| 1. |
Krishnan HB,
Kim WS,
Sun-Hyung J,
Kim KY,
Jiang G,
( 2003 ) Citrate synthase mutants of Sinorhizobium fredii USDA257 form ineffective nodules with aberrant ultrastructure. PMID : 12788763 : DOI : 10.1128/aem.69.6.3561-3568.2003 PMC : PMC161545 Abstract >>
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.
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2. |
Krishnan HB,
Lorio J,
Kim WS,
Jiang G,
Kim KY,
DeBoer M,
Pueppke SG,
( 2003 ) Extracellular proteins involved in soybean cultivar-specific nodulation are associated with pilus-like surface appendages and exported by a type III protein secretion system in Sinorhizobium fredii USDA257. PMID : 12848427 : DOI : 10.1094/MPMI.2003.16.7.617 Abstract >>
Several gram-negative plant and animal pathogenic bacteria have evolved a type III secretion system (TTSS) to deliver effector proteins directly into the host cell cytosol. Sinorhizobium fredii USDA257, a symbiont of soybean and many other legumes, secretes proteins called Nops (nodulation outer proteins) into the extracellular environment upon flavonoid induction. Mutation analysis and the nucleotide sequence of a 31.2-kb symbiosis (sym) plasmid DNA region of USDA257 revealed the existence of a TTSS locus in this symbiotic bacterium. This locus includes rhc (rhizobia conserved) genes that encode components of a TTSS and proteins that are secreted into the environment (Nops). The genomic organization of the TTSS locus of USDA257 is remarkably similar to that of another broad-host range symbiont, Rhizobium sp. strain NGR234. Flavonoids that activate the transcription of the nod genes of USDA257 also stimulate the production of novel filamentous appendages known as pili. Electron microscope examination of isolated pili reveals needle-like filaments of 6 to 8 nm in diameter. The production of the pili is dependent on a functional nodD1 and the presence of a nod gene-inducing compound. Mutations in several of the TTSS genes negate the ability of USDA257 to elaborate pili. Western blot analysis using antibodies raised against purified NopX, Nop38, and Nop7 reveals that these proteins were associated with the pili. Mutations in rhcN, rhcJ, rhcC, and ttsI alter the ability of USDA257 to form nodules on Glycine max and Macroptilium atropurpureum.
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3. |
Gaunt MW,
Turner SL,
Rigottier-Gois L,
Lloyd-Macgilp SA,
Young JP,
( 2001 ) Phylogenies of atpD and recA support the small subunit rRNA-based classification of rhizobia. PMID : 11760945 : DOI : 10.1099/00207713-51-6-2037 Abstract >>
The current classification of the rhizobia (root-nodule symbionts) assigns them to six genera. It is strongly influenced by the small subunit (16S, SSU) rRNA molecular phylogeny, but such single-gene phylogenies may not reflect the evolution of the genome as a whole. To test this, parts of the atpD and recA genes have been sequenced for 25 type strains within the alpha-Proteobacteria, representing species in Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium, Azorhizobium, Agrobacterium, Phyllobacterium, Mycoplana and Brevundimonas. The current genera Sinorhizobium and Mesorhizobium are well supported by these genes, each forming a distinct phylogenetic clade with unequivocal bootstrap support. There is good support for a Rhizobium clade that includes Agrobacterium tumefaciens, and the very close relationship between Agrobacterium rhizogenes and Rhizobium tropici is confirmed. There is evidence for recombination within the genera Mesorhizobium and Sinorhizobium, but the congruence of the phylogenies at higher levels indicates that the genera are genetically isolated. rRNA provides a reliable distinction between genera, but genetic relationships within a genus may be disturbed by recombination.
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4. |
Jiang G,
Krishnan AH,
Kim YW,
Wacek TJ,
Krishnan HB,
( 2001 ) A functional myo-inositol dehydrogenase gene is required for efficient nitrogen fixation and competitiveness of Sinorhizobium fredii USDA191 to nodulate soybean (Glycine max [L.] Merr.). PMID : 11274120 : DOI : 10.1128/JB.183.8.2595-2604.2001 PMC : PMC95177 Abstract >>
Inositol derivative compounds provide a nutrient source for soil bacteria that possess the ability to degrade such compounds. Rhizobium strains that are capable of utilizing certain inositol derivatives are better colonizers of their host plants. We have cloned and determined the nucleotide sequence of the myo-inositol dehydrogenase gene (idhA) of Sinorhizobium fredii USDA191, the first enzyme responsible for inositol catabolism. The deduced IdhA protein has a molecular mass of 34,648 Da and shows significant sequence similarity with protein sequences of Sinorhizobium meliloti IdhA and MocA; Bacillus subtilis IolG, YrbE, and YucG; and Streptomyces griseus StrI. S. fredii USDA191 idhA mutants revealed no detectable myo-inositol dehydrogenase activity and failed to grow on myo-inositol as a sole carbon source. Northern blot analysis and idhA-lacZ fusion expression studies indicate that idhA is inducible by myo-inositol. S. fredii USDA191 idhA mutant was drastically affected in its ability to reduce nitrogen and revealed deteriorating bacteroids inside the nodules. The number of bacteria recovered from such nodules was about threefold lower than the number of bacteria isolated from nodules initiated by S. fredii USDA191. In addition, the idhA mutant was also severely affected in its ability to compete with the wild-type strain in nodulating soybean. Under competitive conditions, nodules induced on soybean roots were predominantly occupied by the parent strain, even when the idhA mutant was applied at a 10-fold numerical advantage. Thus, we conclude that a functional idhA gene is required for efficient nitrogen fixation and for competitive nodulation of soybeans by S. fredii USDA191.
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5. |
Sy A,
Giraud E,
Jourand P,
Garcia N,
Willems A,
de Lajudie P,
Prin Y,
Neyra M,
Gillis M,
Boivin-Masson C,
Dreyfus B,
( 2001 ) Methylotrophic Methylobacterium bacteria nodulate and fix nitrogen in symbiosis with legumes. PMID : 11114919 : DOI : 10.1128/JB.183.1.214-220.2001 PMC : PMC94868 Abstract >>
Rhizobia described so far belong to three distinct phylogenetic branches within the alpha-2 subclass of Proteobacteria. Here we report the discovery of a fourth rhizobial branch involving bacteria of the Methylobacterium genus. Rhizobia isolated from Crotalaria legumes were assigned to a new species, "Methylobacterium nodulans," within the Methylobacterium genus on the basis of 16S ribosomal DNA analyses. We demonstrated that these rhizobia facultatively grow on methanol, which is a characteristic of Methylobacterium spp. but a unique feature among rhizobia. Genes encoding two key enzymes of methylotrophy and nodulation, the mxaF gene, encoding the alpha subunit of the methanol dehydrogenase, and the nodA gene, encoding an acyltransferase involved in Nod factor biosynthesis, were sequenced for the type strain, ORS2060. Plant tests and nodA amplification assays showed that "M. nodulans" is the only nodulating Methylobacterium sp. identified so far. Phylogenetic sequence analysis showed that "M. nodulans" NodA is closely related to Bradyrhizobium NodA, suggesting that this gene was acquired by horizontal gene transfer.
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6. |
Jiang G,
Krishnan HB,
( 2000 ) Sinorhizobium fredii USDA257, a cultivar-specific soybean symbiont, carries two copies of y4yA and y4yB, two open reading frames that are located in a region that encodes the type III protein secretion system. PMID : 10975657 : DOI : 10.1094/MPMI.2000.13.9.1010 Abstract >>
Sinorhizobium fredii USDA257 forms nitrogen-fixing nodules on primitive soybean (Glycine max) cultivar Peking but fails to nodulate the improved cultivar McCall. Cultivar specificity is governed by a plasmid-borne locus, nolXBTUV. By DNA sequence analysis, we have identified two open reading frames, y4yA and y4yB, immediately downstream of nolX. Northern (RNA) blot analysis indicated that the expression of both y4yA and y4yB is inducible by isoflavonoids, and an intact copy of nolX is required. Two copies each of y4yA and y4yB are present in S. fredii USDA257, one on the sym plasmid (y4yAsp and y4yBsp), and the other on the chromosome (y4yAc and y4yBc). The cultivar-nonspecific strain USDA191 lacks y4yAc and y4yBc. Introduction of y4yAc plus y4yBc from USDA257 into USDA191 did not influence the ability of the latter strain to nodulate McCall soybean plants. Unlike nolX, the inactivation of y4yAsp and y4yBsp of USDA257 did not extend the host range of this strain. A double mutant, in which both the plasmid and chromosomal copies of y4yA and y4yB were mutated, had no observable effect on symbiotic ability of USDA257. The y4yAsp and y4yBsp mutants did not influence flavonoid-dependent extracellular protein production. Rhizobium sp. strain NGR234 and S. saheli USDA4893 both contain sequences similar to S. fredii USDA257 y4yAsp and y4yBsp; however, Bradyrhizobium spp., the traditional soybean symbionts, lack these genes.
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7. |
Turner SL,
( 2000 ) The glutamine synthetases of rhizobia: phylogenetics and evolutionary implications. PMID : 10677854 : DOI : 10.1093/oxfordjournals.molbev.a026311 Abstract >>
Glutamine synthetase exists in at least two related forms, GSI and GSII, the sequences of which have been used in evolutionary molecular clock studies. GSI has so far been found exclusively in bacteria, and GSII has been found predominantly in eukaryotes. To date, only a minority of bacteria, including rhizobia, have been shown to express both forms of GS. The sequences of equivalent internal fragments of the GSI and GSII genes for the type strains of 16 species of rhizobia have been determined and analyzed. The GSI and GSII data sets do not produce congruent phylogenies with either neighbor-joining or maximum-likelihood analyses. The GSI phylogeny is broadly congruent with the 16S rDNA phylogeny for the same bacteria; the GSII phylogeny is not. There are three striking rearrangements in the GSII phylograms, all of which might be explained by horizontal gene transfer to Bradyrhizobium (probably from Mesorhizobium), to Rhizobium galegae (from Rhizobium), and to Mesorhizobium huakuii (perhaps from Rhizobium). There is also evidence suggesting intrageneric DNA transfer within Mesorhizobium. Meta-analysis of both GS genes from the different genera of rhizobia and other reference organisms suggests that the divergence times of the different rhizobium genera predate the existence of legumes, their host plants.
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8. |
Thomas-Oates J,
Tejero-Mateo P,
Ruiz-Sainz JE,
Spaink HP,
Pueppke SG,
Ollero FJ,
Megias M,
Krishnan HB,
Gil A,
( 1999 ) Mutation in GDP-fucose synthesis genes of Sinorhizobium fredii alters Nod factors and significantly decreases competitiveness to nodulate soybeans. PMID : 10065558 : DOI : 10.1094/MPMI.1999.12.3.207 Abstract >>
We mutagenized Sinorhizobium fredii HH103-1 with Tn5-B20 and screened about 2,000 colonies for increased beta-galactosidase activity in the presence of the flavonoid naringenin. One mutant, designated SVQ287, produces lipochitooligosaccharide Nod factors (LCOs) that differ from those of the parental strain. The nonreducing N-acetylglucosamine residues of all of the LCOs of mutant SVQ287 lack fucose and 2-O-methylfucose substituents. In addition, SVQ287 synthesizes an LCO with an unusually long, C20:1 fatty acyl side chain. The transposon insertion of mutant SVQ287 lies within a 1.1-kb HindIII fragment. This and an adjacent 2.4-kb HindIII fragment were sequenced. The sequence contains the 3' end of noeK, nodZ, and noeL (the gene interrupted by Tn5-B20), and the 5' end of nolK, all in the same orientation. Although each of these genes has a similarly oriented counterpart on the symbiosis plasmid of the broad-host-range Rhizobium sp. strain NGR234, there are significant differences in the noeK/nodZ intergenic region. Based on amino acid sequence homology, noeL encodes GDP-D-mannose dehydratase, an enzyme involved in the synthesis of GDP-L-fucose, and nolK encodes a NAD-dependent nucleotide sugar epimerase/dehydrogenase. We show that expression of the noeL gene is under the control of NodD1 in S. fredii and is most probably mediated by the nod box that precedes nodZ. Transposon insertion into neoL has two impacts on symbiosis with Williams soybean: nodulation rate is reduced slightly and competitiveness for nodulation is decreased significantly. Mutant SVQ287 retains its ability to form nitrogen-fixing nodules on other legumes, but final nodule number is attenuated on Cajanus cajan.
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9. |
Silva C,
Kan FL,
Martínez-Romero E,
( 2007 ) Population genetic structure of Sinorhizobium meliloti and S. medicae isolated from nodules of Medicago spp. in Mexico. PMID : 17386032 : DOI : 10.1111/j.1574-6941.2007.00301.x Abstract >>
We studied the genetic structure of 176 bacterial isolates from nodules of Medicago sativa, M. lupulina and M. polymorpha in fifteen sites distributed in three localities in Mexico. The strains were characterized by multilocus enzyme electrophoresis, plasmid profiles, PCR restriction fragment length polymorphism of 16S rRNA genes and of the intergenic spacer between 16S and 23S rRNA genes, and partial sequences of glnII, recA and nodB. Most of the strains were classified as Sinorhizobium meliloti, and a high genetic diversity was recorded. Six strains were classified as Sinorhizobium medicae, with no genetic variation. Phylogenetic and population genetic analyses revealed evidence of frequent recombination and migration within species.
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10. |
Martens M,
Delaere M,
Coopman R,
De Vos P,
Gillis M,
Willems A,
( 2007 ) Multilocus sequence analysis of Ensifer and related taxa. PMID : 17329774 : DOI : 10.1099/ijs.0.64344-0 Abstract >>
Multilocus sequence analysis (MLSA) was performed on representatives of Ensifer (including species previously assigned to the genus Sinorhizobium) and related taxa. Neighbour-joining (NJ), maximum-parsimony (MP) and maximum-likelihood (ML) phylogenies of dnaK, gltA, glnA, recA, thrC and 16S rRNA genes were compared. The data confirm that the potential for discrimination of Ensifer species is greater using MLSA of housekeeping genes than 16S rRNA genes. In incongruence-length difference tests, the 16S rRNA gene was found to be significantly incongruent with the other genes, indicating that this gene should not be used as a single indicator of relatedness in this group. Significant congruence was detected for dnaK, glnA and thrC. Analyses of concatenated sequences of dnaK, glnA and thrC genes yielded very similar NJ, MP and ML trees, with high bootstrap support. In addition, analysis of a concatenation of all six genes essentially produced the same result, levelling out potentially conflicting phylogenetic signals. This new evidence supports the proposal to unite Ensifer and Sinorhizobium in a single genus. Support for an alternative solution preserving the two genera is less strong. In view of the opinions expressed by the Judicial Commission, the name of the genus should be Ensifer, as proposed by Young [Young, J. M. (2003). Int J Syst Evol Microbiol 53, 2107-2110]. Data obtained previously and these new data indicate that Ensifer adhaerens and 'Sinorhizobium morelense' are not heterotypic synonyms, but represent separate species. However, transfer to the genus Ensifer is not possible at present because the species name is the subject of a pending Request for an Opinion, which would affect whether a novel species in the genus Ensifer or a new combination based on a basonym would be created.
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11. |
Lloret L,
Ormeño-Orrillo E,
Rincón R,
Martínez-Romero J,
Rogel-Hernández MA,
Martínez-Romero E,
( 2007 ) Ensifer mexicanus sp. nov. a new species nodulating Acacia angustissima (Mill.) Kuntze in Mexico. PMID : 17293074 : DOI : 10.1016/j.syapm.2006.12.002 Abstract >>
A new lineage of Ensifer nodulating the American legume Acacia angustissima in the tropical forest of Chiapas and Morelos, Mexico is described. Bacteria were identified as Ensifer with ssb or nolR specific primers. Phylogenetic analysis with partial sequences of the five chromosomal genes gyrA, nolR, recA, rpoB and rrs revealed that this new lineage is related to African Ensifer terangae. The results of total DNA-DNA hybridization and selected phenotypic tests among the A. angustissima strains and E. terangae indicated that they belong to different species. The phylogeny with the symbiotic nifH gene also separates this group as a different clade but with close affinities to bacteria belonging to the genus Ensifer isolated from American hosts. ITTG R7(T) (=CFN ER1001, HAMBI 2910, CIP 109033, ATCC BAA-1312, DSM18446) is the type strain of a new species for which the name Ensifer mexicanus sp. nov. is proposed.
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12. |
Martens M,
Weidner S,
Linke B,
de Vos P,
Gillis M,
Willems A,
( 2007 ) A prototype taxonomic microarray targeting the rpsA housekeeping gene permits species identification within the rhizobial genus Ensifer. PMID : 17291704 : DOI : 10.1016/j.syapm.2007.01.002 Abstract >>
To develop a reliable tool for the identification and classification of the different Ensifer species, without the need for sequencing, a prototype DNA microarray that targets the rpsA housekeeping gene was designed and tested. Internal segments of the rpsA gene from 34 reference strains, representing the different Ensifer species, were sequenced and the sequences were used to select 44 diagnostic oligonucleotides that served as probes for the identification microarray. Both, genomic DNA and specific rpsA PCR-products were tested as a target in hybridisation experiments. Experimental conditions were optimised and the diagnostic oligonucleotides were validated. Hybridisation results with the rpsA PCR-products showed reliable identification of the reference strains to species and genomovar level. Our data indicate that a microarray targeting housekeeping genes is a promising, accurate and relatively simple genotyping technique that would also be applicable for the identification and characterization of other bacterial groups of interest.
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13. |
Barcellos FG,
Menna P,
da Silva Batista JS,
Hungria M,
( 2007 ) Evidence of horizontal transfer of symbiotic genes from a Bradyrhizobium japonicum inoculant strain to indigenous diazotrophs Sinorhizobium (Ensifer) fredii and Bradyrhizobium elkanii in a Brazilian Savannah soil. PMID : 17308185 : DOI : 10.1128/AEM.01823-06 PMC : PMC1855619 Abstract >>
The importance of horizontal gene transfer (HGT) in the evolution and speciation of bacteria has been emphasized; however, most studies have focused on genes clustered in pathogenesis and very few on symbiosis islands. Both soybean (Glycine max [L.] Merrill) and compatible Bradyrhizobium japonicum and Bradyrhizobium elkanii strains are exotic to Brazil and have been massively introduced in the country since the early 1960s, occupying today about 45% of the cropped land. For the past 10 years, our group has obtained several isolates showing high diversity in morphological, physiological, genetic, and symbiotic properties in relation to the putative parental inoculant strains. In this study, parental strains and putative natural variants isolated from field-grown soybean nodules were genetically characterized in relation to conserved genes (by repetitive extragenic palindromic PCR using REP and BOX A1R primers, PCR-restriction fragment length polymorphism, and sequencing of the 16SrRNA genes), nodulation, and N(2)-fixation genes (PCR-RFLP and sequencing of nodY-nodA, nodC, and nifH genes). Both genetic variability due to adaptation to the stressful environmental conditions of the Brazilian Cerrados and HGT events were confirmed. One strain (S 127) was identified as an indigenous B. elkanii strain that acquired a nodC gene from the inoculant B. japonicum. Another one (CPAC 402) was identified as an indigenous Sinorhizobium (Ensifer) fredii strain that received the whole symbiotic island from the B. japonicum inoculant strain and maintained an extra copy of the original nifH gene. The results highlight the strategies that bacteria may commonly use to obtain ecological advantages, such as the acquisition of genes to establish effective symbioses with an exotic host legume.
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14. |
Yoshida K,
Kim WS,
Kinehara M,
Mukai R,
Ashida H,
Ikeda H,
Fujita Y,
Krishnan HB,
( 2006 ) Identification of a functional 2-keto-myo-inositol dehydratase gene of Sinorhizobium fredii USDA191 required for myo-inositol utilization. PMID : 17151471 : DOI : 10.1271/bbb.60362 Abstract >>
Sinorhizobium fredii USDA191 is a Gram-negative bacterium capable of forming nitrogen-fixing nodules on soybean roots. The USDA191 idhA gene encoding myo-inositol dehydrogenase, an enzyme necessary for myo-inositol utilization, is known to be involved in competitive nodulation and nitrogen fixation. In Bacillus subtilis, myo-inositol dehydrogenase catalyzes the first step of the myo-inositol catabolic pathway. Recently iolE was identified as the gene encoding 2-keto-myo-inositol dehydratase, which catalyzes the second step in the pathway. Here we report the presence of 2-keto-myo-inositol dehydratase activity in free-living USDA191 cells cultured in a medium containing myo-inositol. An iolE ortholog was cloned from USDA191. USDA191 iolE was expressed in Escherichia coli as a His(6)-tag fusion and purified to exhibit 2-keto-myo-inositol dehydratase activity. Inactivation of USDA191 iolE led to defective myo-inositol utilization. USDA191 iolE partially complemented a B. subtilis iolE deficient mutant. These results suggest that S. fredii USDA191 utilizes a myo-inositol catabolic pathway, analogous to that of B. subtilis, involving at least idhA and iolE.
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15. |
Krishnan HB,
Pueppke SG,
( 1991 ) nolC, a Rhizobium fredii gene involved in cultivar-specific nodulation of soybean, shares homology with a heat-shock gene. PMID : 1646377 : DOI : 10.1111/j.1365-2958.1991.tb00744.x Abstract >>
Rhizobium fredii strain USDA257 does not nodulate soybean (Glycine max (L.) Merr.) cultivar McCall. Mutant 257DH5, which contains a Tn5 insert in the bacterial chromosome, forms nodules on this cultivar, but acetylene-reduction activity is absent. We have sequenced the region corresponding to the site of Tn5 insertion in this mutant and find that it lies within a 1176bp open reading frame that we designate nolC. nolC encodes a protein of deduced molecular weight 43564. Nucleotide sequences homologous to nolC are present in several other Rhizobium strains, as well as Agrobacterium tumefaciens, but not in Pseudomonas syringae pathovar glycinea. nolC lacks significant sequence homology with known genes that function in nodulation, but is 61% homologous to dnaJ, an Escherichia coli gene that encodes a 41 kDa heat-shock protein. Both R. fredii USDA257 and mutant 257DH5 produce heat-shock proteins of 78, 70, 22, and 16kDa. A 4.3kb EcoRI-HindIII subclone containing nolC expresses a single 43kDa polypeptide in mini-cells. A longer, 9.4kb EcoRI fragment expresses both the 43kDa polypeptide and a 78kDa polypeptide that corresponds in size to that of the largest heat-shock protein. Thus, although nolC has strong sequence homology to dnaJ and appears to be linked to another heat-shock gene, it does not directly function in the heat-shock response.
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16. |
Parada M,
Vinardell JM,
Ollero FJ,
Hidalgo A,
Gutiérrez R,
Buendía-Clavería AM,
Lei W,
Margaret I,
López-Baena FJ,
Gil-Serrano AM,
Rodríguez-Carvajal MA,
Moreno J,
Ruiz-Sainz JE,
( 2006 ) Sinorhizobium fredii HH103 mutants affected in capsular polysaccharide (KPS) are impaired for nodulation with soybean and Cajanus cajan. PMID : 16404952 : DOI : 10.1094/MPMI-19-0043 Abstract >>
The Sinorhizobium fredii HH103 rkp-1 region, which is involved in capsular polysaccharides (KPS) production, was isolated and sequenced. The organization of the S. fredii genes identified, rkpUAGHIJ and kpsF3, was identical to that described for S. meliloti 1021 but different from that of S. meliloti AK631. The long rkpA gene (7.5 kb) of S. fredii HH103 and S. meliloti 1021 appears as a fusion of six clustered AK631 genes, rkpABCDEF. S. fredii HH103-Rif(r) mutants affected in rkpH or rkpG were constructed. An exoA mutant unable to produce exopolysaccharide (EPS) and a double mutant exoA rkpH also were obtained. Glycine max (soybean) and Cajanus cajan (pigeon pea) plants inoculated with the rkpH, rkpG, and rkpH exoA derivatives of S. fredii HH103 showed reduced nodulation and severe symptoms of nitrogen starvation. The symbiotic capacity of the exoA mutant was not significantly altered. All these results indicate that KPS, but not EPS, is of crucial importance for the symbiotic capacity of S. fredii HH103-Rif(r). S. meliloti strains that produce only EPS or KPS are still effective with alfalfa. In S. fredii HH103, however, EPS and KPS are not equivalent, because mutants in rkp genes are symbiotically impaired regardless of whether or not EPS is produced.
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17. |
Jiang JQ,
Wei W,
Du BH,
Li XH,
Wang L,
Yang SS,
( 2004 ) Salt-tolerance genes involved in cation efflux and osmoregulation of Sinorhizobium fredii RT19 detected by isolation and characterization of Tn5 mutants. PMID : 15451112 : DOI : 10.1016/j.femsle.2004.08.029 Abstract >>
Salt-tolerance genes of Sinorhizobium fredii RT19 were identified by the construction and screening of a transposon Tn5-1063 library containing over 30,000 clones. Twenty-one salt-sensitive mutants were obtained and five different genes were identified by sequencing. Eight mutants were found with disruptions in the phaA2 gene, which encodes a cation efflux system protein, while mutations in genes encoding other cation effux system proteins were found in seven (phaD2), two (phaF2) and two (phaG2) mutants. A mutation in the metH gene, encoding 5' methyltetrahydrofolate homocysteine methyltransferase, was found in two of the salt sensitive strains. Growth experiments showed that phaA2, phaD2, phaF2 and phaG2 mutants were hypersensitive to Na+/Li+ and slightly sensitive to K+ and not sensitive to sucrose and that metH mutants were highly sensitive to any of Na+, Li+, K+ and sucrose. Na+ intracellular content measurements established that phaA2, phaD2, phaF2 and phaG2 are mainly involved in the Na+ efflux in S. fredii RT19. Recovery of growth of the metH mutants incubated with different concentrations of NaCl could be obtained by additions of methionine, choline and betaine, which showed that the metH gene is probably involved in osmoregulation in S. fredii RT19.
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18. |
Nanba K,
King GM,
Dunfield K,
( 2004 ) Analysis of facultative lithotroph distribution and diversity on volcanic deposits by use of the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase. PMID : 15066819 : DOI : 10.1128/aem.70.4.2245-2253.2004 PMC : PMC383153 Abstract >>
A 492- to 495-bp fragment of the gene coding for the large subunit of the form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) (rbcL) was amplified by PCR from facultatively lithotrophic aerobic CO-oxidizing bacteria, colorless and purple sulfide-oxidizing microbial mats, and genomic DNA extracts from tephra and ash deposits from Kilauea volcano, for which atmospheric CO and hydrogen have been previously documented as important substrates. PCR products from the mats and volcanic sites were used to construct rbcL clone libraries. Phylogenetic analyses showed that the rbcL sequences from all isolates clustered with form IC rbcL sequences derived from facultative lithotrophs. In contrast, the microbial mat clone sequences clustered with sequences from obligate lithotrophs representative of form IA rbcL. Clone sequences from volcanic sites fell within the form IC clade, suggesting that these sites were dominated by facultative lithotrophs, an observation consistent with biogeochemical patterns at the sites. Based on phylogenetic and statistical analyses, clone libraries differed significantly among volcanic sites, indicating that they support distinct lithotrophic assemblages. Although some of the clone sequences were similar to known rbcL sequences, most were novel. Based on nucleotide diversity and average pairwise difference, a forested site and an 1894 lava flow were found to support the most diverse and least diverse lithotrophic populations, respectively. These indices of diversity were not correlated with rates of atmospheric CO and hydrogen uptake but were correlated with estimates of respiration and microbial biomass.
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19. |
Gil-Serrano AM,
Ruiz-Sainz JE,
Vinardell JM,
Ollero FJ,
Hidalgo A,
López-Baena FJ,
Medina C,
Ivanov-Vangelov K,
Parada M,
Madinabeitia N,
Espuny Mdel R,
Bellogín RA,
Camacho M,
Rodríguez-Navarro DN,
Soria-Díaz ME,
( 2004 ) NolR regulates diverse symbiotic signals of Sinorhizobium fredii HH103. PMID : 15195950 : DOI : 10.1094/MPMI.2004.17.6.676 Abstract >>
We have investigated in Sinorhizobium fredii HH103-1 (=HH103 Str(r)) the influence of the nolR gene on the production of three different bacterial symbiotic signals: Nod factors, signal responsive (SR) proteins, and exopolysaccharide (EPS). The presence of multiple copies of nolR (in plasmid pMUS675) repressed the transcription of all the flavonoid-inducible genes analyzed: nodA, nodD1, nolO, nolX, noeL, rhcJ, hesB, and y4pF. Inactivation of nolR (mutant SVQ517) or its overexpression (presence of pMUS675) altered the amount of Nod factors detected. Mutant SVQ517 produced Nod factors carrying N-methyl residues at the nonreducing N-acetyl-glucosamine, which never have been detected in S. fredii HH103. Plasmid pMUS675 increased the amounts of EPS produced by HH103-1 and SVQ517. The flavonoid genistein repressed EPS production of HH103-1 and SVQ517 but the presence of pMUS675 reduced this repression. The presence of plasmid pMUS675 clearly decreased the secretion of SR proteins. Inactivation, or overexpression, of nolR decreased the capacity of HH103 to nodulate Glycine max. However, HH103-1 and SVQ517 carrying plasmid pMUS675 showed enhanced nodulation capacity with Vigna unguiculata. The nolR gene was positively identified in all S. fredii strains investigated, S. xinjiangense CCBAU110, and S. saheli USDA4102. Apparently, S. teranga USDA4101 does not contain this gene.
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20. |
Krishnan HB,
Lewin A,
Fellay R,
Broughton WJ,
Pueppke SG,
( 1992 ) Differential expression of nodS accounts for the varied abilities of Rhizobium fredii USDA257 and Rhizobium sp. strain NGR234 to nodulate Leucaena spp. PMID : 1484488 : DOI : 10.1111/j.1365-2958.1992.tb02200.x Abstract >>
Transfer of a cosmid containing nodSU from Rhizobium sp. NGR234 to Rhizobium fredii USDA257 expands the host range for nodulation to include the perennial tropical legumes, Leucaena leucocephala and Leucaena diversifolia. Complementation experiments with a series of subclones established that nodS and its associated nod-box promoter from NGR234 are sufficient to confer this extended host-range phenotype to L. leucocephala. Strain USDA257 contains its own copy of nodSU, including upstream nod-box sequences. Although both nucleotide and deduced amino acid sequences of the reading frames are homologous between the two strains, there are gaps within the promoter region and the 5'-end of nodS of USDA257. Consequently, the deduced NodS protein of USDA257 is shorter than its counterpart from NGR234, and the distance between the nod-box and the initiation codon is greater. A 36 bp deletion encompasses the extreme right border of the USDA257 nod-box and extends into the upstream leader sequence. Transcriptional fusions with both nod-boxes confirmed that the promoter from NGR234 is flavonoid-inducible, and that the nod-box from USDA257 is not. These observations were corroborated by Northern analysis with a nodS-containing Xhol fragment as hybridization probe. Flavonoid-induced cells of NGR234 gave an intense signal, but those of USDA257 yielded only a weak trace of hybridization. EcoRI fragments with homology to nodSU of USDA257 are present in 17 of 35 tested strains, including several representatives of Bradyrhizobium japonicum, Rhizobium sp., R. loti, and R. fredii. Two wild-type, leucaena-nodulating strains of Rhizobium sp. lack this homology. We conclude that a genetic defect in expression of nodS accounts for the inability of USDA257 to nodulate leucaena and that diverse rhizobia may have evolved alternative mechanisms to nodulate this legume species.
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21. |
Vinardell JM,
López-Baena FJ,
Hidalgo A,
Ollero FJ,
Bellogín R,
del Rosario Espuny M,
Temprano F,
Romero F,
Krishnan HB,
Pueppke SG,
Ruiz-Sainz JE,
( 2004 ) The effect of FITA mutations on the symbiotic properties of Sinorhizobium fredii varies in a chromosomal-background-dependent manner. PMID : 14689165 : DOI : 10.1007/s00203-003-0635-3 Abstract >>
nodD1 of Sinorhizobium fredii HH103, which is identical to that of S. fredii USDA257 and USDA191, repressed its own expression. Spontaneous flavonoid-independent transcription activation (FITA) mutants of S. fredii HH103 M (=HH103 RifR pSym::Tn 5-Mob) showing constitutive expression of nod genes were isolated. No differences were found among soybean cultivar Williams plants inoculated with FITA mutants SVQ250 or SVQ253 or with the parental strain HH103M. Soybean plants inoculated with mutant SVQ255 formed more nodules, and those inoculated with mutant SVQ251 had symptoms of nitrogen starvation. Sequence analyses showed that all of the FITA mutants carried a point mutation in their nodD1 coding region. Mutants SVQ251 and SVQ253 carried the same mutation, but only the former was symbiotically impaired, which indicated the presence of an additional mutation elsewhere in the genome of mutant SVQ251. Mutants SVQ251 and SVQ255 were outcompeted by the parental strain for nodulation of soybean cultivar Williams. The symbiotic plasmids of mutants SVQ251 and SVQ255 (pSym251 and pSym255, respectively) and that (pSymHH103M) of the parental strain were transferred to pSym-cured derivatives of S. fredii USDA192 and USDA193 (USDA192C and USDA193C, respectively). Soybean responses to inoculation with S. fredii USDA192C and USDA193C transconjugants carrying pSym251 and pSymHH103M were not significantly different, whereas more nodules were formed after inoculation with transconjugants carrying pSym255. Only transconjugant USDA192C(pSym255) produced a significant increase in soybean dry weight.
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22. |
Pérez-Montaño F,
Guasch-Vidal B,
González-Barroso S,
López-Baena FJ,
Cubo T,
Ollero FJ,
Gil-Serrano AM,
Rodríguez-Carvajal M?,
Bellogín RA,
Espuny MR,
( 2011 ) Nodulation-gene-inducing flavonoids increase overall production of autoinducers and expression of N-acyl homoserine lactone synthesis genes in rhizobia. PMID : 21600980 : DOI : 10.1016/j.resmic.2011.05.002 Abstract >>
Legume-nodulating rhizobia use N-acyl homoserine lactones (AHLs) to regulate several physiological traits related to the symbiotic plant-microbe interaction. In this work, we show that Sinorhizobium fredii SMH12, Rhizobium etli ISP42 and Rhizobium sullae IS123, three rhizobial strains with different nodulation ranges, produced a similar pattern of AHL molecules, sharing, in all cases, production of N-octanoyl homoserine lactone and its 3-oxo and/or 3-hydroxy derivatives. Interestingly, production of AHLs was enhanced when these three rhizobia were grown in the presence of their respective nod-gene-inducing flavonoid, while a new molecule, C14-HSL, was produced by S. fredii SMH12 upon genistein induction. In addition, expression of AHL synthesis genes traI from S. fredii SMH12 and cinI and raiI from R. etli ISP42 increased when induced with flavonoids, as demonstrated by qRT-PCR analysis.
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23. |
Zhang YM,
Li Y,
Chen WF,
Wang ET,
Tian CF,
Li QQ,
Zhang YZ,
Sui XH,
Chen WX,
( 2011 ) Biodiversity and biogeography of rhizobia associated with soybean plants grown in the North China Plain. PMID : 21784912 : DOI : 10.1128/AEM.00542-11 PMC : PMC3187167 Abstract >>
As the putative center of origin for soybean and the second largest region of soybean production in China, the North China Plain covers temperate and subtropical regions with diverse soil characteristics. However, the soybean rhizobia in this plain have not been sufficiently studied. To investigate the biodiversity and biogeography of soybean rhizobia in this plain, a total of 309 isolates of symbiotic bacteria from the soybean nodules collected from 16 sampling sites were studied by molecular characterization. These isolates were classified into 10 genospecies belonging to the genera Sinorhizobium and Bradyrhizobium, including four novel groups, with S. fredii (68.28%) as the dominant group. The phylogeny of symbiotic genes nodC and nifH defined four lineages among the isolates associated with Sinorhizobium fredii, Bradyrhizobium elkanii, B. japonicum, and B. yuanmingense, demonstrating the different origins of symbiotic genes and their coevolution with the chromosome. The possible lateral transfer of symbiotic genes was detected in several cases. The association between soil factors (available N, P, and K and pH) and the distribution of genospecies suggest clear biogeographic patterns: Sinorhizobium spp. were superdominant in sampling sites with alkaline-saline soils, while Bradyrhizobium spp. were more abundant in neutral soils. This study clarified the biodiversity and biogeography of soybean rhizobia in the North China Plain.
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24. |
Li QQ,
Wang ET,
Zhang YZ,
Zhang YM,
Tian CF,
Sui XH,
Chen WF,
Chen WX,
( 2011 ) Diversity and biogeography of rhizobia isolated from root nodules of Glycine max grown in Hebei Province, China. PMID : 21340735 : DOI : 10.1007/s00248-011-9820-0 Abstract >>
A total of 215 rhizobial strains were isolated and analyzed with 16S rRNA gene, 16S-23S intergenic spacer, housekeeping genes atpD, recA, and glnII, and symbiotic genes nifH and nodC to understand the genetic diversity of soybean rhizobia in Hebei province, China. All the strains except one were symbiotic bacteria classified into nine genospecies in the genera of Bradyrhizobium and Sinorhizobium. Surveys on the distribution of these rhizobia in different regions showed that Bradyrhizobium japonicum and Bradyrhizobium elkanii strains were found only in neutral to slightly alkaline soils whereas Bradyrhizobium yuanmingense, Bradyrhizobium liaoningense-related strains and strains of five Sinorhizobium genospecies were found in alkaline-saline soils. Correspondence and canonical correspondence analyses on the relationship of rhizobial distribution and their soil characteristics reveal that high soil pH, electrical conductivity, and potassium content favor distribution of the B. yuanmingense and the five Sinorhizobium species but inhibit B. japonicum and B. elkanii. High contents of available phosphorus and organic matters benefit Sinorhizobium fredii and B. liaoningense-related strains and inhibit the others groups mentioned above. The symbiotic gene (nifH and nodC) lineages among B. elkanii, B. japonicum, B. yuanmingense, and Sinorhizobium spp. were observed in the strains, signifying that vertical gene transfer was the main mechanism to maintain these genes in the soybean rhizobia. However, lateral transfer of symbiotic genes commonly in Sinorhizobium spp. and rarely in Bradyrhizobium spp. was also detected. These results showed the genetic diversity, the biogeography, and the soil determinant factors of soybean rhizobia in Hebei province of China.
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25. |
Wu LJ,
Wang HQ,
Wang ET,
Chen WX,
Tian CF,
( 2011 ) Genetic diversity of nodulating and non-nodulating rhizobia associated with wild soybean (Glycine soja Sieb. & Zucc.) in different ecoregions of China. PMID : 21303397 : DOI : 10.1111/j.1574-6941.2011.01064.x Abstract >>
A total of 99 bacterial isolates that originated from root nodules of Glycine soja were characterized with restriction analyses of amplified 16S ribosomal DNA and 16S-23S rDNA intergenic spacers (ITS), and sequence analyses of 16S rRNA, rpoB, atpD, recA and nodC genes. When tested for nodulation of G. soja, 72 of the isolates were effective symbionts, and these belonged to five species: Bradyrhizobium japonicum, Bradyrhizobium elkanii, Bradyrhizobium yuanmingense, Bradyrhizobium liaoningense and Sinorhizobium fredii. All of these, except some B. yuanmingense strains, also formed effective nodules on the domesticated soybean Glycine max. The remaining 27 isolates did not nodulate either host, but were identified as Rhizobium. Phylogeny nodC in the G. soja symbionts suggested that this symbiosis gene was mainly maintained by vertical gene transfer. Different nodC sublineages and rrs-ITS clusters reflected the geographic origins of isolates in this study.
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26. |
Zhao L,
Deng Z,
Yang W,
Cao Y,
Wang E,
Wei G,
( 2010 ) Diverse rhizobia associated with Sophora alopecuroides grown in different regions of Loess Plateau in China. PMID : 20965680 : DOI : 10.1016/j.syapm.2010.08.004 Abstract >>
A total of seventy-five symbiotic bacterial strains isolated from root nodules of wild Sophora alopecuroides grown in different regions of China's Loess Plateau were characterized. Based on the combined RFLP patterns, thirty-five genotypes were defined among the rhizobia and they were classified into nine genomic species, including Mesorhizobium alhagi and M. gobiense as the main groups, as well as Agrobacterium tumefaciens, M. amorphae, Phyllobacterium trifolii, Rhizobium giardinii, R. indigoferae, Sinorhizobium fredii and S. meliloti as the minor groups according to the 16S rRNA and recA gene analyses. Five and three lineages of nodA and nifH were found, respectively, in these strains, implying that the symbiotic genes of the S. alopecuroides rhizobia had different origins or had divergently evolved. Results of correspondence analysis showed that there was a correlation between rhizobial genotypes and the geographic origins. Possible lateral transfer of the recA and 16S rRNA genes between the P. trifolii and A. tumefaciens strains, and that of symbiotic genes (nodA, nifH) between different genera, was shown by discrepancies of the phylogenetic relationships of the four gene loci. These results revealed diverse rhizobia associated with wild S. alopecuroides grown in different regions of China's Loess Plateau, and demonstrated for the first time the existence of symbiotic A. tumefaciens strains in root nodules of S. alopecuroides.
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27. |
Li QQ,
Wang ET,
Chang YL,
Zhang YZ,
Zhang YM,
Sui XH,
Chen WF,
Chen WX,
( 2011 ) Ensifer sojae sp. nov., isolated from root nodules of Glycine max grown in saline-alkaline soils. PMID : 20851917 : DOI : 10.1099/ijs.0.025049-0 Abstract >>
Thirteen bacterial isolates from root nodules of soybean grown in saline-alkaline soils in the Chinese province of Hebei were identified as a unique group in the genus Ensifer based upon BOX-PCR patterns, sequencing analyses of 16S rRNA and housekeeping genes and DNA-DNA hybridization. Phenotypically, positive tests for acid production and negative results for reduction in litmus milk and sensitivity to 50 ?g ampicillin ml(-1), as well as some other features, could differentiate the novel group from defined species of the Ensifer-Sinorhizobium group. The novel group had symbiotic gene sequences (nodC and nifH) that were identical or very similar to those of Ensifer (Sinorhizobium) fredii, and formed effective nodules with Glycine max (soybean), Vigna unguiculata and Glycine soja. Based upon the consensus of these analyses, a novel species, Ensifer sojae sp. nov., is proposed, with CCBAU 05684(T) (= LMG 25493(T) = HAMBI 3098(T)) as the type strain. The DNA G+C content of strain CCBAU 05684(T) was 60.9 mol% (T(m)).
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28. |
Lorio JC,
Kim WS,
Krishnan AH,
Krishnan HB,
( 2010 ) Disruption of the glycine cleavage system enables Sinorhizobium fredii USDA257 to form nitrogen-fixing nodules on agronomically improved North American soybean cultivars. PMID : 20453144 : DOI : 10.1128/AEM.00437-10 PMC : PMC2897462 Abstract >>
The symbiosis between Sinorhizobium fredii USDA257 and soybean [Glycine max (L.) Merr.] exhibits a high degree of cultivar specificity. USDA257 nodulates primitive soybean cultivars but fails to nodulate agronomically improved cultivars such as McCall. In this study we provide evidence for the involvement of a new genetic locus that controls soybean cultivar specificity. This locus was identified in USDA257 by Tn5 transposon mutagenesis followed by nodulation screening on McCall soybean. We have cloned the region corresponding to the site of Tn5 insertion and found that it lies within a 1.5-kb EcoRI fragment. DNA sequence analysis of this fragment and an adjacent 4.4-kb region identified an operon made up of three open reading frames encoding proteins of deduced molecular masses of 41, 13, and 104 kDa, respectively. These proteins revealed significant amino acid homology to glycine cleavage (gcv) system T, H, and P proteins of Escherichia coli and other organisms. Southern blot analysis revealed the presence of similar sequences in diverse rhizobia. Measurement of beta-galactosidase activity of a USDA257 strain containing a transcriptional fusion of gcvT promoter sequences to the lacZ gene revealed that the USDA257 gcvTHP operon was inducible by glycine. Inactivation of either gcvT or gcvP of USDA257 enabled the mutant to nodulate several agronomically improved North American soybean cultivars. These nodules revealed anatomical features typical of determinate nodules, with numerous bacteroids within the infected cells. Unlike for the previously characterized soybean cultivar specificity locus nolBTUVW, inactivation of the gcv locus had no discernible effect on the secretion of nodulation outer proteins of USDA257.
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29. |
Zhang YF,
Sui XH,
Chen WF,
Chen WX,
Han LL,
Wang ET,
Lu YL,
( 2009 ) Bradyrhizobium spp. and Sinorhizobium fredii are predominant in root nodules of Vigna angularis, a native legume crop in the subtropical region of China. PMID : 19557346 : DOI : 10.1007/s12275-009-0001-5 Abstract >>
Adzuki bean (Vigna angularis) is an important legume crop native to China, but its rhizobia have not been well characterized. In the present study, a total of 60 rhizobial strains isolated from eight provinces of China were analyzed with amplified 16S rRNA gene RFLP, IGS-RFLP, and sequencing analyses of 16S rRNA, atpD, recA, and nodC genes. These strains were identified as genomic species within Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium, and Ochrobactrum. The most abundant groups were Bradyrhizobium species and Sinorhizobium fredii. Diverse nodC genes were found in these strains, which were mainly co-evolved with the housekeeping genes, but a possible lateral transfer of nodC from Sinorhizobium to Rhizobium was found. Analyses of the genomic and symbiotic gene backgrounds showed that adzuki bean shared the same rhizobial gene pool with soybean (legume native to China) and the exotic Vigna species. All of these data demonstrated that nodule formation is the interaction of rhizobia, host plants, and environment characters.
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30. |
Crespo-Rivas JC,
Margaret I,
Hidalgo A,
Buendía-Clavería AM,
Ollero FJ,
López-Baena FJ,
del Socorro Murdoch P,
Rodríguez-Carvajal MA,
Soria-Díaz ME,
Reguera M,
Lloret J,
Sumpton DP,
Mosely JA,
Thomas-Oates JE,
van Brussel AA,
Gil-Serrano A,
Vinardell JM,
Ruiz-Sainz JE,
( 2009 ) Sinorhizobium fredii HH103 cgs mutants are unable to nodulate determinate- and indeterminate nodule-forming legumes and overproduce an altered EPS. PMID : 19348575 : DOI : 10.1094/MPMI-22-5-0575 Abstract >>
Sinorhizobium fredii HH103 produces cyclic beta glucans (CG) composed of 18 to 24 glucose residues without or with 1-phosphoglycerol as the only substituent. The S. fredii HH103-Rifr cgs gene (formerly known as ndvB) was sequenced and mutated with the lacZ-gentamicin resistance cassette. Mutant SVQ562 did not produce CG, was immobile, and grew more slowly in the hypoosmotic GYM medium, but its survival in distilled water was equal to that of HH103-Rifr. Lipopolysaccharides and K-antigen polysaccharides produced by SVQ562 were not apparently altered. SVQ562 overproduced exopolysaccharides (EPS) and its exoA gene was transcribed at higher levels than in HH103-Rifr. In GYM medium, the EPS produced by SVQ562 was of higher molecular weight and carried higher levels of substituents than that produced by HH103-Rifr. The expression of the SVQ562 cgsColon, two colonslacZ fusion was influenced by the pH and the osmolarity of the growth medium. The S. fredii cgs mutants SVQ561 (carrying cgs::Omega) and SVQ562 only formed pseudonodules on Glycine max (determinate nodules) and on Glycyrrhiza uralensis (indeterminate nodules). Although nodulation factors were detected in SVQ561 cultures, none of the cgs mutants induced any macroscopic response in Vigna unguiculata roots. Thus, the nodulation process induced by S. fredii cgs mutants is aborted at earlier stages in V. unguiculata than in Glycine max.
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31. |
Zhao CT,
Wang ET,
Chen WF,
Chen WX,
( 2008 ) Diverse genomic species and evidences of symbiotic gene lateral transfer detected among the rhizobia associated with Astragalus species grown in the temperate regions of China. PMID : 18657113 : DOI : 10.1111/j.1574-6968.2008.01282.x Abstract >>
Based on the analyses of ribosomal DNA and housekeeping genes, a total of 118 bacterial isolates obtained from 13 Astragalus species grown in the temperate region of China were identified as 19 genomic species of Mesorhizobium, Rhizobium, Sinorhizobium and Bradyrhizobium, two of them being putatively new species. Phylogenetic comparison of symbiotic genes (nodC and nifH) and housekeeping genes showed that the symbiotic genes of the Astragalus rhizobia were maintained by both vertical and horizontal transfer. The results demonstrated that the Astragalus species were very promiscuous hosts for rhizobia and that their rhizobia had very diverse genomic and symbiotic gene backgrounds.
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32. |
López-Baena FJ,
Vinardell JM,
Pérez-Montaño F,
Crespo-Rivas JC,
Bellogín RA,
Espuny Mdel R,
Ollero FJ,
( 2008 ) Regulation and symbiotic significance of nodulation outer proteins secretion in Sinorhizobium fredii HH103. PMID : 18524937 : DOI : 10.1099/mic.0.2007/016337-0 Abstract >>
In this work we show that the Sinorhizobium fredii HH103 ttsI gene is essential for the expression of the tts genes and secretion of nodulation outer proteins (Nops). Moreover, we demonstrate for the first time, to our knowledge, that the nod box preceding ttsI is necessary for Nops secretion. TtsI is responsible for the transcriptional activation of nopX, nopA, rhcJ and rhcQ. We confirm that the S. fredii HH103 ttsI gene is activated by NodD1 and repressed by NolR. In contrast, NodD2 is not involved in the regulation of ttsI expression. Despite the dependence of expression of both ttsI and nodA on NodD1 and flavonoids, clear differences in the capacity of some flavonoids to activate these genes were found. The expression of the ttsI and nodA genes was also sensitive to differences in the pH of the media. Secretion of Nops in the ttsI mutant could not be complemented with a DNA fragment containing the ttsI gene and its nod box, but it was restored when a plasmid harbouring the ttsI, rhcC2 and y4xK genes was transferred to the mutant strain. The symbiotic effect of Nops secretion was host-dependent but independent of the type of nodule formed by the host legume. Nops are beneficial in the symbiosis with Glycine max and Glycyrrhiza uralensis, and detrimental in the case of the tropical legume Erythrina variegata.
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33. |
Lu ZJ,
Cao YQ,
Long WJ,
Long ZD,
Chen G,
Ma QS,
Wu B,
( 2008 ) Isolation and characterization of an operon involved in sulfate and sulfite metabolism in Sinorhizobium fredii. PMID : 18336549 : DOI : 10.1111/j.1574-6968.2008.01102.x Abstract >>
A gene cluster ORFabcd from a Sinorhizobium fredii HN01 mutant strain HSMRalpha was isolated. We showed that it was an operon involved in sulfur metabolism. Functional studies revealed that, except for ORFb, the three genes ORFa, ORFc and ORFd were involved in sulfite reduction. ORFa and ORFc were similar to the cysG and cysI from Sinorhizobium meliloti 1021 and Rhizobium etli CFN 42, respectively. ORFd encodes a conserved hypothetical protein in other bacteria. We demonstrate here, for the first time, that it was a new locus involved in sulfate assimilation in S. fredii HN01 and we designated it as cysII.
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34. |
Crespo-Rivas JC,
Margaret I,
Pérez-Montaño F,
López-Baena FJ,
Vinardell JM,
Ollero FJ,
Moreno J,
Ruiz-Sainz JE,
Buendía-Clavería AM,
( 2007 ) A pyrF auxotrophic mutant of Sinorhizobium fredii HH103 impaired in its symbiotic interactions with soybean and other legumes. PMID : 18075998 : Abstract >>
Transposon Tn5-Mob mutagenesis allowed the selection of a Sinorhizobium fredii HH103 mutant derivative (SVQ 292) that requires the presence of uracil to grow in minimal media. The mutated gene, pyrF, codes for an orotidine-5 - monophosphate decarboxylase (EC 4.1.1.23). Mutant SVQ 292 and its parental prototrophic mutant HH103 showed similar Nod-factor and lipopolysaccharide profiles. The symbiotic properties of mutant SVQ 292 were severely impaired with all legumes tested. Mutant SVQ 292 formed small ineffective nodules on Cajanus cajan and abnormal nodules (pseudonodules) unable to fix nitrogen on Glycine max (soybean), Macroptitlium atropurpureum, Indigofera tinctoria, and Desmodium canadense. It also did not induce any macroscopic response in Macrotyloma axillare roots. The symbiotic capacity of SVQ 292 with soybean was not enhanced by the addition of uracil to the plant nutritive solution.
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35. |
Lee SG,
Krishnan HB,
Jez JM,
( 2014 ) Structural basis for regulation of rhizobial nodulation and symbiosis gene expression by the regulatory protein NolR. PMID : 24733893 : DOI : 10.1073/pnas.1402243111 PMC : PMC4035926 Abstract >>
The symbiosis between rhizobial microbes and host plants involves the coordinated expression of multiple genes, which leads to nodule formation and nitrogen fixation. As part of the transcriptional machinery for nodulation and symbiosis across a range of Rhizobium, NolR serves as a global regulatory protein. Here, we present the X-ray crystal structures of NolR in the unliganded form and complexed with two different 22-base pair (bp) double-stranded operator sequences (oligos AT and AA). Structural and biochemical analysis of NolR reveals protein-DNA interactions with an asymmetric operator site and defines a mechanism for conformational switching of a key residue (Gln56) to accommodate variation in target DNA sequences from diverse rhizobial genes for nodulation and symbiosis. This conformational switching alters the energetic contributions to DNA binding without changes in affinity for the target sequence. Two possible models for the role of NolR in the regulation of different nodulation and symbiosis genes are proposed. To our knowledge, these studies provide the first structural insight on the regulation of genes involved in the agriculturally and ecologically important symbiosis of microbes and plants that leads to nodule formation and nitrogen fixation.
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36. |
Zhao L,
Fan M,
Zhang D,
Yang R,
Zhang F,
Xu L,
Wei X,
Shen Y,
Wei G,
( 2014 ) Distribution and diversity of rhizobia associated with wild soybean (Glycine soja Sieb. & Zucc.) in Northwest China. PMID : 25052953 : DOI : 10.1016/j.syapm.2014.05.011 Abstract >>
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.
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37. |
Sadowsky MJ,
Olson ER,
Foster VE,
Kosslak RM,
Verma DP,
( 1988 ) Two host-inducible genes of Rhizobium fredii and characterization of the inducing compound. PMID : 2447061 : DOI : 10.1128/jb.170.1.171-178.1988 PMC : PMC210622 Abstract >>
Random transcription fusions with Mu d1(Kan lac) generated three mutants in Rhizobium fredii (strain USDA 201) which showed induction of beta-galactosidase when grown in root exudate of the host plants Glycine max, Phaseolus vulgaris, and Vigna ungliculata. Two genes were isolated from a library of total plasmid DNA of one of the mutants, 3F1. These genes, present in tandem on a 4.2-kilobase HindIII fragment, appear in one copy each on the symbiotic plasmid and do not hybridize to the Rhizobium meliloti common nodulation region. They comprise two separate transcriptional units coding for about 450 and 950 nucleotides, both of which are transcribed in the same direction. The two open reading frames are separated by 586 base pairs, and the 5H regions of the two genes show a common sequence. No similarity was found with the promoter areas of Rhizobium trifolii, R. meliloti, or Bradyrhizobium japonicum nif genes and with any known nodulation genes. Regions homologous to both sequences were detected in EcoRI digests of genomic DNAs from B. japonicum USDA 110, USDA 122, and 61A76, but not in genomic DNA from R. trifolii, Rhizobium leguminosarum, or Rhizobium phaseoli. Mass spectrometry and nuclear magnetic resonance analysis indicated that the inducing compound has properties of 4',7-dihydroxyisoflavone, daidzein. These results suggest that, in addition to common nodulation genes, several other genes appear to be specifically induced by compounds in the root exudate of the host plants.
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38. |
( 1993 ) Molecular cloning and characterization of a sym plasmid locus that regulates cultivar-specific nodulation of soybean by Rhizobium fredii USDA257. PMID : 8412662 : DOI : 10.1111/j.1365-2958.1993.tb01665.x Abstract >>
Rhizobium fredii strain USDA257 produces nitrogen-fixing nodules on primitive soybean cultivars such as Peking but fails to nodulate agronomically improved cultivars such as McCall. Transposon-mutant 257DH4 has two new phenotypes: it nodulates McCall, and its ability to do so is sensitive to the presence of parental strain USDA257, i.e. it is subject to competitive nodulation blocking. We have isolated a cosmid containing DNA that corresponds to the site of transposon insertion in 257DH4 and have localized Tn5 on an 8.0 kb EcoRI fragment. The 5596 bp DNA sequence that surrounds the insertion site contains seven open reading frames. Five of these, designated nolBTU, ORF4, and nolV, are closely spaced and of the same polarity. nolW and nolX are of the opposite polarity. The initiation codon for nolW lies 155 bp upstream from that of nolB, and its is separated from nolX by 281 bp. The predicted NolT and NolW proteins have putative membrane-spanning regions. The N-terminus of the hypothetical NolW protein also has limited homology to NodH of Rhizobium meliloti, but none of the deduced protein sequences has significant homology to known nodulation gene products. Site-directed mutagenesis with mudII1734 confirms that inactivation of nolB, nolT, nolU, nolV, nolW, or nolX extends host range for nodulation to McCall soybean. This phenotype could not be genetically dissected from sensitivity to competitive nodulation blocking. Expression of nolBTU and nolX is induced as much as 30-fold by flavonoid signal molecules, even though these genes lack nod-box promoters. Histochemical staining of McCall roots inoculated with nolB-, nolU-, or nolX-lacZ fusions verifies that these genes are expressed continuously from preinfection to the stage of the functional nodule. Although a nolU-ORF4-nolV clone hybridizes to a single 8.0 kb EcoRI fragment from 10 strains of R. fredii and broad-host-range Rhizobium sp. NGR234, hybridizing sequences are not detectable in other rhizobia.
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( 1995 ) Transcriptional organization and expression of noIXWBTUV, a locus that regulates cultivar-specific nodulation of soybean by Rhizobium fredii USDA257. PMID : 8596441 : DOI : 10.1111/j.1365-2958.1995.mmi_17050923.x Abstract >>
Rhizobium fredii is a nitrogen-fixing bacterial symbiont of soybean and a number of other legume species. We have studied the transcriptional organization of a Sym plasmid locus that restricts the host range of R. fredii USDA257 at both the host species and cultivar level. The genes of this host-specificity locus, noIXWBTUV, are transcribed from three promoters. Two of these, which are upstream of noIW and noIBTUV, are oriented face to face and initiate transcription at sites that are 14 bp apart. The third lies upstream from noIX. The noIW promoter is constitutive, whereas the noIB and noIX promoters are inducible by flavonoid signals. We have attempted to express genes from this locus in Escherichia coli systems, both in vivo and in vitro. We detected the insert- and orientation-specific expression of two genes, noIX and noIW, but we were unable to obtain expression of noIBTUV. Antiserum raised against NoIT nevertheless detected an abundantly expressed polypeptide of the predicted size in protein extracts of USDA257. This observation, as well as RNA dot blot data from a series of mutants, indicates that noIBTUV is expressed as a single transcriptional unit in R. fredii. Immunological detection of NoIT, and of a second protein, NoIX, was strictly dependent on flavonoid induction. The NoIX protein was larger than the size predicted from the previously published nucleotide sequence, and this led to resequencing and revision of the open reading frame.
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( 1993 ) Characterization of RFRS9, a second member of the Rhizobium fredii repetitive sequence family from the nitrogen-fixing symbiont R. fredii USDA257. PMID : 8382462 : PMC : PMC202070 Abstract >>
The genome of the nitrogen-fixing symbiont, Rhizobium fredii USDA257, contains nine copies of repetitive sequences known as the R. fredii repetitive sequence (RFRS) family. We previously sequenced RFRS3, which is linked to symbiosis plasmid-borne nodulation genes of this organism and has substantial homology to the T-DNA of Agrobacterium rhizogenes and lesser homology to reiterated sequences of Bradyrhizobium japonicum. Here we characterize a second family member, RFRS9. The EcoRI fragment containing RFRS9 is 1,248 bp in length and contains a single 666-bp open reading frame that is flanked by perfect 8-bp inverted repeats. Nucleic and amino acid sequences corresponding to the C terminus of the putative RFRS9 protein are nearly identical to those of RFRS3, and they retain homology to DNA from A. rhizogenes. The central portion of the RFRS9 protein also appears to be related to the S locus-specific glycoprotein family of pollen stigma incompatibility glycoproteins from Brassica oleracea, which are involved in signal perception. Sequences that define the RFRS family are restricted to the open reading frame of RFRS9 and associated upstream sequences. These regions also contain a second group of repetitive sequences, which is present in four copies within the genome of USDA257. Both families of repetitive sequences are ubiquitous in R. fredii, and they are preferentially localized on symbiosis plasmids. Southern hybridization confirms that sequences homologous to RFRS9 are present in broad-host-range Rhizobium sp. strain NGR234, in A. rhizogenes, and in two biotype 3 strains of Agrobacterium tumefaciens.
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Yang SH,
Chen WH,
Wang ET,
Chen WF,
Yan J,
Han XZ,
Tian CF,
Sui XH,
Singh RP,
Jiang GM,
Chen WX,
( 2018 ) Rhizobial biogeography and inoculation application to soybean in four regions across China. PMID : 29719942 : DOI : 10.1111/jam.13897 Abstract >>
The aim of the study was to survey rhizobial biogeography and to inoculate soybean with selected rhizobia in China to enhance symbiotic nitrogen fixation (SNF). Biogeography, genetic diversity and phylogeny of soybean rhizobia were surveyed. Inocula were prepared and applied to soybean. Results showed that Bradyrhizobium elkanii and Ensifer fredii were widely distributed in acid and alkaline soils respectively. Available iron was detected as the first determinant for distribution of the two rhizobia and the soybean varieties did not greatly affect the rhizobial compatibility. Geographical latitude and precipitation in June were the main geographical and climatic factors affecting the rhizobial distribution. Inoculation with selected rhizobia increased the nodule number, fresh weight, occupation ratio, seed protein content and soybean yields. Selection and application of effective soybean rhizobia across China according to biogeography were clarified to promote the SNF, thereby improving soybean yield. Rhizobial diversity and biogeography were evaluated systematically in six sites across China. Available iron and soil pH are found to be the most important determinants for the distribution of soybean rhizobia. Inoculation to soybean enhances SNF, positively correlating to the increase in soybean yield and seed protein content.
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( 2013 ) Mutualistic co-evolution of type III effector genes in Sinorhizobium fredii and Bradyrhizobium japonicum. PMID : 23468637 : DOI : 10.1371/journal.ppat.1003204 PMC : PMC3585131 Abstract >>
Two diametric paradigms have been proposed to model the molecular co-evolution of microbial mutualists and their eukaryotic hosts. In one, mutualist and host exhibit an antagonistic arms race and each partner evolves rapidly to maximize their own fitness from the interaction at potential expense of the other. In the opposing model, conflicts between mutualist and host are largely resolved and the interaction is characterized by evolutionary stasis. We tested these opposing frameworks in two lineages of mutualistic rhizobia, Sinorhizobium fredii and Bradyrhizobium japonicum. To examine genes demonstrably important for host-interactions we coupled the mining of genome sequences to a comprehensive functional screen for type III effector genes, which are necessary for many Gram-negative pathogens to infect their hosts. We demonstrate that the rhizobial type III effector genes exhibit a surprisingly high degree of conservation in content and sequence that is in contrast to those of a well characterized plant pathogenic species. This type III effector gene conservation is particularly striking in the context of the relatively high genome-wide diversity of rhizobia. The evolution of rhizobial type III effectors is inconsistent with the molecular arms race paradigm. Instead, our results reveal that these loci are relatively static in rhizobial lineages and suggest that fitness conflicts between rhizobia mutualists and their host plants have been largely resolved.
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Cardinet GH,
Holliday TA,
( 1979 ) Neuromuscular diseases of domestic animals: a summary of muscle biopsies from 159 cases. PMID : 289312 : Abstract >>
N/A
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( 2012 ) Robust markers reflecting phylogeny and taxonomy of rhizobia. PMID : 23028691 : DOI : 10.1371/journal.pone.0044936 PMC : PMC3444505 Abstract >>
Genomic ANI (Average Nucleotide Identity) has been found to be able to replace DNA-DNA hybridization in prokaryote taxonomy. The ANI of each of the core genes that has a phylogeny congruent with the reference species tree of rhizobia was compared to the genomic ANI. This allowed us to identify three housekeeping genes (SMc00019-truA-thrA) whose ANI reflected the intraspecies and interspecies genomic ANI among rhizobial strains, revealing an ANI gap (?2%) between the inter- and intra-species comparisons. The intraspecies (96%) and interspecies (94%) ANI boundaries calculated from three genes (SMc00019-truA-thrA) provided a criterion for bacterial species definition and confirmed 621/629 of known interspecies relationships within Bradyrhizobium, Mesorhizobium, Sinorhizobium and Rhizobium. Some widely studied strains should be renamed. The SMc00019-truA-thrA ANI also correlates well with the genomic ANI of strains in Agrobacterium, Methylobacterium, Ralstonia, Rhodopseudomonas, Cupriavidus and Burkholderia, suggesting their wide applicability in other bacteria.
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( 1998 ) Three phylogenetic groups of nodA and nifH genes in Sinorhizobium and Mesorhizobium isolates from leguminous trees growing in Africa and Latin America. PMID : 9464375 : PMC : PMC106060 Abstract >>
The diversity and phylogeny of nodA and nifH genes were studied by using 52 rhizobial isolates from Acacia senegal, Prosopis chilensis, and related leguminous trees growing in Africa and Latin America. All of the strains had similar host ranges and belonged to the genera Sinorhizobium and Mesorhizobium, as previously determined by 16S rRNA gene sequence analysis. The restriction patterns and a sequence analysis of the nodA and nifH genes divided the strains into the following three distinct groups: sinorhizobia from Africa, sinorhizobia from Latin America, and mesorhizobia from both regions. In a phylogenetic tree also containing previously published sequences, the nodA genes of our rhizobia formed a branch of their own, but within the branch no correlation between symbiotic genes and host trees was apparent. Within the large group of African sinorhizobia, similar symbiotic gene types were found in different chromosomal backgrounds, suggesting that transfer of symbiotic genes has occurred across species boundaries. Most strains had plasmids, and the presence of plasmid-borne nifH was demonstrated by hybridization for some examples. The nodA and nifH genes of Sinorhizobium teranga ORS1009T grouped with the nodA and nifH genes of the other African sinorhizobia, but Sinorhizobium saheli ORS609T had a totally different nodA sequence, although it was closely related based on the 16S rRNA gene and nifH data. This might be because this S. saheli strain was originally isolated from Sesbania sp., which belongs to a different cross-nodulation group than Acacia and Prosopis spp. The factors that appear to have influenced the evolution of rhizobial symbiotic genes vary in importance at different taxonomic levels.
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