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1.
PLoS One ; 15(8): e0238452, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32866191

RESUMO

The filamentous fungus Acremonium chrysogenum is the main industrial producer of cephalosporin C (CPC), one of the major precursors for manufacturing of cephalosporin antibiotics. The plasma membrane H+-ATPase (PMA) plays a key role in numerous fungal physiological processes. Previously we observed a decrease of PMA activity in A. chrysogenum overproducing strain RNCM 408D (HY) as compared to the level the wild-type strain A. chrysogenum ATCC 11550. Here we report the relationship between PMA activity and CPC biosynthesis in A. chrysogenum strains. The elevation of PMA activity in HY strain through overexpression of PMA1 from Saccharomyces cerevisiae, under the control of the constitutive gpdA promoter from Aspergillus nidulans, results in a 1.2 to 10-fold decrease in CPC production, shift in beta-lactam intermediates content, and is accompanied by the decrease in cef genes expression in the fermentation process; the characteristic colony morphology on agar media is also changed. The level of PMA activity in A. chrysogenum HY OE::PMA1 strains has been increased by 50-100%, up to the level observed in WT strain, and was interrelated with ATP consumption; the more PMA activity is elevated, the more ATP level is depleted. The reduced PMA activity in A. chrysogenum HY strain may be one of the selected events during classical strain improvement, aimed at elevating the ATP content available for CPC production.


Assuntos
Acremonium/metabolismo , Membrana Celular/metabolismo , Cefalosporinas/biossíntese , Cefalosporinas/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Adenosina Trifosfatases/metabolismo , Meios de Cultura/metabolismo , Fermentação/fisiologia , Regulação Fúngica da Expressão Gênica/fisiologia , beta-Lactamas/metabolismo
2.
PLoS One ; 15(8): e0236164, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32760085

RESUMO

Hyaluronan (HA) is a nonsulfated glycosaminoglycan that has been widely used for biomedical applications. Here, we have analyzed the effect of HA on the rescue of primary cells under stress as well as its potential to recover muscle atrophy and validated the developed model in vitro using primary muscle cells derived from rats. The potentials of different HAs were elucidated through comparative analyses using pharmaceutical grade a) high (HHA) and b) low molecular weight (LHA) hyaluronans, c) hybrid cooperative complexes (HCC) of HA in three experimental set-ups. The cells were characterized based on the expression of myogenin, a muscle-specific biomarker, and the proliferation was analyzed using Time-Lapse Video Microscopy (TLVM). Cell viability in response to H2O2 challenge was evaluated by 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, and the expression of the superoxide dismutase enzyme (SOD-2) was assessed by western blotting. Additionally, in order to establish an in vitro model of atrophy, muscle cells were treated with tumor necrosis factor-alpha (TNF-α), along with hyaluronans. The expression of Atrogin, MuRF-1, nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-kB), and Forkhead-box-(Fox)-O-3 (FoxO3a) was evaluated by western blotting to elucidate the molecular mechanism of atrophy. The results showed that HCC and HHA increased cell proliferation by 1.15 and 2.3 folds in comparison to un-treated cells (control), respectively. Moreover, both pre- and post-treatments of HAs restored the cell viability, and the SOD-2 expression was found to be reduced by 1.5 fold in HA-treated cells as compared to the stressed condition. Specifically in atrophic stressed cells, HCC revealed a noteworthy beneficial effect on the myogenic biomarkers indicating that it could be used as a promising platform for tissue regeneration with specific attention to muscle cell protection against stressful agents.


Assuntos
Ácido Hialurônico/farmacologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Atrofia Muscular/terapia , Medicina Regenerativa/métodos , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultura/metabolismo , Géis , Humanos , Ácido Hialurônico/química , Peróxido de Hidrogênio/toxicidade , Microscopia Intravital , Peso Molecular , Fibras Musculares Esqueléticas/patologia , Atrofia Muscular/patologia , Miogenina/análise , Miogenina/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Cultura Primária de Células , Ratos , Superóxido Dismutase/análise , Superóxido Dismutase/metabolismo , Imagem com Lapso de Tempo , Fator de Necrose Tumoral alfa/metabolismo
3.
Nat Commun ; 11(1): 3557, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32678091

RESUMO

Bacteria of the genus Bacteroides are common members of the human intestinal microbiota and important degraders of polysaccharides in the gut. Among them, the species Bacteroides thetaiotaomicron has emerged as the model organism for functional microbiota research. Here, we use differential RNA sequencing (dRNA-seq) to generate a single-nucleotide resolution transcriptome map of B. thetaiotaomicron grown under defined laboratory conditions. An online browser, called 'Theta-Base' ( www.helmholtz-hiri.de/en/datasets/bacteroides ), is launched to interrogate the obtained gene expression data and annotations of ~4500 transcription start sites, untranslated regions, operon structures, and 269 noncoding RNA elements. Among the latter is GibS, a conserved, 145 nt-long small RNA that is highly expressed in the presence of N-acetyl-D-glucosamine as sole carbon source. We use computational predictions and experimental data to determine the secondary structure of GibS and identify its target genes. Our results indicate that sensing of N-acetyl-D-glucosamine induces GibS expression, which in turn modifies the transcript levels of metabolic enzymes.


Assuntos
Bacteroides thetaiotaomicron/genética , Microbioma Gastrointestinal , Pequeno RNA não Traduzido/genética , Transcriptoma , Acetilglucosamina/metabolismo , Proteínas de Bactérias/genética , Bacteroides thetaiotaomicron/crescimento & desenvolvimento , Bacteroides thetaiotaomicron/metabolismo , Meios de Cultura/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , RNA Bacteriano/genética , Pequeno RNA não Traduzido/química , RNA não Traduzido/genética , Reprodutibilidade dos Testes , Navegador
4.
PLoS One ; 15(7): e0236739, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32730333

RESUMO

Rhodopseudomonas palustris PS3 is one of the purple phototrophic non-sulfur bacteria (PNSB), which have plant growth-promoting effects on various plants. To expand the scale of PS3 fermentation in a time- and cost-effective fashion, the purpose of this work was to evaluate the use of low-cost materials as culture media and to optimize the culture conditions via response surface methodology. Corn steep liquor (CSL) and molasses were identified as potential materials to replace the nitrogen and carbon sources, respectively, in the conventional growth medium. The optimum culture conditions identified through central composite design were CSL, 39.41 mL/L; molasses, 32.35 g/L; temperature, 37.9°C; pH, 7.0; and DO 30%. Under the optimized conditions, the biomass yield reached 2.18 ± 0.01 g/L at 24 hours, which was 7.8-fold higher than that under the original medium (0.28 ± 0.01 g/L). The correlation between the predicted and experimental values of the model was over 98%, which verified the validity of the response models. Furthermore, we verified the effectiveness of the R. palustris PS3 inoculant grown under the newly developed culture conditions for plant growth promotion. This study provides a potential strategy for improving the fermentation of R. palustris PS3 in low-cost media for large-scale industrial production.


Assuntos
Carbono/metabolismo , Meios de Cultura/química , Meios de Cultura/economia , Nitrogênio/metabolismo , Desenvolvimento Vegetal , Rodopseudomonas/crescimento & desenvolvimento , Meios de Cultura/metabolismo , Fermentação , Microbiologia Industrial , Rodopseudomonas/metabolismo
5.
Nat Commun ; 11(1): 3135, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561713

RESUMO

It is commonly thought that when multiple carbon sources are available, bacteria metabolize them either sequentially (diauxic growth) or simultaneously (co-utilization). However, this view is mainly based on analyses in relatively simple laboratory settings. Here we show that a heterotrophic marine bacterium, Pseudoalteromonas haloplanktis, can use both strategies simultaneously when multiple possible nutrients are provided in the same growth experiment. The order of nutrient uptake is partially determined by the biomass yield that can be achieved when the same compounds are provided as single carbon sources. Using transcriptomics and time-resolved intracellular 1H-13C NMR, we reveal specific pathways for utilization of various amino acids. Finally, theoretical modelling indicates that this metabolic phenotype, combining diauxie and co-utilization of substrates, is compatible with a tight regulation that allows the modulation of assimilatory pathways.


Assuntos
Carbono/metabolismo , Processos Heterotróficos/fisiologia , Modelos Biológicos , Pseudoalteromonas/fisiologia , Biomassa , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Meios de Cultura/metabolismo , Cinética , Espectroscopia de Prótons por Ressonância Magnética
6.
Nat Commun ; 11(1): 3120, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561727

RESUMO

Hyaluronan is widely used in cosmetics and pharmaceutics. Development of robust and safe cell factories and cultivation approaches to efficiently produce hyaluronan is of many interests. Here, we describe the metabolic engineering of Corynebacterium glutamicum and application of a fermentation strategy to manufacture hyaluronan with different molecular weights. C. glutamicum is engineered by combinatorial overexpression of type I hyaluronan synthase, enzymes of intermediate metabolic pathways and attenuation of extracellular polysaccharide biosynthesis. The engineered strain produces 34.2 g L-1 hyaluronan in fed-batch cultures. We find secreted hyaluronan encapsulates C. glutamicum, changes its cell morphology and inhibits metabolism. Disruption of the encapsulation with leech hyaluronidase restores metabolism and leads to hyper hyaluronan productions of 74.1 g L-1. Meanwhile, the molecular weight of hyaluronan is also highly tunable. These results demonstrate combinatorial optimization of cell factories and the extracellular environment is efficacious and likely applicable for the production of other biopolymers.


Assuntos
Corynebacterium glutamicum/enzimologia , Glucose/metabolismo , Ácido Hialurônico/biossíntese , Engenharia Metabólica/métodos , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas de Cultura Celular por Lotes/métodos , Metabolismo dos Carboidratos/genética , Corynebacterium glutamicum/genética , Meios de Cultura/metabolismo , Hialuronan Sintases/genética , Hialuronan Sintases/metabolismo , Hialuronoglucosaminidase/metabolismo , Redes e Vias Metabólicas/genética , Polissacarídeos Bacterianos/biossíntese
7.
J Biosci Bioeng ; 130(2): 195-199, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32370929

RESUMO

Ectoine production using inexpensive and renewable biomass resources has attracted great interest among the researchers due to the low yields of ectoine in current fermentation approaches that complicate the large-scale production of ectoine. In this study, ectoine was produced from corn steep liquor (CSL) and soybean hydrolysate (SH) in replacement to yeast extract as the nitrogen sources for the fermentation process. To enhance the bacterial growth and ectoine production, biotin was added to the Halomonas salina fermentation media. In addition, the effects addition of surfactants such as Tween 80 and saponin on the ectoine production were also investigated. Results showed that both the CSL and SH can be used as the nitrogen source substitutes in the fermentation media. Higher amount of ectoine (1781.9 mg L-1) was produced in shake flask culture with SH-containing media as compared to CSL-containing media. A total of 2537.0 mg L-1 of ectoine was produced at pH 7 when SH-containing media was applied in the 2 L batch fermentation. Moreover, highest amount of ectoine (1802.0 mg L-1) was recorded in the SH-containing shake flask culture with addition of 0.2 µm mL-1 biotin. This study demonstrated the efficacy of industrial waste as the nutrient supplement for the fermentation of ectoine production.


Assuntos
Diamino Aminoácidos/metabolismo , Fermentação , Halomonas/metabolismo , Microbiologia Industrial/métodos , Técnicas de Cultura Celular por Lotes , Biomassa , Biotina/metabolismo , Meios de Cultura/química , Meios de Cultura/metabolismo , Resíduos Industriais , Nitrogênio/metabolismo , Soja/química , Zea mays/química
8.
Can J Microbiol ; 66(4): 303-312, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32118486

RESUMO

Herein we describe a highly structured, filamentous growth phenotype displayed by an isolate of the food spoilage microorganism Brochothrix thermosphacta. The growth morphology of this B. thermosphacta strain (strain BII) was dependent on environmental factors such as the growth media, incubation temperatures, and the inoculum concentration. Inoculation of cultures in highly dilute suspensions resulted in the formation of isolated, tight aggregates resembling fungal growth in liquid media. This same strain also formed stable, mesh-like structures in 6-well tissue culture plates under specific growth conditions. The complex growth phenotype does not appear to be unique to strain BII but was common among B. thermosphacta strains isolated from chicken. Light and electron micrographs showed that the filaments of multiple BII cells can organize into complex, tertiary structures resembling multistranded cables. Time-lapse microscopy was employed to monitor the development of such aggregates over 18 h and revealed growth originating from short filaments into compact ball-like clusters that appeared fuzzy due to protruding filaments or cables. This report is the first to document this complex filamentous growth phenotype in a wild-type bacterial isolate of B. thermosphacta.


Assuntos
Brochothrix/crescimento & desenvolvimento , Galinhas/microbiologia , Animais , Brochothrix/classificação , Brochothrix/isolamento & purificação , Brochothrix/metabolismo , Meios de Cultura/química , Meios de Cultura/metabolismo , Contaminação de Alimentos/análise , Carne/microbiologia , Temperatura
9.
J Vis Exp ; (156)2020 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-32150164

RESUMO

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype called mucoidy. Alginate is linked to chronic lung infections resulting in poor prognosis in patients with cystic fibrosis (CF). Understanding the pathways that regulate the production of alginate can aid in the development of novel therapeutic strategies targeting the alginate formation. Another disease-related phenotype is the small colony variant (SCV). SCV is due to the slow growth of bacteria and often associated with increased resistance to antimicrobials. In this paper, we first show a method of culturing a genetically defined form of P. aeruginosa SCV due to pyrimidine biosynthesis mutations. Supplementation of nitrogenous bases, uracil or cytosine, returns the normal growth to these mutants, demonstrating the presence of a salvage pathway that scavenges free bases from the environment. Next, we discuss two methods for the measurement of bacterial alginate. The first method relies on the hydrolysis of the polysaccharide to its uronic acid monomer followed by derivatization with a chromogenic reagent, carbazole, while the second method uses an ELISA based on a commercially available, alginate-specific mAb. Both methods require a standard curve for quantitation. We also show that the immunological method is specific for alginate quantification and may be used for the measurement of alginate in the clinical specimens.


Assuntos
Alginatos/análise , Técnicas Bacteriológicas/métodos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Alginatos/metabolismo , Meios de Cultura/metabolismo , Fibrose Cística/microbiologia , Humanos , Mutação , Fenótipo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Pirimidinas/metabolismo
10.
J Agric Food Chem ; 68(8): 2485-2492, 2020 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-32049524

RESUMO

Employing isotope incubation studies, the biosynthetic pathway leading to a series of benzylic derivatives was elucidated in the fermentation broth of the edible mushroom Ischnoderma resinosum (P. Karst). Twenty-six hydroxy- and methoxy- benzylic derivatives were screened by gas chromatography-mass spectrometry (GC-MS) of which 13 were detected in the culture media. Results from the isotope incubation studies showed the transformation of both benzyl alcohol and benzoic acid into benzaldehyde. Benzaldehyde was then converted into 4-methoxybenzaldehyde via hydroxylation and subsequent methylation of the 4-C position. The resulting 4-methoxybenzaldehyde was then hydroxylated in the 3-C position followed by methylation into 3,4-dimethoxybenzaldehyde. Based on these findings, a novel metabolic scheme for the biosynthesis of benzylic derivatives in I. resinosum was proposed. The knowledge of the biosynthetic pathway was utilized to produce 4-hydroxy-3-methoxybenzaldehyde (vanillin) from 4-hydroxy-3-methoxybenzoic acid (vanillic acid). This is the first report to elucidate the biosynthetic pathway of benzyl derivatives and production of vanillin from I. resinosum.


Assuntos
Benzaldeídos/metabolismo , Polyporales/metabolismo , Benzaldeídos/análise , Ácido Benzoico/análise , Ácido Benzoico/metabolismo , Álcool Benzílico/análise , Álcool Benzílico/metabolismo , Biotransformação , Meios de Cultura/química , Meios de Cultura/metabolismo , Fermentação , Cromatografia Gasosa-Espectrometria de Massas , Polyporales/química , Polyporales/genética , Ácido Vanílico/análise , Ácido Vanílico/metabolismo
11.
Int J Food Microbiol ; 322: 108562, 2020 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-32109682

RESUMO

Shiga toxin-producing Escherichia coli (STEC) in sprouts have caused large scale outbreaks in the past involving severe illness. The combination of this very diverse pathogen and a food matrix with high numbers of background microbiota poses a particular challenge for detection and isolation. An acid treatment of the enrichment before plating on agar has been shown to improve the recovery of STEC from sprouts. After enrichment in buffered peptone water (BPW) at 37 °C we applied an acid treatment, followed by plating on tryptone bile x-glucuronide (TBX) agar (acid bile method). An inter-laboratory study was organized with 21 laboratories taking part to evaluate the performance parameters and applicability of the acid bile method. A sample set of six sprout samples was prepared consisting of two uninoculated samples and four spiked samples, each containing one of two STEC strains at one of two concentrations (low and high). Analyzing a set of six samples at the National Reference Laboratory (NRL E. coli), we determined the relative abundance of STEC without, after acid-, after bile- and after acid-bile treatment using real-time PCR. The participating laboratories successfully applied the acid bile method and were better able to detect (sensitivity 92.9% vs. 70.0%) and isolate (sensitivity 87.5% vs. 31.3%) STEC from positive samples using the acid bile method compared to non-acid methods. The relative limit of detection (RLOD) after isolation using the acid bile method (vs. non-acid method) was <1 for both STEC strains used, BfR-EC-14434 O133:H25 (0.146) and BfR-EC-16015 O26:H11 (0.073). A collection of STEC (n = 71) of diverse type and characteristics was assessed for their resistance towards the acid bile treatment selection. The majority (n = 65) of STEC strains could be recovered after acid treatment on TBX plates. However, a few strains (n = 6), among them clinical isolates were (partly) sensitive. These results suggest that an acid bile method is a rapid and reasonable approach to improve the recovery of STEC from sprouts when used in combination with methods targeting other selection markers.


Assuntos
Ácidos e Sais Biliares/metabolismo , Microbiologia de Alimentos/métodos , Ácido Clorídrico/metabolismo , Escherichia coli Shiga Toxigênica/isolamento & purificação , Verduras/microbiologia , Ágar , Animais , Meios de Cultura/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Plântula/microbiologia , Escherichia coli Shiga Toxigênica/metabolismo
12.
J Food Sci ; 85(3): 639-646, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32078749

RESUMO

Peanut sprouts are a functional food material rich in phytochemicals, including trans-resveratrol. This study aimed to optimize the recovery of trans-resveratrol from peanut sprouts using a combination of peanut varieties and sawdust medium through accelerated solvent extraction (ASE) and the response surface method (RSM). We also aimed to determine the antioxidant activity of this trans-resveratrol extract. Optimal fermentation periods of sawdust and peanut variety for cultivating peanut sprouts were determined on the basis of trans-resveratrol content via high-performance liquid chromatography. The extraction variables temperature, static time, and ethanol concentration were used to create a 20-sample set fit to a second-order polynomial equation through multiple regression analysis (R2 = 0.8787, P < 0.01). Trans-resveratrol content (19.62 ± 2.33 µg/g) peaked in the Palgwang variety cultured in sawdust medium fermented for 45 days. Optimal conditions for ASE were determined regarding the extraction temperature (90.29 °C), static time (3.95 min), and solvent (81.54% EtOH/water), and the predicted trans-resveratrol content under optimal conditions was 30.23 µg/g. Sawdust medium was more effective in increasing the trans-resveratrol content than conventional hydroponics, and the optimized process of combining fermented sawdust cultivation for harvesting peanut sprouts with ASE has potential as an efficient method of obtaining mass quantities of trans-resveratrol from peanut sprouts with improved nutritional and functional properties. PRACTICAL APPLICATION: This study showed that sawdust medium is more effective than hydroponics in increasing the trans-resveratrol content in peanut sprouts. The recovery of trans-resveratrol from peanut sprouts and its antioxidant activity were optimized via accelerated solvent extraction (ASE) and the response surface methodology (RSM). The optimized process of combining fermented sawdust cultivation for harvesting peanut sprouts with ASE potentially provides an efficient method to obtain mass quantities of trans-resveratrol from peanut sprouts with improved nutritional and functional properties.


Assuntos
Arachis/química , Resveratrol/análise , Sementes/crescimento & desenvolvimento , Antioxidantes/análise , Antioxidantes/metabolismo , Arachis/crescimento & desenvolvimento , Arachis/metabolismo , Cromatografia Líquida de Alta Pressão , Meios de Cultura/química , Meios de Cultura/metabolismo , Fermentação , Hidroponia/instrumentação , Resveratrol/metabolismo , Sementes/química , Sementes/metabolismo
13.
Nat Commun ; 11(1): 764, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034154

RESUMO

Our understanding of the signalling pathways regulating early human development is limited, despite their fundamental biological importance. Here, we mine transcriptomics datasets to investigate signalling in the human embryo and identify expression for the insulin and insulin growth factor 1 (IGF1) receptors, along with IGF1 ligand. Consequently, we generate a minimal chemically-defined culture medium in which IGF1 together with Activin maintain self-renewal in the absence of fibroblast growth factor (FGF) signalling. Under these conditions, we derive several pluripotent stem cell lines that express pluripotency-associated genes, retain high viability and a normal karyotype, and can be genetically modified or differentiated into multiple cell lineages. We also identify active phosphoinositide 3-kinase (PI3K)/AKT/mTOR signalling in early human embryos, and in both primed and naïve pluripotent culture conditions. This demonstrates that signalling insights from human blastocysts can be used to define culture conditions that more closely recapitulate the embryonic niche.


Assuntos
Autorrenovação Celular/fisiologia , Células-Tronco Embrionárias Humanas/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Ativinas/metabolismo , Animais , Blastocisto/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Meios de Cultura/química , Meios de Cultura/metabolismo , Meios de Cultura/farmacologia , Endoderma/citologia , Endoderma/metabolismo , Membranas Extraembrionárias/citologia , Membranas Extraembrionárias/metabolismo , Fibroblastos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Humanas/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/fisiologia , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Transcriptoma , Inativação do Cromossomo X/fisiologia
14.
Microbes Environ ; 35(1)2020.
Artigo em Inglês | MEDLINE | ID: mdl-32037377

RESUMO

Although the bioavailability of rare earth elements (REEs, including scandium, yttrium, and 15 lanthanides) has not yet been examined in detail, methane-oxidizing bacteria (methanotrophs) were recently shown to harbor specific types of methanol dehydrogenases (XoxF-MDHs) that contain lanthanides in their active site, whereas their well-characterized counterparts (MxaF-MDHs) were Ca2+-dependent. However, lanthanide dependency in methanotrophs has not been demonstrated, except in acidic environments in which the solubility of lanthanides is high. We herein report the isolation of a lanthanide-dependent methanotroph from a circumneutral environment in which lanthanides only slightly dissolved. Methanotrophs were enriched and isolated from pond sediment using mineral medium supplemented with CaCl2 or REE chlorides. A methanotroph isolated from the cerium (Ce) chloride-supplemented culture, Methylosinus sp. strain Ce-a6, was clearly dependent on lanthanide. Strain Ce-a6 only required approximately 30 nM lanthanide chloride for its optimal growth and exhibited the ability to utilize insoluble lanthanide oxides, which may enable survival in circumneutral environments. Genome and gene expression analyses revealed that strain Ce-a6 lost the ability to produce functional MxaF-MDH, and this may have been due to a large-scale deletion around the mxa gene cluster. The present results provide evidence for lanthanide dependency as a novel survival strategy by methanotrophs in circumneutral environments.


Assuntos
Genoma Bacteriano/genética , Elementos da Série dos Lantanídeos/metabolismo , Proteobactérias/genética , Proteobactérias/isolamento & purificação , Oxirredutases do Álcool/genética , Proteínas de Bactérias/genética , Meios de Cultura/metabolismo , Sedimentos Geológicos/microbiologia , Metais Terras Raras/metabolismo , Metano/metabolismo , Methylosinus/classificação , Methylosinus/genética , Methylosinus/isolamento & purificação , Methylosinus/metabolismo , Tanques/microbiologia , Proteobactérias/classificação , Proteobactérias/fisiologia , RNA Ribossômico 16S/genética
15.
Food Microbiol ; 87: 103393, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31948634

RESUMO

Four wild strains of Saccharomyces cerevisiae and the collection strain S. cerevisiae var. boulardii ATCC MYA-796 were used as test organisms to study the effect of some environmental conditions on the formation of biofilm by potentially probiotic yeasts. In a first step, the formation of biofilm was studied in four different media (YPD-Yeast Peptone Glucose; diluted YPD; 2% BP, a medium containing only bacteriological peptone; 2% GLC, a medium containing only glucose). Then, the dilution of YPD was combined with pH and temperature through a mixture design to assess the weight of the interaction of the variables; the experiments were done on S. boulardii and on S. cerevisiae strain 4. The dilution of nutrients generally determined an increased biofilm formation, whereas the effect of pH relied upon the strain. For S. cerevisiae strain 4, the highest level of sessile cells was found at pH 4-5, while S. boulardii experienced an enhanced biofilm formation at pH 6.0. Concerning temperature, the highest biofilm formation was found at 25-30 °C for both strains. The importance of this work lies in its extension of our knowledge of the effect of different environmental conditions on biofilm formation by potentially probiotic S. cerevisiae strains, as a better understanding of this trait could be an important screening tool into the selection of new multifunctional yeasts.


Assuntos
Biofilmes , Saccharomyces cerevisiae/fisiologia , Meios de Cultura/química , Meios de Cultura/metabolismo , Concentração de Íons de Hidrogênio , Probióticos/química , Saccharomyces cerevisiae/crescimento & desenvolvimento
16.
PLoS One ; 15(1): e0227816, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31935268

RESUMO

In the context of research for new cytotoxic compounds, obtaining bioactive molecules from renewable sources remain a big challenge. Microorganisms and more specifically Actinobacteria from original sources are well known for their biotechnological potential and are hotspots for the discovery of new bioactive compounds. The strain DP94 studied here had shown an interesting cytotoxic activity of its culture broth (HaCaT: IC50 = 8.0 ± 1.5 µg/mL; B16: IC50 = 4.6 ± 1.8 µg/mL), which could not been explained by the compounds isolated in a previous work. The increase of the cytotoxic activity of extracts was investigated, based on a Taguchi L9 orthogonal array design, after DP94 culture in TY medium using two different vessels (bioreactor or Erlenmeyer flasks). Various culture parameters such as temperature, pH and inoculum ratio (%) were studied. For experiments conducted in a bioreactor, stirring speed was included as an additional parameter. Significant differences in the cytotoxic activities of different extracts on B16 melanoma cancer cell lines, highlighted the influence of culture temperature on the production of cytotoxic compound(s) using a bioreactor. A culture in Erlenmeyer flasks was also performed and afforded an increase of the production of the active compounds. The best conditions for the highest cytotoxicity (IC50 on B16: 6 ± 0.5 µg/mL) and the highest yield (202.0 mg/L) were identified as: pH 6, temperature 37°C and 5% inoculum.


Assuntos
Sobrevivência Celular/efeitos dos fármacos , Meios de Cultura/toxicidade , Citotoxinas/toxicidade , Nocardia/metabolismo , Animais , Reatores Biológicos , Linhagem Celular , Meios de Cultura/química , Meios de Cultura/metabolismo , Citotoxinas/isolamento & purificação , Citotoxinas/metabolismo , Humanos , Microbiologia Industrial , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Camundongos , Nocardia/química , Nocardiose/microbiologia
17.
Talanta ; 209: 120574, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31892056

RESUMO

The paper outlines the first report of application of a differential pulse voltammetry for simultaneous quantification of clinically important molecular markers - tryptophan and its metabolite - kynurenine. The analytes were determined in less than 60 s at the boron-doped diamond electrode modified in situ with bismuth film (BiF/BDDE). Proper adjustment of a supporting electrolyte composition allowed to obtain good separation of tryptophan and kynurenine oxidation peaks that appeared at potential of 0.88 and 1.05 V (vs. Ag/AgCl), respectively. Studies using an optical profilometer have confirmed an increase in electrode surface area after deposition of Bi film. At the optimized conditions, the obtained detection limits of tryptophan and kynurenine were at 30 nM concentrations. The method was validated for linearity, precision, accuracy, selectivity and recovery. We have investigated an impact of numerous relevant interfering organic compounds (including amino acids and different tryptophan metabolites of kynurenine pathway) on voltammetric signals of the measured analytes. Finally, for proof-of-technology, the sensor was used for tryptophan and kynurenine quantification in culture medium collected from human cancer cell lines (breast MDA-MB-231 and ovary SK-OV-3). The target molecules were analyzed directly, without any sample preparation step. The sensor showed good accuracy in presence of the sample matrix components that was confirmed by high performance liquid chromatography measurements. Our work emphasizes the advantages of application of the herein proposed, easy to fabricate voltammetric sensor, instead of popular chromatographic assays or previously proposed potentiometric immunosensor. The method might serve for rapid assessment of kynurenine pathway activity in cancer cells.


Assuntos
Meios de Cultura/metabolismo , Cinurenina/metabolismo , Neoplasias/metabolismo , Triptofano/metabolismo , Técnicas Biossensoriais , Boro/química , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Meios de Cultura/química , Diamante/química , Técnicas Eletroquímicas , Eletrodos , Humanos , Cinurenina/análise , Neoplasias/química , Triptofano/análise
18.
Int J Mol Sci ; 21(2)2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31952279

RESUMO

To investigate environmental factors that contribute to ultraviolet A (UVA)-induced oxidative stress, which accelerates the senescence and toxicity of skin cells, we irradiated human fibroblasts cultured in commonly used essential media with UVA and evaluated their viability and production of reactive oxygen species. The viability of fibroblasts exposed to a single dose of 3.6 J/cm2 UVA was not reduced when cultured in Hanks balanced salt solution, but it was significantly decreased when cultured in Dulbecco's modified Eagle's medium (DMEM), which contains various amino acids and vitamins. Furthermore, cell viability was not reduced when fibroblasts were cultured in DMEM and treated with a hydrogen peroxide (H2O2) scavenger such as glutathione or catalase added after UVA irradiation. In addition, we confirmed that the production of H2O2 was dramatically increased by UVA photosensitization when riboflavin (R) coexisted with amino acids such as tryptophan (T), and found that R with folic acid (F) produced high levels of H2O2 after UVA irradiation. Furthermore, we noticed that R and F or R and T have different photosensitization mechanisms since NaN3, which is a singlet oxygen quencher, suppressed only R and T photosensitization. Lastly, we examined the effects of antioxidants (L-ascorbic acid, trolox, L-cysteine, and L-histidine), which are singlet oxygen or superoxide or H2O2 scavengers, on R and F or on R and T photosensitization, and found that 1 mM ascorbic acid, Trolox, and L-histidine were strongly photosensitized with R, and produced significant levels of H2O2 during UVA exposure. However, 1 mM L-cysteine dramatically suppressed H2O2 production by UVA photosensitization. These data suggest that a low concentration of R-derived photosensitization is elicited by different mechanisms depending on the coexisting vitamins and amino acids, and regulates cellular oxidative stress by producing H2O2 during UVA exposure.


Assuntos
Aminoácidos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Peróxido de Hidrogênio/metabolismo , Riboflavina/farmacologia , Raios Ultravioleta , Vitaminas/farmacologia , Aminoácidos/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Meios de Cultura/metabolismo , Meios de Cultura/farmacologia , Fibroblastos/citologia , Prepúcio do Pênis/citologia , Humanos , Recém-Nascido , Masculino , Oxirredução/efeitos dos fármacos , Oxirredução/efeitos da radiação , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Fármacos Fotossensibilizantes/metabolismo , Fármacos Fotossensibilizantes/farmacologia , Riboflavina/metabolismo , Vitaminas/metabolismo
19.
Appl Microbiol Biotechnol ; 104(5): 2097-2108, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31900554

RESUMO

The xylose oxidative pathway (XOP) is continuously gaining prominence as an alternative for the traditional pentose assimilative pathways in prokaryotes. It begins with the oxidation of D-xylose to D-xylonic acid, which is further converted to α-ketoglutarate or pyruvate + glycolaldehyde through a series of enzyme reactions. The persistent drawback of XOP is the accumulation of D-xylonic acid intermediate that causes culture media acidification. This study addresses this issue through the development of a novel pH-responsive synthetic genetic controller that uses a modified transmembrane transcription factor called CadCΔ. This genetic circuit was tested for its ability to detect extracellular pH and to control the buildup of D-xylonic acid in the culture media. Results showed that the pH-responsive genetic sensor confers dynamic regulation of D-xylonic acid accumulation, which adjusts with the perturbation of culture media pH. This is the first report demonstrating the use of a pH-responsive transmembrane transcription factor as a transducer in a synthetic genetic circuit that was designed for XOP. This may serve as a benchmark for the development of other genetic controllers for similar pathways that involve acidic intermediates.


Assuntos
Meios de Cultura/química , Escherichia coli/metabolismo , Xilose/análogos & derivados , Xilose/metabolismo , Meios de Cultura/metabolismo , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Oxirredução
20.
Appl Microbiol Biotechnol ; 104(5): 2007-2015, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31927760

RESUMO

Rhodovulum sulfidophilum DSM-1374 is a potential producer of polyester when growing in phototrophic conditions. The present study investigated on a polyester product (P3HB) by culturing Rhodovulum sulfidophilum DSM-1374 in two different photobioreactors (PBR-1 and PBR-2) both with 4-L working volumes. PBR-1 is equipped with an internal rotor having 4 paddles to mix the bacterial culture while PBR-2 has an internal coil-shaped rotor. After selecting PBR-1, which best performed in the preliminary experiment, the effect of different stressing growth conditions as pH (7.0, 8.0, and 9.0), temperature (25, 30, and 35 °C), and medium salinity (1.5, 2.5, 3.5, and 4.5%) were tested. When the pH of the culture was set to 8.0, the capability of the bacterium to synthetize the polyester increased significantly reaching a concentration of 412 mg (P3HB)/L; the increase of the pH at 9.0 caused a reduction of the P3HB concentration in the culture. The medium salinity of 4.5% was the best stress-growth condition to reach the highest concentration of polyester in the culture (820 ± 50 mg (P3HB)/L) with a P3HB mass fraction in the dry biomass of 33 ± 1.5%. Stresses caused by culture temperature are another potential parameter that could increase the synthesis of P3HB.


Assuntos
Meios de Cultura/química , Poliésteres/metabolismo , Rhodovulum/metabolismo , Biomassa , Meios de Cultura/metabolismo , Concentração de Íons de Hidrogênio , Rhodovulum/crescimento & desenvolvimento , Salinidade , Temperatura
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