Sulfur-regulated control of the met-2 + gene of Neurospora crassa encoding cystathionine β-lyase
© Reveal and Paietta; licensee BioMed Central Ltd. 2013
Received: 9 January 2013
Accepted: 3 July 2013
Published: 8 July 2013
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.
The cystathionine β-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 Δcys-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.
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 Δcys-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.
Neurospora crassa provides a useful model system to dissect the regulation and dynamics of the transsulfuration pathway [6–8]. Early work with met-2 mutants of N. crassa confirmed that they lacked cystathionine β-lyase activity . In N. crassa, cystathionine β-lyase has been purified and enzymatic activity assayed under several growth conditions [9, 10]. The most significant difference reported  was that the level of cystathionine β-lyase activity was reduced in wild-type by one-fourth over the reported baseline when grown with methionine supplemented at the highest level tested (i.e., 5 μmoles/ml) with little change noted at lower levels of supplementation. N. crassa microarray expression data collected at different stages of development demonstrate only modest changes in met-2+ expression during, for example, conidial germination or colony development (e.g., 1.4× higher early in germination versus 15 hr of growth) [11, 12]. Limited data is available regarding the regulation of cystathionine β-lyase in other fungi. In Aspergillus nidulans, the cystathionine β-lyase gene is encoded by the metG locus and the gene has been cloned and characterized . Analysis of the transcription of metG demonstrated constitutive expression with no apparent regulation by sulfur source . Cystathionine β-lyase activity in A. nidulans was found to be repressed by methionine , but constitutively present in another study . For Saccharomyes cerevisiae, comparable expression data for growth under different levels of sulfur supplementation is not available for cystathionine β-lyase, which is encoded by the STR3 gene . Based on microarray expression data, STR3 expression does increase by 12-fold under fermentation stress response conditions . In addition, the suggestion has been made that there is a role for Gto1 (omega-class glutathione transferase) in the redox regulation of S. cerevisiae cystathionine β-lyase .
An important question is whether there is a regulatory connection of the transsulfuration-related genes, including cystathionine β-lyase, to the N. crassa sulfur regulatory system which is made up of a set of trans-acting regulatory genes and a set of genes encoding a variety of sulfur-related enzymes and transporters. When N. crassa is cultured under sulfur-limiting conditions (i.e., derepressing conditions) then the coordinate expression of this set of sulfur-related genes occurs. The regulated genes include arylsulfatase, choline sulfatase, sulfate permease I and II, among others [6, 8]. Recently, cystathionine γ-lyase has been added to the list of genes confirmed to be under control of this key regulatory circuit . CYS3, a bZIP transcriptional activator, is the key regulator and necessary for regulation expression of these sulfur-related genes . Based on binding-site studies, a consensus sequence for CYS3 binding has been determined .
We present here the cloning and regulatory analysis of the N. crassa met-2 + cystathionine β- lyase gene. This report demonstrates a new connection of cystathionine β-lyase to the CYS3-controlled regulatory network and provides us with important data for the continued dissection of the regulatory dynamics of transsulfuration.
Results and discussion
Sequence and characterization of the cystathionine β-lyase gene
Analysis of met-2 + gene expression
In order to test whether met-2 + is subject to regulation by CYS3, Northern blot analysis was carried out using poly(A)+ mRNA isolated from a cys-3 mutant deletion mutant (Δcys-3) and probed with the met-2 + gene. For this case, the met-2 + transcript was detectable only at a very low level under either low- or high-sulfur culture conditions. In fact, the levels observed in Δcys-3 were slightly lower than in wild-type under repressing conditions (based on phosphorimager quantification). The reduction in met-2 + transcript level in the Δcys-3 background is typical of genes that have been demonstrated to be part of the N. crassa sulfur regulatory system in previous studies [6, 8].
Gel mobility shift analysis of the met-2 + promoter
The expression of the N. crassa met-2 + gene, which encodes cystathionine β-lyase, was found to increase in level under culture conditions of sulfur limitation. Further, the reduced in vivo expression of met-2 + seen in a Δcys-3 background along with the in vitro detection of CYS3 binding sites within the met-2 + promoter supports a role for CYS3-mediated regulation of this gene. The data add to the continuing analysis of the regulatory dynamics of transsulfuration in this model system.
Strains, plasmids and culture conditions
The met-2 mutant strain FGSC#4061 and Orbach/Sachs N. crassa genomic library  that was constructed with the cosmid pMOCosX  were obtained from the Fungal Genetics Stock Center (University of Missouri-Kansas City). The ∆cys-3 (18–4) deletion strain was constructed as described previously . The strain 74OR23-1a was used as wild-type for these experiments. Vogel minimal medium , with supplements as required, was used. Sulfur repression and derepression experiments were carried out as described previously by culturing mycelia at 25°C on Vogel-minus-sulfur medium plus high sulfur (5.0 mM methionine) and low-sulfur medium (0.25 mM methionine) medium, respectively .
Gene cloning and sequencing
The N. crassa cosmid library was subdivided into chromosome IV specific clones which were then used in individual transformations of met- 2 with selection for methionine prototrophy and hygromycin resistance. A single clone designated G8F01 was found that was capable of transforming met- 2. Subsequent met- 2 transformation tests identified a 4.1 kb fragment which contained the met-2 + gene. Following subcloning into pSPORT, the segment was subjected to automated sequencing (Cleveland Genomics; Cleveland, OH) by primer walking. The genomic sequence was submitted to GenBank as AF401237. The met-2 + gene has been assigned the locus designation NCU07987 in the Broad Institute Neurospora crassa database .
Total RNA was isolated by the phenol extraction procedure described previously . Following sodium acetate washes, poly(A)+ mRNA was isolated by oligo(dT) cellulose chromatography. Oligolabeling of DNA fragments was used to prepare 32P-labeled probes and used to probe Northern blots prepared as outlined elsewhere . Quantitation of Northern blots was accomplished by using a Molecular Dynamics Phosphorimager.
Gel mobility shifts
Initially, the met-2 + promoter was subjected to restriction endonuclease digestion to generate a series of overlapping fragments tested in gel mobility shift assays. Subsequent identification of fragments that bound CYS3 allowed for the synthesis of short segments (24 bp) that retained binding activity. The 24 bp oligonucleotides representing the binding sites to be further tested were synthesized with an Applied Biosystems 391EP synthesizer, labeled using [ϒ-32P]ATP, annealed and gel purified as described previously [20, 30]. Four oligonucleotides (and complementary strands) representing putative CYS3 binding sites on the met-2 + promoter were analyzed: Site 1 [ 5′ GAAAAGGATGGCGAATTTTAGTGA 3′], Site 2 [ 5′ GGTCTAGGTGTTATCATCTGGTGG 3′], Site 3 [5′GGCCCTGATTTCGCCATTTTCTTT 3′], and Site 4 [5′ TTGACTCATCACACCATCGGCCTC 3′]. Mutated CYS3 binding sites had a purine to pyrimidine substitution at the sixth position of the 10 bp core of the CYS3 consensus binding site: Site 1 M [ 5′ GAAAAGGATGGCC AATTTTAGTGA 3′], Site 2 M [5′ GGTCTAGGTGTTC TCATCTGGTGG 3′], Site 3 M [5′ GGCCCTGATTTCT CCATTTTCTTT 3′], and Site 4 M [5′ TTGACTCATCACC CCATCGGCCTC 3′]. E. coli produced CYS3 protein  was used for gel shift assays as we have described [20, 30]. A Molecular Dynamics Phosphorimager was used to quantify the gel shift binding assays.
Availability of supporting data
The sequence data supporting the results of this article is available in the GenBank respository [AF401237, http://www.ncbi.nlm.nih.gov/nuccore/AF401237].
The work was supported by a Medical Innovations Grant from the Wright State University Boonshoft School of Medicine.
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