Phylogenetic and characterization of salt-tolerant rhizobial strain nodulating faba bean plants

Improvement of faba bean production in the new reclamation land in Egypt requires isolation and selection of effective abiotic stress tolerant rhizobial strains. Three rhizobial strains were isolated from healthy faba bean plants growing in different geographic areas in Egypt. These isolates were adapted against different concentrations of NaCl (100, 150 and 200 mM) by using the enrichment method. They were evaluated by measuring the symbiotic N2-fixation parameters under greenhouse and field conditions during two seasons (2010/2011 and 2011/2012). One rhizobial strain exhibited the highest values of symbiotic N2-parameters, nitrogenase activity and proline content. Based on 16S rDNA and nifH gene sequence, this strain was shown to belong to the Rhizobium leguminosarum bv. viciae. A strong similarity was found between the 16S rDNA and nifH gene sequence of the strain E15 and R. leguminosarum bv. viciae 3841 (100% similarity for 16S rDNA and 95% similarity for nifH gene). The results show that the maximum growth of this strain was obtained at pH 7 and 30°C. This strain was tolerant to drought stress till 20% polyethylene glycol and it yielded the highest concentrations of indole-3-acetic acid (IAA) at the end of the logarithmic phase. This strain solubilized inorganic phosphorus. R. leguminosarum bv. viciae was able to survive, persist, grow and effectively nodulated faba bean plants at high salt concentrations under greenhouse and field conditions and it could be used for biofertilization to reduce the severe effects of salinity and drought stress in the new reclamation land in Egypt.Keywords: Vicia faba, Rhizobium leguminosarum bv. viciae, abiotic stress, nifH geneAfrican Journal of Biotechnology Vol. 12(27), pp. 4324-433

Organization and partial sequence of a DNA region of the Rhizobium leguminosarum symbiotic plasmid pRL6JI containing the genes fixABC, nifA, nifB and a novel open reading frame

Grönger P, Manian SS, Reiländer H, O´Connell M, Priefer UB, Pühler A. Organization and partial sequence of a DNA region of the Rhizobium leguminosarum symbiotic plasmid pRL6JI containing the genes fixABC, nifA, nifB and a novel open reading frame. Nucleic Acids Research. 1987;15(1):31-49.By hybridization and heteroduplex studies the fixABC and nifA genes of the Rhizobium leguminosarum symbiotic plasmid pRL6JI have been identified. DNA sequencing of the region containing nifA showed an open reading frame of 1557 bp encoding a protein of 56,178 D. Based on sequence homology, this ORF was confirmed to correspond to the nifA gene. Comparison of three nifA proteins (Klebsiella pneumoniae, Rhizobium meliloti, Rhizobium leguminosarum) revealed only a weak relationship in their N-terminal regions, whereas the C-terminal parts exhibited strong homology. Sequence analysis also showed that the R. leguminosarum nifA gene is followed by nifB and preceded by fixC with an open reading frame inserted in between. This novel ORF of 294 bp was found to be highly conserved also in R. meliloti. No known promoter and termination signals could be defined on the sequenced R. leguminosarum fragment

Characterisation of the fixABCX operon in symbiotic nitrogen fixation

The fixABCX genes are essential for nitrogen fixation in R. leguminosarum bv. viciae 3841. Comparison of FixABCX to homologous proteins across the Kingdoms of Life suggests a role in electron transport to nitrogenase, which requires eight electrons per molecule of dinitrogen fixed. Mutation of this operon leads to bacteroids unable to fix nitrogen in symbiosis with P. sativum (pea). Electron microscopy revealed a drastically altered bacteroid morphology in fixAB mutants, revealing insights into the developmental response of both plant and bacteria to a lack of nitrogen fixation. Observations from electron microscopy were coupled to data obtained using single-cell Raman microscopy in order to understand metabolite production in nitrogen fixing and non-fixing bacteroids. The promoter controlling fixA has been characterised to a minimal region consisting of binding sites for NifA, the general transcriptional activator of nitrogen fixation, and RpoN (σ54), its cognate sigma factor. Mutation analysis reveals that fixABCX is part of a larger operon including the nifA gene. Promoter analysis of the downstream genes has identified a set of basal promoters found within the fixCX region, which control expression of the nifA gene. Control of nitrogen fixation occurs at the post-transcriptional level, whereby NifA is able to activate nitrogen fixation genes, including the fixABCXnifA operon, autoregulating its own expression under nitrogen-fixing conditions. Pull-down assays have revealed protein-protein interactions between FixAB and nitrogenase, as well as an interaction with both pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase. FixAB may interact with these dehydrogenases and via electron bifurcation couple the exergonic reduction of the quinone pool to the endergonic reduction of ferredoxin and subsequently nitrogenase. Furthermore, FixAB, nitrogenase and pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase may form a supra-molecular complex within nitrogen fixing bacteroids