Produção e consumo de acetato em cultivos de Salmonella typhimurium em condições aeróbias

Linhagens atenuadas de Salmonella typhimurium têm sido amplamente estudadas para o desenvolvimento de diversas vacinas e no tratamento do câncer. No entanto, ainda há poucas informações a respeito de seu metabolismo. S. typhimurium e Escherichia coli apresentam grande similaridade em seus genomas e redes metabólicas. Com o intuito de avaliar o metabolismo da Salmonella sob condições aeróbias, realizou-se seis cultivos contínuos nas taxas de diluição de 0,1 a 0,67 h-1 utilizando glicose como substrato. Estes resultados demonstraram um desvio de carbono para a formação de acetato, a partir da taxa de diluição de 0,1 h-1, diferentemente do que é relatado para E. coli, onde o acúmulo de acetato é detectado somente em taxas de diluição acima de 0,4 h-1. Investigou-se ainda a assimilação do acetato em cultivos em batelada, utilizando ácido acético como substrato principal. Os resultados obtidos confirmaram que a Salmonella possui um fluxo de assimilação de acetato menor do que a E. coli. Apesar de ambos os microrganismos apresentarem as mesmas reações no metabolismo central do carbono, este trabalho apresenta evidências experimentais que demonstram a menor capacidade de S. typhimurium em direcionar os fluxos de carbono para biomassa e produção de energia, caracterizada pela maior formação de acetato. A produção de acetato é considerada um desperdício de carbono, diminuindo a produtividade e os rendimentos, e comprometendo a obtenção de alta densidade celular essencial para o desenvolvimento de vacinas recombinantes de baixo custo. A análise dos fluxos metabólicos pode contribuir para minimizar a formação deste subproduto indesejado, tornando o metabolismo mais eficiente

Cooperative WebLab in chemical engineering between France and Brazil: Validation of the methodology

A WebLab is an experiment operated remotely via Internet. Besides the strictly technical aspects of such an experiment, which may contribute to the learning of Chemical Engineering fundamentals, there is also important feedback when teams of students of two different countries are working together: the WebLab becomes an intercultural experience, enhancing the communication skills of the students. A WebLab between Universidade Federal de São Carlos (DEQ/UFSCar) and the Ecole Nationale Supérieure d’Ingénieurs en Arts Chimiques et Technologiques (ENSIACET) is presented in this work. A mass transfer experiment in a bench scale reactor (stirred and aerated) had to be studied by mixed teams, thus emulating challenges that will be common in future working environments. In order to perform the experiment, students in Brazil and in France were put into groups. The students had to make decisions about the procedure for executing the experiments. All the students were able to control the equipment, no matter where they were physically. Students communicated using video conference software. The students' and teachers' opinions of this experience were very positive. This methodology is an important contribution to the education of engineers in a world integrated by modern communication technologies

Mapping Salmonella typhimurium pathways using ¹³C metabolic flux analysis

In the last years, Salmonella has been extensively studied not only due to its importance as a pathogen, but also as a host to produce pharmaceutical compounds. However, the full exploitation of Salmonella as a platform for bioproduct delivery has been hampered by the lack of information about its metabolism. Genome-scale metabolic models can be valuable tools to delineate metabolic engineering strategies as long as they closely represent the actual metabolism of the target organism. In the present study, a 13C-MFA approach was applied to map the fluxes at the central carbon pathways of S. typhimurium LT2 growing at glucose-limited chemostat cultures. The experiments were carried out in a 2L bioreactor, using defined medium enriched with 20% 13C-labeled glucose. Metabolic flux distributions in central carbon pathways of S. typhimurium LT2 were estimated using OpenFLUX2 based on the labeling pattern of biomass protein hydrolysates together with biomass composition. The results suggested that pentose phosphate is used to catabolize glucose, with minor fluxes through glycolysis. In silico simulations, using Optflux and pFBA as simulation method, allowed to study the performance of the genome-scale metabolic model. In general, the accuracy of in silico simulations was improved by the superimposition of estimated intracellular fluxes to the existing genome-scale metabolic model, showing a better fitting to the experimental extracellular fluxes, whereas the intracellular fluxes of pentose phosphate and anaplerotic reactions were poorly described.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil (CAPES) – Finance code 001. The authors also acknowledge the support of CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil); International cooperation project CAPES-FCT (Coordenacão de Aperfeiçoamento de Pessoal de Nível Superior/Brazil - Fundação para a Ciência e Tecnologia/Portugal - Process 315/11); CAPES, Brazil (Atração de Jovens Talentos - Process 064922/2014-01); Fundação para a Ciência e Tecnologia, Portugal (strategic funding of UID/BIO/04469 unit) and COMPETE 2020, Portugal (POCI-01-0145-FEDER-006684), in addition to the BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norteinfo:eu-repo/semantics/publishedVersio

Salmonella typhimurium and Escherichia coli dissimilarity: closely related bacteria with distinct metabolic profiles

Live attenuated strains of Salmonella typhimurium have been extensively investigated as vaccines for a number of infectious diseases. However, there is still little information available concerning aspects of their metabolism. S. typhimurium and Escherichia coli show a high degree of similarity in terms of their genome contents and metabolic networks. However, this work presents experimental evidence showing that significant differences exist in their abilities to direct carbon fluxes to biomass and energy production. It is important to study the metabolism of Salmonella in order to elucidate the formation of acetate and other metabolites involved in optimizing the production of biomass, essential for the development of recombinant vaccines. The metabolism of Salmonella under aerobic conditions was assessed using continuous cultures performed at dilution rates ranging from 0.1 to 0.67 h1, with glucose as main substrate. Acetate assimilation and glucose metabolism under anaerobic conditions were also investigated using batch cultures. Chemostat cultivations showed deviation of carbon towards acetate formation, starting at dilution rates above 0.1 h1. This differed from previous findings for E. coli, where acetate accumulation was only detected at dilution rates exceeding 0.4 h1, and was due to the lower rate of acetate assimilation by S. typhimurium under aerobic conditions. Under anaerobic conditions, both microorganisms mainly produced ethanol, acetate, and formate. A genome-scale metabolic model, reconstructed for Salmonella based on an E. coli model, provided a poor description of the mixed fermentation pattern observed during Salmonella cultures, reinforcing the different patterns of carbon utilization exhibited by these closely related bacteria. This article is protected by copyright. All rights reserved.Special thanks to Amadeus Azevedo for the HPLC analyses and technical assistance. The authors acknowledge the national funding received from CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Brazil), the international cooperation project CAPES-FCT (Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior/Brazil-Fundacao para a Ciencia e a Tecnologia/Portugal-Process 315/11), CAPES (Atracao de Jovens Talentos-Process 064922/2014-01) and to Fundacao para a Ciencia e Tecnologia the strategic funding of UID/BIO/04469/2013 unit

Evaluation of immobilized lipases on poly-hydroxybutyrate beads to catalyze biodiesel synthesis

Five microbial lipase preparations from several sources were immobilized by hydrophobic adsorption on small or large poly-hydroxybutyrate (PHB) beads and the effect of the support particle size on the biocatalyst activity was assessed in the hydrolysis of olive oil, esterification of butyric acid with butanol and transesterification of babassu oil (Orbignya sp.) with ethanol. The catalytic activity of the immobilized lipases in both olive oil hydrolysis and biodiesel synthesis was influenced by the particle size of PHB and lipase source. In the esterification reaction such influence was not observed. Geobacillus thermocatenulatus lipase (BTL2) was considered to be inadequate to catalyze biodiesel synthesis, but displayed high esterification activity. Butyl butyrate synthesis catalyzed by BTL2 immobilized on small PHB beads gave the highest yield (approximate to 90 mmol L-1). In biodiesel synthesis, the catalytic activity of the immobilized lipases was significantly increased in comparison to the free lipases. Full conversion of babassu oil into ethyl esters was achieved at 72 h in the presence of Pseudozyma antarctica type B (CALB), Thermomyces lanuginosus lipase (Lipex (R) 100L) immobilized on either small or large PHB beads and Pseudomonas fluorescens (PFL) immobilized on large PHB beads. The latter preparation presented the highest productivity (40.9 mg of ethyl esters mg(-1) immobilized protein h(-1)). (C) 2012 Elsevier B.V. All rights reserved.FAPESP (Brazil) [04/14593-4

Competition between the rearrangement of electrodeposited overlayers of copper on platinum and the hydrogen electrode reactions

The electrodeposition of Cu2+ ions on polycrystalline Pt from acid aqueous CuSO4, solutions was studied in the underpotential (upd) and overpotential (opd) ranges by using potentiostatic and potentiodynamic techniques. In the upd region, monotonic current transients were recorded for potentials (Ed) more positive than the reversible potential (Er) of the Cu2+/Cu electrode. For Cu electrodeposits involving a charge density close to that of the complete Cu monolayer, the hydrogen electrode reactions were strongly inhibited. Otherwise, non-monotonic current transients were recorded for Ed → Er. These transients suggest rearrangement of the monolayer structure when additional Cu atoms are deposited. In the opd region where the early stages of threodimensional(3D) nucleation and growth take place, the contribution of the hydrogen electrode reaction reappeared due to bare platinum sites which are present in the early stages of 3D growth. The present results in the limiting case can be interpreted in terms of a 2D to 3D transition at the overlayer level implying a certain distribution of clusters and the reappearance of the bare substrate surface. This process, in principle, accounts for the anomalies observed systematicly in vohammetric measurements for this system.Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicada

A functional biological network centered on XRCC3: a new possible marker of chemoradiotherapy resistance in rectal cancer patients

Preoperative chemoradiotherapy is widely used to improve local control of disease, sphincter preservation and to improve survival in patients with locally advanced rectal cancer. Patients enrolled in the present study underwent preoperative chemoradiotherapy, followed by surgical excision. Response to chemoradiotherapy was evaluated according to Mandard's Tumor Regression Grade (TRG). TRG 3, 4 and 5 were considered as partial or no response while TRG 1 and 2 as complete response. From pretherapeutic biopsies of 84 locally advanced rectal carcinomas available for the analysis, only 42 of them showed 70% cancer cellularity at least. By determining gene expression profiles, responders and non-responders showed significantly different expression levels for 19 genes (P < 0.001). We fitted a logistic model selected with a stepwise procedure optimizing the Akaike Information Criterion (AIC) and then validated by means of leave one out cross validation (LOOCV, accuracy = 95%). Four genes were retained in the achieved model: ZNF160, XRCC3, HFM1 and ASXL2. Real time PCR confirmed that XRCC3 is overexpressed in responders group and HFM1 and ASXL2 showed a positive trend. In vitro test on colon cancer resistant/susceptible to chemoradioterapy cells, finally prove that XRCC3 deregulation is extensively involved in the chemoresistance mechanisms. Protein-protein interactions (PPI) analysis involving the predictive classifier revealed a network of 45 interacting nodes (proteins) with TRAF6 gene playing a keystone role in the network. The present study confirmed the possibility that gene expression profiling combined with integrative computational biology is useful to predict complete responses to preoperative chemoradiotherapy in patients with advanced rectal cance

Metabolic profiling of secreted fermentative byproducts by Salmonella typhimurium

Salmonella typhimurium is a pathogenic bacterium that has been studied to be used as a platform for vaccine development as a live bacterial vector (LBV). It is closely related to other enterobacteria like E. coli, but presents some metabolic differences regarding the efficiency of biomass and energy production. This work presents a metabolic profile of the main byproducts excreted by S. typhimurium aiming to evaluate the distribution of carbon between respiratory and fermentative pathways for this bacterium. A genome scale metabolic model (GSMM) of S. typhimurium was also used to simulate the phenotypic behavior of this bacterium under the studied conditions and compare the experimental results to the model predictions. Simulations were performed using the free software Optflux and biomass maximization was used as the objective function for Flux Balance Analysis (FBA). Experimental cultivations were conducted in 2 L bioreactors with a working volume of 0.8 L under varying dilution rates (D) of 0.1, 0.24, 0.5, 0.58 and 0.67 h-1, using 10.0 g/L of glucose as the sole carbon source and M9 minimum medium. The dissolved oxygen concentration was kept at 30 % of its saturation, and pH and temperature were kept constant at 7.0 and 37°C, respectively. Supernatant samples were analyzed by HPLC to measure glucose consumption rate and fermentative products formation. Our initial results showed an intense production of organic acids by S. typhimurium, mainly acetic acid, at increasing amounts for higher dilution rates, and starting from the lower dilution rate studied. From D of 0.5 to D = 0.67 h-1 formate was also produced, and the secretion rates of both by-products increased linearly with the growth rate. These results may reflect some metabolic limitations of the species under investigation concerning its respiratory capacity, as the fermentative pathways seem to be active even under low growth rates and aerobic conditions, what is not observed for E. coli. It is also noteworthy that a considerable fraction of the carbon consumed is directed to the formation of these metabolites, compromising the biomass formation. Simulation results employing the free software Optflux 3.0.7 and the GSMM STM_v1.0 predicted some aspects of the fermentative behavior observed in the experiments, but with low accuracy, indicating that the model employed (that was constructed based on an E. coli model) does not reflect the differences between the metabolism of these two closed related bacteria. Results from this work will be incorporated in the model to increase its predictive capacity regarding the organic acids formation by Salmonella cells