| 1. |
Bott M,
Preisig O,
Hennecke H,
( 1992 ) Genes for a second terminal oxidase in Bradyrhizobium japonicum. PMID : 1444719 : DOI : 10.1007/bf00245362 Abstract >>
Bradyrhizobium japonicum possesses a mitochondria-like respiratory chain terminating with an aa3-type cytochrome c oxidase. The gene for subunit I of this enzyme (coxA) had been identified and cloned previously via heterologous hybridization using a Paracoccus denitrificans DNA probe. In the course of these studies, another B. japonicum DNA region was discovered which apparently encoded a second terminal oxidase that was different from cytochrome aa3 but also belonged to the superfamily of heme/copper oxidases. Nucleotide sequence analysis revealed a cluster of at least four genes, coxMNOP, organized most probably in an operon. The predicted coxM gene product shared significant similarity with subunit II of cytochrome c oxidases from other organisms: in particular, all of the proposed CuA ligands were conserved as well as three of the four acidic amino acid residues that might be involved in the binding of cytochrome c. The coxN gene encoded a polypeptide with about 40% sequence identity with subunit I representatives including the previously found CoxA protein: the six presumed histidine ligands of the prosthetic groups (two hemes and CuB) were strictly conserved. A remarkable feature of the DNA sequence was the presence of two genes, coxO and coxP, whose products were both homologous to subunit III proteins. A B. japonicum coxN mutant strain was created by marker exchange mutagenesis which, however, exhibited no obvious defects in free-living, aerobic growth or in root nodule symbiosis with soybean. This shows that the coxMNOP genes are not essential for respiration in the N2 fixing bacteroid.
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2. |
Göttfert M,
Holzhäuser D,
Bäni D,
Hennecke H,
( N/A ) Structural and functional analysis of two different nodD genes in Bradyrhizobium japonicum USDA110. PMID : 1421512 : Abstract >>
Bradyrhizobium japonicum has two closely linked homologs of the nodulation regulatory gene, nodD; these homologs are located upstream of and in divergent orientation to the nodYABCSUIJ gene cluster. We report here the nucleotide sequence and mutational analyses of both nodD copies. The predicted NodD1 and NodD2 proteins shared 62% identical amino acid residues at corresponding positions and exhibited different degrees of homology with NodD proteins of other Bradyrhizobium, Azorhizobium, and Rhizobium strains. Induction of the nodYABCSUIJ operon, as measured by expression of a translational nodC'-'lacZ fusion, required the nodD1 gene, but not nodD2. A B. japonicum mutant deleted for both nodD copies (strain delta 1267) still showed residual nodulation activity; however, nodulation of soybean was significantly delayed, and nodulation of mung bean and siratro resulted in strongly reduced nodule numbers. Fully efficient nodulation of mung bean and siratro by strain delta 1267 was restored by genetic complementation with the nodD1 gene, but not with nodD2. We conclude from these data that nodD1 is the critical gene that contributes to maximal nodulation efficiency, whereas the nodD2 gene does not play any obvious role in nodulation of the host plants tested.
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3. |
Ruan X,
Peters NK,
( 1992 ) Isolation and characterization of rhizobitoxine mutants of Bradyrhizobium japonicum. PMID : 1317377 : DOI : 10.1128/jb.174.11.3467-3473.1992 PMC : PMC206029 Abstract >>
To explore the role of rhizobitoxine in Bradyrhizobium-legume symbiosis, 11 rhizobitoxine mutants of B. japonicum USDA61 were isolated on the basis of their inability to synthesize the toxin in culture. Each mutant is prototrophic and symbiotically effective on soybean, cowpea, siratro, and Glycine soja. The rhizobitoxine mutants differ in their chlorosis phenotypes and rhizobitoxine production in planta. As expected, one group of mutant fail to make toxin in planta, resulting in the absence of chlorosis. Another group of mutants causes severe chlorosis on all cultivars of soybean tested. Surprisingly, this group of mutants makes more rhizobitoxine in soybean nodules than the wild-type strain does. This phenotype is only observed on soybean and not on other hosts such as cowpea, siratro, or G. soja. The remaining mutants all produce rhizobitoxine in planta but vary in the amount of toxin they produce and the severity of chlorosis they induce in soybean plants. Biochemical analysis of mutants demonstrates that one mutant is unable to synthesize serinol, a molecule hypothesized to be an intermediate in rhizobitoxine biosynthesis. By using these mutants, it was found that rhizobitoxine plays no apparent role in the nodulation of rj1 soybeans. Recently, it was found that inhibition of ethylene biosynthesis allows Rhizobium meliloti to overcome nitrate inhibition of nodule formation on alfalfa. Because rhizobitoxine also inhibits ethylene biosynthesis, we tested the ability of mutants which accumulate high levels of toxin in planta to overcome nitrate inhibition of nodule formation on soybean plants and found that the nodule formation induced by the wild type and that induced by mutant strains were equally suppressed in the presence of nitrate.
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4. |
Shin S,
Yun YS,
Koo HM,
Kim YS,
Choi KY,
Oh BH,
( 2003 ) Characterization of a novel Ser-cisSer-Lys catalytic triad in comparison with the classical Ser-His-Asp triad. PMID : 12711609 : DOI : 10.1074/jbc.M302156200 Abstract >>
Amidase signature family enzymes, which are widespread in nature, contain a newly identified Ser-cisSer-Lys catalytic triad in which the peptide bond between Ser131 and the preceding residue Gly130 is in a cis configuration. In order to characterize the property of the novel triad, we have determined the structures of five mutant malonamidase E2 enzymes that contain a Cys-cisSer-Lys, Ser-cisAla-Lys, or Ser-cisSer-Ala triad or a substitution of Gly130 with alanine. Cysteine cannot replace the role of Ser155 due to a hyper-reactivity of the residue, which results in the modification of the cysteine to cysteinyl sulfinic acid, most likely inside the expression host cells. The lysine residue plays a structural as well as a catalytic role, since the substitution of the residue with alanine disrupts the active site structure completely. The two observations are in sharp contrast with the consequences of the corresponding substitutions in the classical Ser-His-Asp triad. Structural data on the mutant containing the Ser-cisAla-Lys triad convincingly suggest that Ser131 plays an analogous catalytic role as the histidine of the Ser-His-Asp triad. The unusual cis configuration of Ser131 appears essential for the precise contacts of this residue with the other triad residues, as indicated by the near invariance of the preceding glycine residue (Gly130), structural data on the G130A mutant, and by a modeling experiment. The data provide a deep understanding of the role of each residue of the new triad at the atomic level and demonstrate that the new triad is a catalytic device distinctively different from the classical triad or its variants.
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5. |
Chen R,
Bhagwat AA,
Yaklich R,
Keister DL,
( 2002 ) Characterization of ndvD, the third gene involved in the synthesis of cyclic beta-(1 --> 3),(1 --> 6)-D-glucans in Bradyrhizobium japonicum. PMID : 12556128 : DOI : 10.1139/w02-099 Abstract >>
Previously, we identified two genes in Bradyrhizobium japonicum (ndvB, ndvC) that are required for cyclic beta-(1 --> 3),(1 --> 6)-D-glucan synthesis and successful symbiotic interaction with soybean (Glycine max). In this study, we report a new open reading frame (ORF1) located in the intergenic region between ndvB and ndvC, which is essential for beta-glucan synthesis and effective nodulation of G. max. This new gene is designated ndvD (nodule development). The ndvD translation product has a predicted molecular mass of 26.4 kDa with one transmembrane domain. Genetic experiments involving gene deletion, Tn5 insertion, and gene complementation revealed that the mutation of ndvD generated pleiotropic phenotypes, including hypoosmotic sensitivity, reduced motility, and defects in conjugative gene transfer, in addition to symbiotic ineffectiveness. Although deficient in in vivo beta-glucan synthesis, membrane preparations from the ndvD mutant synthesized neutral beta-glucans in vitro. Therefore, ndvD does not appear to be a structural gene for beta-glucan synthesis. Our hypothesis for the mechanism of beta-(1 --> 3),(1 --> 6)-D-glucan synthesis is presented.
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6. |
Mesa S,
Velasco L,
Manzanera ME,
Delgado MJ,
Bedmar EJ,
( 2002 ) Characterization of the norCBQD genes, encoding nitric oxide reductase, in the nitrogen fixing bacterium Bradyrhizobium japonicum. PMID : 12427946 : DOI : 10.1099/00221287-148-11-3553 Abstract >>
The genes norCBQD that encode the bc-type nitric oxide reductase from Bradyrhizobium japonicum USDA110 have been isolated and characterized. norC and norB encode the cytochrome c-containing subunit II and cytochrome b-containing subunit I of nitric oxide reductase, respectively. norQ encodes a protein with an ATP/GTP-binding motif, and the predicted norD gene product shows similarity with NorD from other denitrifiers. Mutational analysis indicates that the two structural norC and norB genes are required for microaerobic growth under nitrate-respiring conditions. A mutant strain lacking a functional norC gene also lacked the 16 kDa c-type cytochrome that is normally detectable by haem-staining of proteins from membranes of microaerobically grown wild-type cells. Expression of a transcriptional fusion of the nor promoter region to the reporter gene lacZ (P(norC)-lacZ) was not detected in aerobically grown cells of USDA110, but the fusion was induced threefold when the cells were cultured under microaerobic conditions (1% O(2)) with either nitrite or nitric oxide, and about 18-fold when nitrate was the N oxide present in the medium. The P(norC)-lacZ fusion was not expressed in the B. japonicum fixK(2) mutant strain 9043, but complementation of the mutant with the fixK(2) gene restored beta-galactosidase activity to levels similar to those found in the parental strain. The promoter region of the norCBQD genes has been characterized by primer extension. A major transcript initiates 45.5 bp downstream of the centre of a putative binding site for the transcription factor FixK(2).
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7. |
Parker MA,
Lafay B,
Burdon JJ,
van Berkum P,
( 2002 ) Conflicting phylogeographic patterns in rRNA and nifD indicate regionally restricted gene transfer in Bradyrhizobium. PMID : 12177349 : DOI : 10.1099/00221287-148-8-2557 Abstract >>
Major differences in evolutionary relationships of the 16S rRNA gene and the nitrogenase alpha-subunit gene (nifD) were observed among 38 strains of Bradyrhizobium sp. nodule bacteria from North America, Central America, Asia and Australia. Two lineages were evident in the 16S rRNA phylogeny representing strains related to Bradyrhizobium japonicum (29 isolates) or Bradyrhizobium elkanii (9 isolates). Both clades were distributed across most or all of the geographic regions sampled. By contrast, in the nifD tree almost all isolates were placed into one of three groups each exclusively composed of taxa from a single geographic region (North Temperate, Central America or Australia). Isolates that were closely related or identical in gene sequence at one locus often had divergent sequences at the other locus and a partition homogeneity test indicated that the 16S rRNA and nifD phylogenies were significantly incongruent. No evidence for any gene duplication of nifD was found by Southern hybridization analysis on a subset of the strains, so unrecognized paralogy is not likely to be responsible for the discrepancy between 16S rRNA and nifD tree topologies. These results are consistent with a model whereby geographic areas were initially colonized by several diverse 16S rRNA lineages, with subsequent horizontal gene transfer of nifD leading to increased nifD sequence homogeneity within each regional population.
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8. |
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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9. |
Velasco L,
Mesa S,
Delgado MJ,
Bedmar EJ,
( 2001 ) Characterization of the nirK gene encoding the respiratory, Cu-containing nitrite reductase of Bradyrhizobium japonicum. PMID : 11690645 : DOI : 10.1016/s0167-4781(01)00279-2 Abstract >>
The structural gene, nirK, for the respiratory Cu-containing nitrite reductase from Bradyrhizobium japonicum USDA110 has been isolated and sequenced. The deduced amino acid sequence exhibited a high degree of similarity to other Cu-containing nitrite reductases from various sources. The full-length protein included a signal peptide for protein export. Analysis of the sequence upstream from the structural nirK gene revealed the presence of an anaerobox located 83 base pairs from the putative translational start codon. Cells of strain GRK308, a nitrite reductase-deficient derivative of strain USDA110, were unable to grow when cultured under microaerobic conditions (1% O(2)) in the presence of either nitrate or nitrite. Maximal expression of a nirK-lacZ fusion in strain USDA110 required simultaneously both low level oxygen conditions and the presence of nitrate. Expression of beta-galactosidase activity was not detected in the B. japonicum fixL 7403, fixJ 7360 and fixK(2) 9043 mutants transformed with the nirK-lacZ fusion after incubation of the cells under oxygen-limiting conditions either with or without nitrate. Complementation of B. japonicum 9043 with the fixK(2) gene restored beta-galactosidase activity to levels similar to those found in the parental strain. These results suggest that nirK expression depends on the low-oxygen-responsive two-component regulatory system FixLJ and on the Fnr/FixK-like DNA binding protein FixK(2).
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10. |
Minder AC,
de Rudder KE,
Narberhaus F,
Fischer HM,
Hennecke H,
Geiger O,
( 2001 ) Phosphatidylcholine levels in Bradyrhizobium japonicum membranes are critical for an efficient symbiosis with the soybean host plant. PMID : 11251836 : Abstract >>
Phosphatidylcholine (PC), the major membrane phospholipid in eukaryotes, is found in only some bacteria including members of the family Rhizobiaceae. For this reason, it has long been speculated that rhizobial PC might be required for a successful interaction of rhizobia with their legume host plants in order to allow the formation of nitrogen-fixing root nodules. A major pathway for PC formation in prokaryotes involves a threefold methylation of the precursor phosphatidylethanolamine (PE). Here, we report on the isolation of a Bradyrhizobium japonicum gene (pmtA) encoding the phospholipid N-methyltransferase PmtA. Upon expression of the bradyrhizobial pmtA gene in Escherichia coli, predominantly monomethylphosphatidylethanolamine was formed from PE. PmtA-deficient B. japonicum mutants still produced low levels of PC by a second methylation pathway. The amount of PC formed in such mutants (6% of total phospholipids) was greatly decreased compared with the wild type (52% of total phospholipids). Root nodules of soybean plants infected with B. japonicum pmtA mutants showed a nitrogen fixation activity of only 18% of the wild-type level. The interior colour of the nodules was beige instead of red, suggesting decreased amounts of leghaemoglobin. Moreover, ultrastructure analysis of these nodules demonstrated a greatly reduced number of bacteroids within infected plant cells. These data suggest that the biosynthesis of wild-type amounts of PC are required to allow for an efficient symbiotic interaction of B. japonicum with its soybean host plant.
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11. |
Louch HA,
Miller KJ,
( 2001 ) Synthesis of a low-molecular-weight form of exopolysaccharide by Bradyrhizobium japonicum USDA 110. PMID : 11157281 : DOI : 10.1128/AEM.67.2.1011-1014.2001 PMC : PMC92685 Abstract >>
A novel extracellular low-molecular-weight polysaccharide was detected as a contaminant within extracellular cyclic beta-1,6-beta-1,3-glucan preparations from Bradyrhizobium japonicum USDA 110 cultures. Compositional analysis, methylation analysis, and nuclear magnetic resonance analysis revealed that this low-molecular-weight polysaccharide was composed of the same pentasaccharide repeating unit previously described for the high-molecular-weight form of the exopolysaccharide (EPS) synthesized by B. japonicum strains. Mass spectrometry analysis indicated that the size of this low-molecular-weight form of EPS was consistent with a dimeric form of the pentasaccharide repeating unit.
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12. |
King ND,
O'Brian MR,
( 2001 ) Evidence for direct interaction between enzyme I(Ntr) and aspartokinase to regulate bacterial oligopeptide transport. PMID : 11287431 : DOI : 10.1074/jbc.M101982200 Abstract >>
Bradyrhizobium japonicum transports oligopeptides and the heme precursor delta-aminolevulinic acid (ALA) by a common mechanism. Two Tn5-induced mutants disrupted in the lysC and ptsP genes were identified based on the inability to use prolyl-glycyl-glycine as a proline source and were defective in [(14)C]ALA uptake activity. lysC and ptsP were shown to be proximal genes in the B. japonicum genome. However, RNase protection and in trans complementation analysis showed that lysC and ptsP are transcribed separately, and that both genes are involved in oligopeptide transport. Aspartokinase, encoded by lysC, catalyzes the phosphorylation of aspartate for synthesis of three amino acids, but the lysC strain is not an amino acid auxotroph. The ptsP gene encodes Enzyme I(Ntr) (EI(Ntr)), a paralogue of Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase (PTS) system. In vitro pull-down experiments indicated that purified recombinant aspartokinase and EI(Ntr) interact directly with each other. Expression of ptsP in trans from a multicopy plasmid complemented the lysC mutant, suggesting that aspartokinase normally affects Enzyme I(Ntr) in a manner that can be compensated for by increasing the copy number of the ptsP gene. ATP was not a phosphoryl donor to purified EI(Ntr), but it was phosphorylated by ATP in the presence of cell extracts. This phosphorylation was inhibited in the presence of aspartokinase. The findings demonstrate a role for a PTS protein in the transport of a non-sugar solute and suggest an unusual regulatory function for aspartokinase in regulating the phosphorylation state of EI(Ntr).
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13. |
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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14. |
Fischer HM,
Velasco L,
Delgado MJ,
Bedmar EJ,
Schären S,
Zingg D,
Göttfert M,
Hennecke H,
( 2001 ) One of two hemN genes in Bradyrhizobium japonicum is functional during anaerobic growth and in symbiosis. PMID : 11157943 : DOI : 10.1128/JB.183.4.1300-1311.2001 PMC : PMC95004 Abstract >>
Previously, we screened the symbiotic gene region of the Bradyrhizobium japonicum chromosome for new NifA-dependent genes by competitive DNA-RNA hybridization (A. Nienaber, A. Huber, M. G?ttfert, H. Hennecke, and H. M. Fischer, J. Bacteriol. 182:1472-1480, 2000). Here we report more details on one of the genes identified, a hemN-like gene (now called hemN(1)) whose product exhibits significant similarity to oxygen-independent coproporphyrinogen III dehydrogenases involved in heme biosynthesis in facultatively anaerobic bacteria. In the course of these studies, we discovered that B. japonicum possesses a second hemN-like gene (hemN(2)), which was then cloned by using hemN(1) as a probe. The hemN(2) gene maps outside of the symbiotic gene region; it is located 1.5 kb upstream of nirK, the gene for a Cu-containing nitrite reductase. The two deduced HemN proteins are similar in size (445 and 450 amino acids for HemN(1) and HemN(2), respectively) and share 53% identical (68% similar) amino acids. Expression of both hemN genes was monitored with the help of chromosomally integrated translational lacZ fusions. No significant expression of either gene was detected in aerobically grown cells, whereas both genes were strongly induced (> or = 20-fold) under microaerobic or anaerobic conditions. Induction was in both cases dependent on the transcriptional activator protein FixK(2). In addition, maximal anaerobic hemN(1) expression was partially dependent on NifA, which explains why this gene had been identified by the competitive DNA-RNA hybridization approach. Strains were constructed carrying null mutations either in individual hemN genes or simultaneously in both genes. All mutants showed normal growth in rich medium under aerobic conditions. Unlike the hemN(1) mutant, strains lacking a functional hemN(2) gene were unable to grow anaerobically under nitrate-respiring conditions and largely failed to fix nitrogen in symbiosis with the soybean host plant. Moreover, these mutants lacked several c-type cytochromes which are normally detectable by heme staining of proteins from anaerobically grown wild-type cells. Taken together, our results revealed that B. japonicum hemN(2), but not hemN(1), encodes a protein that is functional under the conditions tested, and this conclusion was further corroborated by the successful complementation of a Salmonella enterica serovar Typhimurium hemF hemN mutant with hemN(2) only.
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15. |
King ND,
Hojnacki D,
O'Brian MR,
( 2000 ) The Bradyrhizobium japonicum proline biosynthesis gene proC is essential for symbiosis. PMID : 11097929 : DOI : 10.1128/aem.66.12.5469-5471.2000 PMC : PMC92483 Abstract >>
Plant host-derived proline is proposed to serve as an energy source for rhizobia in the rhizosphere and in symbiotic root nodules. The Bradyrhizobium japonicum proC gene was isolated, and a proC mutant strain that behaved as a strict proline auxotroph in culture was constructed. The proC strain elicited undeveloped nodules on soybeans that lacked nitrogen fixation activity and plant hemoglobin. We conclude that the proC gene is essential for symbiosis and suggest that the mutant does not obtain an exogenous supply of proline in association with soybeans sufficient to satisfy its auxotrophy.
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16. |
Göttfert M,
Röthlisberger S,
Kündig C,
Beck C,
Marty R,
Hennecke H,
( 2001 ) Potential symbiosis-specific genes uncovered by sequencing a 410-kilobase DNA region of the Bradyrhizobium japonicum chromosome. PMID : 11157954 : DOI : 10.1128/JB.183.4.1405-1412.2001 PMC : PMC95015 Abstract >>
The physical and genetic map of the Bradyrhizobium japonicum chromosome revealed that nitrogen fixation and nodulation genes are clustered. Because of the complex interactions between the bacterium and the plant, we expected this chromosomal sector to contain additional genes that are involved in the maintenance of an efficient symbiosis. Therefore, we determined the nucleotide sequence of a 410-kb region. The overall G+C nucleotide content was 59.1%. Using a minimum gene length of 150 nucleotides, 388 open reading frames (ORFs) were selected as coding regions. Thirty-five percent of the predicted proteins showed similarity to proteins of rhizobia. Sixteen percent were similar only to proteins of other bacteria. No database match was found for 29%. Repetitive DNA sequence-derived ORFs accounted for the rest. The sequenced region contained all nitrogen fixation genes and, apart from nodM, all nodulation genes that were known to exist in B. japonicum. We found several genes that seem to encode transport systems for ferric citrate, molybdate, or carbon sources. Some of them are preceded by -24/-12 promoter elements. A number of putative outer membrane proteins and cell wall-modifying enzymes as well as a type III secretion system might be involved in the interaction with the host.
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17. |
Koo HM,
( 2000 ) Identification of active-site residues in Bradyrhizobium japonicum malonyl-coenzyme A synthetase. PMID : 10871057 : DOI : 10.1006/abbi.2000.1813 Abstract >>
Malonyl-CoA synthetase (MCS) has been previously purified and characterized from Bradyrhizobium japonicum USDA 110. The gene encoding this enzyme is now cloned, sequenced, and expressed in Escherichia coli. The enzyme contains 509 amino acid residues, with a calculated molecular mass of 55,239 Da. The recombinant enzyme was also purified from the transformed E. coli. The enzyme was essentially indistinguishable from the MCS of B. japonicum by the criteria of polyacrylamide gel electrophoresis and biochemical properties. Based on inhibitor studies of Rhizobium trifolii MCS reported previously and database analysis, Arg173, Lys175, His211, and Glu308 were selected for site-directed mutagenesis in order to identify amino acid residues essential for substrate binding and/or catalysis. Five different mutant enzymes (R173G, K175M, H211L, K175M/H211L, and E308Q) were prepared and then subjected to steady-state kinetic studies. The kinetic data measured for the mutants suggest that Lys175 and His211 participate in the formation of malonyl-AMP, whereas Glu308 may play a role in malonate binding.
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18. |
So JS,
Kim WS,
Stacey G,
( 2000 ) Molecular characterization of a gene region involved in lipopolysaccharide biosynthesis in Bradyrhizobium japonicum: cloning, sequencing and expression of rfaf gene. PMID : 10981699 : DOI : 10.1111/j.1574-6968.2000.tb09271.x Abstract >>
A 3.0-kb region involved in lipopolysaccharide biosynthesis in Bradyrhizobium japonicum was sequenced. One complete open reading frame was identified which encodes a polypeptide of 354 amino acid residues with a predicted molecular mass of 38 209 Da. Expression of the protein using a T7 gene expression system revealed a band of similar molecular mass after sodium dodecyl sulfate polyacrylamide gel electrophoresis. A database search against known gene sequences revealed a significant sequence similarity to the rfaF gene cloned from several Gram-negative bacteria. The rfaF gene is known to encode heptosyltransferase II that transfers a second heptose to the inner core of lipopolysaccharide. The cloned B. japonicum open reading frame was able to functionally complement a rfaF mutant of Salmonella typhimurium SL3789. Transformation of this mutant with the B. japonicum gene restored production of an intact lipopolysaccharide and resistance to the hydrophobic antibiotic, novobiocin. An additional open reading frame having a significant sequence similarity to the rfaD gene was found to be divergently oriented to the rfaF gene.
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19. |
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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20. |
Urech C,
Narberhaus F,
( 1999 ) Characterization of the Bradyrhizobium japonicum ftsH gene and its product. PMID : 10572147 : PMC : PMC103706 Abstract >>
The Bradyrhizobium japonicum ftsH gene was cloned by using a set of widely applicable degenerated oligonucleotides. Western blot experiments indicated that the FtsH protein was produced under standard growth conditions and that it was not heat inducible. Attempts to delete the ftsH gene in B. japonicum failed, suggesting a pivotal cellular function of this gene. The expression of B. japonicum ftsH in an ftsH-negative Escherichia coli strain significantly enhanced the fitness of this mutant and reduced the steady-state level of sigma(32).
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21. |
Krummenacher P,
( 2000 ) Two genes encoding a putative multidrug efflux pump of the RND/MFP family are cotranscribed with an rpoH gene in Bradyrhizobium japonicum. PMID : 10675036 : DOI : 10.1016/s0378-1119(99)00490-4 Abstract >>
The rpoH3 gene of Bradyrhizobium japonicum codes for one of three sigma32-type transcription factors in this organism and is flanked by rag (rpoH3-associated) genes comprising the chromosomal arrangement ragABrpoH3ragCD. The first genes in this cluster code for a classical two-component regulatory system with an unknown function (Narberhaus et al., 1997. Mol. Microbiol. 24, 93-104). The deduced proteins of the last two genes display a high sequence similarity to heavy metal or multidrug efflux pumps of the RND (Resistance/Nodulation/cell Division)-MFP (Membrane Fusion Protein) family. Reverse transcription and polymerase chain reaction (RT-PCR) analysis demonstrated that ragC is cotranscribed with rpoH3. Mutant strains carrying disrupted rag genes or an extented deletion of the rag locus exhibited neither an apparent growth defect nor a deficiency in the symbiotic interaction with soybean roots. The minimal inhibitory concentrations (MICs) of various metals and organic compounds were identical for the wild-type and mutant strains. Moreover, translational lacZ fusions to each of the first four genes of the rag cluster showed a very low expression under all conditions tested; hence, the substrate for the putative efflux pump has not been uncovered.
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22. |
Fischer HM,
Minder AC,
( 2000 ) Role of HrcA and CIRCE in the heat shock regulatory network of Bradyrhizobium japonicum. PMID : 10613857 : DOI : 10.1128/jb.182.1.14-22.2000 PMC : PMC94234 Abstract >>
A large number of bacteria regulate chaperone gene expression by the CIRCE-HrcA system in which a DNA element called CIRCE serves as binding site for the repressor protein HrcA under non-heat-shock conditions. We have cloned the two consecutive genes hrcA and grpE of Bradyrhizobium japonicum by using a complementation approach that screened for GrpE function. In vivo and in vitro transcript mapping demonstrated that both genes are transcribed separately from RpoH (sigma(32))-dependent promoters. To investigate the supposed negative regulatory function of HrcA, we compared the expression of putative target genes in the wild type with that in an hrcA mutant. Transcription of the CIRCE-associated chaperonin operons groESL(4) and groESL(5), as well as the beta-galactosidase activity derived from corresponding groE-lacZ fusions, was strongly elevated in the hrcA mutant even at physiological temperatures. Expression of other heat shock regulons (RpoH or ROSE dependent) was not affected. To study the activity of HrcA in vitro, we purified a histidine-tagged version of the protein under nondenaturing conditions. Specific binding to the CIRCE element was obtained with a soluble fraction of HrcA in gel retardation experiments.
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23. |
Huber A,
Göttfert M,
Nienaber A,
( 2000 ) Three new NifA-regulated genes in the Bradyrhizobium japonicum symbiotic gene region discovered by competitive DNA-RNA hybridization. PMID : 10692350 : DOI : 10.1128/jb.182.6.1472-1480.2000 PMC : PMC94442 Abstract >>
The so-called symbiotic region of the Bradyrhizobium japonicum chromosome (C. K?ndig, H. Hennecke, and M. G?ttfert, J. Bacteriol. 175:613-622, 1993) was screened for the presence of genes controlled by the nitrogen fixation regulatory protein NifA. Southern blots of restriction enzyme-digested cosmids that represent an ordered, overlapping library of the symbiotic region were competitively hybridized with in vitro-labeled RNA from anaerobically grown wild-type cells and an excess of RNA isolated either from anaerobically grown nifA and rpoN mutant cells or from aerobically grown wild-type cells. In addition to the previously characterized nif and fix gene clusters, we identified three new NifA-regulated genes that were named nrgA, nrgB, and nrgC (nrg stands for NifA-regulated gene). The latter two probably form an operon, nrgBC. The proteins encoded by nrgC and nrgA exhibited amino acid sequence similarity to bacterial hydroxylases and N-acetyltransferases, respectively. The product of nrgB showed no significant similarity to any protein with a database entry. Primer extension experiments and expression studies with translational lacZ fusions revealed the presence of a functional -24/-12-type promoter upstream of nrgA and nrgBC and proved the NifA- and RpoN (sigma(54))-dependent transcription of the respective genes. Null mutations introduced into nrgA and nrgBC resulted in mutant strains that exhibited wild-type-like symbiotic properties, including nitrogen fixation, when tested on soybean, cowpea, or mung bean host plants. Thus, the discovery of nrgA and nrgBC further emphasizes the previously suggested role of NifA as an activator of anaerobically induced genes other than the classical nitrogen fixation genes.
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24. |
Hassett R,
Hamza I,
( 1999 ) Identification of a functional fur gene in Bradyrhizobium japonicum. PMID : 10482529 : PMC : PMC94108 Abstract >>
The recent identification of the iron response regulator (Irr) in Bradyrhizobium japonicum raised the question of whether the global regulator Fur is present in that organism. A fur gene homolog was isolated by the functional complementation of an Escherichia coli fur mutant. The B. japonicum Fur bound to a Fur box DNA element in vitro, and a fur mutant grown in iron-replete medium was derepressed for iron uptake activity. Thus, B. japonicum expresses at least two regulators of iron metabolism.
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25. |
Sameshima R,
Mitsui H,
Minamisawa K,
( 1999 ) IS1631 occurrence in Bradyrhizobium japonicum highly reiterated sequence-possessing strains with high copy numbers of repeated sequences RSalpha and RSbeta. PMID : 10427040 : PMC : PMC91525 Abstract >>
From Bradyrhizobium japonicum highly reiterated sequence-possessing (HRS) strains indigenous to Niigata and Tokachi in Japan with high copy numbers of the repeated sequences RSalpha and RSbeta (K. Minamisawa, T. Isawa, Y. Nakatsuka, and N. Ichikawa, Appl. Environ. Microbiol. 64:1845-1851, 1998), several insertion sequence (IS)-like elements were isolated by using the formation of DNA duplexes by denaturation and renaturation of total DNA, followed by treatment with S1 nuclease. Most of these sequences showed structural features of bacterial IS elements, terminal inverted repeats, and homology with known IS elements and transposase genes. HRS and non-HRS strains of B. japonicum differed markedly in the profiles obtained after hybridization with all the elements tested. In particular, HRS strains of B. japonicum contained many copies of IS1631, whereas non-HRS strains completely lacked this element. This association remained true even when many field isolates of B. japonicum were examined. Consequently, IS1631 occurrence was well correlated with B. japonicum HRS strains possessing high copy numbers of the repeated sequence RSalpha or RSbeta. DNA sequence analysis indicated that IS1631 is 2,712 bp long. In addition, IS1631 belongs to the IS21 family, as evidenced by its two open reading frames, which encode putative proteins homologous to IstA and IstB of IS21, and its terminal inverted repeat sequences with multiple short repeats.
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26. |
Guerinot ML,
( 1999 ) Succinate dehydrogenase (Sdh) from Bradyrhizobium japonicum is closely related to mitochondrial Sdh. PMID : 10419971 : PMC : PMC103604 Abstract >>
The sdhCDAB operon, encoding succinate dehydrogenase, was cloned from the soybean symbiont Bradyrhizobium japonicum. Sdh from B. japonicum is phylogenetically related to Sdh from mitochondria. This is the first example of a mitochondrion-like Sdh functionally expressed in Escherichia coli.
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27. |
Gu CT,
Wang ET,
Sui XH,
Chen WF,
Chen WX,
( 2007 ) Diversity and geographical distribution of rhizobia associated with Lespedeza spp. in temperate and subtropical regions of China. PMID : 17530227 : DOI : 10.1007/s00203-007-0256-3 Abstract >>
Eighty-eight root-nodule isolates from Lespedeza spp. grown in temperate and subtropical regions of China were characterized by a polyphasic approach. Nine clusters were defined in numerical taxonomy and SDS-PAGE analysis of whole cell proteins. Based upon further characterizations of amplified 16S rDNA restriction analysis (ARDRA), PCR-based restriction fragment length polymorphism of ribosomal IGS, 16S rDNA sequence analysis and DNA-DNA hybridization, these isolates were identified as Bradyrhizobium japonicum, B. elkanii, B. yuanmingense, Mesorhizobium amorphae, M. huakuii, Sinorhizobium meliloti and three genomic species related to B. yuanmingense, Rhizobium gallicum and R. tropici. The Bradyrhizobium species and R. tropici-related rhizobia were mainly isolated from the subtropical region and the species of Mesorhizobium, S. meliloti and R. gallicum-related species were all isolated from the temperate region. Phylogenetic analyses of nifH and nodC indicated that the symbiotic genes of distinct rhizobial species associated with Lespedeza spp. might have different origins and there was no evidence for lateral gene transfer of symbiotic genes. The results obtained in the present study and in a previous report demonstrated that Lespedeza spp. are nodulated by rhizobia with diverse genomic backgrounds and these Lespedeza-nodulating rhizobia were not specific to the host species, but specific to their geographic origins.
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28. |
Stepkowski T,
Hughes CE,
Law IJ,
Markiewicz ?,
Gurda D,
Chlebicka A,
Moulin L,
( 2007 ) Diversification of lupine Bradyrhizobium strains: evidence from nodulation gene trees. PMID : 17400786 : DOI : 10.1128/AEM.02125-06 PMC : PMC1907101 Abstract >>
Bradyrhizobium strains isolated in Europe from Genisteae and serradella legumes form a distinct lineage, designated clade II, on nodulation gene trees. Clade II bradyrhizobia appear to prevail also in the soils of Western Australia and South Africa following probably accidental introduction with seeds of their lupine and serradella hosts. Given this potential for dispersal, we investigated Bradyrhizobium isolates originating from a range of native New World lupines, based on phylogenetic analyses of nodulation (nodA, nodZ, noeI) and housekeeping (atpD, dnaK, glnII, recA) genes. The housekeeping gene trees revealed considerable diversity among lupine bradyrhizobia, with most isolates placed in the Bradyrhizobium japonicum lineage, while some European strains were closely related to Bradyrhizobium canariense. The nodA gene tree resolved seven strongly supported groups (clades I to VII) that correlated with strain geographical origins and to some extent with major Lupinus clades. All European strains were placed in clade II, whereas only a minority of New World strains was placed in this clade. This work, as well as our previous studies, suggests that clade II diversified predominately in the Old World, possibly in the Mediterranean. Most New World isolates formed subclade III.2, nested in a large "pantropical" clade III, which appears to be New World in origin, although it also includes strains originating from nonlupine legumes. Trees generated using nodZ and noeI gene sequences accorded well with the nodA tree, but evidence is presented that the noeI gene may not be required for nodulation of lupine and that loss of this gene is occurring.
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29. |
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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30. |
Batista JS,
Hungria M,
Barcellos FG,
Ferreira MC,
Mendes IC,
( 2007 ) Variability in Bradyrhizobium japonicum and B. elkanii seven years after introduction of both the exotic microsymbiont and the soybean host in a cerrados soil. PMID : 17265000 : DOI : 10.1007/s00248-006-9149-2 Abstract >>
The plasticity of rhizobial genomes is far greater than previously thought, with complex genomic recombination events that may be accelerated by the often stressful environmental conditions of the tropics. This study aimed at evaluating changes in soybean rhizobia due to adaptation to inhospitable environmental conditions (high temperatures, drought, and acid soils) in the Brazilian Cerrados. Both the host plant and combinations of four strains of soybean Bradyrhizobium were introduced in an uncropped soil devoid of rhizobia capable of nodulating soybean. After the third year, seeds were not reinoculated. Two hundred and sixty-three isolates were obtained from nodules of field-grown soybean after the seventh year, and their morphological, physiological, serological, and symbiotic properties determined, followed by genetic analysis of conserved and symbiotic genes. B. japonicum strain CPAC 15 (same serogroup as USDA 123) was characterized as having high saprophytic capacity and competitiveness and by the seventh year represented up to 70% of the cultivable population, in contrast to the poor survival and competitiveness of B. japonicum strain CPAC 7 (same serogroup as CB 1809). In general, adapted strains had increased mucoidy, and up to 43% of the isolates showed no serological reaction. High variability, presumably resulting from the adaptation to the harsh environmental conditions, was verified in rep-PCR (polymerase chain reaction) profiles, being lower in strain CPAC 15, intermediate in B. elkanii, and higher in CPAC 7. Restriction fragment length polymorphism (RFLP)-PCR types of the 16S rDNA corresponded to the following: one type for B. elkanii species, two for B. japonicum, associated to CPAC 15 and CPAC 7, and unknown combinations of profiles. However, when nodC sequences and RFLP-PCR of the nifH region data were considered, only two clusters were observed having full congruence with B. japonicum and B. elkanii species. Combining the results, variability was such that even within a genetically more stable group (such as that of CPAC 15), only 6.4% of the isolates showed high similarity to the inoculant strain, whereas none was similar to CPAC 7. The genetic variability in our study seems to result from a variety and combination of events including strain dispersion, genomic recombination, and horizontal gene transfer. Furthermore, the genetic variability appears to be mainly associated with adaptation, saprophytic capacity, and competitiveness, and not with symbiotic effectiveness, as the similarity of symbiotic genes was higher than that of conserved regions of the DNA.
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31. |
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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32. |
Ormeño-Orrillo E,
Vinuesa P,
Zúñiga-Dávila D,
Martínez-Romero E,
( 2006 ) Molecular diversity of native bradyrhizobia isolated from lima bean (Phaseolus lunatus L.) in Peru. PMID : 16564961 : DOI : 10.1016/j.syapm.2005.09.002 Abstract >>
The diversity of a collection of 21 bradyrhizobial isolates from Lima bean (Phaseolus lunatus L.) was assayed by molecular methods. Moderately high to high genetic diversity was revealed by multilocus enzyme electrophoresis (MLEE) analysis of seven enzyme loci and genomic fingerprints with ERIC and BOX primers. Two groups with differences in growth rate were found among the isolates and their differentiation as two divergent bradyrhizobial lineages was supported by PCR-RFLP of the rpoB gene and sequence analysis of the 16S rDNA and dnaK genes. Isolates with slow growth (SG) were identified as Bradyrhizobium yuanmingense, while extra-slow growing isolates (ESG) constitute a new lineage different from all described Bradyrhizobium species. Three distinct symbiotic genotypes were detected among Lima bean bradyrhizobia by PCR-RFLP and sequence analysis of the nifH and nodB genes. One genotype was found in the ESG lineage and two in B. yuanmingense. Another symbiotic genotype was detected in B. yuamingense isolated from Lespedeza plants. The identified bradyrhizobial lineages constitute sympatric species effectively nodulating Lima bean on the coast of Peru.
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33. |
Delgado MJ,
Tresierra-Ayala A,
Talbi C,
Bedmar EJ,
( 2006 ) Functional characterization of the Bradyrhizobium japonicum modA and modB genes involved in molybdenum transport. PMID : 16385130 : DOI : 10.1099/mic.0.28347-0 Abstract >>
A modABC gene cluster that encodes an ABC-type, high-affinity molybdate transporter from Bradyrhizobium japonicum has been isolated and characterized. B. japonicum modA and modB mutant strains were unable to grow aerobically or anaerobically with nitrate as nitrogen source or as respiratory substrate, respectively, and lacked nitrate reductase activity. The nitrogen-fixing ability of the mod mutants in symbiotic association with soybean plants grown in a Mo-deficient mineral solution was severely impaired. Addition of molybdate to the bacterial growth medium or to the plant mineral solution fully restored the wild-type phenotype. Because the amount of molybdate required for suppression of the mutant phenotype either under free-living or under symbiotic conditions was dependent on sulphate concentration, it is likely that a sulphate transporter is also involved in Mo uptake in B. japonicum. The promoter region of the modABC genes has been characterized by primer extension. Reverse transcription and expression of a transcriptional fusion, P(modA)-lacZ, was detected only in a B. japonicum modA mutant grown in a medium without molybdate supplementation. These findings indicate that transcription of the B. japonicum modABC genes is repressed by molybdate.
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34. |
Vinuesa P,
Silva C,
Werner D,
Martínez-Romero E,
( 2005 ) Population genetics and phylogenetic inference in bacterial molecular systematics: the roles of migration and recombination in Bradyrhizobium species cohesion and delineation. PMID : 15579380 : DOI : 10.1016/j.ympev.2004.08.020 Abstract >>
A combination of population genetics and phylogenetic inference methods was used to delineate Bradyrhizobium species and to uncover the evolutionary forces acting at the population-species interface of this bacterial genus. Maximum-likelihood gene trees for atpD, glnII, recA, and nifH loci were estimated for diverse strains from all but one of the named Bradyrhizobium species, and three unnamed "genospecies," including photosynthetic isolates. Topological congruence and split decomposition analyses of the three housekeeping loci are consistent with a model of frequent homologous recombination within but not across lineages, whereas strong evidence was found for the consistent lateral gene transfer across lineages of the symbiotic (auxiliary) nifH locus, which grouped strains according to their hosts and not by their species assignation. A well resolved Bayesian species phylogeny was estimated from partially congruent glnII+recA sequences, which is highly consistent with the actual taxonomic scheme of the genus. Population-level analyses of isolates from endemic Canarian genistoid legumes based on REP-PCR genomic fingerprints, allozyme and DNA polymorphism analyses revealed a non-clonal and slightly epidemic population structure for B. canariense isolates of Canarian and Moroccan origin, uncovered recombination and migration as significant evolutionary forces providing the species with internal cohesiveness, and demonstrated its significant genetic differentiation from B. japonicum, its sister species, despite their sympatry and partially overlapped ecological niches. This finding provides strong evidence for the existence of well delineated species in the bacterial world. The results and approaches used herein are discussed in the context of bacterial species concepts and the evolutionary ecology of (brady)rhizobia.
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35. |
Becker BU,
Bonnard N,
Boiffin V,
Mörschel E,
Tresierra A,
Müller P,
( 2004 ) A novel genetic locus outside the symbiotic island is required for effective symbiosis of Bradyrhizobium japonicum with soybean Glycine max. PMID : 15501655 : DOI : 10.1016/j.resmic.2004.06.010 Abstract >>
In order to investigate the symbiotic interaction between soybean and Bradyrhizobium japonicum, TnphoA mutagenesis of the microsymbiont was performed. Mutant strain 2-10 was found to induce a strongly reduced number of ineffective nodules. Ultrastructural analysis of the soybean nodule central tissue revealed the presence of numerous starch granules and vacuoles in the infected cells. In addition, the number of symbiosomes was extremely low, indicating an impaired interaction between the plant and invading bacteria. Cloning and sequencing of the mutated DNA region uncovered four open reading frames (ORFs) lacking any data base similarities. ORFs srrA1 and srrA2, the 2-10 TnphoA insertion site, are encoded in the same reading frame. A 35-kDa expression product in Escherichia coli indicated the presence of a common protein, called SrrA (symbiotically relevant region) in B. japonicum 110spc4, encoded by combined srrA1 and srrA2 genes. The analysis of gene disruption mutants revealed that srrB and srrC were also required for effective symbiosis with soybeans. Further downstream the gene for a putative inner membrane protein (pipA) of unknown function was encoded on the opposite strand. Primer extension studies led to the conclusion that the organization of genes differed from the RhizoBase annotation in this particular region of B. japonicum USDA110.
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36. |
Cantera JJ,
Kawasaki H,
Seki T,
( 2004 ) The nitrogen-fixing gene (nifH) of Rhodopseudomonas palustris: a case of lateral gene transfer? PMID : 15256566 : DOI : 10.1099/mic.0.26940-0 Abstract >>
Nitrogen fixation is catalysed by some photosynthetic bacteria. This paper presents a phylogenetic comparison of a nitrogen fixation gene (nifH) with the aim of elucidating the processes underlying the evolutionary history of Rhodopseudomonas palustris. In the NifH phylogeny, strains of Rps. palustris were placed in close association with Rhodobacter spp. and other phototrophic purple non-sulfur bacteria belonging to the alpha-Proteobacteria, separated from its close relatives Bradyrhizobium japonicum and the phototrophic rhizobia (Bradyrhizobium spp. IRBG 2, IRBG 228, IRBG 230 and BTAi 1) as deduced from the 16S rRNA phylogeny. The close association of the strains of Rps. palustris with those of Rhodobacter and Rhodovulum, as well as Rhodospirillum rubrum, was supported by the mol% G+C of their nifH gene and by the signature sequences found in the sequence alignment. In contrast, comparison of a number of informational and operational genes common to Rps. palustris CGA009, B. japonicum USDA 110 and Rhodobacter sphaeroides 2.4.1 suggested that the genome of Rps. palustris is more related to that of B. japonicum than to the Rba. sphaeroides genome. These results strongly suggest that the nifH of Rps. palustris is highly related to those of the phototrophic purple non-sulfur bacteria included in this study, and might have come from an ancestral gene common to these phototrophic species through lateral gene transfer. Although this finding complicates the use of nifH to infer the phylogenetic relationships among the phototrophic bacteria in molecular diversity studies, it establishes a framework to resolve the origins and diversification of nitrogen fixation among the phototrophic bacteria in the alpha-Proteobacteria.
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37. |
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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38. |
Dedysh SN,
Ricke P,
Liesack W,
( 2004 ) NifH and NifD phylogenies: an evolutionary basis for understanding nitrogen fixation capabilities of methanotrophic bacteria. PMID : 15133093 : DOI : 10.1099/mic.0.26585-0 Abstract >>
The ability to utilize dinitrogen as a nitrogen source is an important phenotypic trait in most currently known methanotrophic bacteria (MB). This trait is especially important for acidophilic MB, which inhabit acidic oligotrophic environments, highly depleted in available nitrogen compounds. Phylogenetically, acidophilic MB are most closely related to heterotrophic dinitrogen-fixing bacteria of the genus BEIJERINCKIA: To further explore the phylogenetic linkage between these metabolically different organisms, the sequences of nifH and nifD gene fragments from acidophilic MB of the genera Methylocella and Methylocapsa, and from representatives of Beijerinckia, were determined. For reference, nifH and nifD sequences were also obtained from some type II MB of the alphaproteobacterial Methylosinus/Methylocystis group and from gammaproteobacterial type I MB. The trees constructed for the inferred amino acid sequences of nifH and nifD were highly congruent. The phylogenetic relationships among MB in the NifH and NifD trees also agreed well with the corresponding 16S rRNA-based phylogeny, except for two distinctive features. First, different methods used for phylogenetic analysis grouped the NifH and NifD sequences of strains of the gammaproteobacterial MB Methylococcus capsulatus within a clade mainly characterized by Alphaproteobacteria, including acidophilic MB and type II MB of the Methylosinus/Methylocystis group. From this and other genomic data from Methylococcus capsulatus Bath, it is proposed that an ancient event of lateral gene transfer was responsible for this aberrant branching. Second, the identity values of NifH and NifD sequences between Methylocapsa acidiphila B2 and representatives of Beijerinckia were clearly higher (98.5 and 96.6 %, respectively) than would be expected from their 16S rRNA-based relationships. Possibly, these two bacteria originated from a common acidophilic dinitrogen-fixing ancestor, and were subject to similar evolutionary pressure with regard to nitrogen acquisition. This interpretation is corroborated by the observation that, in contrast to most other diazotrophs, M. acidiphila B2 and Beijerinckia spp. are capable of active growth on nitrogen-free media under fully aerobic conditions.
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39. |
Wiedemann G,
Müller P,
( 2004 ) Use of Tn KPK2 for sequencing a 10.6-kb PstI DNA fragment of Bradyrhizobium japonicum and for the construction of aspA and ndvA mutants. PMID : 15188087 : DOI : 10.1007/s00203-004-0673-5 Abstract >>
Transposon Tn KPK2 was used to saturate a randomly cloned Bradyrhizobium japonicum PstI fragment and the insertions were used as starting points for the sequence determination. The first gene of the 10.6-kb DNA insert encodes a homologue to ndvA, the product of which is known to be involved in the formation of periplasmic cyclic glucans. Selected Tn KPK2 insertions were introduced into the B. japonicum wild-type strain. The resulting mutants were subsequently tested for their symbiotic interactions with soybeans. As in Sinorhizobium meliloti, a B. japonicum ndvA mutant was affected in salt-stress tolerance and exhibited symbiotic defects in that it induced the formation of ineffective soybean nodules. The central nodule tissue was infected by bacteroids, but within the infected cells the mutant was not properly maintained. Another gene was found to be highly similar to bacterial aspartases and thus was named aspA. The putative function of the product of this gene was confirmed by genetic complementation of aspartase-less Escherichia coli strain TK237. The symbiotic phenotype of a B. japonicum aspA:Tn KPK2 mutant consisted of enlarged symbiosomes that made the system ineffective. In general, Tn KPK2 is a suitable means for fast sequencing. In combination with pJQ200SK, the resulting recombinant plasmids can be directly used to create genetically defined mutants.
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40. |
Benson HP,
LeVier K,
Guerinot ML,
( 2004 ) A dominant-negative fur mutation in Bradyrhizobium japonicum. PMID : 14973020 : DOI : 10.1128/jb.186.5.1409-1414.2004 PMC : PMC344408 Abstract >>
In many bacteria, the ferric uptake regulator (Fur) protein plays a central role in the regulation of iron uptake genes. Because iron figures prominently in the agriculturally important symbiosis between soybean and its nitrogen-fixing endosymbiont Bradyrhizobium japonicum, we wanted to assess the role of Fur in the interaction. We identified a fur mutant by selecting for manganese resistance. Manganese interacts with the Fur protein and represses iron uptake genes. In the presence of high levels of manganese, bacteria with a wild-type copy of the fur gene repress iron uptake systems and starve for iron, whereas fur mutants fail to repress iron uptake systems and survive. The B. japonicum fur mutant, as expected, fails to repress iron-regulated outer membrane proteins in the presence of iron. Unexpectedly, a wild-type copy of the fur gene cannot complement the fur mutant. Expression of the fur mutant allele in wild-type cells leads to a fur phenotype. Unlike a B. japonicum fur-null mutant, the strain carrying the dominant-negative fur mutation is unable to form functional, nitrogen-fixing nodules on soybean, mung bean, or cowpea, suggesting a role for a Fur-regulated protein or proteins in the symbiosis.
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41. |
Cobbe N,
Heck MM,
( 2004 ) The evolution of SMC proteins: phylogenetic analysis and structural implications. PMID : 14660695 : DOI : 10.1093/molbev/msh023 Abstract >>
The SMC proteins are found in nearly all living organisms examined, where they play crucial roles in mitotic chromosome dynamics, regulation of gene expression, and DNA repair. We have explored the phylogenetic relationships of SMC proteins from prokaryotes and eukaryotes, as well as their relationship to similar ABC ATPases, using maximum-likelihood analyses. We have also investigated the coevolution of different domains of eukaryotic SMC proteins and attempted to account for the evolutionary patterns we have observed in terms of available structural data. Based on our analyses, we propose that each of the six eukaryotic SMC subfamilies originated through a series of ancient gene duplication events, with the condensins evolving more rapidly than the cohesins. In addition, we show that the SMC5 and SMC6 subfamily members have evolved comparatively rapidly and suggest that these proteins may perform redundant functions in higher eukaryotes. Finally, we propose a possible structure for the SMC5/SMC6 heterodimer based on patterns of coevolution.
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42. |
King GM,
( 2003 ) Molecular and culture-based analyses of aerobic carbon monoxide oxidizer diversity. PMID : 14660374 : DOI : 10.1128/aem.69.12.7257-7265.2003 PMC : PMC309980 Abstract >>
Isolates belonging to six genera not previously known to oxidize CO were obtained from enrichments with aquatic and terrestrial plants. DNA from these and other isolates was used in PCR assays of the gene for the large subunit of carbon monoxide dehydrogenase (coxL). CoxL and putative coxL fragments were amplified from known CO oxidizers (e.g., Oligotropha carboxidovorans and Bradyrhizobium japonicum), from novel CO-oxidizing isolates (e.g., Aminobacter sp. strain COX, Burkholderia sp. strain LUP, Mesorhizobium sp. strain NMB1, Stappia strains M4 and M8, Stenotrophomonas sp. strain LUP, and Xanthobacter sp. strain COX), and from several well-known isolates for which the capacity to oxidize CO is reported here for the first time (e.g., Burkholderia fungorum LB400, Mesorhizobium loti, Stappia stellulata, and Stappia aggregata). PCR products from several taxa, e.g., O. carboxidovorans, B. japonicum, and B. fungorum, yielded sequences with a high degree (>99.6%) of identity to those in GenBank or genome databases. Aligned sequences formed two phylogenetically distinct groups. Group OMP contained sequences from previously known CO oxidizers, including O. carboxidovorans and Pseudomonas thermocarboxydovorans, plus a number of closely related sequences. Group BMS was dominated by putative coxL sequences from genera in the Rhizobiaceae and other alpha-PROTEOBACTERIA: PCR analyses revealed that many CO oxidizers contained two coxL sequences, one from each group. CO oxidation by M. loti, for which whole-genome sequencing has revealed a single BMS-group putative coxL gene, strongly supports the notion that BMS sequences represent functional CO dehydrogenase proteins that are related to but distinct from previously characterized aerobic CO dehydrogenases.
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43. |
Delgado MJ,
Bonnard N,
Tresierra-Ayala A,
Bedmar EJ,
Müller P,
( 2003 ) The Bradyrhizobium japonicum napEDABC genes encoding the periplasmic nitrate reductase are essential for nitrate respiration. PMID : 14663073 : DOI : 10.1099/mic.0.26620-0 Abstract >>
The napEDABC gene cluster that encodes the periplasmic nitrate reductase from Bradyrhizobium japonicum USDA110 has been isolated and characterized. napA encodes the catalytic subunit, and the napB and napC gene products are predicted to be a soluble dihaem c and a membrane-anchored tetrahaem c-type cytochrome, respectively. napE encodes a transmembrane protein of unknown function, and the napD gene product is a soluble protein which is assumed to play a role in the maturation of NapA. Western blots of the periplasmic fraction from wild-type cells grown anaerobically with nitrate revealed the presence of a protein band with a molecular size of about 90 kDa corresponding to NapA. A B. japonicum mutant carrying an insertion in the napA gene was unable to grow under nitrate-respiring conditions, lacked nitrate reductase activity, and did not show the 90 kDa protein band. Complementation of the mutant with a plasmid bearing the napEDABC genes restored both nitrate-dependent anaerobic growth of the cells and nitrate reductase activity. A membrane-bound and a periplasmic c-type cytochrome, with molecular masses of 25 kDa and 15 kDa, respectively, were not detected in the napA mutant strain incubated anaerobically with nitrate, which identifies those proteins as the NapC and the NapB components of the B. japonicum periplasmic nitrate reductase enzyme. These results suggest that the periplasmic nitrate reductase is the enzyme responsible for anaerobic growth of B. japonicum under nitrate-respiring conditions. The promoter region of the napEDABC genes has been characterized by primer extension. A major transcript initiates 66.5 bp downstream of the centre of a putative FNR-like binding site.
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44. |
Khamis A,
Colson P,
Raoult D,
Scola BL,
( 2003 ) Usefulness of rpoB gene sequencing for identification of Afipia and Bosea species, including a strategy for choosing discriminative partial sequences. PMID : 14602635 : DOI : 10.1128/aem.69.11.6740-6749.2003 PMC : PMC262318 Abstract >>
Bacteria belonging to the genera Afipia and Bosea are amoeba-resisting bacteria that have been recently reported to colonize hospital water supplies and are suspected of being responsible for intensive care unit-acquired pneumonia. Identification of these bacteria is now based on determination of the 16S ribosomal DNA sequence. However, the 16S rRNA gene is not polymorphic enough to ensure discrimination of species defined by DNA-DNA relatedness. The complete rpoB sequences of 20 strains were first determined by both PCR and genome walking methods. The percentage of homology between different species ranged from 83 to 97% and was in all cases lower than that observed with the 16S rRNA gene; this was true even for species that differed in only one position. The taxonomy of Bosea and Afipia is discussed in light of these results. For strain identification that does not require the complete rpoB sequence (4,113 to 4,137 bp), we propose a simple computerized method that allows determination of nucleotide positions of high variability in the sequence that are bordered by conserved sequences and that could be useful for design of universal primers. A fragment of 740 to 752 bp that contained the most highly variable area (positions 408 to 420) was amplified and sequenced with these universal primers for 47 strains. The variability of this sequence allowed identification of all strains and correlated well with results of DNA-DNA relatedness. In the future, this method could be also used for the determination of variability "hot spots" in sets of housekeeping genes, not only for identification purposes but also for increasing the discriminatory power of sequence typing techniques such as multilocus sequence typing.
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45. |
Gueneau de Novoa P,
Williams KP,
( 2004 ) The tmRNA website: reductive evolution of tmRNA in plastids and other endosymbionts. PMID : 14681369 : DOI : 10.1093/nar/gkh102 PMC : PMC308836 Abstract >>
tmRNA combines tRNA- and mRNA-like properties and ameliorates problems arising from stalled ribosomes. Research on the mechanism, structure and biology of tmRNA is served by the tmRNA website (http://www.indiana.edu/~ tmrna), a collection of sequences, alignments, secondary structures and other information. Because many of these sequences are not in GenBank, a BLAST server has been added; another new feature is an abbreviated alignment for the tRNA-like domain only. Many tmRNA sequences from plastids have been added, five found in public sequence data and another 10 generated by direct sequencing; detection in early-branching members of the green plastid lineage brings coverage to all three primary plastid lineages. The new sequences include the shortest known tmRNA sequence. While bacterial tmRNAs usually have a lone pseudoknot upstream of the mRNA segment and a string of three or four pseudoknots downstream, plastid tmRNAs collectively show loss of pseudoknots at both postions. The pseudoknot-string region is also too short to contain the usual pseudoknot number in another new entry, the tmRNA sequence from a bacterial endosymbiont of insect cells, Tremblaya princeps. Pseudoknots may optimize tmRNA function in free-living bacteria, yet become dispensible when the endosymbiotic lifestyle relaxes selective pressure for fast growth.
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46. |
Chang YL,
Wang ET,
Sui XH,
Zhang XX,
Chen WX,
( 2011 ) Molecular diversity and phylogeny of rhizobia associated with Lablab purpureus (Linn.) grown in Southern China. PMID : 21498018 : DOI : 10.1016/j.syapm.2010.12.004 Abstract >>
As an introduced plant, Lablab purpureus serves as a vegetable, herbal medicine, forage and green manure in China. In order to investigate the diversity of rhizobia associated with this plant, a total of 49 rhizobial strains isolated from ten provinces of Southern China were analyzed in the present study with restriction fragment length polymorphism and/or sequence analyses of housekeeping genes (16S rRNA, IGS, atpD, glnII and recA) and symbiotic genes (nifH and nodC). The results defined the L. purpureus rhizobia as 24 IGS-types within 15 rrs-IGS clusters or genomic species belonging to Bradyrhizobium, Rhizobium, Ensifer (synonym of Sinorhizobium) and Mesorhizobium. Bradyrhizobium spp. (81.6%) were the most abundant isolates, half of which were B. elkanii. Most of these rhizobia induced nodules on L. purpureus, but symbiotic genes were only amplified from the Bradyrhizobium and Rhizobium leguminosarum strains. The nodC and nifH phylogenetic trees defined five lineages corresponding to B. yuanmingense, B. japonicum, B. elkanii, B. jicamae and R. leguminosarum. The coherence of housekeeping and symbiotic gene phylogenies demonstrated that the symbiotic genes of the Lablab rhizobia were maintained mainly through vertical transfer. However, a putative lateral transfer of symbiotic genes was found in the B. liaoningense strain. The results in the present study clearly revealed that L. purpureus was a promiscuous host that formed nodules with diverse rhizobia, mainly Bradyrhizobium species, harboring different symbiotic genes.
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47. |
St?pkowski T,
Zak M,
Moulin L,
Króliczak J,
Goli?ska B,
Narożna D,
Safronova VI,
M?drzak CJ,
( 2011 ) Bradyrhizobium canariense and Bradyrhizobium japonicum are the two dominant rhizobium species in root nodules of lupin and serradella plants growing in Europe. PMID : 21514760 : DOI : 10.1016/j.syapm.2011.03.002 Abstract >>
Forty three Bradyrhizobium strains isolated in Poland from root nodules of lupin species (Lupinus albus, L. angustifolius and L. luteus), and pink serradella (Ornithopus sativus) were examined based on phylogenetic analyses of three housekeeping (atpD, glnII and recA) and nodulation (nodA) gene sequences. Additionally, seven strains originating from root-nodules of yellow serradella (O. compressus) from Asinara Island (Italy) were included in this study. Phylogenetic trees revealed that 15 serradella strains, including all yellow serradella isolates, and six lupin strains grouped in Bradyrhizobium canariense (BC) clade, whereas eight strains from pink serradella and 15 lupin strains were assigned to Bradyrhizobium japonicum (BJ1). Apparently, these species are the two dominant groups in soils of central Europe, in the nodules of lupin and serradella plants. Only three strains belonged to other chromosomal lineages: one formed a cluster that was sister to B. canariense, one strain grouped outside the branch formed by B. japonicum super-group, and one strain occupied a distant position in the genus Bradyrhizobium, clustering with strains of the Rhodopseudomonas genus. All strains in nodulation nodA gene tree grouped in a cluster referred to as Clade II, which is in line with earlier data on this clade dominance among Bradyrhizobium strains in Europe. The nodA tree revealed four well-supported subgroups within Clade II (II.1-II.4). Interestingly, all B. canariense strains clustered in subgroup II.1 whereas B. japonicum strains dominated subgroups II.2-II.4.
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48. |
Menna P,
Hungria M,
( 2011 ) Phylogeny of nodulation and nitrogen-fixation genes in Bradyrhizobium: supporting evidence for the theory of monophyletic origin, and spread and maintenance by both horizontal and vertical transfer. PMID : 21357454 : DOI : 10.1099/ijs.0.028803-0 Abstract >>
Bacteria belonging to the genus Bradyrhizobium are capable of establishing symbiotic relationships with a broad range of plants belonging to the three subfamilies of the family Leguminosae (=Fabaceae), with the formation of specialized structures on the roots called nodules, where fixation of atmospheric nitrogen takes place. Symbiosis is under the control of finely tuned expression of common and host-specific nodulation genes and also of genes related to the assembly and activity of the nitrogenase, which, in Bradyrhizobium strains investigated so far, are clustered in a symbiotic island. Information about the diversity of these genes is essential to improve our current poor understanding of their origin, spread and maintenance and, in this study, we provide information on 40 Bradyrhizobium strains, mostly of tropical origin. For the nodulation trait, common (nodA), Bradyrhizobium-specific (nodY/K) and host-specific (nodZ) nodulation genes were studied, whereas for fixation ability, the diversity of nifH was investigated. In general, clustering of strains in all nod and nifH trees was similar and the Bradyrhizobium group could be clearly separated from other rhizobial genera. However, the congruence of nod and nif genes with ribosomal and housekeeping genes was low. nodA and nodY/K were not detected in three strains by amplification or hybridization with probes using Bradyrhizobium japonicum and Bradyrhizobium elkanii type strains, indicating the high diversity of these genes or that strains other than photosynthetic Bradyrhizobium must have alternative mechanisms to initiate the process of nodulation. For a large group of strains, the high diversity of nod genes (with an emphasis on nodZ), the low relationship between nod genes and the host legume, and some evidence of horizontal gene transfer might indicate strategies to increase host range. On the other hand, in a group of five symbionts of Acacia mearnsii, the high congruence between nod and ribosomal/housekeeping genes, in addition to shorter nodY/K sequences and the absence of nodZ, highlights a co-evolution process. Additionally, in a group of B. japonicum strains that were symbionts of soybean, vertical transfer seemed to represent the main genetic event. In conclusion, clustering of nodA and nifH gives additional support to the theory of monophyletic origin of the symbiotic genes in Bradyrhizobium and, in addition to the analysis of nodY/K and nodZ, indicates spread and maintenance of nod and nif genes through both vertical and horizontal transmission, apparently with the dominance of one or other of these events in some groups of strains.
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49. |
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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50. |
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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51. |
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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52. |
Chahboune R,
Carro L,
Peix A,
Barrijal S,
Velázquez E,
Bedmar EJ,
( 2011 ) Bradyrhizobium cytisi sp. nov., isolated from effective nodules of Cytisus villosus. PMID : 21257682 : DOI : 10.1099/ijs.0.027649-0 Abstract >>
Several strains isolated from Cytisus villosus nodules have been characterized based on their diverse genetic, phenotypic and symbiotic characteristics. According to 16S rRNA gene sequence analysis, the isolates formed a group that was closely related to Bradyrhizobium canariense BTA-1(T) with 99.4% similarity. Analysis of three housekeeping genes, recA, atpD and glnII, suggested that the C. villosus strains represent a novel Bradyrhizobium species most closely related to B. canariense BTA-1(T) with similarities of 94.2, 96.7 and 94.5%, respectively. All these differences were congruent with DNA-DNA hybridization analysis, which revealed 31% relatedness between a representative strain (CTAW11(T)) isolated from C. villosus nodules and B. canariense BTA-1(T). Phenotypic differences among the strains isolated from C. villosus and B. canariense were based on assimilation of carbon and nitrogen sources. The nodC and nifH genes of strain CTAW11(T) were phylogenetically related to those of strains belonging to bv. genistearum and divergent from those of bv. glycinearum and, accordingly, they do not nodulate soybean. Based on the genotypic and phenotypic data obtained in this study, our strains should be classified as representatives of a novel species for which the name Bradyrhizobium cytisi sp. nov. is proposed; the type strain is CTAW11(T) (=LMG 25866(T)=CECT 7749(T)).
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53. |
Risal CP,
Yokoyama T,
Ohkama-Ohtsu N,
Djedidi S,
Sekimoto H,
( 2010 ) Genetic diversity of native soybean bradyrhizobia from different topographical regions along the southern slopes of the Himalayan Mountains in Nepal. PMID : 20851547 : DOI : 10.1016/j.syapm.2010.06.008 Abstract >>
Soybean-nodulating bradyrhizobia are genetically diverse and are classified into different species. In this study, the genetic diversity of native soybean bradyrhizobia isolated from different topographical regions along the southern slopes of the Himalayan Mountains in Nepal was explored. Soil samples were collected from three different topographical regions with contrasting climates. A local soybean cultivar, Cobb, was used as a trap plant to isolate bradyrhizobia. A total of 24 isolates selected on the basis of their colony morphology were genetically characterized. For each isolate, the full nucleotide sequence of the 16S rRNA gene and ITS region, and partial sequences of the nifD and nodD1 genes were determined. Two lineages were evident in the conserved gene phylogeny; one representing Bradyrhizobium elkanii (71% of isolates), and the other representing Bradyrhizobium japonicum (21%) and Bradyrhizobium yuanmingense (8%). Phylogenetic analyses revealed three novel lineages in the Bradyrhizobium elkanii clade, indicating high levels of genetic diversity among Bradyrhizobium isolates in Nepal. B. japonicum and B. yuanmingense strains were distributed in areas from 2420 to 2660 m above sea level (asl), which were mountain regions with a temperate climate. The B. elkanii clade was distributed in two regions; hill regions ranging from 1512 to 1935 m asl, and mountain regions ranging from 2420 to 2660 m asl. Ten multi-locus genotypes were detected; seven among B. elkanii, two among B. japonicum, and one among B. yuanmingense-related isolates. The results indicated that there was higher species-level diversity of Bradyrhizobium in the temperate region than in the sub-tropical region along the southern slopes of the Himalayan Mountains in Nepal.
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54. |
Velázquez E,
Valverde A,
Rivas R,
Gomis V,
Peix A,
Gantois I,
Igual JM,
León-Barrios M,
Willems A,
Mateos PF,
Martínez-Molina E,
( 2010 ) Strains nodulating Lupinus albus on different continents belong to several new chromosomal and symbiotic lineages within Bradyrhizobium. PMID : 20135225 : DOI : 10.1007/s10482-010-9415-7 Abstract >>
In this work we analysed different chromosomal and symbiotic markers in rhizobial strains nodulating Lupinus albus (white lupin) in several continents. Collectively the analysis of their rrs and atpD genes, and 16S-23S intergenic spacers (ITS), showed that they belong to at least four chromosomal lineages within the genus Bradyrhizobium. Most isolates from the Canary Islands (near to the African continent) grouped with some strains isolated on mainland Spain and were identified as Bradyrhizobium canariense. These strains are divided into two ITS subgroups coincident with those previously described from isolates nodulating Ornithopus. The remaining strains isolated on mainland Spain grouped with most isolates from Chile (American continent) forming a new lineage related to Bradyrhizobium japonicum. The strains BLUT2 and ISLU207 isolated from the Canary Islands and Chile, respectively, formed two new lineages phylogenetically close to different species of Bradyrhizobium depending on the marker analyzed. The analysis of the nodC gene showed that all strains nodulating L. albus belong to the biovar genistearum; nevertheless they form four different nodC lineages of which lineage C is at present exclusively formed by L. albus endosymbionts isolated from different continents.
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55. |
Menna P,
Barcellos FG,
Hungria M,
( 2009 ) Phylogeny and taxonomy of a diverse collection of Bradyrhizobium strains based on multilocus sequence analysis of the 16S rRNA gene, ITS region and glnII, recA, atpD and dnaK genes. PMID : 19628593 : DOI : 10.1099/ijs.0.009779-0 Abstract >>
The genus Bradyrhizobium encompasses a variety of bacteria that can live in symbiotic and endophytic associations with legumes and non-legumes, and are characterized by physiological and symbiotic versatility and broad geographical distribution. However, despite indications of great genetic variability within the genus, only eight species have been described, mainly because of the highly conserved nature of the 16S rRNA gene. In this study, 169 strains isolated from 43 different legumes were analysed by rep-PCR with the BOX primer, by sequence analysis of the 16S rRNA gene and the 16S-23S rRNA intergenic transcribed spacer (ITS) and by multilocus sequence analysis (MLSA) of four housekeeping genes, glnII, recA, atpD and dnaK. Considering a cut-off at a level of 70 % similarity, 80 rep-PCR profiles were distinguished, which, together with type strains, were clustered at a very low level of similarity (24 %). In both single and concatenated analyses of the 16S rRNA gene and ITS sequences, two large groups were formed, with bootstrap support of 99 % in the concatenated analysis. The first group included the type and/or reference strains of Bradyrhizobium japonicum, B. betae, B. liaoningense, B. canariense and B. yuanmingense and B. japonicum USDA 110, and the second group included strains related to Bradyrhizobium elkanii USDA 76(T), B. pachyrhizi PAC48(T) and B. jicamae PAC68(T). Similar results were obtained with MLSA of glnII, recA, atpD and dnaK. Greatest variability was observed when the atpD gene was amplified, and five strains related to B. elkanii revealed a level of variability never reported before. Another important observation was that a group composed of strains USDA 110, SEMIA 5080 and SEMIA 6059, all isolated from soybean, clustered in all six trees with high bootstrap support and were quite distinct from the clusters that included B. japonicum USDA 6(T). The results confirm that MLSA is a rapid and reliable way of providing information on phylogenetic relationships and of identifying rhizobial strains potentially representative of novel species.
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56. |
Anthamatten D,
Hennecke H,
( 1991 ) The regulatory status of the fixL- and fixJ-like genes in Bradyrhizobium japonicum may be different from that in Rhizobium meliloti. PMID : 2000090 : DOI : 10.1007/bf00282640 Abstract >>
The cloning, sequencing and mutational analysis of the Bradyrhizobium japonicum symbiotic nitrogen fixation genes fixL and fixJ are reported here. The two genes were adjacent and probably formed an operon, fixLJ. The predicted FixL and FixJ proteins, members of the two-component sensor/regulator family, were homologous over almost their entire lengths to the corresponding Rhizobium meliloti proteins (approx. 50% identity). Downstream of the B. japonicum fixJ gene was found an open reading frame with 138 codons (ORF138) whose product shared 36% homology with the N-terminal part of FixJ. Deletion and insertion mutations within fixL and fixJ led to a loss of approximately 90% wild-type symbiotic nitrogen fixation (Fix) activity, whereas an ORF138 mutant was Fix+. In fixL, fixJ and ORF138 mutant backgrounds, the aerobic expression of the fixR-nifA operon was not affected. NifA itself did not regulate the expression of the fixJ gene. Thus, the B. japonicum FixL and FixJ proteins were neither involved in the regulation of aerobic nifA gene expression nor in the anaerobic NifA-dependent autoregulation of the fixRnifA operon, rather they appeared to control symbiotically important genes other than those whose expression was dependent on the NifA protein. The fixL and fixJ mutant strains were unable to grow anaerobically with nitrate as the terminal electron acceptor. Therefore, some of the FixJ-dependent genes in B. japonicum may be concerned with anaerobic respiration.
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57. |
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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58. |
Bott M,
Bolliger M,
Hennecke H,
( 1990 ) Genetic analysis of the cytochrome c-aa3 branch of the Bradyrhizobium japonicum respiratory chain. PMID : 1965217 : DOI : 10.1111/j.1365-2958.1990.tb00576.x Abstract >>
Further genetic evidence is provided here that Bradyrhizobium japonicum possesses a mitochondria-like electron-transport pathway: 2[H]----UQ----bc1----c----aa3----O2. Two Tn5-induced mutants, COX122 and COX132, having cytochrome c oxidase-negative phenotypes, were obtained and characterized. Mutant COX122 was defective in a novel gene, named cycM, which was responsible for the synthesis of a c-type cytochrome with an Mr of 20,000 (20K). This 20K cytochrome c appeared to catalyse electron transport from the cytochrome bc1 complex to the aa3-type terminal oxidase and, unlike mitochondrial cytochrome c, was membrane-bound in B. japonicum. The Tn5 insertion of mutant COX132 was localized in coxA, the structural gene for subunit I of cytochrome aa3. This finding also led to the cloning and sequencing of the corresponding wild-type coxA gene that encoded a 541-amino-acid protein with a predicted Mr of 59,247. The CoxA protein shared about 60% sequence identity with the cytochrome aa3 subunit I of mitochondria. The B. japonicum cycM and coxA mutants were able to fix nitrogen in symbiosis with soybean (Fix+). In contrast, mutants described previously which lacked the bc1 complex did not develop into endosymbiotic bacteroids and were thus Fix-. The data suggest that a symbiosis-specific respiratory chain exists in B. japonicum in which the electrons branch off at the bc1 complex.
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59. |
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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60. |
Vinuesa P,
Rojas-Jiménez K,
Contreras-Moreira B,
Mahna SK,
Prasad BN,
Moe H,
Selvaraju SB,
Thierfelder H,
Werner D,
( 2008 ) Multilocus sequence analysis for assessment of the biogeography and evolutionary genetics of four Bradyrhizobium species that nodulate soybeans on the asiatic continent. PMID : 18791003 : DOI : 10.1128/AEM.00875-08 PMC : PMC2583495 Abstract >>
A highly supported maximum-likelihood species phylogeny for the genus Bradyrhizobium was inferred from a supermatrix obtained from the concatenation of partial atpD, recA, glnII, and rpoB sequences corresponding to 33 reference strains and 76 bradyrhizobia isolated from the nodules of Glycine max (soybean) trap plants inoculated with soil samples from Myanmar, India, Nepal, and Vietnam. The power of the multigene approach using multiple strains per species was evaluated in terms of overall tree resolution and phylogenetic congruence, representing a practical and portable option for bacterial molecular systematics. Potential pitfalls of the approach are highlighted. Seventy-five of the isolates could be classified as B. japonicum type Ia (USDA110/USDA122-like), B. liaoningense, B. yuanmingense, or B. elkanii, whereas one represented a novel Bradyrhizobium lineage. Most Nepalese B. japonicum Ia isolates belong to a highly epidemic clone closely related to strain USDA110. Significant phylogenetic evidence against the monophyly of the of B. japonicum I and Ia lineages was found. Analysis of their DNA polymorphisms revealed high population distances, significant genetic differentiation, and contrasting population genetic structures, suggesting that the strains in the Ia lineage are misclassified as B. japonicum. The DNA polymorphism patterns of all species conformed to the expectations of the neutral mutation and population equilibrium models and, excluding the B. japonicum Ia lineage, were consistent with intermediate recombination levels. All species displayed epidemic clones and had broad geographic and environmental distribution ranges, as revealed by mapping climate types and geographic origins of the isolates on the species tree.
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61. |
Yokoyama T,
( 2008 ) Flavonoid-responsive nodY-lacZ expression in three phylogenetically different Bradyrhizobium groups. PMID : 18449225 : DOI : 10.1139/w08-021 Abstract >>
Previously, restriction fragment length polymorphism analysis using the nodD1YABC gene probe showed the genetic diversity of common nodD1ABC gene regions of Bradyrhizobium japonicum, Bradyrhizobium elkanii, and the Thai soybean Bradyrhizobium. The nodD1 sequences of representative strains of the 3 groups differed phylogenetically, suggesting that responses of NodD1 proteins of the 3 Bradyrhizobium groups to diverse flavonoids may differ. To confirm this hypothesis, 6 representative strains were chosen from the 3 Bradyrhizobium groups. Six reporter strains were constructed, all carrying the pZB32 plasmid, which contains a nod box and the nodY-lacZ fusion of B. japonicum USDA 110. Differences in nodY-lacZ expression among the strains in response to 37 flavonoid compounds at various concentrations were evaluated. Of those compounds, prunetin (4',5-dihydroxy-7-methoxyisoflavone) and esculetin (6,7-dihydroxycoumarin) were identified as Bradyrhizobium group-specific nod gene inducers. Esculetin showed nod gene induction activities unique to Thai Bradyrhizobium strains. The levels of nodY-lacZ induction among B. japonicum and Thai Bradyrhizobium strains increased with increasing concentration of prunetin, whereas, those in B. elkanii strains did not.
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62. |
Appunu C,
N'Zoue A,
Laguerre G,
( 2008 ) Genetic diversity of native bradyrhizobia isolated from soybeans (Glycine max L.) in different agricultural-ecological-climatic regions of India. PMID : 18676699 : DOI : 10.1128/AEM.01320-08 PMC : PMC2565974 Abstract >>
Fifty isolates from root nodules of soybean plants sampled in five agricultural-ecological-climatic regions of India were analyzed by PCR-restriction fragment length polymorphism analysis of the 16S rRNA gene, the intergenic spacer region between the 16S and 23S rRNA genes (IGS), and the nifH and nodC genes. Eight haplotypes assigned to the Bradyrhizobium genus were identified, and the genetic diversity was conserved across regions. Sequence analyses of the IGS and the dnaK, glnII, recA, and nifH genes revealed three groups. One of them (26% of isolates) was assigned to Bradyrhizobium liaoningense. A second group (36% of isolates) was identified as B. yuanmingense but likely forms a new biovar able to nodulate soybean plants. The third lineage (38% of isolates) was different from all described Bradyrhizobium species but showed the same symbiotic genotype as B. liaoningense and B. japonicum bv. glycinearum.
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63. |
Chang WS,
Park KM,
Koh SC,
So JS,
( 2008 ) Characterization of the Bradyrhizobium japonicum galE gene: its impact on lipopolysaccharide profile and nodulation of soybean. PMID : 18266738 : DOI : 10.1111/j.1574-6968.2008.01066.x Abstract >>
The galE gene from Bradyrhizobium japonicum 61A101C, a soybean endosymbiont, was cloned and characterized. Its deduced amino-acid sequence showed a high similarity with that of other rhizobia. Functional identification of the galE gene was achieved by complementation of a galE mutant strain, PL2, with a series of pKM subclones. Disruption of the B. japonicum galE gene affects the lipopolysaccharide profile compared with that of the wild type, suggesting that galE is responsible for alteration of lipopolysaccharide structure. Examination of nodule formation by the wild-type and galE mutant revealed that the former displayed normal nodule development on soybean roots, whereas the latter showed no nodule formation at all time points examined except for 20 days after inoculation when <10% of soybean formed pseudo-nodules.
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64. |
Appelbaum ER,
Thompson DV,
Idler K,
Chartrain N,
( 1988 ) Rhizobium japonicum USDA 191 has two nodD genes that differ in primary structure and function. PMID : 2826389 : DOI : 10.1128/jb.170.1.12-20.1988 PMC : PMC210599 Abstract >>
Several Rhizobium genes (designated nod genes) are involved in early steps in nodule formation. Here we present the results of DNA sequence and functional analysis of two nodD genes from the symbiotic plasmid of USDA 191, a fast-growing strain that forms nitrogen-fixing nodules on soybeans. Both genes encoded full-length nodD-related polypeptides, which were 69% homologous to each other. One of these genes, nodD1, complemented a Rhizobium trifolii nodD::Tn5 mutant for clover nodulation; the other gene, nodD2, did not. The nodD1 coding region was preceded by a conserved DNA sequence previously noted in other rhizobia, but no such sequence was found in front of nodD2. Plants inoculated with a nodD1 insertion mutant appeared to be nitrogen starved and had a greatly reduced nodule number. Plants inoculated with a nodD2 mutant had a partially nitrogen-starved appearance and normal nodule number, were slightly delayed in nodule formation, and formed nodules that contained reduced levels of nodulin-35 and had fewer bacteroids per infected plant cell. Thus, both of these genes are involved in symbiosis. USDA 191 carrying extra copies of nodD2 on a plasmid vector had an altered colony morphology that suggested inhibition of exopolysaccharide synthesis. The predicted gene products of nodD1 and nodD2 both showed homology to LysR, an E. coli regulatory protein. We conclude that nodD1 probably has the same function as nodD in temperate rhizobia, namely, activation of nodABC transcription in the presence of plant signals. nodD2 may be involved in regulation of exopolysaccharide synthetic genes.
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65. |
Yan J,
Chen W,
Han X,
Wang E,
Zou W,
Zhang Z,
( 2017 ) Genetic diversity of indigenous soybean-nodulating rhizobia in response to locally-based long term fertilization in a Mollisol of Northeast China. PMID : 27848139 : DOI : 10.1007/s11274-016-2170-9 Abstract >>
The influences of five different fertilizer treatments on diversity of rhizobia in soybean nodule were investigated in a long-term experiment with with four replicates: (1) control (without fertilization), (2) balanced NPK fertilizer (NPK), and (3-5) unbalanced chemical fertilizers without one of the major elements (NP, PK, and NK) in Mollisol in Northeast China. The highest soybean yield was observed in the NPK treatment. Total of 200 isolates were isolated and grouped into four Bradyrhizobium genospecies corresponding to B. japonicum, B. diazoefficiens, B. ottawaense and Bradyrhizobium sp. I, based upon the multilocus sequence analysis of 6 housekeeping genes. The Bradyrhizobium sp. I was extensively distributed throughout the study site and was recorded as the dominant soybean rhizobia (82.5-87.5%). Except the NK treatment, the other fertilizer treatments had no effect on rhizobial species composition. Compared with the CK treatment, all the fertilizer treatments decreased species richness, diversity and evenness. The soil organic carbon contents, available N content and pH were the key soil factors to rhizobial community structure. Results suggest that long-term fertilization can decrease rhizobial species diversity, while balanced fertilization with NPK is the most suitable fertilization regime if taking both soybean yields and rhizobial diversity into account.
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66. |
Sekine M,
Watanabe K,
Syono K,
( 1989 ) Nucleotide sequence of a gene for indole-3-acetamide hydrolase from Bradyrhizobium japonicum. PMID : 2771653 : DOI : 10.1093/nar/17.15.6400 PMC : PMC318300 Abstract >>
N/A
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67. |
Noh JG,
Jeon HE,
So JS,
Chang WS,
( 2015 ) Effects of the Bradyrhizobium japonicum waaL (rfaL) Gene on Hydrophobicity, Motility, Stress Tolerance, and Symbiotic Relationship with Soybeans. PMID : 26213919 : DOI : 10.3390/ijms160816778 PMC : PMC4581169 Abstract >>
We cloned and sequenced the waaL (rfaL) gene from Bradyrhizobium japonicum, which infects soybean and forms nitrogen-fixing nodules on soybean roots. waaL has been extensively studied in the lipopolysaccharide (LPS) biosynthesis of enteric bacteria, but little is known about its function in (brady)rhizobial LPS architecture. To characterize its role as O-antigen ligase in the LPS biosynthesis pathway, we constructed a waaL knock-out mutant and its complemented strain named JS015 and CS015, respectively. LPS analysis showed that an LPS structure of JS015 is deficient in O-antigen as compared to that of the wild type and complemented strain CS015, suggesting that WaaL ligates the O-antigen to lipid A-core oligosaccharide to form a complete LPS. JS015 also revealed increased cell surface hydrophobicity, but it showed decreased motility in soft agar plates. In addition to the alteration in cell surface properties, disruption of the waaL gene caused increased sensitivity of JS015 to hydrogen peroxide, osmotic pressure, and novobiocin. Specifically, plant tests revealed that JS015 failed to nodulate the host plant soybean, indicating that the rhizobial waaL gene is responsible for the establishment of a symbiotic relationship between soybean and B. japonicum.
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68. |
Gubler M,
Zürcher T,
Hennecke H,
( 1989 ) The Bradyrhizobium japonicum fixBCX operon: identification of fixX and of a 5' mRNA region affecting the level of the fixBCX transcript. PMID : 2503674 : DOI : 10.1111/j.1365-2958.1989.tb01803.x Abstract >>
The Bradyrhizobium japonicum fixX gene was identified and shown to be essential for symbiotic and free-living, microaerobic nitrogen fixation. The fixX gene encodes a ferredoxin-like protein which may be involved in a redox process (electron transport?) essential for nitrogenase activity. This gene was localized downstream of fixC and its expression was dependent on the fixB promoter, providing evidence for the existence of a fixBCX operon. Mutagenesis and sequence analysis of the unusually long, 709bp leader region between the fixB promoter and the fixB structural gene did not reveal the presence of a nif or fix gene that was absolutely essential for nitrogen fixation. However, a short open reading frame (ORF) within this region encoding a polypeptide of 35 amino acids (ORF35) was shown to be efficiently translated. Chromosomal deletion of a 400bp DNA fragment covering ORF35 resulted in a three-fold reduction of the fixBCX mRNA level, which in turn also reduced the nitrogen fixation activity of this mutant. This suggests a possible post-transcriptional control mechanism for the expression of the fixBCX operon involving the stabilization of fixBCX mRNA by ribosomes actively translating ORF35.
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69. |
Yan J,
Han XZ,
Ji ZJ,
Li Y,
Wang ET,
Xie ZH,
Chen WF,
( 2014 ) Abundance and diversity of soybean-nodulating rhizobia in black soil are impacted by land use and crop management. PMID : 24951780 : DOI : 10.1128/AEM.01135-14 PMC : PMC4136101 Abstract >>
To investigate the effects of land use and crop management on soybean rhizobial communities, 280 nodule isolates were trapped from 7 fields with different land use and culture histories. Besides the known Bradyrhizobium japonicum, three novel genospecies were isolated from these fields. Grassland (GL) maintained a higher diversity of soybean bradyrhizobia than the other cultivation systems. Two genospecies (Bradyrhizobium spp. I and III) were distributed widely in all treatments, while Bradyrhizobium sp. II was found only in GL treatment. Cultivation with soybeans increased the rhizobial abundance and diversity, except for the soybean monoculture (S-S) treatment. In monoculture systems, soybeans favored Bradyrhizobium sp. I, while maize and wheat favored Bradyrhizobium sp. III. Fertilization decreased the rhizobial diversity indexes but did not change the species composition. The organic carbon (OC) and available phosphorus (AP) contents and pH were the main soil parameters positively correlated with the distribution of Bradyrhizobium spp. I and II and Bradyrhizobium japonicum and negatively correlated with Bradyrhizobium sp. III. These results revealed that different land uses and crop management could not only alter the diversity and abundance of soybean rhizobia, but also change interactions between rhizobia and legume or nonlegume plants, which offered novel information about the biogeography of rhizobia.
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70. |
Wang JY,
Wang R,
Zhang YM,
Liu HC,
Chen WF,
Wang ET,
Sui XH,
Chen WX,
( 2013 ) Bradyrhizobium daqingense sp. nov., isolated from soybean nodules. PMID : 22544787 : DOI : 10.1099/ijs.0.034280-0 Abstract >>
Thirteen slow-growing rhizobial strains isolated from root nodules of soybean (Glycine max L.) grown in Daqing city in China were classified in the genus Bradyrhizobium based on 16S rRNA gene sequence analysis. Multilocus sequence analysis of IGS, atpD, glnII and recA genes revealed that the isolates represented a novel clade in this genus. DNA-DNA relatedness lower than 42.5 % between the representative strain CCBAU 15774(T) and the type strains of the closely related species Bradyrhizobium liaoningense USDA 3622(T), Bradyrhizobium yuanmingense CCBAU 10071(T) and Bradyrhizobium betae LMG 21987(T), further confirmed that this group represented a novel species. CCBAU 15774(T) shared seven cellular fatty acids with the three above-mentioned species, but the fatty acids 15 : 0 iso and summed feature 5 (18 : 2�s6,9c and/or 18 : 0 anteiso) were unique for this strain. The respiratory quinone in CCBAU 15774(T) was ubiquinone-10 and the cellular polar lipids were phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, cardiolipin and unknown aminolipid, polar lipid and phospholipid. In addition, some phenotypic features could be used to differentiate the novel group from the related species. On basis of these results, we propose the name Bradyrhizobium daqingense sp. nov., with CCBAU 15774(T) (= LMG 26137(T) = HAMBI 3184(T) = CGMCC 1.10947(T)) as the type strain. The DNA G+C content of the type strain is 61.2 mol% (T(m)).
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71. |
Mishra RP,
Tisseyre P,
Melkonian R,
Chaintreuil C,
Miché L,
Klonowska A,
Gonzalez S,
Bena G,
Laguerre G,
Moulin L,
( 2012 ) Genetic diversity of Mimosa pudica rhizobial symbionts in soils of French Guiana: investigating the origin and diversity of Burkholderia phymatum and other beta-rhizobia. PMID : 22093060 : DOI : 10.1111/j.1574-6941.2011.01235.x Abstract >>
The genetic diversity of 221 Mimosa pudica bacterial symbionts trapped from eight soils from diverse environments in French Guiana was assessed by 16S rRNA PCR-RFLP, REP-PCR fingerprints, as well as by phylogenies of their 16S rRNA and recA housekeeping genes, and by their nifH, nodA and nodC symbiotic genes. Interestingly, we found a large diversity of beta-rhizobia, with Burkholderia phymatum and Burkholderia tuberum being the most frequent and diverse symbiotic species. Other species were also found, such as Burkholderia mimosarum, an unnamed Burkholderia species and, for the first time in South America, Cupriavidus taiwanensis. The sampling site had a strong influence on the diversity of the symbionts sampled, and the specific distributions of symbiotic populations between the soils were related to soil composition in some cases. Some alpha-rhizobial strains taxonomically close to Rhizobium endophyticum were also trapped in one soil, and these carried two copies of the nodA gene, a feature not previously reported. Phylogenies of nodA, nodC and nifH genes showed a monophyly of symbiotic genes for beta-rhizobia isolated from Mimosa spp., indicative of a long history of interaction between beta-rhizobia and Mimosa species. Based on their symbiotic gene phylogenies and legume hosts, B. tuberum was shown to contain two large biovars: one specific to the mimosoid genus Mimosa and one to South African papilionoid legumes.
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72. |
van Berkum P,
Elia P,
Song Q,
Eardly BD,
( 2012 ) Development and application of a multilocus sequence analysis method for the identification of genotypes within genus Bradyrhizobium and for establishing nodule occupancy of soybean (Glycine max L. Merr). PMID : 22074348 : DOI : 10.1094/MPMI-09-11-0241 Abstract >>
A multilocus sequence typing (MLST) method based on allelic variation of seven chromosomal loci was developed for characterizing genotypes (GT) within the genus Bradyrhizobium. With the method, 29 distinct multilocus GT were identified among 190 culture collection soybean strains. The occupancy of 347 nodules taken from uninoculated field-grown soybean plants also was determined. The bacteroid GT were either the same as or were closely related to GT identified among strains in the culture collection. Double-nodule occupancy estimates of 2.9% were much lower than values published based on serology. Of the 347 nodules examined, 337 and 10 were occupied by Bradyrhizobium japonicum and B. elkanii, respectively. The collection strains within the species B. japonicum and B. elkaniialso were compared with Bradyrhizobium cultures from other legumes. In many cases, the observed GT varied more according to their geographic origin than by their trap hosts of isolation. In other cases, there were no apparent relationships with either the legume or geographic source. The MLST method that was developed should be a useful tool in determining the influence of geographic location, temperature, season, soil type, and host plant cultivar on the distribution of GT of Bradyrhizobium spp.
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73. |
Lorite MJ,
Videira e Castro I,
Muñoz S,
Sanjuán J,
( 2012 ) Phylogenetic relationship of Lotus uliginosus symbionts with bradyrhizobia nodulating genistoid legumes. PMID : 22092879 : DOI : 10.1111/j.1574-6941.2011.01230.x Abstract >>
Lotus species are legumes with potential for pastures in soils with low-fertility and environmental constraints. The aim of this work was to characterize bacteria that establish efficient nitrogen-fixing symbiosis with the forage species Lotus uliginosus. A total of 39 isolates were obtained from nodules of L. uliginosus naturally growing in two different locations of Portugal. Molecular identification of the isolates plus the commercial inoculant strain NZP2039 was performed by REP-PCR, 16S rRNA RFLP, and 16S rRNA, glnII and recA sequence analyses. Limited genetic diversity was found among the L. uliginosus symbionts, which showed a close phylogenetic relationship with the species Bradyrhizobium japonicum. The symbiotic nifH, nodA and nodC gene sequences were closely related with the corresponding genes of various Bradyrhizobium strains isolated from Lupinus and other genistoid legumes and therefore were phylogenetically separated from other Lotus spp. rhizobia. The L. uliginosus bradyrhizobia were able to nodulate and fix nitrogen in association with L. uliginosus, could nodulate Lotus corniculatus with generally poor nitrogen-fixing efficiency, formed nonfixing nodules in Lotus tenuis and Lupinus luteus roots and were unable to nodulate Glycine soja or Glycine max. Thus, L. uliginosus rhizobia seem closely related to B. japonicum biovar genistearum strains.
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74. |
Ormeño-Orrillo E,
Rogel-Hernández MA,
Lloret L,
López-López A,
Martínez J,
Barois I,
Martínez-Romero E,
( 2012 ) Change in land use alters the diversity and composition of Bradyrhizobium communities and led to the introduction of Rhizobium etli into the tropical rain forest of Los Tuxtlas (Mexico). PMID : 22109095 : DOI : 10.1007/s00248-011-9974-9 Abstract >>
Nitrogen-fixing bacteria of the Bradyrhizobium genus are major symbionts of legume plants in American tropical forests, but little is known about the effects of deforestation and change in land use on their diversity and community structure. Forest clearing is followed by cropping of bean (Phaseolus vulgaris) and maize as intercropped plants in Los Tuxtlas tropical forest of Mexico. The identity of bean-nodulating rhizobia in this area is not known. Using promiscuous trap plants, bradyrhizobia were isolated from soil samples collected in Los Tuxtlas undisturbed forest, and in areas where forest was cleared and land was used as crop fields or as pastures, or where secondary forests were established. Rhizobia were also trapped by using bean plants. Bradyrhizobium strains were classified into genospecies by dnaK sequence analysis supported by recA, glnII and 16S-23S rDNA IGS loci analyses. A total of 29 genospecies were identified, 24 of which did not correspond to any described taxa. A reduction in Bradyrhizobium diversity was observed when forest was turned to crop fields or pastures. Diversity seemed to recover to primary forest levels in secondary forests that derived from abandoned crop fields or pastures. The shifts in diversity were not related to soil characteristics but seemingly to the density of nodulating legumes present at each land use system (LUS). Bradyrhizobium community composition in soils was dependent on land use; however, similarities were observed between crop fields and pastures but not among forest and secondary forest. Most Bradyrhizobium genospecies present in forest were not recovered or become rare in the other LUS. Rhizobium etli was found as the dominant bean-nodulating rhizobia present in crop fields and pastures, and evidence was found that this species was introduced in Los Tuxtlas forest.
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75. |
( 1997 ) Molecular studies on a new genetic locus linked to the common nodulation genes in Bradyrhizobium japonicum. PMID : 9084141 : DOI : 10.1111/j.1574-6968.1997.tb10280.x Abstract >>
ORFA, an actively transcribed genetic locus linked to the common nodulation genes in Bradyrhizobium japonicum USDA110, was sequenced and analysed. The expression of ORFA is neither dependent on the regulatory proteins NifA, NtrC, NtrB and NodD1 nor on either copy of sigma 54, RpoN1 and RpoN2. The transcriptional start site of ORFA was determined and found to overlap the oppositely transcribed nodZ gene by 224 nucleotides. An appropriately located -10 sequence identical to the consensus proposed for rhizobia and a homologous -35 region were identified upstream of the transcriptional start site. ORFA showed no significant homologies to known sequences in gene databases, and its mutational inactivation had no effect on the nodulation of five legume species. Nevertheless, ORFA seems to be conserved among bradyrhizobia, since an ORFA probe hybridised to total DNA extracted from other Bradyrhizobium strains.
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76. |
( 1997 ) Three disparately regulated genes for sigma 32-like transcription factors in Bradyrhizobium japonicum. PMID : 9140968 : DOI : 10.1046/j.1365-2958.1997.3141685.x Abstract >>
Bradyrhizobium japonicum possesses a subclass of heat-shock genes whose members are transcribed from a sigma 32 consensus promoter. Having identified previously one gene (rpoH1) encoding a sigma 32-like RNA polymerase transcription factor, we report here the characterization of two additional rpoH-like genes (rpoH2 and rpoH3). B. japonicum thus represents the first example of an organism possessing an rpoH multigene family. All three rpoH genes encode functional proteins that are able to initiate transcription from the Escherichia coli groE promoter. Each rpoH gene is apparently regulated by a different mechanism. Although both rpoH1 and rpoH2 are transcribed from sigma 70-type promoters, transcription of the rpoH1 operon was found to be heat inducible by an unknown mechanism, whereas the level of rpoH2 mRNA decreased after heat shock. At extreme temperatures (48 degrees C), rpoH2 was transcribed from a second promoter that resembled the E. coli sigma E-type promoter. The rpoH3 gene was found to be associated with two upstream genes, ragA and ragB, coding for a classical two-component regulatory system. Transcription initiated from a promoter that mapped in front of the putative response regulator gene ragA, suggesting that ragA, ragB and rpoH3 are organized in an operon. The ragA promoter was similar to a sigma 32 consensus promoter. The three B. japonicum rpoH genes also varied in their significance to support growth of the organism. While the rpoH2 gene could not be eliminated by mutation, knock-out mutants of rpoH1 and/ or rpoH3 were readily obtained and shown to be indistinguishable from the wild type under aerobic growth conditions or during root-nodule symbiosis. We conclude that rpoH2 is essential for the synthesis of cellular proteins under physiological growth conditions, whereas rpoH1, and probably also rpoH3, are involved in their synthesis during the stress response.
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77. |
( 1997 ) The dnaKJ operon belongs to the sigma32-dependent class of heat shock genes in Bradyrhizobium japonicum. PMID : 9108282 : DOI : 10.1007/s004380050408 Abstract >>
The dnaKJ genes of Bradyrhizobium japonicum were cloned and sequenced. They map adjacent to each other, as in other proteobacteria of the alpha and gamma subgroups. Primer extension experiments identified two strongly heat-inducible transcripts starting 99 bp (T1) and 204 bp (T2) upstream of dnaK. Synthesis of the shorter transcript T1 in Escherichia coli required the presence of a recently characterized sigma32 homologue (RpoH1) from B. japonicum. The -35 and -10 regions of the promoters associated with the transcription start sites T1 and T2 displayed nucleotide sequence motifs that are characteristic for sigma32-dependent promoters in E. coli and alpha-proteobacteria. Heat shock regulation of dnaK expression was confirmed by immunoblot analysis of DnaK protein. All of these results put dnaK into the sigma32-dependent class, not the CIRCE-dependent class, of heat shock genes in B. japonicum. At normal growth temperature dnaK was expressed at a significant basal level. All attempts to eliminate dnaK function by insertion or deletion mutagenesis failed. By contrast, dnaJ null mutants and insertions in the dnaKJ intergenic region were easily obtained. The growth rate of dnaJ mutants was reduced but the final cell density reached in rich medium and their symbiotic properties were indistinguishable from the wild type.
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78. |
( 1997 ) The sequences of hypF, hypC and hypD complete the hyp gene cluster required for hydrogenase activity in Bradyrhizobium japonicum. PMID : 9358044 : DOI : 10.1016/s0378-1119(97)00352-1 Abstract >>
A region of DNA 6 kb downstream of the hydrogenase (H2ase) structural genes and directly downstream of the hypB gene of Bradyrhizobium japonicum was shown by mutational analysis to be necessary for H2ase synthesis. Sequencing of this region revealed two complete open reading frames, and the 5' fragment of a third ORF. They encode proteins with homologies to the HypF, HypC and the N-terminus of HypD from other H2ase-containing organisms. The hypF of B. japonicum encodes a 753-aa protein with a predicted molecular mass of 80.3 kDa that contains the two zinc-finger motifs characteristic of other HypF proteins. The hypC encodes a 85-aa protein with a predicted molecular mass of 8.4 kDa. The 5' portion of hypD, which encodes the first 35 aa, upon combining with the previously reported C-terminus of HypD, designated HypD' (Van Soom et al. (1993) Mol. Gen. Genet. 239, 235-240) encodes a protein with a predicted molecular mass of 42.4 kDa. Complementation studies on a H2 uptake defective strain of B. japonicum containing a polar mutation in the hyp operon revealed that the products of the hyp F, C, D, E genes are required for H2ase production. Evidence is also presented that the hyp genes are co-transcribed from a large operon together with the downstream genes hupGHIJK, making a polycistronic message of 11 genes.
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79. |
( 1997 ) Squalene-hopene cyclase from Bradyrhizobium japonicum: cloning, expression, sequence analysis and comparison to other triterpenoid cyclases. PMID : 9141686 : DOI : 10.1099/00221287-143-4-1235 Abstract >>
With the help of a PCR-based screening method, the gene encoding squalenehopene cyclase (SHC) of Bradyrhizobium japonicum USDA 110 was isolated from a cosmid library. The SHC catalyses the cyclization of squalene to hopanoids, a class of triterpenoid lipids recently discovered in nitrogen-fixing, root-nodule-forming Bradyrhizobium bacteria. Hybridization experiments showed that the gene is present in bacteria of all Bradyrhizobium strains tested and in photosynthetic bacteria forming stem nodules on tropical legumes of the genus Aeschynomene. The Bradyrhizobium shc gene is 1983 bp in length and encodes a protein of 660 amino acid residues with a calculated molecular mass of 73671 Da. Comparison of the deduced amino acid sequence with the sequences of other SHCs revealed highest similarity (70%) to the SHC from the Gram-negative Zymomonas mobilis and lower similarity (48%) to the SHCs from the Gram-positive Alicyclobacillus acidocaldarius and Alicyclobacillus acidoterrestris. Bradyrhizobium SHC also showed similarity (38-43%) to eukaryotic oxidosqualene cyclases. The B. japonicum shc gene was expressed in Escherichia coli. The recombinant SHC catalysed the cyclization of squalene to the hopanoids hopene and diplopterol in vitro. However, the formation of the gammacerane derivative tetrahymanol, which is produced in addition to hopanoids in B. japonicum strains in vivo, could not be detected in vitro. Therefore, the presence of a second squalene cyclase in B. japonicum can be assumed. Sequence analysis of 0.5 kb upstream from the shc gene identified a partial ORF with significant similarity to the C-terminus of an ORF located immediately upstream from the shc gene in Z. mobilis. Both ORFs also showed similarity to phytoene desaturases from cyanobacteria and plants. The 3'-end of this ORF from B. japonicum overlaps with 13 bp at the 5'-end of shc. The close proximity of this ORF to shc suggests that shc and this ORF may be part of an operon.
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( 1996 ) The Bradyrhizobium japonicum fegA gene encodes an iron-regulated outer membrane protein with similarity to hydroxamate-type siderophore receptors. PMID : 8955412 : DOI : 10.1128/jb.178.24.7265-7275.1996 PMC : PMC178643 Abstract >>
Iron is important in the symbiosis between soybean and its nitrogen-fixing endosymbiont Bradyrhizobium japonicum, yet little is known about rhizobial iron acquisition strategies. Analysis of outer membrane proteins (OMPs) from B. japonicum 61A152 identified three iron-regulated OMPs in the size range of several known receptors for Fe(III)-scavenging siderophores. One of the iron-regulated proteins, FegA, was purified and microsequenced, and a reverse genetics approach was used to clone a fegA-containing DNA fragment. Sequencing of this fragment revealed a single open reading frame of 750 amino acids. A putative N-terminal signal sequence of 14 amino acids which would result in a mature protein of 736 amino acids with a molecular mass of 80,851 Da was predicted. FegA shares significant amino acid similarity with several Fe(III)-siderophore receptors from gram-negative bacteria and has greater than 50% amino acid similarity and 33% amino acid identity with two [corrected] bacterial receptors for hydroxamate-type Fe(III)-siderophores. A dendrogram describing total inferred sequence similarity among 36 TonB-dependent OMPs was constructed; FegA grouped with Fe(III)-hydroxamate receptors. The transcriptional start site of fegA was mapped by primer extension analysis, and a putative Fur-binding site was found in the promoter. Primer extension and RNA slot blot analysis demonstrated that fegA was expressed only in cells grown under iron-limiting conditions. This is the first report of the cloning of a gene encoding a putative Fe(III)-siderophore receptor from nitrogen-fixing rhizobia.
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( 1997 ) Identification of the lrp gene in Bradyrhizobium japonicum and its role in regulation of delta-aminolevulinic acid uptake. PMID : 9045849 : DOI : 10.1128/jb.179.5.1828-1831.1997 PMC : PMC178902 Abstract >>
The heme precursor delta-aminolevulinic acid (ALA) is taken up by the dipeptide permease (Dpp) system in Escherichia coli. In this study, we identified a Bradyrhizobium japonicum genomic library clone that complemented both ALA and dipeptide uptake activities in E. coli dpp mutants. The complementing B. japonicum DNA encoded a product with 58% identity to the E. coli global transcriptional regulator Lrp (leucine-responsive regulatory protein), implying the presence of Dpp-independent ALA uptake activity in those cells. Data support the conclusion that the Lrp homolog induced the oligopeptide permease system in the complemented cells by interfering with the repressor activity of the endogenous Lrp, thus conferring oligopeptide and ALA uptake activities. ALA uptake by B. japonicum was effectively inhibited by a tripeptide and, to a lesser extent, by a dipeptide, and a mutant strain that expressed the lrp homolog from a constitutive promoter was deficient in ALA uptake activity. The data show that Lrp negatively affects ALA uptake in E. coli and B. japonicum. Furthermore, the product of the isolated B. japonicum gene is both a functional and structural homolog of E. coli Lrp, and thus the regulator is not restricted to enteric bacteria.
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( 1997 ) Dissection of the transcription machinery for housekeeping genes of Bradyrhizobium japonicum. PMID : 8990287 : DOI : 10.1128/jb.179.2.364-369.1997 PMC : PMC178705 Abstract >>
By using a PCR approach, the Bradyrhizobium japonicum sigA gene, which encodes the primary RNA polymerase sigma factor, sigma80, was cloned and its nucleotide sequence was established. The deduced protein is highly homologous to the SigA protein of Rhizobium meliloti (72% amino acid sequence identity) but less so to RpoD of Escherichia coli (51% identity). Well conserved is the C-terminal end of the protein, which is probably involved in promoter recognition and binding of the RNA polymerase core enzyme. A remarkable feature of the primary sequence is an alanine- and proline-rich segment of 24 amino acids between conserved regions 1 and 2, which might function as an interdomain linker. We purified the B. japonicum RNA polymerase holoenzyme. One of the subunits had an apparent molecular mass of 90 kDa and corresponded to the sigA gene product, as judged by N-terminal amino acid sequencing. The purified RNA polymerase was used in an in vitro transcription system to determine the transcription start sites of the rrn and groESL4 operons. They were identical to those previously identified in vivo. The rrn promoter was cloned upstream of a rho-independent terminator, yielding a transcript of about 240 bases. This served as a suitable template to analyze promoter activity. Then mutant derivatives of the rrn promoter were constructed and tested in in vitro transcription experiments. Several base pairs essential for promoter activity were thus identified. The results suggest that the well-characterized -35/-10 promoter class is predominantly used in B. japonicum for the expression of "housekeeping" genes.
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( 1997 ) Bradyrhizobium japonicum does not require alpha-ketoglutarate dehydrogenase for growth on succinate or malate. PMID : 8981998 : DOI : 10.1128/jb.179.1.194-201.1997 PMC : PMC178679 Abstract >>
The sucA gene, encoding the E1 component of alpha-ketoglutarate dehydrogenase, was cloned from Bradyrhizobium japonicum USDA110, and its nucleotide sequence was determined. The gene shows a codon usage bias typical of non-nif and non-fix genes from this bacterium, with 89.1% of the codons being G or C in the third position. A mutant strain of B. japonicum, LSG184, was constructed with the sucA gene interrupted by a kanamycin resistance marker. LSG184 is devoid of alpha-ketoglutarate dehydrogenase activity, indicating that there is only one copy of sucA in B. japonicum and that it is completely inactivated in the mutant. Batch culture experiments on minimal medium revealed that LSG184 grows well on a variety of carbon substrates, including arabinose, malate, succinate, beta-hydroxybutyrate, glycerol, formate, and galactose. The sucA mutant is not a succinate auxotroph but has a reduced ability to use glutamate as a carbon or nitrogen source and an increased sensitivity to growth inhibition by acetate, relative to the parental strain. Because LSG184 grows well on malate or succinate as its sole carbon source, we conclude that B. japonicum, unlike most other bacteria, does not require an intact tricarboxylic acid (TCA) cycle to meet its energy needs when growing on the four-carbon TCA cycle intermediates. Our data support the idea that B. japonicum has alternate energy-yielding pathways that could potentially compensate for inhibition of alpha-ketoglutarate dehydrogenase during symbiotic nitrogen fixation under oxygen-limiting conditions.
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( 1995 ) A TnphoA insertion within the Bradyrhizobium japonicum sipS gene, homologous to prokaryotic signal peptidases, results in extensive changes in the expression of PBM-specific nodulins of infected soybean (Glycine max) cells. PMID : 8825087 : DOI : 10.1111/j.1365-2958.1995.18050831.x Abstract >>
Bradyrhizobium japonicum mutant 132 was obtained by random TnphoA mutagenesis of strain 110spc4. A 6.5 kb BamHI kanamycin-resistance-coding DNA fragment of mutant 132 was used as a hybridization probe to clone the corresponding wild-type fragment. DNA sequence analysis of a 3213 bp BamHI-ClaI fragment revealed that three open reading frames (ORFs) were encoded in the same orientation. Based on sequence similarities to other proteins in the database, the second ORF was called sipS (signal peptidase). The TnphoA insertion in mutant 132 was found to be in frame near the 3' end of sipS. The resulting SipS-PhoA hybrid polypeptide was shown to be expressed in free-living B. japonicum and in Escherichia coli cultures. An immunoblot analysis with a polyclonal antibody directed against the alkaline phosphatase revealed the appearance of a weak signal of a 70 kDa polypeptide both in B. japonicum and in E. coli, in agreement with the calculated size of the hybrid polypeptide. A much stronger 52 kDa band was also detected. Mutant 132 was specifically disturbed in the interaction with soybean (Glycine max) when the bacteria were released from the infection threads. The bacteroids were not stably maintained within the symbiosome. Numerous vesicles were found in the plant cytosol, which finally resulted in the formation of large vacuoles within the infected nodule cells. Immunohistochemical analyses with antibodies directed against nodulins of the peribacteroid membrane indicated a lower expression of these proteins. The mutant phenotype was genetically complemented by a 4.4 kb BamHI fragment including sipS. Subfragments thereof also complemented a temperature-sensitive E. coli lepB mutant, demonstrating that the B. japonicum fragment was functionally replacing Lepts in E. coli. Genetic studies suggested that the three genes are organized in one common operon which is expressed from a promoter upstream of the sequenced region. Inactivation of the gene downstream of sipS did not result in a detectable phenotype.
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( 1996 ) The Bradyrhizobium japonicum rpoH1 gene encoding a sigma 32-like protein is part of a unique heat shock gene cluster together with groESL1 and three small heat shock genes. PMID : 8808920 : DOI : 10.1128/jb.178.18.5337-5346.1996 PMC : PMC178348 Abstract >>
The heat shock response of Bradyrhizobium japonicum is controlled by a complex network involving two known regulatory systems. While some heat shock genes are controlled by a highly conserved inverted-repeat structure (CIRCE), others depend on a sigma 32-type heat shock sigma factor. Using Western blot (immunoblot) analysis, we confirmed the presence of a sigma 32-like protein in B. japonicum and defined its induction pattern after heat shock. A B. japonicum rpoH-like gene (rpoH1) was cloned by complementation of an Escherichia coli strain lacking sigma 32. A knockout mutation in rpoH1 did not abolish sigma 32 production in B. japonicum, and the rpoH1 mutant showed the wild-type growth phenotype, suggesting the presence of multiple rpoH homologs in this bacterium. Further characterization of the rpoH1 gene region revealed that the rpoH1 gene is located in a heat shock gene cluster together with the previously characterized groESL1 operon and three genes encoding small heat shock proteins in the following arrangement: groES1, groEL1, hspA, rpoH1, hspB, and hspC. Three heat-inducible promoters are responsible for transcription of the six genes as three bicistronic operons. A sigma 32-dependent promoter has previously been described upstream of the groESL1 operon. Although the hspA-rpoH1 and hspBC operons were clearly heat inducible, they were preceded by sigma 70-like promoters. Interestingly, a stretch of about 100 bp between the transcription start site and the start codon of the first gene in each of these two operons was nearly identical, making it a candidate for a regulatory element potentially allowing heat shock induction of sigma 70-dependent promoters.
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( 1993 ) Genes for a microaerobically induced oxidase complex in Bradyrhizobium japonicum are essential for a nitrogen-fixing endosymbiosis. PMID : 8386371 : DOI : 10.1073/pnas.90.8.3309 PMC : PMC46289 Abstract >>
We report the discovery of a Bradyrhizobium japonicum gene cluster (fixNOQP) in which mutations resulted in defective soybean root-nodule bacteroid development and symbiotic nitrogen fixation. The predicted, DNA-derived protein sequences suggested that FixN is a heme b and copper-binding oxidase subunit, FixO a monoheme cytochrome c, FixQ a polypeptide of 54 amino acids, and FixP a diheme cytochrome c and that they are all membrane-bound. The isolation and analysis of membrane proteins from B. japonicum wild-type and mutant cells revealed two c-type cytochromes of 28 and 32 kDa as the likely products of the fixO and fixP genes and showed that both were synthesized only under oxygen-limited growth conditions. Furthermore, fixN insertion and fixNO deletion mutants grown microaerobically or anaerobically (with nitrate) exhibited a strong decrease in whole-cell oxidase activity as compared with the wild type. The data suggest that the fixNOQP gene products are induced at low oxygen concentrations and constitute a member of the bacterial heme/copper cytochrome oxidase superfamily. The described features are compatible with the postulate that this oxidase complex is specifically required to support bacterial respiration in endosymbiosis.
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( 1996 ) The Bradyrhizobium japonicum fixGHIS genes are required for the formation of the high-affinity cbb3-type cytochrome oxidase. PMID : 8661920 : Abstract >>
We report structural and functional analyses of the Bradyrhizobium japonicum fixGHIS genes, which map immediately downstream of the fixNOQP operon for the symbiotically essential cbb3-type heme-copper oxidase complex. Expression of fixGHIS, like that of fixNOQP, is strongly induced in cells grown microaerobically or anaerobically. A fixGHI deletion led to the same prominent phenotypes as those known from a fixNOQP deletion: defective symbiotic nitrogen fixation (Fix-) and decreased cytochrome oxidase activity in cells grown under oxygen deprivation. Only traces, if any, of cytochrome cbb3 subunits were present in membranes isolated from the delta fixGHI strain, as revealed by Western blot analysis with subunit-specific antibodies. This effect was not due to lack of fixNOQP transcription. The results suggested a critical involvement of the fixGHIS gene products in the assembly and/or stability of the cbb3-type heme-copper oxidase. On the basis of sequence similarities between the FixI protein and a Cu-transporting P-type ATPase (CopA) of Enterococcus hirae, and between FixG and a membrane-bound oxidoreductase (RdxA) of Rhodobacter sphaeroides, we postulate that a membrane-bound FixGHIS complex might play a role in uptake and metabolism of copper required for the cbb3-type heme-copper oxidase.
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( 1993 ) nolMNO genes of Bradyrhizobium japonicum are co-transcribed with nodYABCSUIJ, and nolO is involved in the synthesis of the lipo-oligosaccharide nodulation signals. PMID : 8262943 : Abstract >>
A host-inducible lacZ fusion was mapped down-stream of the nodYABCSUIJ operon in Bradyrhizobium japonicum strain USDA110. Sequencing of this region identified three novel genes, nolMNO. RNA dot blot analysis showed that nolO transcription is nodD1-dependent and that a polar mutation in nodS, located 5 kilobases upstream of nolO, blocks the transcription of nolO. Coupled with the host-inducible nature of nolO expression, these results indicate that nolMNO are part of a 9-kilobase operon, nodYABCSUIJnolMNO. The lipo-oligosaccharide nodulation signals produced by strains SL67 (nolO-) and SL65(nolNO-) were purified, and their chemical structures were determined. In addition to the wild-type signal molecules, both mutants produced modified compounds that are not produced by the parent strain USDA110. The most prevalent difference observed was the absence of the 2-O-methylfucosyl residue from the mutant structures. In addition, metabolites were found in which the N-acetylglucosamine residue at the reducing end was glycosidically linked to glycerol. These alterations in the profiles of nodulation signals produced by strains SL67 and SL65 were accompanied by reduced nodulation efficiency on all hosts tested.
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( 1993 ) A novel response-regulator is able to suppress the nodulation defect of a Bradyrhizobium japonicum nodW mutant. PMID : 8264528 : DOI : 10.1007/bf00279895 Abstract >>
The two-component regulatory system Nod-VW of Bradyrhizobium japonicum is essential for the nodulation of the legume host plants Vigna radiata, V. unguiculata and Macroptilium atropurpureum. The NodV protein shares homology with the sensor-kinases, whereas the NodW protein is a member of the response-regulator class. We report here the identification of a new B. japonicum DNA region that is able to suppress the phenotypic defect of a nodW mutant, provided that this region is expressed from a foreign promoter. The minimal complementing region, which itself is not essential for nodulation in a nodW+ background, consists of one gene designated nwsB (nodW-suppressor). The deduced amino acid sequence of the nwsB gene product shows a high degree of homology to NodW. The nws B gene is preceded by a long open reading frame, nwsA, whose putative product appears to be a sensor-kinase. Downstream of nwsB, an open reading frame encoding a second putative response-regulator was identified. Interspecies hybridization revealed the presence of nwsAB-like DNA also in other Bradyrhizobium strains. Using nwsB'-'lacZ fusions, the nwsB gene was found to be expressed rather weakly in B. japonicum. This low level of expression is obviously not sufficient to compensate for a nodW- defect, whereas strong overexpression of nwsB is a condition that leads to suppression of the nodW- mutation.
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( 1994 ) nodZ, a unique host-specific nodulation gene, is involved in the fucosylation of the lipooligosaccharide nodulation signal of Bradyrhizobium japonicum. PMID : 8300517 : DOI : 10.1128/jb.176.3.620-633.1994 PMC : PMC205098 Abstract >>
The nodulation genes of rhizobia are regulated by the nodD gene product in response to host-produced flavonoids and appear to encode enzymes involved in the production of a lipo-chitose signal molecule required for infection and nodule formation. We have identified the nodZ gene of Bradyrhizobium japonicum, whose product is required for the addition of a 2-O-methylfucose residue to the terminal reducing N-acetylglucosamine of the nodulation signal. This substitution is essential for the biological activity of this molecule. Mutations in nodZ result in defective nodulation of siratro. Surprisingly, although nodZ clearly codes for nodulation function, it is not regulated by NodD and, indeed, shows elevated expression in planta. Therefore, nodZ represents a unique nodulation gene that is not under the control of NodD and yet is essential for the synthesis of an active nodulation signal.
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( 1994 ) Sequences and characterization of hupU and hupV genes of Bradyrhizobium japonicum encoding a possible nickel-sensing complex involved in hydrogenase expression. PMID : 7961478 : DOI : 10.1128/jb.176.22.7102-7106.1994 PMC : PMC197088 Abstract >>
A 2.7-kb DNA fragment of Bradyrhizobium japonicum previously shown to be involved in hydrogenase expression has been sequenced. The area is located just upstream of the hupSLCDF operon and was found to contain two open reading frames, designated hupU and hupV; these encode proteins of 35.4 and 51.8 kDa, respectively. These proteins are homologous to Rhodobacter capsulatus HupU, a possible repressor of hydrogenase expression in that organism. B. japonicum HupU is 54% identical to the N terminus of R. capsulatus HupU, and HupV is 50% identical to the C terminus of R. capsulatus HupU. HupU and HupV also show homology to the [Ni-Fe] hydrogenase small and large subunits, respectively. Notably, HupV contains the probable nickel-binding sites RxCGxC and DPCxxCxxH, which are located in the N- and C-terminal portions, respectively, of the large subunit of hydrogenases. Hydrogenase activity assays, immunological assays for hydrogenase subunits, and beta-galactosidase assays on mutant strain JHCS2 (lacking a portion of HupV) were all indicative that HupV is necessary for transcriptional activation of hydrogenase. A physiological role as a possible nickel- or other environmental (i.e., oxygen or hydrogen)-sensing complex is proposed for HupU and HupV.
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( 1996 ) Beta-glucan synthesis in Bradyrhizobium japonicum: characterization of a new locus (ndvC) influencing beta-(1-->6) linkages. PMID : 8755895 : DOI : 10.1128/jb.178.15.4635-4642.1996 PMC : PMC178234 Abstract >>
Bradyrhizobium japonicum synthesizes periplasmic cyclic beta-(1-->3),beta-(1-->6)-D-glucans during growth in hypoosmotic environments, and evidence is growing that these molecules may have a specific function during plant-microbe interactions in addition to osmoregulation. Site-directed Tn5 mutagenesis of the DNA region upstream of ndvB resulted in identification of a new gene (ndvC) involved in beta-(1--> 3), beta-(1-->6)-glucan synthesis and in nodule development. The predicted translation product was a polypeptide (ca. 62 kDa) with several transmembrane domains. It contained a sequence characteristic of a conserved nucleoside-sugar-binding motif found in many bacterial enzymes and had 51% similarity with a beta-glucanosyltransferase from Candida albicans. B. japonicum carrying a Tn5 insertion in ndvC resulted in synthesis of altered cyclic beta-glucans composed almost entirely of beta-(1--> 3)-glycosyl linkages. The mutant strain was only slightly sensitive to hypoosmotic growth conditions compared with the ndvB mutant, but it was severely impaired in symbiotic interactions with soybean (Glycine max). Nodulation was delayed by 8 to 10 days, and many small nodule-like structures apparently devoid of viable bacteria were formed. This finding suggests that the structure of the beta-glucan molecule is important for a successful symbiotic interaction, and beta-glucans may have a specific function in addition to their role in hypoosmotic adaptation.
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( 1994 ) Identification and characterization of a novel Bradyrhizobium japonicum gene involved in host-specific nitrogen fixation. PMID : 7961425 : DOI : 10.1128/jb.176.21.6717-6729.1994 PMC : PMC197029 Abstract >>
To understand the genetic mechanism of host specificity in the interaction between rhizobia and their hosts, it is important to identify genes that influence both early and late steps in symbiotic development. This paper focuses on the little-understood genetics of host-specific nitrogen fixation. A deletion mutant of Bradyrhizobium japonicum, strain NAD163, was found to induce effective, nitrogen-fixing nodules on soybean and siratro plants but produced ineffective nodules on cowpea plants. Additional transposon and deletion mutants defined a small region that conferred this phenotype, and this region was sequenced to identify two putative open reading frames (ORFs). Data indicate that only one of these ORFs is detectable in bacteroids. This ORF was termed hsfA, with a predicted protein product of 11 kDa. The transcriptional start site of hsfA was determined and found to coincide with a predicted RpoN-dependent promoter. Microscopic studies of nodules induced by the wild type and hsfA mutants on cowpea and soybean plants indicate that the cowpea mutant nodules are slow to develop. The data indicate that hsfA appears to play a crucial role in bacteroid development on cowpea but does not appear to be essential for nitrogen fixation on the other hosts tested.
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( N/A ) A Bradyrhizobium japonicum gene essential for nodulation competitiveness is differentially regulated from two promoters. PMID : 8012043 : Abstract >>
We report the identification and nucleotide sequence of a new symbiotic gene (nfeC) from the soybean root nodule bacterium, Bradyrhizobium japonicum. A Tn5 insertion (NAD14) in this gene did not affect nitrogen fixation but caused a significant delay in soybean nodulation. In addition, this mutant exhibited a reduction in its competitive ability to nodulate soybean when coinoculated with the wild type. Sequence analysis of the mutated region revealed that the NAD14 Tn5 insertion mapped within an open reading frame of 825 bp. Primer extension using B. japonicum mRNA from three different growth conditions, aerobic, anaerobic, and bacteroids (i.e., symbiotic form) indicated that the upstream region of the gene contained two promoters, which were differentially regulated in response to the growth conditions. One promoter was expressed in bacteroids, but not under aerobic or anaerobic free-living conditions. The other promoter was expressed only under aerobic conditions.
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( N/A ) Identification and characterization of the nolYZ genes of Bradyrhizobium japonicum. PMID : 8012039 : Abstract >>
Characterization of an isoflavone-inducible locus closely linked to the common nod genes of Bradyrhizobium japonicum USDA110 led to the discovery of two open reading frames, designated nolY and nolZ. These open reading frames are preceded by a sequence with strong similarity to a consensus NodD-binding site (nod box). Studies utilizing a nolZ'-'lacZ fusion indicated that inducible expression is dependent upon both NodD1 and NodW, transcriptional regulators that are required for the expression of the common nodulation genes (e.g., nodYABC) of B. japonicum. A deletion mutation within nolY produced only slight defects in nodulation of soybeans, siratro, and cowpeas, but stronger defects were observed in nodulation of mung beans. An insertion mutation within nolZ showed no nodulation defects in the host plants tested. Competition assays for nodule occupancy in soybeans did not show any decrease in the competitiveness of a nolY mutant, nor did a nolY mutant show any detectable alteration in the production of lipooligosaccharide nodulation signals.
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( 1996 ) Isolation, DNA sequence analysis, and mutagenesis of a proline dehydrogenase gene (putA) from Bradyrhizobium japonicum. PMID : 8572700 : PMC : PMC167790 Abstract >>
We report here the cloning and sequencing of the gene for proline dehydrogenase (putA) of Bradyrhizobium japonicum. An open reading frame coding for 1,016 amino acids was identified. The B. japonicum gene codes for a bifunctional protein with proline dehydrogenase and pyrroline-5-carboxylate (P5C) dehydrogenase activities, as it does in Escherichia coli and Salmonella typhimurium. Comparison of the sequences of these proteins with other proline and P5C dehydrogenase sequences identified proline dehydrogenase and P5C dehydrogenase catalytic domains. Within the proline dehydrogenation domain, several areas of high identity were observed between B. japonicum, E. coli, S. typhimurium, Saccharomyces cerevisiae put1, and Drosophila melanogaster slgA. Within the P5C dehydrogenase domain, several areas of high identity were observed between B. japonicum, E. coli, S. typhimurium, Bacillus subtilis ipa76d, and S. cerevisiae put2. A consensus catalytic site for semialdehyde dehydrogenase was observed in the P5C dehydrogenase domain. This suggests that the substrate for this domain may be the open-chain gamma-glutamylsemialdehyde, not its cyclized form, P5C. Unlike the gene isolated from E. coli, S. typhimurium, and K. pneumoniae, the B. japonicum putA gene does not appear to be part of an operon with the proline porter gene (putP). Additionally, the B. japonicum gene lacks the putative C-terminal regulatory domain present in the E. coli and S. typhimurium genes. The gene was disrupted by insertion of antibiotic resistance gene cassettes, which were then recombined into the bacterial chromosome. Symbiotically active mutant strains that were devoid of putA activity were isolated. With this proline dehydrogenase clone, we will test the hypothesis that putA in symbiotic nitrogen-fixing B. japonicum bacteroids is transcriptionally regulated by drought and other stresses.
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( 1993 ) Bradyrhizobium japonicum rhizobitoxine genes and putative enzyme functions: expression requires a translational frameshift. PMID : 8464870 : DOI : 10.1073/pnas.90.7.2641 PMC : PMC46151 Abstract >>
Some strains of Bradyrhizobium japonicum produce rhizobitoxine, a phytotoxin that causes foliar chlorosis on susceptible host plants. We have previously obtained Tn5-induced rhizobitoxine null mutants of B. japonicum. DNA sequence analysis of the region surrounding two Tn5 insertions identifies two overlapping open reading frames. The first open reading frame (rtxA) predicts a 54-kDa protein for which the N-terminal 280 residues have sequence similarity to serine: pyruvate aminotransferase. The sequence homology to aminotransferase is consistent with the involvement of this gene in serinol production, a likely intermediate in rhizobitoxine biosynthesis. Previously, a mutant in this open reading frame was shown not to make serinol. The predicted amino acid sequence of the second open reading frame (rtxB) has similarity to yeast O-acetylhomoserine sulfhydrolase. This enzyme function is similar to that required for dihydrorhizobitoxine synthase. The DNA sequence shows that the rtxB open reading frame overlaps rtxA, suggesting that expression of rtxB requires a -1 translational frameshift. Protein expression experiments demonstrate production of an RtxAB fusion protein. The ability of the overlapping rtxA and rtxB sequences to promote a translational frameshift was confirmed in a heterologous expression system. In Escherichia coli, this frameshift appears to be unusually efficient, occurring at a frequency of 80-90%.
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( 1994 ) Genetic regulation of nitrogen fixation in rhizobia. PMID : 7968919 : PMC : PMC372973 Abstract >>
This review presents a comparison between the complex genetic regulatory networks that control nitrogen fixation in three representative rhizobial species, Rhizobium meliloti, Bradyrhizobium japonicum, and Azorhizobium caulinodans. Transcription of nitrogen fixation genes (nif and fix genes) in these bacteria is induced primarily by low-oxygen conditions. Low-oxygen sensing and transmission of this signal to the level of nif and fix gene expression involve at least five regulatory proteins, FixL, FixJ, FixK, NifA, and RpoN (sigma 54). The characteristic features of these proteins and their functions within species-specific regulatory pathways are described. Oxygen interferes with the activities of two transcriptional activators, FixJ and NifA. FixJ activity is modulated via phosphorylation-dephosphorylation by the cognate sensor hemoprotein FixL. In addition to the oxygen responsiveness of the NifA protein, synthesis of NifA is oxygen regulated at the level of transcription. This type of control includes FixLJ in R. meliloti and FixLJ-FixK in A. caulinodans or is brought about by autoregulation in B. japonicum. NifA, in concert with sigma 54 RNA polymerase, activates transcription from -24/-12-type promoters associated with nif and fix genes and additional genes that are not directly involved in nitrogen fixation. The FixK proteins constitute a subgroup of the Crp-Fnr family of bacterial regulators. Although the involvement of FixLJ and FixK in nifA regulation is remarkably different in the three rhizobial species discussed here, they constitute a regulatory cascade that uniformly controls the expression of genes (fixNOQP) encoding a distinct cytochrome oxidase complex probably required for bacterial respiration under low-oxygen conditions. In B. japonicum, the FixLJ-FixK cascade also controls genes for nitrate respiration and for one of two sigma 54 proteins.
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Kalita M,
Ma?ek W,
( 2019 ) The ftsA gene as a molecular marker for phylogenetic studies in Bradyrhizobium and identification of Bradyrhizobium japonicum. PMID : 30417315 : DOI : 10.1007/s13353-018-0479-9 PMC : PMC6373400 Abstract >>
The use of ftsA gene sequences for taxonomic studies of the genus Bradyrhizobium bacteria was assessed. The ftsA gene codes for an actin-like protein involved in prokaryotic cell division. Up to now, this gene has not been used as a phylogenetic marker for analysis of bacteria establishing root nodule symbiosis with Fabaceae plants. In this study, the ftsA gene sequences obtained for bradyrhizobia forming N2 fixing symbiosis with four Genisteae tribe plants growing in Poland and most of the type strains of the genus Bradyrhizobium species were analyzed and evaluated as molecular markers for phylogenetic studies of these bacteria for the first time. The ftsA gene sequences of all bradyrhizobial strains with completely or partially sequenced genomes, available in the GenBank database, were also included into the analysis. The phylogeny of the ftsA gene was compared to the phylogenies of other chromosomal genes commonly used in the studies of Bradyrhizobium bacteria. The results showed that the phylogenies of ftsA and the core genes recA and glnII were congruent, making the ftsA gene useful as a phylogenetic marker. Analysis of the ftsA gene sequences revealed a single-nucleotide polymorphism unique to Bradyrhizobium japonicum strains, and the potential use of this SNP for identification of this species was discussed.
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100. |
Nieuwkoop AJ,
Banfalvi Z,
Deshmane N,
Gerhold D,
Schell MG,
Sirotkin KM,
Stacey G,
( 1987 ) A locus encoding host range is linked to the common nodulation genes of Bradyrhizobium japonicum. PMID : 3584066 : DOI : 10.1128/jb.169.6.2631-2638.1987 PMC : PMC212140 Abstract >>
By using cloned Rhizobium meliloti, Rhizobium leguminosarum, and Rhizobium sp. strain MPIK3030 nodulation (nod) genes as hybridization probes, homologous regions were detected in the slow-growing soybean symbiont Bradyrhizobium japonicum USDA 110. These regions were found to cluster within a 25-kilobase (kb) region. Specific nod probes from R. meliloti were used to identify nodA-, nodB-, nodC-, and nodD-like sequences clustered on two adjacent HindIII restriction fragments of 3.9 and 5.6 kb. A 785-base-pair sequence was identified between nodD and nodABC. This sequence contained an open reading frame of 420 base pairs and was oriented in the same direction as nodABC. A specific nod probe from R. leguminosarum was used to identify nodIJ-like sequences which were also contained within the 5.6-kb HindIII fragment. A nod probe from Rhizobium sp. strain MPIK3030 was used to identify hsn (host specificity)-like sequences essential for the nodulation of siratro (Macroptilium atropurpureum) on a 3.3-kb HindIII fragment downstream of nodIJ. A transposon Tn5 insertion within this region prevented the nodulation of siratro, but caused little or no delay in the nodulation of soybean (Glycine max).
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101. |
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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102. |
Bastard K,
Perret A,
Mariage A,
Bessonnet T,
Pinet-Turpault A,
Petit JL,
Darii E,
Bazire P,
Vergne-Vaxelaire C,
Brewee C,
Debard A,
Pellouin V,
Besnard-Gonnet M,
Artiguenave F,
Médigue C,
Vallenet D,
Danchin A,
Zaparucha A,
Weissenbach J,
Salanoubat M,
de Berardinis V,
( 2017 ) Parallel evolution of non-homologous isofunctional enzymes in methionine biosynthesis. PMID : 28581482 : DOI : 10.1038/nchembio.2397 Abstract >>
Experimental validation of enzyme function is crucial for genome interpretation, but it remains challenging because it cannot be scaled up to accommodate the constant accumulation of genome sequences. We tackled this issue for the MetA and MetX enzyme families, phylogenetically unrelated families of acyl-L-homoserine transferases involved in L-methionine biosynthesis. Members of these families are prone to incorrect annotation because MetX and MetA enzymes are assumed to always use acetyl-CoA and succinyl-CoA, respectively. We determined the enzymatic activities of 100 enzymes from diverse species, and interpreted the results by structural classification of active sites based on protein structure modeling. We predict that >60% of the 10,000 sequences from these families currently present in databases are incorrectly annotated, and suggest that acetyl-CoA was originally the sole substrate of these isofunctional enzymes, which evolved to use exclusively succinyl-CoA in the most recent bacteria. We also uncovered a divergent subgroup of MetX enzymes in fungi that participate only in L-cysteine biosynthesis as O-succinyl-L-serine transferases.
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103. |
Kaluza K,
Hahn M,
Hennecke H,
( 1985 ) Repeated sequences similar to insertion elements clustered around the nif region of the Rhizobium japonicum genome. PMID : 2985537 : PMC : PMC218881 Abstract >>
Two different repeated sequences (RSs) were discovered in the Rhizobium japonicum genome: RSRj alpha is 1126 base pairs long and is repeated 12 times; RSRj beta is approximately 950 base pairs long and is repeated at least 6 times. Their arrangement in root nodule bacteroid DNA is the same as in DNA from bacteria grown in culture. Deletion analysis showed that many copies of alpha and beta are clustered around the nitrogenase genes nifDK and nifH, or, in general, they are found within a genomic region harboring genes that are nonessential for growth. One copy each of alpha and beta are located upstream of nifDK and are adjacent to each other. Neither of them, however, is involved in the expression of nifDK. Nucleotide sequence analysis of three copies of RS alpha revealed many characteristics of procaryotic insertion sequence elements: potential inverted repeats at their ends, potential target site duplication, and large open reading frames. Despite this, their genomic positions appear to be stable. One possible function of these RSs is in deletion formation probably via recombination between them.
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104. |
( 2013 ) The structure of Bradyrhizobium japonicum transcription factor FixK2 unveils sites of DNA binding and oxidation. PMID : 23546876 : DOI : 10.1074/jbc.M113.465484 PMC : PMC3656280 Abstract >>
FixK2 is a regulatory protein that activates a large number of genes for the anoxic and microoxic, endosymbiotic, and nitrogen-fixing life styles of the �\-proteobacterium Bradyrhizobium japonicum. FixK2 belongs to the cAMP receptor protein (CRP) superfamily. Although most CRP family members are coregulated by effector molecules, the activity of FixK2 is negatively controlled by oxidation of its single cysteine (Cys-183) located next to the DNA-binding domain and possibly also by proteolysis. Here, we report the three-dimensional x-ray structure of FixK2, a representative of the FixK subgroup of the CRP superfamily. Crystallization succeeded only when (i) an oxidation- and protease-insensitive protein variant (FixK2(C183S)-His6) was used in which Cys-183 was replaced with serine and the C terminus was fused with a hexahistidine tag and (ii) this protein was allowed to form a complex with a 30-mer double-stranded target DNA. The structure of the FixK2-DNA complex was solved at a resolution of 1.77 ?, at which the protein formed a homodimer. The precise protein-DNA contacts were identified, which led to an affirmation of the canonical target sequence, the so-called FixK2 box. The C terminus is surface-exposed, which might explain its sensitivity to specific cleavage and degradation. The oxidation-sensitive Cys-183 is also surface-exposed and in close proximity to DNA. Therefore, we propose a mechanism whereby the oxo acids generated after oxidation of the cysteine thiol cause an electrostatic repulsion, thus preventing specific DNA binding.
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105. |
( 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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106. |
Chibeba AM,
Kyei-Boahen S,
Guimarães MF,
Nogueira MA,
Hungria M,
( 2017 ) Isolation, characterization and selection of indigenous Bradyrhizobium strains with outstanding symbiotic performance to increase soybean yields in Mozambique. PMID : 28775390 : DOI : 10.1016/j.agee.2017.06.017 PMC : PMC5521954 Abstract >>
Soybean inoculation with effective rhizobial strains makes unnecessary the use of N-fertilizers in the tropics. A frequently reported problem is the failure of the inoculant strains to overcome the competition imposed by indigenous rhizobial populations. The screening of indigenous rhizobia, already adapted to local conditions, searching for highly effective strains for use as inoculants represents a promising strategy in overcoming inoculation failure. The objective of this study was to isolate and characterize indigenous rhizobia and to identify strains that hold potential to be included in inoculant formulations for soybean production, with both promiscuous and non-promiscuous soybean cultivars, in Mozambican agro-climatic conditions. A total of 105 isolates obtained from nodules of promiscuous soybean grown at 15 sites were screened for N2-fixation effectiveness in the greenhouse along with five commercial strains. Eighty-seven isolates confirmed the ability to form effective nodules on soybean and were used for genetic characterization by rep-PCR (BOX) and sequencing of the 16S rRNA gene, and also for symbiotic effectiveness. BOX-PCR fingerprinting revealed remarkable genetic diversity, with 41 clusters formed, considering a similarity level of 65%. The 16S rRNA analysis assigned the isolates to the genera Bradyrhizobium (75%) and Agrobacterium/Rhizobium (25%). Great variability in symbiotic effectiveness was detected among the indigenous rhizobia from Mozambique, with ten isolates performing better than the commercial strain B. diazoefficiens USDA 110, the best reference strain, and 51 isolates with lower performance than all reference strains. Thirteen of the best isolates from the first greenhouse trial were evaluated, along with the five commercial strains, in two promiscuous (TGx 1963-3F and TGx 1835-10E) and one non-promiscuous (BRS 284) soybean cultivars in a second greenhouse trial. In general the promiscous soybeans responded better to inoculation. The 13 isolates were also characterized for tolerance to acidity and alkalinity (pH 3.5 and 9.0, respectively), salinity (0.1, 0.3 and 0.5 mol L-1 of NaCl) and high temperatures (35, 40 and 45 �XC) in vitro. Five isolates, three (Moz 4, Moz 19 and Moz 22) belonging to the superclade B. elkanii and two (Moz 27 and Moz 61) assigned to the superclade B. japonicum, consistently showed high symbiotic effectiveness, suggesting that the inoculation with indigenous rhizobia adapted to local conditions represents a possible strategy for increasing soybean yields in Mozambique. Phylogenetic position of the five elite isolates was confirmed by the MLSA with four protein-coding housekeeping genes, dnaK, glnII, gyrB and recA.
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107. |
( 2012 ) Microevolution of symbiotic Bradyrhizobium populations associated with soybeans in east North America. PMID : 23301163 : DOI : 10.1002/ece3.404 PMC : PMC3538991 Abstract >>
Microevolution and origins of Bradyrhizobium populations associated with soybeans at two field sites (A and B, 280 km apart in Canada) with contrasting histories of inoculation was investigated using probabilistic analyses of six core (housekeeping) gene sequences. These analyses supported division of 220 isolates in five lineages corresponding either to B. japonicum groups 1 and 1a or to one of three novel lineages within the genus Bradyrhizobium. None of the isolates from site A and about 20% from site B (the only site with a recent inoculation history) were attributed to inoculation sources. The data suggest that most isolates were of indigenous origin based on sequence analysis of 148 isolates of soybean-nodulating bacteria from native legumes (Amphicarpaea bracteata and Desmodium canadense). Isolates from D. canadense clustered with B. japonicum group 1, whereas those from A. bracteata were placed in two novel lineages encountered at soybean field sites. One of these novel lineages predominated at soybean sites and exhibited a significant clonal expansion likely reflecting selection by the plant host. Homologous recombination events detected in the 35 sequence types from soybean sites had an effect on genetic diversification that was approximately equal to mutation. Interlineage transfer of core genes was infrequent and mostly attributable to gyrB that had a history of frequent recombination. Symbiotic gene sequences (nodC and nifH) of isolates from soybean sites and native legumes clustered in two lineages corresponding to B. japonicum and B. elkani with the inheritance of these genes appearing predominantly by vertical transmission. The data suggest that soybean-nodulating bacteria associated with native legumes represent a novel source of ecologically adapted bacteria for soybean inoculation.
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108. |
( 2013 ) The type III Secretion System of Bradyrhizobium japonicum USDA122 mediates symbiotic incompatibility with Rj2 soybean plants. PMID : 23204412 : DOI : 10.1128/AEM.03297-12 PMC : PMC3568557 Abstract >>
The rhcJ and ttsI mutants of Bradyrhizobium japonicum USDA122 for the type III protein secretion system (T3SS) failed to secrete typical effector proteins and gained the ability to nodulate Rj2 soybean plants (Hardee), which are symbiotically incompatible with wild-type USDA122. This suggests that effectors secreted via the T3SS trigger incompatibility between these two partners.
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109. |
( 2013 ) Native bradyrhizobia from Los Tuxtlas in Mexico are symbionts of Phaseolus lunatus (Lima bean). PMID : 23280323 : DOI : 10.1016/j.syapm.2012.10.006 Abstract >>
Los Tuxtlas is the northernmost rain forest in North America and is rich in Bradyrhizobium with an unprecedented number of novel lineages. ITS sequence analysis of legumes in polycultures from Los Tuxtlas led to the identification of Phaseolus lunatus and Vigna unguiculata in addition to Phaseolus vulgaris as legumes associated with maize in crops. Bacterial diversity of isolates from nitrogen-fixing nodules of P. lunatus and V. unguiculata was revealed using ERIC-PCR and PCR-RFLP of rpoB genes, and sequencing of recA, nodZ and nifH genes. P. lunatus and V. unguiculata nodule bacteria corresponded to bradyrhizobia closely related to certain native bradyrhizobia from the Los Tuxtlas forest and novel groups were found. This is the first report of nodule bacteria from P. lunatus in its Mesoamerican site of origin and domestication.
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110. |
( 1998 ) Exopolysaccharide (EPS) synthesis in Bradyrhizobium japonicum: sequence, operon structure and mutational analysis of an exo gene cluster. PMID : 9747707 : DOI : 10.1007/s004380050801 Abstract >>
The nucleotide sequence of a 8330-bp DNA fragment from Bradyrhizobium japonicum 110spc4 was determined. Sequence analysis revealed that six ORFs were present and the deduced amino acid sequences were homologous to enzymes involved in exopolysaccharide (EPS) biosynthesis. The genes appear to be organized into at least four different operons. One gene was found to be homologous to exoB, which encodes a UDP-galactose 4'-epimerase. Other ORFs were homologous to UDP-hexose transferases and one ORF showed similarity to Sinorhizobium (Rhizobium) meliloti ExoP, which has been suggested to be involved in EPS chain-length determination. A set of deletion and insertion mutants was constructed and the resulting B. japonicum strains were tested for their symbiotic traits. Deletion mutant deltaP22, which lacks the C-terminal part of ExoP, the UDP-hexose transferase ExoT and the N-terminal part of ExoB, shows a delayed nodulation phenotype and induces symptoms of plant defense reactions; its EPS does not contain galactose and no high molecular weight fraction is synthesized. In contrast, insertion mutant EH3, which expresses an exoP gene product that is truncated in its putative periplasmic domain, produced an EPS containing both HMW and LMW fractions. However, the interaction of EH3 with soybeans was severely perturbed. As a rule, only the initial steps of nodule formation were observed.
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111. |
( 1998 ) Bradyrhizobium japonicum FixK2, a crucial distributor in the FixLJ-dependent regulatory cascade for control of genes inducible by low oxygen levels. PMID : 9748464 : PMC : PMC107567 Abstract >>
Bradyrhizobium japonicum possesses a second fixK-like gene, fixK2, in addition to the previously identified fixK1 gene. The expression of both genes depends in a hierarchical fashion on the low-oxygen-responsive two-component regulatory system FixLJ, whereby FixJ first activates fixK2, whose product then activates fixK1. While the target genes for control by FixK1 are unknown, there is evidence for activation of the fixNOQP, fixGHIS, and rpoN1 genes and some heme biosynthesis and nitrate respiration genes by FixK2. FixK2 also regulates its own structural gene, directly or indirectly, in a negative way.
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112. |
( 1998 ) A novel DNA element that controls bacterial heat shock gene expression. PMID : 9622356 : DOI : 10.1046/j.1365-2958.1998.00794.x Abstract >>
The hspArpoH1 and hspBCdegP heat shock operons of Bradyrhizobium japonicum are preceded by a novel, conserved DNA element of approximately 100 bp, which is responsible for the temperature-regulated transcription of their sigma70-type promoters. We designated this motif ROSE for repression of heat shock gene expression and found additional ROSE elements upstream of two newly identified heat shock operons. A critical core region in the hspA-associated ROSE1 was defined by introducing insertions or deletions. While four mutants retained the ability to repress transcription of the hspArpoH1 operon, five deletion mutants produced elevated hspA mRNA levels under low-temperature growth conditions. Derepression was confirmed by increased RpoH1 levels in non-heat-shocked cells from one of these mutants and by strains that contained a translational hspA-lacZ fusion associated with mutated ROSE1 elements. The hspArpoH1 operon was efficiently transcribed in vitro, and a deletion of ROSE1 did not impair this activity. Gel retardation experiments demonstrated that a protein in non-heat-shocked cells specifically binds to the intact ROSE1 element but not to a mutated element lacking the core region. Taken together, these results indicate that a central region of ROSE serves as a binding site for a repressor protein under standard growth conditions in order to prevent the undesired transcription of heat shock genes.
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113. |
( 1998 ) Expression of the fixR-nifA operon in Bradyrhizobium japonicum depends on a new response regulator, RegR. PMID : 9683482 : PMC : PMC107369 Abstract >>
Many nitrogen fixation-associated genes in the soybean symbiont Bradyrhizobium japonicum are regulated by the transcriptional activator NifA, whose activity is inhibited by aerobiosis. NifA is encoded in the fixR-nifA operon, which is expressed at a low level under aerobic conditions and induced approximately fivefold under low-oxygen tension. This induction depends on a -24/-12-type promoter (fixRp1) that is recognized by the sigma54 RNA polymerase and activated by NifA. Low-level aerobic expression and part of the anaerobic expression originates from a second promoter (fixRp2) that overlaps with fixRp1 and depends on an upstream DNA region (UAS) located around position -68 (H. Barrios, H. M. Fischer, H. Hennecke, and E. Morett, J. Bacteriol. 177:1760-1765, 1995). A protein binding to the UAS was previously postulated to act as an activator. This protein has now been purified, and the corresponding gene (regR) has been cloned. On the basis of the predicted amino acid sequence, RegR belongs to the family of response regulators of two-component regulatory systems. We identified upstream of the regR gene an additional gene (regS) encoding a putative sensor kinase. A regR mutant was constructed in which neither a specific UAS-binding activity nor fixRp2-dependent transcript formation and fixR'-'lacZ expression was detected in aerobically grown cells. Anaerobic fixR'-'lacZ expression was also decreased in regR mutants to about 10% of the level observed in the wild type. Similarly, regR mutants showed only about 2% residual nitrogen fixation activity, but unlike nodules induced by nifA mutants, the morphology of those nodules was normal, displaying no signs of necrosis. While regR mutants grew only slightly slower in free-living, aerobic conditions, they displayed a strong growth defect under anaerobic conditions. The phenotypic properties of regS mutants differed only marginally, if at all, from those of the wild type, suggesting the existence of a compensating sensor activity in these strains. The newly identified RegR protein may be regarded as a master regulator in the NifA-dependent network controlling nif and fix gene expression in B. japonicum.
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114. |
( 1998 ) The Bradyrhizobium japonicum phoB gene is required for phosphate-limited growth but not for symbiotic nitrogen fixation. PMID : 9561731 : DOI : 10.1111/j.1574-6968.1998.tb12927.x Abstract >>
We identified by cloning and DNA sequence analysis the phosphate regulatory gene phoB of Bradyrhizobium japonicum. The deduced gene product displayed pronounced similarity to the PhoB protein of Sinorhizobium meliloti (71.4% identical amino acids). Escherichia coli (50.2%) and other bacterial species. Insertion of a kanamycin resistance cassette into phoB led to impaired growth of the B. japonicum mutant in media containing approximately 25 microM phosphate or less. A standard plant infection test using wild-type and phoB-defective B. japonicum strains showed that the phoB mutation had no effect on the symbiotic properties of B. japonicum with its soybean host plant.
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115. |
( 1999 ) Multiple small heat shock proteins in rhizobia. PMID : 9864316 : PMC : PMC103535 Abstract >>
Seven genes coding for small heat shock proteins (sHsps) in Bradyrhizobium japonicum have been identified. They are organized in five operons that are coordinately regulated by ROSE, a negatively cis-acting DNA element. The deduced sHsps can be divided into two separate classes: class A, consisting of proteins that show similarity to Escherichia coli IbpA and IbpB, and class B, whose members display significant similarity to other sHsps from prokaryotes and eukaryotes. Two-dimensional gel electrophoresis and Edman sequencing revealed the presence of at least 12 sHsps in B. japonicum, indicating a remarkable abundance of sHsps in this organism. Three additional members of class A and two potentially novel heat shock proteins were identified on the basis of their amino termini. The presence of multiple sHsps was also demonstrated for a variety of Rhizobium and Bradyrhizobium species by immunoblot analysis and two-dimensional gel electrophoresis. An extensive database survey revealed that, in contrast to the rhizobia, other bacteria contain maximally two sHsps whereas many plants have been reported to possess a sHsp superfamily.
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116. |
( 1998 ) A second gene for type I signal peptidase in Bradyrhizobium japonicum, sipF, is located near genes involved in RNA processing and cell division. PMID : 9870699 : DOI : 10.1007/pl00008631 Abstract >>
The TnphoA-induced Bradyrhizobium japonicum mutant 184 shows slow growth and aberrant colonization of soybean nodules. Using a DNA fragment adjacent to the transposon insertion site as a probe, a 3.4-kb BglII fragment of B. japonicum 110spc4 DNA was identified and cloned. Sequence analysis indicated that two truncated ORFs and three complete ORFs were encoded on this fragment. A database search revealed homologies to several other prokaryotic proteins: PdxJ (an enzyme involved in vitamin B6 biosynthesis), AcpS (acyl carrier protein synthase), Lep or Sip (prokaryotic type I signal peptidase), RNase III (an endoribonuclease which processes double-stranded rRNA precursors and mRNA) and Era (a GTP-binding protein required for cell division). The mutation in strain 184 was found to lie within the signal peptidase gene, which was designated sipF. Therefore, sipF is located in a region that encodes gene products involved in posttranscriptional and posttranslational processing processes. By complementation of the lep(ts) E. coli mutant strain IT41 it was demonstrated that sipF indeed encodes a functional signal peptidase, and genetic complementation of B. japonicum mutant 184 by a 2.8-kb SalI fragment indicated that sipF is expressed from a promoter located directly upstream of sipF. Using a non-polar kanamycin resistance cassette, a specific sipF mutant was constructed which exhibited defects in symbiosis similar to those of the original mutant 184.
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117. |
( 1998 ) Characterization of Bradyrhizobium japonicum pcaBDC genes involved in 4-hydroxybenzoate degradation. PMID : 9582432 : DOI : 10.1016/s0167-4781(98)00048-7 Abstract >>
The pca structural genes encode enzymes that participate in the conversion of protocatechuate to succinate and acetylcoenzyme A. A 3. 05-kb region of the Bradyrhizobium japonicum strain USDA110 genome has been characterized, which contains the pcaB, pcaD and pcaC genes. The predicted protein sequences of the three genes have extensive homologies with beta-carboxy-cis,cis-muconate cycloisomerase (PcaB), beta-ketodiapate enol-lactone hydrolase (PcaD), and gamma-carboxymuconolactone decarboxylase (PcaC), respectively, from Acinetobacter calcoaceticus and Pseudomonas putida. The DNA sequence revealed that the pca genes are probably arranged in a single transcriptional unit, pcaBDC, similar to that described in P. putida. A pcaB deletion mutant constructed by marker exchange mutagenesis lost the ability to use 4-hydroxybenzoate or protocatechuate as the only carbon source, demonstrating functionality of the characterized genes in catabolism of hydroxyaromatics by B. japonicum. Furthermore, 4-hydroxybenzoate and protocatechuate became toxic for the pcaB mutant, indicating that hydroxyaromatics catabolism serves both nutritional and detoxifying purposes.
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118. |
( 1998 ) The bacterial irr protein is required for coordination of heme biosynthesis with iron availability. PMID : 9705301 : DOI : 10.1074/jbc.273.34.21669 Abstract >>
Heme is a ubiquitous macromolecule that serves as the active group of proteins involved in many cellular processes. The multienzyme pathway for heme formation culminates with the insertion of iron into a protoporphyrin ring. The cytotoxicity of porphyrins suggests the need for coordination of its biosynthesis with iron availability. We isolated a mutant strain of the bacterium Bradyrhizobium japonicum that, under iron limitation, accumulated protoporphyrin and showed aberrantly high expression of hemB, an iron-regulated gene encoding the heme synthesis enzyme delta-aminolevulinic acid dehydratase. The strain carries a loss of function mutation in irr, a newly described gene that encodes a putative member of the GntR family of bacterial transcriptional regulators. Irr accumulated only under iron limitation, and turned over rapidly upon an increase in iron availability. A separate role for Irr in controlling the cellular iron level was inferred based on a deficiency in high affinity iron transport activity in the irr strain, and suggests that regulation of the heme pathway is coordinated with iron homeostasis. A high level of protoporphyrin accumulation is not a normal consequence of nutritional iron deprivation, thus a mechanism for iron-dependent control of heme biosynthesis may be present in other organisms.
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119. |
( 1998 ) Identification of the Bradyrhizobium japonicum degP gene as part of an operon containing small heat-shock protein genes. PMID : 9446679 : Abstract >>
A degP (htrA)-like gene of Bradyrhizobium japonicum was identified immediately downstream of two genes (hspB and hspC) coding for small heat-shock proteins. All three genes are oriented in the same direction and are separated by only 85 and 72 bp, and a heat-inducible transcript covering hspB, hspC, and degP was detected by RT-PCR. These results show that the genes are organized in an operon. Two mutants, a degP insertion mutant and a DeltahspBCdegP mutant, were constructed by marker replacement mutagenesis. Immunoblot analysis performed with a serum raised against the amino-terminal end of IbpA, an HspB homolog of Escherichia coli, identified three heat-inducible protein bands in B. japonicum extract, one of which was missing in the deletion mutant. None of the mutants showed an obvious defect during growth at different temperatures, after heat-shock treatment, or in the presence of solvents. Moreover, they were not affected in root-nodule symbiosis, indicating that the small heat-shock proteins HspB and HspC and the DegP homolog of B. japonicum are not required under a wide range of growth conditions.
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