene. Chitinase-producing S. fredii USDA191 formed nodules on soybean cultivar McCall. However, there was a delay in nodule formation and a marked decrease in the total number of nodules formed by the chitinase-producing S. fredii in comparison with the wild-type strain. Expression of chitinase in S. meliloti RCR2011 also impeded alfalfa nodulation. Thin-layer chromatography of 14C-labeled Nod factors from chitinase-producing S. fredii USDA191 revealed hydrolysis of lipochitooligosaccharides.     

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.     

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.     

Decreased Exopolysaccharide Synthesis by Anaerobic and Symbiotic Cells of Bradyrhizobium japonicum

Experiments were conducted to determine whether symbiotic bacteroids of Bradyrhizobium japonicum produce exopolysaccharide within soybean (Glycine max [L.] Merr. cv `Lee 74') nodules. B. japonicum strains RT2, a derivative of USDA 110 with resistance to streptomycin and rifampicin, and RT176-1, a mutant deficient in exopolysaccharide synthesis, were used. Although aerobically cultured RT2 produced 1550 micrograms of exopolysaccharide per 10¹⁰ cells, root nodules formed by RT2 contained only 55.7 micrograms of polysaccharide per 10¹⁰ bacteroids, indicating that little exopolysaccharide synthesis occurred within the nodules. The polysaccharide level of RT2 nodules was about equal to that of nodules containing the exopolysaccharide mutant RT176-1 (61.0 micrograms per 10¹⁰ bacteroids). Gas chromatographic analysis showed that the sugar composition of polysaccharide from nodules of RT2 or RT176-1 was almost the same as that of polysaccharide from unnodulated root tissue, but differed strikingly from that of rhizobial exopolysaccharide from aerobic cultures. Thus, the host plant and not the bacteroids was probably the source of most or all of the polysaccharide in the nodule extracts. Also, bacteroids from nodules failed to bind soybean lectin, confirming the absence of an exopolysaccharide capsule.