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

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

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

Analysis of the role of the two flagella of Bradyrhizobium japonicum in competition for nodulation of soybean

Bradyrhizobium japonicum has two types of flagella. One has thin filaments consisting of the 33-kDa flagellins FliCI and FliCII (FliCI-II) and the other has thick filaments consisting of the 65-kDa flagellins FliC1, FliC2, FliC3, and FliC4 (FliC1-4). To investigate the roles of each flagellum in competition for nodulation, we obtained mutants deleted in fliCI-II and/or fliC1-4 in the genomic backgrounds of two derivatives from the reference strain USDA 110: the streptomycin-resistant derivative LP 3004 and its more motile derivative LP 3008. All mutations diminished swimming motility. When each mutant was co-inoculated with the parental strain on soybean plants cultivated in vermiculite either at field capacity or flooded, their competitiveness differed according to the flagellin altered. ΔfliCI-II mutants were more competitive, occupying 64-80% of the nodules, while ΔfliC1-4 mutants occupied 45-49% of the nodules. Occupation by the nonmotile double mutant decreased from 55% to 11% as the water content of the vermiculite increased from 85% to 95% field capacity to flooding. These results indicate that the influence of motility on competitiveness depended on the water status of the rooting substrate.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.Instituto de Biotecnologia y Biologia Molecula

Soybean lectin enhances biofilm formation by Bradyrhizobium japonicum in the absence of plants

Soybean lectin (SBL) purified fromsoybean 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.Facultad de Ciencias ExactasInstituto de Biotecnologia y Biologia Molecula

Strain selection for improvement of Bradyrhizobium japonicum competitiveness for nodulation of soybean

A Bradyrhizobium japonicum USDA 110-derived strain able to produce wider halos in soft-agar medium than its parental strain was obtained by recurrent selection. It was more chemotactic than the wild type towards mannitol and three amino acids. When cultured in minimal medium with mannitol as a single carbon-source, it had one thick subpolar flagellum as the wild type, plus several other flagella that were thinner and sinusoidal. Root adsorption and infectivity in liquid media were 50-100% higher for the selected strain, but root colonization in water-unsaturated vermiculite was similar to the wild type. A field experiment was then carried out in a soil with a naturalized population of 1.8 × 105 soybean-nodulating rhizobia g of soil -1. Bradyrhizobium japonicum strains were inoculated either on the soybean seeds or in the sowing furrows. Nodule occupation was doubled when the strains were inoculated in the sowing furrows with respect to seed inoculation (significant with P<0.05). On comparing strains, nodule occupation with seed inoculation was 6% or 10% for the wild type or selected strains, respectively, without a statistically significant difference, while when inoculated in the sowing furrows, nodule occupation increased to 12% and 22%, respectively (differences significant with P<0.05).Instituto de Biotecnologia y Biologia Molecula

Analysis of the role of the two flagella of Bradyrhizobium japonicum in competition for nodulation of soybean

Bradyrhizobium japonicum has two types of flagella. One has thin filaments consisting of the 33-kDa flagellins FliCI and FliCII (FliCI-II) and the other has thick filaments consisting of the 65-kDa flagellins FliC1, FliC2, FliC3, and FliC4 (FliC1-4). To investigate the roles of each flagellum in competition for nodulation, we obtained mutants deleted in fliCI-II and/or fliC1-4 in the genomic backgrounds of two derivatives from the reference strain USDA 110: the streptomycin-resistant derivative LP 3004 and its more motile derivative LP 3008. All mutations diminished swimming motility. When each mutant was co-inoculated with the parental strain on soybean plants cultivated in vermiculite either at field capacity or flooded, their competitiveness differed according to the flagellin altered. ΔfliCI-II mutants were more competitive, occupying 64-80% of the nodules, while ΔfliC1-4 mutants occupied 45-49% of the nodules. Occupation by the nonmotile double mutant decreased from 55% to 11% as the water content of the vermiculite increased from 85% to 95% field capacity to flooding. These results indicate that the influence of motility on competitiveness depended on the water status of the rooting substrate.Instituto de Biotecnologia y Biologia Molecula