Modulation of the complex regulatory network for methionine biosynthesis in fungi

Abstract

The assimilation of inorganic sulfate and the synthesis of the sulfur-containing amino acids methionine and cysteine is mediated by a multibranched biosynthetic pathway. We have investigated this circuitry in the fungal pathogen Candida albicans, which is phylogenetically intermediate between the filamentous fungi and Saccharomyces cerevisiae. In S. cerevisiae, this pathway is regulated by a collection of five transcription factors (Met4, Cbf1, Met28, and Met31/Met32), while in the filamentous fungi the pathway is controlled by a single Met4-like factor. We found that in C. albicans, the Met4 ortholog is also a core regulator of methionine biosynthesis, where it functions together with Cbf1. While C. albicans encodes this Met4 protein, a Met4 paralog designated Met28 (Orf19.7046), and a Met31 protein, deletion, and activation constructs suggest that of these proteins only Met4 is actually involved in the regulation of methionine biosynthesis. Both Met28 and Met31 are linked to other functions; Met28 appears essential, and Met32 appears implicated in the regulation of genes of central metabolism. Therefore, while S. cerevisiae and C. albicans share Cbf1 and Met4 as central elements of the methionine biosynthesis control, the other proteins that make up the circuit in S. cerevisiae are not members of the C. albicans control network, and so the S. cerevisiae circuit likely represents a recently evolved arrangement.

Keywords: genetics; methionine biosynthesis; regulatory complexes; rewiring; transcription factor.

Figure 1

Phylogenetic tree. (A) The tree is constructed based on the sequence similarities of Met4 and Met28 across the fungal kingdom. A block diagram showing all the domains of the transcription factors. (B) The sequence alignment of subdomain that are highlighted by star with respective color shows that the disrupted B-zip domain, AUX and INT domain are present only in S. cerevisiae and K. lactis clade whereas in the rest of fungi complete B-Zip domain is conserved.

Figure 2

Activation and deletion of Met4 and Met32 in C. albicans. (A) Methionine starvation of strain SC5314, Met4-deleted strain and Met32 deleted strain. The Met4 deleted strain shows no growth during methionine starvation. (B) Transcriptomic profile of top 50 upregulated genes in the Met4 activated strain show upregulation of methionine biosynthesis-related genes along with some important SCF^Met30-related genes. There is no obvious functional enrichment within the Met4 upregulated genes that are not linked to methionine regulation.

Figure 3

Genes upregulated by Met28 activation. (A) Top 50 upregulated genes generated by activation of the CaMet28 transcription factor. There are 9 essential genes (in red color), 10 genes that have the AnMet28 DNA binding motif 5ʹTGRYRYCA 3ʹ, 23 genes that are annotated as biological function unknown, and 7 genes that encode candidate secreted or cell wall associated proteins (in blue color). (B) AnMet28 DNA binding motif 5ʹTGRYRYCA 3ʹ. (C) The location of the AnMet28 DNA binding motif in the upstream regions of specific upregulated genes.

Figure 4

Search for the Met4 Motif. (A) We used the Meme-suit online tool to look for potential regulatory binding sites. This approach identifies the Cbf1 motif in the upstream region of the upregulated methionine genes. The second-best motif we found is 5ʹCAACTCCAAR 3ʹ that may represent a Met4-binding motif. (B) A diagram highlighting the potential binding motifs of Cbf1 and Met4 throughout the methionine biosynthesis circuit.

Figure 5

Phylogenetic analysis of methionine regulatory transcriptional factors across the ascomycetes. In the phylogenetic tree, a Met4 ortholog is involved with the methionine biosynthesis in most of the fungi. Cbf1 was rewired and connects to methionine regulation in the CTG clade, whereas Met32 and Met28 are connected to the pathway later in the protoploids. The designation “I” for involved in methionine biosynthesis, “NI” for not involved, and “-” for not present in the genome, represent the situation for all species in the group identified.