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El aceite de oliva es obtenido por molturación mecánica, batido y centrifugación del fruto del “olivo” Olea europea (Oleaceae), para la denominación de aceite extra virgen es necesario que la acidez sea menor o igual al 0,8% expresada en ácido oleico y una puntuación menor o igual a 6,5 puntos, conservando los compuestos polifenólicos originarios, que le confieren la gran calidad de gusto y aroma. En este contexto, el presente estudio tiene como objetivo determinar la estabilidad de la calidad sensorial de aceite de oliva extra virgen variedad Farga y Frantoio con Farga en el periodo de marzo a octubre, de acuerdo, con el análisis de varianza unidireccional no existe una diferencia estadística significativa en los resultados químicos de acidez e índice de peróxidos en el aceite de oliva extra virgen variedad Frantoio con Farga, a diferencia del aceite de oliva extra virgen variedad Farga que presentó valores dispares al iniciar y final del periodo de evaluación. Se determinó que existe una correlación significativa entre el índice de peróxido y los resultados sensoriales, por tanto, los autores consideran que el aceite de oliva extra virgen variedad Frantoio con Farga presenta mayor estabilidad que el aceite de oliva extra virgen variedad Farga. “Olive” Olea europaea (Oleaceae) oil is obtained by mechanical grinding, whipping and centrifugation of the fruit of the olive tree, for the denomination of extra virgin oil it is necessary that the acidity is less than or equal to 0.8% expressed in oleic acid and a score less than or equal to 6.5 points, preserving the original polyphenolic compounds, which confer the great quality of taste and aroma. In this context, the present study aims to determine the stability of the sensory quality of extra virgin olive oil variety Farga and Frantoio with Farga in the period from March to October, according to the unidirectional analysis of variance there is no difference Statistical significance in the chemical results of acidity and peroxide index in the extra virgin olive oil of the Frantoio with Farga variety, unlike the extra virgin olive oil of the Farga variety that presented different values at the beginning and end of the evaluation period. It was determined that there is a significant correlation between the peroxide index and the sensory results, therefore, the authors consider that the extra virgin olive oil of the Frantoio with Farga variety presents greater stability than the extra virgin olive oil of the Farga variety.

En Perú, el olivo es cultivado para la producción de aceite y/o aceituna, con un crecimiento ydesarrollo sostenible, sustentado por mejoras agronómicas, incorporación de capital y mejoramientoproductivo. La producción olivícola se concentra principalmente en el Sur del país, siendo la regiónTacna la zona de mayor crecimiento, especialmente en las áreas de La Yarada, Los Palos, Magollo ySama. Por tanto, la industria olivícola requiere cultivares de mayor producción, que implica que solovariedades comerciales sean cultivas, mientras que las menos comerciales se encuentren restringidas,de ahí la importancia de la conservación, debido a que varios rasgos adaptativos podrían usarse enmejoramiento genético. En este contexto, se implementó el banco de germoplasma de olivo en laEstación Experimental Agraria Tacna, el objetivo de esta investigación fue realizar la caracterizaciónagromorfológica de las plantas madre del banco de germoplasma de olivo. Los resultados obtenidosen la evaluación de fruto, endocarpio, hoja y árbol en 34 plantas madre, se ajustan a las característicasrepresentativas de las variedades en cuestión. El resultado estadístico mostró que el endocarpioes la característica con mayor poder discriminante y heredable. Se asumió la posibilidad de dossinonimias, una entre la variedad Cornezuelo y Cornicabra, la segunda entre Arauco y SevillanaTacneña, sin embargo, los autores recomiendan realizar caracterización molecular para descartaresta posibilidad. Por último, es necesario realizar evaluaciones en las accesiones correspondientesdel banco de germoplasma.

La “quinua” Chenopocium quinoa (Amaranthaceae) es un alimento básico en la nutrición humana con diferentes aplicaciones, siendo el malteado de gran interés en la industria de alimentos. En el norte de Perú, una de las principales regiones productoras de “quinua” es La Libertad, y sus principales provincias productoras de quinua son Julcán, Sánchez Carrión y Santiago de Chuco. Por tal motivo, el objetivo de la investigación fue identificar variedades de quinua nativa en la región La Libertad, Perú con potencial para el malteado. Las variedades colectadas fueron Blanca Junín, Blanca Huancayo y Hualhuas, que fueron liberadas por el Instituto Nacional de Innovación Agraria. Se colectaron 52 muestras; que fueron analizadas en dos fases. La primera fue el análisis del porcentaje de humedad, proteína y almidón toral del grano maduro. La segunda fue la determinación del porcentaje de proteína, amilasa, amilopectina y almidón total en los granos malteados. Posteriormente se aplicó un análisis de varianza unidireccional a los promedios de los resultados. Se encontró diferencia estadística significativa entre las variedades de quinua en el grano maduro, sin embargo, no se encontró una diferencia estadística significativa en el grano malteado. Por tanto, para la industria de bebidas fermentadas e industria de alimentos se recomienda la variedad Hualhuas. The “quinoa” Chenopocium quinoa (Amaranthaceae) is a basic food in human nutrition with different applications, being the malting of great interest in the food industry. In northern Peru, one of the main quinoa producing regions is La Libertad, and its main quinoa producing provinces are Julcán, Sánchez Carrión and Santiago de Chuco. Therefore, the objective of the research was to identify varieties of native quinoa in the La Libertad region, Peru with potential for malting. The varieties collected were Blanca Junín, Blanca Huancayo and Hualhuas, which were released by the National Institute of Agrarian Innovation. 52 samples were collected; which were analyzed in two phases. The first was the analysis of the percentage of moisture, protein and total starch of the mature grain. The second was the determination of the percentage of protein, amylase, amylopectin and total starch in the malted grains. Subsequently, a one-way analysis of variance was applied to the averages of the results. A statistically significant difference was found between the varieties of quinoa in the mature grain, however, a statistically significant difference was not found in the malted grain. Therefore, for the fermented beverage industry and the food industry, the Hualhuas variety is recommended.

El malteado de trigo presenta un gran interés en la industria de alimentos y bebidas alcohólicas.Por tal motivo, el objetivo de la investigación fue identificar variedades de trigo nativo en la regiónLa Libertad, Perú con potencial para el malteado. Las variedades colectadas fueron Andino INIA,INIA 405 San Isidro y Centenario que se emplean en la industria de panificación, que fueronliberadas por el Instituto Nacional de Innovación Agraria. Se recolectaron 55 muestras; que fueronanalizadas en dos fases. La primera fue el análisis del porcentaje de humedad, proteína y almidóntotal del grano maduro. La segunda fue la determinación del porcentaje de proteína, amilasa,amilopectina y almidón total en los granos malteados. Posteriormente se aplicó un análisis devarianza unidireccional a los promedios de los resultados. Sin encontrar una diferencia estadísticasignificativa entre las variedades respecto al porcentaje de humedad, almidón total, sin embargo,se encontró una diferencia estadística significativa entre las variedades respecto al porcentaje deproteína, siendo la variedad Andino INIA la de mayor concentración. Por tanto, para la industriade bebidas fermentadas se recomiendan las variedades INIA 405 San Isidro y Centenario. Para laindustria de alimentos, la variedad Andino INIA.

Proton transport is indispensable for cell life. It is believed that molecular mechanisms of proton movement through different types of proton-conducting molecules have general universal features. However, elucidation of such mechanisms is a challenge. It requires true-atomic-resolution structures of all key proton-conducting states. Here we present a comprehensive function-structure study of a light-driven bacterial inward proton pump, xenorhodopsin, from Bacillus coahuilensis in all major proton-conducting states. The structures reveal that proton translocation is based on proton wires regulated by internal gates. The wires serve as both selectivity filters and translocation pathways for protons. The cumulative results suggest a general concept of proton translocation. We demonstrate the use of serial time-resolved crystallography at a synchrotron source with sub-millisecond resolution for rhodopsin studies, opening the door for principally new applications. The results might also be of interest for optogenetics since xenorhodopsins are the only alternative tools to fire neurons.

Non-coding RNAs (crRNAs) produced from clustered regularly interspaced short palindromic repeats (CRISPR) loci and CRISPR-associated (Cas) proteins of the prokaryotic CRISPR-Cas systems form complexes that interfere with the spread of transmissible genetic elements through Cas-catalysed cleavage of foreign genetic material matching the guide crRNA sequences. The easily programmable targeting of nucleic acids enabled by these ribonucleoproteins has facilitated the implementation of CRISPR-based molecular biology tools for in vivo and in vitro modification of DNA and RNA targets. Despite the diversity of DNA-targeting Cas nucleases so far identified, native and engineered derivatives of the Streptococcus pyogenes SpCas9 are the most widely used for genome engineering, at least in part due to their catalytic robustness and the requirement of an exceptionally short motif (5′-NGG-3′ PAM) flanking the target sequence. However, the large size of the SpCas9 variants impairs the delivery of the tool to eukaryotic cells and smaller alternatives are desirable. Here, we identify in a metagenome a new CRISPR-Cas9 system associated with a smaller Cas9 protein (EHCas9) that targets DNA sequences flanked by 5′-NGG-3′ PAMs. We develop a simplified EHCas9 tool that specifically cleaves DNA targets and is functional for genome editing applications in prokaryotes and eukaryotic cells. Conselleria de Innovación, Universidades, Ciencia y Sociedad Digital, Generalitat Valenciana (CIUCSD), Grant/Award Numbers: PROMETEO/2017/129 and PROMETEO/2021/057. Ministerio de Ciencia e Innovación (MICINN), Grant/Award Numbers: RTI2018-101223-B-I00 and PID2021-127644OB-I00.

International audience; All living organisms include a set of signaling devices that confer the ability to dynamically perceive and adapt to the fluctuating environment. Two-component systems are part of this sensory machinery that regulates the execution of different genetic and/or biochemical programs in response to specific physical or chemical signals. In the last two decades, there has been tremendous progress in our molecular understanding on how signals are detected, the allosteric mechanisms that control intramolecular information transmission and the specificity determinants that guarantee correct wiring. All this information is starting to be exploited in the development of new synthetic networks. Connecting multiple molecular players, analogous to programming lines of code, can provide the resources to build new sophisticated biocomputing systems. The Synthetic Biology field is starting to revolutionize several scientific fields, such as biomedicine and agriculture, propelling the development of new solutions. Expanding the spectrum of available nanodevices in the toolbox is key to unleash its full potential. This review aims to discuss, from a structural perspective, how to take advantage of the vast array of sensor and effector protein modules involved in two-component systems for the construction of new synthetic circuits.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 is an effector protein that targets invading DNA and plays a major role in the prokaryotic adaptive immune system. Although Streptococcus pyogenes CRISPR–Cas9 has been widely studied and repurposed for applications including genome editing, its origin and evolution are poorly understood. Here, we investigate the evolution of Cas9 from resurrected ancient nucleases (anCas) in extinct firmicutes species that last lived 2.6 billion years before the present. We demonstrate that these ancient forms were much more flexible in their guide RNA and protospacer-adjacent motif requirements compared with modern-day Cas9 enzymes. Furthermore, anCas portrays a gradual palaeoenzymatic adaptation from nickase to double-strand break activity, exhibits high levels of activity with both single-stranded DNA and single-stranded RNA targets and is capable of editing activity in human cells. Prediction and characterization of anCas with a resurrected protein approach uncovers an evolutionary trajectory leading to functionally flexible ancient enzymes. This work has been supported by grant nos. PID2019-109087RB-I00 (to R.P.-J.) and RTI2018-101223-B-I00 and PID2021-127644OB-I00 (to L.M.) from the Spanish Ministry of Science and Innovation. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 964764 (to R.P.-J.). The content presented in this document represents the views of the authors, and the European Commission has no liability in respect to the content. We acknowledge financial support from the Spanish Foundation for the Promotion of Research of Amyotrophic Lateral Sclerosis. A.F. acknowledges Spanish Center for Biomedical Network Research on Rare Diseases (CIBERE) intramural funds (no. ER19P5AC756/2021). F.J.M.M. acknowledges research support by Conselleria d’Educació, Investigació, Cultura i Esport from Generalitat Valenciana, research project nos. PROMETEO/2017/129 and PROMETEO/2021/057. M.M. acknowledges funding from CIBERER (grant no. ER19P5AC728/2021). The work has received funding from the Regional Government of Madrid (grant no. B2017/BMD3721 to M.A.M.-P.) and from Instituto de Salud Carlos III, cofounded with the European Regional Development Fund ‘A way to make Europe’ within the National Plans for Scientific and Technical Research and Innovation 2017–2020 and 2021–2024 (nos. PI17/1659, PI20/0429 and IMP/00009; to M.A.M.-P. B.P.K. was supported by an MGH ECOR Howard M. Goodman Award and NIH P01 HL142494.

The Cas1 protein is essential for the functioning of CRISPR-Cas adaptive systems. However, despite the high prevalence of CRISPR-Cas systems in thermophilic microorganisms, few studies have investigated the occurrence and diversity of Cas1 across hot spring microbial communities. Phylogenomic analysis of 2,150 Cas1 sequences recovered from 48 metagenomes representing hot springs (42–80°C, pH 6–9) from three continents, revealed similar ecological diversity of Cas1 and 16S rRNA associated with geographic location. Furthermore, phylogenetic analysis of the Cas1 sequences exposed a broad taxonomic distribution in thermophilic bacteria, with new clades of Cas1 homologs branching at the root of the tree or at the root of known clades harboring reference Cas1 types. Additionally, a new family of casposases was identified from hot springs, which further completes the evolutionary landscape of the Cas1 superfamily. This ecological study contributes new Cas1 sequences from known and novel locations worldwide, mainly focusing on under-sampled hot spring microbial mat taxa. Results herein show that circumneutral hot springs are environments harboring high diversity and novelty related to adaptive immunity systems. This work was financed in part by FONDECYT regular N° 1190998 (ANID) and Iniciativa de Investigación UnACh 2021-157-Unach. OS and JT-L were supported in part by ANID National Doctoral Scholarship (Beca de Doctorado Nacional ANID) N° 21172022 and 21171048, respectively. SG-L was supported by ANID FONDECYT Postdoctoral N° 3210547. AM-B and RQ were supported by Centro Ciencia and Vida, FB210008, Financiamiento Basal para Centros Científicos y Tecnológicos de Excelencia de ANID, and FONDECYT regular N° 1221035 (ANID). FJMM acknowledged research support by the Conselleria d’Innovació, Universitats, Ciència i Societat Digital from Generalitat Valenciana, research project PROMETEO/2021/057. BD acknowledged the Millennium Institute Center for Genome Regulation, Project ICN2021-044 supported by the ANID Millennium Scientific Initiative (Chile).

A long time has passed since regularly interspaced DNA repeats were discovered in prokaryotes. Today, those enigmatic repetitive elements termed clustered regularly interspaced short palindromic repeats (CRISPR) are acknowledged as an emblematic part of multicomponent CRISPR-Cas (CRISPR associated) systems. These systems are involved in a variety of roles in bacteria and archaea, notably, that of conferring protection against transmissible genetic elements through an adaptive immune-like response. This review summarises the present knowledge on the diversity, molecular mechanisms and biology of CRISPR-Cas. We pay special attention to the most recent findings related to the determinants and consequences of CRISPR-Cas activity. Research on the basic features of these systems illustrates how instrumental the study of prokaryotes is for understanding biology in general, ultimately providing valuable tools for diverse fields and fuelling research beyond the mainstream. The authors are supported by grant PROMETEO/2017/129 (Conselleria d’Educació, Investigació, Cultura i Esport, Generalitat Valenciana, Spain).

International audience; A case series study was conducted to determine the frequency of causes of abortion in dairy cattle in Uruguay. The sample size of 102 cases was composed of 53 fetuses, 35 fetuses with placentas, and 14 placentas without an associated fetus. All cases underwent gross and microscopic pathologic examinations as well as microbiological and serological testing. The etiology was determined in 54 (53%) of cases, 51 of which were caused by infectious agents. Within the observed 102 cases, 30 (29%) were caused by Neospora caninum, six (6%) by Coxiella burnetii and two (2%) by Campylobacter fetus subsp. venerealis. Bovine Parainfluenza-3 virus and Salmonella enterica serovar Newport caused one abortion each. Opportunistic bacteria (Escherichia coli, Streptococcus sp., Staphylococcus sp., Mannheimia sp., Trueperella pyogenes, and Providencia stuartii) were associated with 11 abortions. In two cases the fetal death was attributed to dystocia, and in one case the fetus had a congenital mesothelioma. Bovine viral diarrhea virus (BVDV) infection was identified in three fetuses; two of which were co-infected with and had typical lesions of N. caninum. No lesions were observed in the other fetus infected by BVDV. Leptospira interrogans was identified in one fetus without lesions. Despite the relatively low overall success rate in establishing an etiological diagnosis in cases of abortion in cattle, a systemic workup of bovine abortion is necessary to establish prevention and control strategies. This also facilitates monitoring and surveillance of reproductive diseases in dairy cattle, some of which represent a risk to public health.; Uma série de casos foi estudada para determinar a frequência de causas do aborto em bovinos leiteiros no Uruguai. A amostra, de 102 casos, foi composta por 53 fetos, 35 fetos com placentas e 14 placentas sem feto associado. Todos os casos foram submetidos a exames patológicos macroscópicos e microscópicos, além de testes microbiológicos e sorológicos. A etiologia foi determinada em 54 (53%) dos casos, 51 dos quais foram causados por agentes infecciosos. Nos 102 casos observados, 30 (29%) foram causados por Neospora caninum, seis (6%) por Coxiella burnetii e dois (2%) por Campylobacter fetus subsp. venerealis. O vírus da Parainfluenza-3 e Salmonella enterica serovar Newport causaram um aborto cada. Bactérias oportunistas (Escherichia coli, Streptococcus sp., Staphylococcus sp., Mannheimia sp., Trueperella pyogenes e Providencia stuartii) foram associadas a 11 abortos. Em dois casos, a morte fetal foi atribuída a distocia e, em um caso, o feto apresentava mesotelioma congênito. A infecção pelo vírus da diarreia viral bovina (BVDV) foi identificada em três fetos; dois dos quais foram co-infectados e apresentavam lesões típicas de N. caninum. Não foram observadas lesões no outro feto infectado pelo BVDV. Leptospira interrogans foi identificada em um feto sem lesões. Apesar da relativamente baixa taxa de sucesso no diagnóstico etiológico nos casos de aborto em bovinos, é necessário o diagnóstico sistemático dos abortos para estabelecer estratégias de prevenção e controle. Isso também facilita o monitoramento e a vigilância de doenças reprodutivas em bovinos leiteiros, algumas das quais representam um risco para a saúde pública.

Several Leptospira species are bacterial agents of leptospirosis, a neglected tropical disease responsible for ~1 million cases and 50,000 deaths each year worldwide. Leptospira, like other members of the Spirochaeta phylum, possess specially adapted flagella that remain confined within the periplasm. These appendages drive a unique, corkscrew-like swimming style that enables efficient motility and pathogenesis. However, the composition, function, and molecular architecture of spirochetal flagellar filaments remain poorly understood. We solved single-particle cryo-EM structures of isolated Leptospira flagellar filaments, comparing the wild-type form to two mutant forms with different missing components and abrogated motilities. The structures reveal a complex proteinaceous sheath surrounding a conserved core composed of the FlaB flagellin homolog. Sheath proteins were found to fall into two distinct categories, both of which are required for motility. Filament ‘coiling’ proteins, FcpA and FcpB, exert force on the filament when they bind its surface, causing the filament to stretch. In contrast, we identify sheath components FlaAP (newly discovered in this study) and FlaA2 as ‘template’ factors, which have little effect on filament shape by themselves, but partition the coiling proteins to one side of the filament. In this way, the two types of Leptospira sheath factors operate collectively on the flagellar filament to bend it from a ‘relaxed’ form associated with cell immobility, to a motility-competent shape that is tightly supercoiled. Our structures also indicate that core-sheath interactions are largely mediated by carbohydrate moieties from flagellin core side chain O-glycosylations. The supercoiling mechanism presented here provides a benchmark for studies with other bacteria, for which near-atomic resolution structures of flagellar filament in native supercoiled forms, are still lacking.

The long-term fate of plastics in the ocean and their interactions with marine microorganisms remain poorly understood. In particular, the role of sinking plastic particles as a transport vector for surface microbes towards the deep sea has not been investigated. Here, we present the first data on the composition of microbial communities on floating and suspended plastic particles recovered from the surface to the bathypelagic water column (0-2000 m water depth) of the North Pacific Subtropical Gyre. Microbial community composition of suspended plastic particles differed from that of plastic particles afloat at the sea surface. However, in both compartments, a diversity of hydrocarbon-degrading bacteria was identified. These findings indicate that microbial community members initially present on floating plastics are quickly replaced by microorganisms acquired from deeper water layers, thus suggesting a limited efficiency of sinking plastic particles to vertically transport microorganisms in the North Pacific Subtropical Gyre. HN, AV were financed through the European Research Council (ERC-CoG Grant Nr 772923, project VORTEX). PDM was supported by NWO (VI.Veni.212.040).

The number and diversity of known CRISPR–Cas systems have substantially increased in recent years. Here, we provide an updated evolutionary classification of CRISPR–Cas systems and cas genes, with an emphasis on the major developments that have occurred since the publication of the latest classification, in 2015. The new classification includes 2 classes, 6 types and 33 subtypes, compared with 5 types and 16 subtypes in 2015. A key development is the ongoing discovery of multiple, novel class 2 CRISPR–Cas systems, which now include 3 types and 17 subtypes. A second major novelty is the discovery of numerous derived CRISPR–Cas variants, often associated with mobile genetic elements that lack the nucleases required for interference. Some of these variants are involved in RNA-guided transposition, whereas others are predicted to perform functions distinct from adaptive immunity that remain to be characterized experimentally. The third highlight is the discovery of numerous families of ancillary CRISPR-linked genes, often implicated in signal transduction. Together, these findings substantially clarify the functional diversity and evolutionary history of CRISPR–Cas. K.S.M., Y.I.W., J.I., S.A.S. and E.V.K. are supported through the Intramural Research Program of the US National Institutes of Health; F.J.M.M. was supported by grants BIO2014-53029-P (Ministerio de Ciencia, Innovación y Universidades, Spain), and 291815 Era- Net ANIHWA (7th Framework Programme, European Commission) and PROMETEO/2017/129 (Conselleria d'Educació, Investigació, Cultura i Esport, Generalitat Valenciana, Spain); S.A.S. was supported by RFBR (research project 18-34-00012) and a Systems Biology Fellowship from Philip Morris Sales and Marketing; S.M. was funded by funding from the Natural Sciences and Engineering Research Council of Canada (Discovery program) and holds a Tier 1 Canada Research Chair in Bacteriophages.

Haloferax mediterranei is an extremely halophilic archaeon, able to live in hypersaline environments with versatile nutritional requirements, whose study represents an excellent basis in the field of biotechnology. The transcriptional machinery in Archaea combines the eukaryotic basal apparatus and the bacterial regulation mechanisms. However, little is known about molecular mechanisms of gene expression regulation compared with Bacteria, particularly in Haloarchaea. The genome of Hfx. mediterranei contains a gene, lrp (HFX_RS01210), which encodes a transcriptional factor belonging to Lrp/AsnC family. It is located downstream of the glutamine synthetase gene (HFX_RS01205), an enzyme involved in ammonium assimilation and amino acid metabolism. To study this transcriptional factor more deeply, the lrp gene has been homologously overexpressed and purified under native conditions by two chromatographic steps, namely nickel affinity and gel filtration chromatography, showing that Lrp behaves asa tetrameric protein of approximately 67 kDa. Its promoter region has been characterized under different growth conditions using bgaH as a reporter gene. The amount of Lrp protein was also analyzed by Western blotting in different nitrogen sources and under various stress conditions. To sum up, regarding its involvement in the nitrogen cycle, it has been shown that its expression profile does not change in response to the nitrogen sources tested. Differences in its expression pattern have been observed under different stress conditions, such as in the presence of hydrogen peroxide or heavy metals. According to these results, the Lrp seems to be involved in a general response against stress factors, acting as a first-line transcriptional regulator. This research was funded by Universidad de Alicante, VIGROB-016.

International audience; Most cancers become more dangerous by the outgrowth of malignant subclones with additional DNA mutations that favor proliferation or survival. Using chronic lymphocytic leukemia (CLL), a disease exemplary of this process, and a model for neoplasms in general, we created transgenic mice overexpressing the enzyme, activation-induced deaminase (AID), whose normal function is to induce DNA mutations in B lymphocytes. AID allows normal B lymphocytes to develop more effective immunoglobulin (Ig)-mediated immunity, but also is able to mutate non-Ig genes, predisposing to cancer. In chronic lymphocytic leukemia (CLL), AID expression correlates with poor prognosis suggesting a role for this enzyme in disease progression. Nevertheless, direct experimental evidence identifying the specific genes that are mutated by AID and indicating that those genes are associated with disease progression is not available. To address this point, we overexpressed Aicda in a murine model of CLL (Em-TCL1). Analyses of TCL1/AID mice demonstrate a role for AID in disease kinetics, CLL-cell proliferation, and the development of cancer-related target mutations with canonical AID signatures in non-Igs genes. Notably, our mouse models can accumulate mutations in the same genes that are mutated in human cancers. Moreover, some of these mutations occur at homologous positions, leading to identical or chemically-similar amino acid substitutions as in human CLL and lymphoma.Together, these findings support a direct link between aberrant AID activity and CLL driver mutations that are then selected for their oncogenic effects, whereby AID promotes aggressiveness in CLL and other B-cell neoplasms.

Pangenome analyses reveal major clues on evolutionary instances and critical genome core conservation. The order Rhizobiales encompasses several families with rather disparate ecological attitudes. Among them, Rhizobiaceae, Bradyrhizobiaceae, Phyllobacteriacreae and Xanthobacteriaceae, include members proficient in mutualistic symbioses with plants based on the bacterial conversion of N2 into ammonia (nitrogen-fixation). The pangenome of 12 nitrogen-fixing plant symbionts of the Rhizobiales was analyzed yielding total 37,364 loci, with a core genome constituting 700 genes. The percentage of core genes averaged 10.2% over single genomes, and between 5% to 7% were found to be plasmid-associated. The comparison between a representative reference genome and the core genome subset, showed the core genome highly enriched in genes for macromolecule metabolism, ribosomal constituents and overall translation machinery, while membrane/periplasm-associated genes, and transport domains resulted under-represented. The analysis of protein functions revealed that between 1.7% and 4.9% of core proteins could putatively have different functions. This work was supported in part by grant “Progetto di Ateneo PRAT CPDA154841/15” from the University of Padova.

La evaluación de los aprendizajes en competencias transversales requiere la aplicación de metodologías que favorezcan las oportunidades para aprender. El principal propósito de este proyecto ha sido el desarrollo de procedimientos de evaluación colaborativos aplicables a la evaluación de acciones formativas grupales y de sus productos, en la asignatura Iniciación a la Investigación en Biología (Grado en Biología). Para ello hemos trabajado a lo largo de cinco ejes principales: i) desarrollar rúbricas digitales colaborativas para la evaluación de productos de actividades grupales; ii) aplicar las rúbricas a trabajos colaborativos de estudiantes; iii) elaboración de cuestionarios de evaluación de las rúbricas de evaluación de las actividades formativas por el alumnado; iv) administrar la encuesta de evaluación para valorar la percepción del alumnado sobre el proceso de evaluación del trabajo grupal; y v) analizar y valorar los resultados y proponer posibles propuestas de mejora. Mayoritariamente, los estudiantes han valorado muy positivamente la utilización del sistema de e-rúbricas para la evaluación de las entregas realizadas durante el curso, y se han mostrado útiles para la autoevaluación de sus competencias, así como para la autocorrección de errores y la valoración de los indicadores de evaluación.

The DNA damage checkpoint protein DisA and the branch migration translocase RecG are implicated in the preservation of genome integrity in reviving haploid Bacillus subtilis spores. DisA synthesizes the essential cyclic 3′, 5′-diadenosine monophosphate (c-di-AMP) second messenger and such synthesis is suppressed upon replication perturbation. In vitro, c-di-AMP synthesis is suppressed when DisA binds DNA structures that mimic stalled or reversed forks (gapped forks or Holliday junctions [HJ]). RecG, which does not form a stable complex with DisA, unwinds branched intermediates, and in the presence of a limiting ATP concentration and HJ DNA, it blocks DisA-mediated c-di-AMP synthesis. DisA pre-bound to a stalled or reversed fork limits RecG-mediated ATP hydrolysis and DNA unwinding, but not if RecG is pre-bound to stalled or reversed forks. We propose that RecG-mediated fork remodeling is a genuine in vivo activity, and that DisA, as a molecular switch, limits RecG-mediated fork reversal and fork restoration. DisA and RecG might provide more time to process perturbed forks, avoiding genome breakage. This work was supported by the Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación (MCIU/AEI)/FEDER PGC2018-097054-B-I00 to S.A. and J.C.A.

La Red MICROcer0 se crea con la intención principal de ayudar a los alumnos del Grado de Gastronomía y Artes culinarias en la asignatura de Microbiología Alimentaria. Su alumnado es heterogéneo en cuanto a edad y origen académico y muchos carecen de una base sólida de conocimientos en Microbiología y en general, de Biología para cursar la asignatura “Microbiología Alimentaria”. Para conocer qué flaquezas de conceptos existen, se han diseñado “test” previos que el alumno realizará al principio de la asignatura (curso 2021-2022) y en función de sus resultados, sabrá qué temas son los que ha de repasar-aprender previamente al desarrollo de la asignatura. Todo este material se hará llegar a través de Moodle vía UA Cloud. Se ha realizado una encuesta a los alumnos del presente año (tras cursas la misma) y se muestran los resultados principales. Entre otros aspectos, un alto porcentaje de los alumnos usaría este tipo de material, lo que haría que éstos entendiesen mejor la importancia de la Microbiología en sus carreras profesionales, algo que los alumnos de este Grado no acaban de visualizar.

Mycobacterium tuberculosis is a pathogen with a unique cell envelope including very long fatty acids, implicated in bacterial resistance and host immune modulation. FasR is a TetR-like transcriptional activator that plays a central role in sensing mycobacterial long-chain fatty acids and regulating lipid biosynthesis. Here we disclose crystal structures of M. tuberculosis FasR in complex with acyl effector ligands and with DNA, uncovering its molecular sensory and switching mechanisms. A long tunnel traverses the entire effector-binding domain, enabling long fatty acyl effectors to bind. Only when the tunnel is entirely occupied, the protein dimer adopts a rigid configuration with its DNA-binding domains in an open state, leading to DNA dissociation. The protein-folding hydrophobic core connects the two domains, and is completed into a continuous spine when the effector binds. Such a transmission spine is conserved in a large number of TetR-like regulators, offering insight into effector-triggered allosteric functional control., FasR is a TetR-like transcriptional activator that plays a central role in sensing mycobacterial long-chain fatty acids and regulating lipid biosynthesis in Mycobacterium tuberculosis. Here authors present crystal structures of M. tuberculosis FasR in complex with acyl effector ligands and with DNA, uncovering its molecular sensory and switching mechanisms.

CRISPR-Cas represents a prokaryotic defense mechanism against invading genetic elements. Although there is a diversity of CRISPR-Cas systems, they all share similar, essential traits. In general, a CRISPR-Cas system consists of one or more groups of DNA repeats named CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), regularly separated by unique sequences referred to as spacers, and a set of functionally associated cas (CRISPR associated) genes typically located next to one of the repeat arrays. The origin of spacers is in many cases unknown but, when ascertained, they usually match foreign genetic molecules. The proteins encoded by some of the cas genes are in charge of the incorporation of new spacers upon entry of a genetic element. Other Cas proteins participate in generating CRISPR-spacer RNAs and perform the task of destroying nucleic acid molecules carrying sequences similar to the spacer. In this way, CRISPR-Cas provides protection against genetic intruders that could substantially affect the cell viability, thus acting as an adaptive immune system. However, this defensive action also hampers the acquisition of potentially beneficial, horizontally transferred genes, undermining evolution. Here we cover how the model bacterium Escherichia coli deals with CRISPR-Cas to tackle this major dilemma, evolution versus survival. The authors are supported by grants BIO2014-53029-P (Ministerio de Economía y Competitividad, Spain), 291815 Era-Net ANIHWA (7th Framework Programme, European Commission) and PROMETEO/2017/129 (Conselleria d'Ed-ucació, Investigació, Cultura i Esport, Generalitat Valenciana, Spain).

La asignatura Iniciación a la Investigación en Biología del Grado en Biología se desarrolla a partir de una experiencia de aprendizaje basada en un proyecto de investigación bibliográfica y bibliométrica. Se presentan los resultados de una serie de encuestas de autoevaluación y de satisfacción del alumnado sobre experiencias para mejorar la motivación (elección de tema de investigación) y participación, y sobre la herramienta de planificación del trabajo colaborativo. Los resultados indican los aspectos positivos y cierto margen de mejora en la percepción y conocimientos del alumnado sobre la organización y planificación del trabajo.

The ability to perceive the environment, an essential attribute in living organisms, is linked to the evolution of signalling proteins that recognize specific signals and execute predetermined responses. Such proteins constitute concerted systems that can be as simple as a unique protein, able to recognize a ligand and exert a phenotypic change, or extremely complex pathways engaging dozens of different proteins which act in coordination with feedback loops and signal modulation. To understand how cells sense their surroundings and mount specific adaptive responses, we need to decipher the molecular workings of signal recognition, internalization, transfer and conversion into chemical changes inside the cell. Protein allostery and dynamics play a central role. Here, we review recent progress on the study of two- component systems, important signalling machineries of prokaryotes and lower eukaryotes. Such systems implicate a sensory histidine-kinase and a separate response regulator protein. Both components exploit protein flexibility to effect specific conformational rearrangements, modulating protein:protein interactions, and ultimately transmitting information accurately. Recent work has revealed how histidine-kinases switch between discrete functional states according to the presence or absence of the signal, shifting key amino acid positions that define their catalytic activity. In concert with the cognate response regulator’s allosteric changes, the phosphoryl-transfer flow during the signalling process is exquisitely fine-tuned for proper specificity, efficiency and directionality. Agencia Nacional de Investigación e Innovación

Pathogenic Leptospira species represent a major concern for livestock but also for human health, as they cause zoonotic infections. Forty strains representing L. interrogans , L. borgpetersenii , and L. noguchii were isolated from naturally infected cattle in Uruguay. Here, we report the whole-genome sequences for these strains.

Perceiving environmental and internal information and reacting in adaptive ways are essential attributes of living organisms. Two-component systems are relevant protein machineries from prokaryotes and lower eukaryotes that enable cells to sense and process signals. Implicating sensory histidine kinases and response regulator proteins, both components take advantage of protein phosphorylation and flexibility to switch conformations in a signal-dependent way. Dozens of two component systems act simultaneously in any given cell, challenging our understanding about the means that ensure proper connectivity. This review dives into the molecular level, attempting to summarize an emerging picture of how histidine kinases and cognate response regulators achieve required efficiency, specificity, and directionality of signaling pathways, properties that rely on protein:protein interactions. α helices that carry information through long distances, the fine combination of loose and specific kinase/regulator interactions, and malleable reaction centers built when the two components meet emerge as relevant universal principles. Agencia Nacional de Investigación e Innovación IMiZA Joint International Unit (Institut Pasteur–Institut Pasteur de Montevideo)

In C4 grasses of agronomical interest, malate shuttled into the bundle sheath cells is decarboxylated mainly by nicotinamide adenine dinucleotide phosphate (NADP)-malic enzyme (C4-NADP-ME). The activity of C4-NADP-ME was optimized by natural selection to efficiently deliver CO2 to Rubisco. During its evolution from a plastidic non-photosynthetic NADP-ME, C4-NADP-ME acquired increased catalytic efficiency, tetrameric structure and pH-dependent inhibition by its substrate malate. Here, we identified specific amino acids important for these C4 adaptions based on strict differential conservation of amino acids, combined with solving the crystal structures of maize and sorghum C4-NADP-ME. Site-directed mutagenesis and struc-tural analyses show that Q503, L544 and E339 are involved in catalytic efficiency; E339 confers pH-dependent regulation by malate, F140 is critical for the stabilization of the oligomeric structure and the N-terminal region is involved in tetramerization. Together, the identified molecular adaptations form the basis for the efficient catalysis and regulation of one of the central biochemical steps in C4 metabolism

International audience; Glucose-6-phosphate dehydrogenase (G6PDH) is the key enzyme supplying reducing power (NADPH) to the cells, by oxidation of glucose-6-phosphate (G6P), and in the process providing a precursor of ribose-5-phosphate. G6PDH is also a virulence factor of pathogenic trypanosomatid parasites. To uncover the biochemical and structural features that distinguish TcG6PDH from its human homolog, we have solved and analyzed the crystal structures of the G6PDH from Trypanosoma cruzi (TcG6PDH), alone and in complex with G6P. TcG6PDH crystallized as a tetramer and enzymatic assays further indicated that the tetramer is the active form in the parasite, in contrast to human G6PDH, which displays higher activity as a dimer. This quaternary structure was shown to be particularly stable. The molecular reasons behind this disparity were unveiled by structural analyses: a TcG6PDH-specific residue, R323, is located at the dimer-dimer interface, critically contributing with two salt bridges per subunit that are absent in the human enzyme. This explains why TcG6PDH dimerization impaired enzyme activity. The parasite protein is also distinct in displaying a 37-amino-acid extension at the N-terminus, which comprises the non-conserved C8 and C34 involved in the covalent linkage of two neighboring protomers. In addition, a cysteine triad (C53, C94 and C135) specific of Kinetoplastid G6PDHs proved critical for stabilization of TcG6PDH active site. Based on the structural and biochemical data, we posit that the N-terminal region and the catalytic site are highly dynamic. The unique structural features of TcG6PDH pave the way toward the design of efficacious and highly specific anti-trypanosomal drugs.

International audience; Glycan-protein interactions control numerous biological events from cell-cell recognition and signaling to pathogen host cell attachment for infections. To infect cells, some viruses bind to immune cells with the help of DC-SIGN (dendritic cell [DC]-specific ICAM3-grabbing nonintegrin) C-type lectin expressed on dendritic and macrophage cell membranes, via their envelope protein. Prevention of this infectious interaction is a serious therapeutic option. Here, we describe the synthesis of the first water-soluble tetravalent fucocluster pseudopeptide-based 1,3-alternate thiacalixarenes as viral antigen mimics designed for the inhibition of DC-SIGN, to prevent viral particle uptake. Their preparation exploits straightforward convergent strategies involving one-pot Ugi four-component (Ugi-4CR) and azido-alkyne click chemistry reactions as key steps. Surface plasmon resonance showed strong inhibition of DC-SIGN interaction properties by tetravalent ligands designed with high relative potencies and β avidity factors. All ligands block DC-SIGN active sites at nanomolar IC50 preventing cis-cell infection by Ebola viral particles pseudotyped with EBOV glycoprotein (Zaire species of Ebola virus) on Jurkat cells that express DC-SIGN. In addition, we observed strong inhibition of DC-SIGN/human cytomegalovirus (HCMV)-gB recombinant glycoprotein interaction. This finding opens the way to the simple development of new models of water-soluble glycocluster-based thia-calixarenes with wide-ranging antimicrobial activities.

Protein Kinase A (PKA) is a widespread enzyme that plays a key role in many signaling pathways from lower eukaryotes to metazoans. In mammals, the regulatory (R) subunits sequester and target the catalytic (C) subunits to proper subcellular locations. This targeting is accomplished by the dimerization and docking (D/D) domain of the R subunits. The activation of the holoenzyme depends on the binding of the second messenger cAMP. The only available structures of the D/D domain proceed from mammalian sources. Unlike dimeric mammalian counterparts, the R subunit from Saccharomyces cerevisiae (Bcy1) forms tetramers in solution. Here we describe the first high-resolution structure of a non-mammalian D/D domain. The tetramer in the crystals of the Bcy1 D/D domain is a dimer of dimers that retain the classical D/D domain fold. By using phylogenetic and structural analyses combined with site-directed mutagenesis, we found that fungal R subunits present an insertion of a single amino acid at the D/D domain that shifts the position of a downstream, conserved arginine. This residue participates in intra-dimer interactions in mammalian D/D domains, while due to this insertion it is involved in inter-dimer contacts in Bcy1, which are crucial for the stability of the tetramer. This surprising finding challenges well-established concepts regarding the oligomeric state within the PKAR protein family and provides important insights into the yet unexplored structural diversity of the D/D domains and the molecular determinants of R subunit oligomerization.

CRISPR-Cas are self-/nonself-discriminating systems found in prokaryotic cells. They represent a remarkable example of molecular memory that is hereditarily transmitted. Their discovery can be considered as one of the first fruits of the systematic exploration of prokaryotic genomes. Although this genomic feature was serendipitously discovered in molecular biology studies, it was the availability of multiple complete genomes that shed light about their role as a genetic immune system. Here we tell the story of how this discovery originated and was slowly and painstakingly advanced to the point of understating the biological role of what initially was just an odd genomic feature. FJMM is funded by the Spanish Ministerio de Economía y Competitividad (BIO2014-53029P) and the European Commission/Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (291815 Era-Net ANIHWA). FR-V is funded by projects MEDIMAX BFPU2013-48007-P from the Spanish Ministerio de Economía y Competitividad, MaCuMBA Project 311975 of the European Commission FP7 and PROMETEO II/2014/012 project AQUAMET from the Generalitat Valenciana.

Hepatitis C virus (HCV) is a highly variable infectious agent, classified into 8 genotypes and 86 subtypes. Our laboratory has implemented an in-house developed high-resolution HCV subtyping method based on next-generation sequencing (NGS) for error-free classification of the virus using phylogenetic analysis and analysis of genetic distances in sequences from patient samples compared to reference sequences. During routine diagnostic, a sample from an Equatorial Guinea patient could not be classified into any of the existing subtypes. The whole genome was analyzed to confirm that the new isolate could be classified as a new HCV subtype. In addition, naturally occurring resistance-associated substitutions (RAS) were analyzed by NGS. Whole-genome analysis based on p-distances suggests that the sample belongs to a new HCV genotype 1 subtype. Several RAS in the NS3 (S122T, D168E and I170V) and NS5A protein (Q(1b)24K, R(1b)30Q and Y93L+Y93F) were found, which could limit the use of some inhibitors for treating this subtype. RAS studies of new subtypes are of great interest for tailoring treatment, as no data on treatment efficacy are reported. In our case, the patient has not yet been treated, and the RAS report will be used to design the most effective treatment., This study was supported by the Spanish Ministry of Health, Consumption and Social Welfare grant name Plan Estrategico Nacional contra la Hepatitis C. This study was funded by Instituto de Salud Carlos III grants, PI15/00856, PI15/00829 and PI16/00337, and cofinanced by CIBERehd (Consorcio Centro de Investigacion en Red de Enfermedades Hepaticas y Digestivas), which is funded by Instituto de Salud Carlos III and Centro para el Desarrollo Tecnologico Industrial-CDTI from the Spanish Ministry of Economy and Business, grant number, IDI-20151125.

Este trabajo se propone mejorar la adquisición de competencias transversales del Módulo Básico del Grado en Biología, a través de una mejora en la experiencia docencia-aprendizaje de la asignatura Iniciación a la Investigación en Biología del alumnado que cursa primero del Grado en Biología. Para ello hemos realizado un diagnóstico de la situación centrándonos en cuatro objetivos principales: el análisis de la experiencia del alumnado en el trabajo grupal y el método de aprendizaje basado en proyectos a través del análisis de rúbricas; el análisis de la adquisición de competencias trasversales del Módulo Básico del Grado en Biología; el análisis de las preferencias del alumnado sobre la selección de temas de investigación; y la evaluación de los métodos de evaluación individual formativa de conocimientos en tiempo real en clases de teoría. Los resultados obtenidos nos permiten asegurar que la adopción progresiva de nuevas estrategias de docencia-aprendizaje basadas en la retroalimentación de la propia Red Docente está consiguiendo mejorar los resultados de adquisición de competencias transversales de los estudiantes.

SIMBAD is a sequence-independent molecular-replacement pipeline for solving difficult molecular-replacement cases where contaminants have been crystallized. It can also be used to find structurally related search models where no obvious homologue can be found through sequence-based searching., The conventional approach to finding structurally similar search models for use in molecular replacement (MR) is to use the sequence of the target to search against those of a set of known structures. Sequence similarity often correlates with structure similarity. Given sufficient similarity, a known structure correctly positioned in the target cell by the MR process can provide an approximation to the unknown phases of the target. An alternative approach to identifying homologous structures suitable for MR is to exploit the measured data directly, comparing the lattice parameters or the experimentally derived structure-factor amplitudes with those of known structures. Here, SIMBAD, a new sequence-independent MR pipeline which implements these approaches, is presented. SIMBAD can identify cases of contaminant crystallization and other mishaps such as mistaken identity (swapped crystallization trays), as well as solving unsequenced targets and providing a brute-force approach where sequence-dependent search-model identification may be nontrivial, for example because of conformational diversity among identifiable homologues. The program implements a three-step pipeline to efficiently identify a suitable search model in a database of known structures. The first step performs a lattice-parameter search against the entire Protein Data Bank (PDB), rapidly determining whether or not a homologue exists in the same crystal form. The second step is designed to screen the target data for the presence of a crystallized contaminant, a not uncommon occurrence in macromolecular crystallography. Solving structures with MR in such cases can remain problematic for many years, since the search models, which are assumed to be similar to the structure of interest, are not necessarily related to the structures that have actually crystallized. To cater for this eventuality, SIMBAD rapidly screens the data against a database of known contaminant structures. Where the first two steps fail to yield a solution, a final step in SIMBAD can be invoked to perform a brute-force search of a nonredundant PDB database provided by the MoRDa MR software. Through early-access usage of SIMBAD, this approach has solved novel cases that have otherwise proved difficult to solve.

Phytoplasmas are wall-less phytopathogenic bacteria that produce devastating effects in a wide variety of plants. Reductive evolution has shaped their genome, with the loss of many genes, limiting their metabolic capacities. Owing to the high concentration of C4 compounds in plants, and the presence of malic enzyme (ME) in all phytoplasma genomes so far sequenced, the oxidative decarboxylation of l-malate might represent an adaptation to generate energy. Aster yellows witches’-broom (Candidatus Phytoplasma) ME (AYWB-ME) is one of the smallest of all characterized MEs, yet retains full enzymatic activity. Here, the crystal structure of AYWB-ME is reported, revealing a unique fold that differs from those of ‘canonical’ MEs. AYWB-ME is organized as a dimeric species formed by intertwining of the N-terminal domains of the protomers. As a consequence of such structural differences, key catalytic residues such as Tyr36 are positioned in the active site of each protomer but are provided by the other protomer of the dimer. A Tyr36Ala mutation abolishes the catalytic activity, indicating the key importance of this residue in the catalytic process but not in the dimeric assembly. Phylogenetic analyses suggest that larger MEs (largesubunit or chimeric MEs) might have evolved from this type of smaller scaffold by gaining small sequence cassettes or an entire functional domain. The Candidatus Phytoplasma AYWB-ME structure showcases a novel minimal structure design comprising a fully functional active site, making this enzyme an attractive starting point for rational genetic design. Fil: Alvarez, Clarisa Ester. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; Argentina Fil: Trajtenberg, F.. Instituto Pasteur de Montevideo; Uruguay Fil: Larrieux, N.. Instituto Pasteur de Montevideo; Uruguay Fil: Saigo, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; Argentina Fil: Golic, Adrián Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; Argentina Fil: Andreo, Carlos Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; Argentina Fil: Hogenhout, S. A.. John Innes Institute; Reino Unido Fil: Mussi, María Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; Argentina Fil: Drincovich, Maria Fabiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; Argentina Fil: Buschiazzo, A.. Instituto Pasteur de Montevideo; Uruguay

Bacterial canker caused by Clavibacter michiganensis subspecies michiganensis (Cmm) has been a threat to tomato production in Uruguay for several years. In this study, 39 Cmm strains were collected from several outbreaks and production areas in Uruguay, and were identified by molecular assays and pathogenicity tests on a susceptible cultivar of tomato. In addition, a TaqMan assay targeting a putative two-component system sensor kinase gene demonstrated good specificity with all strains tested and gave no false negative results. The first epidemiological study of Cmm in this country was carried out in order to elucidate the origin of outbreaks and sources of infection and dissemination of the pathogen. Strains from Uruguay showed high genetic diversity based on a Multi Locus Sequence Typing analysis of five housekeeping genes. This approach revealed 36 sequence types (STs) within a worldwide collection of 108 Cmm strains. Ten STs correspond to strains solely isolated in Uruguay, including eight novel STs for the subspecies michiganensis. This high diversity reflects the introduction of new strains from different origins that most probably results from seed importation. This study provides relevant information about the distribution and origin of strains causing bacterial canker in Uruguay and will pave the way for the establishment of preventive measures to control the disease.

En esta investigación se propone diagnosticar y mejorar la adquisición de competencias transversales del Módulo Básico del Grado en Biología, a través de la mejora de la la experiencia de docencia-aprendizaje de la asignatura Iniciación a la Investigación. Para ello se realiza un diagnóstico analizando las preferencias del alumnado respecto a la elección de temas de investigación, el análisis de los conocimientos previos y la adquisición de competencias tras cursar la asignatura, así como su comparación, aplicando la mejora de las estrategias y metodologías de aprendizaje mediante el uso de TICs en clases de Teoría, y la identificación de las propuestas de mejora de la experiencia docente por parte del alumnado.

Unveiling virus-host interactions are relevant for understanding the biology and evolution of microbes globally, but in particular, it has also a paramount impact on the manufacture of fermented dairy products. In this study, we aim at characterizing phages infecting the commonly used heterofermentative Leuconostoc spp. on the basis of host range patterns and genome analysis. Host range of six Leuconostoc phages was investigated using three methods (efficiency of plaquing, spot and turbidity tests) against Ln. mesenteroides and Ln. pseudomesenteroides strains. Complete genome sequencing from four out of the six studied Leuconostoc phages were obtained in this work, while the remaining two have been sequenced previously. According to our results, cross-species host specificity was demonstrated, as all phages tested were capable of infecting both Ln. pseudomesenteroides and Ln. mesenteroides strains, although with different efficiency of plaquing (EOP). Phage adsorption rates and ability of low-EOP host strains to propagate phages by crossing the Leuconostoc species' barrier confirm results. At the genome level, phages CHA, CHB, Ln-7, Ln-8 and Ln-9 revealed high similarity with previously characterized phages infecting mostly Ln. mesenteroides strains, while phage LDG was highly similar to phages infecting Ln. pseudomesenteroides. Additionally, correlation between receptor binding protein (RBP) and host range patterns allowed us to unveil a finer clustering of Leuconostoc phages studied into four groups. This is the first report of overlapped phage host ranges between Leuconostoc species. This work was supported by the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET; Project PIP 112-201201-00046; Argentina), the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT; Project PICT 2010-0138; Argentina) and the Universidad Nacional del Litoral (UNL, Project CAI+D PI 501 201101 00039 LI; Argentina). S.A.P. was the recipient of an international scholarship awarded by BEC.AR (Becas de formación en el exterior en Ciencia y Tecnología, Presidencia de la Nación, Argentina). M.M.G. thanks Ministerio de Economía y Competitividad (refs. CGL2013-40564-R and SAF2013-49267-EXP), Generalitat Valenciana (ACOMP/2015/133) and Gordon and Betty Moore Foundation (Grant award ref. 5334). F.J.M.M. is funded by the Spanish Ministerio de Economía y Competitividad (BIO2014-53029P) and the European Commission/Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (291815 Era-Net ANIHWA).

Antibiotics started to be used almost 90 years ago to eradicate life-threatening infections. The urgency of the problem required rapid, broad-spectrum elimination of infectious agents. Since their initial discovery, these antimicrobials have saved millions of lives. However, they are not exempt from side effects, which include the indiscriminate disruption of the beneficial microbiota. Recent technological advances have enabled the development of antimicrobials that can selectively target a gene, a cellular process, or a microbe of choice. These strategies bring us a step closer to developing personalized therapies that exclusively remove disease-causing infectious agents. Here, we advocate the preservation of our beneficial microbes and provide an overview of promising alternatives to broad-spectrum antimicrobials. Specifically, we emphasize nucleic acid and peptide-based systems as a foundation for next-generation alternatives to antibiotics that do not challenge our microbiota and may help to mitigate the spread of resistance. Cesar de la Fuente-Nunez is a Ramon Areces Foundation Fellow (Spain). Marcelo Der Torossian Torres is supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (Brazil). Francisco J. M. Mojica is funded by the Spanish Ministerio de Economía y Competitividad (BIO2014-53029P) and the European Commission/Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (291815 Era-Net ANIHWA). Timothy Lu is supported by the National Institutes of Health, National Science Foundation, Defense Threat Reduction Agency, Defense Advanced Research Projects Agency, Center for Microbiome Informatics and Therapeutics, Kenneth Rainin Foundation, Koch Institute for Integrative Cancer Research, and Broad Institute of MIT and Harvard.

Background: Blood infections are serious complex conditions that generally require rapid diagnosis and treatment. The big challenge is to reduce the time necessary to make a diagnosis with current clinical microbiological methods so as to improve the treatment given to patients. Methods: In this study, we assess for the first time the Sepsis Flow Chip assay, which is a novel diagnostic assay for simultaneous rapid-detection of the vast majority of bloodstream pathogens, including Gram-positive and Gram-negative bacteria and fungi, in the same assay, and for the detection of most common antibiotic resistance genes. The SFC assay is based on multiplex PCR and low density DNA arrays. Results: Positive blood cultures from 202 consecutive bacteremia patients were analyzed by SFC assay and the results were compared with the results obtained by the gold standard methodology used in clinical microbiology diagnostic laboratories (EUCAST guidelines). SFC assay overall sensitivity and specificity for bacterial identification were 93.3% and 100% respectively and sensitivity and specificity for the identification of antibiotic genetic resistance determinants were 93.6% and 100% respectively. Conclusions: This is the first evaluation of SFC assay in clinical samples. This new method appears to be very promising by combining the high number of distinct pathogens and genetic resistance determinants identified in a single assay. Further investigations should be done to evaluate the usefulness of this assay in combination with clinical multidisciplinary groups (stewardship), in order for the results to be applied appropriately to the management of patients ‘infectious processes. This work was supported by Hospital General Universitario de Alicante (UGP-14-270) http://alicante.san.gva.es/; Fundación Soria Melguizo (no number) http://www.f-soria.es/; and FISABIO (UGP-14-216) http://fisabio.san.gva.es/.

International audience; We have developed protocols to generate site-specific variants of the histidine-kinase DesK and its cognate response regulator DesR, conducive to trapping different signaling states of the proteins. Co-expression of both partners in E. coli, ensuring an excess of the regulator, was essential for soluble production of the DesK:DesR complexes and further purification. The 3D structures of the complex trapped in the phosphotransferase and in the phosphatase reaction steps, were solved by X-ray crystallography using molecular replacement. The solution was not trivial, and we found that in silico-generated models used as search probes, were instrumental to succeeding in placing a large portion of the complex in the asymmetric unit. Electron density maps were then clear enough to allow for manual model building attaining complete atomic models. These methods contribute to tackling a major challenge in the bacterial signaling field, namely obtaining stable kinase:regulator complexes, in distinct conformational states, amenable for high-resolution crystallographic studies.

The protein FcpA is a unique component of the flagellar filament of spirochete bacteria belonging to the genusLeptospira. Although it plays an essential role in translational motility and pathogenicity, no structures of FcpA homologues are currently available in the PDB. Its three-dimensional structure will unveil the novel motility mechanisms that render pathogenicLeptospiraparticularly efficient at invading and disseminating within their hosts, causing leptospirosis in humans and animals. FcpA fromL. interroganswas purified and crystallized, but despite laborious attempts no useful X ray diffraction data could be obtained. This challenge was solved by expressing a close orthologue from the related saprophytic speciesL. biflexa. Three different crystal forms were obtained: a primitive and a centred monoclinic form, as well as a hexagonal variant. All forms diffracted X-rays to suitable resolutions for crystallographic analyses, with the hexagonal type typically reaching the highest limits of 2.0 Å and better. A variation of the quick-soaking procedure resulted in an iodide derivative that was instrumental for single-wavelength anomalous diffraction methods.

PURPOSE Antimicrobial resistance is a worldwide public health threat and Salmonella enterica subsp. enterica serotype Typhimurium phage type DT104B multiresistant strains with additional quinolone resistance have been responsible for global outbreaks and high mortality. Quinolone resistance is known to be multifactorial but is still far from a complete understanding. To give new insights about the resistance mechanisms involved, this work aimed to evaluate subproteome changes between an S. Typhimurium DT104B clinical strain that acquired fluoroquinolone resistance after treatment (Se20) and its pretreatment parental strain (Se6), and also subproteome variations in Se20 under ciprofloxacin (CIP) stress (Se20+CIP). EXPERIMENTAL DESIGN The proteomes were compared at the intracellular and membrane levels by a 2-DE∼LC-MS/MS and a shotgun LC-MS/MS approach, respectively. RESULTS In total, 14 differentially abundant proteins were identified when comparing Se6 with Se20 and 91 were identified between Se20 and Se20+CIP. Several proteins with known and possible roles in quinolone resistance (AAC(6')-Ib-cr4, OmpD, OmpX, GlmS, GlmU, H-NS, etc.) were identified and discussed. CONCLUSIONS AND CLINICAL RELEVANCE The great number of proteins identified in this study provides important information about mechanism-related differential protein expression which supports the current knowledge and might lead to new testable hypotheses on the mechanism of action of fluoroquinolone drugs.

La asignatura Iniciación a la investigación en Biología, que se imparte en el segundo semestre de primer curso del grado en Biología recoge parte de las competencias transversales del módulo Básico del Grado. En esta investigación se propone mejorar la metodología docente de la misma, a partir del análisis crítico de las actividades realizadas y de los resultados de la evaluación de cursos anteriores. Para ello se realiza un diagnóstico y se compara los resultados de evaluación de estudiantes de siete cursos, se mejoran y amplían las competencias específicas, se mejoran los contenidos de las diferentes actividades, se elaboran una serie de documentos de coordinación y se mejora la asignación de criterios y procedimientos de evaluación del alumnado, según las características de cada una de las actividades prácticas y de la tipología del estudiante.

Prokaryotes immunize themselves against transmissible genetic elements by the integration (acquisition) in clustered regularly interspaced short palindromic repeats (CRISPR) loci of spacers homologous to invader nucleic acids, defined as protospacers. Following acquisition, mono-spacer CRISPR RNAs (termed crRNAs) guide CRISPR-associated (Cas) proteins to degrade (interference) protospacers flanked by an adjacent motif in extrachomosomal DNA. During acquisition, selection of spacer-precursors adjoining the protospacer motif and proper orientation of the integrated fragment with respect to the leader (sequence leading transcription of the flanking CRISPR array) grant efficient interference by at least some CRISPR-Cas systems. This adaptive stage of the CRISPR action is poorly characterized, mainly due to the lack of appropriate genetic strategies to address its study and, at least in Escherichia coli, the need of Cas overproduction for insertion detection. In this work, we describe the development and application in Escherichia coli strains of an interference-independent assay based on engineered selectable CRISPR-spacer integration reporter plasmids. By using this tool without the constraint of interference or cas overexpression, we confirmed fundamental aspects of this process such as the critical requirement of Cas1 and Cas2 and the identity of the CTT protospacer motif for the E. coli K12 system. In addition, we defined the CWT motif for a non-K12 CRISPR-Cas variant, and obtained data supporting the implication of the leader in spacer orientation, the preferred acquisition from plasmids harboring cas genes and the occurrence of a sequential cleavage at the insertion site by a ruler mechanism. This work was funded by the Ministerio de Economía y Competitividad (BIO2011-24417).

International audience; Bacterial two-component systems consist of a sensor histidine kinase (HK) and a response regulator (RR). HKs are homodimers that catalyze the autophosphorylation of a histidine residue and the subsequent phosphoryl transfer to its RR partner, triggering an adaptive response. How the HK autokinase and phosphotransferase activities are coordinated remains unclear. Here, we report X-ray structures of the prototypical HK CpxA trapped as a hemi-phosphorylated dimer, and of the receiver domain from the RR partner, CpxR. Our results reveal that the two catalytic reactions can occur simultaneously, one in each protomer of the asymmetric CpxA dimer. Furthermore, the increase of autokinase activity in the presence of phosphotransfer-impaired CpxR put forward the idea of an allosteric switching mechanism, according to which CpxR binding to one CpxA protomer triggers autophosphorylation in the second protomer. The ensuing dynamical model provides a mechanistic explanation of how HKs can efficiently orchestrate two catalytic reactions involving large-scale protein motions.

Two-component systems (TCS) are protein machineries that enable cells to respond to input signals. Histidine kinases (HK) are the sensory component, transferring information toward downstream response regulators (RR). HKs transfer phosphoryl groups to their specific RRs, but also dephosphorylate them, overall ensuring proper signaling. The mechanisms by which HKs discriminate between such disparate directions, are yet unknown. We now disclose crystal structures of the HK:RR complex DesK:DesR from Bacillus subtilis, comprising snapshots of the phosphotransfer and the dephosphorylation reactions. The HK dictates the reactional outcome through conformational rearrangements that include the reactive histidine. The phosphotransfer center is asymmetric, poised for dissociative nucleophilic substitution. The structural bases of HK phosphatase/phosphotransferase control are uncovered, and the unexpected discovery of a dissociative reactional center, sheds light on the evolution of TCS phosphotransfer reversibility. Our findings should be applicable to a broad range of signaling systems and instrumental in synthetic TCS rewiring. DOI: http://dx.doi.org/10.7554/eLife.21422.001