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

BACKGROUND: 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. FINDINGS: 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. 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 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.