Uso del nitrógeno en algas: desvelando piezas del rompecabezas de la asimilación del nitrógeno y su regulación en el alga modelo Chlamydomonas reinhardtii

Las algas, formando parte de la base de la cadena trófica de ecosistemas marinos y de agua dulce, son clave para la vida acuática. Estos organismos fotosintéticos, sometidos a fluctuaciones constantes de disponibilidad de nutrientes, muestran un alto nivel de adaptabilidad a estos ambientes dinámicos. Aunque el nitrógeno (N), nutriente esencial para la vida, es comúnmente usado por las algas en su forma inorgánica, algunas especies de algas pueden usar compuestos de N orgánico, los cuales pueden ser especialmente abundantes debido a la escorrentía y filtrado de áreas fertilizadas de forma intensiva. El alga modelo Chlamydomonas reinhardtii (Chlamydomonas) puede consumir fuentes de nitrógeno inorgánico (amonio, nitrato y nitrito), así como L-arginina y urea. Además, este alga presenta una Laminoácido oxidasa extracelular (LAO1) que desamina un amplio rango de aminoácidos. En este trabajo hemos estudiado el control de la señalización que da lugar a la preferencia de nitrato sobre N orgánico en Chlamydomonas, el papel clave de LAO1 en el uso de aminoácidos y péptidos, así como el establecimiento de nuevas interacciones mutualistas con bacterias que promueven el crecimiento en N orgánico. Capítulo 1 El factor de transcripción NIT2 es el regulador clave de los genes de la asimilación de nitrato en Chlamydomonas. En primer lugar, comparamos el transcriptoma de una estirpe silvestre y otra mutante nit2 de Chlamydomonas en respuesta a nitrato. Observamos que nitrato y NIT2 reprimen los genes involucrados en el uso de fuentes de N orgánicas, incluyendo LAO1. Mediante el uso de mutantes de Chlamydomonas demostramos que tanto el nitrato como el nitrito afectan negativamente el uso de aminoácidos por este alga. Capítulo 2 Las enzimas L-aminoácido oxidasa (LAAO, L-Amino Acid Oxidase) están ampliamente distribuidas en la naturaleza y se propone que su papel principal en hongos y algas es la captación de nitrógeno. Mediante búsquedas genómicas comparativas, no pudimos encontrar ningún ortólogo de LAO1 en ningún alga verde ni en plantas, pero identificamos ortólogos en 10 de otras 27 especies de algas, incluyendo Rhodophyta, Alveolata, Heterokonta, Haptophyta y Dinophyta. La construcción de un árbol filogenético de enzimas LAAO reveló que las secuencias identificadas como ortólogas de LAO1 -denominadas aquí como ALAAOs (Algal LAAOs)-, se agrupaban en la misma rama evolutiva. Observamos que en Chlamydomonas el gen LAO1 está situado adyacente a un gen que codifica una putativa proteína RidA, que resultó estar evolutivamente cercana a la de cianobacterias. Nuestro análisis filogenético apoya la idea de que las proteínas ALAAOs pueden tener un origen en el ancestro común de las algas, el cual se originó por la endosimbiosis de una cianobacteria por un protista. Mediante el uso de un mutante lao1 hemos mostrado que LAO1 era crucial para el crecimiento de Chlamydomonas en 16 de 20 aminoácidos proteinogénicos, así como para algunos di-/tri-péptidos. Además de amonio, las enzimas LAAO producen el correspondiente cetoácido y peróxido de hidrógeno. Hemos demostrado que la reacción espontánea de los productos derivados de la desaminación por LAO1 de Lalanina -ácido pirúvico y peróxido de hidrógeno- genera ácido acético. Capítulo 3 Aunque Chlamydomonas puede crecer en la mayoría de los L-aminoácidos y en algunos di-/tri-péptidos como únicas fuentes de N, este crecimiento es mucho menos eficiente que en fuentes de N inorgánicas, y además, algunos aminoácidos y péptidos no pueden ser usados por este alga. De forma fortuita descubrimos una contaminación Methylobacterium sp. que permitió el crecimiento de Chlamydomonas en un di-péptido que no puede asimilar. Las especies de Methylobacterium están incluidas en el grupo de bacterias promotoras del crecimiento de plantas (PGPB, del inglés Plant Growth- Promoting Bacteria), las cuales mejoran el crecimiento de las plantas. Tras el muestreo en campo, aislamiento e identificación de bacterias, encontramos que algunas especies salvajes, incluidas en los géneros Methylobacterium, Sphingomonas, Deinococcus y Chitinophagaceae, mejoran el crecimiento de Chlamydomonas en L-serina. Además, algunas especies de Methylobacterium permitieron el crecimiento de Chlamydomonas en algunos aminoácidos y péptidos que este alga no puede usar. Hemos demostrado un nuevo mutualismo basado en un intercambio metabólico de carbono y nitrógeno entre Chlamydomonas y M. aquaticum. Por otro lado, algunas especies de Methylobacterium mejoraron el crecimiento de Chlamydomonas en aminoácidos asimilables. Para esta mejora, la enzima LAO1 fue esencial para el crecimiento del consorcio con algunas estirpes de Methylobacterium, incluyendo M. extorquens, M. hispanicum y M. organophilum. La comunicación química en la interacción entre organismos diferentes media las relaciones simbióticas. Entre estas moléculas de señalización, el ácido indolacético es una de las más estudiadas. Descubrimos que la producción de índoles dependiente de L-triptófano por Chlamydomonas, observada aquí por primera vez, disminuyó significativamente en el mutante lao1. Además, observamos que altas concentraciones de ácido indolacético (> 30 μM) inhibe el crecimiento de Chlamydomonas y que esta inhibición se puede reducir por la presencia de especies de Methylobacterium.Algae, lying on the basis of food webs in marine and freshwater ecosystems, are key for aquatic life. These photosynthetic organisms live under continuously fluctuating nutrients availability, showing a high level of adaptability to these dynamic environments. Although the essential nutrient Nitrogen (N) is usually used by algae in its inorganic form, some algal species can use organic N compounds, which may become especially abundant due to terrestrial leaking and runoff of highly fertilized areas. The model alga Chlamydomonas reinhardtii (Chlamydomonas) uptakes inorganic N sources (i.e. ammonium, nitrate and nitrite), as well as L-arginine and urea. Moreover, this alga presents an extracellular L-amino acid oxidase (LAO1) with a broad substrate specificity that scavenges N from L-amino acids. In this work we studied the signaling control that leads the preference for nitrate over organic N in Chlamydomonas, the key role of LAO1 in the use of amino acids and peptides, as well as the establishment of new mutualistic interactions with bacteria to facilitate growth on organic N. Chapter 1 The transcription factor NIT2 is the key regulator of nitrate assimilation genes in Chlamydomonas. First, we compared the transcriptome of Chlamydomonas wild type (WT) and a nit2 mutant in response to nitrate. We observed that nitrate and NIT2 down-regulated genes involved in organic N scavenging, including LAO1. By the use of Chlamydomonas mutant strains we demonstrated that both nitrate and nitrite negatively impact the use of amino acids by this alga. Chapter 2 L-amino acid oxidase (LAAO) enzymes are widely present in nature and a major role as N scavenger has been proposed in fungal and algal LAAOs. By comparative genomic searches, we could not find any LAO1 ortholog in any green plant or plant, but we identified orthologs in 10 out of 27 other algal species, including Rhodophyta, Alveolata, Heterokonta, Haptophyta and Dinophyta algae. The construction of a LAAO phylogenetic tree revealed that algal protein sequences identified as LAO1 orthologs -named here as ALAAOs (Algal LAAOs)-, clustered on the same evolutionary branch. We observed that Chlamydomonas LAO1 gene is clustered to a putative RidA gene (LAO2), which resulted to be closely related to cyanobacterial members. Our phylogenetic analysis favoured the idea that ALAAOs may have a common origin in the archaeplastidan ancestor, originated by a protist engulfing cyanobacteria. By the use of a lao1 mutant, we showed that LAO1 was crucial for Chlamydomonas growth on 16 out of 20 proteinogenic amino acids, as well as for some di- and tripeptides. Besides ammonium, LAAO produces keto acids and hydrogen peroxide. We have demonstrated that the spontaneous reaction of the LAO1-derived products generated by L-alanine deamination, pyruvic acid and hydrogen peroxide, generates acetic acid. Chapter 3 Although Chlamydomonas can grow on most amino acids and some di-/tripeptides as the sole N sources, this growth is far less efficient than that on inorganic N, and yet, there are some amino acids and peptides that cannot be used by this alga. We serendipitously found a contaminating Methylobacterium sp. that allowed Chlamydomonas growth on a dipeptide that is not readily assimilated by this alga. Methylobacterium spp. are included in the PGPB group of bacteria (Plant Growth-Promoting Bacteria), which improve plant growth and fitness. After field sampling, isolation and identification of some bacteria, we found that some wild species, included in Methylobacterium, Sphingomonas, Deinococcus, and Chitinophagaceae genera, promoted Chlamydomonas growth on L-serine. Moreover, some Methylobacterium spp. allowed Chlamydomonas growth on amino acids and peptides that are not used by this alga. We have demonstrated a new mutualism based on carbon-nitrogen metabolic exchange between Chlamydomonas and M. aquaticum. Otherwise, some Methylobacterium spp. improved Chlamydomonas growth on assimilable amino acids. For this growth promotion, LAO1 was crucial for consortia growth with some Methylobacterium spp., including M. extorquens, M. hispanicum and M. organophilum. The chemical cross-talk between interacting organisms mediates the beneficial and pathogenic symbiotic relationships. Within these inter-kingdom signal molecules IAA (Indole-3-Acetic Acid) is one of the best studied. We found that L-tryptophan-dependent indoles production in Chlamydomonas, observed here for the first time, was significantly reduced in the lao1 mutant. Moreover, we observed that high levels of exogenously added IAA (> 30 μM) inhibits Chlamydomonas growth and that this inhibition may be relieved by the presence of Methylobacterium spp

Identification of the MAPK Cascade and its Relationship with Nitrogen Metabolism in the Green Alga Chlamydomonasreinhardtii

The mitogen activated protein kinases (MAPKs) form part of a signaling cascade through phosphorylation reactions conserved in all eukaryotic organisms. The MAPK cascades are mainly composed by threeproteins, MAPKKKs, MAPKKs and MAPKs. Some signals induce MAPKKK-mediated phosphorylation and activation of MAPKK that phosphorylate and activate MAPK. Afterward, MAPKs can act either in the cytoplasm or be imported into the nucleus to activate other proteins or transcription factors. In the green microalga Chlamydomonasreinhardtii the pathway for nitrogen (N) assimilation is well characterized, yet its regulation still has many unknown features. Nitric oxide (NO) is a fundamental signal molecule for N regulation, where nitrate reductase (NR) plays a central role in its synthesis. The MAPK cascades could be regulating N assimilation, since it has been described that the phosphorylation of NR by MAPK6 promotes NO production in Arabidopsis thaliana. We have identified the proteins involved in the MAPK cascades in Chlamydomonasreinhardtii, finding 17 MAPKs, 2 MAPKKs and 108 MAPKKKs (11 MEKK-, 94 RAF- and 3 ZIK-type) that have been structurally and phylogenetically characterized. The genetic expressions of MAPKs and the MAPKK were slightly regulated by N. However, the genetic expressions of MAPKKKs RAF14 and RAF79 showed a very strong repression by ammonium, which suggests that they may have a key role in the regulation of N assimilation, encouraging to further analyze in detail the role of MAPK cascades in the regulation of N metabolism

Chlamydomonas reinhardtii, a Reference Organism to Study Algal–Microbial Interactions: Why Can’t They Be Friends?

The stability and harmony of ecological niches rely on intricate interactions between their members. During evolution, organisms have developed the ability to thrive in different environments, taking advantage of each other. Among these organisms, microalgae are a highly diverse and widely distributed group of major primary producers whose interactions with other organisms play essential roles in their habitats. Understanding the basis of these interactions is crucial to control and exploit these communities for ecological and biotechnological applications. The green microalga Chlamydomonas reinhardtii, a well-established model, is emerging as a model organism for studying a wide variety of microbial interactions with ecological and economic significance. In this review, we unite and discuss current knowledge that points to C. reinhardtii as a model organism for studying microbial interactions

Nitrous Oxide Emissions from Nitrite Are Highly Dependent on Nitrate Reductase in the Microalga Chlamydomonas reinhardtii

Nitrous oxide (N2O) is a powerful greenhouse gas and an ozone-depleting compound whose synthesis and release have traditionally been ascribed to bacteria and fungi. Although plants and microalgae have been proposed as N2O producers in recent decades, the proteins involved in this process have been only recently unveiled. In the green microalga Chlamydomonas reinhardtii, flavodiiron proteins (FLVs) and cytochrome P450 (CYP55) are two nitric oxide (NO) reductases responsible for N2O synthesis in the chloroplast and mitochondria, respectively. However, the molecular mechanisms feeding these NO reductases are unknown. In this work, we use cavity ring-down spectroscopy to monitor N2O and CO2 in cultures of nitrite reductase mutants, which cannot grow on nitrate or nitrite and exhibit enhanced N2O emissions. We show that these mutants constitute a very useful tool to study the rates and kinetics of N2O release under different conditions and the metabolism of this greenhouse gas. Our results indicate that N2O production, which was higher in the light than in the dark, requires nitrate reductase as the major provider of NO as substrate. Finally, we show that the presence of nitrate reductase impacts CO2 emissions in both light and dark conditions, and we discuss the role of NO in the balance between CO2 fixation and release

From the Eukaryotic Molybdenum Cofactor Biosynthesis to the Moonlighting Enzyme mARC

All eukaryotic molybdenum (Mo) enzymes contain in their active site a Mo Cofactor (Moco), which is formed by a tricyclic pyranopterin with a dithiolene chelating the Mo atom. Here, the eukaryotic Moco biosynthetic pathway and the eukaryotic Moco enzymes are overviewed, including nitrate reductase (NR), sulfite oxidase, xanthine oxidoreductase, aldehyde oxidase, and the last one discovered, the moonlighting enzyme mitochondrial Amidoxime Reducing Component (mARC). The mARC enzymes catalyze the reduction of hydroxylated compounds, mostly N-hydroxylated (NHC), but as well of nitrite to nitric oxide, a second messenger. mARC shows a broad spectrum of NHC as substrates, some are prodrugs containing an amidoxime structure, some are mutagens, such as 6-hydroxylaminepurine and some others, which most probably will be discovered soon. Interestingly, all known mARC need the reducing power supplied by different partners. For the NHC reduction, mARC uses cytochrome b5 and cytochrome b5 reductase, however for the nitrite reduction, plant mARC uses NR. Despite the functional importance of mARC enzymatic reactions, the structural mechanism of its Moco-mediated catalysis is starting to be revealed. We propose and compare the mARC catalytic mechanism of nitrite versus NHC reduction. By using the recently resolved structure of a prokaryotic MOSC enzyme, from the mARC protein family, we have modeled an in silico three-dimensional structure of a eukaryotic homologue

NRT2.4 and NRT2.5 Are Two Half-Size Transporters from the Chlamydomonas NRT2 Family

The NRT2 transporters mediate High Affinity Nitrate/NitriteTransport (HAN/NiT), which are essential for nitrogen acquisition from these inorganic forms. The NRT2 proteins are encoded by a multigene family in plants, and contain 12 transmembrane-spanning domains. Chlamydomonas reinhardtii has six NRT2, two of which (NRT2.5 and NRT2.4) are located in Chromosome III, in tandem head to tail. cDNAs for these genes were isolated and their sequence revealed that they correspond to half-size NRT2 transporters each containing six transmembrane domains. NRT2.5 has long N- and C- termini sequences without known homology. NRT2.4 also contains long termini sequences but smaller than NRT2.5. Expression of both studied genes occurred at a very low level, slightly in darkness, and was not modified by the N or C source. Silencing of NRT2.4 by specific artificial miRNA resulted in the inhibition of nitrite transport in the absence of other HANNiT (NRT2.1/NAR2) in the cell genetic background. Nitrite transport activity in the Hansenula polymorpha Dynt::URA3 Leu2 mutant was restored by expressing CrNRT2.4. These results indicate that half-size NRT2 transporters are present in photosynthetic organisms and that NRT2.4 is a HANiT

Chlamydomonas reinhardtii, an Algal Model in the Nitrogen Cycle

Nitrogen (N) is an essential constituent of all living organisms and the main limiting macronutrient. Even when dinitrogen gas is the most abundant form of N, it can only be used by fixing bacteria but is inaccessible to most organisms, algae among them. Algae preferentially use ammonium (NH4+) and nitrate (NO3−) for growth, and the reactions for their conversion into amino acids (N assimilation) constitute an important part of the nitrogen cycle by primary producers. Recently, it was claimed that algae are also involved in denitrification, because of the production of nitric oxide (NO), a signal molecule, which is also a substrate of NO reductases to produce nitrous oxide (N2O), a potent greenhouse gas. This review is focused on the microalga Chlamydomonas reinhardtii as an algal model and its participation in different reactions of the N cycle. Emphasis will be paid to new actors, such as putative genes involved in NO and N2O production and their occurrence in other algae genomes. Furthermore, algae/bacteria mutualism will be considered in terms of expanding the N cycle to ammonification and N fixation, which are based on the exchange of carbon and nitrogen between the two organisms

Eduarda Mansilla: la Biografía, Redes familiares y amicales. Los Epistolarios. Los escritos dispersos. Hacia un estudio crítico integral

El estudio de la vida y obra de Eduarda Mansilla se enmarca en la crítica sobre literatura argentina decimonónica escrita por mujeres, así como en la historia de la vida privada y su interconexión con el mapa político-social de la época, especialmente visible en las grandes familias de la clase dirigente. El corpus específicamente literario de Mansilla aún no había sido efectivamente cerrado y determinado. Se sabía, sobre todo por las memorias de su hijo Daniel García-Mansilla (1950), de la existencia de obras varias, y de escritos de crítica de arte que no han podido ubicarse. Nos constaba además, por compulsas ya realizadas, que diversos textos de la autora subsisten dispersos en publicaciones periódicas de la época, nacionales y en el extranjero. Tanto ese material, como cuanto se pudiera conseguir en lo que hace a epistolarios, crónicas, memorias, diarios y escritos testimoniales de todo tipo guardados en archivos públicos y privados, era precario y se hallaba expuesto a la desaparición y al deterioro. Hasta el presente los asedios a la vida/obra de nuestra autora se han mantenido dentro de marcos más o menos acotados, subordinados a un objetivo mayor: una edición textual, la presentación de un panorama femenino (“argentinas”, “mujer romántica”), la historia de la composición musical en la Argentina, en el que esta figura singular se engarza, pero no constituyeron investigaciones de envergadura autónoma. Lo antedicho confirmó la necesidad de localizar y rescatar la producción total de la autora a través de la compulsa de archivos públicos y privados, lo que nos permitió establecer una biografía confiable de Eduarda Mansilla que, aspiramos, se constituya en obra de consulta. Los resultados de esta investigación se publicaron en la Colección EALA de la editorial Corregidor.Our study of Eduarda Mansilla´s life and works is carried on within the field of criticism about Argentine Nineteenth-Century literature written by women, as well as in the history of private lifeand its interconnection with the political and social map of that period, which was especially visiblein certain traditional families. The specifically literary corpus of Mansilla has not yet been closednor clearly determined. Thanks to the memories written by one of her sons, Daniel García-Mansilla(1950), it is known that Mansilla has produced several literary pieces that disappeared and are notavailable to us nowadays. In addition, there are many other diverse literary works that have beenoriginally published nationally and abroad in a series of periodicals, newspapers and literary journalsthat are still inaccessible to the researchers. These materials, along with memoires, travel journals,personal notes, and any other disperse writing production by Eduarda are scattered, hidden inlibraries, unpublished and in danger of material destruction. Until today, research about Mansillahas been framed within certain fields such as the study of romanticism, Women writings, musicalcomposition, always with the goal of pursuing a critical edition. But none of them constitutes initself an independent research project. The above-mentioned confirmed the necessity of localizingand rescuing the total production of this writer nowadays scattered in public as well as in privatearchives, with the goal of achieving a serious and reliable biography of Eduarda Mansilla which,we expect, will become a referent in our discipline. The results of this research were published inthe EALA Collection Corregidor publisher

COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P < 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men