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1.
PeerJ ; 12: e17282, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38666083

RESUMO

This study investigated the potential of using steam-exploded oil palm empty fruit bunches (EFB) as a renewable feedstock for producing fumaric acid (FA), a food additive widely used for flavor and preservation, through a separate hydrolysis and fermentation process using the fungal isolate K20. The efficiency of FA production by free and immobilized cells was compared. The maximum FA concentration (3.25 g/L), with 0.034 g/L/h productivity, was observed after incubation with the free cells for 96 h. Furthermore, the production was scaled up in a 3-L air-lift fermenter using oil palm EFB-derived glucose as the substrate. The FA concentration, yield, and productivity from 100 g/L initial oil palm EFB-derived glucose were 44 g/L, 0.39 g/g, and 0.41 g/L/h, respectively. The potential for scaling up the fermentation process indicates favorable results, which could have significant implications for industrial applications.


Assuntos
Células Imobilizadas , Fermentação , Fumaratos , Fumaratos/metabolismo , Células Imobilizadas/metabolismo , Óleo de Palmeira , Frutas/microbiologia , Frutas/química , Arecaceae/microbiologia , Arecaceae/química , Óleos de Plantas/metabolismo , Hidrólise , Glucose/metabolismo
2.
Appl Environ Microbiol ; 89(7): e0086823, 2023 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-37367298

RESUMO

Shewanella oneidensis MR-1 is a facultative anaerobe that grows by respiration using a variety of electron acceptors. This organism serves as a model to study how bacteria thrive in redox-stratified environments. A glucose-utilizing engineered derivative of MR-1 has been reported to be unable to grow in glucose minimal medium (GMM) in the absence of electron acceptors, despite this strain having a complete set of genes for reconstructing glucose to lactate fermentative pathways. To gain insights into why MR-1 is incapable of fermentative growth, this study examined a hypothesis that this strain is programmed to repress the expression of some carbon metabolic genes in the absence of electron acceptors. Comparative transcriptomic analyses of the MR-1 derivative were conducted in the presence and absence of fumarate as an electron acceptor, and these found that the expression of many genes involved in carbon metabolism required for cell growth, including several tricarboxylic acid (TCA) cycle genes, was significantly downregulated in the absence of fumarate. This finding suggests a possibility that MR-1 is unable to grow fermentatively on glucose in minimal media owing to the shortage of nutrients essential for cell growth, such as amino acids. This idea was demonstrated in subsequent experiments that showed that the MR-1 derivative fermentatively grows in GMM containing tryptone or a defined mixture of amino acids. We suggest that gene regulatory circuits in MR-1 are tuned to minimize energy consumption under electron acceptor-depleted conditions, and that this results in defective fermentative growth in minimal media. IMPORTANCE It is an enigma why S. oneidensis MR-1 is incapable of fermentative growth despite having complete sets of genes for reconstructing fermentative pathways. Understanding the molecular mechanisms behind this defect will facilitate the development of novel fermentation technologies for the production of value-added chemicals from biomass feedstocks, such as electro-fermentation. The information provided in this study will also improve our understanding of the ecological strategies of bacteria living in redox-stratified environments.


Assuntos
Aminoácidos , Shewanella , Fermentação , Aminoácidos/metabolismo , Shewanella/metabolismo , Glucose/metabolismo , Fumaratos/metabolismo , Suplementos Nutricionais
3.
Meat Sci ; 201: 109176, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37023594

RESUMO

Long-term feeding of high-concentrate (HC) diet causes the decrease of rumen pH, and induces subacute rumen acidosis (SARA), which results in metabolic disorders in sheep. This not only reduces animal performance, but also increases the risk of oxidative stress and inflammatory reaction. Disodium fumarate can improve the rumen buffering capacity and increase rumen pH. This experiment was conducted to investigate the effects of high concentrate diet on muscle quality, chemical composition, oxidative damage and lipid metabolism of Hu sheep, and the regulating effect of disodium fumarate. The results showed that HC diet induced SARA by reducing rumen pH value, thus causing oxidative stress and lipid metabolism disorder in longissimus lumborum (LL) muscle of Hu sheep, which also reduced meat quality by increasing shear force, drip loss, cooking loss, chewiness and hardness, and reducing the contents of crude fat and crude protein in LL muscle. However, disodium fumarate can improve meat quality of SARA Hu sheep by regulating rumen pH, inhibiting muscle oxidative stress and promoting lipid metabolism.


Assuntos
Fumaratos , Metabolismo dos Lipídeos , Ovinos , Animais , Fumaratos/análise , Fumaratos/metabolismo , Fumaratos/farmacologia , Dieta/veterinária , Rúmen/química , Músculos/metabolismo , Suplementos Nutricionais , Estresse Oxidativo , Ração Animal/análise , Concentração de Íons de Hidrogênio
4.
mBio ; 14(1): e0330222, 2023 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-36625597

RESUMO

Aminoglycosides (AG) have been used against Gram-negative bacteria for decades. Yet, how bacterial metabolism and environmental conditions modify AG toxicity is poorly understood. Here, we show that the level of AG susceptibility varies depending on the nature of the respiratory chain that Escherichia coli uses for growth, i.e., oxygen, nitrate, or fumarate. We show that all components of the fumarate respiratory chain, namely, hydrogenases 2 and 3, the formate hydrogenlyase complex, menaquinone, and fumarate reductase are required for AG-mediated killing under fumarate respiratory conditions. In addition, we show that the AAA+ ATPase RavA and its Von Wildebrand domain-containing partner, ViaA, are essential for AG to act under fumarate respiratory conditions. This effect was true for all AG that were tested but not for antibiotics from other classes. In addition, we show that the sensitizing effect of RavA-ViaA is due to increased gentamicin uptake in a proton motive force-dependent manner. Interestingly, the sensitizing effect of RavA-ViaA was prominent in poor energy conservation conditions, i.e., with fumarate, but dispensable under high energy conservation conditions, i.e., in the presence of nitrate or oxygen. We propose that RavA-ViaA can facilitate uptake of AG across the membrane in low-energy cellular states. IMPORTANCE Antibiotic resistance is a major public health, social, and economic problem. Aminoglycosides (AG) are known to be highly effective against Gram-negative bacteria, but their use is limited to life-threatening infections because of their nephrotoxicity and ototoxicity at therapeutic dose. Elucidation of AG-sensitization mechanisms in bacteria would allow reduced effective doses of AG. Here, we have identified the molecular components involved in anaerobic fumarate respiration that are required for AG to kill. In addition to oxidoreductases and menaquinone, this includes new molecular players, RavA, an AAA+ ATPase, and ViaA, its partner that has the VWA motif. Remarkably, the influence of RavA-ViaA on AG susceptibility varies according to the type of bioenergetic metabolism used by E. coli. This is a significant advance because anaerobiosis is well known to reduce the antibacterial activity of AG. This study highlights the critical importance of the relationship between culture conditions, metabolism, and antibiotic susceptibility.


Assuntos
Proteínas de Escherichia coli , Escherichia coli , Aminoglicosídeos/farmacologia , Nitratos/metabolismo , Vitamina K 2/metabolismo , Vitamina K 2/farmacologia , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Metabolismo Energético , Succinato Desidrogenase , Bactérias/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Fumaratos/farmacologia , Fumaratos/metabolismo , Anaerobiose , Adenosina Trifosfatases/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo
5.
Microbiol Spectr ; 10(5): e0235422, 2022 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-36106752

RESUMO

We recently described a new member of the CRP (cyclic AMP receptor protein)/FNR (fumarate and nitrate reductase regulatory protein) family called RedB, an acronym for redox brake, that functions to limit the production of ATP and NADH. This study shows that the RedB regulon significantly overlaps the FnrL regulon, with 199 genes being either directly or indirectly regulated by both of these global regulatory proteins. Among these 199 coregulated genes, 192 are divergently regulated, indicating that RedB functions as an antagonist of FnrL. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis indicates that RedB and Fnr directly coregulate only 4 out of 199 genes. The primary mechanism for the divergent regulation of target genes thus involves indirect regulation by both RedB and FnrL (156 cases). Additional regulation involves direct binding by RedB and indirect regulation by FnrL (36 cases) or direct binding by FnrL and indirect regulation by RedB (3 cases). Analysis of physiological pathways under direct and indirect control by these global regulators demonstrates that RedB functions primarily to limit energy production, while FnrL functions to enhance energy production. This regulation includes glycolysis, gluconeogenesis, photosynthesis, hydrogen oxidation, electron transport, carbon fixation, lipid biosynthesis, and protein synthesis. Finally, we show that 75% of genomes from diverse species that code for RedB proteins also harbor genes coding for FNR homologs. This cooccurrence indicates that RedB likely has an important role in buffering FNR-mediated energy production in a broad range of species. IMPORTANCE The CRP/FNR family of regulatory proteins constitutes a large collection of related transcription factors, several of which globally regulate cellular energy production. A well-characterized example is FNR (called FnrL in Rhodobacter capsulatus), which is responsible for regulating the expression of numerous genes that promote maximal energy production and growth under anaerobic conditions. In a companion article (N. Ke, J. E. Kumka, M. Fang, B. Weaver, et al., Microbiol Spectr 10:e02353-22, 2022, https://doi.org/10.1128/Spectrum02353-22), we identified a new subgroup of the CRP/FNR family and demonstrated that a member of this new subgroup, called RedB, has a role in limiting cellular energy production. In this study, we show that numerous genes encompassing the RedB regulon significantly overlap genes that are members of the FnrL regulon. Furthermore, 97% of the genes that are members of both the RedB and FnrL regulons are divergently regulated by these two transcription factors. RedB thus functions as a buffer limiting the amount of energy production that is promoted by FnrL.


Assuntos
Rhodobacter capsulatus , Rhodobacter sphaeroides , Trifosfato de Adenosina/metabolismo , Anaerobiose , Proteínas de Bactérias/metabolismo , Proteína Receptora de AMP Cíclico/metabolismo , Fumaratos/metabolismo , Regulação Bacteriana da Expressão Gênica , Hidrogênio/metabolismo , Lipídeos , NAD/genética , NAD/metabolismo , Oxirredução , Rhodobacter capsulatus/genética , Rhodobacter capsulatus/metabolismo , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
6.
Biotechnol Bioeng ; 119(9): 2471-2481, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35665482

RESUMO

Filamentous fungi occupy a uniquely favorable position in the bioproduction of organic acids. Intracellular stress is the main stimulator in filamentous fungi to produce and accumulate organic acids with high flux. However, stress can affect the physiological activities of filamentous fungi, thereby deteriorating their fermentation performance. Herein, we report that peptide supplementation during Rhizopus oryzae fermentation significantly improved fumaric acid production. Specifically, fumaric acid productivity was elevated by approximately 100%, fermentation duration was shortened from 72 to 36 h, while maintaining the final titer. Furthermore, transcriptome profile analysis and biochemical assays indicated that the overall capabilities of the stress defense systems (enzymatic and nonenzymatic) were significantly improved in R. oryzae. Consequently, glycolytic metabolism was distinctly enhanced, which eventually resulted in improved fumaric acid production and reduced fermentation duration. We expect our findings and efforts to provide essential insights into the optimization of the fermentation performance of filamentous fungi in industrial biotechnology and fermentation engineering.


Assuntos
Fumaratos , Rhizopus , Ácidos/metabolismo , Suplementos Nutricionais , Fermentação , Fumaratos/metabolismo , Fungos/metabolismo , Peptídeos/metabolismo
7.
BMC Microbiol ; 20(1): 369, 2020 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-33272193

RESUMO

BACKGROUND: Pasteurella multocida is responsible for a highly infectious and contagious disease in birds, leading to heavy economic losses in the chicken industry. However, the pathogenesis of this disease is poorly understood. We recently identified an aspartate ammonia-lyase (aspA) in P. multocida that was significantly upregulated under iron-restricted conditions, the protein of which could effectively protect chicken flocks against P. multocida. However, the functions of this gene remain unclear. In the present study, we constructed aspA mutant strain △aspA::kan and complementary strain C△aspA::kan to investigate the function of aspA in detail. RESULT: Deletion of the aspA gene in P. multocida resulted in a significant reduction in bacterial growth in LB (Luria-Bertani) and MH (Mueller-Hinton) media, which was rescued by supplementation with 20 mM fumarate. The mutant strain △aspA::kan showed significantly growth defects in anaerobic conditions and acid medium, compared with the wild-type strain. Moreover, growth of △aspA::kan was more seriously impaired than that of the wild-type strain under iron-restricted conditions, and this growth recovered after supplementation with iron ions. AspA transcription was negatively regulated by iron conditions, as demonstrated by quantitative reverse transcription-polymerase chain reaction. Although competitive index assay showed the wild-type strain outcompetes the aspA mutant strain and △aspA::kan was significantly more efficient at producing biofilms than the wild-type strain, there was no significant difference in virulence between the mutant and the wild-type strains. CONCLUSION: These results demonstrate that aspA is required for bacterial growth in complex medium, and under anaerobic, acid, and iron-limited conditions.


Assuntos
Aspartato Amônia-Liase/metabolismo , Proteínas de Bactérias/metabolismo , Pasteurella multocida/enzimologia , Ácidos/metabolismo , Anaerobiose , Animais , Aspartato Amônia-Liase/genética , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Galinhas , Fumaratos/metabolismo , Ferro/metabolismo , Mutação , Infecções por Pasteurella/microbiologia , Infecções por Pasteurella/veterinária , Pasteurella multocida/crescimento & desenvolvimento
8.
J Dairy Sci ; 103(12): 11449-11460, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33222857

RESUMO

Our objective was to determine the temporal effects of increasing supply of propionate on propionate metabolism in liver tissue of dairy cows in the postpartum (PP) period. A total of 6 dairy cows [primiparous: n = 3, 9.00 ± 1.00 d PP (mean ± SD) and multiparous: n = 3; 4.67 ± 1.15 d PP] were biopsied for liver explants in a block-design experiment. Explants were treated with 3 concentrations of [13C3]sodium propionate of 1, 2, or 4 mM. Explants were incubated in 2 mL of Medium 199 supplemented with 1% BSA, 0.6 mM oleic acid, 2 mM sodium l-lactate, 0.2 mM sodium pyruvate, and 0.5 mMl-glutamine at 38°C and sampled at 0.5, 15, and 60 min. Increasing the concentration of [13C3]propionate increased total 13C% enrichment of propionyl coenzyme A (CoA), succinate, fumarate, malate, and citrate with time. Concentration of propionate did not affect total 13C% enrichment of hepatic glucose or acetyl CoA, but total 13C% enrichment increased with time for hepatic glucose. The 13C labeling from propionate was incorporated into acetyl CoA, but increased concentrations of propionate did not result in greater labeling of acetyl CoA. However, increases in 13C% enrichment of [M+4]citrate and [M+5]citrate concentrations of [13C3]propionate indicate propionate conversion to acetyl CoA and subsequent entry of acetyl CoA into the tricarboxylic acid cycle in dairy cows in the PP period. This research presents evidence that despite an increase in hepatic acetyl CoA concentration and general consensus on the upregulation of gluconeogenesis of dairy cows during the PP period, carbon derived from propionate contributes to the pool of acetyl CoA, which increases as concentration of propionate increases, in addition to stimulating oxidation of acetyl CoA from other sources. Because of the hypophagic effects of propionate, but importance of propionate as a glucose precursor, a balance of propionate supply to dairy cows could lead to improvements in dry matter intake, and subsequently, health and production in dairy cows.


Assuntos
Bovinos/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Período Pós-Parto/metabolismo , Propionatos/administração & dosagem , Acetilcoenzima A/metabolismo , Animais , Ácido Cítrico/metabolismo , Ciclo do Ácido Cítrico , Suplementos Nutricionais , Relação Dose-Resposta a Droga , Feminino , Fumaratos/metabolismo , Gluconeogênese , Glucose/metabolismo , Lactação/fisiologia , Malatos/metabolismo , Propionatos/metabolismo
9.
Plant J ; 103(6): 2193-2210, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32579242

RESUMO

Potato (Solanum tuberosum L.) is one of the world's most important crops, but it is facing major challenges due to climatic changes. To investigate the effects of intermittent drought on the natural variability of plant morphology and tuber metabolism in a novel potato association panel comprising 258 varieties we performed an augmented block design field study under normal irrigation and under water-deficit and recovery conditions in Ica, Peru. All potato genotypes were profiled for 45 morphological traits and 42 central metabolites via nuclear magnetic resonance. Statistical tests and norm of reaction analysis revealed that the observed variations were trait specific, that is, genotypic versus environmental. Principal component analysis showed a separation of samples as a result of conditional changes. To explore the relational ties between morphological traits and metabolites, correlation-based network analysis was employed, constructing one network for normal irrigation and one network for water-recovery samples. Community detection and difference network analysis highlighted the differences between the two networks, revealing a significant correlational link between fumarate and plant vigor. A genome-wide association study was performed for each metabolic trait. Eleven single nucleotide polymorphism (SNP) markers were associated with fumarate. Gene Ontology analysis of quantitative trait loci regions associated with fumarate revealed an enrichment of genes regulating metabolic processes. Three of the 11 SNPs were located within genes, coding for a protein of unknown function, a RING domain protein and a zinc finger protein ZAT2. Our findings have important implications for future potato breeding regimes, especially in countries suffering from climate change.


Assuntos
Característica Quantitativa Herdável , Solanum tuberosum/metabolismo , Aminoácidos/metabolismo , Desidratação , Fumaratos/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Estudo de Associação Genômica Ampla , Espectroscopia de Ressonância Magnética , Filogenia , Polimorfismo de Nucleotídeo Único/genética , Locos de Características Quantitativas/genética , Solanum tuberosum/anatomia & histologia , Solanum tuberosum/genética , Solanum tuberosum/fisiologia , Clima Tropical , Água/metabolismo
10.
Microbiology (Reading) ; 166(2): 199-211, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31774392

RESUMO

Purple non-sulfur bacteria (PNSB) use light for energy and organic substrates for carbon and electrons when growing photoheterotrophically. This lifestyle generates more reduced electron carriers than are required for biosynthesis, even during consumption of some of the most oxidized organic substrates like malate and fumarate. Reduced electron carriers not used in biosynthesis must still be oxidized for photoheterotrophic growth to occur. Diverse PNSB commonly rely on the CO2-fixing Calvin cycle to oxidize reduced electron carriers. Some PNSB also produce H2 or reduce terminal electron acceptors as alternatives to the Calvin cycle. Rhodospirillum rubrum Calvin-cycle mutants defy this trend by growing phototrophically on malate or fumarate without H2 production or access to terminal electron acceptors. We used 13C-tracer experiments to examine how a Rs. rubrum Calvin-cycle mutant maintains electron balance under such conditions. We detected the reversal of some tricarboxylic acid cycle enzymes, carrying reductive flux from malate or fumarate to αKG. This pathway and the reductive synthesis of αKG-derived amino acids are likely important for electron balance, as supplementing the growth medium with αKG-derived amino acids prevented Rs. rubrum Calvin-cycle-mutant growth unless a terminal electron acceptor was provided. Flux estimates also suggested that the Calvin-cycle mutant preferentially synthesized isoleucine using the reductive threonine-dependent pathway instead of the less-reductive citramalate-dependent pathway. Collectively, our results suggest that alternative biosynthetic pathways can contribute to electron balance within the constraints of a relatively constant biomass composition.


Assuntos
Aminoácidos/biossíntese , Ciclo do Ácido Cítrico/fisiologia , Elétrons , Fotossíntese/genética , Rhodospirillum rubrum/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biomassa , Vias Biossintéticas , Fumaratos/metabolismo , Isoleucina/biossíntese , Ácidos Cetoglutáricos/metabolismo , Malatos/metabolismo , Mutação , Oxirredução , Rhodospirillum rubrum/genética , Rhodospirillum rubrum/crescimento & desenvolvimento
11.
Artigo em Inglês | MEDLINE | ID: mdl-31332064

RESUMO

In the last decade, carbon monoxide-releasing molecules (CORMs) have been shown to act against several pathogens and to be promising antimicrobials. However, the understanding of the mode of action and reactivity of these compounds on bacterial cells is still deficient. In this work, we used a metabolomics approach to probe the toxicity of the ruthenium(II) complex Ru(CO)3Cl(glycinate) (CORM-3) on Escherichia coli By resorting to 1H nuclear magnetic resonance, mass spectrometry, and enzymatic activities, we show that CORM-3-treated E. coli accumulates larger amounts of glycolytic intermediates, independently of the oxygen growth conditions. The work provides several evidences that CORM-3 inhibits glutamate synthesis and the iron-sulfur enzymes of the tricarboxylic acid (TCA) cycle and that the glycolysis pathway is triggered in order to establish an energy and redox homeostasis balance. Accordingly, supplementation of the growth medium with fumarate, α-ketoglutarate, glutamate, and amino acids cancels the toxicity of CORM-3. Importantly, inhibition of the iron-sulfur enzymes glutamate synthase, aconitase, and fumarase is only observed for compounds that liberate carbon monoxide. Altogether, this work reveals that the antimicrobial action of CORM-3 results from intracellular glutamate deficiency and inhibition of nitrogen and TCA cycles.


Assuntos
Antibacterianos/farmacologia , Monóxido de Carbono/farmacologia , Ciclo do Ácido Cítrico/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica , Nitrogênio/metabolismo , Compostos Organometálicos/farmacologia , Aconitato Hidratase/antagonistas & inibidores , Aconitato Hidratase/genética , Aconitato Hidratase/metabolismo , Antibacterianos/química , Monóxido de Carbono/química , Ciclo do Ácido Cítrico/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Fumarato Hidratase/antagonistas & inibidores , Fumarato Hidratase/genética , Fumarato Hidratase/metabolismo , Fumaratos/metabolismo , Glutamato Sintase/antagonistas & inibidores , Glutamato Sintase/genética , Glutamato Sintase/metabolismo , Ácido Glutâmico/metabolismo , Glicólise/efeitos dos fármacos , Glicólise/genética , Ácidos Cetoglutáricos/metabolismo , Espectroscopia de Ressonância Magnética , Metabolômica/métodos , Compostos Organometálicos/química , Oxirredução
12.
Appl Environ Microbiol ; 85(16)2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31175188

RESUMO

Shewanella oneidensis strain MR-1, a facultative anaerobe and model organism for dissimilatory metal reduction, uses a periplasmic flavocytochrome, FccA, both as a terminal fumarate reductase and as a periplasmic electron transfer hub for extracellular respiration of a variety of substrates. It is currently unclear how maturation of FccA and other periplasmic flavoproteins is achieved, specifically in the context of flavin cofactor loading, and the fitness cost of flavin secretion has not been quantified. We demonstrate that deletion of the inner membrane flavin adenine dinucleotide (FAD) exporter Bfe results in a 23% slower growth rate than that of the wild type during fumarate respiration and an 80 to 90% loss in fumarate reductase activity. Exogenous flavin supplementation does not restore FccA activity in a Δbfe mutant unless the gene encoding the periplasmic FAD hydrolase UshA is also deleted. We demonstrate that the small Bfe-independent pool of FccA is sufficient for anaerobic growth with fumarate. Strains lacking Bfe were unable to grow using urocanate as the sole electron acceptor, which relies on the periplasmic flavoprotein UrdA. We show that periplasmic flavoprotein maturation occurs in careful balance with periplasmic FAD hydrolysis, and that the current model for periplasmic flavin cofactor loading must account for a Bfe-independent mechanism for flavin transport. Finally, we determine that the metabolic burden of flavin secretion is not significant during growth with flavin-independent anaerobic electron acceptors. Our work helps frame the physiological motivations that drove evolution of flavin secretion by ShewanellaIMPORTANCEShewanella species are prevalent in marine and aquatic environments, throughout stratified water columns, in mineral-rich sediments, and in association with multicellular marine and aquatic organisms. The diversity of niches shewanellae can occupy are due largely to their respiratory versatility. Shewanella oneidensis is a model organism for dissimilatory metal reduction and can respire a diverse array of organic and inorganic compounds, including dissolved and solid metal oxides. The fumarate reductase FccA is a highly abundant multifunctional periplasmic protein that acts to bridge the periplasm and temporarily store electrons in a variety of respiratory nodes, including metal, nitrate, and dimethyl sulfoxide respiration. However, maturation of this central protein, particularly flavin cofactor acquisition, is poorly understood. Here, we quantify the fitness cost of flavin secretion and describe how free flavins are acquired by FccA and a homologous periplasmic flavoprotein, UrdA.


Assuntos
Flavinas/metabolismo , Fumaratos/metabolismo , Shewanella/metabolismo , Anaerobiose , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte de Elétrons , Flavina-Adenina Dinucleotídeo/metabolismo , Periplasma , Shewanella/genética , Shewanella/crescimento & desenvolvimento , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismo
13.
Plant Physiol Biochem ; 136: 188-195, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30685698

RESUMO

This work examines the effect of a treatment with 1 mM of γ-aminobutyric acid (GABA) on zucchini fruit during postharvest cold storage. Specifically, the effect of GABA on postharvest quality was measured, as well as its implication in the GABA shunt and other related metabolic pathways. The treatments were performed in Sinatra, a variety of zucchini highly sensitive to low-temperature storage. The application of GABA improved the quality of zucchini fruit stored at 4 °C, with a reduction of chilling-injury index, weight loss, and cell death, as well as a lower rate of electrolyte leakage. GABA content was significantly higher in the treated fruit than in the control fruit at all times analyzed. At the end of the storage period, GABA-treated fruit had higher contents of both proline and putrescine. The catabolism of this polyamine was not affected by exogenous GABA. Also, over the long term, the treatment induced the GABA shunt by increasing the activities of the enzymes GABA transaminase (GABA-T) and glutamate decarboxylase (GAD). GABA-treated fruit contained higher levels of fumarate and malate than did non-treated fruit, as well as higher ATP and NADH contents. These results imply that the GABA shunt is involved in providing metabolites to produce energy, reduce power, and help the fruit to cope with cold stress over the long term.


Assuntos
Cucurbita/efeitos dos fármacos , Armazenamento de Alimentos , Frutas/efeitos dos fármacos , 4-Aminobutirato Transaminase/metabolismo , Trifosfato de Adenosina/metabolismo , Alanina/metabolismo , Amina Oxidase (contendo Cobre)/metabolismo , Morte Celular/efeitos dos fármacos , Temperatura Baixa , Cucurbita/metabolismo , Armazenamento de Alimentos/métodos , Frutas/metabolismo , Fumaratos/metabolismo , Glutamato Descarboxilase/metabolismo , Ácido Glutâmico/metabolismo , Malatos/metabolismo , NAD/metabolismo , Prolina/metabolismo , Putrescina/metabolismo , Ácido gama-Aminobutírico/farmacologia
14.
Appl Biochem Biotechnol ; 187(1): 163-175, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29911265

RESUMO

Accumulation of succinate as a fermentation product of Fusobacterium varium was enhanced when the anaerobic bacterium was grown on complex peptone medium supplemented with fumarate. Residual substrates and fermentation products were determined by proton NMR spectroscopy. Cells collected from the fumarate-supplemented medium (8-10 h after inoculation) supported the conversion of fumarate to succinate when suspended with fumarate and a co-substrate (glucose, sorbitol, or glycerol). Succinate production was limited by the availability of fumarate or reducing equivalents supplied by catabolism of a co-substrate via the Embden-Meyerhof-Parnas (EMP) pathway. The choice of reducing co-substrate influenced the yield of acetate and lactate as side products. High conversions of fumarate to succinate were achieved over pH 6.6-8.2 and initial fumarate concentrations up to 300 mM. However, at high substrate concentrations, intracellular retention of succinate reduced extracellular yields. Overall, the efficient utilization of fumarate (≤ 400 mM) combined with the significant extracellular accumulation of succinate (corresponding to ≥ 70% conversion) indicated the effective utilization of fumarate as a terminal electron acceptor by F. varium and the potential of the methodology for the bioproduction of succinate.


Assuntos
Fumaratos/metabolismo , Fusobacterium/metabolismo , Ácido Succínico/metabolismo , Oxirredução
15.
Environ Sci Pollut Res Int ; 25(36): 35960-35970, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29654455

RESUMO

The microbial production of fumaric acid by Rhizopus arrhizus NRRL 2582 has been evaluated using soybean cake from biodiesel production processes and very high polarity (VHP) sugar from sugarcane mills. Soybean cake was converted into a nutrient-rich hydrolysate via a two-stage bioprocess involving crude enzyme production via solid state fermentations (SSF) of either Aspergillus oryzae or R. arrhizus cultivated on soybean cake followed by enzymatic hydrolysis of soybean cake. The soybean cake hydrolysate produced using crude enzymes derived via SSF of R. arrhizus was supplemented with VHP sugar and evaluated using different initial free amino nitrogen (FAN) concentrations (100, 200, and 400 mg/L) in fed-batch cultures for fumaric acid production. The highest fumaric acid concentration (27.3 g/L) and yield (0.7 g/g of total consumed sugars) were achieved when the initial FAN concentration was 200 mg/L. The combination of VHP sugar with soybean cake hydrolysate derived from crude enzymes produced by SSF of A. oryzae at 200 mg/L initial FAN concentration led to the production of 40 g/L fumaric acid with a yield of 0.86 g/g of total consumed sugars. The utilization of sugarcane molasses led to low fumaric acid production by R. arrhizus, probably due to the presence of various minerals and phenolic compounds. The promising results achieved through the valorization of VHP sugar and soybean cake suggest that a focused study on molasses pretreatment could lead to enhanced fumaric acid production.


Assuntos
Aspergillus oryzae/enzimologia , Biocombustíveis , Fumaratos , Glycine max , Resíduos Industriais , Saccharum , Açúcares/metabolismo , Técnicas de Cultura Celular por Lotes , Indústria Química , Conservação dos Recursos Naturais , Fermentação , Indústria Alimentícia , Fumaratos/metabolismo , Hidrólise , Rhizopus/enzimologia
16.
Free Radic Biol Med ; 116: 1-10, 2018 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-29289705

RESUMO

Near-infrared photoimmunotherapy (NIR PIT) employs the photoabsorbing dye IR700 conjugated to antibodies specific for cell surface epidermal growth factor receptor (EGFR). NIR PIT has shown highly selective cytotoxicity in vitro and in vivo. Cell necrosis is thought to be the main mode of cytotoxicity based mainly on in vitro studies. To better understand the acute effects of NIR PIT, molecular imaging studies were performed to assess its cellular and vascular effects. In addition to in vitro studies for cytotoxicity of NIR PIT, the in vivo tumoricidal effects and hemodynamic changes induced by NIR PIT were evaluated by 13C MRI using hyperpolarized [1,4-13C2] fumarate, R2* mapping from T2*-weighted MRI, and photoacoustic imaging. In vitro studies confirmed that NIR PIT resulted in rapid cell death via membrane damage, with evidence for rapid cell expansion followed by membrane rupture. Following NIR PIT, metabolic MRI using hyperpolarized fumarate showed the production of malate in EGFR-expressing A431 tumor xenografts, providing direct evidence for photosensitized tumor necrosis induced by NIR PIT. R2* mapping studies showed temporal changes in oxygenation, with an accompanying increase of deoxyhemoglobin at the start of light exposure followed by a sustained decrease after cessation of light exposure. This result suggests a rapid decrease of blood flow in EGFR-expressing A431 tumor xenografts, which is supported by the results of the photoacoustic imaging experiments. Our findings suggest NIR PIT mediates necrosis and hemodynamic changes in tumors by photosensitized oxidation pathways and that these imaging modalities, once translated, may be useful in monitoring clinical treatment response.


Assuntos
Imunoterapia/métodos , Panitumumabe/uso terapêutico , Fármacos Fotossensibilizantes/uso terapêutico , Fototerapia/métodos , Neoplasias Cutâneas/terapia , Animais , Linhagem Celular Tumoral , Receptores ErbB/imunologia , Feminino , Fumaratos/metabolismo , Hemodinâmica/efeitos dos fármacos , Humanos , Raios Infravermelhos , Imageamento por Ressonância Magnética , Camundongos , Camundongos Nus , Necrose , Oxirredução , Técnicas Fotoacústicas , Neoplasias Cutâneas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Br J Nutr ; 118(11): 959-970, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29173237

RESUMO

n-3 PUFA are lipids that play crucial roles in immune-regulation, cardio-protection and neurodevelopment. However, little is known about the role that these essential dietary fats play in modulating caecal microbiota composition and the subsequent production of functional metabolites. To investigate this, female C57BL/6 mice were assigned to one of three diets (control (CON), n-3 supplemented (n3+) or n-3 deficient (n3-)) during gestation, following which their male offspring were continued on the same diets for 12 weeks. Caecal content of mothers and offspring were collected for 16S sequencing and metabolic phenotyping. n3- male offspring displayed significantly less % fat mass than n3+ and CON. n-3 Status also induced a number of changes to gut microbiota composition such that n3- offspring had greater abundance of Tenericutes, Anaeroplasma and Coriobacteriaceae. Metabolomics analysis revealed an increase in caecal metabolites involved in energy metabolism in n3+ including α-ketoglutaric acid, malic acid and fumaric acid. n3- animals displayed significantly reduced acetate, butyrate and total caecal SCFA production. These results demonstrate that dietary n-3 PUFA regulate gut microbiota homoeostasis whereby n-3 deficiency may induce a state of disturbance. Further studies are warranted to examine whether these microbial and metabolic disturbances are causally related to changes in metabolic health outcomes.


Assuntos
Fenômenos Fisiológicos da Nutrição Animal , Ceco/microbiologia , Ácidos Graxos Ômega-3/deficiência , Microbioma Gastrointestinal , Animais , Composição Corporal , DNA Bacteriano/isolamento & purificação , Dieta , Suplementos Nutricionais , Ácidos Graxos/metabolismo , Ácidos Graxos Ômega-3/sangue , Feminino , Fumaratos/metabolismo , Ácidos Cetoglutáricos/metabolismo , Malatos/metabolismo , Masculino , Metaboloma , Metabolômica , Camundongos , Camundongos Endogâmicos C57BL , RNA Ribossômico 16S/isolamento & purificação , Análise de Sequência de DNA
18.
Toxicol Appl Pharmacol ; 316: 83-94, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28041784

RESUMO

The lichen-forming fungi Cetraria islandica has been largely used in folk medicines, and it has recently showed promising in vitro antioxidant effects in glial-like cells. Current work aimed at investigating the neuroprotective potential of its major isolated secondary metabolite: the depsidone fumarprotocetraric acid (FUM). H2O2 was used herein to induce oxidative stress (OS)-mediated cytotoxicity in two models of neurons and astrocytes cells (SH-SY5Y and U373-MG cell lines). We found that a pre-treatment with FUM significantly enhanced cell viability compared to H2O2-treated cells, and we selected the optimal concentrations in each model (1 and 25µg/ml, respectively) for assessing its cytoprotective mechanisms. FUM, which exerted effective peroxyl radical scavenging effect in the chemical oxygen radical antioxidant capacity (ORAC) assay, alleviated the alterations in OS markers provoked by H2O2. It attenuated intracellular ROS formation, lipid peroxidation and GSH depletion. At mitochondrial level, FUM prevented from the dissipation of mitochondrial membrane potential and the increase in mitochondrial calcium, implying a protective role against oxidative damage in mitochondrial membrane. Similarly, FUM pre-treatment diminished H2O2-induced apoptosis, as evidenced by the reduction in caspase-3 activity and expression; inmunoblot analysis also revealed a decrease in Bax and an increase in Bcl-2 proteins levels. Furthermore, FUM up-regulated the expression of the antioxidant enzymes catalase, superoxide dismutase-1, and hemeoxigenase-1. These findings and the activation of Nrf2 binding activity in nuclear extracts suggest a plausible involvement of Nrf2 signaling pathway in the cytoprotection by FUM. In conclusion, FUM emerges as a potential drug candidate in the therapy of OS-related diseases, such as the neurodegenerative disorders.


Assuntos
Fumaratos/farmacologia , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/metabolismo , Fármacos Neuroprotetores/farmacologia , Parmeliaceae , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Fumaratos/isolamento & purificação , Fumaratos/metabolismo , Humanos , Peróxido de Hidrogênio/toxicidade , Líquens , Fármacos Neuroprotetores/isolamento & purificação , Fármacos Neuroprotetores/metabolismo , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo
19.
Bioresour Technol ; 222: 24-32, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27697734

RESUMO

In order to relieve the toxicity of furfural on Rhizopus oryzae fermentation, the molecular mechanism of R. oryzae responding to furfural stress for fumaric acid-production was investigated by omics-based approaches. In metabolomics analysis, 29 metabolites including amino acid, sugars, polyols and fatty acids showed significant changes for maintaining the basic cell metabolism at the cost of lowering fumaric acid production. To further uncover the survival mechanism, lipidomics was carried out, revealing that phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol and polyunsaturated acyl chains might be closely correlated with R. oryzae's adapting to furfural stress. Based on the above omics analysis, lecithin, inositol and soybean oil were exogenously supplemented separately with an optimized concentration in the presence of furfural, which increased fumaric acid titer from 5.78g/L to 10.03g/L, 10.05g/L and 12.13g/L (increased by 73.5%, 73.8% and 110%, respectively). These findings provide a methodological guidance for hemicellulose-fumaric acid development.


Assuntos
Fumaratos/metabolismo , Furaldeído/toxicidade , Metabolômica/métodos , Fosfolipídeos/metabolismo , Rhizopus/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Xilose/metabolismo , Análise Discriminante , Fermentação/efeitos dos fármacos , Furaldeído/metabolismo , Espaço Intracelular/metabolismo , Análise dos Mínimos Quadrados , Metaboloma/efeitos dos fármacos , Fatores de Tempo
20.
Cell Physiol Biochem ; 39(4): 1307-16, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27606686

RESUMO

BACKGROUND/AIMS: Motility is a feature of many pathogens that contributes to the migration and dispersion of the infectious agent. Whether gentamycin has a post-antibiotic effect (PAE) on the swarming and swimming motility of Escherichia coli (E. coli) remains unknown. In this study, we aimed to examine whether short-term pretreatment of sub-inhibitory concentrations of gentamycin alter motility of E. coli and the mechanisms involved therein. METHODS: After exposure to sub-inhibitory concentrations (0.8 µg/ml) of gentamicin, the swarming and swimming motility of E. coli was tested in semi-solid media. Real-time PCR was used to detect the gene expression of succinate dehydrogenase (SDH). The production of SDH and fumarate by E. coli pretreated with or without gentamycin was measured. Fumarate was added to swarming agar to determine whether fumarate could restore the swarming motility of E. coli. RESULTS: After pretreatment of E. coli with sub-inhibitory concentrations of gentamycin, swarming motility was repressed in the absence of growth inhibition. The expression of all four subunits of SDH was down-regulated, and the intracellular concentration of SDH and fumarate, produced by E. coli, were both decreased. Supplementary fumarate could restore the swarming motility inhibited by gentamycin. A selective inhibitor of SDH (propanedioic acid) could strongly repress the swarming motility. CONCLUSION: Sub-inhibitory concentrations of gentamycin inhibits the swarming motility of E. coli. This effect is mediated by a reduction in cellular fumarate caused by down-regulation of SDH. Gentamycin may be advantageous for treatment of E. coli infections.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Escherichia coli/efeitos dos fármacos , Flavoproteínas/antagonistas & inibidores , Regulação Bacteriana da Expressão Gênica , Gentamicinas/farmacologia , Subunidades Proteicas/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Flavoproteínas/genética , Flavoproteínas/metabolismo , Fumaratos/metabolismo , Malonatos/farmacologia , Testes de Sensibilidade Microbiana , Movimento/efeitos dos fármacos , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transdução de Sinais , Ácido Succínico/metabolismo , Fatores de Tempo
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