Identification of a novel ATP-binding cassette transporter involved in long-chain fatty acid import and its role in triacylglycerol accumulation in Rhodococcus jostii RHA1

Members of Rhodococcus genus are specialists in the biosynthesis and accumulation of triacylglycerols (TAG). Since no transport protein related with TAG metabolism has been characterized until now in these bacteria, we used the available genomic information of Rhodococcus jostii RHA1 to perform a broad survey of genes coding for putative lipid transporter proteins in this oleaginous microorganism. Among the seven genes encoding for putative lipid transporters, ro05645 (now called ltp1: lipid transporter protein) coding for an ABC protein was found clustered with others genes encoding for enzymes catalyzing the three putative acylation reactions of the Kennedy pathway for TAG synthesis. Overexpression of ltp1 gene in the RHA1 strain led to an increase of approximately six- and three-folds in biomass and TAG production; respectively, when cells were cultivated on palmitic acid and oleic acid. Moreover, overexpression of ltp1 also promoted a significant increase in the uptake of a fluorescently labeled long-chain fatty acid in comparison with the wild type strain RHA1, and its further incorporation into the TAG fraction. Gluconate-grown cells showed increasing amounts of intracellular free fatty acids, but not of TAG, after overexpressing ltp1 gene. Thus, for the first time to our knowledge, a transporter functionally related to TAG metabolism was identified in oleaginous rhodococci. Our results suggest that Ltp1 is an importer of long chain fatty acids, which plays a functional role in lipid homeostasis of R. jostii RHA1.Fil: Villalba, María S.. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales - Sede Comodoro; ArgentinaFil: Alvarez, Hector Manuel. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales - Sede Comodoro; Argentina. Oil m&s; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Nacional Patagónico; Argentin

Identification of genes coding for putative wax ester synthase/diacylglycerol acyltransferase enzymes in terrestrial and marine environments

Synthesis of neutral lipids such as triacylglycerols (TAG) and wax esters (WE) is catalyzed in bacteria by wax ester synthase/diacylglycerol acyltransferase enzymes (WS/DGAT). We investigated the diversity of genes encoding this enzyme in contrasting natural environments from Patagonia (Argentina). The content of petroleum hydrocarbons in samples collected from oil-producing areas was measured. PCR-based analysis covered WS/DGAT occurrence in marine sediments and soil. No product was obtained in seawater samples. All clones retrieved from marine sediments affiliated with gammaproteobacterial sequences and within them, most phylotypes formed a unique cluster related to putative WS/DGAT belonging to marine OM60 clade. In contrast, soils samples contained phylotypes only related to actinomycetes. Among them, phylotypes affiliated with representatives largely or recently reported as oleaginous bacteria, as well as with others considered as possible lipid-accumulating bacteria based on the analysis of their annotated genomes. Our study shows for the first time that the environment could contain a higher variety of ws/ dgat than that reported from bacterial isolates. The results of this study highlight the relevance of the environment in a natural process such as the synthesis and accumulation of neutral lipids. Particularly, both marine sediments and soil may serve as a useful source for novel WS/DGAT with biotechnological interest.Fil: Lanfranconi, Mariana Patricia. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales - Sede Comodoro; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Alvarez, Adrián F.. Universidad Nacional Autónoma de México; MéxicoFil: Alvarez, Hector Manuel. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales - Sede Comodoro; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

The pleiotropic transcriptional regulator NlpR contributes to the modulation of nitrogen metabolism, lipogenesis and triacylglycerol accumulation in oleaginous rhodococci

The regulatory mechanisms involved in lipogenesis and triacylglycerol (TAG) accumulation are largely unknown in oleaginous rhodococci. In this study a regulatory protein (here called NlpR: Nitrogen lipid Regulator), which contributes to the modulation of nitrogen metabolism, lipogenesis and triacylglycerol accumulation in oleaginous rhodococci was identified. Under nitrogen deprivation conditions, in which TAG accumulation is stimulated, the nlpR gene was significantly upregulated, whereas a significant decrease of its expression and TAG accumulation occurred when cerulenin was added. The nlpR disruption negatively affected the nitrate/nitrite reduction as well as lipid biosynthesis under nitrogen-limiting conditions. In contrast, its overexpression increased TAG production during cultivation of cells in nitrogen-rich media. A putative ‘NlpR-binding motif’ upstream of several genes related to nitrogen and lipid metabolisms was found. The nlpR disruption in RHA1 strain led to a reduced transcription of genes involved in nitrate/nitrite assimilation, as well as in fatty acid and TAG biosynthesis. Purified NlpR was able to bind to narK, nirD, fasI, plsC and atf3 promoter regions. It was suggested that NlpR acts as a pleiotropic transcriptional regulator by activating of nitrate/nitrite assimilation genes and others genes involved in fatty acid and TAG biosynthesis, in response to nitrogen deprivation.Fil: Hernández, Martín Alejandro. Universidad Nacional de la Patagonia "san Juan Bosco". Instituto de Biociencias de la Patagonia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto de Biociencias de la Patagonia; ArgentinaFil: Lara, María Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Gago, Gabriela Marisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Gramajo, Hugo Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Alvarez, Hector Manuel. Universidad Nacional de la Patagonia "san Juan Bosco". Instituto de Biociencias de la Patagonia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto de Biociencias de la Patagonia; Argentin

On the kinetic and allosteric regulatory properties of the ADP-glucose pyrophosphorylase from Rhodococcus jostii: An approach to evaluate glycogen metabolism in oleaginous bacteria

Rhodococcus spp. are oleaginous bacteria that accumulate glycogen during exponential growth. Despite the importance of these microorganisms in biotechnology, little is known about the regulation of carbon and energy storage, mainly the relationship between glycogen and triacylglycerols metabolisms. Herein, we report the molecular cloning and heterologous expression of the gene coding for ADP-glucose pyrophosphorylase (EC 2.7.7.27) of Rhodococcus jostii, strain RHA1. The recombinant enzyme was purified to electrophoretic homogeneity to accurately characterize its oligomeric, kinetic, and regulatory properties. The R. jostii ADP-glucose pyrophosphorylase is a homotetramer of 190 kDa exhibiting low basal activity to catalyze synthesis of ADP-glucose, which is markedly influenced by different allosteric effectors. Glucose-6P, mannose-6P, fructose-6P, ribose-5P, and phosphoenolpyruvate were major activators; whereas, NADPH and 6P-gluconate behaved as main inhibitors of the enzyme. The combination of glucose-6P and other effectors (activators or inhibitors) showed a cross-talk effect suggesting that the different metabolites could orchestrate a fine regulation of ADP-glucose pyrophosphorylase in R. jostii. The enzyme exhibited some degree of affinity toward ATP, GTP, CTP, and other sugar-1P substrates. Remarkably, the use of glucosamine-1P was sensitive to allosteric activation. The relevance of the fine regulation of R. jostii ADP-glucose pyrophosphorylase is further analyzed in the framework of proteomic studies already determined for the bacterium. Results support a critical role for glycogen as a temporal reserve that provides a pool of carbon able of be re-routed to produce long-term storage of lipids under certain conditions.Fil: Cereijo, Antonela Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Asención Diez, Matías Damián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Dávila Costa, José Sebastián. Universidad Nacional de la Patagonia Austral. Centro de Investigaciones y Transferencia Golfo San Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia Golfo San Jorge. Universidad Nacional de la Patagonia ; ArgentinaFil: Alvarez, Hector Manuel. Universidad Nacional de la Patagonia Austral. Centro de Investigaciones y Transferencia Golfo San Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia Golfo San Jorge. Universidad Nacional de la Patagonia ; ArgentinaFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentin

Label-free and redox proteomic analyses of the triacylglycerol-accumulating Rhodococcus jostii RHA1

The bacterium Rhodococcus jostii RHA1 synthesizes large amounts of triacylglycerols (TAG) under conditions of nitrogen starvation. To better understand the molecular mechanisms behind this process, we performed proteomic studies in this oleaginous bacterium. Upon nitrogen starvation, we observed a re-routing of the carbon flux towards the formation of TAG. Under these conditions, the cellular lipid content made up more than half of the cell?s dry weight. On the proteome level, this coincided with a shift towards non-glycolytic carbohydrate-metabolizing pathways. These pathways (Entner-Doudoroff and pentose-phosphate shunt) contribute NADPH and precursors of glycerol-3-phosphate and acetyl-CoA to lipogenesis. The expression of proteins involved in the degradation of branched-chain-amino acids and the methyl malonyl-CoA pathway probably provided propionyl-CoA for the biosynthesis of odd-numbered fatty acids, which make up almost 30% of RHA1 fatty acid composition. Additionally, lipolytic and glycerol-degrading enzymes increased in abundance, suggesting a dynamic cycling of cellular lipids. Conversely, abundance of proteins involved in consuming intermediates of lipogenesis decreased. Furthermore, we identified another level of lipogenesis regulation through redox-mediated thiol modification in R. jostii. Enzymes affected included acetyl-CoA carboxylase and a β-ketoacyl-[ACP] synthase II (FabF). An integrative metabolic model for the oleaginous RHA1 strain is proposed based on our results.Fil: Dávila Costa, José Sebastián. Universidad Nacional de la Patagonia "San Juan Bosco"; ArgentinaFil: Herrero, O. Marisa. Universidad Nacional de la Patagonia "san Juan Bosco". Facultad de Ciencias Naturales - Sede Comodoro; ArgentinaFil: Alvarez, Hector Manuel. Universidad Nacional de la Patagonia "san Juan Bosco". Facultad de Ciencias Naturales - Sede Comodoro; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Nacional Patagónico; ArgentinaFil: Leichert, Lars. Ruhr-Universitat Bochum; Alemani

Tratamiento de la esquizofrenia en el primer episodio psicótico. Revisión de la literatura

La esquizofrenia es un trastorno mental psicótico, de etiología en estudio, caracterizado por alteraciones del pensamiento, del estado de ánimo y del comportamiento. Objetivo: Analizar la literatura del 2006 al 2011 acerca del manejo del primer episodio psicótico en la esquizofrenia. Material y método: Revisión sistemática de la literatura de acuerdo con criterios de la Medicina Basada en Evidencias de acuerdo con el Centre for Evidence-Based Medicine (CEBM) de Oxford. Resultados: De un total de 50 artículos, 23 de ellos (46%) hacen referencia a aspectos farmacológicos y 27 artículos (54%) describen intervenciones psicosociales, terapia molecular y seguimiento de tratamientos farmacológicos por estudios de neuro-imagen. Un solo artículo correspondió a nivel de evidencia 1 y grado de recomendación A. Conclusiones: El manejo de la esquizofrenia está con antipsicóticos de segunda generación y la disminución del problema se ve al final del primer a˜no de manejo. La adición de antagonistas alfa 2 mejora la eficacia de los fármacos antipsicóticos al reducir los síntomas negativos. © 2014 Universidad Autónoma del Estado de México. Publicado por Masson Doyma México S.A. Todos los derechos reservado

El control contable de las cuentas por cobrar y su incidencia en la liquidez de Gafumex E.I.R.L., Comas, 2018

Hoy en día, las empresas se encuentran en una constante competencia, es por ello, que para poder estar al nivel y si es posible marcar la diferencia con el resto de las empresas, tienen que buscar procesos o instrumentos que les posibiliten resaltar u obtener ventajas ante las demás organizaciones. La presente tesis tiene por objetivo determinar la incidencia que existe entre el control contable de las cuentas por cobrar y la liquidez de GAFUMEX E.I.R.L., Comas, 2018. Se comenzó planteando la realidad problemática de la empresa y luego a la recopilación de información para nuestra tesis. Fue muy importante la contribución proporcionada por los autores de los libros y trabajos de investigación, en relación con cada una de nuestras variables. Por tanto, nos ayuda a sostener nuestro tema de investigación. La metodología fue definida según su propósito aplicada porque está encaminada a resolver problemas prácticos de la organización. Asimismo, según el diseño de investigación es no experimental, porque no vamos a alterar ninguna de las variables, puesto que se va observar dentro de su contexto natural en un periodo determinado, habiendo elegido como técnica e instrumento de recolección el análisis documental y cuestionario. En nuestros resultados, hemos analizado los estados financieros, ratios de liquidez para ver la incidencia de una variable respecto a la otra variable. Además, realizamos una entrevista al gerente general y encardo del área administrativa de la empresa. Terminamos elaborando nuestra discusión para que sea consistente con las hipótesis y las conclusiones con los objetivos. Se concluyó demostrando la incidencia del control contable de las cuentas por cobrar sobre la liquidez.Atualmente, as empresas estão em constante competição, por isso, para estar no nível e se é possível fazer a diferença com o resto das empresas, elas precisam procurar processos ou instrumentos que lhes permitam se destacar ou obter vantagens. antes das outras organizações. O objetivo desta tese é determinar a incidência existente entre o controle contábil das contas a receber e a liquidez do GAFUMEX E.I.R.L., Comas, 2018. Começou colocando a realidade problemática da empresa e depois a coleta de informações para nossa tese. A contribuição dos autores dos livros e trabalhos de pesquisa, em relação a cada uma de nossas variáveis, foi muito importante. Portanto, nos ajuda a apoiar nosso tópico de pesquisa. A metodologia foi definida de acordo com sua finalidade aplicada, pois visa solucionar problemas práticos da organização. Da mesma forma, de acordo com o desenho da pesquisa, não é experimental, pois não alteraremos nenhuma das variáveis, pois será observada em seu contexto natural em um determinado período, tendo escolhido a análise documental e o questionário como técnica e instrumento de coleta. Em nossos resultados, analisamos as demonstrações financeiras e os índices de liquidez para ver a incidência de uma variável em relação à outra variável. Além disso, realizamos uma entrevista com o gerente geral e o gerente da área administrativa da empresa. Acabamos elaborando nossa discussão para ser consistente com as hipóteses e as conclusões com os objetivos. Foi concluído demonstrando o impacto do controle contábil das contas a receber na liquidez

The atf2 gene is involved in triacylglycerol biosynthesis and accumulation in the oleaginous Rhodococcus opacus PD630

Rhodococcus opacus PD630 is an oleaginous bacterium able to accumulate large amounts of triacylglycerols (TAG) in different carbon sources. The last reaction for TAG biosynthesis is catalyzed by the bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT) enzymes encoded by atf genes. R. opacus PD630 possesses at least 17 putative atf homologous genes in its genome, but only atf1 and atf2 exhibited a significant DGAT activity when expressed in E. coli, as revealed in a previous study. The contribution of atf1 gene to TAG accumulation by strain PD630 has been demonstrated previously, although additional Atfs may also contribute to lipid accumulation, since the atf1-disrupted mutant is still able to produce significant amounts of TAG (Alvarez et al. 2008). In this study, we investigated the in vivo role of atf2 gene in TAG accumulation by R.opacus PD630 by using different genetic strategies. The atf2-disrupted mutant exhibited a decrease in TAG accumulation (up to 25-30 %, w/w) and an approximately tenfold increase in glycogen formation in comparison with the wild type strain. Surprisingly, in contrast to the single mutants, a double mutant generated by the disruption of atf1 and atf2 genes, only showed a very low effect in TAG and in glycogen accumulation under lipid storage conditions. Over-expression of atf1 and atf2 genes in strain PD630 promoted an increase of approximately 10 % (w/w) in TAG accumulation; while heterologous expression of atf2 gene in Mycobacterium smegmatis caused an increase in TAG accumulation during cultivation in nitrogen-rich media. This study demonstrated that, in addition to atf1 gene, atf2 is actively involved in TAG accumulation by the oleaginous R. opacus PD630.Fil: Hernández, Martín Alejandro. Universidad Nacional de la Patagonia "San Juan Bosco"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Arabolaza, Ana Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Rodriguez, Eduardo Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Gramajo, Hugo Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Alvarez, Hector Manuel. Universidad Nacional de la Patagonia "San Juan Bosco"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Rhodococcus as Biofactories for Microbial Oil Production

Bacteria belonging to the Rhodococcus genus are frequent components of microbial communitiesin diverse natural environments. Some rhodococcal species exhibit the outstanding ability toproduce significant amounts of triacylglycerols (TAG) (>20% of cellular dry weight) in the presenceof an excess of the carbon source and limitation of the nitrogen source. For this reason, they canbe considered as oleaginous microorganisms. As occurs as well in eukaryotic single-cell oil (SCO)producers, these bacteria possess specific physiological properties and molecular mechanisms that differentiate them from other microorganisms unable to synthesize TAG. In this review, we summarized several of the well-characterized molecular mechanisms that enable oleaginous rhodococci to produce significant amounts of SCO. Furthermore, we highlighted the ability of these microorganisms to degrade a wide range of carbon sources coupled to lipogenesis. The qualitative and quantitative oil production by rhodococci from diverse industrial wastes has also been included. Finally, we summarized the genetic and metabolic approaches applied to oleaginous rhodococci to improve SCO production. This review provides a comprehensive and integrating vision on the potential of oleaginous rhodococci to be considered as microbial biofactories for microbial oil production.Fil: Alvarez, Hector Manuel. Universidad Nacional de la Patagonia "San Juan Bosco". Instituto de Biociencias de la Patagonia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto de Biociencias de la Patagonia; ArgentinaFil: Hernández, Martín Alejandro. Universidad Nacional de la Patagonia "San Juan Bosco". Instituto de Biociencias de la Patagonia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto de Biociencias de la Patagonia; ArgentinaFil: Lanfranconi, Mariana Patricia. Universidad Nacional de la Patagonia "San Juan Bosco". Instituto de Biociencias de la Patagonia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto de Biociencias de la Patagonia; ArgentinaFil: Silva, Roxana Alejandra. Universidad Nacional de la Patagonia "San Juan Bosco". Instituto de Biociencias de la Patagonia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto de Biociencias de la Patagonia; ArgentinaFil: Villalba, María Soledad. Universidad Nacional de la Patagonia "San Juan Bosco". Instituto de Biociencias de la Patagonia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto de Biociencias de la Patagonia; Argentin