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1.
Nat Commun ; 12(1): 2116, 2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33837188

RESUMO

Nicotinamide adenine dinucleotide (NAD) and its reduced form are indispensable cofactors in life. Diverse NAD mimics have been developed for applications in chemical and biological sciences. Nicotinamide cytosine dinucleotide (NCD) has emerged as a non-natural cofactor to mediate redox transformations, while cells are fed with chemically synthesized NCD. Here, we create NCD synthetase (NcdS) by reprograming the substrate binding pockets of nicotinic acid mononucleotide (NaMN) adenylyltransferase to favor cytidine triphosphate and nicotinamide mononucleotide over their regular substrates ATP and NaMN, respectively. Overexpression of NcdS alone in the model host Escherichia coli facilitated intracellular production of NCD, and higher NCD levels up to 5.0 mM were achieved upon further pathway regulation. Finally, the non-natural cofactor self-sufficiency was confirmed by mediating an NCD-linked metabolic circuit to convert L-malate into D-lactate. NcdS together with NCD-linked enzymes offer unique tools and opportunities for intriguing studies in chemical biology and synthetic biology.


Assuntos
Coenzimas/biossíntese , Proteínas de Escherichia coli/genética , Niacinamida/biossíntese , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Engenharia de Proteínas , Coenzimas/química , Citidina Trifosfato/metabolismo , Ensaios Enzimáticos , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ensaios de Triagem em Larga Escala , Ácido Láctico/metabolismo , Malatos/metabolismo , Niacinamida/química , Mononucleotídeo de Nicotinamida/metabolismo , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Oxirredução , Especificidade por Substrato/genética , Biologia Sintética/métodos
2.
Int J Mol Sci ; 22(9)2021 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-33924853

RESUMO

Low phosphorus (P) availability is one of the major constraints to plant growth, particularly in acidic soils. A possible mechanism for enhancing the use of sparsely soluble P forms is the secretion of malate in plants by the aluminum-activated malate transporter (ALMT) gene family. Despite its significance in plant biology, the identification of the ALMT gene family in oilseed rape (Brassica napus; B. napus), an allotetraploid crop, is unveiled. Herein, we performed genome-wide identification and characterization of ALMTs in B. napus, determined their gene expression in different tissues and monitored transcriptional regulation of BnaALMTs in the roots and leaves at both a sufficient and a deficient P supply. Thirty-nine BnaALMT genes were identified and were clustered into five branches in the phylogenetic tree based on protein sequences. Collinearity analysis revealed that most of the BnaALMT genes shared syntenic relationships among BnaALMT members in B. napus, which suggested that whole-genome duplication (polyploidy) played a major driving force for BnaALMTs evolution in addition to segmental duplication. RNA-seq analyses showed that most BnaALMT genes were preferentially expressed in root and leaf tissues. Among them, the expression of BnaC08g13520D, BnaC08g15170D, BnaC08g15180D, BnaC08g13490D, BnaC08g13500D, BnaA08g26960D, BnaC05g14120D, BnaA06g12560D, BnaC05g20630D, BnaA07g02630D, BnaA04g15700D were significantly up-regulated in B. napus roots and leaf at a P deficient supply. The current study analyzes the evolution and the expression of the ALMT family in B. napus, which will help in further research on their role in the enhancement of soil P availability by secretion of organic acids.


Assuntos
Alumínio/metabolismo , Brassica napus/genética , Evolução Molecular , Malatos/metabolismo , Transportadores de Ânions Orgânicos/genética , Fosfatos/metabolismo , Proteínas de Arabidopsis/genética , Cromossomos de Plantas , Genoma de Planta , Família Multigênica , Transportadores de Ânions Orgânicos/metabolismo
3.
Int J Food Microbiol ; 344: 109115, 2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33662901

RESUMO

Development of bloater defect in cucumber fermentations is the result of carbon dioxide (CO2) production by the indigenous microbiota. The amounts of CO2 needed to cause bloater defect in cucumber fermentations brined with low salt and potential microbial contributors of the gas were identified. The carbonation of acidified cucumbers showed that 28.68 ± 6.04 mM (12%) or higher dissolved CO2 induces bloater defect. The microbiome and biochemistry of cucumber fermentations (n = 9) brined with 25 mM calcium chloride (CaCl2) and 345 mM sodium chloride (NaCl) or 1.06 M NaCl were monitored on day 0, 2, 3, 5, 8, 15 and 21 using culture dependent and independent microbiological techniques and High-Performance Liquid Chromatography. Changes in pH, CO2 concentrations and the incidence of bloater defect were also followed. The enumeration of Enterobacteriaceae on Violet Red Bile Glucose agar plates detected a cell density of 5.2 ± 0.7 log CFU/g on day 2, which declined to undetectable levels by day 8. A metagenomic analysis identified Leuconostocaceae in all fermentations at 10 to 62%. The presence of both bacterial families in fermentations brined with CaCl2 and NaCl coincided with a bloater index of 24.0 ± 10.3 to 58.8 ± 23.9. The prevalence of Lactobacillaceae in a cucumber fermentation brined with NaCl with a bloater index of 41.7 on day 5 suggests a contribution to bloater defect. This study identifies the utilization of sugars and malic acid by the cucumber indigenous Lactobacillaceae, Leuconostocaceae and Enterobacteriaceae as potential contributors to CO2 production during cucumber fermentation and the consequent bloater defect.


Assuntos
Dióxido de Carbono/análise , Cucumis sativus/microbiologia , Enterobacteriaceae/metabolismo , Lactobacillaceae/metabolismo , Leuconostocaceae/metabolismo , Cloreto de Cálcio , Fermentação , Concentração de Íons de Hidrogênio , Malatos/metabolismo , Microbiota/fisiologia , Sais , Cloreto de Sódio/análise
4.
Int J Mol Sci ; 22(5)2021 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-33668723

RESUMO

The biotechnological production of dicarboxylic acids (C4) from renewable carbon sources represents an attractive approach for the provision of these valuable compounds by green chemistry means. Glycerol has become a waste product of the biodiesel industry that serves as a highly reduced carbon source for some microorganisms. Escherichia coli is capable of consuming glycerol to produce succinate under anaerobic fermentation, but with the deletion of some tricarboxylic acid (TCA) cycle genes, it is also able to produce succinate and malate in aerobiosis. In this study, we investigate possible rate-limiting enzymes by overexpressing the C-feeding anaplerotic enzymes Ppc, MaeA, MaeB, and Pck in a mutant that lacks the succinate dehydrogenase (Sdh) enzyme. The overexpression of the TCA enzyme Mdh and the activation of the glyoxylate shunt was also examined. Using this unbiased approach, we found that phosphoenol pyruvate carboxylase (Ppc) overexpression enhances an oxidative pathway that leads to increasing succinate, while phosphoenol pyruvate carboxykinase (Pck) favors a more efficient reductive branch that produces mainly malate, at 57.5% of the theoretical maximum molar yield. The optimization of the culture medium revealed the importance of bicarbonate and pH in the production of malate. An additional mutation of the ppc gene highlights its central role in growth and C4 production.


Assuntos
Escherichia coli/enzimologia , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Glicerol/metabolismo , Malatos/metabolismo , Aerobiose , Bicarbonatos/metabolismo , Escherichia coli/crescimento & desenvolvimento , Cinética , Malato Desidrogenase/metabolismo , Mutação/genética , Fosfoenolpiruvato Carboxilase/metabolismo
5.
Proc Natl Acad Sci U S A ; 118(3)2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33431667

RESUMO

A plant pathway that initiates with the formation of citramalate from pyruvate and acetyl-CoA by citramalate synthase (CMS) is shown to contribute to the synthesis of α-ketoacids and important odor-active esters in apple (Malus × domestica) fruit. Microarray screening led to the discovery of a gene with high amino acid similarity to 2-isopropylmalate synthase (IPMS). However, functional analysis of recombinant protein revealed its substrate preference differed substantially from IPMS and was more typical of CMS. MdCMS also lacked the regulatory region present in MdIPMS and was not sensitive to feedback inhibition. 13C-acetate feeding of apple tissue labeled citramalate and α-ketoacids in a manner consistent with the presence of the citramalate pathway, labeling both straight- and branched-chain esters. Analysis of genomic DNA (gDNA) revealed the presence of two nearly identical alleles in "Jonagold" fruit (MdCMS_1 and MdCMS_2), differing by two nonsynonymous single-nucleotide polymorphisms (SNPs). The mature proteins differed only at amino acid 387, possessing either glutamine387 (MdCMS_1) or glutamate387 (MdCMS_2). Glutamate387 was associated with near complete loss of activity. MdCMS expression was fruit-specific, increasing severalfold during ripening. The translated protein product was detected in ripe fruit. Transient expression of MdCMS_1 in Nicotiana benthamiana induced the accumulation of high levels of citramalate, whereas MdCMS_2 did not. Domesticated apple lines with MdCMS isozymes containing only glutamate387 produced a very low proportion of 2-methylbutanol- and 2-methylbutanoate (2MB) and 1-propanol and propanoate (PROP) esters. The citramalate pathway, previously only described in microorganisms, is shown to function in ripening apple and contribute to isoleucine and 2MB and PROP ester biosynthesis without feedback regulation.


Assuntos
Vias Biossintéticas/genética , Ésteres/metabolismo , Malatos/metabolismo , Proteínas de Plantas/metabolismo , Aminoácidos/metabolismo , Frutas/enzimologia , Frutas/metabolismo , Regulação da Expressão Gênica de Plantas , Isoleucina/metabolismo , Malus/enzimologia , Malus/metabolismo , Tabaco/genética
6.
Int J Food Microbiol ; 339: 109020, 2021 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-33360296

RESUMO

Extra ingredients are often used in traditional sourdough production recipes by artisan bakeries. These ingredients may be the source of microorganisms or stimulate the growth and/or the metabolic activities of the microorganisms added to or naturally present in the flour-water mixture. The present study examined the influence of the addition of lemon juice or apple juice as source of citrate or malate, respectively, on the growth and activity of the citrate- and malate-positive Companilactobacillus crustorum LMG 23699 strain (formerly known as Lactobacillus crustorum LMG 23699), used to initiate firm (dough yield of 200) wheat sourdough productions, and on the flavour of the baked goods produced. Three fermentation strategies were applied, namely one-step long fermentation sourdough production processes with the addition of juice at the start (Type 2) and backslopped fermentations with the addition of juice either only at the start of the sourdough productions or at the start of the sourdough productions and at the beginning of each subsequent refreshment step during the whole backslopping process (both Type 3). It turned out that the starter culture strain used prevailed during all sourdough productions performed. Yeasts were particularly present in Type 3 sourdough productions, although lemon juice retarded their growth. Due to high yeast activity, high concentrations of ethanol and glycerol were produced toward the end of the sourdough productions. Addition of lemon juice stimulated the production of lactic acid, acetic acid, and the buttery flavour compounds acetoin and diacetyl, because of citrate conversion, during the Type 2 and Type 3 sourdough productions. In Type 3 sourdough productions, these compounds were found in higher concentrations only when lemon juice was added at each backslopping step. Alternatively, the addition of apple juice led to high concentrations of lactic acid because of malolactic fermentation in both Type 2 and Type 3 sourdough productions. Moreover, the addition of apple juice increased the initial concentrations of the carbohydrates (fructose, glucose, and sucrose) and sugar alcohols (mannitol and sorbitol), which were exhausted upon backslopping or accumulated in the sourdough matrix, respectively. Baked goods produced using sourdoughs obtained from the Type 2 and Type 3 sourdough productions with the addition of juice at each backslopping step were significantly different in flavour from doughs supplemented with the respective juices and lactic acid and/or Type 3 sourdough productions with the addition of juice only at the start.


Assuntos
Pão/microbiologia , Ácido Cítrico/metabolismo , Microbiologia de Alimentos , Sucos de Frutas e Vegetais , Lactobacillus/metabolismo , Pão/análise , Citrus/química , Fermentação , Farinha/microbiologia , Frutas/química , Malatos/metabolismo , Malus/química , Odorantes/análise
7.
Plant Sci ; 302: 110711, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33288018

RESUMO

To identify unknown regulatory mechanisms leading to aluminium (Al)-induction of the Al tolerance gene ALS3, we conducted an expression genome-wide association study (eGWAS) for ALS3 in the shoots of 95 Arabidopsis thaliana accessions in the presence of Al. The eGWAS was conducted using a mixed linear model with 145,940 genome-wide single nucleotide polymorphisms (SNPs) and the association results were validated using reverse genetics. We found that many SNPs from the eGWAS were associated with genes related to phosphatidylinositol metabolism as well as stress signal transduction, including Ca2+signals, inter-connected in a co-expression network. Of these, PLC9, CDPK32, ANAC071, DIR1, and a hypothetical protein (AT4G10470) possessed amino acid sequence/ gene expression level polymorphisms that were significantly associated with ALS3 expression level variation. Furthermore, T-DNA insertion mutants of PLC9, CDPK32, and ANAC071 suppressed shoot ALS3 expression in the presence of Al. This study clarified the regulatory mechanisms of ALS3 expression in the shoot and provided genetic evidence of the involvement of phosphatidylinositol-derived signal transduction under Al stress.


Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Alumínio/toxicidade , Proteínas de Arabidopsis/fisiologia , Arabidopsis/genética , Fosfatidilinositóis/metabolismo , Brotos de Planta/metabolismo , Transdução de Sinais , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Estudo de Associação Genômica Ampla , Malatos/metabolismo , Brotos de Planta/fisiologia , Polimorfismo de Nucleotídeo Único/genética , Estresse Fisiológico , Transcriptoma
8.
Int J Food Microbiol ; 337: 108954, 2021 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-33202298

RESUMO

Non-Saccharomyces yeasts have increasingly been used in vinification recently. This is particularly true of Torulaspora delbrueckii and Metschnikowia pulcherrima, which are inoculated before S. cerevisiae, to complete a sequential alcoholic fermentation. This paper aims to study the effects of these two non-Saccharomyces yeasts on malolactic fermentation (MLF) carried out by two strains of Oenococcus oeni, under cellar conditions. Oenological parameters, and volatile and phenolic compounds were analysed in wines. The wines were tasted, and the microorganisms identified. In general, non-Saccharomyces created more MLF friendly conditions, largely because of lower concentrations of SO2 and medium chain fatty acids. The most favourable results were observed in wines inoculated with T. delbrueckii, that seemed to promote the development of O. oeni and improve MLF performance.


Assuntos
Metschnikowia/metabolismo , Oenococcus/metabolismo , Torulaspora/metabolismo , Vinho/análise , Vinho/microbiologia , Fermentação , Ácido Láctico/metabolismo , Malatos/metabolismo , Oenococcus/crescimento & desenvolvimento , Fenóis/análise , Fenóis/metabolismo , Compostos Orgânicos Voláteis/análise , Compostos Orgânicos Voláteis/metabolismo
9.
Food Chem ; 334: 127479, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-32688181

RESUMO

Calcium treatment effects on malate metabolism and the GABA pathway in 'Cripps Pink' apple fruit during storage were investigated. Postharvest apple fruit treated with 1% and 4% calcium chloride solutions were stored at 25 ± 1 °C. The 4% calcium treatment suppressed declines in titratable acidity and malate content and increased succinate and oxalate concentrations. Calcium treatment also reduced the respiration rate and decreased ethylene production peak during storage. Moreover, 4% calcium treatment significantly enhanced cyNAD-MDH and PEPC activities and upregulated MdMDH1, MdMDH2, MdPEPC1 and MdPEPC2 expression while inhibiting cyNADP-ME and PEPCK activities and downregulating MdME1, MdME4 and MdPEPCK2 expression. Surprisingly, calcium treatment changed the content of some free amino acids (GABA, proline, alanine, aspartic acid and glutamate), two of which (glutamate and GABA) are primary metabolites of the GABA pathway. Furthermore, calcium application enhanced GABA pathway activity by increasing MdGAD1, MdGAD2, MdGABA-T1/2 and MdSSADH transcript levels.


Assuntos
Cálcio/farmacologia , Frutas/efeitos dos fármacos , Malatos/metabolismo , Malus/efeitos dos fármacos , Malus/metabolismo , Ácido gama-Aminobutírico/metabolismo , Aminoácidos/análise , Aminoácidos/metabolismo , Etilenos/metabolismo , Qualidade dos Alimentos , Frutas/química , Frutas/genética , Frutas/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Malus/química , Malus/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
10.
Food Chem ; 334: 127567, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-32707362

RESUMO

Fruit acidity is an important determinant of peach organoleptic quality, but its regulatory mechanism remains elusive. Measurement of organic acids in ripe fruits of seventy-five peach cultivars revealed the predominant components malate and citrate, accompanied by quinate. Organic acid accumulation increased at early stages of fruit growth, but exhibited a more dramatic reduction in low-acid cultivar during later stages of fruit development compared to high-acid cultivars. Low-acid cultivars showed citrate degradation and less transport of malate into the vacuole due to up- and down-regulation of a GABA pathway gene GAD and a malate transporter gene ALMT9, respectively. The NAD-MDH1 gene might control the rate-limiting step in malate synthesis, while three genes, PDK, PK, and ADH, could affect citrate synthesis through the pyruvate-to-acetyl-CoA-to-citrate pathway. Altogether, these results suggested that malate accumulation is controlled at the level of metabolism and vacuolar storage, while metabolism is crucial for citrate accumulation in peach.


Assuntos
Ácidos Carboxílicos/metabolismo , Frutas/metabolismo , Malatos/metabolismo , Proteínas de Plantas/genética , Prunus persica/metabolismo , Acetilcoenzima A/metabolismo , Ácido Cítrico/metabolismo , Frutas/genética , Frutas/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo , Prunus persica/genética , Prunus persica/crescimento & desenvolvimento , Ácido Pirúvico/metabolismo , Vacúolos/metabolismo
11.
Proc Natl Acad Sci U S A ; 117(36): 22452-22461, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32820073

RESUMO

Carbon fixation via the Calvin cycle is constrained by the side activity of Rubisco with dioxygen, generating 2-phosphoglycolate. The metabolic recycling of phosphoglycolate was extensively studied in photoautotrophic organisms, including plants, algae, and cyanobacteria, where it is referred to as photorespiration. While receiving little attention so far, aerobic chemolithoautotrophic bacteria that operate the Calvin cycle independent of light must also recycle phosphoglycolate. As the term photorespiration is inappropriate for describing phosphoglycolate recycling in these nonphotosynthetic autotrophs, we suggest the more general term "phosphoglycolate salvage." Here, we study phosphoglycolate salvage in the model chemolithoautotroph Cupriavidus necator H16 (Ralstonia eutropha H16) by characterizing the proxy process of glycolate metabolism, performing comparative transcriptomics of autotrophic growth under low and high CO2 concentrations, and testing autotrophic growth phenotypes of gene deletion strains at ambient CO2 We find that the canonical plant-like C2 cycle does not operate in this bacterium, and instead, the bacterial-like glycerate pathway is the main route for phosphoglycolate salvage. Upon disruption of the glycerate pathway, we find that an oxidative pathway, which we term the malate cycle, supports phosphoglycolate salvage. In this cycle, glyoxylate is condensed with acetyl coenzyme A (acetyl-CoA) to give malate, which undergoes two oxidative decarboxylation steps to regenerate acetyl-CoA. When both pathways are disrupted, autotrophic growth is abolished at ambient CO2 We present bioinformatic data suggesting that the malate cycle may support phosphoglycolate salvage in diverse chemolithoautotrophic bacteria. This study thus demonstrates a so far unknown phosphoglycolate salvage pathway, highlighting important diversity in microbial carbon fixation metabolism.


Assuntos
Crescimento Quimioautotrófico/fisiologia , Glicolatos/metabolismo , Fotossíntese/fisiologia , Acetilcoenzima A/metabolismo , Proteínas de Bactérias/metabolismo , Ciclo do Carbono/fisiologia , Cupriavidus necator/genética , Cupriavidus necator/metabolismo , Malato Sintase/metabolismo , Malatos/metabolismo , Oxirredução
12.
Food Chem ; 332: 127382, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32619943

RESUMO

Sea buckthorn (Hippophaë rhamnoides L.) berries have high biological value as a rich source of phenolic compounds, fatty acids and vitamins A, C, E. Due to the high organic acid content and sour taste, the fruits are rarely used in juice production. Therefore, the study aimed to determine the metabolic activity of Lactobacillus plantarum, Lactobacillus plantarum subsp. argentoratensis and Oenococcus oeni strains along with the dynamics of changes in organic acids, sugars, phenolic compounds, and antioxidant activity during 72-h fermentation of 100% sea buckthorn and mixed with apple (1:1) juices. The strongest malolactic conversion was in mixed juices (to 75.0%). The most efficient strains were L. plantarum DSM 10492, 20174 and 6872. L. plantarum strains caused an increase in flavonols and antioxidant activity of sea buckthorn-apple juices. The results can be used to select conditions and strains in industrial-scale fermentation, to produce novel sea buckthorn products and increase their consumption.


Assuntos
Antioxidantes/química , Sucos de Frutas e Vegetais/análise , Ácido Láctico/metabolismo , Malatos/metabolismo , Fenóis/metabolismo , Açúcares/metabolismo , Fermentação , Flavonóis/química , Flavonóis/metabolismo , Frutas/química , Frutas/metabolismo , Hippophae/química , Hippophae/metabolismo , Concentração de Íons de Hidrogênio , Lactobacillus/crescimento & desenvolvimento , Lactobacillus/metabolismo , Malus/química , Malus/metabolismo , Oenococcus/crescimento & desenvolvimento , Oenococcus/metabolismo , Fenóis/química
13.
PLoS One ; 15(6): e0235066, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32584859

RESUMO

Shiga toxin-producing Escherichia coli (STEC) are a leading cause of foodborne outbreaks of human disease, but they reside harmlessly as an asymptomatic commensal in the ruminant gut. STEC serogroup O145 are difficult to isolate as routine diagnostic methods are unable to distinguish non-O157 serogroups due to their heterogeneous metabolic characteristics, resulting in under-reporting which is likely to conceal their true prevalence. In light of these deficiencies, the purpose of this study was a twofold approach to investigate enhanced STEC O145 diagnostic culture-based methods: firstly, to use a genomic epidemiology approach to understand the genetic diversity and population structure of serogroup O145 at both a local (New Zealand) (n = 47) and global scale (n = 75) and, secondly, to identify metabolic characteristics that will help the development of a differential media for this serogroup. Analysis of a subset of E. coli serogroup O145 strains demonstrated considerable diversity in carbon utilisation, which varied in association with eae subtype and sequence type. Several carbon substrates, such as D-serine and D-malic acid, were utilised by the majority of serogroup O145 strains, which, when coupled with current molecular and culture-based methods, could aid in the identification of presumptive E. coli serogroup O145 isolates. These carbon substrates warrant subsequent testing with additional serogroup O145 strains and non-O145 strains. Serogroup O145 strains displayed extensive genetic heterogeneity that was correlated with sequence type and eae subtype, suggesting these genetic markers are good indicators for distinct E. coli phylogenetic lineages. Pangenome analysis identified a core of 3,036 genes and an open pangenome of >14,000 genes, which is consistent with the identification of distinct phylogenetic lineages. Overall, this study highlighted the phenotypic and genotypic heterogeneity within E. coli serogroup O145, suggesting that the development of a differential media targeting this serogroup will be challenging.


Assuntos
Carbono/metabolismo , Infecções por Escherichia coli , Genótipo , Filogenia , Sorogrupo , Escherichia coli Shiga Toxigênica , Animais , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/metabolismo , Humanos , Malatos/metabolismo , Nova Zelândia/epidemiologia , Serina/genética , Serina/metabolismo , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/isolamento & purificação , Escherichia coli Shiga Toxigênica/metabolismo
14.
Nat Commun ; 11(1): 3238, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591540

RESUMO

The challenge of monitoring in planta dynamic changes of NADP(H) and NAD(H) redox states at the subcellular level is considered a major obstacle in plant bioenergetics studies. Here, we introduced two circularly permuted yellow fluorescent protein sensors, iNAP and SoNar, into Arabidopsis thaliana to monitor the dynamic changes in NADPH and the NADH/NAD+ ratio. In the light, photosynthesis and photorespiration are linked to the redox states of NAD(P)H and NAD(P) pools in several subcellular compartments connected by the malate-OAA shuttles. We show that the photosynthetic increases in stromal NADPH and NADH/NAD+ ratio, but not ATP, disappear when glycine decarboxylation is inhibited. These observations highlight the complex interplay between chloroplasts and mitochondria during photosynthesis and support the suggestions that, under normal conditions, photorespiration supplies a large amount of NADH to mitochondria, exceeding its NADH-dissipating capacity, and the surplus NADH is exported from the mitochondria to the cytosol through the malate-OAA shuttle.


Assuntos
Arabidopsis/fisiologia , Arabidopsis/efeitos da radiação , Luz , Proteínas Luminescentes/metabolismo , NADP/metabolismo , NAD/metabolismo , Fotossíntese/efeitos da radiação , Respiração Celular/efeitos da radiação , Cloroplastos/metabolismo , Citosol/metabolismo , Transporte de Elétrons/efeitos da radiação , Malatos/metabolismo , Mitocôndrias/metabolismo , Modelos Biológicos , Oxirredução , Peroxissomos/metabolismo , Plântula/metabolismo , Plântula/efeitos da radiação
15.
Biochim Biophys Acta Proteins Proteom ; 1868(9): 140462, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32485238

RESUMO

Malic enzymes participate in key metabolic processes, the MaeB-like malic enzymes carry a catalytic inactive phosphotransacetylase domain whose function remains elusive. Here we show that acetyl-CoA directly binds and inhibits MaeB-like enzymes with a saturable profile under physiological relevant acetyl-CoA concentrations. A MaeB-like enzyme from the nitrogen-fixing bacterium Azospirillum brasilense, namely AbMaeB1, binds both acetyl-CoA and unesterified CoASH in a way that inhibition of AbMaeB1 by acetyl-CoA is relieved by increasing CoASH concentrations. Hence, AbMaeB1 senses the acetyl-CoA/CoASH ratio. We revisited E. coli MaeB regulation to determine the inhibitory constant for acetyl-CoA. Our data support that the phosphotransacetylase domain of MaeB-like enzymes senses acetyl-CoA to dictate the fate of carbon distribution at the phosphoenol-pyruvate / pyruvate / oxaloacetate metabolic node.


Assuntos
Acetilcoenzima A/metabolismo , Coenzima A/metabolismo , Malato Desidrogenase/metabolismo , Malatos/metabolismo , NADP/metabolismo , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Malato Desidrogenase/genética , Fosfato Acetiltransferase/metabolismo
16.
Trends Plant Sci ; 25(5): 446-454, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32304657

RESUMO

In photosynthetic cells, chloroplasts and mitochondria are the sites of the core redox reactions underpinning energy metabolism. Such reactions generate reactive oxygen species (ROS) when oxygen is partially reduced. ROS signaling leads to responses by cells which enable them to adjust to changes in redox status. Recent studies in Arabidopsis thaliana reveal that chloroplast NADH can be used to generate malate which is exported to the mitochondrion where its oxidation regenerates NADH. Oxidation of this NADH produces mitochondrial ROS (mROS) which can activate signaling systems to modulate energy metabolism, and in certain cases can lead to programmed cell death (PCD). We propose the term 'malate circulation' to describe such redistribution of reducing equivalents to mediate energy homeostasis in the cell.


Assuntos
Arabidopsis , Malatos , Cloroplastos/metabolismo , Malatos/metabolismo , Mitocôndrias/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo
17.
J Food Sci ; 85(4): 1070-1081, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32125714

RESUMO

Wines produced from grapes cultivated in cool climate areas are characterized by high levels of organic acids. One method to correct this is malolactic fermentation (MLF). The aim of this study was to determine the effectiveness of different strains of Oenococcus oeni bacteria (Viniflora CH11, Viniflora CH16, Viniflora CH35, Viniflora Oenos, SIHA LACT Oeno) during the biological acidity reduction process. Red wine from Rondo and Regent cultivars was obtained by ethanol fermentation of the pulp, at 20 °C for 14 days. The profile of organic acids was examined with a particular focus on changes in the content of l-malic and l-lactic acids. Additionally, the impact on profile and quantity of phenolic compounds and antioxidant capacity was measured. The results showed that MLF had a positive influence on content of organic acids through the reduction of l-malic acid content with a simultaneous increase of the amount of l-lactic acid. The best effect was obtained with the CH11 and CH35 bacterial strains. The biological acidity reduction process had no significant (P > 0.05) impact on phenolic content or antioxidant capacity. However, the wine making process (ethanol fermentation, maturation) contributed to the reduction of polyphenols and in consequence lower antioxidant capacity of the final tested wines. PRACTICAL APPLICATION: The present study provides useful information on the impact of different Oenococcus oeni bacterial strains on MLF in red wines, reduction of l-malic to l-lactic acid, and stability of phenolic compounds during MLF and the maturation period. Also, this article provides information about phenolic compounds and antioxidant capacity during malolactic fermentation and maturity of red wines made from hybrids of Vitis vinifera such as Rondo and Regent cultivars.


Assuntos
Ácido Láctico/metabolismo , Malatos/metabolismo , Oenococcus/metabolismo , Fenóis/metabolismo , Vitis/microbiologia , Etanol/análise , Etanol/metabolismo , Fermentação , Ácido Láctico/análise , Malatos/análise , Oenococcus/classificação , Oenococcus/genética , Oenococcus/isolamento & purificação , Fenóis/análise , Vitis/química , Vinho/análise , Vinho/microbiologia
18.
PLoS One ; 15(3): e0229738, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32160222

RESUMO

Ultrasound has many uses, such as in medical imaging, monitoring of crystallization, characterization of emulsions and suspensions, and disruption of cell membranes in the food industry. It can also affect microbial cells by promoting or slowing their growth and increasing the production of some metabolites. However, the exact mechanism explaining the effect of ultrasound has not been identified yet. Most equipment employed to study the effect of ultrasound on microorganisms has been designed for other applications and then only slightly modified. This results in limited control over ultrasound frequency and input power, or pressure distribution in the reactor. The present study aimed to obtain a well-defined reactor by simulating the pressure distribution of a sonobioreactor. Specifically, we optimized a sonotrode to match the bottle frequency and compared it to measured results to verify the accuracy of the simulation. The measured pressure distribution spectrum presented the same overall trend as the simulated spectrum. However, the peaks were much less intense, likely due to non-linear events such as the collapse of cavitation bubbles. To test the application of the sonobioreactor in biological systems, two biotechnologically interesting microorganisms were assessed: an electroactive bacterium, Geobacter sulfurreducens, and a lignocellulose-degrading fungus, Fusarium oxysporum. Sonication resulted in increased malate production by G. sulfurreducens, but no major effect on growth. In comparison, morphology and growth of F. oxysporum were more sensitive to ultrasound intensity. Despite considerable morphological changes at 4 W input power, the growth rate was not adversely affected; however, at 12 W, growth was nearly halted. The above findings indicate that the novel sonobioreactor provides an effective tool for studying the impact of ultrasound on microorganisms.


Assuntos
Reatores Biológicos/microbiologia , Fusarium/crescimento & desenvolvimento , Geobacter/crescimento & desenvolvimento , Análise Numérica Assistida por Computador , Sonicação , Calorimetria , Simulação por Computador , Fusarium/ultraestrutura , Geobacter/metabolismo , Malatos/metabolismo , Metaboloma , Pressão , Vibração
19.
Plant Biol (Stuttg) ; 22(4): 701-708, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32174006

RESUMO

Worldwide, 50% of soil is acidic, which induces aluminium (Al) toxicity in plants, as the phyto-availability of Al3+ increases in acidic soil. Plants responds to Al3+ toxicity by exuding organic acids into the rhizosphere. The organic acid responsible for Al3+ stress response varies from species to species, which in the case of blackgram (Vigna mungo L.) is citrate. In blackgram, an Arabidopsis malate transporter, AtALMT1, was overexpressed with the motive of inducing enhanced exudation of malate. Transgenics were generated using cotyledon node explants through Agrobacterium tumefaciens-mediated transformation. The putative transgenics were initially screened by AtALMT1-specific genomic DNA PCR, followed by quantitative PCR. Two independent transgenic events were identified and functionally characterized in the T3 generation. The transgenic lines, Line 1 and 2, showed better root growth, relative water content and chlorophyll content under Al3+ stress. Both lines also accounted for less oxidative damage, due to reduced accumulation of ROS molecules. Photosynthetic efficiency, as measured in terms of Fv /Fm , NPQ and Y(II), increased when compared to the wild type (WT). Relative expression of genes (VmSTOP1, VmALS3, VmMATE) responsible for Al3+ stress response in blackgram showed that overexpression of a malate transporter did not have any effect on their expression. Malate exudation increased whereas citrate exudation did not show any divergence from the WT. A pot stress assay found that the transgenics showed better adaptation to acidic soil. This report demonstrates that the overexpression of a malate transporter in a non-malate exuding species improves adaptation to Al3+ toxicity in acidic soil without effecting its stress response mechanism.


Assuntos
Proteínas de Arabidopsis , Malatos , Rizosfera , Vigna , Alumínio/toxicidade , Proteínas de Arabidopsis/genética , Tolerância a Medicamentos/genética , Expressão Gênica , Malatos/metabolismo , Transportadores de Ânions Orgânicos/genética , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Vigna/genética
20.
Theor Appl Genet ; 133(3): 993-1008, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31932953

RESUMO

KEY MESSAGE: In a grapevine segregating population, genomic regions governing berry pH were identified, paving the way for breeding new grapevine varieties best adapted to a warming climate. As a consequence of global warming, grapevine berry acidity is expected to dramatically decrease. Adapting grapevine (Vitis vinifera L.) varieties to the climatic conditions of the future requires a better understanding of the genetic architecture of acidity-related traits. For this purpose, we studied during five growing seasons 120 individuals from a grapevine biparental cross. Each offspring was genotyped by simple sequence repeats markers and by hybridization on a 20-K Grapevine Illumina® SNP chip. Quantitative trait loci (QTLs) for pH colocalized with QTLs for the ratio between potassium and tartaric acid concentrations, on chromosomes 10, 11 and 13. Strong QTLs for malic acid concentration or for the malic acid-to-tartaric acid ratio, on chromosomes 6 and 8, were not associated with variations of pH but can be useful for controlling pH stability under high temperatures. Our study highlights the interdependency between acidity parameters and consequently the constraints and degrees of freedom for designing grapevine genotypes better adapted to the expected warmer climatic conditions. In particular, it is possible to create grapevine genotypes with a high berry acidity as the result of both high tartaric acid concentrations and low K+ accumulation capacities.


Assuntos
Ácidos/metabolismo , Frutas/genética , Genes de Plantas , Potássio/metabolismo , Vitis/genética , Alelos , Mapeamento Cromossômico , Mudança Climática , Variação Genética , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Temperatura Alta , Concentração de Íons de Hidrogênio , Malatos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Locos de Características Quantitativas
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