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.     

Phylogeny of fast-growing soybean-nodulating rhizobia support synonymy of Sinorhizobium and Rhizobium and assignment to Rhizobium fredii.

We determined the sequences for a 260-base segment amplified by the polymerase chain reaction (corresponding to positions 44 to 337 in the Escherichia coli 16S rRNA sequence) from seven strains of fast-growing soybean-nodulating rhizobia (including the type strains of Rhizobium fredii chemovar fredii, Rhizobium fredii chemovar siensis, Sinorhizobium fredii, and Sinorhizobium xinjiangensis) and broad-host-range Rhizobium sp. strain NGR 234. These sequences were compared with the corresponding previously published sequences of Rhizobium leguminosarum, Rhizobium meliloti, Agrobacterium tumefaciens, Azorhizobium caulinodans, and Bradyrhizobium japonicum. All of the sequences of the fast-growing soybean rhizobia, including strain NGR 234, were identical to the sequence of R. meliloti and similar to the sequence of R. leguminosarum. These results are discussed in relation to previous findings; we concluded that the fast-growing soybean-nodulating rhizobia belong in the genus Rhizobium and should be called Rhizobium fredii.     

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.     

A chromosomal genetic map of Rhizobium sp. NGR234 generated with Tn5-Mob

Summary Rhizobium sp. NGR234 in a fast-growing Rhizobium strain with a broad host range. The location and role of chromosomal genes involved in cellular metabolism or in the legume symbioses is unknown. We isolated a series of auxotrophic and antibiotic resistant mutants of NGR234 and utilized a chromosome mobilization system based on Tn5-Mob and pJB3JI; Tn5-Mob donor strains behaved like Hfr strains, transferring the chromosome polarly at high frequency from a fixed point of insertion. The use of four different strains with Tn5-Mob located at different nutritional loci in crosses with double auxotrophic recipients, allowed us to build up a circular linkage map of NGR234 based on relative recombination frequencies. Also, symbiotically important genes identified by site-directed mutagenesis, such as hemA and ntrA, could be located and mapped on the chromosome.Abbreviations Tc tetracycline - Sp spectinomycin - Rif rifampicin - Km kanamycin     

Genetic diversity of indigenous tropical fast-growing rhizobia isolated from soybean nodules

This study characterized genetically 30 fast-growing rhizobial strains isolated from nodules of Asian and modern soybean genotypes that had been inoculated with soils from disparate regions of Brazil. Analyses by rep-PCR (ERIC and REP) and RAPD indicated a high level of genetic diversity among the strains. The RFLP-PCR and sequencing analysis of the 16S rRNA genes indicated that none of the strains was related to Sinorhizobium (Ensifer) fredii, whereas most were related to Rhizobium tropici (although they were unable to nodulate Phaseolus vulgaris) and to Rhizobium genomic species Q. One strain was related to Rhizobium sp. OR 191, while two others were closely related to Agrobacterium (Rhizobium) spp.; furthermore, symbiotic effectiveness with soybean was maintained in those strains. Five strains were related to Bradyrhizobium japonicum and B. elkanii, with four of them being similar to strains carried in Brazilian inoculants, therefore modifications in physiological properties, as a shorter doubling time might have resulted from adaptation to local conditions. Phospholipid fatty acid analysis (PFLA) was less precise in delineating taxonomic relationships. The strains fit into eight Nod-factor profiles that were related to rhizobial species, but not to N₂-fixation capacity or competitiveness. The data obtained highlight the diversity and promiscuity of rhizobia in the tropics, being capable of nodulating exotic legumes and might reflect ecological strategies to survive in N-poor soils; in addition, the diversity could also represent an important source of efficient and competitive rhizobial strains for the tropics. Putative new rhizobial species were detected only in undisturbed soils. Three species (R. tropici, B. japonicum and B. elkanii) were found under the more sustainable management system known as no-till, while the only species isolated from soils under conventional till was R. tropici. Those results emphasize that from the moment that agriculture was introduced into undisturbed soils rhizobial diversity has changed, being drastically reduced when a less sustainable soil management system was adopted.     

The Rhizobium sp. strain NGR234 systemically suppresses arbuscular mycorrhizal root colonization in a split‐root system of barley (Hordeum vulgare)

Nitrogen‐fixing bacteria (rhizobia) form a nodule symbiosis with legumes, but also induce certain effects on non‐host plants. Here, we used a split‐root system of barley to examine whether inoculation with Rhizobium sp. strain NGR234 on one side of a split‐root system systemically affects arbuscular mycorrhizal (AM) root colonization on the other side. Mutant strains of NGR234 deficient in Nod factor production (strain NGRΔnodABC), perception of flavonoids (strain NGRΔnodD1) and secretion of type 3 effector proteins (strain NGRΩrhcN) were included in this study. Inoculation resulted in a systemic reduction of AM root colonization with all tested strains. However, the suppressive effect of strain NGRΩrhcN was less pronounced. Moreover, levels of salicylic acid, an endogenous molecule related to plant defense, were increased in roots challenged with rhizobia. These data indicate that barley roots perceived NGR234 and that a systemic regulatory mechanism of AM root colonization was activated. The suppressive effect appears to be Nod factor independent, but enhanced by type 3 effector proteins of NGR234.     

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.     

采用PCR-RFLP技术在不同水平上鉴定大豆根瘤菌

采用16S rRNA基因PCR扩增与限制性酶切片段多态性分析(RFLP)技术对选自弗氏中华根瘤菌(S.fredii)、大豆慢生根瘤菌(B.japonicum)和埃氏慢生根瘤菌(B.elkanii)的19株代表菌进行了比较分析,根据用3种限制性内切酶的RFLP分析结果,可将供试菌株分为S.fredii,B.japonicum, B.elkanii Ⅱ和B.elkanii Ⅱa等4种基因型。各类菌株之间没有交叉,因此本研究采用的PCR-RFLP技术不失为一种快速鉴别大豆根瘤菌的新方法。采用本技术已将分离自中国的22株快生菌和19株慢生菌分别鉴定为S.fredii和B.japonicum。对供试参比菌株和野生型菌株进行的16S～23S基因间隔DNA(IGS)的PCR-RFLP分析结果表明：S.fredii和B.japonicum菌株的IGS长度不同,所有供试S.fredii菌株的IGS为2.1 kb,而供试B.japonicum菌株则为2.0 kb。依据RFLP的差异,可将来自中国两个不同地区的S.fredii株区分为2个基因型,而来自中国东北黑龙江地区的19株B.japonicum菌株则可分为11个基因型。对上述野生型菌株还进行了REP-PCR和ERIC-PCR分析并确定其具有菌株水平的特异性。     

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.     

Conserved plasmid/chromosome sequences in fast- and slow-growing rhizobia that nodulate the same plant

Apart from the ability to nodulate legumes, fast-and slow-growing rhizobia have few bacteriological traits in common. Given that there is only one pathway to nodulation, DNA sequences conserved in fast- and slow-growing organisms that nodulate the same host should be strongly enriched in infectivity genes. We tested this hypothesis with seven fast-growing and five slow-growing strains that produced responses varying from fully effective nodulation through various ineffective associations to non-nodulation on four different hosts (Lotus pedunculatus, Lupinus nanus, Macroptilium atropurpureum, and Vigna unguiculata). When restriction enzyme digested total DNA from 10 of the strains was separately hybridized with nick-translated plasmid DNA isolated from 4 fast-growing strains, variable but significant homologies were found with all 10 strains. Part of this homology was shown to be associated with the nifKDH genes for nitrogenase and part with putative nodulation genes carried on pC2, a cosmid clone containing a 37 kbp region of the large sym plasmid present in the fast-growing broad-host range Rhizobium sp. strain NGR234. Analysis of the extent of homology between the plasmids of 3 fastgrowing strains (NGR234, TAL 996 and UMKL 19) able to effectively nodulate Vigna unguiculata showed them to have homologous DNA fragments totalling 47 kbp. This core homology represents less than 12% of the total coding capacity of the sym plasmid present in each of these strains.Abbreviations Sym symbiotic sequences/plasmids - nod genes required for nodulation - nod putative nod genes - nif genes required for the synthesis of the enzyme nitrogenase     

兰坪铅锌尾矿区豆科根瘤菌遗传多样性ARDRA分析

通过16S rDNA扩增产物限制性片段长度多态性分析(ARDRA),对兰坪铅锌尾矿区豆科植物根瘤菌的遗传多样性进行了研究。采用限制性内切酶Hae Ⅲ、Hind Ⅲ、Hinf Ⅰ和Taq Ⅰ对16S rDNA扩增产物进行了酶切分型,根据ARDRA酶切图谱的不同,进行树状聚类。结果表明:49株根瘤菌在40%的相似水平上按氮含量不同及铅锌含量的采集地不同分别聚为OTU1、OTU2和OTU33个群,说明根瘤菌的遗传多样性及分布与土壤中的氮含量和铅锌含量有关。代表菌株的16S rDNA测序结果分析表明,它们在系统发育树上属于Rhizobium sp.、Sinorhizobium sp.和Bradyrhizobium sp.3个系统发育分支,进一步说明兰坪铅锌尾矿区豆科植物根瘤菌多样性较丰富。     

Effect of phosphorus supply on periplasmic protein profiles in Rhizobia

Lysozyme/EDTA treatment of four fast-growing rhizobia released repeatable protein profiles after polyacrylamide slab gel electrophoresis. Similar treatment of slow-growing rhizobia failed to release such periplasmic proteins.For the four-fast-growing rhizobia, both P-repressible and P-inducible protein bands occurred. The only P-repressible protein identified was alkaline phosphatase, which showed strain differences in both electrophoretic mobility and activation by Mg²⁺.The derepression of the P-repressible periplasmic proteins in cowpea Rhizobium NGR234 correlated with derepression of both phosphate and glycerol 1-phosphate uptake.Abbreviation HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid     

Exopolysaccharide-deficient mutants of Astragali rhizobia are symbiotically effective on Astragalus sinicus, an indeterminate nodulating host

Five exopolysaccharide-deficient mutants were isolated after rhizobial strain 107 was subjected to transposon Tn5 mutagenesis. The amount of EPS produced by the mutants was dramatically decreased to between 3% and 6% of wild-type level. All mutants carried a singel copy of Tn5. Two mutants (NA3 and NA10) were complemented by the R. meliloti exoA gene and the functionally equivalent exoD gene of Rhizobium sp. strain NGR234. Two other mutants (NA7 and NA8) were complemented by the R. meliloti exoB gene and the functionally equivalent NGR234 exoC gene. The remaining mutant (NA11) was not complemented by any exo genes of R. meliloti or Rhizobium NGR234. All mutants induced normal nitrogen-fixing nodules on Astragalus sinicus, an indeterminate nodulating host.     

Sinorhizobium fredii cultured in media supplemented with Amaranthus cruentus L. seed meal and bacterial cell survival in liquid and peat based inoculum

Nine different growth media were evaluated to determine the best growth conditions to achieve cultures of a high cell number of fast-growing rhizobia to produce inoculants. We found that Sinorhizobium fredii strains have complex nutritional requirements that were fulfilled by adding to the media 4 g Amaranthus cruentus L. seed meal/l. The survival of fast growing strains is a variable trait, but those strains that survived at high levels even after 6-month storage, hypernodulated soybeans and fixed atmospheric nitrogen at levels as high as those of Bradyrhizobium japonicum.     

Novel associations between rhizobial populations and legume species within the genera Lathyrus and O xytropis grown in the temperate region of China

Fifty rhizobial isolates of Lathyrus and Oxytropis collected from northern regions of China were studied in their genotypic characterization based upon analyses of ARDRA, 16S-23S IGS PCR-RFLP, TP-RAPD, MLEE, sequences of 16S rDNA gene and housekeeping genes of atpD, recA and glnII. The results demonstrated that most of the Lathyrus rhizobia belonged to Rhizobium and most of the Oxytropis rhizobia belonged to Sinorhizobium. A novel group of Rhizobium sp. I and S. meliloti were identified as the main microsymbionts respectively associated with Lathyrus and Oxytropis species in the collection area, which were new associations between rhizobia and the mentioned hosts. This study also provides new evidence for biogeography of rhizobia. Supported by the National Program for Basic S&T Platform Construction (Grant No. 2005DKA21201-1), the National Natural Science Foundation of China (Grant No. 30670001), and the National Basic Research Program of China (Grant No. 2006CB100206)     

The uptake and hydrolysis of disaccharides by fast-and slow-growing species of Rhizobium

Slow growing strains of rhizobia appear to lack both uptake systems and catabolic enzymes for disaccharides. In the fast-growing strains of rhizobia there are uptake mechanisms and catabolic enzymes for disaccharide metabolism. In Rhizobium leguminosarum WU 163 and WU235 and R. trifolii WU290, sucrose and maltose uptake appears to be constitutive whereas in R. meliloti WU60 and in cowpea Rhizobium NGR234 uptake of these disaccharides is inducible. There is evidence that there are at least two distinct disaccharide uptake systems in fast-growing rhizobia, one transporting sucrose, maltose and trehalose and the other, lactose. Disaccharide uptake is via an active process since uptake is inhibited by azide, dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone but not by arsenate. Bacteroids of R. leguminosarum WU235 and R. lupini WU8 are unable to accumulate disaccharides.     

<Emphasis Type="Italic">Rhizobium helanshanense</Emphasis> sp. nov., a bacterium that nodulates <Emphasis Type="Italic">Sphaerophysa salsula</Emphasis> (Pall.) DC. in China

Studying rhizobia in the root nodules of Sphaerophysa salsula (Pall.) DC in the northwest of China, we obtained five strains classified as genus Rhizobium on the basis of their 16S rRNA gene sequences. The sequence similarity of strain CCNWQTX14^T with the most related species was 99.0%. Further phylogenetic analysis of housekeeping genes (recA and atpD) suggested the five strains comprised a novel lineage within Rhizobium. The nifH and nodD gene sequences of CCNWQTX14^T were phylogenetically closely related with those of Sinorhizobium kummerowiae and R. sphaerophysae, respectively. The five strains isolated from different places were also distinct from related Rhizobium species using ERIC fingerprint profiles. The DNA–DNA hybridization value was 41.8% between CCNWQTX14^T and Rhizobium sphaerophysae CCNWGS0238^T. Our novel strains were only able to form effective nodules on its original host Sphaerophysa salsula. Our data showed that the five Rhizobium strains formed a unique genomic species, for which a novel species Rhizobium helanshanense sp. nov. is proposed. The type strain is CCNWQTX14^T (=ACCC 16237^T =HAMBI 3083^T).     

Comparison of the host range of fast-growingR. japonicum strains with a fast-growing isolate from Lablab

Summary Fast-growingRhizobium japnicum strains derived from the People's Republic of China were compared with a fast-growingRhizobium isolate from Lablab for their ability to nodulate tropical legumes grown in Leonard-jars and test tube culture. Fast-growingR. japonicum strains were all effective to varying degrees in their symbiosis withVigna unguiculata. Two strains USDA 192 and USDA 201, effectively nodulatedGlycine whightii and one strain, USDA 193, effectively nodulatedMacroptilium atropurpureum. Other nodulation responses in tropical legumes were ineffective. The fast-growing isolate from Lablab was more promiscuous, effectively nodulating with a larger host range. The fast-growing Lablab strain was considered more akin, on a symbiotic basis, to the slow-growing cowpea type rhizobia than the fast-growing China strains ofR. japonicum whilst maintaining physiological characteristics of other fast-growing rhizobia.     

Characterization of symbiotic and endophytic bacteria isolated from root nodules of herbaceous legumes grown in Qinghai–Tibet plateau and in other zones of China

Qinghai-Tibet plateau is the highest place in the world and the environment in that plateau is hard for animals and plants, with low temperature, low concentration of oxygen and high solar radiation. In this study, 61 root nodule isolates from Vicia, Oxytropis, Medicago, Melilotus and Onobrychis species grown in Qinghai-Tibet plateau and in loess plateau were comparatively characterized. Based upon the results of numerical taxonomy, ARDRA, AFLP, DNA-DNA hybridization and 16S rDNA sequencing, the isolates were classified as Rhizobium leguminosarum, Sinorhizobium meliloti, Sinorhizobium fredii, Mesorhizobium sp., Phyllobacterium sp., Stenotrophomonas sp. and two non-symbiotic groups related to Agrobacterium and Enterobacteriaceae. The strains isolated from Qinghai-Tibet plateau and from the loess plateau were mixed in these species or groups. Oxytropis spp. and Medicago archiducis-nicolai grown in Qinghai-Tibet plateau were recorded as new hosts for R. leguminosarum, as well as Oxytropis glabra and Medicago lupulina for S. fredii. In addition, strains resistant to high alkaline (pH 11) and high concentration of NaCl (3-5%, w/v) were found in each of the rhizobial species. This was the first systematic study of rhizobia isolated from Qinghai-Tibet plateau.     

Nod factors of Rhizobium are a key to the legume door

Symbiotic interactions between rhizobia and legumes are largely controlled by reciprocal signal exchange. Legume roots excrete flavonoids which induce rhizobial nodulation genes to synthesize and excrete lopo-oligosaccharide Nod factors. In turn, Nod factors provoke deformation of the root hairs and nodule primordium formation. Normally, rhizobia enter roots through infection threads in markedly curled root hairs. If Nod factors are responsible for symbiosis-specific root hair deformation, they could also be the signal for entry of rhizobia into legume roots. We tested this hypothesis by adding, at inoculation, NodNGR-factors to signal-production-deficient mutants of the broad-host-range Rhizobium sp. NGR234 and Bradyrhizobium japorticum strain USDA110. Between 10 ⁻⁷ M and 10⁻⁶ M NodNGR factors permitted these NodABC mutants to penetrate, nodulate and fix nitrogen on Vigna unguiculata and Glycine max, respectively. NodNGR factors also allowed Rhizobium fredii strain USDA257 to enter and fix nitrogen on Calopogonium caeruleum, a non-host. Detailed cytological investigations of V. unguiculata showed that the NodABC mutant UGR AnodABC, in the presence of NodNGR factors, entered roots in the same way as the wild-type bacterium. Since infection threads were also present in the resulting nodules, we conclude that Nod factors are the signals that permit rhizobia to penetrate legume roots via infection threads.