Biological nitrogen fixation by the Rhizobium -legume symbiosis allows the conversion of atmospheric nitrogen into ammonia within root nodules mediated by the nitrogenase enzyme. Nitrogenase activity results in the evolution of hydrogen as a result of a side reaction intrinsic to the activity of this enzyme. Some rhizobia, and also other nitrogen fixers, induce a NiFe uptake hydrogenase (Hup) to recycle hydrogen produced by nitrogenase, thus improving the efficiency of the nitrogen fixation process. In this work we report the generation and symbiotic behavior of hydrogenase-positive Rhizobium leguminosarum and Mesorhizobium loti strains effective in vetch ( Vicia sativa) and birsfoot trefoil ( Lotus corniculatus ) forage crops, respectively. The ability of hydrogen recycling was transferred to these strains through the incorporation of hup minitransposon Tn HB100 , thus leading to full recycling of hydrogen in nodules. Inoculation of Vicia and Lotus plants with these engineered strains led to significant increases in the levels of nitrogen incorporated into the host legumes. The level of improvement of symbiotic performance was dependent on the recipient strain and also on the legume host. These results indicate that hydrogen recycling has the potential to improve symbiotic nitrogen fixation in forage plants.

EDITORIAL article Front. Agron., 23 November 2021 | https://doi.org/10.3389/fagro.2021.796717

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).

Los rizobios son alfa-proteobacterias capaces de infectar las raices de las leguminosas e inducir en las mismas la formacion de un nuevo organo, el nodulo radicular. En dicho nodulo las celulas bacterianas, diferenciadas en bacteroides especializados en la fijacion de nitrogeno, estan rodeadas de una membrana peribacteroidal a traves de cual la planta controla el intercambio de nutrientes hacia y desde el bacteroide. La adaptacion de las bacterias al estilo de vida simbiotico es el resultado de un proceso de co-evolucion entre ambos socios en el que se produce el intercambio de fuentes carbonadas y nitrogeno fijado en forma de amonio. En el proceso de establecimiento de la simbiosis se han descrito compuestos de diversa naturaleza quimica (flavonoides, lipoquitooligosacaridos, EPS) que median un reconocimiento especifico entre el rizobio y la leguminosa.1 Sin embargo, el intercambio de senales no termina con la formacion del nodulo. El funcionamiento de la simbiosis Rhizobium-leguminosa supone el ajuste metabolico de ambos componentes simbioticos en proceso cuyos detalles aun se desconocen. Uno de los objetivos de nuestro laboratorio se centra en el estudio de la adaptacion de Rhizobium a la simbiosis analizando como la bacteria responde al ambiente nodular proporcionado por la planta. Recientemente se ha descrito que en el caso de las leguminosas que inducen nodulos indeterminados (con actividad meristematica persistente) como Medicago, Pisum, o Vicia, la planta envia al bacteroide una bateria de multiples peptidos denominados NCR (Nodule-specific Cystein-Rich). de los que no se conoce la funcion concreta, aunque se ha demostrado que algunos de ellos son capaces de inducir modificaciones en celulas en cultivo similares a las descritas en bacteroides (inhibicion de la division celular, endorreduplicacion y alteraciones en la permeabilidd de la membrana).2 La hipotesis actual es que la accion combinada de los NCR controla parcial o totalmente la fisiologia de la bacteria induciendo su diferenciacion en bacteroide y convirtiendole en algo similar a un ?esclavo metabolico? cuya funcion esencial es la fijacion de nitrogeno para su aporte a la planta, interfiriendo con multiples procesos fisiologicos. En el caso de rizobios capaces de establecer simbiosis con distintas leguminosas, como es el caso de Rhizobium leguminosarum bv viciae con Pisum, Lens, Vicia y Lathyrus, es de esperar que los bacteroides inducidos en cada planta encuentren un habitat intracelular distinto si cada planta aporta un complemento de peptidos diferente. En esas condiciones el estudio de la respuesta de la bacteria a cada uno de esos habitats podria aportar informacion relevante sobre los caracteres que permiten la adaptacion de Rhizobium al estilo de vida intracelular en los nodulos de las leguminosas. En este trabajo se trata de evaluar la importancia de caracteres de adaptacion al hospedador en la asociacion simbiotica entre Rhizobium leguminosarum bv viciae (Rlv) y plantas leguminosas. Para ello se ha realizado la comparacion de los perfiles proteomicos de celulas endosimbioticas de Rlv UPM791 inducidas en nodulos de lenteja (Lens culinaris) y guisante (Pisum sativum). Dichos perfiles se obtuvieron mediante analisis LC-MS de extractos de bacteroides, complementado con marcaje diferencial empleando la metodologia iTRAQ. Este analisis ha revelado la existencia de diferencias en la expresion de un numero significativo de proteinas codificadas en distintas partes del genoma bacteriano. Entre estas proteinas se han identificado proteinas de respuesta a estres, un regulador transcripcional de tipo GntR, y otras proteinas que podrian tener un papel en el metabolismo de C/N en el bacteroide. Estos datos sugieren que las bacterias encuentran ambientes distintos en distintos hospedadores induciendo respuestas de adaptacion diferenciales. Dos de las proteinas identificadas, denominadas DABA y AMYDO, se encuentran codificadas en el plasmido simbiotico de la bacteria

The Rhizobium-legume symbiosis is highly specific and depends on several molecular signals produced by both partners. Some of these signals are bacterial proteins named effectors that are translocated into the plant cells by secretion systems similar to contractile nanomachines also called injectisomes (Deakin and Broughton, 2009). The injectisomes puncture and deliver the effectors into the target cell. One of these nanomachines, known as type VI secretion system (T6SS), was discovered recently and is reminiscent of phage injection machinery (Records, 2011). The role of these systems in legume endosymbiotic bacteria is mostly unknown, and this work presents the initial study of T6SSs from different bradyrhizobia. T6SSs have been identified in draft genomic sequences from Bradyrhizobium strains isolated from Lupinus spp. thriving in the Iberian Peninsula. In all cases, the genes encoding T6SSs were grouped and showed, in most cases, a high degree of conservation among genes encoding the structural components of the system. Bradyrhizobium sp. strain ISLU101 isolated from L. angustifolius, contains two clusters of genes involved in the formation of T6SS. One of such systems, designated as T6SS-1, contains 17 genes and shows a high degree of conservation regarding genes of B. diazoefficiens USDA110. The other one, T6SS-2, contains 16 genes flanked by insertion element sequences. Amino acid similarity between equivalent proteins encoded in both clusters is only about 40-50 %. A phylogenetic analysis based on the concatenation of sequences of several T6SS proteins was performed, and results indicate a clear separation of T6SS-2 from most rhizobial T6SSs. ISLU101 T6SS mutant derivatives in genes impO, impC1 and impC2 were generated by single homologous recombination of amplified internal fragments from the respective genes cloned into the suicide vector pK18mobsac. The symbiotic behaviour of mutants was examined with L. angustifolius. Results showed no effect of impC1 and impC2 mutations, while the impO mutant generated smaller plants with a mixture of white/red nodules. These results suggest that T6SSs may play a role in the Bradyrhizobium-lupines symbioses

Desde la década de los 80, los odontólogos de las especialidades de Ortodoncia y Cirugía Maxilofacial, integrados a un equipo multidisciplinario, juegan un rol importante en la terapia del ronquido y del Síndrome Apnea-Hipoapnea Obstructiva del Sueño (SAHOS). Este artículo describe los Dispositivos de Avance Mandibular (DAM) como tratamiento del SAHOS, las consideraciones anatómicas, estudios sobre su efectividad y control de su eficiencia. Se clasifican y detalla su mecanismo de acción, criterios de elección y sus efectos secundarios. Se presentan 2 casos clínicos que utilizaron un DAM de diseño nacional durante un mes, donde previo y posterior al uso de este aparato se midió el Índice Apnea e Hipoapnea, ronquidos y saturación de oxígeno con el Apnealink. Como resultado, se obtuvo una disminución en los valores de todos estos índices, calificando el tratamiento con DAM como exitoso en la terapia del SAHOS.

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).

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.

Rhizobium leguminosarum bv viciae (Rlv) es una alfa-proteobacteria capaz de establecer una simbiosis diazotrofica con distintas leguminosas. Uno de los factores implicados en el establecimiento de la simbiosis es el sistema de comunicacion intercelular conocido como Quorum Sensing (QS). Mediante este sistema, las bacterias actuan de manera coordinada en respuesta a cambios en la densidad de poblacion a traves de la produccion y deteccion de senales extracelulares. El genoma de Rlv UPM791 contiene dos sistemas tipo luxRI mediados por senales de tipo N-acyl-homoserina lactonas (AHLs): el sistema rhiRI, codificado en el plasmido simbiotico, 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 regulacion de los sistemas de QS en esta bacteria endosimbiotica se generaron mutantes defectivos en cada uno de los sistemas de QS, y se llevo a cabo un analisis detallado sobre la produccion de AHLs y la simbiosis con plantas de guisante, veza y lenteja. El sistema rhiRI se necesita para un comportamiento simbiotico normal, dado que la mutacion en rhiI reduce considerablemente la eficiencia simbiotica. rhiR es esencial para la fijacion de nitrogeno en ausencia del plasmido pUPM791d. Asimismo, mutaciones en el sistema cinRIS mostraron tambien un importante efecto en simbiosis. El mutante ?cinRIS no produce la senal 3-OH-C14:1-HSL, y da lugar a nodulos blancos e inefectivos, carentes de bacteroides. El mutante ?cinI, incapaz de producir AHLs, no forma nodulos en ninguna de las leguminosas utilizadas. El analisis genetico revelo que dicha mutacion origina la inestabilizacion del plasmido simbiotico 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 estadios de la simbiosis, e indican la existencia de un modelo de regulacion dependiente de QS significativamente distinto a los que se han descrito previamente en otras cepas de R. 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.

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.

Nickel, like other transition metals, can be toxic to cells even at moderate concentration (low microM range) by displacing essential metals from their native binding sites or by generating reactive oxygen species that cause oxidative DNA damage. For this reason, cells have evolved mechanisms to deal with excess nickel. Efflux systems include members of the Resistance-Nodulation-cell Division (RND) protein family, P-type ATPases, cation diffusion facilitators (CDF) and other resistance factors. Nickel-specific exporters have been characterized in Cupravidus metallidurans, Helicobacter pylori, Achromobacter xylosoxidans, Serratia marcenses and Escherichia coli.

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.

La asociacion Rhizobium-leguminosa constituye una interaccion planta-microorganismo particularmente beneficiosa a nivel medioambiental debido a su capacidad promotora del crecimiento vegetal en condiciones de deficiencia de nitrogeno. Se ha demostrado que una excesiva concentracion de metales pesados en el suelo afecta negativamente la competitividad bacteriana y al desarrollo de interacciones diazotroficas 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 biosintesis de enzimas bacterianas relacionadas con el proceso de fijacion de nitrogeno que ocurre en el interior de los nodulos de las leguminosas (Moreau et al., 1995). Con objeto de identificar sistemas genicos implicados en la homeostasis de niquel en bacterias endosimbioticas, se ha llevado a cabo una mutagenesis mediante insercion aleatoria de un minitransposon derivado de Tn5 en Rhizobium leguminosarum bv. viciae UPM1137, una cepa capaz de resistir elevadas concentraciones de niquel y cobalto. Como resultado de esta mutagenesis se han obtenido 14 mutantes incapaces de crecer en medios suplementados con NiCl2. La localizacion de la insercion en estos mutantes muestra que una elevada proporcion de los genes afectados codifican proteinas de membrana o proteinas 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 analisis bioinformatico de los genomas secuenciados y la caracterizacion fenotipica de los mutantes obtenidos permitira identificar potenciales sistemas de resistencia y su contribucion a la homeostasis de metales.