Caracterización de variantes fenotípicos y mutantes de Pseudomonas fluorescens F113: colonización de la rizosfera, formación de biopelículas, movilidad y biocontrol

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de lectura: 22-07-201

BART-based inference for Poisson processes

The effectiveness of Bayesian Additive Regression Trees (BART) has been demonstrated in a variety of contexts including non parametric regression and classification. Here we introduce a BART scheme for estimating the intensity of inhomogeneous Poisson Processes. Poisson intensity estimation is a vital task in various applications including medical imaging, astrophysics and network traffic analysis. Our approach enables full posterior inference of the intensity in a nonparametric regression setting. We demonstrate the performance of our scheme through simulation studies on synthetic and real datasets in one and two dimensions, and compare our approach to alternative approaches

Engineering a hydrogen biosensor: selection of overproducing nitrogenase variants for biohydrogen production

Biologically-produced hydrogen (H2) or ?biohydrogen? is one promising source of renewable energy. A number of microorganisms are being studied as potential producers of biohydrogen through biophotolysis, indirect biophotolysis, photo-fermentations or dark-fermentations. Microorganisms produce H2 by the activity of either hydrogenases or nitrogenases: Hydrogenase enzymes catalyze the reaction: 2H+ + 2e- ? H2 whereas nitrogenases catalyze the reduction N2 with the following limiting stoichiometry: N2 + 8H+ + 8e- ? H2 + 2NH3. In this work, we have coordinated aspects of both pathways to develop optimized biocatalysts for hydrogen overproduction using the following steps: 1. Engineering a hydrogen responsive genetic circuit in the purple non-sulphur nitrogen-fixing bacterium Rhodobacter capsulatus SB1003: R. capsulatus carries nitrogenase and hydrogenase enzymes able to produce H2. It also carries a system to detect H2 that is composed of three proteins: a H2-sensor hydrogenase (HupUV), a histidine kinase (HupT) and a response regulator (NtrC-like transcription factor, HupR) (Vignais et al., 2005). In the presence of H2, this sensor triggers expression of hydrogenase structural and biosynthetic genes. Taking advantage of this system, we have introduced a reporter gene under the control of hupS promoter and removed the uptake hydrogenase, generating a new biological-sensor strain capable of accumulating and detecting the presence of both exogenous H2 and the H2 produced by its own nitrogenase. This biotechnological tool allows us to obtain a measurable and proportional signal when H2 is present in the cell. 2. Generating variants of the molybdenum nitrogenase structural genes nifH, nifD and nifK: we are using in vitro evolution techniques to perform random mutagenesis in these genes with a controlled mutation rate. The resulting variants were cloned under nifH promoter control into a broad-host-range vector (Kovach et al., 1995) optimized for diazotrophic conditions. Libraries obtained (around 4 x 106 clones) were introduced and expressed in the strain carrying the modified biological hydrogen sensor. The suitable combination of both tools results in the development of a genetic circuit for the high-throughput screening of H2 overproducing nitrogenase variants thus allowing detection and isolation of clones that present a significant signal increased, through the use of cell-sorting cytometry. Thus far, around 1500 clones have been successfully selected by this method, confirming the possibility of using the designed system to select hydrogen-overproducing enzymes

NifQ and NifO are essential to express nitrogenase activity in the presence of nitrate in Azotobacter vinelandii

In the presence of nitrate, Azotobacter vinelandii is able to assimilate nitrogen by using nitrogenase and nitrate reductase/nitrite reductase pathways simultaneously. Nitrogenase and nitrate reductase are Mo-enzymes containing FeMo-co and Mo-MGD at their active sites, respectively. In order to optimize the use of Mo, a scarce metal in nature, regulation of Mo distribution between both enzymes must be strictly controlled during nitrogen assimilation processes. The nifO and nifQ genes are grouped together with nifB, fdxN and rhdN in one transcriptional unit. It has been shown that nifO and nifQ expression levels change antagonistically depending on the presence of Mo in the medium (Rodriguez-Quinones 1993). In addition, the nifO mutant exhibits a Nif- phenotype in the presence of nitrate, whereas nifO overexpression lowers nitrate reductase activity (Gutierrez, J.C. 1997). The nifQ mutant is unable to fix N2 unless growth medium is supplemented with 1000-fold excess of Mo. Importantly, NifQ has been characterized as the physiological Mo donor to a NifEN/NifH complex during FeMo-co synthesis. (Hernandez, J.A. 2008). We aimed to understand the relationship between NifO and NifQ during expression of nitrogenase activity in presence of nitrate in A. vinelandii. The nifQ mutant was unable to fix N2 in the presence of nitrate, independently of the level of Mo in the medium. In contrast nifQ mutant showed enhanced nitrate reductase activity. Analysis of nitrogenase and nitrate reductase activities demonstrated that the nifQ overexpressing strain exhibited lower nitrogenase activity and higher nitrate reductase activity than wild-type when grown diazotrophically in the presence of nitrate, a phenotype similar to the nifO mutant (Gutierrez, J.C. 1997). An antagonist effect had been observed in the nifO overexpressing strain (Gutierrez, J.C. 1997). Simultaneous overexpression of both nifQ and nifO yielded nitrogenase and nitrate reductase activities similar to wild-type. The phenotype observed in nifQ overexpressing, but not in nifOQ overexpressing strain, points to NifO as candidate to preserve NifQ as Mo donor to nitrogenase when nitrate reductase is present. Transcriptional expression analysis performed by RT-qPCR showed lower expression of nitrogenase structural genes in the nifO mutant. In contrast increased expression of nitrate and nitrite reductase structural genes was observed for both nifO mutant and nifQ overexpression strains. Comparison between NifQ proteins isolated before and after addition of nitrate to the same culture of a nifQ overexpressing strain grown under diazotrophic conditions, showed NifQ cluster content alteration, resulting in decrease of [Mo-3Fe-4S]3+ and increase of [3Fe-4S]+ clusters. This effect of nitrate is consistent with the inability of NifQ to donate Mo for FeMo-co biosynthesis under nitrate reductase derepressing conditions. These results revealed two Mo pathways to nitrogenase: one that can be sorted by a large excess of Mo in the medium, and a second pathway strictly dependent on NifQ and NifO that would be essential to maintain active nitrogenase while assimilating nitrate through the molybdoenzyme nitrate reductase

Mechanistic insights into arrhythmogenic right ventricular cardiomyopathy caused by desmocollin-2 mutations

Aims: Recent immunohistochemical studies observed the loss of plakoglobin (PG) from the intercalated disc (ID) as a hallmark of arrhythmogenic right ventricular cardiomyopathy (ARVC), suggesting a final common pathway for this disease. However, the underlying molecular processes are poorly understood. Methods and results: We have identified novel mutations in the desmosomal cadherin desmocollin 2 (DSC2 R203C, L229X, T275M, and G371fsX378). The two missense mutations (DSC2 R203C and T275M) have been functionally characterized, together with a previously reported frameshift variant (DSC2 A897fsX900), to examine their pathogenic potential towards PG's functions at the ID. The three mutant proteins were transiently expressed in various cellular systems and assayed for expression, processing, localization, and binding to other desmosomal components in comparison to wild-type DSC2a protein. The two missense mutations showed defects in proteolytic cleavage, a process which is required for the functional activation of mature cadherins. In both cases, this is thought to cause a reduction of functional DSC2 at the desmosomes in cardiac cells. In contrast, the frameshift variant was incorporated into cardiac desmosomes; however, it showed reduced binding to PG. Conclusion: Despite different modes of action, for all three variants, the reduced ability to provide a ligand for PG at the desmosomes was observed. This is in agreement with the reduced intensity of PG at these structures observed in ARVC patients

The Gac-Rsm and SadB Signal Transduction Pathways Converge on AlgU to Downregulate Motility in Pseudomonas fluorescens

Flagella mediated motility in Pseudomonas fluorescens F113 is tightly regulated. We have previously shown that motility is repressed by the GacA/GacS system and by SadB through downregulation of the fleQ gene, encoding the master regulator of the synthesis of flagellar components, including the flagellin FliC. Here we show that both regulatory pathways converge in the regulation of transcription and possibly translation of the algU gene, which encodes a sigma factor. AlgU is required for multiple functions, including the expression of the amrZ gene which encodes a transcriptional repressor of fleQ. Gac regulation of algU occurs during exponential growth and is exerted through the RNA binding proteins RsmA and RsmE but not RsmI. RNA immunoprecipitation assays have shown that the RsmA protein binds to a polycistronic mRNA encoding algU, mucA, mucB and mucD, resulting in lower levels of algU. We propose a model for repression of the synthesis of the flagellar apparatus linking extracellular and intracellular signalling with the levels of AlgU and a new physiological role for the Gac system in the downregulation of flagella biosynthesis during exponential growth

Multinational evaluation of genetic diversity indicators for the Kunming-Montreal Global Biodiversity Framework

DATA AVAILABILITY STATEMENT : Kobo forms and scripts used to collect and analyse data are available on the GitHub repository https://github.com/AliciaMstt/GeneticIndicators (Zenodo: https://zenodo.org/doi/10.5281/zenodo.10620306).The data that support the findings of this study are available in DRYAD (https://doi.org/10.5061/dryad.bk3j9kdkm). Some data that could lead to the geographic identification of endangered species have been obscured.Under the recently adopted Kunming-Montreal Global Biodiversity Framework, 196 Parties committed to reporting the status of genetic diversity for all species. To facilitate reporting, three genetic diversity indicators were developed, two of which focus on processes contributing to genetic diversity conservation: maintaining genetically distinct populations and ensuring populations are large enough to maintain genetic diversity. The major advantage of these indicators is that they can be estimated with or without DNA-based data. However, demonstrating their feasibility requires addressing the methodological challenges of using data gathered from diverse sources, across diverse taxonomic groups, and for countries of varying socio-economic status and biodiversity levels. Here, we assess the genetic indicators for 919 taxa, representing 5271 populations across nine countries, including megadiverse countries and developing economies. Eighty-three percent of the taxa assessed had data available to calculate at least one indicator. Our results show that although the majority of species maintain most populations, 58% of species have populations too small to maintain genetic diversity. Moreover, genetic indicator values suggest that IUCN Red List status and other initiatives fail to assess genetic status, highlighting the critical importance of genetic indicators.Agence Nationale de la Recherche; Svenska Forskningsrådet Formas; Consejo Nacional de Ciencia y Tecnología; Vetenskapsrådet.http://www.wileyonlinelibrary.com/journal/elehj2024Zoology and EntomologySDG-15:Life on lan

Multinational evaluation of genetic diversity indicators for the Kunming‐Montreal Global Biodiversity Framework

Under the recently adopted Kunming‐Montreal Global Biodiversity Framework, 196 Parties committed to reporting the status of genetic diversity for all species. To facilitate reporting, three genetic diversity indicators were developed, two of which focus on processes contributing to genetic diversity conservation: maintaining genetically distinct populations and ensuring populations are large enough to maintain genetic diversity. The major advantage of these indicators is that they can be estimated with or without DNA‐based data. However, demonstrating their feasibility requires addressing the methodological challenges of using data gathered from diverse sources, across diverse taxonomic groups, and for countries of varying socio‐economic status and biodiversity levels. Here, we assess the genetic indicators for 919 taxa, representing 5271 populations across nine countries, including megadiverse countries and developing economies. Eighty‐three percent of the taxa assessed had data available to calculate at least one indicator. Our results show that although the majority of species maintain most populations, 58% of species have populations too small to maintain genetic diversity. Moreover, genetic indicator values suggest that IUCN Red List status and other initiatives fail to assess genetic status, highlighting the critical importance of genetic indicators