Regulation of PTS systems and their interplay with central carbon metabolism in Pseudomonas putida

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 29-06-201

Chemical constituents from Zanthoxylum setulosum (Rutaceae)

Continuando con el estudio fitoquímico de plantas de Costa Rica, en este trabajo informamos el aislamiento e identificación de ocho compuestos de las partes aéreas de Zanthoxylum setulosum (Rutaceae). Los compuestos fueron identificados como el alcaloide skimmianina, los lignanos savinina, kusunokinina, sesamina, siringaresinol y el éter isopentílico del pluviatol, la amida conocida como acetato de aurantiamida, y el triterpeno lupeol. Este es el primer informe del aislamiento de skimmianina en las hojas de Z. setulosum, lo cual confirma que alcaloides quinolínicos y  benzofenantridinicos pueden ser considerados marcadores quimiotaxonómicos en éste género. La estructura de los compuestos aislados fue caracterizada por métodos espectroscópicos (incluyendo 1HNMR, 13C-NMR, HMQC, HMBC y NOESY) y comparación con datos de la literatura. Abstract Following our phytochemical studies of Costa Rican plants, in this work we report the isolation and identification of eight compounds from aerial parts of Zanthoxylum setulosum (Rutaceae). They were identified as the alkaloid skimmianine, the lignans savinin, kusunokinin, sesamin, syringaresinol and the isopentenyl ether of pluviatol, the amide aurantiamide acetate, and the triterpen lupeol. This is the first report of isolation of skimmianine from the leaves of Z. setulosum and its presence confirm that quinoline and benzophenanthridine alkaloids, can be considered as chemotaxonomic markers of  this genus. All the isolated compounds were characterized by spectroscopic methods (including 1H-NMR, 13C-NMR, , HMQC, HMBC and NOESY) and comparison with the literature data. Keywords: Rutaceae, Zanthoxylum, alkaloids, skimmianine, lignans.

Determinación del consumo voluntario de pasto kikuyo en pastoreo utilizando óxido de cromo.

El propósito del presente trabajo fué el comprobar que el uso de indicadores, como el óxido de Cr, pueden suministrar información válida del consumo de forraje por animales en pastoreo y determinar si el uso de ovinos puede suministrar información válida para extrapolar a los bovinos. El experimento se realizó en el CNIA Tibaitatá ICA, Sección de Ganado de Leche. Se utilizaron 4 vacas Holstein secas con peso promedio de 545 kg y 4 ovinos machos Romney Marsh con peso de 46 kg. Dos bovinos y dos ovinos disponían de tístulas rumiales. Se ubicaron en una pradera de kikuyo de más o menos una hectárea. No se encontró diferencia individual en excreción de heces. Se encontró diferencia significativa entre especies (P menor que 0.01) y tratamientos. (P menor que 0.05). Se encontró diferencia significativa en el consumo entre ovinos y bovinos aunque no interacciones entre especie y tratamiento. Los consumos promedios fueron normales 125.2 kg W a la 75 y 150.2 g/kg W a la 75 para la literatura (146 kg W a la 75). El coeficiente de variación fué de 24.5 por ciento para ovinos y bovinos respectivamente. La determinación de consumo en pastoreo se deberá realizar con la especie animal con la que se quiere trabajar en una determinada área ganadera y no extrapolar resultados obtenidos en ovinos y bovinos o viceversa, especialmente cuando se usa marcadores como en el caso del presente estudioAlgodó

The Bacterial Product Violacein Exerts an Immunostimulatory Effect Via TLR8

Violacein, an indole-derived, purple-colored natural pigment isolated from Chromobacterium violaceum has shown multiple biological activities. In this work, we studied the effect of violacein in different immune cell lines, namely THP-1, MonoMac 6, ANA-1, Raw 264.7 cells, as well as in human peripheral blood mononuclear cells (PBMCs). A stimulation of TNF-α production was observed in murine macrophages (ANA-1 and Raw 264.7), and in PBMCs, IL-6 and IL-1β secretion was detected. We obtained evidence of the molecular mechanism of activation by determining the mRNA expression pattern upon treatment with violacein in Raw 264.7 cells. Incubation with violacein caused activation of pathways related with an immune and inflammatory response. Our data utilizing TLR-transfected HEK-293 cells indicate that violacein activates the human TLR8 (hTLR8) receptor signaling pathway and not human TLR7 (hTLR7). Furthermore, we found that the immunostimulatory effect of violacein in PBMCs could be suppressed by the specific hTLR8 antagonist, CU-CPT9a. Finally, we studied the interaction of hTLR8 with violacein in silico and obtained evidence that violacein could bind to hTLR8 in a similar fashion to imidazoquinoline compounds. Therefore, our results indicate that violacein may have some potential in contributing to future immune therapy strategies.Universidad de Costa Rica/[801-B2-519]/UCR/Costa RicaMinisterio de Ciencia, Tecnología y Telecomunicaciones/[FI-497-11]/MICITT/Costa RicaMinisterio de Ciencia, Tecnología y Telecomunicaciones/[DFG-TR84]/MICITT/Costa RicaMinisterio de Ciencia, Tecnología y Telecomunicaciones/[DFG-KFO325]/MICITT/Costa RicaUCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de QuímicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigaciones en Productos Naturales (CIPRONA)UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM)UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto de Investigaciones Farmacéuticas (INIFAR

The urgent need for microbiology literacy in society

Contains fulltext : 204933.pdf (publisher's version ) (Open Access

The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes

The 'Standard European Vector Architecture' database (SEVA-DB, http://seva.cnb.csic.es) was conceived as a user-friendly, web-based resource and a material clone repository to assist in the choice of optimal plasmid vectors for de-constructing and re-constructing complex prokaryotic phenotypes. The SEVA-DB adopts simple design concepts that facilitate the swapping of functional modules and the extension of genome engineering options to microorganisms beyond typical laboratory strains. Under the SEVA standard, every DNA portion of the plasmid vectors is minimized, edited for flaws in their sequence and/or functionality, and endowed with physical connectivity through three inter-segment insulators that are flanked by fixed, rare restriction sites. Such a scaffold enables the exchangeability of multiple origins of replication and diverse antibiotic selection markers to shape a frame for their further combination with a large variety of cargo modules that can be used for varied end-applications. The core collection of constructs that are available at the SEVA-DB has been produced as a starting point for the further expansion of the formatted vector platform. We argue that adoption of the SEVA format can become a shortcut to fill the phenomenal gap between the existing power of DNA synthesis and the actual engineering of predictable and efficacious bacteria

Special Issue: Diversity of Extremophiles in Time and Space

Extreme environments are fascinating ecosystems that have allowed us to increase our knowledge about the evolutionary processes of life [1], develop new biotechnological applications (e.g., industrial applications of lipases [2], and thermostable DNA Polymerases in PCR tests [3]) and establish some fundamental concepts about the origins of life and the search for life in the Universe [1]. Despite the fact that research on the living beings that inhabit these extreme environments (i.e., extremophiles) began more than five decades ago with the pioneering works of Thomas D. Brock [4], nowadays, we still have a lot to learn about microbial diversity, and especially about the metabolism and biochemistry of these microorganisms; therefore, the study of extremophiles, extremozymes and their biotechnological potential remains a hot topic.Universidad de Costa Rica/[809-B6-524]/UCR/Costa RicaEuropean Union’s Horizon/[892961]/EU/Unión EuropeaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigaciones en Productos Naturales (CIPRONA

Accumulation of inorganic polyphosphate enables stress endurance and catalytic vigour in Pseudomonas putida KT2440

[Background] Accumulation of inorganic polyphosphate (polyP), a persistent trait throughout the whole Tree of Life, is claimed to play a fundamental role in enduring environmental insults in a large variety of microorganisms. The share of polyP in the tolerance of the soil bacterium Pseudomonas putida KT2440 to a suite of physicochemical stresses has been studied on the background of its capacity as a host of oxidative biotransformations.[Results] Cells lacking polyphosphate kinase (Ppk), which expectedly presented a low intracellular polyP level, were more sensitive to a number of harsh external conditions such as ultraviolet irradiation, addition of β-lactam antibiotics and heavy metals (Cd2+ and Cu2+). Other phenotypes related to a high-energy phosphate load (e.g., swimming) were substantially weakened as well. Furthermore, the ppk mutant was consistently less tolerant to solvents and its survival in stationary phase was significantly affected. In contrast, the major metabolic routes were not significantly influenced by the loss of Ppk as diagnosed from respiration patterns of the mutant in phenotypic microarrays. However, the catalytic vigour of the mutant decreased to about 50% of that in the wild-type strain as estimated from the specific growth rate of cells carrying the catabolic TOL plasmid pWW0 for m-xylene biodegradation. The catalytic phenotype of the mutant was restored by over-expressing ppk in trans. Some of these deficits could be explained by the effect of the ppk mutation on the expression profile of the rpoS gene, the stationary phase sigma factor, which was revealed by the analysis of a P rpoS  → rpoS‘-’lacZ translational fusion. Still, every stress-related effect of lacking Ppk in P. putida was relatively moderate as compared to some of the conspicuous phenotypes reported for other bacteria.[Conclusions] While polyP can be involved in a myriad of cellular functions, the polymer seems to play a relatively secondary role in the genetic and biochemical networks that ultimately enable P. putida to endure environmental stresses. Instead, the main value of polyP could be ensuring a reservoire of energy during prolonged starvation. This is perhaps one of the reasons for polyP persistence in live systems despite its apparent lack of essentiality.This study was supported by the BIO and FEDER CONSOLIDER-INGENIO programme of the Spanish Ministry of Science and Innovation, the MICROME and ST-FLOW Contracts of the EU, the ERANET Program and the PROMT Project of the CAM. P.I.N. is a researcher from Consejo Nacional de Investigaciones Científicas y Técnicas (Argentina) and holds a Marie Curie Actions Program grant from the EC (ALLEGRO, UE-FP7-PEOPLE-2011-IIF-300508). M.C. was the recipient of a fellowship from the University of Costa Rica.Peer Reviewe

Quantitative Physiology Approaches to Understand and Optimize Reducing Power Availability in Environmental Bacteria

The understanding of how carbon fluxes are distributed through a metabolic network offers an overview of the pathways that a given microorganism uses to produce energy, reducing power, and biomass. These invaluable data are related to the physiological state of the cell and provide information about the metabolic potential of microorganisms for specific environmental and biotechnological applications such as the degradation of toxic compounds (e.g., hydrocarbons) or the targeted production of high value-added products (e.g., lipids). Here, we propose a general approach to explore the pathways involved in NADPH balance in bacteria, which are in turn responsible for maintaining redox homeostasis and endowing the microorganism with the ability to counteract oxidative stress. We focus on the fluxes catalyzed by NADP+-dependent enzymes in the metabolic network of the model soil bacterium Pseudomonas putida KT2440. This environmental microorganism is a promising cell factory for a number of NADPH-dependent biotransformations, including industrial and bioremediation processes. The relevant enzymes involved in redox balance in strain KT2440 are (1) glucose-6-phosphate dehydrogenase, (2) 6-phosphogluconate dehydrogenase, (3) isocitrate dehydrogenase, (4) malic enzyme, and (5) 2-keto-6-phosphogluconate reductase. NADPH can be generated or consumed by other enzymatic reactions depending on the microorganism; however, the first four enzymes listed above are recognized as a major source of reducing power in a wide variety of microorganisms. The present protocol includes a first stage in which the NADPH balance is derived from fluxomic data and in vitro enzymatic assays. A second step is then proposed, where the redox ratios of pyridine dinucleotides and the cell capacity to counter oxidative stress are qualitatively correlated.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigaciones en Productos Naturales (CIPRONA)UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Químic

Four new compounds from the non-polar extract of the plant Amyris brenesii (Rutaceae) from Costa Rica

El estudio fitoquímico de las partes aéreas de Amyris brenesii (Rutaceae) recolectadas en Río Cuarto, Grecia, Alajuela (Costa Rica) mostró la presencia de cuatro nuevos compuestos: la 6-hidroxi-6-O-(3-hidroximetil-3-metilalil)- angelicina 1, el 6-(N-acetil-etanamin) -2,2-dimetil-2Hcromeno 2, el lignano 2,5-deshidrohinokinina 3 y la N-acetil-O-(geranil)-tiramina 4. Adicionalmente se aislaron los lignanos hinokinina 5, y justicidina E 6, las cumarinas escopoletina 7 y marmesina 8, el ácido 24-moretenoico 9 y el O-(3,3-dimetilalil)-halfordinol 10. Las separaciones se llevaron a cabo mediante la aplicación de técnicas cromatográficas y la elucidación de las estructuras se realizó con la ayuda de técnicas espectroscópicas de Resonancia Magnética Nuclear (RMN) de una y dos dimensiones.Fractionation of a non polar extract of the aerial parts of Amyris brenesii collected in Río Cuarto, Grecia, Costa Rica has resulted in the isolation of four new compounds, 6-hidroxy-6-O-(3-hidroxymethyl-3-methylalyl)-angelicin 1, 6-(N-acetyl-2-etanamin)-2,2-dimethyl-2H-cromen 2, the lignan 2,5-dehidrohinokinin 3 and N-acetyl-O-(geranyl)-tiramine 4. In addition, we isolated six previously known compounds: the lignans hinokinin 5 and Justicidin E 6, the coumarins scopoletin 7 and marmesin 8, 24-moretenoic acid 9, and the nitrogen compound O-(3,3-dimethylalyl)-halfordinol 10. All the separations were done with chromatographic techniques and the structures were elucidated by using 1D and 2D NMR techniques.Universidad Nacional, Costa Rica.Escuela de Ciencias Ambientale