scholarly journals Controlled Transcription of Regulator Gene carS by Tet-on or by a Strong Promoter Confirms Its Role as a Repressor of Carotenoid Biosynthesis in Fusarium fujikuroi

2020 ◽  
Vol 9 (1) ◽  
pp. 71
Author(s):  
Julia Marente ◽  
Javier Avalos ◽  
M. Carmen Limón
Keyword(s):  
Wild Strain ◽  
Ring Finger ◽  
Carotenoid Biosynthesis ◽  
Negative Regulator ◽  
Fusarium Fujikuroi ◽  
Structural Genes ◽  
Gene Encoding ◽  
In The Wild ◽  
Set Up

Carotenoid biosynthesis is a frequent trait in fungi. In the ascomycete Fusarium fujikuroi, the synthesis of the carboxylic xanthophyll neurosporaxanthin (NX) is stimulated by light. However, the mutants of the carS gene, encoding a protein of the RING finger family, accumulate large NX amounts regardless of illumination, indicating the role of CarS as a negative regulator. To confirm CarS function, we used the Tet-on system to control carS expression in this fungus. The system was first set up with a reporter mluc gene, which showed a positive correlation between the inducer doxycycline and luminescence. Once the system was improved, the carS gene was expressed using Tet-on in the wild strain and in a carS mutant. In both cases, increased carS transcription provoked a downregulation of the structural genes of the pathway and albino phenotypes even under light. Similarly, when the carS gene was constitutively overexpressed under the control of a gpdA promoter, total downregulation of the NX pathway was observed. The results confirmed the role of CarS as a repressor of carotenogenesis in F. fujikuroi and revealed that its expression must be regulated in the wild strain to allow appropriate NX biosynthesis in response to illumination.

scholarly journals The carP lncRNA Is a carS-Related Regulatory Element with Broad Effects on the Fusarium fujikuroi Transcriptome

2021 ◽  
Vol 7 (3) ◽  
pp. 46
Author(s):  
Javier Pardo-Medina ◽  
Gabriel Gutiérrez ◽  
M. Carmen Limón ◽  
Javier Avalos
Keyword(s):  
Regulatory Element ◽  
Ring Finger ◽  
Carotenoid Biosynthesis ◽  
Fusarium Fujikuroi ◽  
Mrna Levels ◽  
Carotenoid Production ◽  
Cis Acting ◽  
Acting Mechanism ◽  
Cascade Effects ◽  
Genomic Locations

Carotenoid biosynthesis in the fungus Fusarium fujikuroi is regulated by environmental factors, with light being the main stimulating signal. The CarS RING-finger protein plays an important role in the downregulation of structural genes of the carotenoid pathway. A recent transcriptomic analysis on the effect of carS mutation identified a gene for a long non-coding RNA (lncRNA) upstream of carS, called carP, the deletion of which results in increased carS mRNA levels and lack of carotenoid production. We have investigated the function of carP by studying the transcriptomic effect of its deletion and the phenotypes resulting from the reintroduction of carP to a deletion strain. The RNA-seq data showed that the loss of carP affected the mRNA levels of hundreds of genes, especially after illumination. Many of these changes appeared to be cascade effects as a result of changes in carS expression, as suggested by the comparison with differentially expressed genes in a carS mutant. Carotenoid production only recovered when carP was integrated upstream of carS, but not at other genomic locations, indicating a cis-acting mechanism on carS. However, some genes hardly affected by CarS were strongly upregulated in the carP mutant, indicating that carP may have other regulatory functions as an independent regulatory element.

Abstract 400: Role of the 17beta-Estradiol/beta-Catenin Axis in Heart Failure

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Georgios Kararigas ◽  
Laura C Zelarayan ◽  
Karl Toischer ◽  
Gerd Hasenfuss ◽  
Hubertus Jarry ◽  
...  
Keyword(s):  
Heart Failure ◽  
Ring Finger ◽  
Posterior Wall ◽  
Left Ventricular ◽  
Negative Regulator ◽  
Heart Weight ◽  
Beta Catenin ◽  
Cross Sectional ◽  
End Stage

The role of β-catenin and the actions of 17β-estradiol (E2) in cardiovascular (patho)physiology remain unclear. In the diseased heart, E2 is expected to be cardioprotective. However, in the absence of prior disease, complications in pregnancy or transgender individuals indicate that E2 might lead to cardiovascular disease. We aimed at the characterization of the effects of the E2/β-catenin axis in the healthy C57Bl/6N heart. Two-month-old female C57Bl/6N wild-type (WT) and cardiac-specific β-catenin-deleted (β-cat Δex2-6 ) mice were ovariectomized and randomized to an E2-containing or soy-free (control, CON) diet ( n = 7-13/group). The 3-month physiological dose of E2 led to a significant repression of nuclear β-catenin levels vs. CON ( P < 0.001) in WT mice. This was underlain by a significant decrease in the heart weight to tibia length ratio and cardiomyocyte cross-sectional area (E2 vs. CON WT mice P < 0.001). Echocardiography revealed a significant decrease in septum width ( P < 0.001), posterior wall thickness ( P < 0.01) and fractional shortening ( P < 0.05) in E2 vs. CON WT mice. Deletion of β-catenin abrogated these E2-mediated deleterious effects on cardiac structure and function. Mechanistically, we found a significant increase in the levels of the ubiquitin ligase and key regulator of proteasome-dependent protein degradation muscle-specific RING finger protein 1 (MuRF1) in E2 vs. CON WT mice ( P < 0.05), while there was no effect in β-cat Δex2-6 mice. Although we also hypothesized increased autophagic activity, we found no effect on the autophagy-related protein LC3 in WT or β-cat Δex2-6 mice. In a translational approach, we profiled the left ventricular transcriptome of female patients ( n = 5) with end-stage non-ischemic dilated cardiomyopathy (DCM) vs. healthy controls ( n = 8). We found a significant induction in the expression of the genes coding for SFRP5 , a negative regulator of Wnt signaling, DACT2 , an antagonist of β-catenin, and APC2 , which is required for targeted degradation of β-catenin ( P < 0.01), in DCM samples. Our mouse data indicate that repression but not deletion of β-catenin leads to heart failure and our human data suggest that targeting Wnt/β-catenin signaling in heart failure may be of therapeutic value.

scholarly journals Regulation of Carotenogenesis and Secondary Metabolism by Nitrogen in Wild-Type Fusarium fujikuroi and Carotenoid-Overproducing Mutants

2008 ◽  
Vol 75 (2) ◽  
pp. 405-413 ◽  
Author(s):  
Roberto Rodríguez-Ortiz ◽  
M. Carmen Limón ◽  
Javier Avalos
Keyword(s):  
Nitrogen Availability ◽  
Polyketide Synthase ◽  
Nitrogen Starvation ◽  
Carotenoid Biosynthesis ◽  
Gibberella Fujikuroi ◽  
Fusarium Fujikuroi ◽  
Wild Type ◽  
Carotenoid Accumulation ◽  
Gibberellin Production ◽  
In The Wild

ABSTRACT The fungus Fusarium fujikuroi (Gibberella fujikuroi MP-C) produces metabolites of biotechnological interest, such as gibberellins, bikaverins, and carotenoids. Gibberellin and bikaverin productions are induced upon nitrogen exhaustion, while carotenoid accumulation is stimulated by light. We evaluated the effect of nitrogen availability on carotenogenesis in comparison with bikaverin and gibberellin production in the wild type and in carotenoid-overproducing mutants (carS). Nitrogen starvation increased carotenoid accumulation in all strains tested. In carS strains, gibberellin and bikaverin biosynthesis patterns differed from those of the wild type and paralleled the expression of key genes for both pathways, coding for geranylgeranyl pyrophosphate (GGPP) and kaurene synthases for the former and a polyketide synthase for the latter. These results suggest regulatory connections between carotenoid biosynthesis and nitrogen-controlled biosynthetic pathways in this fungus. Expression of gene ggs1, which encodes a second GGPP synthase, was also derepressed in the carS mutants, suggesting the participation of Ggs1 in carotenoid biosynthesis. The carS mutations did not affect genes for earlier steps of the terpenoid pathway, such as fppS or hmgR. Light induced carotenoid biosynthesis in the wild type and carRA and carB levels in the wild-type and carS strains irrespective of nitrogen availability.

scholarly journals l-Lysine Catabolism Is Controlled by l-Arginine and ArgR in Pseudomonas aeruginosa PAO1

2010 ◽  
Vol 192 (22) ◽  
pp. 5874-5880 ◽  
Author(s):  
Han Ting Chou ◽  
Mohamed Hegazy ◽  
Chung-Dar Lu
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Sole Source ◽  
Enzyme Characterization ◽  
Minor Role ◽  
Gene Encoding ◽  
In The Wild ◽  
Tight Connection ◽  
A Minor ◽  
Lysine Catabolism

ABSTRACT In comparison to other pseudomonads, Pseudomonas aeruginosa grows poorly in l-lysine as a sole source of nutrient. In this study, the ldcA gene (lysine de c arboxylase A; PA1818), previously identified as a member of the ArgR regulon of l-arginine metabolism, was found essential for l-lysine catabolism in this organism. LdcA was purified to homogeneity from a recombinant strain of Escherichia coli, and the results of enzyme characterization revealed that this pyridoxal-5-phosphate-dependent decarboxylase takes l-lysine, but not l-arginine, as a substrate. At an optimal pH of 8.5, cooperative substrate activation by l-lysine was depicted from kinetics studies, with calculated Km and V max values of 0.73 mM and 2.2 μmole/mg/min, respectively. Contrarily, the ldcA promoter was induced by exogenous l-arginine but not by l-lysine in the wild-type strain PAO1, and the binding of ArgR to this promoter region was demonstrated by electromobility shift assays. This peculiar arginine control on lysine utilization was also noted from uptake experiments in which incorporation of radioactively labeled l-lysine was enhanced in cells grown in the presence of l-arginine but not l-lysine. Rapid growth on l-lysine was detected in a mutant devoid of the main arginine catabolic pathway and with a higher basal level of the intracellular l-arginine pool and hence elevated ArgR-responsive regulons, including ldcA. Growth on l-lysine as a nitrogen source can also be enhanced when the aruH gene encoding an arginine/lysine:pyruvate transaminase was expressed constitutively from plasmids; however, no growth of the ldcA mutant on l-lysine suggests a minor role of this transaminase in l-lysine catabolism. In summary, this study reveals a tight connection of lysine catabolism to the arginine regulatory network, and the lack of lysine-responsive control on lysine uptake and decarboxylation provides an explanation of l-lysine as a poor nutrient for P. aeruginosa.

scholarly journals GABA, A Primary Metabolite Controlled by the Gac/Rsm Regulatory Pathway, Favors a Planktonic Over a Biofilm Lifestyle in Pseudomonas protegens CHA0

2018 ◽  
Vol 31 (2) ◽  
pp. 274-282 ◽  
Author(s):  
Kasumi Takeuchi
Keyword(s):  
Signal Transduction Pathway ◽  
Succinic Semialdehyde ◽  
Gene Encoding ◽  
Primary Metabolite ◽  
Succinic Semialdehyde Dehydrogenase ◽  
Gaba A ◽  
Semialdehyde Dehydrogenase ◽  
Niche Adaptation ◽  
In The Wild

In Pseudomonas protegens CHA0 and other fluorescent pseudomonads, the Gac/Rsm signal transduction pathway is crucial for the expression of secondary metabolism and the biological control of fungi, nematodes, and insects. Based on the findings of a previous metabolomic study, the role of intracellular γ-aminobutyrate (GABA) as a potential signal in the Gac/Rsm pathway was investigated herein. The function and regulation of a gabDT (c01870-c01880) gene cluster in strain CHA0 were described. The gabT gene encoded GABA transaminase (GABAT) and enabled the growth of the bacterium on GABA, whereas the upstream gabD gene (annotated as a gene encoding succinic semialdehyde dehydrogenase) had an unknown function. A gacA mutant exhibited low GABAT activity, leading to the markedly greater intracellular accumulation of GABA than in the wild type. In the gacA mutant, the RsmA and RsmE proteins caused translational gabD repression, with concomitant gabT repression. Due to very low GABAT activity, the gabT mutant accumulated GABA to high levels. This trait promoted a planktonic lifestyle, reduced biofilm formation, and favored root colonization without exhibiting the highly pleiotropic gacA phenotypes. These results suggest an important role of GABA in the Gac/Rsm-regulated niche adaptation of strain CHA0 to plant roots.

scholarly journals Regulation of Polyhydroxybutyrate Accumulation in Sinorhizobium meliloti by the Trans-Encoded Small RNA MmgR

2017 ◽  
Vol 199 (8) ◽  
Author(s):  
Antonio Lagares ◽  
Germán Ceizel Borella ◽  
Uwe Linne ◽  
Anke Becker ◽  
Claudio Valverde
Keyword(s):  
Small Rnas ◽  
Sinorhizobium Meliloti ◽  
Reducing Power ◽  
Negative Regulator ◽  
Fine Tuning ◽  
Biological Role ◽  
Wild Type ◽  
Content Type ◽  
In The Wild

ABSTRACT Riboregulation has a major role in the fine-tuning of multiple bacterial processes. Among the RNA players, trans-encoded untranslated small RNAs (sRNAs) regulate complex metabolic networks by tuning expression from multiple target genes in response to numerous signals. In Sinorhizobium meliloti, over 400 sRNAs are expressed under different stimuli. The sRNA MmgR (standing for Makes more granules Regulator) has been of particular interest to us since its sequence and structure are highly conserved among the alphaproteobacteria and its expression is regulated by the amount and quality of the bacterium's available nitrogen source. In this work, we explored the biological role of MmgR in S. meliloti 2011 by characterizing the effect of a deletion of the internal conserved core of mmgR (mmgR Δ33–51). This mutation resulted in larger amounts of polyhydroxybutyrate (PHB) distributed into more intracellular granules than are found in the wild-type strain. This phenotype was expressed upon cessation of balanced growth owing to nitrogen depletion in the presence of surplus carbon (i.e., at a carbon/nitrogen molar ratio greater than 10). The normal PHB accumulation was complemented with a wild-type mmgR copy but not with unrelated sRNA genes. Furthermore, the expression of mmgR limited PHB accumulation in the wild type, regardless of the magnitude of the C surplus. Quantitative proteomic profiling and quantitative reverse transcription-PCR (qRT-PCR) revealed that the absence of MmgR results in a posttranscriptional overexpression of both PHB phasin proteins (PhaP1 and PhaP2). Together, our results indicate that the widely conserved alphaproteobacterial MmgR sRNA fine-tunes the regulation of PHB storage in S. meliloti. IMPORTANCE High-throughput RNA sequencing has recently uncovered an overwhelming number of trans-encoded small RNAs (sRNAs) in diverse prokaryotes. In the nitrogen-fixing alphaproteobacterial symbiont of alfalfa root nodules Sinorhizobium meliloti, only four out of hundreds of identified sRNA genes have been functionally characterized. Thus, uncovering the biological role of sRNAs currently represents a major issue and one that is particularly challenging because of the usually subtle quantitative regulation contributed by most characterized sRNAs. Here, we have characterized the function of the broadly conserved alphaproteobacterial sRNA gene mmgR in S. meliloti. Our results strongly suggest that mmgR encodes a negative regulator of the accumulation of polyhydroxybutyrate, the major carbon and reducing power storage polymer in S. meliloti cells growing under conditions of C/N overbalance.

scholarly journals Increased Production of Zeaxanthin and Other Pigments by Application of Genetic Engineering Techniques toSynechocystis sp. Strain PCC 6803

2000 ◽  
Vol 66 (1) ◽  
pp. 64-72 ◽  
Author(s):  
Delphine Lagarde ◽  
Laurent Beuf ◽  
Wim Vermaas
Keyword(s):  
Mutant Strain ◽  
Fold Increase ◽  
Carotenoid Biosynthesis ◽  
Carotenoid Content ◽  
Wild Type ◽  
Gene Encoding ◽  
Genes Encoding ◽  
In The Wild ◽  
Β Carotene ◽  
Pcc 6803

ABSTRACT The psbAII locus was used as an integration platform to overexpress genes involved in carotenoid biosynthesis inSynechocystis sp. strain PCC 6803 under the control of the strong psbAII promoter. The sequences of the genes encoding the yeast isopentenyl diphosphate isomerase (ipi) and theSynechocystis β-carotene hydroxylase (crtR) and the linked Synechocystis genes coding for phytoene desaturase and phytoene synthase (crtP andcrtB, respectively) were introduced intoSynechocystis, replacing the psbAII coding sequence. Expression of ipi, crtR, andcrtP and crtB led to a large increase in the corresponding transcript levels in the mutant strains, showing that the psbAII promoter can be used to drive transcription and to overexpress various genes in Synechocystis. Overexpression of crtP and crtB led to a 50% increase in the myxoxanthophyll and zeaxanthin contents in the mutant strain, whereas the β-carotene and echinenone contents remained unchanged. Overexpression of crtR induced a 2.5-fold increase in zeaxanthin accumulation in the corresponding overexpressing mutant compared to that in the wild-type strain. In this mutant strain, zeaxanthin becomes the major pigment (more than half the total amount of carotenoid) and the β-carotene and echinenone amounts are reduced by a factor of 2. However, overexpression of ipi did not result in a change in the carotenoid content of the mutant. To further alter the carotenoid content of Synechocystis, the crtOgene, encoding β-carotene ketolase, which converts β-carotene to echinenone, was disrupted in the wild type and in the overexpressing strains so that they no longer produced echinenone. In this way, by a combination of overexpression and deletion of particular genes, the carotenoid content of cyanobacteria can be altered significantly.

scholarly journals A Negative Regulator of Carotenogenesis in Blakeslea trispora

2020 ◽  
Vol 86 (6) ◽  
Author(s):  
Wei Luo ◽  
Zunyang Gong ◽  
Na Li ◽  
Yuzheng Zhao ◽  
Huili Zhang ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Carotenoid Biosynthesis ◽  
Negative Regulator ◽  
Blakeslea Trispora ◽  
Regulation Mechanism ◽  
Null Mutant ◽  
Structural Genes ◽  
Wild Type ◽  
Β Carotene

ABSTRACT As an ideal carotenoid producer, Blakeslea trispora has gained much attention due to its large biomass and high production of β-carotene and lycopene. However, carotenogenesis regulation in B. trispora still needs to be clarified, as few investigations have been conducted at the molecular level in B. trispora. In this study, a gene homologous to carotenogenesis regulatory gene (crgA) was cloned from the mating type (−) of B. trispora, and the deduced CrgA protein was analyzed for its primary structure and domains. To clarify the crgA-mediated regulation in B. trispora, we used the strategies of gene knockout and complementation to investigate the effect of crgA expression on the phenotype of B. trispora. In contrast to the wild-type strain, the crgA null mutant (ΔcrgA) was defective in sporulation but accumulated much more β-carotene (31.2% improvement at the end) accompanied by enhanced transcription of three structural genes (hmgR, carB, and carRA) for carotenoids throughout the culture time. When the wild-type copy of crgA was complemented into the crgA null mutant, sporulation, transcription of structural genes, and carotenoid production were restored to those of the wild-type strain. A gas chromatography-mass spectrometry (GC-MS)-based metabolomic approach and multivariate statistical analyses were performed to investigate the intracellular metabolite profiles. The reduced levels of tricarboxylic acid (TCA) cycle components and some amino acids and enhanced levels of glycolysis intermediates and fatty acids indicate that more metabolic flux was driven into the mevalonate (MVA) pathway; thus, the increase of precursors and fat content contributes to the accumulation of carotenoids. IMPORTANCE The zygomycete Blakeslea trispora is an important strain for the production of carotenoids on a large scale. However, the regulation mechanism of carotenoid biosynthesis is still not well understood in this filamentous fungus. In the present study, we sought to investigate how crgA influences the expression of structural genes for carotenoids, carotenoid biosynthesis, and other anabolic phenotypes. This will lead to a better understanding of the global regulation mechanism of carotenoid biosynthesis and facilitate engineering this strain in the future for enhanced production of carotenoids.

scholarly journals Light-Dependent Functions of the Fusarium fujikuroi CryD DASH Cryptochrome in Development and Secondary Metabolism

2013 ◽  
Vol 79 (8) ◽  
pp. 2777-2788 ◽  
Author(s):  
Marta Castrillo ◽  
Jorge García-Martínez ◽  
Javier Avalos
Keyword(s):  
Secondary Metabolism ◽  
Nitrogen Starvation ◽  
Phytopathogenic Fungus ◽  
Fusarium Fujikuroi ◽  
Mrna Levels ◽  
Wild Type ◽  
Gene Encoding ◽  
Content Type ◽  
Gibberellin Production ◽  
In The Wild

ABSTRACTDASH (Drosophila,Arabidopsis,Synechocystis, human) cryptochromes (cry-DASHs) constitute a subgroup of the photolyase cryptochrome family with diverse light-sensing roles, found in most taxonomical groups. The genome ofFusarium fujikuroi, a phytopathogenic fungus with a rich secondary metabolism, contains a gene encoding a putative cry-DASH, named CryD. The expression of thecryDgene is induced by light in the wild type, but not in mutants of the “white collar” genewcoA. Targeted ΔcryDmutants show light-dependent phenotypic alterations, including changes in morphology and pigmentation, which disappear upon reintroduction of a wild-typecryDallele. In addition to microconidia, the colonies of the ΔcryDmutants produced under illumination and nitrogen starvation large septated spores called macroconidia, absent in wild-type colonies. The ΔcryDmutants accumulated similar amounts of carotenoids to the control strain under constant illumination, but produced much larger amounts of bikaverin under nitrogen starvation, indicating a repressing role for CryD in this biosynthetic pathway. Additionally, a moderate photoinduction of gibberellin production was exhibited by the wild type but not by the ΔcryDmutants. The phenotypic alterations of the ΔcryDmutants were only noticeable in the light, as expected from the low expression ofcryDin the dark, but did not correlate with mRNA levels for structural genes of the bikaverin or gibberellin biosynthetic pathways, suggesting the participation of CryD in posttranscriptional regulatory mechanisms. This is the first report on the participation of a cry-DASH protein in the regulation of fungal secondary metabolism.

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