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

Comparative genomics of Bradyrhizobium japonicum CPAC 15 and Bradyrhizobium diazoefficiens CPAC 7: elite model strains for understanding symbiotic performance with soybean.

The soybean-Bradyrhizobium symbiosis can be highly efficient in fixing nitrogen, but few genomic sequences of elite inoculant strains are available. Here we contribute with information on the genomes of two commercial strains that are broadly applied to soybean crops in the tropics. B. japonicum CPAC 15 (=SEMIA 5079) is outstanding in its saprophytic capacity and competitiveness, whereas B. diazoefficiens CPAC 7 (=SEMIA 5080) is known for its high efficiency in fixing nitrogen. Both are well adapted to tropical soils. The genomes of CPAC 15 and CPAC 7 were compared to each other and also to those of B. japonicum USDA 6T and B. diazoefficiens USDA 110T. Differences in genome size were found between species, with B. japonicum having larger genomes than B. diazoefficiens. Although most of the four genomes were syntenic, genome rearrangements within and between species were observed, including events in the symbiosis island. In addition to the symbiotic region, several genomic islands were identified. Altogether, these features must confer high genomic plasticity that might explain adaptation and differences in symbiotic performance. It was not possible to attribute known functions to half of the predicted genes. About 10% of the genomes was composed of exclusive genes of each strain, but up to 98% of them were of unknown function or coded for mobile genetic elements. In CPAC 15, more genes were associated with secondary metabolites, nutrient transport, iron-acquisition and IAA metabolism, potentially correlated with higher saprophytic capacity and competitiveness than seen with CPAC 7. In CPAC 7, more genes were related to the metabolism of amino acids and hydrogen uptake, potentially correlated with higher efficiency of nitrogen fixation than seen with CPAC 15. Several differences and similarities detected between the two elite soybean-inoculant strains and between the two species of Bradyrhizobium provide new insights into adaptation to tropical soils, efficiency of N2 fixation, nodulation and competitiveness

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)

Genome-wide identification of the Phaseolus vulgaris sRNAome using small RNA and degradome sequencing

Background: MiRNAs and phasiRNAs are negative regulators of gene expression. These small RNAs have been extensively studied in plant model species but only 10 mature microRNAs are present in miRBase version 21, the most used miRNA database, and no phasiRNAs have been identified for the model legume Phaseolus vulgaris. Thanks to the recent availability of the first version of the common bean genome, degradome data and small RNA libraries, we are able to present here a catalog of the microRNAs and phasiRNAs for this organism and, particularly, we suggest new protagonists in the symbiotic nodulation events.Results: We identified a set of 185 mature miRNAs, including 121 previously unpublished sequences, encoded by 307 precursors and distributed in 98 families. Degradome data allowed us to identify a total of 181 targets for these miRNAs. We reveal two regulatory networks involving conserved miRNAs: those known to play crucial roles in the establishment of nodules, and novel miRNAs present only in common bean, suggesting a specific role for these sequences. In addition, we identified 125 loci that potentially produce phased small RNAs, with 47 of them having all the characteristics of being triggered by a total of 31 miRNAs, including 14 new miRNAs identified in this study.Conclusions: We provide here a set of new small RNAs that contribute to the broader knowledge of the sRNAome of Phaseolus vulgaris. Thanks to the identification of the miRNA targets from degradome analysis and the construction of regulatory networks between the mature microRNAs, we present here the probable functional regulation associated with the sRNAome and, particularly, in N2-fixing symbiotic nodules.Peer reviewedBiochemistry and Molecular Biolog