Optimización de inoculantes para la agricultura sustentable en soja: rol de los flagelos de Bradyrhizobium japonicum en la competición para la nodulación

Objetivos de este trabajo de tesis Objetivo general: Generar nuevo conocimiento acerca de la movilidad de B. japonicum. A partir de ello procurar una mejora en la competición para la nodulación de las cepas de B. japonicum empleadas como inoculantes. Objetivos específicos: - Analizar el comportamiento de una cepa de mayor movilidad en la competición para la nodulación, seleccionada de forma natural (sin manipulación genética) y mediante una técnica reproducible. - Analizar la estructura de los flagelos de B. japonicum, su expresión y su contribución a la simbiosis con soja en medios porosos a capacidad de campo.Facultad de Ciencias Exacta

Transcriptional control of the lateral-flagellar genes of Bradyrhizobium diazoefficiens

Bradyrhizobium diazoefficiens, a soybean N2-fixing symbiont, possesses a dual flagellar system comprising a constitutive subpolar flagellum and inducible lateral flagella. Here, we analyzed the genomic organization and biosynthetic regulation of the lateral-flagellar genes. We found that these genes are located in a single genomic cluster, organized in two monocistronic transcriptional units and three operons, one possibly containing an internal transcription start site. Among the monocistronic units is blr6846, homologous to the class IB master regulators of flagellum synthesis in Brucella melitensis and Ensifer meliloti and required for the expression of all the lateral-flagellar genes except lafA2, whose locus encodes a single lateral flagellin. We therefore named blr6846 lafR (lateral-flagellar regulator). Despite its similarity to two-component response regulators and its possession of a phosphorylatable Asp residue, lafR behaved as an orphan response regulator by not requiring phosphorylation at this site. Among the genes induced by lafR is flbTL, a class III regulator. We observed different requirements for FlbTL in the synthesis of each flagellin subunit. Although the accumulation of lafA1, but not lafA2, transcripts required FlbTL, the production of both flagellin polypeptides required FlbTL. Moreover, the regulation cascade of this lateral-flagellar regulon appeared to be not as strictly ordered as those found in other bacterial species.Instituto de Biotecnologia y Biologia Molecula

Transcriptional Control of the Lateral-Flagellar Genes of Bradyrhizobium diazoefficiens

Bradyrhizobium diazoefficiens, a soybean N2-fixing symbiont, possesses a dual flagellar system comprising a constitutive subpolar flagellum and inducible lateral flagella. Here, we analyzed the genomic organization and biosynthetic regulation of the lateral-flagellar genes. We found that these genes are located in a single genomic cluster, organized in two monocistronic transcriptional units and three operons, one possibly containing an internal transcription start site. Among the monocistronic units is blr6846, homologous to the class IB master regulators of flagellum synthesis in Brucella melitensis and Ensifer meliloti and required for the expression of all the lateral-flagellar genes except lafA2, whose locus encodes a single lateral flagellin. We therefore named blr6846 lafR (lateral-flagellar regulator). Despite its similarity to two-component response regulators and its possession of a phosphorylatable Asp residue, lafR behaved as an orphan response regulator by not requiring phosphorylation at this site. Among the genes induced by lafR is flbTL, a class III regulator. We observed different requirements for FlbTL in the synthesis of each flagellin subunit. Although the accumulation of lafA1, but not lafA2, transcripts required FlbTL, the production of both flagellin polypeptides required FlbTL. Moreover, the regulation cascade of this lateral-flagellar regulon appeared to be not as strictly ordered as those found in other bacterial species.Fil: Mongiardini, Elias Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Quelas, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Dardis, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Althabegoiti, Maria Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Lodeiro, Anibal. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentin

Swimming performance of Bradyrhizobium diazoefficiens is an emergent property of its two flagellar systems

Many bacterial species use flagella for self-propulsion in aqueous media. In the soil, which is a complex and structured environment, water is found in microscopic channels where viscosity and water potential depend on the composition of the soil solution and the degree of soil water saturation. Therefore, the motility of soil bacteria might have special requirements. An important soil bacterial genus is Bradyrhizobium, with species that possess one flagellar system and others with two different flagellar systems. Among the latter is B. diazoefficiens, which may express its subpolar and lateral flagella simultaneously in liquid medium, although its swimming behaviour was not described yet. These two flagellar systems were observed here as functionally integrated in a swimming performance that emerged as an epistatic interaction between those appendages. In addition, each flagellum seemed engaged in a particular task that might be required for swimming oriented toward chemoattractants near the soil inner surfaces at viscosities that may occur after the loss of soil gravitational water. Because the possession of two flagellar systems is not general in Bradyrhizobium or in related genera that coexist in the same environment, there may be an adaptive tradeoff between energetic costs and ecological benefits among these different species.Fil: Quelas, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Althabegoiti, Maria Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Jiménez Sánchez, Celia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentina. Instituto de Recursos Naturales y Agrobiología de Sevilla; EspañaFil: Melgarejo, Augusto. Universidad Nacional de La Plata. Facultad de Ingeniería; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Marconi, Veronica Iris. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Física Enrique Gaviola. Universidad Nacional de Córdoba. Instituto de Física Enrique Gaviola; ArgentinaFil: Mongiardini, Elias Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Trejo, Sebastian Alejandro. Universitat Autònoma de Barcelona; España. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mengucci, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Ortega Calvo, José Julio. Instituto de Recursos Naturales y Agrobiología de Sevilla; EspañaFil: Lodeiro, Anibal. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentin

Dual control of flagellar synthesis and exopolysaccharide production by Flbd-Flix Class II regulatory proteins in bradyrhizobium diazoefficiens

Bradyrhizobium diazoefficiens, the N2-fixing symbiont of soybean, has two independent flagellar systems: A single subpolar flagellum and several lateral flagella. Each flagellum is a very complex organelle composed of 30 to 40 different proteins located inside and outside the cell whereby flagellar gene expression must be tightly controlled. Such control is achieved by a hierarchy of regulators that ensure the timing of synthesis and the allocation of the different flagellar substructures. Previously, we analyzed the gene organization, expression, and function of the lateral flagellar system. Here, we studied the role of the response regulator FlbD and its trans-acting regulator FliX in the regulation of subpolar flagellar genes. We found that the LP-ring, distal rod, and hook of the subpolar flagellum were tightly controlled by FlbD and FliX. Furthermore, we obtained evidence for the existence of cross-regulation between these gene products and the expression of LafR, the master regulator of lateral flagella. In addition, we observed that extracellular polysaccharide production and biofilm formation also responded to these flagellar regulators. In this regard, FlbD might contribute to the switch between the planktonic and sessile states.Fil: Dardis, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Quelas, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Mengucci, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Althabegoiti, Maria Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Lodeiro, Anibal. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Agrarias y Forestales. Departamento de Ciencias Biológicas. Laboratorio de Genética; ArgentinaFil: Mongiardini, Elias Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentin

Characterization of Rhizobium grahamii extrachromosomal replicons and their transfer among rhizobia

Background: Rhizobium grahamii belongs to a new phylogenetic group of rhizobia together with Rhizobium mesoamericanum and other species. R. grahamii has a broad-host-range that includes Leucaena leucocephala and Phaseolus vulgaris, although it is a poor competitor for P. vulgaris nodulation in the presence of Rhizobium etli or Rhizobium phaseoli strains. This work analyzed the genome sequence and transfer properties of R. grahamii plasmids. Results: Genome sequence was obtained from R. grahamii CCGE502 type strain isolated from Dalea leporina in Mexico. The CCGE502 genome comprises one chromosome and two extrachromosomal replicons (ERs), pRgrCCGE502a and pRgrCCGE502b. Additionally, a plasmid integrated in the CCGE502 chromosome was found. The genomic comparison of ERs from this group showed that gene content is more variable than average nucleotide identity (ANI). Well conserved nod and nif genes were found in R. grahamii and R. mesoamericanum with some differences. R. phaseoli Ch24-10 genes expressed in bacterial cells in roots were found to be conserved in pRgrCCGE502b. Regarding conjugative transfer we were unable to transfer the R. grahamii CCGE502 symbiotic plasmid and its megaplasmid to other rhizobial hosts but we could transfer the symbiotic plasmid to Agrobacterium tumefaciens with transfer dependent on homoserine lactones. Conclusion: Variable degrees of nucleotide identity and gene content conservation were found among the different R. grahamii CCGE502 replicons in comparison to R. mesoamericanum genomes. The extrachromosomal replicons from R. grahamii were more similar to those found in phylogenetically related Rhizobium species. However, limited similarities of R. grahamii CCGE502 symbiotic plasmid and megaplasmid were observed in other more distant Rhizobium species. The set of conserved genes in R. grahamii comprises some of those that are highly expressed in R. phaseoli on plant roots, suggesting that they play an important role in root colonization.Instituto de Biotecnologia y Biologia Molecula

An inoculant Bradyrhizobium strain with increased motility improves yield of soybean crops

Poster y resumenSoybean production is very important in Argentina, where more than 20 million hectares are sowed annually with this crop. Since soybean plants possess a very high N-demand, it is crucial that this agricultural activity is developed in a sustainable way because otherwise, this crop could deplete Nnutrition from the soils, leading to erosion, compaction, and flooding. Soybean roots are nodulated by Bradyrhizobium spp., which may fix atmospheric N2 in symbiosis with the plant, thus contributing to keep the N-status of the soil. For this reason and due to their low cost, Bradyrhizobium spp. are widely used in inoculants for soybean crops. However, the efficiency of inoculants is low due to the competition exerted by bradyrhizobia resident in the soil. Among factors that affect the competition for nodulation is the self-propelled motility of the rhizobia. Previously, we developed an artificial selection method to obtain bradyrhizobial strains with higher motility. B. japonicum E109 is the strain recommend by INTA for inoculants production. Therefore, we used E109 to increase its motility, and hereby we obtained the derived B. japonicum E109 m+ strain. This strain possesses 50% more motility than its parental strain in semisolid agar medium, and has the same growth kinetics as the wild type, ruling out the possibility that the increased spreading of E109 m+ in semisolid agar be due to faster growth. B. diazoefficiens have two flagella systems, one subpolar and another lateral, characterized by flagellins of different molecular weights. The subpolar flagellum has constitutive expression and the lateral is inducible with L-arabinose as carbon source, but not with D-mannitol as carbon source. However, E109 m+ expressed both flagella with D-mannitol, as observed with SDS-PAGE of purified flagellins, in agreement with previous results obtained with B. diazoefficiens USDA 110. B. japonicum E109 m+ nodulated soybean and after that, bacteria recovered from nodules maintained the higher motility phenotype. Field trials were performed to estimate yield when the soybean plots were inoculated with E109 m+ or the E109 parental strain. Experiments were carried out in San Antonio de Areco, Province of Buenos Aires, in a soil with a competitive resident Bradyrhizobium spp. population, employing a randomized complete block design that included uninoculated controls. Grain yields were compared by ANOVA, which indicated that inoculation with E109 m+ led to significantly higher yield than inoculation with E109 wild type. Our results suggested that inoculation of soybean with improved motility strains could increase soybean yield by enhancing competition for nodulation in a sustainable way.Instituto de Microbiología y Zoología Agrícola (IMYZA)Fil: Colla, Delfina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biotecnología y Biología Molecular; Argentina. Universidad Nacional de La Plata. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Brignoli, Damián.Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biotecnología y Biología Molecular; Argentina. Universidad Nacional de La Plata. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Iturralde, Esteban Tomás.Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biotecnología y Biología Molecular; Argentina. Universidad Nacional de La Plata. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Covelli, Julieta Mariana. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Bioquímica, Microbiología e Interacciones Biológicas en el Suelo; ArgentinaFil: Althabegoiti, María Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biotecnología y Biología Molecular; Argentina. Universidad Nacional de La Plata. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Perticari, Alejandro. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Lodeiro, Aníbal Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biotecnología y Biología Molecular; Argentina. Universidad Nacional de La Plata. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Lodeiro, Aníbal Roberto. Universidad Nacional de La Plata. Facultad de Ciencias Agrarias y Forestales. Laboratorio de Genética; Argentin

The rhizobial adhesion protein RapA1 is involved in adsorption of rhizobia to plant roots but not in nodulation

The effect of the rhizobium adhesion protein RapA1 on Rhizobium leguminosarum bv. trifolii adsorption to Trifolium pratense (red clover) roots was investigated. We altered RapA1 production by cloning its encoding gene under the plac promoter into the stable vector pHC60. After introducing this plasmid in R. leguminosarum bv. trifolii, three to four times more RapA1 was produced, and two to five times higher adsorption to red clover roots was obtained, as compared with results for the empty vector. Enhanced adsorption was also observed on soybean and alfalfa roots, not related to R. leguminosarum cross inoculation groups. Although the presence of 1 mM Ca2+ during rhizobial growth enhanced adsorption, it was unrelated to RapA1 level. Similar effects were obtained when the same plasmid was introduced in Rhizobium etli for its adsorption to bean roots. Although root colonization by the RapA1-overproducing strain was also higher, nodulation was not enhanced. In addition, in vitro biofilm formation was similar to the wild-type both on polar and on hydrophobic surfaces. These results suggest that RapA1 receptors are present in root but not on inert surfaces, and that the function of this protein is related to rhizosphere colonization.Facultad de Ciencias Exacta

Soybean Lectin Enhances Biofilm Formation by Bradyrhizobium japonicum in the Absence of Plants

Soybean lectin (SBL) purified from soybean seeds by affinity chromatography strongly bound to Bradyrhizobium japonicum USDA 110 cell surface. This lectin enhanced biofilm formation by B. japonicum in a concentration-dependent manner. Presence of galactose during biofilm formation had different effects in the presence or absence of SBL. Biofilms were completely inhibited in the presence of both SBL and galactose, while in the absence of SBL, galactose was less inhibitory. SBL was very stable, since its agglutinating activity of B. japonicum cells as well as of human group A+ erythrocytes was resistant to preincubation for one week at 60°C. Hence, we propose that plant remnants might constitute a source of this lectin, which might remain active in soil and thus favor B. japonicum biofilm formation in the interval between soybean crop seasons

Soybean Seed Lectin Prevents the Accumulation of S-Adenosyl Methionine Synthetase and the S1 30S Ribosomal Protein in <i>Bradyrhizobium japonicum</i> Under C and N Starvation

Soybean lectin (SBL) participates in the recognition between Bradyrhizobium japonicum and soybean although its role remains unknown. To search for changes in the proteome in response to SBL, B. japonicum USDA 110 was incubated for 12 h in a C- and N-free medium with or without SBL (10 μg ml ⁻¹), and the soluble protein profiles were compared. Two polypeptides, S-adenosyl-methionine synthetase (MetK) and the 30S ribosomal protein S1 (RpsA), were found only in the fractions from rhizobia incubated without SBL. Transcript levels of metK and rpsA were not correlated with polypeptide levels, indicating that there was regulation at translation. In support of this proposal, the 5′ translation initiation-region of rpsA mRNA contained folding elements as those involved in regulation of its translation in other species. Disappearance of MetK and RpsA from the soluble protein fractions of SBL-treated rhizobia suggests that SBL might have attenuated the nutritional stress response of B. japonicum.Instituto de Biotecnología y Biología Molecula