Targeted bacterial conjugation mediated by synthetic cell-to-cell adhesions

Genetic interventions on microbiomes, for clinical or biotechnological purposes, remain challenging. Conjugation-based delivery of genetic cargo is still unspecific and limited by low conjugation rates. Here we report an approach to overcome these problems, based on a synthetic bacterial adhesion system. Mating assemblers consist on a synthetic adhesion formed by the expression on the surface of donor and target cells of specific nanobodies (Nb) and their cognate antigen (Ag). The Nb?Ag bridge increased 1?3 logs transfer of a variety of plasmids, especially in liquid media, confirming that cell-cell docking is a main determinant limiting mating efficiency. Synthetic cell-to-cell adhesion allows efficient conjugation to targeted recipients, enhancing delivery of desired genes to a predefined subset of prey species, or even specific pathogenic strains such as enterohemorrhagic Escherichia coli (EHEC), within a bacterial community. The synthetic conjugation enhancer presented here optimizes plasmid delivery by selecting the target hosts with high selectivity.FUNDING: Spanish Science and Innovation Ministry (MCIN) [PID2020-117923GB-I00 to F.d.l.C.]; MCIN/AEI and FEDER [BIO2017-89081-R]; MCIN/AEI and NextGeneration EU/PRTR [PLEC2021-007739 to L.A.F.]. Conflict of interest statement. None declared ACKNOWLEDGEMENTS: We thank R. Fernandez-López for providing us with the BWmKate2 strain and, along with M. P. Garcillan-Barcia, for helpful discussions. We also thank E. Zechner for providing the pAR106 plasmid, and I. Rosenshine for EHEC strains and V. Campa for technical assistance in setting up the microscopy assay

A chromosome-level genome assembly enables the identification of the follicule stimulating hormone receptor as the master sex-determining gene in the flatfish Solea senegalensis

Sex determination (SD) shows huge variation among fish and a high evolutionary rate, as illustrated by the Pleuronectiformes (flatfishes). This order is characterized by its adaptation to demersal life, compact genomes and diversity of SD mechanisms. Here, we assembled the Solea senegalensis genome, a flatfish of great commercial value, into 82 contigs (614 Mb) combining long- and short-read sequencing, which were next scaffolded using a highly dense genetic map (28,838 markers, 21 linkage groups), representing 98.9% of the assembly. Further, we established the correspondence between the assembly and the 21 chromosomes by using BAC-FISH. Whole genome resequencing of six males and six females enabled the identification of 41 single nucleotide polymorphism variants in the follicle stimulating hormone receptor (fshr) consistent with an XX/XY SD system. The observed sex association was validated in a broader independent sample, providing a novel molecular sexing tool. The fshr gene displayed differential expression between male and female gonads from 86 days post-fertilization, when the gonad is still an undifferentiated primordium, concomitant with the activation of amh and cyp19a1a, testis and ovary marker genes, respectively, in males and females. The Y-linked fshr allele, which included 24 nonsynonymous variants and showed a highly divergent 3D protein structure, was overexpressed in males compared to the X-linked allele at all stages of gonadal differentiation. We hypothesize a mechanism hampering the action of the follicle stimulating hormone driving the undifferentiated gonad toward testisEuropean Union's Horizon 2020 research and innovation programme under grant agreement (AQUA-FAANG). Grant Number: 81792. Junta de Andalucía-FEDER Grant. Grant Number: P20-00938. Spanish Ministry of Economy and Competitiveness, FEDER Grants. Grant Numbers: RTI2018-096847-B-C21, RTI2018-096847-B-C22S

Whole genome sequencing of turbot (Scophthalmus maximus; Pleuronectiformes):a fish adapted to demersal life

12 páginas, 5 figuras.-- Antonio Figueras ... et al.-- This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly citedThe turbot is a flatfish (Pleuronectiformes) with increasing commercial value, which has prompted active genomic research aimed at more efficient selection. Here we present the sequence and annotation of the turbot genome, which represents a milestone for both boosting breeding programmes and ascertaining the origin and diversification of flatfish. We compare the turbot genome with model fish genomes to investigate teleost chromosome evolution. We observe a conserved macrosyntenic pattern within Percomorpha and identify large syntenic blocks within the turbot genome related to the teleost genome duplication. We identify gene family expansions and positive selection of genes associated with vision and metabolism of membrane lipids, which suggests adaptation to demersal lifestyle and to cold temperatures, respectively. Our data indicate a quick evolution and diversification of flatfish to adapt to benthic life and provide clues for understanding their controversial origin. Moreover, we investigate the genomic architecture of growth, sex determination and disease resistance, key traits for understanding local adaptation and boosting turbot production, by mapping candidate genes and previously reported quantitative trait loci. The genomic architecture of these productive traits has allowed the identification of candidate genes and enriched pathways that may represent useful information for future marker-assisted selection in turbotThis work was funded by the Spanish Government: projects Consolider Ingenio: Aquagenomics (CSD2007-00002) and Metagenoma de la Península Ibérica (CSD2007-00005), Ministerio de Economía y Competitividad and European Regional Development Funds (AGL2012-35904), and Ministerio de Economía y Competitividad (AGL2014-51773 and AGL2014-57065-R); and Local Government Xunta de Galicia (GRC2014/010). P.P. and D.R. gratefully acknowledge the Spanish Ministerio de Educación for their FPU fellowships (AP2010-2408, AP2012-0254). Funding to pay the Open Access publication charges for this article was provided by the Ministerio de Economía y Competitividad (AGL2014-51773) and Xunta de Galicia (GRC2014/010)Peer reviewe

CIBERER : Spanish national network for research on rare diseases: A highly productive collaborative initiative

Altres ajuts: Instituto de Salud Carlos III (ISCIII); Ministerio de Ciencia e Innovación.CIBER (Center for Biomedical Network Research; Centro de Investigación Biomédica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mission is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low-prevalence diseases, in line with the International Rare Diseases Research Consortium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this article, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Análisis molecular y funcional de la celulasa celc2 en la simbiosis rhizobia-leguminosas

[ES] La endosimbiosis Rhizobium-leguminosa constituye la fuente biológica de nitrógeno más eficiente en agricultura. El proceso de infección entre ambos está estrechamente regulado para asegurar la correcta penetración del microsimbionte en las células vegetales. En este trabajo, comprobamos que la celulasa CelC2 de Rhizobium leguminosarum bv. trifolii es esencial en el proceso de infección canónico en trébol ya que genera un orificio específicamente en el ápice no cristalino del pelo radical, por el que penetra la bacteria. Por otra parte, el gen celC, se encuentra muy conservado dentro del género Rhizobium y su sobreexpresión provoca la formación de canales de infección y nódulos aberrantes y un aumento de la competitividad de las bacterias. Estos fenotipos son resultado de la hidrólisis incontrolada de la celulosa no cristalina, cuya distribución en la planta se restringe a los puntos de infección primaria y secundaria. Por otra parte, la expresión heteróloga de la celulasa CelC2 en Sinorhizobium meliloti 1021, provoca un retraso en la nodulación y en la fijación de nitrógeno en plantas de Medicago, lo que sugiere que la infección mediada por endoglucanasas no se limita a Trifolium. Además, la aplicación del enzima purificado altera el patrón de los pulsos de calcio inducidos por los factores de nodulación y a la expresión de las nodulinas tempranas. Por tanto, estos datos indican la importancia de una estrecha regulación en la producción de enzimas degradativos de la pared celular vegetal en los rizobios para que la nodulación sea efectiva. Por último, la celulasa CelC2 está involucrada en la biosíntesis de celulosa en Rhizobium, que actúa a su vez en la formación de biopelículas tanto in planta como in vitro. Todos estos resultados confirman la funcionalidad de la celulasa CelC2 en la colonización y la infección durante el establecimiento de la simbiosis fijadora de nitrógeno en leguminosas.[EN] Rhizobium-legume endosymbiosis is the biological source of nitrogen more efficiently in agriculture. The infection process between the two is tightly regulated to ensure proper penetration of micro organisms in plant cells. In this study, we found that the cellulase CelC2 of Rhizobium leguminosarum bv. trifolii is essential in the infection process canonical clover because it generates a specific hole in the noncrystalline apex of root hair, which penetrates the bacteria. Moreover, the gene Celco, is highly conserved in Rhizobium and its overexpression causes the formation of channels of infection and aberrant nodules and increased competitiveness of the bacteria. These phenotypes are the result of uncontrolled hydrolysis of crystalline cellulose, which are distributed in the plant is restricted to points of primary and secondary infection. Moreover, heterologous expression of cellulase in Sinorhizobium meliloti CelC2 1021, causing a delay in nodulation and nitrogen fixation in Medicago plants, suggesting that infection mediated by endoglucanases not limited to Trifolium. In addition, the application of purified enzyme alters the pattern of pulses of calcium-induced nodulation factors and the expression of early nodulinas. Therefore, these data indicate the importance of tight regulation in the production of degradative enzymes in the plant cell wall in the rhizobia for nodulation effective. Finally, CelC2 cellulase is involved in cellulose biosynthesis in Rhizobium, which in turn acts in biofilm formation both in planta and in vitro. All these results confirm the functionality of cellulase CelC2 in colonization and infection during the establishment of nitrogen-fixing symbiosis in legumes

The noncoding RNA CcnA modulates the master cell cycle regulators CtrA and GcrA in Caulobacter crescentus

International audienceBacteria are powerful models for understanding how cells divide and accomplish global regulatory programs. In Caulobacter crescentus, a cascade of essential master regulators supervises the correct and sequential activation of DNA replication, cell division, and development of different cell types. Among them, the response regulator CtrA plays a crucial role coordinating all those functions. Here, for the first time, we describe the role of a novel factor named CcnA (cell cycle noncoding RNA A), a cell cycle-regulated noncoding RNA (ncRNA) located at the origin of replication, presumably activated by CtrA, and responsible for the accumulation of CtrA itself. In addition, CcnA may be also involved in the inhibition of translation of the S-phase regulator, GcrA, by interacting with its 5' untranslated region (5' UTR). Performing in vitro experiments and mutagenesis, we propose a mechanism of action of CcnA based on liberation (ctrA) or sequestration (gcrA) of their ribosome-binding site (RBS). Finally, its role may be conserved in other alphaproteobacterial species, such as Sinorhizobium meliloti, representing indeed a potentially conserved process modulating cell cycle in Caulobacterales and Rhizobiales

A chromosome-level genome assembly enables the identification of the follicule stimulating hormone receptor as the master sex determining gene in the flatfish Solea senegalensis

Sex determination (SD) shows huge variation among fish and a high evolutionary rate, as illustrated by the Pleuronectiformes (flatfishes). This order is characterized by its adaptation to demersal life, compact genomes and diversity of SD mechanisms. Here, we assembled the Solea senegalensis genome, a flatfish of great commercial value, into 82 contigs (614 Mb) combining long- and short-read sequencing, which were next scaffolded using a highly dense genetic map (28,838 markers, 21 linkage groups), representing 98.9% of the assembly. Further, we established the correspondence between the assembly and the 21 chromosomes by using BAC-FISH. Whole genome resequencing of six males and six females enabled the identification of 41 SNP variants in the follicle stimulating hormone receptor (fshr) consistent with an XX / XY SD system. The observed sex association was validated in a broader independent sample, providing a novel molecular sexing tool. Fshr displayed differential gene expression between male and female gonads from 86 days post-fertilization, when the gonad is still an undifferentiated primordium, concomitant with the activation of amh and cyp19a1a, testis and ovary marker genes, respectively, in males and females. The Y-linked fshr allele, which included 24 non-synonymous variants and showed a highly divergent 3D protein structure, was overexpressed in males compared to the X-linked allele at all stages of gonadal differentiation. We hypothesize a mechanism hampering the action of the follicle stimulating hormone driving the undifferentiated gonad toward testis.info:eu-repo/semantics/acceptedVersio

Rediscovering Public Administration. Creating images from University

Con un enfoque novedoso, este libro recoge las originales aportaciones que un grupo de universitarios españoles han elaborado acerca de la Administración pública en España. El objetivo es transmitir conocimientos desde el ámbito académico a cualquier ciudadano, para alentar la reflexión con criterio nuestras administraciones, sus cambios y sus previsibles dinámicas. También se dirige especialmente al funcionario, que no deja de ser una parte representativa de la sociedad, como una oportunidad para repensar su propia labor y desempeño en los servicios que ofrecen, que garantizan el bienestar de toda la ciudadanía.This book collects, with a novel focus, the original contributions that a group of Spanish university students have produced about Public Administration in Spain. Its goal is to transmit knowledge from the academic sphere to any citizen, in order to encourage a discerning reflection on our administration, its changes and its foreseeable dynamics. It is also aimed to the public servant, who is a representative part of society, as an opportunity to rethink his own labour and performance on the provided services that guarantee the welfare of society.Avec une nouvelle approche, ce livre recueille les contributions originales élaborées par un groupe d’universitaires espagnols à propos de l’Administration Publique en Espagne. Le but est de diffuser des connaissances à n’importe quel citoyen, depuis le cercle académique, afin d’encourager la réflexion sur nos administrations, leurs changements et leurs dynamiques prévisibles. De la même manière, le livre se dirige particulièrement au fonctionnaire, qui n’arrête pas d’être une partie représentative de la société, ainsi qu’une opportunité pour repenser à son propre travail et à son accomplissement sur les services qu’il accomplit, lesquels garantissent le bien-être à toute la citoyenneté.Der vorliegende Text stellt kein Buch zum Gebrauch da, weder in seinem Kern noch in seiner Verfassung, da es beabsichtigt, das positive Konzept der Öffentlichen Verwaltung angesichts von Stereotypen und Vorurteilen wieder herzustellen. Und in der Art der Verfassung, weil aus einer Liste von „vieltalentierten“ Mitteln, oder Bildern, zusammengestellt ist, welche von Studenten erarbeitet wurden, die in der Gesellschaft engagiert sind.Instituto Nacional de Administración PúblicaDepto. de Ciencia Política y de la AdministraciónFac. de Ciencias Políticas y SociologíaTRUEpu