Optimización del tiempo de esterilización de soportes basados en suelo y compost en la producción de inoculentes para leguminosas

Se elaboraron tres soportes consistentes en una mezcla de suelo y compost, y se sometieron a tres tratamientos de esterilización fraccionada en autoclave en dos días consecutivos. La eficiencia de los tratamientos de esterilización se evaluó monitoreando la disminución en las poblaciones de hongos y bacterias mediante recuentos estándar en placa. La esterilización de todos los soportes se logró mediante un tratamiento por 45 minutos el primer día y 30 minutos el segundo día

Optimización del tiempo de esterilización de soportes basados en suelo y compost en la producción de inoculentes para leguminosas

Three soil-compost based carrriers were elaborated and autoclaved by three different treatments of fractionated sterilization in two consecutive days. The efficiency of the sterilization treatments was evaluated monitoring the reduction in bacterial and fungi populations by standard plate count methods. Sterilization of all carriers was possible by the treatment of 45 minutes the first day and 30 minutes the second day.Se elaboraron tres soportes consistentes en una mezcla de suelo y compost, y se sometieron a tres tratamientos de esterilización fraccionada en autoclave en dos días consecutivos. La eficiencia de los tratamientos de esterilización se evaluó monitoreando la disminución en las poblaciones de hongos y bacterias mediante recuentos estándar en placa. La esterilización de todos los soportes se logró mediante un tratamiento por 45 minutos el primer día y 30 minutos el segundo día

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.Fil: Althabegoiti, Maria Julia. Universidad Nacional Autónoma de México; México. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ormeño Orrillo, Ernesto. Universidad Nacional Autónoma de México; MéxicoFil: Lozano, Luis. Universidad Nacional Autónoma de México; MéxicoFil: Torres Tejerizo, Gonzalo Arturo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional Autónoma de México; MéxicoFil: Rogel, Marco Antonio. Universidad Nacional Autónoma de México; MéxicoFil: Mora, Jaime. Universidad Nacional Autónoma de México; MéxicoFil: Martinez Romero, Esperanza. Universidad Nacional Autónoma de México; Méxic

Genome sequences of Burkholderia sp. Strains CCGE1002 and H160, isolated from legume nodules in Mexico and Brazil.

The genome sequences of Burkholderia sp. strains CCGE1002 from Mexico and H160 from Brazil, isolated from legume nodules, are reported. Their gene contents in relation to plant-microbe interactions and xenobiotic degradation are discussed

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

Genome Sequence of the Symbiotic Type Strain Rhizobium tibeticum CCBAU85039T

Rhizobium tibeticum was originally isolated from root nodules of Trigonella archiducis-nicolai grown in Tibet, China. This species is also able to nodulate Medicago sativa and Phaseolus vulgaris. The whole-genome sequence of the type strain, R. tibeticum CCBAU85039T, is reported in this study.Facultad de Ciencias Exacta

Genome sequence of the symbiotic type strain Rhizobium tibeticum CCBAU85039T

Rhizobium tibeticum was originally isolated from root nodules of Trigonella archiducis-nicolai grown in Tibet, China. This species is also able to nodulate Medicago sativa and Phaseolus vulgaris. The whole-genome sequence of the type strain, R. tibeticum CCBAU85039T, is reported in this study.Fil: Torres Tejerizo, Gonzalo Arturo. 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. Universitat Bielefeld; AlemaniaFil: Wibberg, Daniel. Universitat Bielefeld; AlemaniaFil: Winkler, Anika. Universitat Bielefeld; AlemaniaFil: Ormeño Orrillo, Ernesto. Universidad Nacional Agraria La Molina; PerúFil: Martínez Romero, Esperanza. Universidad Nacional Autónoma de México; MéxicoFil: Niehaus, Karsten. Universitat Bielefeld; AlemaniaFil: Pühler, Alfred. Universitat Bielefeld; AlemaniaFil: Kalinowski, Jörn. Universitat Bielefeld; AlemaniaFil: Lagares, Antonio. 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: Schlüter, Andreas. Universitat Bielefeld; AlemaniaFil: Pistorio, Mariano. 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

Caracterización del sistema de secreción de tipo VI en Rhizobium etli Mim1

La simbiosis rizobio-leguminosa es altamente específica. La translocación de proteínas denominadas efectores desde el citoplasma bacteriano a la célula vegetal es un elemento relacionado con dicha especificidad. Los efectores pueden ser translocados a través de diferentes sistemas de secreción. El análisis de genomas de rizobios ha permitido identificar en algunos la presencia de sistemas de secreción de tipo VI (T6SS). El T6SS tiene como componente principal una nanoestructura similar a las que utilizan los bacteriófagos1 para inyectar su ADN y que las bacterias usan para secretar proteínas. Los genes implicados en la formación de T6SS están agrupados y los que codifican para componentes estructurales del sistema presentan mayor grado de conservación entre rizobios y frente a otras bacterias en comparación a los genes que codifican para efectores y reguladores del sistema. En nuestro grupo se está estudiando el T6SS de Rhizobium etli bv mimosae Mim12 aislada de nódulos de Mimosa affinis y capaz de nodular además Phaseolus vulgaris y Leucaena leucocephala. La cepa Mim1 contiene una agrupación de 28 genes en el plásmido f no simbiótico, relacionados con la formación de un T6SS, presentando una organización similar a la descrita en Agrobacterium tumefaciens C583 que consiste en dos operones divergentes. Se ha descrito para varios microorganismos que cuando el T6SS está activo, las proteínas Hcp y VgrG que forman parte del aparato de secreción pueden detectarse en el medio extracelular3. Los genes que codifican proteínas estructurales en las dos bacterias presentan una gran similitud, así Hcp muestra un 94% de identidad entre ambas permitiendo que los anticuerpos que detectan Hcp de Agrobacterium3 también reaccionen con Hcp de Mim1. Utilizando anticuerpos contra Hcp de Agrobacterium se ha identificado esta proteína en el medio extracelular de cultivos de Mim1 en fase estacionaria y débilmente en fase exponencial. También se ha demostrado su presencia en nódulos de judía y en cultivos crecidos en presencia de exudados de L. leucocephala, P. vulgaris y Pisum sativum. Además, con el fin de conocer en qué condiciones se activa el T6SS de Mim1, se analizó una región de ADN presumiblemente promotora comprendida entre las dos agrupaciones de genes orientados de forma divergente de Mim1. Esta región se fusionó transcripcionalmente a un gen b-gal delator sin promotor del vector pMP220 en las dos posibles orientaciones, una de las orientaciones (P1) controlaría la expresión de genes como hcp y posibles efectores y la otra (P2) de otros genes estructurales. Los resultados mostraron que ambas orientaciones se expresaban a altas DO600 (0,8-1) aunque los valores de P1 fueron entre dos y tres veces superiores a los de P2. Sin embargo a bajas DO600 (0,1-0,2) la actividad de P1 ser redujo a la mitad y la de P2 a niveles del control sin promotor. Con el objetivo de conocer el papel del T6SS en simbiosis se han realizado 3 mutantes que afectan a genes estructurales del T6SS de Mim1, uno en el gen hcp, otro en tssM y el tercero es una deleción de todos los genes presumiblemente dependientes de P2. Se examinó el fenotipo producido en P. vulgaris y L. leucocephala y se observó que los tres mutantes produjeron nódulos blancos y plantas con un porte similar a plantas no inoculadas, con menor tamaño que las inoculadas con la cepa parental y con un color más amarillento. En este trabajo se ha mostrado por primera vez que la presencia de un T6SS en rizobios tiene un efecto beneficioso en la simbiosis con varios hospedadores. En estos momentos se esta trabajando en la caracterización de posibles efectores. Referencias. 1. Records AR. 2011 The type VI secretion system: a multipurpose delivery sustem with a phage-like machinery. Mol Plant Microbe Interact 24: 751-757. 2. Rogel MA et al. 2014. Genomic basis of symbiovar mimosae in Rhizobium etli. BMC Genomics 15: 575 3. Wu, HY et al. 2012. Acid-induced type VI secretion system is regulated by ExoR-ChvG/Chv

Genomic analysis of three Bradyrhizobium geno(species) nodulating Lima bean (Phaseolus lunatus L.) in Peru

The Lima bean (Phaseolus lunatus), also known as pallar, ibes, garrofón or butter bean in Peru, México, Spain and USA, respectively, is the second most economically important species of Phaseolus. Peru is a centre of origin and domestication of Lima bean. This crop is cultivated mainly in the Central coast of Peru under a subtropical arid climate. In contrast to the common bean (Phaseolus vulgaris) which forms nodules with fast growing Rhizobium strains, the Lima bean forms nodules with slow growing bacteria of the Bradyrhizobium genus (López-López et al. 2013, Ormeño-Orrillo et al. 2006). We found strains of Bradyrhizobium yuanmingense and of three novel Bradyrhizobium genospecies inside P. lunatus nodules in Peru (Ormeño- Orrillo et al. 2006). Strains of the three novel genospecies were characterized by showing an extra-slow growing phenotype (generation time > 10 h-1) and strong alkali production in yeast extract mannitol medium. Two of the novel genospecies were recently named as Bradyrhizobium paxllaeri and Bradyrhizobium icense (Durán et al. 2014). B. paxllaeri strains dominate nodule occupancy followed by those of B. icense and then the third and yet-unnamed genospecies. With the aim to gain insights into this differential competitive ability, we sequenced the genome of one representative strain of each species. Sequencing was performed with the Illumina HiSeq or MiSeq platform and genome assembly with the SPAdes program. Gene prediction and automated annotation was performed with Prokka and RAST. Annotation of genes putatively involved in competitiveness was manually curated. Assemblies had from 55 to 175 contigs, with N50 sizes > 131 kb. Genome sizes of B. paxllaeri and B. icense were similar (8.2 Mb) and larger than that of the third genospecies (7.8 Mb). Preliminary analysis revealed differences between B. paxllaeri and the other two genospecies such as more genes for type IV pilus and two nodA genes. A comparative genomic analysis of P. lunatus symbionts will be presented at the meeting

Characterization of type VI secretion systems (T6SS) of endosymbionts from mimosa or lupine

The T6SS is a nanosyringe that injects proteins into prokaryotic or eukaryotic cells, and it is encoded in the genomes of more than 25% of Gram-negative bacteria (1). We are studying the T6SS of Rhizobium etli Mim1 and Bradyrhizobium sp. LmicA16, symbionts of Phaseolus vulgaris/Leucaena leucocephala and Lupinus micranthus/Lupinus angustifolius/Spartium junceum, respectively. R. etli Mim1 contains a T6SS gene cluster organized in two divergent operons. When the T6SS is active, Hcp, a constituent of the secretory apparatus, can be detected in the extracellular medium (2). Hcp has been immunologically detected in the supernatant of Mim1 cultures. This protein was also detected in bean nodule extracts and in cultures grown in the presence of different legumes exudates. The putative divergent promoters located between the two T6SS gene clusters were analysed by ?- gal fusions. The results showed high levels of expression of the two promoters at high OD and low values at lower ODs. Mutants affected in structural genes induced white nodules with P. vulgaris and L. leucocephala. On the other hand, mutagenesis of T6SS structural genes from LmicA16 strain produced different symbiotic phenotypes. An LmicA16 tssC mutant showed reduced levels of nitrogen fixation on L. micranthus, whereas the same mutant induced the formation of few white, non-fixing nodules on L. angustifolius and S. junceum. (1) Ho et al. (2013) Cell Host Microbe 15:9-21. (2) Wu et al. (2012) PLoS Pathog. 8:1-18 Funded by grants BIO2013-43040-P (MINECO), CGL2011-26932 (MICINN) and AL16-PID-06 (UPM)