| 1. |
Vinuesa P,
Neumann-Silkow F,
Pacios-Bras C,
Spaink HP,
Martínez-Romero E,
Werner D,
( 2003 ) Genetic analysis of a pH-regulated operon from Rhizobium tropici CIAT899 involved in acid tolerance and nodulation competitiveness. PMID : 12575750 : DOI : 10.1094/MPMI.2003.16.2.159 Abstract >>
Rhizobium tropici CIAT899 is highly acid tolerant and a good competitor for Phaseolus vulgaris nodule occupancy at low pH values. Using Tn5 mutagenesis, we identified an operon required for acid tolerance and nodulation competitiveness. The insertion was mapped to the 5' end of atvA, encoding a product with high sequence identity to the agro-bacterial AcvB virulence protein. Complementation analyses indicated that atvA is an ortholog of acvB, both genes being required for acid tolerance. A Ser/Ala substitution in the LIPASE_SER motif of AtvA resulted in an acid sensitive Fix+ but very poorly competing strain, demonstrating that Ser-313 is essential for AtvA function. atvA is the second gene in an operon that is transcriptionally upregulated by acid shock. The acid-responsive promoter was mapped to a 469-bp intergenic region located upstream of lpiA, the first gene in the operon. lpiA-like genes are found in several alpha, beta, and gamma Proteobacteria that interact with eukaryotic host cells, and they are predicted to encode membrane proteins related to the FmtC/MprF family from low G+C Firmicutes. The latter proteins are involved in resistance to cationic antimicrobial peptides. A nonpolar deletion in lpiA caused a sevenfold decrease in relative nodulation competitiveness.
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2. |
Acosta-Durán C,
Martínez-Romero E,
( 2002 ) Diversity of rhizobia from nodules of the leguminous tree Gliricidia sepium, a natural host of Rhizobium tropici. PMID : 12115061 : DOI : 10.1007/s00203-002-0433-3 Abstract >>
The Rhizobium species that nodulate the legume tree Gliricidia sepium were analyzed by phenotypic characteristics (including nodule formation in different hosts), PCR-RFLP patterns and sequences of 16S rRNA genes, multilocus enzyme electrophoresis, and plasmid patterns. Strains of Rhizobium tropici type A and B, Sinorhizobium spp., and Rhizobium etli bv. phaseoli were encountered in G. sepium nodules and their presence depended on the site sampled.
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3. |
Baginsky C,
Brito B,
Imperial J,
Palacios JM,
Ruiz-Argüeso T,
( 2002 ) Diversity and evolution of hydrogenase systems in rhizobia. PMID : 12324339 : DOI : 10.1128/aem.68.10.4915-4924.2002 PMC : PMC126442 Abstract >>
Uptake hydrogenases allow rhizobia to recycle the hydrogen generated in the nitrogen fixation process within the legume nodule. Hydrogenase (hup) systems in Bradyrhizobium japonicum and Rhizobium leguminosarum bv. viciae show highly conserved sequence and gene organization, but important differences exist in regulation and in the presence of specific genes. We have undertaken the characterization of hup gene clusters from Bradyrhizobium sp. (Lupinus), Bradyrhizobium sp. (Vigna), and Rhizobium tropici and Azorhizobium caulinodans strains with the aim of defining the extent of diversity in hup gene composition and regulation in endosymbiotic bacteria. Genomic DNA hybridizations using hupS, hupE, hupUV, hypB, and hoxA probes showed a diversity of intraspecific hup profiles within Bradyrhizobium sp. (Lupinus) and Bradyrhizobium sp. (Vigna) strains and homogeneous intraspecific patterns within R. tropici and A. caulinodans strains. The analysis also revealed differences regarding the possession of hydrogenase regulatory genes. Phylogenetic analyses using partial sequences of hupS and hupL clustered R. leguminosarum and R. tropici hup sequences together with those from B. japonicum and Bradyrhizobium sp. (Lupinus) strains, suggesting a common origin. In contrast, Bradyrhizobium sp. (Vigna) hup sequences diverged from the rest of rhizobial sequences, which might indicate that those organisms have evolved independently and possibly have acquired the sequences by horizontal transfer from an unidentified source.
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4. |
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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5. |
Manyani H,
Sousa C,
Soria Díaz ME,
Gil-Serrano A,
Megías M,
( 2001 ) Regulation of nod factor sulphation genes in Rhizobium tropici CIAT899. PMID : 11467733 : DOI : 10.1139/w01-032 Abstract >>
Rhizobium tropici CIAT899 is a tropical symbiont able to nodulate various legumes such as Leucaena, Phaseolus, and Macroptilium. Broad host range of this species is related to its Nod factors wide spectrum. R. tropici contains Nod factors sulphation nod genes, nodHPQ genes, which control nodulation efficiency in Leucaena. To study nodHPQ regulation, we carried out different interposon insertions in its upstream region. One of these generated interruptions, nodI mutant produced nonsulphated Nod factors suggesting a possible dependence of these genes on nodI upstream region. Moreover, analysis results of lacZ transcriptional fusions with these genes in symbiotic plasmid showed dependence of these genes on NodD protein. In order to determine nodHPQ organization, we studied the effect of interposon insertion upstream of each lacZ transcriptional fusion, and the data obtained was used to indicate that nodHPQ belong to the nodABCSUIJ operon. However, comparison between nodP::lacZ beta-galactosidase activity in the symbiotic plasmid and in the pHM500 plasmid (containing nodHPQ genes) suggested constitutive expression in free living, and flavonoid inducible expression in symbiotic conditions. Constitutive nodHPQ expression may play a role in bacterial house-keeping metabolism. On the other hand, the transference of R. tropici nodHPQ genes to other rhizobia that do not present sulphated substitutions demonstrated that NodH protein sulphotransference is specific to C6 at the reducing end.
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6. |
Marroquí S,
Zorreguieta A,
Santamaría C,
Temprano F,
Soberón M,
Megías M,
Downie JA,
( 2001 ) Enhanced symbiotic performance by Rhizobium tropici glycogen synthase mutants. PMID : 11208782 : DOI : 10.1128/JB.183.3.854-864.2001 PMC : PMC94951 Abstract >>
We isolated a Tn5-induced Rhizobium tropici mutant that has enhanced capacity to oxidize N,N-dimethyl-p-phenylendiamine (DMPD) and therefore has enhanced respiration via cytochrome oxidase. The mutant had increased levels of the cytochromes c(1) and CycM and a small increase in the amount of cytochrome aa(3). In plant tests, the mutant increased the dry weight of Phaseolus vulgaris plants by 20 to 38% compared with the control strain, thus showing significantly enhanced symbiotic performance. The predicted product of the mutated gene is homologous to glycogen synthases from several bacteria, and the mutant lacked glycogen. The DNA sequence of the adjacent gene region revealed six genes predicted to encode products homologous to the following gene products from Escherichia coli: glycogen phosphorylase (glgP), glycogen branching enzyme (glgB), ADP glucose pyrophosphorylase (glgC), glycogen synthase (glgA), phosphoglucomutase (pgm), and glycogen debranching enzyme (glgX). All six genes are transcribed in the same direction, and analysis with lacZ gene fusions suggests that the first five genes are organized in one operon, although pgm appears to have an additional promoter; glgX is transcribed independently. Surprisingly, the glgA mutant had decreased levels of high-molecular-weight exopolysaccharide after growth on glucose, but levels were normal after growth on galactose. A deletion mutant was constructed in order to generate a nonpolar mutation in glgA. This mutant had a phenotype similar to that of the Tn5 mutant, indicating that the enhanced respiration and symbiotic nitrogen fixation and decreased exopolysaccharide were due to mutation of glgA and not to a polar effect on a downstream gene.
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7. |
Riccillo PM,
Collavino MM,
Grasso DH,
England R,
de Bruijn FJ,
Aguilar OM,
( 2000 ) A guaB mutant strain of Rhizobium tropici CIAT899 pleiotropically defective in thermal tolerance and symbiosis. PMID : 11059489 : DOI : 10.1094/MPMI.2000.13.11.1228 Abstract >>
Rhizobium tropici strain CIAT899 displays a high intrinsic thermal tolerance, and had been used in this work to study the molecular basis of bacterial responses to high temperature. We generated a collection of R. tropici CIAT899 mutants affected in thermal tolerance using TnS-luxAB mutagenesis and described the characterization of a mutant strain, CIAT899-10T, that fails to grow under conditions of high temperature. Strain CIAT899-10T carries a single transposon insertion in a gene showing a high degree of similarity with the guaB gene of Escherichia coli and other organisms, encoding the enzyme inosine monophosphate dehydrogenase. The guaB strain CIAT899-10T does not require guanine for growth due to an alternative pathway via xanthine dehydrogenase and, phenotypically, in addition to the thermal sensitivity, the mutant is also defective in symbiosis with beans, forming nodules that lack rhizobial content. Guanine and its precursors restore wild-type tolerance to grow at high temperature. Our data show that, in R. tropici, the production of guanine via inosine monophosphate dehydrogenase is essential for growth at extreme temperatures and for effective nodulation.
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8. |
Muglia CI,
de Bruijn FJ,
Roe AJ,
Riccillo PM,
( 2000 ) Glutathione is involved in environmental stress responses in Rhizobium tropici, including acid tolerance. PMID : 10692382 : DOI : 10.1128/jb.182.6.1748-1753.2000 PMC : PMC94474 Abstract >>
The isolation of rhizobial strains which exhibit an intrinsic tolerance to acidic conditions has been reported and has facilitated studies on the basic mechanisms underlying acid tolerance. Rhizobium tropici strain CIAT899 displays a high intrinsic tolerance to acidity and therefore was used in this work to study the molecular basis of bacterial responses to acid conditions and other environmental stresses. We generated a collection of R. tropici CIAT899 mutants affected in acid tolerance using Tn5-luxAB mutagenesis, and one mutant strain (CIAT899-13T2), which fails to grow under acid conditions, was characterized in detail. Strain CIAT899-13T2 was found to contain a single Tn5-luxAB insertion in a gene showing a high degree of similarity with the Escherichia coli gshB gene, encoding the enzyme glutathione synthetase. Intracellular potassium pools and intracellular pH levels were found to be lower in the mutant than in the parent. The glutathione-deficient mutant was shown to be sensitive to weak organic acids, osmotic and oxidative stresses, and the presence of methylglyoxal. Glutathione restores responses to these stresses almost to wild-type levels. Our data show that in R. tropici the production of glutathione is essential for growth in extreme environmental conditions. The mutant strain CIAT899-13T2 induced effective nodules; however, it was found to be outcompeted by the wild-type strain in coinoculation experiments.
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9. |
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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10. |
Valverde A,
Igual JM,
Peix A,
Cervantes E,
Velázquez E,
( 2006 ) Rhizobium lusitanum sp. nov. a bacterium that nodulates Phaseolus vulgaris. PMID : 17082403 : DOI : 10.1099/ijs.0.64402-0 Abstract >>
The species Phaseolus vulgaris is a promiscuous legume nodulated by several species of the family Rhizobiaceae. During a study of rhizobia nodulating this legume in Portugal, we isolated several strains that nodulate P. vulgaris effectively and also Macroptilium atropurpureum and Leucaena leucocephala, but they form ineffective nodules in Medicago sativa. According to phylogenetic analysis of the 16S rRNA gene sequence, the strains from this study belong to the genus Rhizobium, with Rhizobium rhizogenes and Rhizobium tropici as the closest related species, with 99.9 and 99.2% similarity, respectively, between the type strains of these species and strain P1-7T. The nodD and nifH genes carried by strain P1-7T are phylogenetically related to those of other species nodulating Phaseolus. This strain does not carry virulence genes present in the type strain of R. rhizogenes, ATCC 11325T. Analysis of the recA and atpD genes confirms this phylogenetic arrangement, showing low similarity with respect to those of R. rhizogenes ATCC 11325T (91.9 and 94.1% similarity, respectively) and R. tropici IIB CIAT 899T (90.6% and 91.8% similarity, respectively). The intergenic spacer (ITS) of the strains from this study is phylogenetically divergent from those of R. rhizogenes ATCC 11235T and R. tropici CIAT 899T, with 85.9 and 82.8% similarity, respectively, with respect to strain P1-7T. The tRNA profile and two-primer random amplified polymorphic DNA pattern of strain P1-7T are also different from those of R. rhizogenes ATCC 11235T and R. tropici CIAT 899T. The strains isolated in this study can be also differentiated from R. rhizogenes and R. tropici by several phenotypic characteristics. The results of DNA-DNA hybridization showed means of 28 and 25% similarity between strain P1-7T and R. rhizogenes ATCC 11235T and R. tropici CIAT 899T, respectively. All these data showed that the strains isolated in this study belong to a novel species of the genus Rhizobium, for which we propose the name Rhizobium lusitanum sp. nov.; the type strain is P1-7T (=LMG 22705T=CECT 7016T).
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11. |
Hernandez-Lucas I,
Ramirez-Trujillo JA,
Gaitan MA,
Guo X,
Flores M,
Martinez-Romero E,
Perez-Rueda E,
Mavingui P,
( 2006 ) Isolation and characterization of functional insertion sequences of rhizobia. PMID : 16842354 : DOI : 10.1111/j.1574-6968.2006.00319.x Abstract >>
Rhizobia are a group of bacteria that form nodules on the roots of legume host plants. The sequenced genomes of the rhizobia are characterized by the presence of many putative insertion sequences (IS) elements. However, it is unknown whether these IS elements are functional and it is therefore relevant to assess their transposition activity. In this work, several functional insertion sequences belonging to the IS1256, IS3, IS5, IS166, and IS21 families were captured from Rhizobium tropici, Rhizobium sp. NGR234 and Sinorhizobium meliloti, using pGBG1 as a trapping system. In silico analysis shows that homologs of rhizobia mobile elements are present in distantly related genomes, suggesting that Rhizobium IS elements are prone to genetic transfer.
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12. |
Rojas-Jiménez K,
Sohlenkamp C,
Geiger O,
Martínez-Romero E,
Werner D,
Vinuesa P,
( 2005 ) A ClC chloride channel homolog and ornithine-containing membrane lipids of Rhizobium tropici CIAT899 are involved in symbiotic efficiency and acid tolerance. PMID : 16353552 : DOI : 10.1094/MPMI-18-1175 Abstract >>
Rhizobium tropici CIAT899 is highly tolerant to several environmental stresses and is a good competitor for nodule occupancy of common bean plants in acid soils. Random transposon mutagenesis was performed to identify novel genes of this strain involved in symbiosis and stress tolerance. Here, we present a genetic analysis of the locus disrupted by the Tn5 insertion in mutant 899-PV9, which lead to the discovery of sycA, a homolog of the ClC family of chloride channels and Cl-/H+ exchange transporters. A nonpolar deletion in this gene caused serious deficiencies in nodule development, nodulation competitiveness, and N2 fixation on Phaseolus vulgaris plants, probably due to its reduced ability to invade plant cells and to form stable symbiosomes, as judged by electron transmission microscopy. A second gene (olsC), found downstream of sycA, is homologous to aspartyl/asparaginyl beta-hydroxylases and modifies two species of ornithine-containing lipids in vivo, presumably by hydroxylation at a still-unknown position. A mutant carrying a nonpolar deletion in olsC is symbiotically defective, whereas overexpressed OlsC in the complemented strain provokes an acid-sensitive phenotype. This is the first report of a ClC homolog being essential for the establishment of a fully developed N2-fixing root nodule symbiosis and of a putative beta-hydroxylase that modifies ornithine-containing membrane lipids of R. tropici CIAT899, which, in turn, are contributing to symbiotic performance and acid tolerance.
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13. |
Sarita S,
Sharma PK,
Priefer UB,
Prell J,
( 2005 ) Direct amplification of rhizobial nodC sequences from soil total DNA and comparison to nodC diversity of root nodule isolates. PMID : 16329967 : DOI : 10.1016/j.femsec.2005.02.015 Abstract >>
A group-specific primer set was developed using nodC as a target gene for the amplification of rhizobial sequence diversity from nodule isolates and total soil DNA preparations. The primer set was tested on 209 nodule isolates, recovered from six different trap plant species which were grown in two soil samples collected from a chickpea and a wheat field site in India. We also amplified and cloned PCR products from total DNA isolated from the same soil samples. The total diversity within the resulting clone libraries (Sigma 218 clones) was higher than that recovered from trap plants, but differed depending on the PCR protocols and primers used. However, some plant-selected genotypes could not be obtained using the community approach, probably due to variable detection limits and limited clone library sizes.
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14. |
Silva C,
Vinuesa P,
Eguiarte LE,
Souza V,
Martínez-Romero E,
( 2005 ) Evolutionary genetics and biogeographic structure of Rhizobium gallicum sensu lato, a widely distributed bacterial symbiont of diverse legumes. PMID : 16262857 : DOI : 10.1111/j.1365-294X.2005.02721.x Abstract >>
We used phylogenetic and population genetics approaches to evaluate the importance of the evolutionary forces on shaping the genetic structure of Rhizobium gallicum and related species. We analysed 54 strains from several populations distributed in the Northern Hemisphere, using nucleotide sequences of three 'core' chromosomal genes (rrs, glnII and atpD) and two 'auxiliary' symbiotic genes (nifH and nodB) to elucidate the biogeographic history of the species and symbiotic ecotypes (biovarieties) within species. The analyses revealed that strains classified as Rhizobium mongolense and Rhizobium yanglingense belong to the chromosomal evolutionary lineage of R. gallicum and harbour symbiotic genes corresponding to a new biovar; we propose their reclassification as R. gallicum bv. orientale. The comparison of the chromosomal and symbiotic genes revealed evidence of lateral transfer of symbiotic information within and across species. Genetic differentiation analyses based on the chromosomal protein-coding genes revealed a biogeographic pattern with three main populations, whereas the 16S rDNA sequences did not resolve that biogeographic pattern. Both the phylogenetic and population genetic analyses showed evidence of recombination at the rrs locus. We discuss our results in the light of the contrasting views of bacterial species expressed by microbial taxonomist and evolutionary biologists.
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15. |
Eardly BD,
Nour SM,
van Berkum P,
Selander RK,
( 2005 ) Rhizobial 16S rRNA and dnaK genes: mosaicism and the uncertain phylogenetic placement of Rhizobium galegae. PMID : 15746335 : DOI : 10.1128/AEM.71.3.1328-1335.2005 PMC : PMC1065159 Abstract >>
The phylogenetic relatedness among 12 agriculturally important species in the order Rhizobiales was estimated by comparative 16S rRNA and dnaK sequence analyses. Two groups of related species were identified by neighbor-joining and maximum-parsimony analysis. One group consisted of Mesorhizobium loti and Mesorhizobium ciceri, and the other group consisted of Agrobacterium rhizogenes, Rhizobium tropici, Rhizobium etli, and Rhizobium leguminosarum. Although bootstrap support for the placement of the remaining six species varied, A. tumefaciens, Agrobacterium rubi, and Agrobacterium vitis were consistently associated in the same subcluster. The three other species included Rhizobium galegae, Sinorhizobium meliloti, and Brucella ovis. Among these, the placement of R. galegae was the least consistent, in that it was placed flanking the A. rhizogenes-Rhizobium cluster in the dnaK nucleotide sequence trees, while it was placed with the other three Agrobacterium species in the 16S rRNA and the DnaK amino acid trees. In an effort to explain the inconsistent placement of R. galegae, we examined polymorphic site distribution patterns among the various species. Localized runs of nucleotide sequence similarity were evident between R. galegae and certain other species, suggesting that the R. galegae genes are chimeric. These results provide a tenable explanation for the weak statistical support often associated with the phylogenetic placement of R. galegae, and they also illustrate a potential pitfall in the use of partial sequences for species identification.
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16. |
Izquierdo J,
Venkova-Canova T,
Ramírez-Romero MA,
Téllez-Sosa J,
Hernández-Lucas I,
Sanjuan J,
Cevallos MA,
( 2005 ) An antisense RNA plays a central role in the replication control of a repC plasmid. PMID : 16005966 : DOI : 10.1016/j.plasmid.2005.05.003 Abstract >>
The widespread replicons of repABC and repC families from alpha-proteobacteria share high similarity in their replication initiator proteins (RepC). Here we describe the minimal region required for stable replication of a member of the repC family, the low copy-number plasmid pRmeGR4a from Sinorizobium meliloti GR4. This region contains only two genes: one encoding the initiator protein RepC (46.8 kDa) and other, an antisense RNA (67 nt). Mapping of transcriptional start sites and promoter regions of both genes showed that the antisense RNA is nested within the repC mRNA leader. The constitutively expressed countertranscribed RNA (ctRNA) forms a single stem-loop structure that acts as an intrinsic rho-independent terminator. The ctRNA is a strong trans-incompatibility factor and negative regulator of repC expression. Based on structural and functional similarities between members of the repC and repABC families we propose a model of their evolutionary relationship.
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17. |
Stepkowski T,
Czapli?ska M,
Miedzinska K,
Moulin L,
( 2003 ) The variable part of the dnaK gene as an alternative marker for phylogenetic studies of rhizobia and related alpha Proteobacteria. PMID : 14666974 : DOI : 10.1078/072320203770865765 Abstract >>
DnaK is the 70 kDa chaperone that prevents protein aggregation and supports the refolding of damaged proteins. Due to sequence conservation and its ubiquity this chaperone has been widely used in phylogenetic studies. In this study, we applied the less conserved part that encodes the so-called alpha-subdomain of the substrate-binding domain of DnaK for phylogenetic analysis of rhizobia and related non-symbiotic alpha-Proteobacteria. A single 330 bp DNA fragment was routinely amplified from DNA templates isolated from the species of the genera, Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium, but also from some non-symbiotic alpha Proteobacteria such as Blastochloris, Chelatobacter and Chelatococcus. Phylogenetic analyses revealed high congruence between dnaK sequences and 16S rDNA trees, but they were not identical. In contrast, the partition homogeneity tests revealed that dnaK sequence data could be combined with other housekeeping genes such as recA, atpD or glnA. The dnaK trees exhibited good resolution in the cases of the genera Mesorhizobium, Sinorhizobium and Rhizobium, even better than usually shown by 16S rDNA phylogeny. The dnaK phylogeny supported the close phylogenetic relationship of Rhizobium galegae and Agrobacterium tumefaciens (R. radiobacter) C58, which together formed a separate branch within the fast-growing rhizobia, albeit closer to the genus Sinorhizobium. The Rhizobium and Sinorhizobium genera carried an insertion composed of two amino acids, which additionally supported the phylogenetic affinity of these two genera, as well as their distinctness from the Mesorhizobium genus. Consistently with the phylogeny shown by 16S-23S rDNA intergenic region sequences, the dnaK trees divided the genus Bradyrhizobium into three main lineages, corresponding to B. japonicum, B. elkanii, and photosynthetic Bradyrhizobium strains that infect Aeschynomene plants. Our results suggest that the 330 bp dnaK sequences could be used as an additional taxonomic marker for rhizobia and related species (alternatively to the 16S rRNA gene phylogeny).
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18. |
Ribeiro RA,
Rogel MA,
López-López A,
Ormeño-Orrillo E,
Barcellos FG,
Martínez J,
Thompson FL,
Martínez-Romero E,
Hungria M,
( 2012 ) Reclassification of Rhizobium tropici type A strains as Rhizobium leucaenae sp. nov. PMID : 21742822 : DOI : 10.1099/ijs.0.032912-0 Abstract >>
Rhizobium tropici is a well-studied legume symbiont characterized by high genetic stability of the symbiotic plasmid and tolerance to tropical environmental stresses such as high temperature and low soil pH. However, high phenetic and genetic variabilities among R. tropici strains have been largely reported, with two subgroups, designated type A and B, already defined within the species. A polyphasic study comprising multilocus sequence analysis, phenotypic and genotypic characterizations, including DNA-DNA hybridization, strongly supported the reclassification of R. tropici type A strains as a novel species. Type A strains formed a well-differentiated clade that grouped with R. tropici, Rhizobium multihospitium, Rhizobium miluonense, Rhizobium lusitanum and Rhizobium rhizogenes in the phylogenies of the 16S rRNA, recA, gltA, rpoA, glnII and rpoB genes. Several phenotypic traits differentiated type A strains from all related taxa. The novel species, for which the name Rhizobium leucaenae sp. nov. is proposed, is a broad host range rhizobium being able to establish effective root-nodule symbioses with Leucaena leucocephala, Leucaena esculenta, common beans (Phaseolus vulgaris) and Gliricidia sepium. Strain CFN 299(T) (= USDA 9039(T) = LMG 9517(T) = CECT 4844(T) = JCM 21088(T) = IAM 14230(T) = SEMIA 4083(T) = CENA 183(T) = UMR1026(T) = CNPSo 141(T)) is designated the type strain of Rhizobium leucaenae sp. nov.
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19. |
Batista S,
Patriarca EJ,
Tatè R,
Martínez-Drets G,
Gill PR,
( 2009 ) An alternative succinate (2-oxoglutarate) transport system in Rhizobium tropici is induced in nodules of Phaseolus vulgaris. PMID : 19502401 : DOI : 10.1128/JB.00252-09 PMC : PMC2725604 Abstract >>
The rhizobial DctA permease is essential for the development of effective nitrogen-fixing bacteroids, which was correlated with its requirement for growth on C(4)-dicarboxylates. A previously described dctA mutant of Rhizobium tropici CIAT899, strain GA1 (dctA), however, was unexpectedly still able to grow on succinate as a sole carbon source but less efficiently than CIAT899. Like other rhizobial dctA mutants, GA1 was unable to grow on fumarate or malate as a carbon source and induced the formation of ineffective nodules. We report an alternative succinate uptake system identified by Tn5 mutagenesis of strain GA1 that was required for the remaining ability to transport and utilize succinate. The alternative uptake system required a three-gene cluster that is highly characteristic of a dctABD locus. The predicted permease-encoding gene had high sequence similarity with open reading frames encoding putative 2-oxoglutarate permeases (KgtP) of Ralstonia solanacearum and Agrobacterium tumefaciens. This analysis was in agreement with the requirement for this gene for optimal growth on and induction by 2-oxoglutarate. The permease-encoding gene of the alternative system was also designated kgtP in R. tropici. The dctBD-like genes in this cluster were found to be required for kgtP expression and were designated kgtSR. Analysis of a kgtP::lacZ transcriptional fusion indicated that a kgtSR-dependent promoter of kgtP was specifically induced by 2-oxoglutarate. The expression of kgtPp was found in bacteroids of nodules formed with either CIAT899 or GA1 on roots of Phaseolus vulgaris. Results suggested that 2-oxoglutarate might be transported or conceivably exported in nodules induced by R. tropici on roots of P. vulgaris.
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20. |
Elliott GN,
Chou JH,
Chen WM,
Bloemberg GV,
Bontemps C,
Martínez-Romero E,
Velázquez E,
Young JP,
Sprent JI,
James EK,
( 2009 ) Burkholderia spp. are the most competitive symbionts of Mimosa, particularly under N-limited conditions. PMID : 19040456 : DOI : 10.1111/j.1462-2920.2008.01799.x Abstract >>
Bacteria isolated from Mimosa nodules in Taiwan, Papua New Guinea, Mexico and Puerto Rico were identified as belonging to either the alpha- or beta-proteobacteria. The beta-proteobacterial Burkholderia and Cupriavidus strains formed effective symbioses with the common invasive species Mimosa diplotricha, M. pigra and M. pudica, but the alpha-proteobacterial Rhizobium etli and R. tropici strains produced a range of symbiotic phenotypes from no nodulation through ineffective to effective nodulation, depending on Mimosa species. Competition studies were performed between three of the alpha-proteobacteria (R. etli TJ167, R. tropici NGR181 and UPRM8021) and two of the beta-rhizobial symbionts (Burkholderia mimosarum PAS44 and Cupriavidus taiwanensis LMG19424) for nodulation of these invasive Mimosa species. Under flooded conditions, B. mimosarum PAS44 out-competed LMG19424 and all three alpha-proteobacteria to the point of exclusion. This advantage was not explained by initial inoculum levels, rates of bacterial growth, rhizobia-rhizobia growth inhibition or individual nodulation rate. However, the competitive domination of PAS44 over LMG19424 was reduced in the presence of nitrate for all three plant hosts. The largest significant effect was for M. pudica, in which LMG19424 formed 57% of the nodules in the presence of 0.5 mM potassium nitrate. In this host, ammonium also had a similar, but lesser, effect. Comparable results were also found using an N-containing soil mixture, and environmental N levels are therefore suggested as a factor in the competitive success of the bacterial symbiont in vivo.
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21. |
Ormeño-Orrillo E,
Rosenblueth M,
Luyten E,
Vanderleyden J,
Martínez-Romero E,
( 2008 ) Mutations in lipopolysaccharide biosynthetic genes impair maize rhizosphere and root colonization of Rhizobium tropici CIAT899. PMID : 18312393 : DOI : 10.1111/j.1462-2920.2007.01541.x Abstract >>
Three transposon mutants of Rhizobium tropici CIAT899 affected in lipopolysaccharide (LPS) biosynthesis were characterized and their maize rhizosphere and endophytic root colonization abilities were evaluated. The disrupted genes coded for the following putative products: the ATPase component of an O antigen ABC-2 type transporter (wzt), a nucleotide-sugar dehydratase (lpsbeta2) and a bifunctional enzyme producing GDP-mannose (noeJ). Electrophoretic analysis of affinity purified LPS showed that all mutants lacked the smooth LPS bands indicating an O antigen minus phenotype. In the noeJ mutant, the rough LPS band migrated faster than the parental band, suggesting a truncated LPS core. When inoculated individually, the wzt and noeJ mutants colonize the rhizosphere and root to a lower extent than the parental strain while no differences were observed between the lpsbeta2 mutant and the parental strain. All mutants were impaired in competitive rhizosphere and root colonization. Pleiotropic effects of the mutations on known colonization traits such as motility and growth rate were observed, but they were not sufficient to explain the colonization behaviours. It was found that the LPS mutants were sensitive to the maize antimicrobial 6-methoxy-2-benzoxazolinone (MBOA). Only the combined effects of altered growth rate and susceptibility to maize antimicrobials could account for all the observed colonization phenotypes. The results suggest an involvement of the LPS in protecting R. tropici against maize defence response during rhizosphere and root colonization.
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22. |
Rouhrazi K,
Khodakaramian G,
Velázquez E,
( 2016 ) Phylogenetic diversity of rhizobial species and symbiovars nodulating Phaseolus vulgaris in Iran. PMID : 26832644 : DOI : 10.1093/femsle/fnw024 Abstract >>
The phylogenetic diversity of 29 rhizobial strains nodulating Phaseolus vulgaris in Iran was analysed on the basis of their core and symbiotic genes. These strains displayed five 16S rRNA-RFLP patterns and belong to eight ERIC-PCR clusters. The phylogenetic analyses of 16S rRNA, recA and atpD core genes allowed the identification of several strains as Rhizobium sophoriradicis, R. leguminosarum, R. tropici and Pararhizobium giardinii, whereas other strains represented a new phylogenetic lineage related to R. vallis. These strains and those identified as R. sophoriradicis and R. leguminosarum belong to the symbiovar phaseoli carrying the �^ nodC allele distributed in P. vulgaris endosymbionts in America, Europe, Africa and Asia. The strain identified as R. tropici belongs to the symbiovar tropici carried by strains of R. tropici, R. leucaenae, R. lusitanum and R. freirei nodulating P. vulgaris in America, Africa and Asia. The strain identified as P. giardinii belongs to the symbiovar giardinii together with the type strain of this species nodulating P. vulgaris in France. It is remarkable that the recently described species R. sophoriradicis is worldwide distributed in P. vulgaris nodules carrying the �^ nodC allele of symbiovar phaseoli harboured by rhizobia isolated in the American distribution centers of this legume.
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23. |
Dall'Agnol RF,
Ribeiro RA,
Delamuta JR,
Ormeño-Orrillo E,
Rogel MA,
Andrade DS,
Martínez-Romero E,
Hungria M,
( 2014 ) Rhizobium paranaense sp. nov., an effective N2-fixing symbiont of common bean (Phaseolus vulgaris L.) with broad geographical distribution in Brazil. PMID : 24972614 : DOI : 10.1099/ijs.0.064543-0 Abstract >>
Nitrogen (N), the nutrient most required for plant growth, is key for good yield of agriculturally important crops. Common bean (Phaseolus vulgaris L.) can benefit from bacteria collectively called rhizobia, which are capable of fixing atmospheric nitrogen (N2) in root nodules and supplying it to the plant. Common bean is amongst the most promiscuous legume hosts; several described species, in addition to putative novel ones have been reported as able to nodulate this legume, although not always effectively in terms of fixing N2. In this study, we present data indicating that Brazilian strains PRF 35(T), PRF 54, CPAO 1135 and H 52, currently classified as Rhizobium tropici, represent a novel species symbiont of common bean. Morphological, physiological and biochemical properties differentiate these strains from other species of the genus Rhizobium, as do BOX-PCR profiles (less than 60 % similarity), multilocus sequence analysis with recA, gyrB and rpoA (less than 96.4 % sequence similarity), DNA-DNA hybridization (less than 50 % DNA-DNA relatedness), and average nucleotide identity of whole genomes (less than 92.8.%). The novel species is effective in nodulating and fixing N2 with P. vulgaris, Leucaena leucocephala and Leucaena esculenta. We propose the name Rhizobium paranaense sp. nov. for this novel taxon, with strain PRF 35(T) (= CNPSo 120(T) = LMG 27577(T) = IPR-Pv 1249(T)) as the type strain.
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24. |
Ribeiro RA,
Ormeño-Orrillo E,
Dall'Agnol RF,
Graham PH,
Martinez-Romero E,
Hungria M,
( 2013 ) Novel Rhizobium lineages isolated from root nodules of the common bean (Phaseolus vulgaris L.) in Andean and Mesoamerican areas. PMID : 23764913 : DOI : 10.1016/j.resmic.2013.05.002 Abstract >>
The taxonomic affiliations of nineteen root-nodule bacteria isolated from the common bean (Phaseolus vulgaris L.) in Mexico, Ecuador and Brazil were investigated by analyses of 16S rRNA and of four protein-coding housekeeping genes. One strain from Mexico could be assigned to Rhizobium etli and two from Brazil to Rhizobium leucaenae, whereas another from Mexico corresponded to a recently described bean-nodulating species-level lineage related to R. etli and Rhizobium phaseoli. Ten strains isolated in Ecuador and Mexico corresponded to three novel Rhizobium lineages that fall into the R. phaseoli/R. etli/Rhizobium leguminosarum clade. One of those lineages, with representatives isolated mostly from Ecuador, seems to be dominant in beans from that Andean region. Only one of the Mexican strains clustered within the Rhizobium tropici clade, but as an independent lineage. Interestingly, four strains were affiliated with species within the Rhizobium radiobacter clade. The existence of yet non-described native Rhizobium lineages in both the Andean and Mesoamerican areas is discussed in relation to common-bean diversity and environmental conditions.
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25. |
Melkonian R,
Moulin L,
Béna G,
Tisseyre P,
Chaintreuil C,
Heulin K,
Rezkallah N,
Klonowska A,
Gonzalez S,
Simon M,
Chen WM,
James EK,
Laguerre G,
( 2014 ) The geographical patterns of symbiont diversity in the invasive legume Mimosa pudica can be explained by the competitiveness of its symbionts and by the host genotype. PMID : 24131520 : DOI : 10.1111/1462-2920.12286 Abstract >>
Variations in the patterns of diversity of symbionts have been described worldwide on Mimosa pudica, a pan-tropical invasive species that interacts with both �\ and �]-rhizobia. In this study, we investigated if symbiont competitiveness can explain these variations and the apparent prevalence of �]- over �\-rhizobia. We developed an indirect method to measure the proportion of nodulation against a GFP reference strain and tested its reproducibility and efficiency. We estimated the competitiveness of 54 strains belonging to four species of �]-rhizobia and four of �\-rhizobia, and the influence of the host genotype on their competitiveness. Our results were compared with biogeographical patterns of symbionts and host varieties. We found: (i) a strong strain effect on competitiveness largely explained by the rhizobial species, with Burkholderia phymatum being the most competitive species, followed by B. tuberum, whereas all other species shared similar and reduced levels of competitiveness; (ii) plant genotype can increase the competitiveness of Cupriavidus taiwanensis. The latter data support the likelihood of the strong adaptation of C. taiwanensis with the M. pudica var. unijuga and help explain its prevalence as a symbiont of this variety over Burkholderia species in some environments, most notably in Taiwan.
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26. |
Faghire M,
Mandri B,
Oufdou K,
Bargaz A,
Ghoulam C,
Ramírez-Bahena MH,
Velázquez E,
Peix A,
( 2012 ) Identification at the species and symbiovar levels of strains nodulating Phaseolus vulgaris in saline soils of the Marrakech region (Morocco) and analysis of the otsA gene putatively involved in osmotolerance. PMID : 22429391 : DOI : 10.1016/j.syapm.2012.02.003 Abstract >>
Salinity is an increasing problem in Africa affecting rhizobia-legume symbioses. In Morocco, Phaseolus vulgaris is cultivated in saline soils and its symbiosis with rhizobia depends on the presence of osmotolerant strains in these soils. In this study, 32 osmotolerant rhizobial strains nodulating P. vulgaris were identified at the species and symbiovar levels by analysing core and symbiotic genes, respectively. The most abundant strains were closely related to Rhizobium etli and R. phaseoli and belonged to symbiovar phaseoli. A second group of strains was identified as R. gallicum sv gallicum. The remaining strains, identified as R. tropici, belonged to the CIAT 899(T) nodC group, which has not yet been described as a symbiovar. In representative strains, the otsA gene involved in the accumulation of trehalose and putatively in osmotolerance was analysed. The results showed that the phylogeny of this gene was not completely congruent with those of other core genes, since the genus Ensifer was more closely related to some Rhizobium species than others. Although the role of the otsA gene in osmotolerance is not well established, it can be a useful protein-coding gene for phylogenetic studies in the genus Rhizobium, since the phylogenies of otsA and other core genes are coincident at the species level.
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27. |
Geurts R,
Squartini A,
Roelofsen W,
Op den Camp RH,
( 2012 ) Nonlegume Parasponia andersonii deploys a broad rhizobium host range strategy resulting in largely variable symbiotic effectiveness. PMID : 22668002 : DOI : 10.1094/MPMI-11-11-0304 Abstract >>
The non-legume genus Parasponia has evolved the rhizobium symbiosis independent from legumes and has done so only recently. We aim to study the promiscuity of such newly evolved symbiotic engagement and determine the symbiotic effectiveness of infecting rhizobium species. It was found that Parasponia andersonii can be nodulated by a broad range of rhizobia belonging to four different genera, and therefore, we conclude that this non-legume is highly promiscuous for rhizobial engagement. A possible drawback of this high promiscuity is that low-efficient strains can infect nodules as well. The strains identified displayed a range in nitrogen-fixation effectiveness, including a very inefficient rhizobium species, Rhizobium tropici WUR1. Because this species is able to make effective nodules on two different legume species, it suggests that the ineffectiveness of P. andersonii nodules is the result of the incompatibility between both partners. In P. andersonii nodules, rhizobia of this strain become embedded in a dense matrix but remain vital. This suggests that sanctions or genetic control against underperforming microsymbionts may not be effective in Parasponia spp. Therefore, we argue that the Parasponia-rhizobium symbiosis is a delicate balance between mutual benefits and parasitic colonization.
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28. |
Robledo M,
Velázquez E,
Ramírez-Bahena MH,
García-Fraile P,
Pérez-Alonso A,
Rivas R,
Martínez-Molina E,
Mateos PF,
( 2011 ) The celC gene, a new phylogenetic marker useful for taxonomic studies in Rhizobium. PMID : 21621937 : DOI : 10.1016/j.syapm.2011.01.010 Abstract >>
The celC gene codifies for a cellulase that fulfils a very significant role in the infection process of clover by Rhizobium leguminosarum. This gene is located in the celABC operon present in the chromosome of strains representing R. leguminosarum, Rhizobium etli and Rhizobium radiobacter whose genomes have been completely sequenced. Nevertheless, the existence of this gene in other species of the genus Rhizobium had not been investigated to date. In this study, the celC gene was analysed for the first time in several species of this genus isolated from legume nodules and plant tumours, in order to compare the celC phylogeny to those of other chromosomal and plasmidic genes. The results obtained showed that phylogenies of celC and chromosomal genes, such as rrs, recA and atpD, were completely congruent, whereas no relation was found with symbiotic or virulence genes. Therefore, the suitability and usefulness of the celC gene to differentiate species of the genus Rhizobium, especially those with closely related rrs genes, was highlighted. Consequently, the taxonomic status of several strains of the genus Rhizobium with completely sequenced genomes is also discussed.
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29. |
Valverde A,
Velázquez E,
Cervantes E,
Igual JM,
van Berkum P,
( 2011 ) Evidence of an American origin for symbiosis-related genes in Rhizobium lusitanum. PMID : 21705533 : DOI : 10.1128/AEM.02017-10 PMC : PMC3165244 Abstract >>
Randomly amplified polymorphic DNA (RAPD) analysis was used to investigate the diversity of 179 bean isolates recovered from six field sites in the Arcos de Valdevez region of northwestern Portugal. The isolates were divided into 6 groups based on the fingerprint patterns that were obtained. Representatives for each group were selected for sequence analysis of 4 chromosomal DNA regions. Five of the groups were placed within Rhizobium lusitanum, and the other group was placed within R. tropici type IIA. Therefore, the collection of Portuguese bean isolates was shown to include the two species R. lusitanum and R. tropici. In plant tests, the strains P1-7, P1-1, P1-2, and P1-16 of R. lusitanum nodulated and formed nitrogen-fixing symbioses both with Phaseolus vulgaris and Leucaena leucocephala. A methyltransferase-encoding nodS gene identical with the R. tropici locus that confers wide host range was detected in the strain P1-7 as well as 24 others identified as R. lusitanum. A methyltransferase-encoding nodS gene also was detected in the remaining isolates of R. lusitanum, but in this case the locus was that identified with the narrow-host-range R. etli. Representatives of isolates with the nodS of R. etli formed effective nitrogen-fixing symbioses with P. vulgaris and did not nodulate L. leucocephala. From sequence data of nodS, the R. lusitanum genes for symbiosis were placed within those of either R. tropici or R. etli. These results would support the suggestion that R. lusitanum was the recipient of the genes for symbiosis with beans from both R. tropici and R. etli.
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30. |
( 1996 ) Characterization of Rhizobium tropici CIAT899 nodulation factors: the role of nodH and nodPQ genes in their sulfation. PMID : 8850086 : Abstract >>
We have purified and characterized the nodulation factors produced by Rhizobium tropici CIAT899. This strain produces a large variety of nodulation factors, these being a mixture of sulfated or nonsulfated penta- or tetra-chito-oligosaccharides to which any of six different fatty acyl moieties may be attached to nitrogen of the nonreducing terminal residue. In this mixture we have also found methylated or nonmethylated lipo-chitin oligosaccharides. Here we describe a novel lipo-chitin-oligosaccharide consisting of a linear backbone of 4 N-acetylglucosamine residues and one mannose that is the reducing-terminal residue and bearing a C18:1 fatty acyl moiety on the nonreducing terminal residue. In addition, we have identified, cloned, and sequenced R. tropici nodH and nodPQ genes, generated mutations in the nodH and nodQ genes, and tested the mutant strains for nodulation in Phaseolus and Leucaena plants. Our results indicate that the sulfate group present in wild-type Nod factors plays a major role in nodulation of Leucaena plants by strain CIAT899 of R. tropici.
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31. |
( 1996 ) The NodA proteins of Rhizobium meliloti and Rhizobium tropici specify the N-acylation of Nod factors by different fatty acids. PMID : 8930915 : DOI : 10.1046/j.1365-2958.1996.00069.x Abstract >>
Rhizobia synthesize mono-N-acylated chitooligosaccharide signals, called Nod factors, that are required for the specific infection and nodulation of their legume hosts. The biosynthesis of Nod factors is under the control of nodulation (nod) genes, including the nodABC genes present in all rhizobial species. The N-acyl substitution can vary between species and can play a role in host specificity. In Rhizobium meliloti, an alfalfa symbiont, the acyl chain is a C16 unsaturated or a (omega-1) hydroxylated fatty acid, whereas in Rhizobium tropici, a bean symbiont, it is vaccenic acid (C18:1). We constructed R. meliloti derivatives having a non-polar deletion of nodA, and carrying a plasmid with either the R. meliloti or the R. tropici nodA gene. The strain with the R. tropici nodA gene produced Nod factors acylated by vaccenic acid, instead of the C16 unsaturated or hydroxylated fatty acids characteristic of R. meliloti Nod factors, and infected and nodulated alfalfa with a significant delay. These results show that NodA proteins of R. meliloti and R. tropici specify the N-acylation of Nod factors by different fatty acids, and that allelic variation of the common nodA gene can contribute to the determination of host range.
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32. |
( 1993 ) Multiple copies of nodD in Rhizobium tropici CIAT899 and BR816. PMID : 8419293 : DOI : 10.1128/jb.175.2.438-447.1993 PMC : PMC196158 Abstract >>
Rhizobium tropici strains are able to nodulate a wide range of host plants: Phaseolus vulgaris, Leucaena spp., and Macroptilium atropurpureum. We studied the nodD regulatory gene for nodulation of two R. tropici strains: CIAT899, the reference R. tropici type IIb strain, and BR816, a heat-tolerant strain isolated from Leucaena leucocephala. A survey revealed several nodD-hybridizing DNA regions in both strains: five distinct regions in CIAT899 and four distinct regions in BR816. Induction experiments of a nodABC-uidA fusion in combination with different nodD-hybridizing fragments in the presence of root exudates of the different hosts indicate that one particular nodD copy contributes to nodulation gene induction far more than any other nodD copy present. The nucleotide sequences of both nodD genes are reported here and show significant homology to those of the nodD genes of other rhizobia and a Bradyrhizobium strain. A dendrogram based on the protein sequences of 15 different NodD proteins shows that the R. tropici NodD proteins are linked most closely to each other and then to the NodD of Rhizobium phaseoli 8002.
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33. |
( 1996 ) Isolation and characterization of Rhizobium tropici Nod factor sulfation genes. PMID : 8755625 : Abstract >>
Rhizobium tropici produces a mixture of sulfated and non-sulfated Nod factors. The genes responsible for the sulfation process in R. tropici strain CFN299 were cloned and sequenced. These genes are homologous to the nodP, nodQ, and nodH genes from R. meliloti. The identity among the two species is 75% for nodP, 74% for nodQ, and 69% for nodH. NodH resembles sulfotransferases in general and NodQ has the characteristic purine-binding motifs and the PAPS 3'-phosphoadenosine 5'-phosphosulfate) motif. Mutants of NodP and NodH were obtained by site-directed mutagenesis. They are no longer able to synthesize the sulfated Nod factor, as was demonstrated in high-pressure liquid chromatography and thin-layer chromatography assays. The NodP- mutant had a decreased nodulation capacity in Phaseolus vulgaris Negro Xamapa bean plants. In contrast, NodH- and NodP- mutants acquired an increased capacity to nodulate the high-nitrogen-fixing bean cultivars N-8-116 and BAT-477. Nodulation was restored to normal levels when the mutants were complemented with a 16-kb clone carrying the wild-type genes. The role of the sulfate on Nod factors in R. tropici was dependent on the bean cultivar and the conditions assayed.
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34. |
( 1995 ) Rhizobium tropici chromosomal citrate synthase gene. PMID : 8526514 : PMC : PMC167707 Abstract >>
Two genes encoding citrate synthase, a key enzyme in the Krebs cycle, have been found in Rhizobium tropici. One of them is in the bacterial chromosome, while the other is in the symbiotic plasmid. We sequenced the chromosomal gene and found that it is very similar to the previously reported plasmidic gene sequence in its structural region but not in its regulatory region. The chromosomal gene is able to complement an Escherichia coli citrate synthase mutant. In R. tropici, a mutant in the chromosomal citrate synthase gene has a diminished citrate synthase activity (in free-living bacteria), a diminished nodulation capacity, and forms nitrogen-fixing nodules. In contrast, the citrate synthase double mutant forms ineffective nodules devoid of bacteroids and forms less nodules than the single chromosomal mutant. It is inferred that both genes are functional and required during the nodulation process in R. tropici.
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35. |
Waelkens F,
Voets T,
Vlassak K,
Vanderleyden J,
van Rhijn P,
( N/A ) The nodS gene of Rhizobium tropici strain CIAT899 is necessary for nodulation on Phaseolus vulgaris and on Leucaena leucocephala. PMID : 7772799 : Abstract >>
Rhizobium tropici strain CIAT899 induces nitrogen-fixing nodules on the roots of a wide range of tropical legumes, including Phaseolus vulgaris and Leucaena leucocephala. Previously, a DNA region of the CIAT899 pSym plasmid containing the common nodulation genes nodABC and one of the nodD alleles was characterized (P. van Rhijn, B. Feys, C. Verreth, and J. Vanderleyden, J. Bacteriol. 175: 438-447, 1993). As reported here, the region immediately downstream of nodC contains the nodSU genes. The nucleotide sequence of these genes is presented. CIAT899 nodS and nodU mutants were constructed. The nodS mutant was completely deficient in nodulation on the host plants P. vulgaris and L. leucocephala. The nodU mutation caused a decrease in nodulation on Leucaena but resorted no effect on Phaseolus. Introduction of the CIAT899 nodABCSU region in R. etli CE-3, a strain that only nodulates P. vulgaris, caused an extension of the host range of strain CE-3 to L. leucocephala.
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36. |
( 1993 ) A Rhizobium tropici DNA region carrying the amino-terminal half of a nodD gene and a nod-box-like sequence confers host-range extension. PMID : 7934929 : DOI : 10.1111/j.1365-2958.1993.tb01245.x Abstract >>
Rhizobium tropici CIAT899 is a broad-host-range strain that, in addition to Phaseolus, nodulates other plant legumes such as Leucaena and Macroptilium. The narrow-host-range of Rhizobium leguminosarum biovars phaseoli (strain CE3) and trifolii (strain RS1051) can be extended to Leucaena esculenta and Phaseolus vulgaris plants, respectively, by the introduction of a DNA fragment 521 bp long, which carries 128 amino acids of the amino-terminal region of a nodD gene from R. tropici, as well as a putative nod-box-like sequence, divergently oriented. The 521 bp fragment, in the presence of L. esculenta or P. vulgaris root exudates, induced a R. leguminosarum bv. viciae nodA-lacZ fusion in either a CE3 or RS1051 background, respectively.
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37. |
( 1994 ) Nodulating ability of Rhizobium tropici is conditioned by a plasmid-encoded citrate synthase. PMID : 8170393 : DOI : 10.1111/j.1365-2958.1994.tb00311.x Abstract >>
Rhizobium species elicit the formation of nitrogen-fixing root nodules through a complex interaction between bacteria and plants. Various bacterial genes involved in the nodulation and nitrogen-fixation processes have been described and most have been localized on the symbiotic plasmids (pSym). We have found a gene encoding citrate synthase on the pSym plasmid of Rhizobium tropici, a species that forms nitrogen-fixing nodules on the roots of beans (Phaseolus vulgaris) and trees (Leucaena spp.). Citrate synthase is a key metabolic enzyme that incorporates carbon into the tricarboxylic acid cycle by catalysing the condensation of acetyl-CoA and oxaloacetic acid to form citrate. R. tropici pcsA (the plasmid citrate synthase gene) is closely related to the corresponding genes of Proteobacteria. pcsA inactivation by a Tn5-mob insertion causes the bacteria to form fewer nodules (30-50% of the original strain) and to have a decreased citrate synthase activity in minimal medium with sucrose. A clone carrying the pcsA gene complemented all the phenotypic alterations of the pcsA mutant, and conferred Rhizobium leguminosarum bv. phaseoli (which naturally lacks a plasmid citrate synthase gene) a higher nodulation and growth capacity in correlation with a higher citrate synthase activity. We have also found that pcsA gene expression is sensitive to iron availability, suggesting a possible role of pcsA in iron uptake.
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38. |
( 1998 ) Mutants of Rhizobium tropici strain CIAT899 that do not induce chlorosis in plants. PMID : 9782510 : DOI : 10.1099/00221287-144-9-2607 Abstract >>
Type B strains of Rhizobium tropici induce severe foliar chlorosis when applied at planting to seeds of symbiotic host and non-host dicotyledonous plants. A Tn5-induced mutant, designated CT4812, or R. tropici strain CIAT899 that was unable to induce chlorosis was isolated. Cloning and sequencing of the DNA flanking the transposon in CT4812 revealed that the Tn5 insertion is located in a gene similar to glnD, which encodes uridylyltransferase/uridylyl-removing enzyme in enteric bacteria. Two marker-exchange mutants with insertions in glnD also failed to induce chlorosis in bean (Phaseolus vulgaris) plants. The 5'-most insertion in glnD (in mutant strain ME330) abolished the ability of R. tropici to utilize nitrate as a sole carbon source, whereas a mutation in glnD further downstream (in mutant strain ME245) did not have an obvious effect on nitrate utilization. A gene similar to the Salmonella typhimurium virulence gene mviN overlaps the 3' end of the R. tropici glnD homologue. A mutation in mviN had no effect on the ability of CIAT899 to induce chlorosis in bean plants. Therefore the glnD homologue, but not mviN, appears to be required for induction of chlorosis in plants by R. tropici strain CIAT899. A high nitrogen: carbon ratio in the rhizosphere of bean plants also prevented R. tropici from inducing chlorosis in bean plants. Mutations in either the glnD homologue or mviN had no significant effect on root nodule formation or acetylene reduction activity. A mutation in mviN eliminated motility in R. tropici. The sequence data, the inability of the glnD mutant to utilize nitrate, and the role of the R. tropici glnD gene in chlorosis induction in plants, a process that is nitrogen regulated, suggest that glnD plays a role in nitrogen sensing in R. tropici as its homologues do in other organisms.
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39. |
( 1998 ) Isolation and sequencing of a second Rhizobium tropici CFN299 genetic locus that contains genes homologous to amino acid sulphate activation genes. PMID : 9773278 : Abstract >>
A Rhizobium tropici CFN299 DNA region, homologous to genes involved in Nod factor synthesis and amino acid sulphate activation, was isolated from a genome library. DNA sequence analysis revealed two open reading frames, orf1 and orf2. orf1 showed highest sequence similarity to the Escherichia coli cysD gene while orf2 is closely related to Rhizobium sp. N33 nodQ. However, the orf2 deduced peptide is 152 amino acids shorter than Rhizobium sp. N33 NodQ, and lacks the 3'-phosphoadenosine 5'-phosphosulphate-binding motif. A dendrogram based on the alignment of the deduced amino acid sequences of orf2/nodQ/cysN genes separated Escherichia coli cysN and orf2 from the nodQ cluster. Upstream of orf1, partial sequence analysis revealed the 3' part of an orf that is highly similar to E. coli cysH. The G + C content of orf1 and orf2 differs significantly from the G + C content of R. tropici symbiotic sulphate activation nodPQ genes. This data suggests that the isolated R. tropici CFN299 locus contains housekeeping genes for amino acid sulphate activation.
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40. |
( 1998 ) Genes essential for nod factor production and nodulation are located on a symbiotic amplicon (AMPRtrCFN299pc60) in Rhizobium tropici. PMID : 9603874 : PMC : PMC107251 Abstract >>
Amplifiable DNA regions (amplicons) have been identified in the genome of Rhizobium etli. Here we report the isolation and molecular characterization of a symbiotic amplicon of Rhizobium tropici. To search for symbiotic amplicons, a cartridge containing a kanamycin resistance marker that responds to gene dosage and conditional origins of replication and transfer was inserted in the nodulation region of the symbiotic plasmid (pSym) of R. tropici CFN299. Derivatives harboring amplifications were selected by increasing the concentration of kanamycin in the cell culture. The amplified DNA region was mobilized into Escherichia coli and then into Agrobacterium tumefaciens. The 60-kb symbiotic amplicon, which we termed AMPRtrCFN299pc60, contains several nodulation and nitrogen fixation genes and is flanked by a novel insertion sequence ISRtr1. Amplification of AMPRtrCFN299pc60 through homologous recombination between ISRtr1 repeats increased the amount of Nod factors. Strikingly, the conjugal transfer of the amplicon into a plasmidless A. tumefaciens strain confers on the transconjugant the ability to produce R. tropici Nod factors and to nodulate Phaseolus vulgaris, indicating that R. tropici genes essential for the nodulation process are confined to an ampliable DNA region of the pSym.
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