Análisis comparativo de bradyrhizobia aislados de Lupinus angustifolius y Lupinus mariae-josephi.

Los altramuces (Lupinus sp.) son leguminosas con alto contenido proteico y alto valor en alimentación animal. Todas las especies de Lupinus que crecen en la Península Ibérica, incluida L. angustifolius, requieren suelos ácidos, excepto L- mariae-josephi, una especie recientemente descrita, que excepcíonalmente crece en suelos alcalinos. Se han analizado las relaciones filogenéticas entre bacterias endosimbióticas (rizobios) de L. mariae-josephi y de L. angustifolius y su posición evolutiva respecto a rizobios aislados de otras especies de Lupinus y de otras leguminosas. Con este fin se comparó la secuencia aminoacídica de proteínas esenciales ("housekeeping"), RecA, AtpD, Glnl l, y de la proteína simbiótica NodC, así como también la secuencia nucleotídica intergénica 16S-23S (ITS). La caracterización simbiótica de L. angustifolius y L. mariae-josephi implicó la realización de ensayos de inoculación cruzada, empleando plantas cuyos endosimbiontes están filogenéticamente próximos. Además, se estudió por microscopía de fluorescencia la distribución de bacteroides en los nodulos, utilizando rizobios que expresan GFP. Se espera identificar factores de la simbiosis con rizobios que capacitan a L .mariae-josephi para crecer en suelos básicos

Efecto de los sistemas de Quorum Sensing sobre la eficiencia simbiótica de R. leguminosarum UPM791

Rhizobium leguminosarum bv viciae (Rlv) es una alfa-proteobacteria capaz de establecer una simbiosis diazotrófica con distintas leguminosas. Uno de los factores implicados en el establecimiento de la simbiosis es el sistema de comunicación intercelular conocido como Quorum Sensing (QS). Mediante este sistema, las bacterias actúan de manera coordinada en respuesta a cambios en la densidad de población a través de la producción y detección de señales extracelulares. El genoma de Rlv UPM791 contiene dos sistemas tipo luxRI mediados por señales de tipo N-acyl-homoserina lactonas (AHLs): el sistema rhiRI, codificado en el plásmido simbiótico, produce C6-HSL, C7-HSL y C8-HSL; y el sistema cinRI, localizado en el cromosoma, produce 3-OH-C14:1-HSL. Con el fin de analizar el significado y la regulación de los sistemas de QS en esta bacteria endosimbiótica se generaron mutantes defectivos en cada uno de los sistemas de QS, y se llevó a cabo un análisis detallado sobre la producción de AHLs y la simbiosis con plantas de guisante, veza y lenteja. El sistema rhiRI se necesita para un comportamiento simbiótico normal, dado que la mutación en rhiI reduce considerablemente la eficiencia simbiótica. rhiR es esencial para la fijación de nitrógeno en ausencia del plásmido pUPM791d. Asimismo, mutaciones en el sistema cinRIS mostraron también un importante efecto en simbiosis. El mutante ?cinRIS no produce la señal 3-OH-C14:1-HSL, y da lugar a nódulos blancos e inefectivos, carentes de bacteroides. El mutante ?cinI, incapaz de producir AHLs, no forma nódulos en ninguna de las leguminosas utilizadas. El análisis genético reveló que dicha mutación origina la inestabilización del plásmido simbiótico por un mecanismo dependiente de cinI que no ha sido aclarado. Los resultados obtenidos sugieren un papel relevante de los sistemas de Quorum Sensing de Rlv UPM791 en los primeros estadíos de la simbiosis, e indican la existencia de un modelo de regulación dependiente de QS significativamente distinto a los que se han descrito previamente en otras cepas de R. leguminosarum

Quorum Sensing is essential for an effective symbiosis in R. leguminosarum UPM791.

The implications of Quorum Sensing in the establishment of a successful symbiosis of Rhizobium leguminosarum bv. viciae (Rlv) with legume plants are discussed in this work. In order to analyze the significance and regulation of the production of AHL signal molecules, mutants deficient in each of the two QS systems present in Rlv UPM791 were constructed. A detailed analysis of the effect of these mutations on growth, AHL production, biofilm formation and symbiosis with pea, vetch and lentil plants has been carried out

Caracterización simbiótica y filogenética de rizobios que nodulan la nueva especie Lupinus mariae-josephi

Una nueva especie de altramuz, Lupinus mariae-josephi, ha sido identificado recientemente en Valencia (Pascual, H.). Esta especie, a diferencia de las descritas en la Península ibérica y en el viejo mundo, no crece en suelos ácidos sino en suelos alcalinos y con alto contenido en calcio. El objetivo general de este proyecto es investigar si existen diferencias fenotípicas y genéticas entre los rizobios que nodulan Lupinus mariae-josephi y lupinos de suelos ácidos nativos de la Península Ibérica (Lupinus angustifolius, L.luteus y otras cuatro especies). En este proyecto se han aislado bacterias (rizobios) de nódulos de L. maria-josephi a partir de suelos básicos de Valencia (localidad de LLombai) y se está realizando su caracterización a nivel morfológico, nutricional, simbiótico y molecular. A nivel molecular se han comparado los genes “housekeeping” 16S rRNA, recA, atpD, gln2 y el simbiótico, nodC de diversas cepas que nodulan L. mariae-josephi y con cepas de otras especies de rizobios. Por otra parte, también se está evaluando la capacidad de L. mariae-josephi de ser nodulada por diferentes rizobios bajo condiciones bacteriológicamente controladas

Symbiosis of the endangered Lupinus mariae-josephae lupin especies: Successful "in situ" propagation with rhizobial inoculation

Region, in Eastern Spain. This lupine thrives in alkaline soils with high pH, a unique habitat for lupines. In these soils, Lmj grows in just a few defined patches, and previous conservation efforts directed towards controlled plant reproduction have been unsuccessful. A legislative decree (70/2009, page 20156 Anex I) published in the el 'Diario Oficial de la Comunitat Valenciana' shows Lmj in a category corresponding, in the latest version of the Red List of IUCN (IUCN, 2012) (International Union for Conservation of Nature and Nature Resources), to an ?Endangered? legume species not extinct in the wild. Most current IUCN criteria used to define rare, small-range legume species, are based on history of reproductive traits such as number of pods and seeds. We have previously shown that Lmj plants establish a specific root nodule symbiosis with bradyrhizobia present in those soils, and we reasoned that the paucity of these bacteria in soils might contribute to the lack of success in reproducing plants for conservation purposes. Greenhouse experiments using Lmj trap-plants showed an absence, or very low concentration, of Lmj-nodulating bacteria in ?terra rossa? soils of Valencia outside of Lmj plant patches. No Lmj endosymbiotic bacteria were found in ?terra rossa? or alkaline red soils outside the Valencia Lmj endemism region in the Iberian Peninsula or Balearic Islands. Among the rhizobia able to establish an efficient symbiosis with L. mariae-josephae plants, two Bradyrhizobium sp. strains, LmjC and LmjM3, were selected as inocula for seed coating. Two planting experiments were carried out in consecutive years under natural conditions in areas with edapho-climatic characteristics identical to those sustaining natural Lmj populations, and successful reproduction of the plant was achieved. Interestingly, the successful reproductive cycle was absolutely dependent on seedling inoculation with effective bradyrhizobia, and optimal performance was observed in plants inoculated with LmjC, a strain that had previously shown the most efficient behavior under controlled conditions. These results define conditions for L. mariae-josephae conservation and for extension to alkaline-limed soil habitats, where no other known lupine can thrive. Broadly speaking, the work singularly identified the rhizobial symbiosis as a factor affecting the conservation of legumes and often being exceedingly vulnerable to threats. Our results also indicate that seed inoculation with N2-fixing, efficient Rhizobium strains is a strategy to consider in the conservation of endangered legume specie

Endangered Lupinus mariae-josephae species: conservation efforts

A lupin endemism, Lupinus mariae-josephae (Lmj), singularly has been identified in the Valencia province, in Eastern Spain. This lupin thrives in alkaline-limed soils with high pH, a unique habitat for lupins, from a small area in Valencia region. In these soils, Lmj grows in just a few small, defined patches, and previous conservation efforts directed towards controlled plant reproduction have been unsuccessful. This lupin was thought to be extinct in Valencia until 2007, when it was discovered in a limestone patch. The reasons behind Lmj endangered status are presently unknown. This study will focus on the symbiosis between Lmj and rhizobia, and how this relationship might impact the population size of Lmj. We have previously shown that Lmj plants establish a specific root nodule symbiosis with bradyrhizobia present in those soils, and we reasoned that the paucity of these bacteria in soils might contribute to the lack of success in reproducing plants for conservation purposes

Phenotypic and molecular diversity of Lupinus mariae-josephi endosymbiotic bacteria.

Lupinus mariae-josephi is a new Lupinus species recently described in a Southeastern area of Spain (Valencia) in soils of singularly high pH and active lime content. Bacteria from L. mariaejosephi have been isolated and correspond to extra-slow growing bacteria symbiotically and phylogenetically distant to endosymbiotic strains nodulating other Lupinus sp. native of the Iberian Peninsula and adapted to growth in acid soils. Cross-inoculation experiments revealed that the L. mariae-josephi endosymbiotic bacteria are unable to nodulate or efficiently fix nitrogen with well-known Lupinus spp. Their species affiliation was examined by a multilocus sequence analysis of four housekeeping genes (16S rDNA, glnII, recA, atpD) and the symbiotic nodC gene. Single and concatenated phylogenetic analyses of these genes consistently revealed that L. mariae-josephi endosymbiotic bacteria belong to a clade, within the Bradyrhizobium genus, highly differentiated from the Bradyrhizobium clade that includes currently named Bradyrhizobium species as well as the endosymbiotic bacteria from Lupinus species tested in this study. Within this new clade the L. mariae-josephi bacteria nested in several subgroups that may correspond to novel sister species. The phylogenetic analysis based on the nodC gene showed that L. mariae-josephi endosymbiotic bacteria define a novel branch in the nodC Bradyrhizobium tree and likely have a common unique ancestor for the symbiotic genes with nodule isolates from Retama spp. At this moment two draft genome sequences belonging to a Bradyrhizobium isolated from L. angustifolius (ISLU101) and to a Bradyrhizobium isolated from L. mariae-josephi (LmjC) have been obtained. The first analysis showed that both genomes correspond to very large chromosomes (>8000 genes) with a high number of unique proteins. nod genes organization are highly conserved among ISLU101, LmjC and B. japonicum USDA110. LmjC presents a single cluster with nod genes from diverse origins. ISLU101 fix genes are found in a single cluster homologous to that of USDA110. LmjC possess a complete copy of fix genes homologous to that from USDA110 and an incomplete one similar to S. meliloti. LmjC possess TypeIII and TypeIV secretion systems. ISLU101 has a Type IV homologous to that of photosynthetic Bradyrhizobium BTAi1, and two copies of Type VI, one homologous to that of USDA110 and the other to R. leguminosarum. Preliminary data indicate that ISLU101 contains a potential second replicon (~100 genes) with high homology to Bradyrhizobium BTai1 plasmid sequenc

Analysis of a periplasmic thiol oxidoreductase in Rhizobium leguminosarum

In this work the expression and cellular localization of a predicted periplasmic thiol oxidoreductase encoded by Rhizobium leguminosarum 3841, ORF RL1083, were analysed. Based on the homology of the encoded protein with DsbA proteins from other bacteria we named it dsbA. The genetic organization of dsbA region showed that it is a monocystronic gene. A putative σ70 promoter was predicted upstream dsbA gene. The promoter region fused to gusA reporter gene revealed that dsbA is expressed in free-living conditions in different media and also, although at a lower level, in pea bacteroids. R. leguminosarum DsbA contains a potential Tat-dependent signal peptide. To localize this protein in different cellular fractions the protein was labelled by means of a C-terminal Strep tag. The DsbA-Strep protein was localized in the periplasmic fraction. At present three type of experiments are in progress: first, the study of DsbA Tat-dependence by using a tat mutant strain harbouring dsbA-Strep; second, the construction of a dsbA mutant and third the evaluation of periplasmic disulfide oxidoreductase activity of different strains: wild-type, tat mutant and dsbA mutant

Diversity of nickel ligands in nodule cytosol, nickel transport, and expression of a nickel-dependent enzyme in endosymbiotic bacteria as affected by the legume host

Provision of metals to endosymbiotic bacteria represents a potential limitation for metalloenzyme synthesis inside legume nodules. Metal ions are usually bound to organic ligands in the cell cytoplasm, and the nature of such metal-ligand complexes might affect metal availability. We have observed a strong effect of the legume host on hydrogenase synthesis when the same Rhizobium leguminosarum bv. viciae strain establishes a symbiotic interaction with pea (Pisum sativum) or lentil (Lens sculenta) plants. These data, along with the different phenotypes of mutants altered in nickel (Ni) transport in these hosts, suggest a role for the chemical form of Ni on metal provision to the bacteroid. The biochemical analysis of cytosolic fractions of pea and lentil nodules has revealed the different nature and concentration of organic ligands chelating Ni in these host

Bradyrhizobium sp endosymbiont bacteria nodulate several species of spontaneous leguminous plants of Genisteae tribe from northeastern of Algeria.

218 bacterial isolates obtained from different legume species belonging to the Gensisteae tribe (Lupinus micruntus, Retama sphaerocarpa, Retama raetam and Cytisus vilosus) from different sampling sites in Algeria were studied. Cultivated on YMA medium these strains show a slow growth. The phylogenetic analysis based on 16S rDNA and the household genes of the representatives of these strains indicates that they belong to the genus Bradyrhizobium. A variety of Bradyrhizobium sp. (1, 2, 3) is present in this collection with the presence of strains that can lead to new species. (1) Boulila Farida et al., (2009). Syst. Appl. Microbiol. 32:245?255. (2) Ahnia, et al (2014). Antonie van Leeuwenhoek, J.Microbiol. ISSN 0003-6072, Volume 105 [6] 1121?1129. (3) Bourebaba and al., (2016). Syst. Appl. Microbiol. 39 266?274. This study was supported by the AECID project A1/038234/11. Bourebaba acknowledges support of the National Exceptional. Program (PNE), a fellowship from the Ministry of Higher Education and Scientific research of Algeria at CBGP, Madrid