scholarly journals Loss of function of the carbon catabolite repressor CreA leads to low but inducer‐independent expression from the feruloyl esterase B promoter in Aspergillus niger

2021 ◽  
Author(s):  
Jos Reijngoud ◽  
Mark Arentshorst ◽  
Claudine Ruijmbeek ◽  
Ian Reid ◽  
Ebru Demirci Alazi ◽  
...  
Keyword(s):  
Catabolite Repression ◽  
Allyl Alcohol ◽  
Carbon Catabolite Repression ◽  
Constitutive Expression ◽  
Genetic Screen ◽  
Feruloyl Esterase ◽  
Loss Of Function ◽  
Gene Encoding ◽  
Targeted Deletion ◽  
Reporter Strains

Abstract Objective With the aim to decipher the mechanisms involved in the transcriptional regulation of feruloyl esterase encoded by faeB, a genetic screen was performed to isolate A. niger mutants displaying inducer-independent expression from the faeB promoter. Result PfaeB-amdS and PfaeB-lux dual reporter strains were constructed and used to isolate trans-acting mutants in which the expression of both reporters was increased, based on the ability to grow on acetamide plates and higher luciferase activity, respectively. The genetic screen on the non-inducing carbon source D-fructose yielded in total 111 trans-acting mutants. The genome of one of the mutants was sequenced and revealed several SNPs, including a point mutation in the creA gene encoding a transcription factor known to be involved in carbon catabolite repression. Subsequently, all mutants were analyzed for defects in carbon catabolite repression by determining sensitivity towards allyl alcohol. All except four of the 111 mutants were sensitive to allyl alcohol, indicating that the vast majority of the mutants are defective in carbon catabolite repression. The creA gene of 32 allyl alcohol sensitive mutants was sequenced and 27 of them indeed contained a mutation in the creA gene. Targeted deletion of creA in the reporter strain confirmed that the loss of CreA results in constitutive expression from the faeB promoter. Conclusion Loss of function of CreA leads to low but inducer-independent expression from the faeB promoter in A. niger.

scholarly journals Genetic and Functional Investigation of Zn 2 Cys 6 Transcription Factors RSE2 and RSE3 in Podospora anserina

2013 ◽  
Vol 13 (1) ◽  
pp. 53-65 ◽  
Author(s):  
Elodie Bovier ◽  
Carole H. Sellem ◽  
Adeline Humbert ◽  
Annie Sainsard-Chanet
Keyword(s):  
Transcription Factors ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Constitutive Expression ◽  
Podospora Anserina ◽  
Loss Of Function ◽  
Gene Encoding ◽  
Content Type ◽  
Zinc Cluster ◽  
Wide Range ◽  
Genes Encoding

ABSTRACT In Podospora anserina , the two zinc cluster proteins RSE2 and RSE3 are essential for the expression of the gene encoding the alternative oxidase ( aox ) when the mitochondrial electron transport chain is impaired. In parallel, they activated the expression of gluconeogenic genes encoding phosphoenolpyruvate carboxykinase ( pck ) and fructose-1,6-biphosphatase ( fbp ). Orthologues of these transcription factors are present in a wide range of filamentous fungi, and no other role than the regulation of these three genes has been evidenced so far. In order to better understand the function and the organization of RSE2 and RSE3, we conducted a saturated genetic screen based on the constitutive expression of the aox gene. We identified 10 independent mutations in 9 positions in rse2 and 11 mutations in 5 positions in rse3 . Deletions were generated at some of these positions and the effects analyzed. This analysis suggests the presence of central regulatory domains and a C-terminal activation domain in both proteins. Microarray analysis revealed 598 genes that were differentially expressed in the strains containing gain- or loss-of-function mutations in rse2 or rse3 . It showed that in addition to aox , fbp , and pck , RSE2 and RSE3 regulate the expression of genes encoding the alternative NADH dehydrogenase, a Zn 2 Cys 6 transcription factor, a flavohemoglobin, and various hydrolases. As a complement to expression data, a metabolome profiling approach revealed that both an rse2 gain-of-function mutation and growth on antimycin result in similar metabolic alterations in amino acids, fatty acids, and α-ketoglutarate pools.

scholarly journals Use of gene replacement transformation to elucidate gene function in the qa gene cluster of Neurospora crassa.

Genetics ◽  
1992 ◽  
Vol 130 (4) ◽  
pp. 729-736
Author(s):  
M E Case ◽  
R F Geever ◽  
D K Asch
Keyword(s):  
Neurospora Crassa ◽  
Gene Cluster ◽  
Catabolite Repression ◽  
Carbon Catabolite Repression ◽  
Genetic Recombination ◽  
Constitutive Expression ◽  
Quinic Acid ◽  
Gene Replacement ◽  
Growth Characteristics ◽  
Repressor Molecule

Abstract Gene replacement by transformation, employing selective genetic recombination techniques, has been used to delete or disrupt the qa-x, qa-y and qa-1S genes of the qa gene cluster of Neurospora crassa. The growth characteristics of the strain carrying the deletion of the qa-y gene support earlier evidence that this gene encodes a quinic acid permease. The strain containing the deletion of the qa-1S gene (delta qa-1S) was examined with respect to quinic acid induction and carbon catabolite repression. The delta qa-1S strain exhibits constitutive expression of the qa genes supporting earlier evidence that the qa-1S gene codes for a repressor. Several of the qa genes continued to be expressed at high levels even in the presence of glucose in the delta qa-1S strain, which indicates that transcription of these genes is not being affected directly by a repressor molecule in the presence of glucose.

scholarly journals Carbon Catabolite Repression in Lactobacillus pentosus: Analysis of the ccpA Region

2000 ◽  
Vol 66 (1) ◽  
pp. 277-283 ◽  
Author(s):  
Kerstin Mahr ◽  
Wolfgang Hillen ◽  
Fritz Titgemeyer
Keyword(s):  
Catabolite Repression ◽  
Carbon Catabolite Repression ◽  
Carbon Sources ◽  
Carbon Utilization ◽  
Gene Encoding ◽  
Lactobacillus Pentosus ◽  
Reading Frame ◽  
Ammonium Fixation ◽  
Responsive Element ◽  
Genetic Structures

ABSTRACT The catabolite control protein CcpA is a central regulator in low-G+C-content gram-positive bacteria. It confers carbon catabolite repression to numerous genes required for carbon utilization. It also operates as a transcriptional activator of genes involved in diverse phenomena, such as glycolysis and ammonium fixation. We have cloned theccpA region of Lactobacillus pentosus. ccpAencodes a protein of 336 amino acids exhibiting similarity to CcpA proteins of other bacteria and to proteins of the LacI/GalR family of transcriptional regulators. Upstream of ccpA was found an open reading frame with similarity to the pepQ gene, encoding a prolidase. Primer extension experiments revealed two start sites of transcription for ccpA. In wild-type cells grown on glucose, mRNA synthesis occurred only from the promoter proximal toccpA. In a ccpA mutant strain, both promoters were used, with increased transcription from the distant promoter, which overlaps a presumptive CcpA binding site called cre(for catabolite responsive element). This suggests that expression ofccpA is autoregulated. Determination of the expression levels of CcpA in cells grown on repressing and nonrepressing carbon sources revealed that the amounts of CcpA produced did not change significantly, leading to the conclusion that the arrangement of two promoters may ensure constant expression of ccpA under various environmental conditions. A comparison of the genetic structures of ccpA regions revealed that lactic acid bacteria possess the gene order pepQ-ccpA-variable while the genetic structure in bacilli and Staphylococcus xylosusis aroA-ccpA-variable-acuC.

scholarly journals Overexpression of a Gene Encoding a Catabolite Repression Element in Alternaria citri Causes Severe Symptoms of Black Rot in Citrus Fruit

2007 ◽  
Vol 97 (5) ◽  
pp. 557-563 ◽  
Author(s):  
H. Katoh ◽  
K. Ohtani ◽  
H. Yamamoto ◽  
K. Akimitsu
Keyword(s):  
Catabolite Repression ◽  
Wild Type Strain ◽  
Carbon Catabolite Repression ◽  
Citrus Fruit ◽  
Normal Growth ◽  
Fruit Production ◽  
Central Axis ◽  
Black Rot ◽  
Wild Type ◽  
Gene Encoding

A gene (AcCreA) encoding a catabolite repression element (CreA) with (two zinc fingers of the Cys2His2 type was isolated from the postharvest fungal pathogen Alternaria citri. The AcCreA overexpression mutant AcOEC2 of A. citri showed normal growth on pectin medium and on segments of peel or the juice sac area from citrus fruit. Production of endopolygalacturonase, an essential virulence factor of this pathogen, was similar in AcOEC2 and the wild type in pectin-containing media. However, addition of glucose to the medium showed that carbon catabolite repression of endopolygalacturonase gene (Acpg1) expression, as well as endopolygalacturonase production, was lost in AcOEC2. The wild-type strain of A. citri causes rot mainly in the central axis of citrus fruit without development of rotting in the juice sac area; however, AcOEC2 caused severe black rot symptoms in both the central axis and juice sac areas. These results indicate that AcCreA-mediated catabolite repression controls the virulence or infection of this pathogen, and that the wild-type A. citri does not cause symptoms in the juice sac area due to carbon catabolite repression by sugars in the juice of the juice sac area.

scholarly journals Control of Inducer Accumulation Plays a Key Role in Succinate-Mediated Catabolite Repression in Sinorhizobiummeliloti

2002 ◽  
Vol 184 (19) ◽  
pp. 5385-5392 ◽  
Author(s):  
Ryan M. Bringhurst ◽  
Daniel J. Gage
Keyword(s):  
Catabolite Repression ◽  
Double Mutant ◽  
Minimal Medium ◽  
Carbon Sources ◽  
Constitutive Expression ◽  
Dicarboxylic Acids ◽  
Genetic Screen ◽  
Aberrant Expression ◽  
Inducer Exclusion ◽  
Secondary Carbon

ABSTRACT The symbiotic, nitrogen-fixing bacterium Sinorhizobium meliloti favors succinate and related dicarboxylic acids as carbon sources. As a preferred carbon source, succinate can exert catabolite repression upon genes needed for the utilization of many secondary carbon sources, including the α-galactosides raffinose and stachyose. We isolated lacR mutants in a genetic screen designed to find S. meliloti mutants that had abnormal succinate-mediated catabolite repression of the melA-agp genes, which are required for the utilization of raffinose and other α-galactosides. The loss of catabolite repression in lacR mutants was seen in cells grown in minimal medium containing succinate and raffinose and grown in succinate and lactose. For succinate and lactose, the loss of catabolite repression could be attributed to the constitutive expression of β-galactoside utilization genes in lacR mutants. However, the inactivation of lacR did not cause the constitutive expression of α-galactoside utilization genes but caused the aberrant expression of these genes only when succinate was present. To explain the loss of diauxie in succinate and raffinose, we propose a model in which lacR mutants overproduce β-galactoside transporters, thereby overwhelming the inducer exclusion mechanisms of succinate-mediated catabolite repression. Thus, some raffinose could be transported by the overproduced β-galactoside transporters and cause the induction of α-galactoside utilization genes in the presence of both succinate and raffinose. This model is supported by the restoration of diauxie in a lacF lacR double mutant (lacF encodes a β-galactoside transport protein) grown in medium containing succinate and raffinose. Biochemical support for the idea that succinate-mediated repression operates by preventing inducer accumulation also comes from uptake assays, which showed that cells grown in raffinose and exposed to succinate have a decreased rate of raffinose transport compared to control cells not exposed to succinate.

scholarly journals The Transcriptional Activator XlnR Regulates Both Xylanolytic and Endoglucanase Gene Expression inAspergillus niger

1998 ◽  
Vol 64 (10) ◽  
pp. 3615-3619 ◽  
Author(s):  
Noël N. M. E. van Peij ◽  
Marco M. C. Gielkens ◽  
Ronald P. de Vries ◽  
Jaap Visser ◽  
Leo H. de Graaff
Keyword(s):  
Cellulose Degradation ◽  
Mrna Level ◽  
Transcriptional Activator ◽  
Feruloyl Esterase ◽  
Loss Of Function ◽  
Gene Encoding ◽  
Xylan Degradation ◽  
Genes Encoding ◽  
Encoding Genes ◽  
Xylanolytic Enzymes

ABSTRACT The expression of genes encoding enzymes involved in xylan degradation and two endoglucanases involved in cellulose degradation was studied at the mRNA level in the filamentous fungusAspergillus niger. A strain with a loss-of-function mutation in the xlnR gene encoding the transcriptional activator XlnR and a strain with multiple copies of this gene were investigated in order to define which genes are controlled by XlnR. The data presented in this paper show that the transcriptional activator XlnR regulates the transcription of thexlnB, xlnC, and xlnD genes encoding the main xylanolytic enzymes (endoxylanases B and C and β-xylosidase, respectively). Also, the transcription of the genes encoding the accessory enzymes involved in xylan degradation, including α-glucuronidase A, acetylxylan esterase A, arabinoxylan arabinofuranohydrolase A, and feruloyl esterase A, was found to be controlled by XlnR. In addition, XlnR also activates transcription of two endoglucanase-encoding genes, eglA andeglB, indicating that transcriptional regulation by XlnR goes beyond the genes encoding xylanolytic enzymes and includes regulation of two endoglucanase-encoding genes.

scholarly journals The Functional ccpA Gene Is Required for Carbon Catabolite Repression in Lactobacillus plantarum

2001 ◽  
Vol 67 (7) ◽  
pp. 2903-2907 ◽  
Author(s):  
Lidia Muscariello ◽  
Rosangela Marasco ◽  
Maurilio De Felice ◽  
Margherita Sacco
Keyword(s):  
Lactobacillus Plantarum ◽  
Mutant Strain ◽  
Catabolite Repression ◽  
Carbon Catabolite Repression ◽  
Protein A ◽  
Regulatory Region ◽  
Gene Encoding ◽  
Upstream Promoter ◽  
In Vivo Footprinting

ABSTRACT We report the characterization of the ccpA gene ofLactobacillus plantarum, coding for catabolite control protein A. The gene is linked to the pepQ gene, encoding a proline peptidase, in the order ccpA-pepQ, with the two genes transcribed in tandem from the same strand as distinct transcriptional units. Two ccpA transcription start sites corresponding to two functional promoters were found, expression from the upstream promoter being autogenously regulated through a catabolite-responsive element (cre) sequence overlapping the upstream +1 site. During growth on ribose, the upstream promoter showed maximal expression, while growth on glucose led to transcription from the downstream promoter. In a ccpA mutant strain, the gene was transcribed mainly from the upstream promoter in both repressing and non repressing conditions. Expression of two enzyme activities, β-glucosidase and β-galactosidase, was relieved from carbon catabolite repression in the ccpA mutant strain. In vivo footprinting analysis of the catabolite-controlledbglH gene regulatory region in the ccpA mutant strain showed loss of protection of the cre under repressing conditions.

scholarly journals Gain-of-function GABRB3 variants identified in vigabatrin-hypersensitive epileptic encephalopathies

2020 ◽  
Vol 2 (2) ◽  
Author(s):  
Nathan L Absalom ◽  
Vivian W Y Liao ◽  
Kavitha Kothur ◽  
Dinesh C Indurthi ◽  
Bruce Bennetts ◽  
...  
Keyword(s):  
De Novo ◽  
Drug Effects ◽  
Receptor Expression ◽  
Aminobutyric Acid ◽  
Loss Of Function ◽  
Molecular Phenotype ◽  
Gain Of Function ◽  
Gene Encoding ◽  
Epileptic Encephalopathies ◽  
Receptor Phenotype

Abstract Variants in the GABRB3 gene encoding the β3-subunit of the γ-aminobutyric acid type A ( receptor are associated with various developmental and epileptic encephalopathies. Typically, these variants cause a loss-of-function molecular phenotype whereby γ-aminobutyric acid has reduced inhibitory effectiveness leading to seizures. Drugs that potentiate inhibitory GABAergic activity, such as nitrazepam, phenobarbital or vigabatrin, are expected to compensate for this and thereby reduce seizure frequency. However, vigabatrin, a drug that inhibits γ-aminobutyric acid transaminase to increase tonic γ-aminobutyric acid currents, has mixed success in treating seizures in patients with GABRB3 variants: some patients experience seizure cessation, but there is hypersensitivity in some patients associated with hypotonia, sedation and respiratory suppression. A GABRB3 variant that responds well to vigabatrin involves a truncation variant (p.Arg194*) resulting in a clear loss-of-function. We hypothesized that patients with a hypersensitive response to vigabatrin may exhibit a different γ-aminobutyric acid A receptor phenotype. To test this hypothesis, we evaluated the phenotype of de novo variants in GABRB3 (p.Glu77Lys and p.Thr287Ile) associated with patients who are clinically hypersensitive to vigabatrin. We introduced the GABRB3 p.Glu77Lys and p.Thr287Ile variants into a concatenated synaptic and extrasynaptic γ-aminobutyric acid A receptor construct, to resemble the γ-aminobutyric acid A receptor expression by a patient heterozygous for the GABRB3 variant. The mRNA of these constructs was injected into Xenopus oocytes and activation properties of each receptor measured by two-electrode voltage clamp electrophysiology. Results showed an atypical gain-of-function molecular phenotype in the GABRB3 p.Glu77Lys and p.Thr287Ile variants characterized by increased potency of γ-aminobutyric acid A without change to the estimated maximum open channel probability, deactivation kinetics or absolute currents. Modelling of the activation properties of the receptors indicated that either variant caused increased chloride flux in response to low concentrations of γ-aminobutyric acid that mediate tonic currents. We therefore propose that the hypersensitivity reaction to vigabatrin is a result of GABRB3 variants that exacerbate GABAergic tonic currents and caution is required when prescribing vigabatrin. In contrast, drug strategies increasing tonic currents in loss-of-function variants are likely to be a safe and effective therapy. This study demonstrates that functional genomics can explain beneficial and adverse anti-epileptic drug effects, and propose that vigabatrin should be considered in patients with clear loss-of-function GABRB3 variants.

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