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A0A6P1CI42_RHITR, a protein originating from Rhizobium tropici strain CIAT 899, has emerged as a key player in leguminous plant symbiosis and nitrogen fixation processes. Understanding the intricate details of its structure and function holds immense significance for unraveling the molecular mechanisms underlying its biological activities. In this study, we employed molecular modeling and molecular dynamics (MD) simulations to investigate the A0A6P1CI42_RHITR protein, with a specific emphasis on the influence of Fe-atoms, linker structural integrity, and conformational changes within the GAF domain. Our findings unveiled noteworthy conformational changes in the A0A6P1CI42_RHITR protein, particularly within the GAF domain, when Fe-atoms were present compared to its apo form. Significant conformational rearrangements after an initial 20 ns, accompanied by the opening of the ligand substrate accommodating loop in the GAF domain influenced by Fe-atoms was observed. At the residue level, the investigation revealed substantial activity variations in individual residues, particularly in those contributing to the GAF domain from positions 51 to 223. Intriguingly, the presence of Fe-atoms led to controlled movement of conserved cysteine residues at positions 467 and 472, indicating a correlation between interlinker domain motion and the activity of the GAF domain loop responsible for substrate accommodation. Moreover, in the presence of Fe-atoms, the distance between Cys467 and Cys472 residues was maintained, ensuring the overall structural integrity of the interdomain loop necessary for protein activation. Conversely, in the apo form, a sudden flip motion of cysteine residues' thiol groups was observed, leading to a loss of structural integration. Overall, our study utilizing molecular modeling and MD simulations offers valuable insights into the structural dynamics and functional implications of the A0A6P1CI42_RHITR protein.Communicated by Ramaswamy H. Sarma.

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The genome sequences of two nitrogen-fixing type strains of the Rhizobium tropici group were obtained: Rhizobium calliandrae CCGE524T and R. mayense CCGE526T. Genomic analyses confirmed their taxonomic position and identified three complete sequences of the repABC genes, indicative of three plasmids, one of them carrying symbiotic genes.

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Inoculants with beneficial microorganisms comprise both selected strains and carriers that ensure a favorable microenvironment for cell survival and stability. Formulations of inoculants using synthetic polymers as carriers are common. However, only a few studies are available in the literature regarding the formulation of inoculants using natural biomolecules as carriers. Exopolysaccharides (EPS) are biomolecules produced by a vast array of microbial species, including symbiotic nitrogen-fixing bacteria, commonly known as rhizobia. EPS perform several functions, such as the protection against the deleterious effects of diverse environmental soil stresses. Two Rhizobium tropici strains and one Paraburkholderia strain were selected after semiquantitative analysis by scanning electron microscopy (SEM) of their EPS production in liquid YMA medium. Their EPS were characterized through a series of analytical techniques, aiming at their use in the formulation of plant inoculants. In addition, the effect of the carbon source on EPS yield was evaluated. Multi-stage fragmentation analysis showed the presence of xylose, glucose, galactose, galacturonic acid, and glucuronic acid in EPS chemical composition, which was confirmed by FT-IR spectra and 13C NMR spectroscopy. Thermal stability (thermogravimetric) was close to 270 °C and viscosity ranged from 120 to 1053.3 mPa.s. Surface morphology (SEM) was rough and irregular, with a cross-linked spongy matrix, which, together with the hydrophilic functional groups, confers water holding capacity. The present study showed that the three EPS have potential as microorganism carriers for formulation of microbial inoculants to be applied in plants.

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In the symbiotic associations between rhizobia and legumes, the NodD regulators orchestrate the transcription of the specific nodulation genes. This set of genes is involved in the synthesis of nodulation factors, which are responsible for initiating the nodulation process. Rhizobium tropici CIAT 899 is the most successful symbiont of Phaseolus vulgaris and can nodulate a variety of legumes. Among the five NodD regulators present in this rhizobium, only NodD1 and NodD2 seem to have a role in the symbiotic process. However, the individual role of each NodD in the absence of the other proteins has remained elusive. In this work, we show that the CIAT 899 NodD2 does not require activation by inducers to promote the synthesis of nodulation factors. A CIAT 899 strain overexpressing nodD2, but lacking all additional nodD genes, can nodulate three different legumes as efficiently as the wild type. Interestingly, CIAT 899 NodD2-mediated gain of nodulation can be extended to another rhizobial species, since its overproduction in Sinorhizobium fredii HH103 not only increases the number of nitrogen-fixing nodules in two host legumes but also results in nodule development in incompatible legumes. These findings potentially open exciting opportunities to develop rhizobial inoculants and increase legume crop production.

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Rhizobial NodD proteins and appropriate flavonoids induce rhizobial nodulation gene expression. In this study, we show that the nodD1 gene of Sinorhizobium fredii HH103, but not the nodD2 gene, can restore the nodulation capacity of a double nodD1/nodD2 mutant of Rhizobium tropici CIAT 899 in bean plants (Phaseolus vulgaris). S. fredii HH103 only induces pseudonodules in beans. We have also studied whether the mutation of different symbiotic regulatory genes may affect the symbiotic interaction of HH103 with beans: ttsI (the positive regulator of the symbiotic type 3 protein secretion system), and nodD2, nolR and syrM (all of them controlling the level of Nod factor production). Inactivation of either nodD2, nolR or syrM, but not that of ttsI, affected positively the symbiotic behavior of HH103 with beans, leading to the formation of colonized nodules. Acetylene reduction assays showed certain levels of nitrogenase activity that were higher in the case of the nodD2 and nolR mutants. Similar results have been previously obtained by our group with the model legume Lotus japonicus. Hence, the results obtained in the present work confirm that repression of Nod factor production, provided by either NodD2, NolR or SyrM, prevents HH103 to effectively nodulate several putative host plants.

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Rhizobium tropici CIAT 899 is a broad-host-range rhizobial strain that establishes symbiotic interactions with legumes and tolerates different environmental stresses such as heat, acidity, or salinity. This rhizobial strain produces a wide variety of symbiotically active nodulation factors (NF) induced not only by the presence of plant-released flavonoids but also under osmotic stress conditions through the LysR-type transcriptional regulators NodD1 (flavonoids) and NodD2 (osmotic stress). However, the activation of NodD2 under high-osmotic-stress conditions remains elusive. Here, we have studied the role of a new AraC-type regulator (named as OnfD) in the symbiotic interaction of R. tropici CIAT 899 with Phaseolus vulgaris and Lotus plants. We determined that OnfD is required under salt stress conditions for the transcriptional activation of the nodulation genes and therefore the synthesis and export of NF, which are required for a successful symbiosis with P. vulgaris Moreover, using bacterial two-hybrid analysis, we demonstrated that the OnfD and NodD2 proteins form homodimers and OnfD/NodD2 form heterodimers, which could be involved in the production of NF in the presence of osmotic stress conditions since both regulators are required for NF synthesis in the presence of salt. A structural model of OnfD is presented and discussed.IMPORTANCE The synthesis and export of rhizobial NF are mediated by a conserved group of LysR-type regulators, the NodD proteins. Here, we have demonstrated that a non-LysR-type regulator, an AraC-type protein, is required for the transcriptional activation of symbiotic genes and for the synthesis of symbiotically active NF under salt stress conditions.

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Rhizobium tropici CIAT 899 is a facultative symbiotic diazotroph able to deal with stressful concentrations of metals. Nevertheless the molecular mechanisms involved in metal tolerance have not been elucidated. Copper (Cu2+) is a metal component essential for the heme-copper respiratory oxidases and enzymes that catalyse redox reactions, however, it is highly toxic when intracellular trace concentrations are surpassed. In this study, we report that R. tropici CIAT 899 is more tolerant to Cu2+ than other Rhizobium and Sinorhizobium species. Through Tn5 random mutagenesis we identify a R. tropici mutant strain with a severe reduction in Cu2+ tolerance. The Tn5 insertion disrupted the gene RTCIAT899_CH17575, encoding a putative heavy metal efflux P1B-1-type ATPase designated as copA. Phaseolus vulgaris plants inoculated with the copA::Tn5 mutant in the presence of toxic Cu2+ concentrations showed a drastic reduction in plant and nodule dry weight, as well as nitrogenase activity. Nodules induced by the copA::Tn5 mutant present an increase in H2O2 concentration, lipoperoxidation and accumulate 40-fold more Cu2+ than nodules formed by the wild-type strain. The copA::Tn5 mutant complemented with the copA gene recovered the wild-type symbiotic phenotypes. Therefore, the copA gene is essential for R. tropici CIAT 899 to survive in copper-rich environments in both free life and symbiosis with P. vulgaris plants.

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A novel exopolysaccharide (EPS) was produced by a bacterium which was isolated from Psophocarpus tetragonolobus (L) D.C. and identified as 99% Rhizobium tropici SRA1 by 16S rDNA sequencing. The flocculating performances along with emulsifying activity began simultaneously with the growth and the production of EPS and reached its utmost at 28 h. EPS was purified via chilled ethanol precipitation followed by dialysis and lyophilization. The existence of hydroxyl, methoxyl, and carboxylic functional groups were confirmed by Fourier transform infrared (FT-IR) spectrum. EPS was found to be compose of 82.44% neutral sugar and 15.93% uronic acid. The average molecular weight of the exopolysaccharide was estimated as ~ 1.8 × 105. Gas-liquid chromatography indicated the presence of glucose and galactose at a molar ratio of 3:1 in EPS. In the pH range of 3-5 with EPS dosage of 15 mg/l at 30 °C, cation-independent flocculation greater than 90% was observed. Emulsification indices (E24) of EPS were observed as 86.66%, 83.33%, 76.66%, and 73.33% with olive oil, kerosene, toluene, and n-hexane respectively. Biosorption of Cu K [45.69 wt%], Cu L [05.67 wt%], Co K [15.58 wt%], and Co L [11.72 wt%] by EPS was confirmed by energy-dispersive X-ray spectroscopy (EDS). This report on the flocculating, emulsifying, and metal sorption properties of EPS produced by R. tropici SRA1 is unique in the literature.

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Rhizobium tropici CIAT899 is a nodule-forming α-proteobacterium displaying intrinsic resistance to several abiotic stress conditions such as low pH and high temperatures, which are common in tropical environments. It is a good competitor for Phaseolus vulgaris (common bean) nodule occupancy at low pH values, however little is known about the genetic and physiological basis of the tolerance to acidic conditions. To identify genes in R. tropici involved in pH stress response we combined two different approaches: (1) A Tn5 mutant library of R. tropici CIAT899 was screened and 26 acid-sensitive mutants were identified. For 17 of these mutants, the transposon insertion sites could be identified. (2) We also studied the transcriptomes of cells grown under different pH conditions using RNA-Seq. RNA was extracted from cells grown for several generations in minimal medium at 6.8 or 4.5 (adapted cells). In addition, we acid-shocked cells pre-grown at pH 6.8 for 45 min at pH 4.5. Of the 6,289 protein-coding genes annotated in the genome of R. tropici CIAT 899, 383 were differentially expressed under acidic conditions (pH 4.5) vs. control condition (pH 6.8). Three hundred and fifty one genes were induced and 32 genes were repressed; only 11 genes were induced upon acid shock. The acid stress response of R. tropici CIAT899 is versatile: we found genes encoding response regulators and membrane transporters, enzymes involved in amino acid and carbohydrate metabolism and proton extrusion, in addition to several hypothetical genes. Our findings enhance our understanding of the core genes that are important during the acid stress response in R. tropici.

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Amino acid-containing acyloxyacyl lipids are composed of a 3-hydroxy fatty acid amide-bound to the α-amino group of an amino acid. A second fatty acid is ester-linked to the 3-hydroxy group of the first fatty acid. Most commonly, ornithine is the headgroup of these lipids, but glycine, serineglycine, glutamine and lysine have also been described in bacteria. Ornithine lipids (OL) can be synthesized by about 50% of the sequenced bacterial species, and several covalent modifications of its basic structure have been described. The OL hydroxylase OlsE is widespread in Rhizobium and Agrobacterium species and is responsible for introducing a hydroxyl group at a hence unknown position within the ornithine headgroup causing the formation of the OL named S2. Using NMR on purified OL S2, we show that the OlsE-mediated hydroxylation takes place at the C-4 position of the ornithine headgroup. Furthermore, we identify a hydroxylase in the genome of Pseudopedobacter saltans, distantly related to OlsE from α-proteobacteria, able to hydroxylate the headgroup of both ornithine lipids and lysine lipids. A homology search with the amino acid sequence of this hydroxylase allows us to predict that OL headgroup hydroxylation is not restricted to a few α-proteobacteria, but is apparently also common in many genera belonging to the Cytophaga-Flavobacterium-Bacteroidetes (CFB) group of bacteria.

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Reactive oxygen species (ROS) produced by respiratory burst oxidase homologs (RBOHs) regulate numerous plant cell processes, including the symbiosis between legumes and nitrogen-fixing bacteria. Rapid and transient ROS production was reported after Phaseolus vulgaris root hairs were treated with Nod factors, indicating the presence of a ROS-associated molecular signature in the symbiosis signaling pathway. Rboh is a multigene family containing nine members (RbohA-I) in P. vulgaris. RNA interference of RbohB suppresses ROS production and attenuates rhizobial infection thread (IT) progression in P. vulgaris root hairs. However, the roles of other Rboh members in symbiotic interactions are largely unknown. In this study, we characterized the role of the NADPH oxidase-encoding gene RbohA (Phvulv091020621) in the P. vulgaris-Rhizobium tropici symbiosis. The spatiotemporal activity of the RbohA promoter colocalized with growing ITs and was associated with vascular bundles in developing nodules. Subcellular localization studies indicated that RBOHA was localized in the plasma membrane of P. vulgaris root hairs. After rhizobial inoculation, PvRBOHA was mainly distributed in the infection pocket and, to a lesser extent, throughout the IT. In PvRbohA RNAi lines, the rhizobial infection events were significantly reduced and, in successful infections, IT progression was arrested within the root hair, but did not impede cortical cell division. PvRbohA-RNAi nodules failed to fix nitrogen, since the infected cells in the few nodules formed were empty. RbohA-dependent ROS production and upregulation of several antioxidant enzymes was attenuated in rhizobia-inoculated PvRbohA-RNAi roots. These combined results indicate that PvRbohA is crucial for effective Rhizobium infection and its release into the nodule cells. This oxidase is partially or indirectly required to promote nodule organogenesis, altering the expression of auxin- and cyclin-related genes and genes involved in cell growth and division.

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BACKGROUND: Testing rhizobial inoculation of common bean (Phaseolus vulgaris L.) in hydroponics enables accurate quantification of biological N2 fixation (BNF) and provides information about the potential of reducing inorganic N fertilizer use. In view of this background, common bean grown on pumice was inoculated with Rhizobium tropici CIAT899 (Rt) and supplied with either full-N (total nitrogen 11.2 mmol L-1 ), 1/3 of full-N or N-free nutrient solution (NS). BNF was quantified at the early pod-filling stage using the 15 N natural abundance method. RESULTS: Full-N supply to Rt-inoculated plants resulted in markedly smaller nodules than less- or zero-N supply, and no BNF. Rt inoculation of full-N-treated plants did not increase biomass and pod yield compared with non-inoculation. Restriction (1/3 of full-N) or omission of inorganic N resulted in successful nodulation and BNF (54.3 and 49.2 kg N ha-1 , corresponding to 58 and 100% of total plant N content respectively) but suppressed dry shoot biomass from 191.7 (full-N, +Rt) to 107.4 and 43.2 g per plant respectively. Nutrient cation uptake was reduced when inorganic N supply was less or omitted. CONCLUSION: Rt inoculation of hydroponic bean provides no advantage when full-N NS is supplied, while 1/3 of full-N or N-free NS suppresses plant biomass and yield, partly because the restricted NO3- supply impairs cation uptake. © 2017 Society of Chemical Industry.

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Exopolysaccharide (EPS) are produced by a diverse of rhizobia species and has been demonstrated to be a bioemulsifier with potential applications in the degradation of hydrocarbons. In the present study, attempts were made to obtain the new exopolysaccharide production by Rhizobium tropici (SEMIA 4080 and MUTZC3) strains during growth on hydrocarbon substrate. Under the different cultivation conditions, the high molecular weight exopolysaccharides from Rhizobium tropici strains cultivated for 96h mainly consisted of carbohydrates (79-85%) and a low percentage of protein. The EPSC3-D differed from the others, with only 60% of carbohydrate. However, all strains produced polymers with distinct rheology properties, such as viscosity of each EPS sample, suitable for different applications. In addition, RP-HPLC, FTIR and NMR studies revealed EPS produced by rhizobia strains were similar indicating minimal difference between EPS compositions.

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Respiratory oxidative burst homolog (RBOH)-mediated reactive oxygen species (ROS) regulate a wide range of biological functions in plants. They play a critical role in the symbiosis between legumes and nitrogen-fixing bacteria or arbuscular mycorrhizal (AM) fungi. For instance, overexpression of PvRbohB enhances nodule numbers, but reduces mycorrhizal colonization in Phaseolus vulgaris hairy roots and downregulation has the opposite effect. In the present study, we assessed the effect of both rhizobia and AM fungi on electrolyte leakage in transgenic P. vulgaris roots overexpressing (OE) PvRbohB. We demonstrate that elevated levels of electrolyte leakage in uninoculated PvRbohB-OE transgenic roots were alleviated by either Rhizobium or AM fungi symbiosis, with the latter interaction having the greater effect. These results suggest that symbiont colonization reduces ROS elevated electrolyte leakage in P. vulgaris root cells.

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The potential use of rhizobia under controlled fermentation conditions may result in the production of new extracellular polymeric substances (EPS) having novel and superior properties that will open up new areas of industrial applications and thus increase their demand. The production of EPS and the stability of emulsions formed with soybean oil, diesel oil and toluene using different concentrations of purified EPS derived from wild-type and mutant strains of Rhizobium tropici SEMIA 4077 was investigated. The EPS was defined as a heteropolysaccharide composed of six constituent monosaccharides that displayed higher intrinsic viscosity and pseudoplastic non-Newtonian fluid behavior in an aqueous solution. The ratio between the total EPS production and the cellular biomass was 76.70 for the 4077::Z04 mutant strain and only 8.10 for the wild-type strain. The EPS produced by the wild-type R. tropici SEMIA 4077 resulted in more stable emulsions with the tested toluene than xanthan gum, and the emulsification indexes with hydrocarbons and soybean oil were higher than 50%, indicating strong emulsion-stabilizing capacity. These results demonstrate that the EPS of R. tropici strains could be attractive for use in industrial and environmental applications, as it had higher intrinsic viscosity and good emulsification activity.

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Rhizobium tropici, a member of the Rhizobiaceae family, has the ability to synthesize and secrete extracellular polysaccharides (EPS). Rhizobial EPS have attracted much attention from the scientific and industrial communities. Rhizobial isolates and R. tropici mutants that produced higher levels of EPS than the wild-type strain SEMIA4080 were used in the present study. The results suggested a heteropolymer structure for these EPS composed by glucose and galactose as prevailing monomer unit. All EPS samples exhibited a typical non-Newtonian and pseudoplastic fluid flow, and the aqueous solutions apparent viscosities increased in a concentration-dependent manner. These results serve as a foundation for further studies aimed at enhancing interest in the application of the MUTZC3, JAB1 and JAB6 strains with high EPS production and viscosity can be exploited for the large-scale commercial production of Rhizobial polysaccharides.

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The aim of this study was to evaluate the effects of seed inoculation with Rhizobium tropici and nitrogen and molybdenum fertilization on the performance of the common-bean (Phaseolus vulgaris L.). A complete randomized block design with four replicates was used, in a factorial arrangement 2x2x2, with the combinations of absence and presence of seed inoculation and nitrogen (N) and molybdenum (Mo) fertilization. Seeds inoculation was just before sowing with a peat inoculante composed by the strain CIAT 899 (SEMIA 4077), N was provided at sowing (10 kg.ha-1) and coverage (50 kg.ha-1) when the plants showed the third trifoliate sheet expand and Mo was applied by foliar spraying with 60 g.ha-1 when plants were at beginning of flowering . No effect of treatments on the dry mass of roots and the number of pods per plant was observed. N fertilization reduced nodulation of plants, however, N fertilization lead to an increase in height and dry mass of bean shoots. It was also observed that plants fertilized with N but in the absence of Mo shoed heavier grains. The interactions between N and Mo fertilization with inoculation of seed negatively affected the leaf N content and the number of grains per pod. The inoculation of seed in beans provided grain yields similar to those provided by the treatment with N fertilization.

O objetivo deste estudo foi avaliar o desempenho do feijoeiro-comum (Phaseolus vulgaris L) em função da inoculação de sementes com Rhizobium tropici e das adubações nitrogenada e molibdica. Utilizou-se o delineamento em blocos casualizados, com quatro repetições, em arranjo fatorial 2x2x2, constituindo-se das combinações de ausência e presença de inoculação de sementes, da adubação molíbdica e da adubação nitrogenada. A inoculação das sementes foi imediatamente antes da semeadura com inoculante turfoso composto da estirpe CIAT 899 (SEMIA 4077); a adubação nitrogenada foi na semeadura (10 kg.ha-1) e em cobertura (50 kg.ha-1), quando as plantas apresentaram a terceira folha trifoliolada expandida e o molibdênio (Mo) foi aplicado em pulverização foliar na dose de 60 g.ha-1. Não foram observados efeitos dos tratamentos para a massa seca de raízes e o número de vagens por planta. A adubação nitrogenada reduziu a nodulação nos feijoeiros. Todavia, com a adubação nitrogenada foi verificado incremento na altura e na massa seca da parte aérea dos feijoeiros. Grãos de feijão mais pesados foram observados em feijoeiros adubados com N na ausência de Mo. As interações entre adubação nitrogenada e molibdica com inoculação de sementes afetaram, também, o teor de N foliar e o número de grãos por vagem A inoculação de sementes proporcionou nos feijoeiros rendimentos de grãos semelhantes aos fertilizados com N.