Dataset Information

Sulfur-regulated control of the met-2? gene of Neurospora crassa encoding cystathionine ?-lyase.

ABSTRACT: BACKGROUND: Cystathionine ?-lyase performs an essential role in the transsulfuration pathway by its primary reaction of forming homocysteine from cystathionine. Understanding how the Neurospora crassa met-2? gene, which encodes cystathionine ?-lyase, is regulated is important in determining the basis of the cellular control of transsulfuration. The aim of this study was to determine the nature of a potential regulatory connection of met-2? to the Neurospora sulfur regulatory network. FINDINGS: The cystathionine ?-lyase (met-2?) gene was cloned by the identification of a cosmid genomic clone capable of transforming a met-2 mutant to methionine prototrophy and subsequently characterized. The gene contains a single intron and encodes a protein of 457 amino acids with conserved residues predicted to be important for catalysis and pyridoxal-5'-phosphate co-factor binding. The expression of met-2? in wild-type N. crassa increased 3.1-fold under sulfur-limiting growth conditions as compared to the transcript levels seen under high sulfur growth conditions (i.e., repressing conditions). In a ?cys-3 strain, met-2? transcript levels were substantially reduced under either low- or high-sulfur growth conditions. In addition, the presence of CYS3 activator binding sites on the met-2? promoter was demonstrated by gel mobility shift assays. CONCLUSIONS: In this report, we demonstrate the sulfur-regulated expression of the met-2? gene and confirm its connection to the N. crassa sulfur regulatory circuit by the reduced expression observed in a ?cys-3 mutant and the in vitro detection of CYS3 binding sites in the met-2? promoter. The data further adds to our understanding of the regulatory dynamics of transsulfuration.

SUBMITTER: Reveal BS

PROVIDER: S-EPMC3716945 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Sulfur-regulated control of the met-2⁺ gene of Neurospora crassa encoding cystathionine β-lyase.

Reveal Brad S BS Paietta John V JV

BMC research notes 20130708

<h4>Background</h4>Cystathionine β-lyase performs an essential role in the transsulfuration pathway by its primary reaction of forming homocysteine from cystathionine. Understanding how the Neurospora crassa met-2⁺ gene, which encodes cystathionine β-lyase, is regulated is important in determining the basis of the cellular control of transsulfuration. The aim of this study was to determine the nature of a potential regulatory connection of met-2⁺ to the Neurospora sulfur regulatory network.<h4>F ...[more]

PMID: 23835025

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Project description:Cellulolytic fungi have evolved a complex regulatory network to maintain the precise balance of nutrients required for growth and hydrolytic enzyme production. When fungi are exposed to cellulose, the transcript levels of cellulase genes rapidly increase and then decline. However, the mechanisms underlying this bell-shaped expression pattern are unclear. We systematically screened a protein kinase deletion set in the filamentous fungus Neurospora crassa to search for mutants exhibiting aberrant expression patterns of cellulase genes. We observed that the loss of stk-12 (NCU07378) caused a dramatic increase in cellulase production and an extended period of high transcript abundance of major cellulase genes. These results suggested that stk-12 plays a critical role as a brake to turn down the transcription of cellulase genes to repress the overexpression of hydrolytic enzymes and prevent energy wastage. Transcriptional profiling analyses revealed that cellulase gene expression levels were maintained at high levels for 56 h in the ?stk-12 mutant, compared to only 8 h in the wild-type (WT) strain. After growth on cellulose for 3 days, the transcript levels of cellulase genes in the ?stk-12 mutant were 3.3-fold over WT, and clr-2 (encoding a transcriptional activator) was up-regulated in ?stk-12 while res-1 and rca-1 (encoding two cellulase repressors) were down-regulated. Consequently, total cellulase production in the ?stk-12 mutant was 7-fold higher than in the WT. These results strongly suggest that stk-12 deletion results in dysregulation of the cellulase expression machinery. Further analyses showed that STK-12 directly targets IGO-1 to regulate cellulase production. The TORC1 pathway promoted cellulase production, at least partly, by inhibiting STK-12 function, and STK-12 and CRE-1 functioned in parallel pathways to repress cellulase gene expression. Our results clarify how cellulase genes are repressed at the transcriptional level during cellulose induction, and highlight a new strategy to improve industrial fungal strains.

Project description:BackgroundHuman uterus undergoes distinct molecular and functional changes during pregnancy and parturition. Hydrogen sulfide (H(2)S) has recently been shown to play a key role in the control of smooth muscle tension. The role of endogenous H(2)S produced locally in the control of uterine contractility during labour is unknown.Methodology/principal findingsHuman myometrium biopsies were obtained from pregnant women undergoing cesarean section at term. Immunohistochemistry analysis showed that cystathionine-γ-lyase (CSE) and cystathionine-β-synthetase (CBS), the principle enzymes responsible for H(2)S generation, were mainly localized to smooth muscle cells of human pregnant myometrium. The mRNA and protein expression of CBS as well as H(2)S production rate were down-regulated in labouring tissues compared to nonlabouring tissues. Cumulative administration of L-cysteine (10(-7)-10(-2) mol/L), a precursor of H(2)S, caused a dose-dependent decrease in the amplitude of spontaneous contractions in nonlabouring and labouring myometrium strips. L-cysteine at high concentration (10(-3) mol/L) increased the frequency of spontaneous contractions and induced tonic contraction. These effects of L-cysteine were blocked by the inhibitors of CBS and CSE. Pre-treatment of myometrium strips with glibenclamide, an inhibitor of ATP-sensitive potassium (K(ATP)) channels, abolished the inhibitory effect of L-cysteine on spontaneous contraction amplitude. The effects of L-cysteine on the amplitude of spontaneous contractions and baseline muscle tone were less potent in labouring tissues than that in nonlabouring strips.Conclusion/significanceH(2)S generated by CSE and CBS locally exerts dual effects on the contractility of pregnant myometrium. Expression of H(2)S synthetic enzymes is down-regulated during labour, suggesting that H(2)S is one of the factors involved in the transition of pregnant uterus from quiescence to contractile state after onset of parturition.

Dataset Information

Sulfur-regulated control of the met-2? gene of Neurospora crassa encoding cystathionine ?-lyase.

Publications

Sulfur-regulated control of the met-2⁺ gene of Neurospora crassa encoding cystathionine β-lyase.

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