Dual role of HupF in the biosynthesis of [NiFe] hydrogenase in Rhizobium leguminosarum

Background: [NiFe] hydrogenases are enzymes that catalyze the oxidation of hydrogen into protons and electrons, to use H2 as energy source, or the production of hydrogen through proton reduction, as an escape valve for the excess of reduction equivalents in anaerobic metabolism. Biosynthesis of [NiFe] hydrogenases is a complex process that occurs in the cytoplasm, where a number of auxiliary proteins are required to synthesize and insert the metal cofactors into the enzyme structural units. The endosymbiotic bacterium Rhizobium leguminosarum requires the products of eighteen genes (hupSLCDEFGHIJKhypABFCDEX) to synthesize an active hydrogenase. hupF and hupK genes are found only in hydrogenase clusters from bacteria expressing hydrogenase in the presence of oxygen. Results: HupF is a HypC paralogue with a similar predicted structure, except for the C-terminal domain present only in HupF. Deletion of hupF results in the inability to process the hydrogenase large subunit HupL, and also in reduced stability of this subunit when cells are exposed to high oxygen tensions. A ?hupF mutant was fully complemented for hydrogenase activity by a C-terminal deletion derivative under symbiotic, ultra low-oxygen tensions, but only partial complementation was observed in free living cells under higher oxygen tensions (1% or 3%). Co-purification experiments using StrepTag-labelled HupF derivatives and mass spectrometry analysis indicate the existence of a major complex involving HupL and HupF, and a less abundant HupF-HupK complex. Conclusions: The results indicate that HupF has a dual role during hydrogenase biosynthesis: it is required for hydrogenase large subunit processing and it also acts as a chaperone to stabilize HupL when hydrogenase is synthesized in the presence of oxygen.

Functional and expression analysis of the metal-inducible dmeRF system from Rhizobium legumionosarum bv. viciae

A gene encoding a homolog to the cation diffusion facilitator protein DmeF from Cupriavidus metallidurans has been identified in the genome of Rhizobium leguminosarum UPM791. The R. leguminosarum dmeF gene is located downstream of an open reading frame (designated dmeR) encoding a protein homologous to the nickel- and cobalt-responsive transcriptional regulator RcnR from Escherichia coli. Analysis of gene expression showed that the R. leguminosarum dmeRF genes are organized as a transcriptional unit whose expression is strongly induced by nickel and cobalt ions, likely by alleviating the repressor activity of DmeR on dmeRF transcription. An R. leguminosarum dmeRF mutant strain displayed increased sensitivity to Co(II) and Ni(II), whereas no alterations of its resistance to Cd(II), Cu(II), or Zn(II) were observed. A decrease of symbiotic performance was observed when pea plants inoculated with an R. leguminosarum dmeRF deletion mutant strain were grown in the presence of high concentrations of nickel and cobalt. The same mutant induced significantly lower activity levels of NiFe hydrogenase in microaerobic cultures. These results indicate that the R. leguminosarum DmeRF system is a metal-responsive efflux mechanism acting as a key element for metal homeostasis in R. leguminosarum under free-living and symbiotic conditions. The presence of similar dmeRF gene clusters in other Rhizobiaceae suggests that the dmeRF system is a conserved mechanism for metal tolerance in legume endosymbiotic bacteria.

Análisis genómico y funcional de los sistemas de Quorum Sensing en Rhizobium leguminosarum

Rhizobium leguminosarum (Rl) es una alfa-proteobacteria capaz de establecer una simbiosis diazotrófica con distintas leguminosas. A pesar de la importancia de esta simbiosis en el balance global del ciclo del nitrógeno, muy pocos genomas de rhizobios han sido secuenciados, que aporten nuevos conocimientos relacionados con las características genéticas que contribuyen a importantes procesos simbióticos. Únicamente tres secuencias completas de Rl han sido publicadas: Rl bv. viciae 3841 y dos genomas de Rl bv. trifolii (WSM1325 y WSM2304), ambos simbiontes de trébol. La secuencia genómica de Rlv UPM791 se ha determinado por medio de secuenciación 454. Este genoma tiene un tamaño aproximado de 7.8 Mb, organizado en un cromosoma y 5 replicones extracromosómicos, que incluyen un plásmido simbiótico de 405 kb. Este nuevo genoma se ha analizado en relación a las funciones simbióticas y adaptativas en comparación con los genomas completos de Rlv 3841 y Rl bv. trifolii WSM1325 y WSM2304. Mientras que los plásmidos pUPM791a y b se encuentran conservados, el plásmido simbiótico pUPM791c exhibe un grado de conservación muy bajo comparado con aquellos descritos en las otras cepas de Rl. Uno de los factores implicados en el establecimiento de la simbiosis es el sistema de comunicación intercelular conocido como Quorum Sensing (QS). El análisis del genoma de Rlv UPM791 ha permitido la identificación de dos sistemas tipo LuxRI mediados por señales de tipo N-acyl-homoserina lactonas (AHLs). El análisis mediante HPLC-MS ha permitido asociar las señales C6-HSL, C7-HSL y C8-HSL al sistema rhiRI, codificado en el plásmido simbiótico; mientras que el sistema cinRI, localizado en el cromosoma, produce 3OH-C14:1-HSL. Se ha identificado una tercera sintasa (TraI) codificada en el plásmido simbiótico, pero su regulador correspondiente se encuentra truncado debido a un salto de fase. Adicionalmente, se han encontrado tres reguladores de tipo LuxR-orphan que no presentan una sintasa LuxI asociada. El efecto potencial de las señales tipo AHL se ha estudiado mediante una estrategia de quorum quenching, la cual interfiere con los sistemas de QS de la bacteria. Esta estrategia está basada en la introducción del gen aiiA de Bacillus subtilis, que expresa constitutivamente una enzima lactonasa degradadora de AHLs. Para llevar a cabo el análisis en condiciones simbióticas, se ha desarrollado un sistema de doble marcaje que permite la identificación basado en los marcadores gusA y celB, que codifican para una enzima β–glucuronidasa y una β–galactosidasa termoestable, respectivamente. Los resultados obtenidos indican que Rlv UPM791 predomina sobre la cepa Rlv 3841 para la formación de nódulos en plantas de guisante. La baja estabilidad del plásmido que codifica para aiiA, no ha permitido obtener una conclusión definitiva sobre el efecto de la lactonasa AiiA en competitividad. Con el fin de analizar el significado y la regulación de la producción de moléculas señal tipo AHL, se han generado mutantes defectivos en cada uno de los dos sistemas de QS. Se ha llevado a cabo un análisis detallado sobre la producción de AHLs, formación de biofilm y simbiosis con plantas de guisante, veza y lenteja. El efecto de las deleciones de los genes rhiI y rhiR en Rlv UPM791 es más drástico en ausencia del plásmido pUPM791d. Mutaciones en cinI o cinRIS muestran tanto ausencia de señales, como producción exclusivamente de las de bajo peso molecular, respectivamente, producidas por el sistema rhiRI. Estas mutaciones mostraron un efecto importante en simbiosis. El sistema rhiRI se necesita para un comportamiento simbiótico normal. Además, mutantes cinRIS generaron nódulos blancos e ineficientes, mientras que el mutante cinI fue incapaz de producir nódulos en ninguna de las leguminosas utilizadas. Dicha mutación resultó en la inestabilización del plasmido simbiótico por un mecanismo dependiente de cinI que no ha sido aclarado. En general, los resultados obtenidos 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, en las cuales no se había observado ningún fenotipo relevante en simbiosis. La regulación de la producción de AHLs Rlv UPM791 es un proceso complejo que implica genes situados en los plásmidos UPM791c y UPM791d, además de la señal 3-OH-C14:1-HSL. Finalmente, se ha identificado un transportador de tipo RND, homologo a mexAB-oprM de P. aeruginosa e implicado en la extrusión de AHLs de cadena larga. La mutación he dicho transportador no tuvo efectos apreciables sobre la simbiosis. ABSTRACT Rhizobium leguminosarum (Rl) is a soil alpha-proteobacterium that establishes a diazotrophic symbiosis with different legumes. Despite the importance of this symbiosis to the global nitrogen cycling balance, very few rhizobial genomes have been sequenced so far which provide new insights into the genetic features contributing to symbiotically relevant processes. Only three complete sequences of Rl strains have been published: Rl bv. viciae 3841, harboring six plasmids (7.75 Mb) and two Rl bv. trifolii (WSM1325 and WSM2304), both clover symbionts, harboring 5 and 4 plasmids, respectively (7.41 and 6.87 Mb). The genomic sequence of Rlv UPM791 was undertaken by means of 454 sequencing. Illumina and Sanger reads were used to improve the assembly, leading to 17 final contigs. This genome has an estimated size of 7.8 Mb organized in one chromosome and five extrachromosomal replicons, including a 405 kb symbiotic plasmid. Four of these plasmids are already closed, whereas there are still gaps in the smallest one (pUPM791d) due to the presence of insertion elements and repeated sequences, which difficult the assembly. The annotation has been carried out thanks to the Manatee pipeline. This new genome has been analyzed as regarding symbiotic and adaptive functions in comparison to the Rlv 3841 complete genome, and to those from Rl bv. trifolii strains WSM1325 and WSM2304. While plasmids pUPM791a and b are conserved, the symbiotic plasmid pUPM791c exhibited the lowest degree of conservation as compared to those from the other Rl strains. One of the factors involved in the symbiotic process is the intercellular communication system known as Quorum Sensing (QS). This mechanism allows bacteria to carry out diverse biological processes in a coordinate way through the production and detection of extracellular signals that regulate the transcription of different target genes. Analysis of the Rlv UPM791 genome allowed the identification of two LuxRI-like systems mediated by N-acyl-homoserine lactones (AHLs). HPLC-MS analysis allowed the adscription of C6-HSL, C7-HSL and C8-HSL signals to the rhiRI system, encoded in the symbiotic plasmid, whereas the cinRI system, located in the chromosome, produces 3OH-C14:1-HSL, previously described as “bacteriocin small”. A third synthase (TraI) is encoded also in the symbiotic plasmid, but its cognate regulator TraR is not functional due to a fameshift mutation. Three additional LuxR orphans were also found which no associated LuxI-type synthase. The potential effect of AHLs has been studied by means of a quorum quenching approach to interfere with the QS systems of the bacteria. This approach is based upon the introduction into the strains Rl UPM791 and Rl 3841 of the Bacillus subtilis gene aiiA expressing constitutively an AHL-degrading lactonase enzyme which led to virtual absence of AHL even when AiiA-expressing cells were a fraction of the total population. No significant effect of AiiA-mediated AHL removal on competitiveness for growth in solid surface was observed. For analysis under symbiotic conditions we have set up a two-label system to identify nodules produced by two different strains in pea roots, based on the markers gusA and celB, encoding a β–glucuronidase and a thermostable β–galactosidase enzymes, respectively. The results obtained show that Rlv UPM791 outcompetes Rlv 3841 for nodule formation in pea plants, and that the presence of the AiiA plasmid does not significantly affect the relative competitiveness of the two Rlv strains. However, the low stability of the pME6863 plasmid, encoding aiiA, did not lead to a clear conclusion about the AiiA lactonase effect on competitiveness. In order to further analyze the significance and regulation of the production of AHL signal molecules, mutants deficient in each of the two QS systems were constructed. A detailed analysis of the effect of these mutations on AHL production, biofilm formation and symbiosis with pea, vetch and lentil plants has been carried out. The effect of deletions on Rlv UPM791 rhiI and rhiR genes is more pronounced in the absence of plasmid pUPM791d, as no signal is detected in UPM791.1, lacking this plasmid. Mutations in cinI or cinRIS show either no signals, or only the small ones produced by the rhiRI system, suggesting that cinR might be regulating the rhiRI system. These mutations had a strong effect on symbiosis. Analysis of rhi mutants revealed that rhiRI system is required for normal symbiotic performance, as a drastic reduction of symbiotic fitness is observed when rhiI is deleted, and rhiR is essential for nitrogen fixation in the absence of plasmid pUPM791d. Furthermore, cinRIS mutants resulted in white and inefficient nodules, whereas cinI mutant was unable to form nodules on any legume tested. The latter mutation is associated to the instabilization of the symbiotic plasmid through a mechanism still uncovered. Overall, the results obtained indicate the existence of a model of QS-dependent regulation significantly different to that previously described in other R. leguminosarum strains, where no relevant symbiotic phenotype had been observed. The regulation of AHL production in Rlv UPM791 is a complex process involving the symbiotic plasmid (pUPM791c) and the smallest plasmid (pUPM791d), with a key role for the 3-OH-C14:1-HSL signal. Finally, we made a search for potential AHL transporters in Rlv UPM791 genome. These signals diffuse freely across membranes, but in the case of the long-chain AHLs an active efflux system might be required, as it has been described for C12-HSL in the case of Pseudomonas aeruginosa. We have identified a putative AHL transporter of the RND family homologous to P. aeruginosa mexAB-oprM. A mutant strain deficient in this transporter has been generated, and TLC analysis shows absence of 3OH-C14:1-HSL in its supernatant. This deficiency was complemented by the reintroduction of an intact copy of the genes via plasmid transfer. The mutation in mexAB genes had no significant effects on the symbiotic performance of R. leguminosarum bv. viciae.

Los tratados y el jardín renacentista en Italia. Principios, metodologías y técnicas

La tesis destaca la influencia que ejerció la teoría, las técnicas y la metodología de proyecto de los principales tratados de la época renacentista en la concepción, diseño y realización de algunos de los más importantes jardines de la época. Los tratados de Vitruvio, Alberti, Filarete, Di Giorgio, Serlio, Palladio, Colonna y Del Riccio establecen una nueva estética y una nueva visión del mundo, basadas en la geometría elemental y en los números simples. En la Grecia antigua, Platón y Pitágoras intuyeron la necesidad existencial del hombre de apoyarse en unas leyes seguras de orden superior. Sus teorías sobre las leyes del Universo son retomadas y aplicadas en el arte y en el jardín por Vitruvio y por los tratadistas del Renacimiento. Según los tratadistas, la estética es el núcleo generador del que se activan la información y las leyes hacia todos los campos del conocimiento y de la vida social y, por lo tanto, la finalidad de los tratados es buscar, a través de un procedimiento científico y racional, las reglas de la belleza. Las leyes estéticas formuladas por los tratadistas examinados se aplican con el mismo rigor y respeto ya sea al paisaje que al jardín, exaltando el elemento natural al igual que el construido (la ciudad y la arquitectura). De los tratados renacentistas se han extraídos términos y conceptos que ayudan a revalorizar, reinterpretar e integrar en el debate los temas del paisaje y del jardín. La tesis sostiene que: 1) una idea moderna de jardín se tiene que fundar en la historia; 2) que la historia es indispensable incluso para enfrentar el problema del mantenimiento y de la restauración de los jardines históricos; 3) que la teoría y las ideas que tienen valor, así como nos enseña Alberti, producen obras que también tienen valor; 4) la influencia de la estética de los tratadistas se encuentra incluso en algunos importantes jardines de época moderna y contemporánea. El capítulo 1 contiene la definición de tratado y la concepción estética del Renacimiento, analizada por medio de: a) la idea de ciencia y b) a través de las pinturas. Los capítulos 2 y 3 contienen respectivamente los principios teóricos de los tratados y el diseño, la metodología, las técnicas constructivas y los métodos de representación del proyecto según los tratadistas. Estos capítulos resultan propedéuticos de los capítulos siguientes por proporcionar el instrumento de lectura e interpretación de los jardines renacentistas y modernos. El capítulo 4 destaca la gran influencia de los tratados sobre las villas y jardines renacentistas y describe sus elementos. El último capítulo, el quinto, contiene un análisis de algunos jardines modernos. Entre ellos: Chiswick, la Villa “I Tatti”, Sutton Place, y la Villa “Il Roseto” que renuevan y prosiguen, en nuestra opinión, la lección de los tratadistas. El primer anexo contiene la propuesta de líneas de investigación que surgen a la luz de la tesis, y el segundo anexo contiene las noticias biográficas de los tratadistas examinados. ABSTRACT The thesis investigates the influence exercised by the theory, techniques and project methodology contained in the most relevant essays on art and architecture of the Renaissance on the concept, design and making of some of the most important gardens of that time. The essays of Vitruvius, Alberti, Filarete, Di Giorgio, Serlio, Palladio, Colonna y Del Riccio established a new concept of aesthetics and a vision of the world based on basic geometry and simple numbers. In ancient Greece, Plato and Pythagoras understood that humans needed “superior laws” to anchor their existences. Their theories on laws of the Universe were adopted and applied in art and gardening by Vitruvius and Renaissance essays authors. According to the authors of these essays, aesthetics represents the core generating information and laws that branch out toward all the fields of knowledge and social life. Therefore, the purpose of art and architecture essays consists in exploring, through a scientific and rational process, the rules of beauty. The laws of aesthetics proposed by the authors apply to landscaping and gardening with the same rigor and exalt the natural component as much as the artificial one (city and architecture). Several terms and concepts are also extracted that, despite their age, still help us to understand and enrich the contemporary debate revolving around landscaping and gardening. The thesis argues that: 1) the modern idea of gardening must be based on history; 2) history is fundamental even to address the topics of maintenance and restoration of historical gardens; 3) like Leon Battista Alberti maintains, valid theory and ideas produce valuable works; 4) the influence played by aesthetics on authors can be found in many important modern and contemporary gardens. Chapter 1 contains the definition of essay and of the Renaissance concept of aesthetics, analyzed by: a) the idea of science and 2) the analysis of some paintings of the period. Chapters 2 and 3 discuss respectively the theoretical principles of the essays and the design, methodology, construction techniques and the methods of representation of the project according to the authors. These chapters are preparatory toward the following chapters and provide us with the keys necessary to read and interpret Renaissance and modern gardens. Chapter 4 highlights the great influence exercised by those authors on the planning of important villas and gardens from the Renaissance while describing their elements. The 5th and final chapter analyzes some modern gardens among which Chiswick, Villa “I Tatti”, Sutton Place, Villa “Il Roseto”. These villas renew and in our opinion carry on the lesson of the essays written in the Renaissance. The first addendum suggests future lines of research derived from the thesis, while the second addendum contains biographical information regarding the authors examined.

Nitrous oxide emissions from European agriculture - an analysis of variability and drivers of emissions from field experiments

Nitrous oxide emissions from a network of agricultural experiments in Europe were used to explore the relative importance of site and management controls of emissions. At each site, a selection of management interventions were compared within replicated experimental designs in plot-based experiments. Arable experiments were conducted at Beano in Italy, El Encin in Spain, Foulum in Denmark, Logarden in Sweden, Maulde in Belgium CE1, Paulinenaue in Germany, and Tulloch in the UK. Grassland experiments were conducted at Crichton, Nafferton and Peaknaze in the UK, Godollo in Hungary, Rzecin in Poland, Zarnekow in Germany and Theix in France. Nitrous oxide emissions were measured at each site over a period of at least two years using static chambers. Emissions varied widely between sites and as a result of manipulation treatments. Average site emissions (throughout the study period) varied between 0.04 and 21.21 kg N2O-N ha−1yr−1, with the largest fluxes and variability associated with the grassland sites. Total nitrogen addition was found to be the single most important deter- minant of emissions, accounting for 15 % of the variance (using linear regression) in the data from the arable sites (p<0.0001), and 77 % in the grassland sites. The annual emissions from arable sites were significantly greater than those that would be predicted by IPCC default emission fac- tors. Variability of N2O emissions within sites that occurred as a result of manipulation treatments was greater than that resulting from site-to-site and year-to-year variation, highlighting the importance of management interventions in contributing to greenhouse gas mitigation

Computational Study of the Fe(CN)(2)CO Cofactor and Its Binding to HypC Protein.

In the intricate maturation process of [NiFe]-hydrogenases, the Fe(CN)2CO cofactor is first assembled in a HypCD complex with iron coordinated by cysteines from both proteins and CO is added after ligation of cyanides. The small accessory protein HypC is known to play a role in delivering the cofactor needed for assembling the hydrogenase active site. However, the chemical nature of the Fe(CN)2CO moiety and the stability of the cofactor–HypC complex are open questions. In this work, we address geometries, properties, and the nature of bonding of all chemical species involved in formation and binding of the cofactor by means of quantum calculations. We also study the influence of environmental effects and binding to cysteines on vibrational frequencies of stretching modes of CO and CN used to detect the presence of Fe(CN)2CO. Carbon monoxide is found to be much more sensitive to sulfur binding and the polarity of the medium than cyanides. The stability of the HypC–cofactor complex is analyzed by means of molecular dynamics simulation of cofactor-free and cofactor-bound forms of HypC. The results show that HypC is stable enough to carry the cofactor, but since its binding cysteine is located at the N-terminal unstructured tail, it presents large motions in solution, which suggests the need for a guiding interaction to achieve delivery of the cofactor.

Análisis molecular de sistemas génicos implicados en la homeostasis de níquel y eficiencia simbiótica en Rhizobium leguminosarum

Los suelos ultramáficos, que poseen elevadas concentraciones de níquel, cobalto y cromo de manera natural, son fuente de bacterias resistentes a altas concentraciones de metales. Se realizó la caracterización físico-química de seis suelos ultramáficos del suroeste europeo, seleccionándose un suelo de la región de Gorro, Italia, como el más adecuado para aislar bacterias endosimbióticas resistentes a metales. A partir de plantas-trampa de guisante y lenteja inoculados con suspensiones de ese suelo, se obtuvieron 58 aislados de Rhizobium leguminosarum bv. viciae (Rlv) que fueron clasificados en 13 grupos según análisis de PCR-RAPDs. Se determinó la resistencia a cationes metálicos [Ni(II), Co(II), Cu(II), Zn(II)] de una cepa representante de cada grupo, así como la secuencia de los genomas de las cepas que mostraron altos niveles (UPM1137 y UPM1280) y bajos niveles (UPM1131 y UPM1136) de tolerancia a metales. Para identificar mecanismos de resistencia a metales se realizó una mutagénesis al azar en dicha cepa mediante la inserción de un minitransposón. El análisis de 4313 transconjugantes permitió identificar 14 mutantes que mostraron una mayor sensibilidad a Ni(II) que la cepa silvestre. Se determinó el punto de inserción del minitransposón en todos ellos y se analizaron en más detalle dos de los mutantes (D2250 y D4239). En uno de los mutantes (D2250), el gen afectado codifica para una proteína que presenta un 44% de identidad con dmeF (divalent efflux protein) de Cupriavidus metallidurans. Cadena arriba de dmeF se identificó un gen que codifica una proteína con un 39% de identidad con el regulador RcnR de Escherichia coli. Se decidió nombrar a este sistema dmeRF, y se generó un mutante en ambos genes en la cepa Rlv SPF25 (Rlv D15). A partir de experimentos de análisis fenotípico y de regulación se pudo demostrar que el sistema dmeRF tiene un papel relevante en la resistencia a Ni(II) y sobre todo a Co(II) en células en vida libre y en simbiosis con plantas de guisante. Ambos genes forman un operón cuya expresión se induce en respuesta a la presencia de Ni(II) y Co(II). Este sistema se encuentra conservado en distintas especies del género Rhizobium como un mecanismo general de resistencia a níquel y cobalto. Otro de los mutantes identificados (D4239), tiene interrumpido un gen que codifica para un regulador transcripcional de la familia AraC. Aunque inicialmente fue identificado por su sensibilidad a níquel, experimentos posteriores demostraron que su elevada sensibilidad a metales era debida a su sensibilidad al medio TY, y más concretamente a la triptona presente en el medio. En otros medios de cultivo el mutante no está afectado específicamente en su tolerancia a metales. Este mutante presenta un fenotipo simbiótico inusual, siendo inefectivo en guisantes y efectivo en lentejas. Análisis de complementación y de mutagénesis dirigida sugieren que el fenotipo de la mutación podría depender de otros factores distintos del gen portador de la inserción del minitransposón. ABSTRACT Ultramafic soils, having naturally high concentrations of nickel, cobalt and chrome, are potential sources of highly metal-resistant bacteria. A physico-chemical characterization of six ultramafic soils from the European southwest was made. A soil from Gorro, Italy, was chosen as the most appropriated for the isolation of heavy-metal-resistant endosymbiotic bacteria. From pea and lentil trap plants inoculated with soil suspensions, 58 isolates of Rhizobium leguminosarum bv. viciae (Rlv) were obtained and classified into 13 groups based on PCR-RAPDs analysis. The resistance to metallic cations [Ni(II), Co(II), Cu(II), Zn(II)] was analyzed in a representative strain of each group. From the results obtained in the resistance assays, the Rlv UPM1137 strain was selected to identify metal resistance mechanism. A random mutagenesis was made in UPM1137 by using minitransposon insertion. Analysis of 4313 transconjugants allowed to identify 14 mutants with higher sensitivity to Ni(II) than the wild type strain. The insertion point of the minitransposon was determined in all of them, and two mutants (D2250 and D4239) were studied in more detail. In one of the mutants (D2250), the affected gene encodes a protein with 44% identity in compared with DmeF (divalent efflux protein) from Cupriavidus metallidurans. Upstream R. leguminosarum dmeF, a gene encoding a protein with 39% identity with RcnR regulator from E. coli was identified. This protein was named DmeR. A mutant with both genes in the dmeRF deleted was generated and characterized in Rlv SPF25 (Rlv D15). From phenotypic and regulation analysis it was concluded that the dmeRF system is relevant for Ni(II) and specially Co(II) tolerance in both free living and symbiotic forms of the bacteria. This system is conserved in different Rhizobium species like a general mechanism for nickel and cobalt resistance. Other of the identified mutants (D4239) contains the transposon insert on a gene that encodes for an AraC-like transcriptional regulator. Although initially this mutant was identified for its nickel sensitivity, futher experiments demonstrated that its high metal sensitivity is due to its sensitivity to the TY medium, specifically for the tryptone. In other media the mutant is not affected specifically in their tolerance to metals. This mutant showed an unusual symbiotic phenotype, being ineffective in pea and effective in lentil. Complementation analysis and directed mutagenesis suggest that the mutation phenotype could depend of other factors different from the insertion minitransposon gene.

Estudio del comportamiento mecánico y en fractura de un hormigón autocompactante con fibras de poliolefina

El uso de hormigón autocompactante se ha convertido en algo habitual desde su aparición a finales de la década de los 80 gracias a la reducción de costes de mano de obra, la buena calidad del acabado superficial y su uso en piezas fuertemente armadas. Por otro lado, los hormigones reforzados con fibras aportan una mejora en las propiedades mecánicas que puede permitir la reducción de armados y, en general, mejorar la respuesta del material ante todo tipo de solicitaciones, especialmente de tracción. En este trabajo se ha estudiado el comportamiento mecánico de un hormigón autocompactante con fibras de poliolefina. Se han obtenido resultados de caracterización mecánica y de fractura de un hormigón autocompactante de referencia sin fibras y de tres dosificaciones con fibras desde 3 kg/m³ hasta 6 kg/m³. Estos resultados han proporcionado un notable incremento en los valores de resistencia post-fisuración y de energía de fractura sin perjuicio de sus propiedades autocompactantes en estado fresco. Since the development of the first Self-Compacting Concrete in the late 80’s, its use has become widespread due to the reduction of the labor costs, the good finishing quality and the achieving of the necessary fluidity for congested reinforced pieces. Furthermore, Fiber Reinforced Concrete provides improvements of the mechanical properties which may even permit the reduction of the reinforcement. The mechanical behavior of a Self-Compacting Concrete with polyolefin fibers has been explored in this research. Results for mechanical properties and for fracture and post-cracking toughness have been obtained. The experimental campaign has been performed for a plain Self-Compacting Concrete and for three different fiber dosages from 3 kg/m³ to 6 kg/m³. These results show a significant enhancement of the post-cracking strength and the fracture energy without harming in the concrete self-compacting properties in fresh state.

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.

DmeRF system is required for nickel and cobalt resistance in Rhizobium leguminosarum bv. viciae.

A member of the Cation Diffusion Facilitator (CDF) family with high sequence similarity to DmeF (Divalent metal efflux) from Cupridavirus metallidurans was identified in Rhizobium leguminosarum bv. viciae UPM1137. The R. leguminosarum dmeF mutant strain was highly sensitive to Co2+ and moderately sensitive to Ni2+, but its tolerance to other metals such as Zn2+, Cu2+ or Mn2+ was unaffected. An open reading frame located upstream of R. leguminosarum dmeF, designated dmeR, encodes a protein homologous to the nickel and cobalt regulator RcnR from E.coli. Expression of the dmeRF operon was induced by nickel and cobalt ions in free-living cells, likely by alleviating DmeR-mediated transcriptional repression of the operon.

Functional roles of HypC and HupK in the biosynthesis of [NiFe] hydrogenase in Rhizobium leguminosarum.

Some rhizobia induce a hydrogen (H2)-uptake system with a [NiFe] hydrogenase along with nitrogenase to recover part of the energy lost as H2. Biosynthesis of NiFe hydrogenases is a process that ocurrs in the cytoplasm, where a number of auxiliary proteins (products of hup and hyp genes) are required to synthesize and insert the metal cofactors into the enzyme structural units. Although HypC is expressed in all hydrogenase systems, HupF and HupK are found only in bacteria that express the hydrogenase in the presence of oxygen (O2). Co-purification experiments have demonstrated HypC-HupK and HypC-HupL interactions. Results have shown that some conserved residues from HypC and HupK play a protective role of hydrogenase against the presence of O2.

Molecular physiology of nickel and cobalt homeostasis in Rhizobium leguminosarum.

Transition metals such as Fe, Cu, Mn, Ni, or Co are essential nutrients, as they are constitutive elements of a significant fraction of cell proteins. Such metals are present in the active site of many enzymes, and also participate as structural elements in different proteins. From a chemical point of view, metals have a defined order of affinity for binding, designated as the Irving-Williams series (Irving and Williams, 1948) Mg2+ menor que Mn2+ menor que Fe2+ menor que Co2+ menor que Ni2+ menor que Cu2+mayor queZn2+ Since cells contain a high number of different proteins harbouring different metal ions, a simplistic model in which proteins are synthesized and metals imported into a ?cytoplasmic soup? cannot explain the final product that we find in the cell. Instead we need to envisage a complex model in which specific ligands are present in definite amounts to leave the right amounts of available metals and protein binding sites, so specific pairs can bind appropriately. A critical control on the amount of ligands and metal present is exerted through specific metal-responsive regulators able to induce the synthesis of the right amount of ligands (essentially metal binding proteins), import and efflux proteins. These systems are adapted to establish the metal-protein equilibria compatible with the formation of the right metalloprotein complexes. Understanding this complex network of interactions is central to the understanding of metal metabolism for the synthesis of metalloenzymes, a key topic in the Rhizobium-legume symbiosis. In the case of the Rhizobium leguminosarum bv viciae (Rlv) UPM791 -Pisum sativum symbiotic system, the concentration of nickel in the plant nutrient solution is a limiting factor for hydrogenase expression, and provision of high amounts of this element to the plant nutrient solution is required to ensure optimal levels of enzyme synthesis (Brito et al., 1994).

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.

Functional characterization of Rhizobium leguminosarum bv. viciae DmeRF, a cation diffusion facilitator system involved in nickel and cobalt resistance

In prokaryotes, nickel is an essential element participating in the structure of enzymes involved in multiple cellular processes. Nickel transport is a challenge for microorganisms since, although essential, high levels of this metal inside the cell are toxic. For this reason, bacteria have developed high-affinity nickel transporters as well as nickel-specific detoxification systems. Ultramafic soils, and soils contaminated with heavy metals are excellent sources of nickel resistant bacteria. Molecular analysis of strains isolated in the habitats has revealed novel genetic systems involved in adaptation to such hostile conditions.

Identificación y análisis funcional de genes implicados en la homeostasis de níquel en la bacteria endosimbiótica de leguminosas Rhizobium leguminosarum

La asociación Rhizobium-leguminosa constituye una interacción planta-microorganismo particularmente beneficiosa a nivel medioambiental debido a su capacidad promotora del crecimiento vegetal en condiciones de deficiencia de nitrógeno. Se ha demostrado que una excesiva concentración de metales pesados en el suelo afecta negativamente la competitividad bacteriana y al desarrollo de interacciones diazotróficas eficientes (Chaudri et al., 2000; Pereira et al., 2006). Por otro lado, el suministro de metales como Fe, Mo, Ni o Cu es fundamental para la biosíntesis de enzimas bacterianas relacionadas con el proceso de fijación de nitrógeno que ocurre en el interior de los nódulos de las leguminosas (Moreau et al., 1995). Con objeto de identificar sistemas génicos implicados en la homeostasis de níquel en bacterias endosimbióticas, se ha llevado a cabo una mutagénesis mediante inserción aleatoria de un minitransposón derivado de Tn5 en Rhizobium leguminosarum bv. viciae UPM1137, una cepa capaz de resistir elevadas concentraciones de níquel y cobalto. Como resultado de esta mutagénesis se han obtenido 14 mutantes incapaces de crecer en medios suplementados con NiCl2. La localización de la inserción en estos mutantes muestra que una elevada proporción de los genes afectados codifican proteínas de membrana o proteínas secretadas. En paralelo, se ha obtenido la secuencia del genoma de la cepa UPM1137, lo que permite realizar estudios in silico comparando los genomas disponibles de varias cepas de R. leguminosarum bv. viciae, que presentan una menor sensibilidad a metales. El análisis bioinformático de los genomas secuenciados y la caracterización fenotípica de los mutantes obtenidos permitirá identificar potenciales sistemas de resistencia y su contribución a la homeostasis de metales.