Análisis filogenético y genómico del género salinivibrio

The genus Salinivibrio constitutes a separate phylogenetic lineage within the Gammaproteobacteria according to 16S rRNA gene sequence analysis and includes moderate halophilic bacteria. This genus was created to accommodate the species Vibrio costicola based on the significant phenotypic and genotypic differences between this and other species of the genus Vibrio (Mellado et al. 1996). Currently, the genus Salinivibrio comprises four species, one of them with three subspecies: S. costicola subsp. costicola (Smith 1938; Mellado et al., 1996; Huang et al., 2000), S. costicola subsp. vallismortis (Huang et al., 2000), S. costicola subsp. alcaliphilus (Romano et al., 2005), Salinivibrio proteolyticus (Amoozegar et al., 2008), Salinivibrio siamensis (Chamroensaksri et al., 2009) and Salinivibrio sharmensis (Romano et al., 2011). This genus comprises Gram-negative, obligatory halophilic bacteria, most of them growing between 5 and 20 % NaCl, 10 and 45 ºC and pH 5.0 and 9.0. They are facultative anaerobes. Moreover, S. costicola is considered as a model microorganism for studying osmoregulation and other physiological mechanisms in moderate halophiles (Oren 2003; Amoozegar et al., 2008). Therefore, members of this genus have developed cellular mechanisms to thrive in extremophilic conditions, such as hypersalinity (Garcia et al., 1987, Huang et al., 2000). Also, these microorganisms are facultative anaerobes and positive for catalase and oxidase tests. The DNA G+C content ranges from 49.0 to 51.0 mol% (Mellado et al., 1996; Huang et al., 2000; Romano et al., 2005; 2011; Amoozegar et al., 2008; Chamroensaksri et al., 2009). Besides, members of the genus Salinivibrio have been isolated from a wide variety of habitats, such as marine and hypersaline water, lagoons, lake, saline soil, salterns, salted food products, animals such as Artemia spp., etc. The phylogeny of the family Vibrionaceae based on a 16S rRNA gene approach is confusing since closely related species within the family cannot always be distinguished reliably due to the high levels of sequence conservation. Several studies have shown that the 16S rRNA gene does not provide sufficient resolution for differentiation between closely related bacterial species (Thompson et al., 2005; 2008; Staley, 2006; Pascual et al., 2010; Gao et al., 2016). The classification of the genus Salinivibrio needs reappraisal since the three subspecies of Salinivibrio costicola do not form a monophyletic group; this conclusion is supported by antecedent studies carried out in the genus Salinivibrio that have shown one of the subspecies, S. costicola subsp. vallismortis, is not related to the other two, but forms a monophyletic group with another species of the genus, S. proteolyticus (Chamroensaksri et al., 2009; Amoozegar et al., 2008; Romano et al., 2011; Gorriti et al., 2014). So as to resolve these problems, it has been demonstrated in a number of studies that concatenating the sequences of several protein-encoding gene fragments, i.e., a multilocus sequence analysis (MLSA), provides a more robust tree topology and an improved understanding of speciation events compared to a tree based solely on 16S rRNA gene sequences (Thompson et al., 2005; 2007; Sawabe et al., 2013; Dubert et al., 2016; González-Castillo et al., 2016). The purpose of this Doctoral Thesis has been to study in detail the phylogenetic relationships of the species of the genus Salinivibrio to clarify the current classification of this genus by using several molecular approaches such as MLSA studies, DNA-DNA hybridization, phenotypic characteristics and the use of genomic data. To begin with a total of two hundred eighty-eight strains were isolated of several solar salterns of different areas of the Spanish geographic (Alicante, Huelva, Granada, Mallorca) and of Puerto Rico. After the analysis of 16S rRNA a collection of seventy strains belonging to the genus Salinivibrio was obtained. Besides, type strains of this genus were used in this study too. With the objective to clarify the current classification of this genus, a MLSA approach was used basing of gyrB, recA, rpoA and rpoD sequences. These genes were selected based on previous studies in the family Vibrionaceae (Thompson et al., 2005; 2008; Pascual et al., 2010). Phylogenetic trees based on the concatenation of these four housekeeping genes show that these seventy-six strains constituted four different phylogroups, with only one strain belonging to the species Salinivibrio sharmensis (with a high bootstrap value of 100%), which cannot be includedwithin any phylogroup since we could not isolate any strain belonging to this species and, therefore, S. sharmensis constitutes a phylotype. In the case of the phylogroup 1, this is composed for a group of strains of differents places of isolation but not from any species, therefore, this phylogroup can be possible to constitute a new species of the genus Salinivibrio. On the basis of this study, we have carried out a complete polyphasic investigation that has led us to propose the description of new taxa. On the other hand, the phylogroup 4 was constituted by five strains isolated from Huelva and Puerto Rico and by species S. proteolyticus DSM 11403T and subspecies S. costicola subsp. vallismortis DSM 8285T. We performed an exhaustive study to the aim to clarify the phylogenetic position of these species. To validate MLSA study, DNA-DNA hybridization (DDH) was carried out in thirty-three strains, including the type strains. The DDH percentage values for strains within the same phylogroup were always above 70 %, a value established as cut-off for species delineation (Wayne et al., 1987; Stackebrandt and Goebel, 1994), confirming that they belong to the same species. DDH analyses among phylogroups always showed values lower than 70 %, indicating that each phylogroup constitutes a different species. After confirm that there was a correlation between both studies (MLSA and DDH), a value of 97 % was proposed as a cut-off for the genus Salinivibrio for MLSA study. Besides, in this investigation genome fingerprinting analysis was performed to observe if these strains constituted clones. This study showed that the environmental strains do not form clonal populations and did not cluster according to their site of cultivation. After the MLSA analysis, an in-depth study of phylogroup 1 was carried out to the aim to verify these strains constituted a new species of the genus Salinivibrio. For that a polyphasic study was performed; including DNA-DNA hybridization, phenotypic characteristics and genomic data of the strains which genomes were sequenced. Afterward these studies, it was concluded that these strains constituted a new species of this genus, which name S. kushneri sp. nov. was proposed. As it mentioned above, the phylogroup 4 was constituted for the subspecies S. costicola subsp. vallismortis DSM 8285T, species S. proteolyticus DSM 11403T and the strains IB574, IB872, PR5, PR919 and PR932. Following the polyphasic studies and the genomic data; it concluded that both, species and subspecies, they were a single species, which was emended as S. vallismortis com. nov., ascending to range of species the subspecies S. costicola subsp. vallismortis DSM8285T; so the species S. proteolyticus constitute later heterotypic synonym of S. costicola subsp. vallismortis. On the other side, a total of thirty-two strains were selected to sequence their genomes. The study of these genomes was performed, together with the type strain of the genus Salinivibrio, S. costicola subsp. costicola ATCC 33508, which were available from the GenBank database. Based on different index (ANIb, ANIm, OrthoANI) and DDH in silico, these strains were classified as five different species belonging to the genus Salinivibrio. Each species correlated with each phylogroup from the MLSA study. Besides, phylogenomic study based on the core-genome showed a phylogenetic tree in which it was possible differentiates four phylogroup, the same as in the MLSA study. Among the most represented subsystems of the members of the genus Salinivibrio are the genes involved in the synthesis and regulation of the flagellum, this is due to the polar flagellum that these strains have at one end and that allows them mobility. In addition, this study of the genomes has permitted us to determine, using recruitments obtained from metagenomic databases of environments with intermediate salinities, that these strains are not abundant in these habitats, despite growing fast in the laboratory and being easily isolated from hypersaline environments

Assessment of MultiLocus Sequence Analysis As a Valuable Tool for the Classification of the Genus Salinivibrio

The genus Salinivibrio includes obligatory halophilic bacteria and is commonly isolated from hypersaline habitats and salted food products. They grow optimally between 7.5 and 10% salts and are facultative anaerobes. Currently, this genus comprises four species, one of them, S. costicola, with three subspecies. In this study we isolated and characterized an additional 70 strains from solar salterns located in different locations. Comparative 16S rRNA gene sequence analysis identified these strains as belonging to the genus Salinivibrio but could not differentiate strains into species-like groups. To achieve finer phylogenetic resolution, we carried out a MultiLocus Sequence Analysis (MLSA) of the new isolates and the type strains of the species of Salinivibrio based on the individual as well as concatenated sequences of four housekeeping genes: gyrB, recA, rpoA, and rpoD. The strains formed four clearly differentiated species-like clusters called phylogroups. All of the known type and subspecies strains were associated with one of these clusters except S. sharmensis. One phylogroup had no previously described species coupled to it. Further DNA–DNA hybridization (DDH) experiments with selected representative strains from these phylogroups permitted us to validate the MLSA study, correlating the species level defined by the DDH (70%) with a 97% cut-off for the concatenated MLSA gene sequences. Based on these criteria, the novel strains forming phylogroup 1 could constitute a new species while strains constructing the other three phylogroups are members of previously recognized Salinivibrio species. S. costicola subsp. vallismortis co-occurs with S. proteolyticus in phylogroup 4, and separately from other S. costicola strains, indicating its need for reclassification. On the other hand, genome fingerprinting analysis showed that the environmental strains do not form clonal populations and did not cluster according to their site of cultivation. In future studies regarding the classification and identification of new Salinivibrio strains we recommend the following strategy: (i) initial partial sequencing of the 16S rRNA gene for genus-level identification; (ii) sequencing and concatenation of the four before mentioned housekeeping genes for species-level discrimination; (iii) DDH experiments, only required when the concatenated MLSA similarity values among a new isolate and other Salinivibrio strains are above the 97% cut-off.España, MINECO project CGL2013- 46941-

Comparative Genomics and Phylogenomic Analysis of the Genus Salinivibrio

In the genomic era phylogenetic relationship among prokaryotes can be inferred from the core orthologous genes (OGs) or proteins in order to elucidate their evolutionary history and current taxonomy should benefits of that. The genus Salinivibrio belongs to the family Vibrionaceae and currently includes only five halophilic species, in spite the fact that new strains are very frequently isolated from hypersaline environments. Species belonging to this genus have undergone several reclassifications and, moreover, there are many strains of Salinivibrio with available genomes which have not been affiliated to the existing species or have been wrongly designated. Therefore, a phylogenetic study using the available genomic information is necessary to clarify the relationships of existing strains within this genus and to review their taxonomic affiliation. For that purpose, we have also sequenced the first complete genome of a Salinivibrio species, Salinivibrio kushneri AL184T, which was employed as a reference to order the contigs of the draft genomes of the type strains of the current species of this genus, as well as to perform a comparative analysis with all the other available Salinivibrio sp. genomes. The genome of S. kushneri AL184T was assembled in two circular chromosomes (with sizes of 2.84 Mb and 0.60 Mb, respectively), as typically occurs in members of the family Vibrionaceae, with nine complete ribosomal operons, which might explain the fast growing rate of salinivibrios cultured under laboratory conditions. Synteny analysis among the type strains of the genus revealed a high level of genomic conservation in both chromosomes, which allow us to hypothesize a slow speciation process or homogenization events taking place in this group of microorganisms to be tested experimentally in the future. Phylogenomic and orthologous average nucleotide identity (OrthoANI)/average amino acid identity (AAI) analyses also evidenced the elevated level of genetic relatedness within members of this genus and allowed to group all the Salinivibrio strains with available genomes in seven separated species. Genome-scale attribute study of the salinivibrios identified traits related to polar flagellum, facultatively anaerobic growth and osmotic response, in accordance to the phenotypic features described for species of this genus.Spanish Ministry of Economy and Competitiveness Project CGL2017- 83385-PEspaña Junta de Andalucía BIO-213Spanish, University of Seville VIPPIT-US-201

Draft Genome Sequences of Salinivibrio proteolyticus, Salinivibrio sharmensis, Salinivibrio siamensis, Salinivibrio costicola subsp. alcaliphilus, Salinivibrio costicola subsp. vallismortis, and 29 New Isolates Belonging to the Genus Salinivibrio

The draft genome sequences of 5 type strains of species of the halophilic genus Salinivibrio and 29 new isolates from different hypersaline habitats belonging to the genus Salinivibrio have been determined. The genomes have 3,123,148 to 3,641,359 bp, a G+C content of 49.2 to 50.9%, and 2,898 to 3,404 open reading frames (ORFs).España, Ministerio de Ciencia e Innovación CGL2013-46941-

Characterization of Salinivibrio socompensis sp. nov., A new halophilic bacterium isolated from the high-altitude hypersaline lake Socompa, Argentina

The genus Salinivibrio belongs to the family Vibrionaceae and includes Gram-stain-negative, motile by a polar flagellum, and facultatively anaerobic curved rods. They are halophilic bacteria commonly found in hypersaline aquatic habitats and salted foods. This genus includes five species and two subspecies. A presumed novel species, strain S35T, was previously isolated from the high-altitude volcanic, alkaline, and saline lake Socompa (Argentinean Andes). In this study we carried out a complete taxonomic characterization of strain S35T, including the 16S rRNA gene sequence and core-genome analysis, the average nucleotide identity (ANIb, ANIm, and orthoANI), and in silico DNA?DNA hybridization (GGDC), as well as the phenotypic and chemotaxonomic characterization. It grew at 3%?20% (w/v) NaCl, pH 6?10, and 10?42 °C, with optimum growth at 7.0%?7.5% (w/v) NaCl, pH 8.0, and 37 °C, respectively. Strain S35T was oxidase- and catalase-positive, able to produce acid from D-glucose and other carbohydrates. Hydrolysis of DNA, methyl red test, and nitrate and nitrite reduction were positive. Its main fatty acids were C16:0, C16:1 ω7c and C16:1 ω6c, and C18:1 ω7c and/or C18:1 ω6c. ANI, GGDC, and core-genome analysis determined that strain S35T constitutes a novel species of the genus Salinivibrio, for which the name Salinivibrio socompensis sp. nov. is proposed. The type strain is S35T (= CECT 9634T = BNM 0535T)Fil: Galisteo, Cristina. Universidad de Sevilla; EspañaFil: Sánchez Porro,Cristina. Universidad de Sevilla; EspañaFil: de la Haba, Rafael R.. Universidad de Sevilla; EspañaFil: López Hermoso, Clara. Universidad de Sevilla; EspañaFil: Fernández, Ana Belén. Universidad de Sevilla; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Farias, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Ventosa, Antonio. Department Of Microbiology And Parasitology, Faculty Of; Españ

Knock-down of phosphoenolpyruvate carboxylase 3 negatively impacts growth, productivity, and responses to salt stress in sorghum (Sorghum bicolor L.)

Phosphoenolpyruvate carboxylase (PEPC) is a carboxylating enzyme with important roles in plant metabolism. Most studies in C-4 plants have focused on photosynthetic PEPC, but less is known about non-photosynthetic PEPC isozymes, especially with respect to their physiological functions. In this work, we analyzed the precise roles of the sorghum (Sorghum bicolor) PPC3 isozyme by the use of knock-down lines with the SbPPC3 gene silenced (Ppc3 lines). Ppc3 plants showed reduced stomatal conductance and plant size, a delay in flowering time, and reduced seed production. In addition, silenced plants accumulated stress indicators such as Asn, citrate, malate, and sucrose in roots and showed higher citrate synthase activity, even in control conditions. Salinity further affected stomatal conductance and yield and had a deeper impact on central metabolism in silenced plants compared to wild type, more notably in roots, with Ppc3 plants showing higher nitrate reductase and NADH-glutamate synthase activity in roots and the accumulation of molecules with a higher N/C ratio. Taken together, our results show that although SbPPC3 is predominantly a root protein, its absence causes deep changes in plant physiology and metabolism in roots and leaves, negatively affecting maximal stomatal opening, growth, productivity, and stress responses in sorghum plants. The consequences of SbPPC3 silencing suggest that this protein, and maybe orthologs in other plants, could be an important target to improve plant growth, productivity, and resistance to salt stress and other stresses where non-photosynthetic PEPCs may be implicated.This research was supported by the Junta de Andalucía (P12-FQM-489 and PAI group BIO298), the Basque Government (IT932-16), and the Ministerio de Economía, Industria y Competitividad (AGL2012-35708 and AGL2016-75413-P)

Jardins per a la salut

Facultat de Farmàcia, Universitat de Barcelona. Ensenyament: Grau de Farmàcia. Assignatura: Botànica farmacèutica. Curs: 2014-2015. Coordinadors: Joan Simon, Cèsar Blanché i Maria Bosch.Els materials que aquí es presenten són el recull de les fitxes botàniques de 128 espècies presents en el Jardí Ferran Soldevila de l’Edifici Històric de la UB. Els treballs han estat realitzats manera individual per part dels estudiants dels grups M-3 i T-1 de l’assignatura Botànica Farmacèutica durant els mesos de febrer a maig del curs 2014-15 com a resultat final del Projecte d’Innovació Docent «Jardins per a la salut: aprenentatge servei a Botànica farmacèutica» (codi 2014PID-UB/054). Tots els treballs s’han dut a terme a través de la plataforma de GoogleDocs i han estat tutoritzats pels professors de l’assignatura. L’objectiu principal de l’activitat ha estat fomentar l’aprenentatge autònom i col·laboratiu en Botànica farmacèutica. També s’ha pretès motivar els estudiants a través del retorn de part del seu esforç a la societat a través d’una experiència d’Aprenentatge-Servei, deixant disponible finalment el treball dels estudiants per a poder ser consultable a través d’una Web pública amb la possibilitat de poder-ho fer in-situ en el propi jardí mitjançant codis QR amb un smartphone

The evolution of the ventilatory ratio is a prognostic factor in mechanically ventilated COVID-19 ARDS patients

Background: Mortality due to COVID-19 is high, especially in patients requiring mechanical ventilation. The purpose of the study is to investigate associations between mortality and variables measured during the first three days of mechanical ventilation in patients with COVID-19 intubated at ICU admission. Methods: Multicenter, observational, cohort study includes consecutive patients with COVID-19 admitted to 44 Spanish ICUs between February 25 and July 31, 2020, who required intubation at ICU admission and mechanical ventilation for more than three days. We collected demographic and clinical data prior to admission; information about clinical evolution at days 1 and 3 of mechanical ventilation; and outcomes. Results: Of the 2,095 patients with COVID-19 admitted to the ICU, 1,118 (53.3%) were intubated at day 1 and remained under mechanical ventilation at day three. From days 1 to 3, PaO2/FiO2 increased from 115.6 [80.0-171.2] to 180.0 [135.4-227.9] mmHg and the ventilatory ratio from 1.73 [1.33-2.25] to 1.96 [1.61-2.40]. In-hospital mortality was 38.7%. A higher increase between ICU admission and day 3 in the ventilatory ratio (OR 1.04 [CI 1.01-1.07], p = 0.030) and creatinine levels (OR 1.05 [CI 1.01-1.09], p = 0.005) and a lower increase in platelet counts (OR 0.96 [CI 0.93-1.00], p = 0.037) were independently associated with a higher risk of death. No association between mortality and the PaO2/FiO2 variation was observed (OR 0.99 [CI 0.95 to 1.02], p = 0.47). Conclusions: Higher ventilatory ratio and its increase at day 3 is associated with mortality in patients with COVID-19 receiving mechanical ventilation at ICU admission. No association was found in the PaO2/FiO2 variation

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Presentació

Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation