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1.
Microb Biotechnol ; 16(5): 931-946, 2023 05.
Article in English | MEDLINE | ID: mdl-36682039

ABSTRACT

The soil bacterium Pseudomonas putida KT2440 has been shown to produce selenium nanoparticles aerobically from selenite; however, the molecular actors involved in this process are unknown. Here, through a combination of genetic and analytical techniques, we report the first insights into selenite metabolism in this bacterium. Our results suggest that the reduction of selenite occurs through an interconnected metabolic network involving central metabolic reactions, sulphur metabolism, and the response to oxidative stress. Genes such as sucA, D2HGDH and PP_3148 revealed that the 2-ketoglutarate and glutamate metabolism is important to convert selenite into selenium. On the other hand, mutations affecting the activity of the sulphite reductase decreased the bacteria's ability to transform selenite. Other genes related to sulphur metabolism (ssuEF, sfnCE, sqrR, sqr and pdo2) and stress response (gqr, lsfA, ahpCF and sadI) were also identified as involved in selenite transformation. Interestingly, suppression of genes sqrR, sqr and pdo2 resulted in the production of selenium nanoparticles at a higher rate than the wild-type strain, which is of biotechnological interest. The data provided in this study brings us closer to understanding the metabolism of selenium in bacteria and offers new targets for the development of biotechnological tools for the production of selenium nanoparticles.


Subject(s)
Nanoparticles , Pseudomonas putida , Selenium , Pseudomonas putida/genetics , Pseudomonas putida/metabolism , Selenium/metabolism , Nanoparticles/metabolism , Selenious Acid/metabolism , Oxidative Stress , Sulfur/metabolism
2.
Microb Biotechnol ; 14(6): 2463-2480, 2021 11.
Article in English | MEDLINE | ID: mdl-33404203

ABSTRACT

The throwaway culture related to the single-use materials such as polyethylene terephthalate (PET) has created a major environmental concern. Recycling of PET waste into biodegradable plastic polyhydroxyalkanoate (PHA) creates an opportunity to improve resource efficiency and contribute to a circular economy. We sequenced the genome of Pseudomonas umsongensis GO16 previously shown to convert PET-derived terephthalic acid (TA) into PHA and performed an in-depth genome analysis. GO16 can degrade a range of aromatic substrates in addition to TA, due to the presence of a catabolic plasmid pENK22. The genetic complement required for the degradation of TA via protocatechuate was identified and its functionality was confirmed by transferring the tph operon into Pseudomonas putida KT2440, which is unable to utilize TA naturally. We also identified the genes involved in ethylene glycol (EG) metabolism, the second PET monomer, and validated the capacity of GO16 to use EG as a sole source of carbon and energy. Moreover, GO16 possesses genes for the synthesis of both medium and short chain length PHA and we have demonstrated the capacity of the strain to convert mixed TA and EG into PHA. The metabolic versatility of GO16 highlights the potential of this organism for biotransformations using PET waste as a feedstock.


Subject(s)
Polyhydroxyalkanoates , Pseudomonas putida , Polyethylene Terephthalates , Pseudomonas/genetics , Pseudomonas putida/genetics
3.
ISME J ; 15(5): 1330-1343, 2021 05.
Article in English | MEDLINE | ID: mdl-33323977

ABSTRACT

The rapid emergence of antibiotic resistant bacterial pathogens constitutes a critical problem in healthcare and requires the development of novel treatments. Potential strategies include the exploitation of microbial social interactions based on public goods, which are produced at a fitness cost by cooperative microorganisms, but can be exploited by cheaters that do not produce these goods. Cheater invasion has been proposed as a 'Trojan horse' approach to infiltrate pathogen populations with strains deploying built-in weaknesses (e.g., sensitiveness to antibiotics). However, previous attempts have been often unsuccessful because population invasion by cheaters was prevented by various mechanisms including the presence of spatial structure (e.g., growth in biofilms), which limits the diffusion and exploitation of public goods. Here we followed an alternative approach and examined whether the manipulation of public good uptake and not its production could result in potential 'Trojan horses' suitable for population invasion. We focused on the siderophore pyoverdine produced by the human pathogen Pseudomonas aeruginosa MPAO1 and manipulated its uptake by deleting and/or overexpressing the pyoverdine primary (FpvA) and secondary (FpvB) receptors. We found that receptor synthesis feeds back on pyoverdine production and uptake rates, which led to strains with altered pyoverdine-associated costs and benefits. Moreover, we found that the receptor FpvB was advantageous under iron-limited conditions but revealed hidden costs in the presence of an antibiotic stressor (gentamicin). As a consequence, FpvB mutants became the fittest strain under gentamicin exposure, displacing the wildtype in liquid cultures, and in biofilms and during infections of the wax moth larvae Galleria mellonella, which both represent structured environments. Our findings reveal that an evolutionary trade-off associated with the costs and benefits of a versatile pyoverdine uptake strategy can be harnessed for devising a Trojan-horse candidate for medical interventions.


Subject(s)
Oligopeptides , Pseudomonas aeruginosa , Biofilms , Pseudomonas aeruginosa/genetics , Siderophores
4.
Curr Opin Biotechnol ; 62: 29-37, 2020 04.
Article in English | MEDLINE | ID: mdl-31580950

ABSTRACT

Bacterial cells have a limited number of resources that can be allocated for gene expression. The intracellular competition for these resources has an impact on the cell physiology. Bacteria have evolved mechanisms to optimize resource allocation in a variety of scenarios, showing a trade-off between the resources used to maximise growth (e.g. ribosome synthesis) and the rest of cellular functions. Limitations in gene expression also play a role in generating phenotypic diversity, which is advantageous in fluctuating environments, at the expenses of decreasing growth rates. Our current understanding of these trade-offs can be exploited for biotechnological applications benefiting from the selective manipulation of the allocation of resources.


Subject(s)
Bacteria , Bacteria/genetics , Gene Expression
5.
Genes (Basel) ; 10(5)2019 05 16.
Article in English | MEDLINE | ID: mdl-31100963

ABSTRACT

Plastics have become an important environmental concern due to their durability and resistance to degradation. Out of all plastic materials, polyesters such as polyethylene terephthalate (PET) are amenable to biological degradation due to the action of microbial polyester hydrolases. The hydrolysis products obtained from PET can thereby be used for the synthesis of novel PET as well as become a potential carbon source for microorganisms. In addition, microorganisms and biomass can be used for the synthesis of the constituent monomers of PET from renewable sources. The combination of both biodegradation and biosynthesis would enable a completely circular bio-PET economy beyond the conventional recycling processes. Circular strategies like this could contribute to significantly decreasing the environmental impact of our dependence on this polymer. Here we review the efforts made towards turning PET into a viable feedstock for microbial transformations. We highlight current bottlenecks in degradation of the polymer and metabolism of the monomers, and we showcase fully biological or semisynthetic processes leading to the synthesis of PET from sustainable substrates.


Subject(s)
Biodegradable Plastics/chemistry , Polyethylene Terephthalates/chemistry , Recycling/methods , Biodegradation, Environmental , Genes, Microbial/genetics , Hydrolases/chemistry , Hydrolysis , Plastics/chemistry , Polymers/chemistry
6.
Front Microbiol ; 9: 1845, 2018.
Article in English | MEDLINE | ID: mdl-30158907

ABSTRACT

Quantitative RNA sequencing (RNA-seq) and the complementary phenotypic assays were implemented to investigate the transcriptional responses of Chromohalobacter salexigens to osmotic and heat stress. These conditions trigger the synthesis of ectoine and hydroxyectoine, two compatible solutes of biotechnological interest. Our findings revealed that both stresses make a significant impact on C. salexigens global physiology. Apart from compatible solute metabolism, the most relevant adaptation mechanisms were related to "oxidative- and protein-folding- stress responses," "modulation of respiratory chain and related components," and "ion homeostasis." A general salt-dependent induction of genes related to the metabolism of ectoines, as well as repression of ectoine degradation genes by temperature, was observed. Different oxidative stress response mechanisms, secondary or primary, were induced at low and high salinity, respectively, and repressed by temperature. A higher sensitivity to H2O2 was observed at high salinity, regardless of temperature. Low salinity induced genes involved in "protein-folding-stress response," suggesting disturbance of protein homeostasis. Transcriptional shift of genes encoding three types of respiratory NADH dehydrogenases, ATP synthase, quinone pool, Na+/H+ antiporters, and sodium-solute symporters, was observed depending on salinity and temperature, suggesting modulation of the components of the respiratory chain and additional systems involved in the generation of H+ and/or Na+ gradients. Remarkably, the Na+ intracellular content remained constant regardless of salinity and temperature. Disturbance of Na+- and H+-gradients with specific ionophores suggested that both gradients influence ectoine production, but with differences depending on the solute, salinity, and temperature conditions. Flagellum genes were strongly induced by salinity, and further induced by temperature. However, salt-induced cell motility was reduced at high temperature, possibly caused by an alteration of Na+ permeability by temperature, as dependence of motility on Na+-gradient was observed. The transcriptional induction of genes related to the synthesis and transport of siderophores correlated with a higher siderophore production and intracellular iron content only at low salinity. An excess of iron increased hydroxyectoine accumulation by 20% at high salinity. Conversely, it reduced the intracellular content of ectoines by 50% at high salinity plus high temperature. These findings support the relevance of iron homeostasis for osmoadaptation, thermoadaptation and accumulation of ectoines, in C. salexigens.

7.
Microb Cell Fact ; 17(1): 2, 2018 Jan 09.
Article in English | MEDLINE | ID: mdl-29316921

ABSTRACT

BACKGROUND: The halophilic bacterium Chromohalobacter salexigens is a natural producer of ectoines, compatible solutes with current and potential biotechnological applications. As production of ectoines is an osmoregulated process that draws away TCA intermediates, bacterial metabolism needs to be adapted to cope with salinity changes. To explore and use C. salexigens as cell factory for ectoine(s) production, a comprehensive knowledge at the systems level of its metabolism is essential. For this purpose, the construction of a robust and high-quality genome-based metabolic model of C. salexigens was approached. RESULTS: We generated and validated a high quality genome-based C. salexigens metabolic model (iFP764). This comprised an exhaustive reconstruction process based on experimental information, analysis of genome sequence, manual re-annotation of metabolic genes, and in-depth refinement. The model included three compartments (periplasmic, cytoplasmic and external medium), and two salinity-specific biomass compositions, partially based on experimental results from C. salexigens. Using previous metabolic data as constraints, the metabolic model allowed us to simulate and analyse the metabolic osmoadaptation of C. salexigens under conditions for low and high production of ectoines. The iFP764 model was able to reproduce the major metabolic features of C. salexigens. Flux Balance Analysis (FBA) and Monte Carlo Random sampling analysis showed salinity-specific essential metabolic genes and different distribution of fluxes and variation in the patterns of correlation of reaction sets belonging to central C and N metabolism, in response to salinity. Some of them were related to bioenergetics or production of reducing equivalents, and probably related to demand for ectoines. Ectoines metabolic reactions were distributed according to its correlation in four modules. Interestingly, the four modules were independent both at low and high salinity conditions, as they did not correlate to each other, and they were not correlated with other subsystems. CONCLUSIONS: Our validated model is one of the most complete curated networks of halophilic bacteria. It is a powerful tool to simulate and explore C. salexigens metabolism at low and high salinity conditions, driving to low and high production of ectoines. In addition, it can be useful to optimize the metabolism of other halophilic bacteria for metabolite production.


Subject(s)
Amino Acids, Diamino/metabolism , Chromohalobacter/genetics , Chromohalobacter/metabolism , Genome, Bacterial , Models, Biological , Adaptation, Physiological , Amino Acids, Diamino/biosynthesis , Biomass , Chromohalobacter/drug effects , Metabolic Flux Analysis , Salinity , Sodium Chloride/metabolism , Sodium Chloride/pharmacology
8.
Microb Cell Fact ; 16(1): 23, 2017 Feb 08.
Article in English | MEDLINE | ID: mdl-28179004

ABSTRACT

BACKGROUND: The halophilic bacterium Chromohalobacter salexigens has been proposed as promising cell factory for the production of the compatible solutes ectoine and hydroxyectoine. This bacterium has evolved metabolic adaptations to efficiently grow under high salt concentrations by accumulating ectoines as compatible solutes. However, metabolic overflow, which is a major drawback for the efficient conversion of biological feedstocks, occurs as a result of metabolic unbalances during growth and ectoines production. Optimal production of ectoines is conditioned by the interplay of carbon and nitrogen metabolisms. In this work, we set out to determine how nitrogen supply affects the production of ectoines. RESULTS: Chromohalobacter salexigens was challenged to grow in media with unbalanced carbon/nitrogen ratio. In C. salexigens, overflow metabolism and ectoines production are a function of medium composition. At low ammonium conditions, the growth rate decreased importantly, up to 80%. Shifts in overflow metabolism were observed when changing the C/N ratio in the culture medium. 13C-NMR analysis of ectoines labelling revealed a high metabolic rigidity, with almost constant flux ratios in all conditions assayed. Unbalanced C/N ratio led to pyruvate accumulation, especially upon N-limitation. Analysis of an ect - mutant demonstrated the link between metabolic overflow and ectoine biosynthesis. Under non ectoine synthesizing conditions, glucose uptake and metabolic overflow decreased importantly. Finally, in fed-batch cultures, biomass yield was affected by the feeding scheme chosen. High growth (up to 42.4 g L-1) and volumetric ectoine yields (up to 4.21 g L-1) were obtained by minimizing metabolite overflow and nutrient accumulation in high density cultures in a low nitrogen fed-batch culture. Moreover, the yield coefficient calculated for the transformation of glucose into biomass was 30% higher in fed-batch than in the batch culture, demonstrating that the metabolic efficiency of C. salexigens can be improved by careful design of culture feeding schemes. CONCLUSIONS: Metabolic shifts observed at low ammonium concentrations were explained by a shift in the energy required for nitrogen assimilation. Carbon-limited fed-batch cultures with reduced ammonium supply were the best conditions for cultivation of C. salexigens, supporting high density growth and maintaining high ectoines production.


Subject(s)
Amino Acids, Diamino/biosynthesis , Carbon/metabolism , Chromohalobacter/metabolism , Nitrogen/metabolism , Ammonia/pharmacology , Batch Cell Culture Techniques , Biomass , Carbohydrate Metabolism , Chromohalobacter/drug effects , Chromohalobacter/growth & development , Culture Media/chemistry , Glucose/metabolism , Osmotic Pressure , Pyruvic Acid/analysis , Salinity
9.
Essays Biochem ; 60(4): 303-313, 2016 11 30.
Article in English | MEDLINE | ID: mdl-27903818

ABSTRACT

The chassis is the cellular host used as a recipient of engineered biological systems in synthetic biology. They are required to propagate the genetic information and to express the genes encoded in it. Despite being an essential element for the appropriate function of genetic circuits, the chassis is rarely considered in their design phase. Consequently, the circuits are transferred to model organisms commonly used in the laboratory, such as Escherichia coli, that may be suboptimal for a required function. In this review, we discuss some of the properties desirable in a versatile chassis and summarize some examples of alternative hosts for synthetic biology amenable for engineering. These properties include a suitable life style, a robust cell wall, good knowledge of its regulatory network as well as of the interplay of the host components with the exogenous circuits, and the possibility of developing whole-cell models and tuneable metabolic fluxes that could allow a better distribution of cellular resources (metabolites, ATP, nucleotides, amino acids, transcriptional and translational machinery). We highlight Pseudomonas putida, widely used in many different biotechnological applications as a prominent organism for synthetic biology due to its metabolic diversity, robustness and ease of manipulation.


Subject(s)
Pseudomonas putida/physiology , Synthetic Biology , Genetic Engineering , Metabolic Networks and Pathways
10.
Ars pharm ; 57(1): 23-26, ene.-mar. 2016.
Article in Spanish | IBECS | ID: ibc-150940

ABSTRACT

Objetivos: Listar las variables imprescindibles en los cuadros de mando integrales para abarcar todas las áreas básicas de trabajo en una Unidad de Radiofarmacia hospitalaria, cuya adecuada gestión puede ser clave para optimizar los recursos disponibles. En segundo lugar, enumerar los beneficios que redundan en la práctica de trabajo diario tras su integración. Métodos: Revisión de la bibliografía publicada sobre cuadros de mando integrales, seleccionando las variables para que el radiofarmacéutico asuma un papel activo en la mejora de su área de trabajo. Se utilizan programas construídos en Microsoft Access para la gestión integral. Se administran en varios módulos toda la información desde la prescripción y citación de los pacientes (asignándoles un código) hasta que se les realiza la exploración. Se recogen también variables como: fecha y hora límites de tramitación de radiofármaco al proveedor; fecha prueba médica; gestión de eluciones de generadores y kits fríos; turnos de trabajo del personal; registro de incidencias tipificadas y de datos de recepción, marcaje, control de calidad y dispensación de cada radiofármaco (asegurando la trazabilidad); detección de desviaciones entre actividad calibrada y medida; la actividad dispensada y la disponible a tiempo real; gestión de eliminación de residuos radiactivos, de existencias y caducidades; fechas de próximas revisiones de equipos; archivado de PNT; sistemas conversores de unidades y registro de informes clínicos. Resultados: Los programas especializados gestionan la información que se maneja en la Unidad de Radiofarmacia, facilitando tomar decisiones coste-efectivas. Los parámetros analizados son: número de preparaciones elaboradas y actividad manejada; posibles incidencias en cualquiera de los procesos cotidianos; porcentaje de resolución satisfactoria sin que derive en falta de disponibilidad; correcta trazabilidad de los radiofármacos; porcentaje de controles de calidad satisfactorios; evolución en el consumo por tipo de radiofármaco, etc. La mejora en la gestión de pedidos asegura la presencia del radiofármaco necesario para cada exploración. Conclusiones: Estos nuevos cuadros de mando integrales son útiles para optimizar pedidos y radiofármacos, asegurar trazabilidad, gestionar inventario, informes clínicos, residuos radiactivos y para evaluar la eficiencia de la Unidad de radiofarmacia, permitiendo la integración de estos datos con otros softwares de gestión sanitaria. Esta metodología puede aplicarse en Centros Sanitarios de Atención Primaria para enfocar al personal en sus funciones asistenciales y operativas


Objetives: We list the essential variables in Balanced Scorecards to cover all basic areas of work in a Radiopharmacy Hospital Unit, whose proper management can be key in optimizing the resources that are available. Secondly, to list the benefits in the practice of daily work that result after integration. Methods: A review of the published literature about Balanced Scorecards, selecting the variables to be collected for the radiopharmaceutical to take an active role in improving his work area. Applications built in Microsoft Access for comprehensive management are used. They are administered in various modules all the information from the prescription and citation patients (assigning a code) until they perform medical test. Also collected variables such as date and time limits for processing radiopharmaceutical supplier; medical test date; management elutions of generators and cold kits; staff work shifts; typified record incidents and receive data, labeling, quality control and dispensing of each radiopharmaceutical (ensuring traceability); detecting deviations between calibrated and measured activity; to know the dispensed activity and available in real time; management of radioactive waste disposal, inventory and expiration dates; dates of upcoming reviews of equipment; SOP filed; converter systems units and registration of clinical reports. Results: These specialized software manage the information handled in daily practice in Radiopharmacy Hospital Unit, facilitating cost-effective decisions. These parameters analyzed are usually: number of elaborate preparations and managed activity; possible incidents in any of the everyday processes; percentage of satisfactory resolution without resulting in unavailability; correct traceability of radiopharmaceuticals; percentage of satisfactory quality controls; evolution in consumption by type of radiopharmaceutical, etc. The improved order management ensures the presence of the radiopharmaceutical needed for each medical test. Conclusions: These new Balanced Scorecards are useful to optimize orders and radiopharmaceuticals, ensure traceability, inventory management, clinical reports, radioactive waste and to evaluate the efficiency of the Radiopharmacy Hospital Unit, allowing the integration of these data with others software health management. This methodology can be applied in Primary Care Health Centers to focus on its healthcare staff and operational functions


Subject(s)
Humans , Male , Female , Community Pharmacy Services/organization & administration , Pharmacy Service, Hospital/organization & administration , Pharmacy Service, Hospital/standards , Pharmacy Service, Hospital , Clinical Pharmacy Information Systems/organization & administration , Clinical Pharmacy Information Systems/standards , Software/legislation & jurisprudence , Software/standards , Primary Health Care/organization & administration , Primary Health Care/standards
11.
Environ Microbiol Rep ; 7(2): 301-11, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25417903

ABSTRACT

Chromohalobacter salexigens is a halophilic γ-proteobacterium that responds to osmotic and heat stresses by accumulating ectoine and hydroxyectoine respectively. Evolution has optimized its metabolism to support high production of ectoines. We analysed the effect of an rpoS mutation in C. salexigens metabolism and ectoines synthesis. In long-term adapted cells, the rpoS strain was osmosensitive but not thermosensitive and showed unaltered ectoines content, suggesting that RpoS regulates ectoine(s)-independent osmoadaptive mechanisms. RpoS is involved in the regulation of C. salexigens metabolic adaptation to stress, as early steps of glucose oxidation through the Entner-Doudoroff pathway were deregulated in the rpoS mutant, leading to improved metabolic efficiency at low salinity. Moreover, a reduced pyruvate (but not acetate) overflow was displayed by the rpoS strain at low salt, probably linked to a slowdown in gluconate production and/or subsequent metabolism. Interestingly, RpoS does not seem to be the main regulator triggering the immediate transcriptional response of ectoine synthesis to osmotic or thermal upshifts. However, it contributed to the expression of the ect genes in cells previously adapted to low or high salinity.


Subject(s)
Amino Acids, Diamino/biosynthesis , Bacterial Proteins/metabolism , Chromohalobacter/metabolism , Gene Expression Regulation, Bacterial , Heat-Shock Response , Osmotic Pressure , Sigma Factor/metabolism , Bacterial Proteins/genetics , Chromohalobacter/drug effects , Chromohalobacter/radiation effects , Gene Knockout Techniques , Sigma Factor/genetics
12.
J Biol Chem ; 288(24): 17769-81, 2013 Jun 14.
Article in English | MEDLINE | ID: mdl-23615905

ABSTRACT

Bacterial osmoadaptation involves the cytoplasmic accumulation of compatible solutes to counteract extracellular osmolarity. The halophilic and highly halotolerant bacterium Chromohalobacter salexigens is able to grow up to 3 m NaCl in a minimal medium due to the de novo synthesis of ectoines. This is an osmoregulated pathway that burdens central metabolic routes by quantitatively drawing off TCA cycle intermediaries. Consequently, metabolism in C. salexigens has adapted to support this biosynthetic route. Metabolism of C. salexigens is more efficient at high salinity than at low salinity, as reflected by lower glucose consumption, lower metabolite overflow, and higher biomass yield. At low salinity, by-products (mainly gluconate, pyruvate, and acetate) accumulate extracellularly. Using [1-(13)C]-, [2-(13)C]-, [6-(13)C]-, and [U-(13)C6]glucose as carbon sources, we were able to determine the main central metabolic pathways involved in ectoines biosynthesis from glucose. C. salexigens uses the Entner-Doudoroff pathway rather than the standard glycolytic pathway for glucose catabolism, and anaplerotic activity is high to replenish the TCA cycle with the intermediaries withdrawn for ectoines biosynthesis. Metabolic flux ratios at low and high salinity were similar, revealing a certain metabolic rigidity, probably due to its specialization to support high biosynthetic fluxes and partially explaining why metabolic yields are so highly affected by salinity. This work represents an important contribution to the elucidation of specific metabolic adaptations in compatible solute-accumulating halophilic bacteria.


Subject(s)
Chromohalobacter/metabolism , Salt Tolerance , Amino Acids/metabolism , Amino Acids, Diamino/biosynthesis , Bacterial Proteins/genetics , Biomass , Carbohydrate Metabolism , Carboxylic Acids/metabolism , Chromohalobacter/genetics , Chromohalobacter/growth & development , Citric Acid Cycle , Computational Biology , Glucose/metabolism , Metabolic Networks and Pathways , Salinity , Sodium Chloride/metabolism , Staining and Labeling
13.
PLoS One ; 7(3): e33587, 2012.
Article in English | MEDLINE | ID: mdl-22448254

ABSTRACT

The disaccharide trehalose is considered as a universal stress molecule, protecting cells and biomolecules from injuries imposed by high osmolarity, heat, oxidation, desiccation and freezing. Chromohalobacter salexigens is a halophilic and extremely halotolerant γ-proteobacterium of the family Halomonadaceae. In this work, we have investigated the role of trehalose as a protectant against salinity, temperature and desiccation in C. salexigens. A mutant deficient in the trehalose-6-phosphate synthase gene (otsA::Ω) was not affected in its salt or heat tolerance, but double mutants ectoine- and trehalose-deficient, or hydroxyectoine-reduced and trehalose-deficient, displayed an osmo- and thermosensitive phenotype, respectively. This suggests a role of trehalose as a secondary solute involved in osmo- (at least at low salinity) and thermoprotection of C. salexigens. Interestingly, trehalose synthesis was osmoregulated at the transcriptional level, and thermoregulated at the post-transcriptional level, suggesting that C. salexigens cells need to be pre-conditioned by osmotic stress, in order to be able to quickly synthesize trehalose in response to heat stress. C. salexigens was more sensitive to desiccation than E. coli and desiccation tolerance was slightly improved when cells were grown at high temperature. Under these conditions, single mutants affected in the synthesis of trehalose or hydroxyectoine were more sensitive to desiccation than the wild-type strain. However, given the low survival rates of the wild type, the involvement of trehalose and hydroxyectoine in C. salexigens response to desiccation could not be firmly established.


Subject(s)
Chromohalobacter/metabolism , Desiccation , Hot Temperature , Salinity , Trehalose/metabolism , Carbon Radioisotopes , Cells, Cultured , Chromohalobacter/genetics , Chromohalobacter/growth & development , Escherichia coli/genetics , Escherichia coli/metabolism , Glucosyltransferases/genetics , Glucosyltransferases/metabolism , Magnetic Resonance Spectroscopy , Mutation/genetics , Osmolar Concentration , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
14.
Methods Mol Biol ; 824: 167-201, 2012.
Article in English | MEDLINE | ID: mdl-22160899

ABSTRACT

Halophilic gammaproteobacteria of the family Halomonadaceae (including the genera Aidingimonas, Carnimonas, Chromohalobacter, Cobetia, Halomonas, Halotalea, Kushneria, Modicisalibacter, Salinicola, and Zymobacter) have current and promising applications in biotechnology mainly as a source of compatible solutes (powerful stabilizers of biomolecules and cells, with exciting potentialities in biomedicine), salt-tolerant enzymes, biosurfactants, and extracellular polysaccharides, among other products. In addition, they display a number of advantages to be used as cell factories, alternative to conventional prokaryotic hosts like Escherichia coli or Bacillus, for the production of recombinant proteins: (1) their high salt tolerance decreases to a minimum the necessity for aseptic conditions, resulting in cost-reducing conditions, (2) they are very easy to grow and maintain in the laboratory, and their nutritional requirements are simple, and (3) the majority can use a large range of compounds as a sole carbon and energy source. In the last 15 years, the efforts of our group and others have made possible the genetic manipulation of this bacterial group. In this review, the most relevant and recent tools for their genetic manipulation are described, with emphasis on nucleic acid isolation procedures, cloning and expression vectors, genetic exchange mechanisms, mutagenesis approaches, reporter genes, and genetic expression analyses. Complementary sections describing the influence of salinity on the susceptibility of these bacteria to antimicrobials, as well as the growth media most routinely used and culture conditions, for these microorganisms, are also included.


Subject(s)
Biotechnology/methods , Cell Culture Techniques/methods , Halomonadaceae/genetics , Halomonadaceae/metabolism , Salt Tolerance/physiology , Anti-Infective Agents/pharmacology , Blotting, Northern/methods , Cloning, Molecular , Culture Media/chemistry , Drug Resistance, Microbial/physiology , Gene Expression Profiling/methods , Gene Transfer Techniques , Genetic Vectors/genetics , Halomonadaceae/drug effects , Halomonadaceae/growth & development , Mutagenesis/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Species Specificity
15.
Biotechnol Adv ; 28(6): 782-801, 2010.
Article in English | MEDLINE | ID: mdl-20600783

ABSTRACT

Microorganisms produce and accumulate compatible solutes aiming at protecting themselves from environmental stresses. Among them, the wide spread in nature ectoines are receiving increasing attention by the scientific community because of their multiple applications. In fact, increasing commercial demand has led to a multiplication of efforts in order to improve processes for their production. In this review, the importance of current and potential applications of ectoines as protecting agents for macromolecules, cells and tissues, together with their potential as therapeutic agents for certain diseases are analyzed and current theories for the understanding of the molecular basis of their biological activity are discussed. The genetic, biochemical and environmental determinants of ectoines biosynthesis by natural and engineered producers are described. The major limitations of current bioprocesses used for ectoines production are discussed, with emphasis on the different microorganisms, environments, molecular engineering and fermentation strategies used to optimize the production and recovery of ectoines. The combined application of both bioprocess and metabolic engineering strategies, allowing a deeper understanding of the main factors controlling the production process is also stated. Finally, this review aims to summarize and update the state of the art in ectoines uses and applications and industrial scale production using bacteria, emphasizing the importance of reactor design and operation strategies, together with the metabolic engineering aspects and the need for feedback between wet and in silico work to optimize bioproduction.


Subject(s)
Amino Acids, Diamino/biosynthesis , Biotechnology , Cells/metabolism , Cytoprotection , Stress, Physiological , Amino Acids, Diamino/chemistry , Animals , Bioreactors , Humans
16.
Rev. cuba. med ; 48(2)abr.-jun. 2009.
Article in Spanish | LILACS | ID: lil-547147

ABSTRACT

La quimioterapia con paclitaxel es fundamental en el cáncer de mama metastásico (CMM). Se evaluó la eficacia y seguridad del uso del paclitaxel combinado con carboplatino. Se realizó un estudio retrospectivo y descriptivo de 63 pacientes tratadas en el Servicio de Oncología del Hospital Hermanos Ameijeiras, desde enero de 2001 hasta diciembre de 2005, con confirmación histológica de cáncer de mama y enfermedad metastásica, tratadas previamente con antraciclinas (en la adyuvancia). Hubo predominio de éstas en etapa premenopáusica; la mediana de edad fue de 44,2 años. Predominaron los receptores hormonales negativos y la presencia de un solo sitio metastásico, El hueso y el pulmón fueron las localizaciones metastásicas más comunes, 26,4 y 21,8 por ciento, respectivamente. El índice de respuesta global fue 33,3 por ciento. No hubo diferencias significativas en los índices de respuestas según las variables pronósticas. La supervivencia a 1, 2 y 3 años fue de 60, 46,4 y 28,2 por ciento, respectivamente. La mediana de supervivencia fue de 16,2 meses. Las variables asociadas a mejor pronóstico fueron índice de Karnofsky ³ 60 por ciento, receptores hormonales positivos y presencia de un solo sitio metastásico. La enfermedad diseminada en hueso, partes blandas y piel tiene mejor pronóstico. Las principales toxicidades fueron: hematológicas, neurológicas y digestivas. El promedio de procesos adversos por paciente fue de 2,6 y 20 por ciento de estos fueron grado 3-4. El incremento de las enzimas hepßticas provocó mayor cantidad de suspensiones de tratamiento. Los esquemas de combinación con paclitaxel tienen buen perfil de seguridad y eficacia aceptable.


Paclitex chemotherapy is essential in metastatic breast cancer (MBC). We assessed effectiveness and safety of combined use of carboplatine. Methods: We made a descriptive and retrospective study of 63 patients treated in Oncology Service of Hermanos Ameijeiras Surgical Clinical Hospital from January, 2001 to December, 2005 with a histological confirmation of breast cancer, and metastatic disease, previously treated with anthracyclins (in adjuvant therapy). There was a predominance of negative hormonal receptors, and presence of an only metastatic site. Bone and lungs were the more common metastatic locations, 26.4 percent and 21.8 percent, respectively. Global response rate was of 33.3 percent. There were not significant differences in response rate by prognostic variables. Survival at 1, 2, ant 3 years was of 60, 46.4 and 28.2 percent, respectively. Survival average was of 16,2 months. Variables associated with a better prognosis were a Karnofsky rate ³ 60 percent, positive hormonal receptors and presence of an only metastatic site. Disease disseminated in bone, soft tissues, and skin had a better prognosis. Main toxicities were: hematologic, neurologic, and digestives. The adverse processes average by patient was of 2.6 and the 20 percent of these ones were 3-4 degrees. Increment in hepatic enzymes caused a higher level of treatment suspension. Combined schemes with Paclitaxel have a good and acceptable profile of safety and effectiveness.


Subject(s)
Humans , Male , Middle Aged , Breast Neoplasms/drug therapy , Paclitaxel/therapeutic use , Taxoids/therapeutic use , Epidemiology, Descriptive , Retrospective Studies
17.
Rev. cuba. med ; 48(2)abr.-jun. 2009.
Article in Spanish | CUMED | ID: cum-40771

ABSTRACT

La quimioterapia con paclitaxel es fundamental en el cáncer de mama metastásico (CMM). Se evaluó la eficacia y seguridad del uso del paclitaxel combinado con carboplatino. Se realizó un estudio retrospectivo y descriptivo de 63 pacientes tratadas en el Servicio de Oncología del Hospital Hermanos Ameijeiras, desde enero de 2001 hasta diciembre de 2005, con confirmación histológica de cáncer de mama y enfermedad metastásica, tratadas previamente con antraciclinas (en la adyuvancia). Hubo predominio de éstas en etapa premenopáusica; la mediana de edad fue de 44,2 años. Predominaron los receptores hormonales negativos y la presencia de un solo sitio metastásico, El hueso y el pulmón fueron las localizaciones metastásicas más comunes, 26,4 y 21,8 por ciento, respectivamente. El índice de respuesta global fue 33,3 por ciento. No hubo diferencias significativas en los índices de respuestas según las variables pronósticas. La supervivencia a 1, 2 y 3 años fue de 60, 46,4 y 28,2 por ciento, respectivamente. La mediana de supervivencia fue de 16,2 meses. Las variables asociadas a mejor pronóstico fueron índice de Karnofsky ³ 60 por ciento, receptores hormonales positivos y presencia de un solo sitio metastásico. La enfermedad diseminada en hueso, partes blandas y piel tiene mejor pronóstico. Las principales toxicidades fueron: hematológicas, neurológicas y digestivas. El promedio de procesos adversos por paciente fue de 2,6 y 20 por ciento de estos fueron grado 3-4. El incremento de las enzimas hepßticas provocó mayor cantidad de suspensiones de tratamiento. Los esquemas de combinación con paclitaxel tienen buen perfil de seguridad y eficacia aceptable(AU)


Paclitex chemotherapy is essential in metastatic breast cancer (MBC). We assessed effectiveness and safety of combined use of carboplatine. Methods: We made a descriptive and retrospective study of 63 patients treated in Oncology Service of Hermanos Ameijeiras Surgical Clinical Hospital from January, 2001 to December, 2005 with a histological confirmation of breast cancer, and metastatic disease, previously treated with anthracyclins (in adjuvant therapy). There was a predominance of negative hormonal receptors, and presence of an only metastatic site. Bone and lungs were the more common metastatic locations, 26.4 percent and 21.8 percent, respectively. Global response rate was of 33.3 percent. There were not significant differences in response rate by prognostic variables. Survival at 1, 2, ant 3 years was of 60, 46.4 and 28.2 percent, respectively. Survival average was of 16,2 months. Variables associated with a better prognosis were a Karnofsky rate ³ 60 percent, positive hormonal receptors and presence of an only metastatic site. Disease disseminated in bone, soft tissues, and skin had a better prognosis. Main toxicities were: hematologic, neurologic, and digestives. The adverse processes average by patient was of 2.6 and the 20 percent of these ones were 3-4 degrees. Increment in hepatic enzymes caused a higher level of treatment suspension. Combined schemes with Paclitaxel have a good and acceptable profile of safety and effectiveness(AU)


Subject(s)
Humans , Male , Middle Aged , Paclitaxel/therapeutic use , Breast Neoplasms/drug therapy , Taxoids/therapeutic use , Epidemiology, Descriptive , Retrospective Studies
18.
Radiología (Madr., Ed. impr.) ; 45(5): 229-231, sept. 2003. ilus
Article in Es | IBECS | ID: ibc-28910

ABSTRACT

Presentamos un caso de carcinoma apocrino de mama en una mujer de 45 años de edad con prótesis mamaria bilateral de silicona, cuya manifestación clínica y mamográfica fue la de un nódulo palpable, de mayor densidad glandular, redondeado y de bordes imprecisos sin microcalcificaciones visibles. La revisión bibliográfica realizada nos confirma la inusual asociación de este tipo de tumor con la presencia de implantes mamarios de silicona, y es ahí donde creemos que radica su interés radiológico (AU)


Subject(s)
Female , Middle Aged , Humans , Carcinoma , Breast Implantation/adverse effects , Breast Neoplasms , Apocrine Glands/pathology , Silicones/adverse effects , Mammography/methods
19.
Med Clin (Barc) ; 120(12): 441-5, 2003 Apr 05.
Article in Spanish | MEDLINE | ID: mdl-12689549

ABSTRACT

BACKGROUND AND OBJECTIVE: The etiology of neural tube defects (NTDs) is multifactorial. The presence of mutated genotypes of C677T and A1298C polymorphisms, and their combined heterozygosity, have been considered risk factors for the occurrence and recurrence of NTDs in some populations. SUBJECTS AND METHOD: This case-control study included 159 healthy controls, 27 NTDs patients, 28 patients' mothers and 23 siblings. The polymorphism study was performed by PCR. For fragment digestion, we used the restriction enzymes Hinf I (C677T) and Mbo II (A1298C). RESULTS: There was no significant difference (p = 0.991) in C677T genotypes between controls (CC: 35%, CT: 50% and TT: 15%) and patients (37, 52 and 11%, respectively), patients' mothers (39, 50 and 11%, respectively) and siblings (35, 48 and 17%, respectively). The prevalence of A1298C genotypes in controls (AA: 49%, AC: 45% and CC: 6%) was similar (p = 0.917) to the prevalence in patients (41, 56 and 4%, respectively), patients' mothers (43, 50 and 7%, respectively) and siblings (52, 39 and 9%, respectively). CONCLUSIONS: The absence of differences in the two polymorphisms between these groups makes us conclude that there is no association with NTDs in the Spanish population.


Subject(s)
Neural Tube Defects/genetics , Oxidoreductases Acting on CH-NH Group Donors/genetics , Case-Control Studies , Genotype , Humans , Methylenetetrahydrofolate Reductase (NADPH2) , Polymerase Chain Reaction , Polymorphism, Genetic , Spain
20.
Med. clín (Ed. impr.) ; 120(12): 441-445, abr. 2003.
Article in Es | IBECS | ID: ibc-20083

ABSTRACT

FUNDAMENTO Y OBJETIVO: La etiología de los defectos de tubo neural (DTN) es multifactorial. La presencia del genotipo mutado para los polimorfismos C677T y A1298C y su heterocigosidad combinada se han considerado factores de riesgo en determinadas poblaciones. SUJETOS Y MÉTODO: El estudio de casos y controles estuvo formado por 159 controles sanos, 27 pacientes afectados de espina bífida, 28 madres y 23 hermanos de pacientes con espina bífida. El estudio de los polimorfismos se realizó mediante reacción en cadena de la polimerasa (PCR) y digestión de los fragmentos obtenidos con las enzimas de restricción Hind I (C677T) y MboII (A1298C). RESULTADOS: No se han encontrado diferencias estadísticamente significativas (p = 0,991) entre la prevalencia de los genotipos C677T del grupo control (CC: 35 por ciento; CT: 50 por ciento, y TT: 15 por ciento) y los obtenidos en el grupo de pacientes (37, 52 y 11 por ciento, respectivamente), grupo de madres de pacientes (39, 50 y 11 por ciento, respectivamente) y grupo de hermanos de pacientes (35, 48 y 17 por ciento, respectivamente). Asimismo, la prevalencia de los genotipos A1298C dentro del grupo control (AA: 49 por ciento; AC: 45 por ciento, y CC: 6 por ciento) fue similar (p = 0,917) a la encontrada en el grupo de pacientes (41, 56 y 4 por ciento, respectivamente), grupo de madres de pacientes (43, 50 y 7 por ciento, respectivamente) y grupo de hermanos de pacientes (52, 39 y 9 por ciento, respectivamente). CONCLUSIONES: La ausencia de diferencias estadísticamente significativas de los dos polimorfismos estudiados en los distintos grupos hace concluir que éstos no participan en la etiología de los DTN en la población española (AU)


Subject(s)
Adult , Female , Humans , Risk , Spain , Polymerase Chain Reaction , Case-Control Studies , Precancerous Conditions , Neural Tube Defects , Methylenetetrahydrofolate Reductase (NADPH2) , Carcinoma in Situ , Carcinoma, Squamous Cell , Genotype , Papillomaviridae , Polymorphism, Genetic , Uterine Cervical Neoplasms , Oxidoreductases Acting on CH-NH Group Donors , Polymorphism, Genetic
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