Purine biosynthesis, riboflavin production, and trophic-phase span are controlled by a Myb-related transcription factor in the fungus Ashbya gossypii

Abstract

Ashbya gossypii is a natural riboflavin overproducer used in the industrial production of the vitamin. We have isolated an insertional mutant exhibiting higher levels of riboflavin production than the wild type. DNA analysis of the targeted locus in the mutant strain revealed that a syntenic homolog of the Saccharomyces cerevisiae BAS1 gene, a member of the Myb family of transcription factors, was inactivated. Directed gene disruption of AgBAS1 confirmed the phenotype observed for the insertional mutant, and the Deltabas1 mutant also showed auxotrophy for adenine and several growth defects, such as a delay in the germination of the spores and an abnormally prolonged trophic phase. Additionally, we demonstrate that the DNA-binding domain of AgBas1p is able to bind to the Bas1-binding motifs in the AgADE4 promoter; we also show a clear nuclear localization of a green fluorescent protein-Bas1 fusion protein. Real-time quantitative PCR analyses comparing the wild type and the Deltabas1 mutant revealed that AgBAS1 was responsible for the adenine-mediated regulation of the purine and glycine pathways, since the transcription of the ADE4 and SHM2 genes was virtually abolished in the Deltabas1 mutant. Furthermore, the transcription of ADE4 and SHM2 in the Deltabas1 mutant did not diminish during the transition from the trophic to the productive phase did not diminish, in contrast to what occurred in the wild-type strain. A C-terminal deletion in the AgBAS1 gene, comprising a hypothetical regulatory domain, caused constitutive activation of the purine and glycine pathways, enhanced riboflavin overproduction, and prolonged the trophic phase. Taking these results together, we propose that in A. gossypii, AgBAS1 is an important transcription factor that is involved in the regulation of different physiological processes, such as purine and glycine biosynthesis, riboflavin overproduction, and growth.

FIG. 1.

Schematic representation of the purine biosynthetic pathway. Black arrows designate the de novo pathway, and gray arrows indicate the salvage pathways. Gly, glycine; GMP, guanosine 5′-monophosphate; Ade, adenine, Gua, guanine; Xan, xanthine. The gene names are italicized and correspond to the following enzymatic activities: PRS, PRPP synthetase; ADE4, PRPP amidotransferase; SHM1 and SHM2, serine hydroxymethyltransferase; IMD1, IMP dehydrogenase; AMD1, AMP deaminase; APT1, adenine phosphoribosyltransferase; XPT1, xanthine phosphoribosyltransferase; ADK1, adenylate kinase; RIB, riboflavin genes.

FIG. 2.

Characterization of A. gossypii bas1 mutant strains. (A) Different A. gossypii strains were grown on solid SMM or SMM plus adenine (+ade; 0.1 g/liter). The bas1 mutants exhibit a yellow color due to riboflavin accumulation. (B) Right panel, schematic representation of the wild-type BAS1 and disrupted bas1::G418^r loci. Left panel, Southern blot analysis to confirm correct BAS1 disruption. Genomic DNA of the wild-type and Δbas1 strains was digested with PstI. P, PstI; X, XhoI; B, BamHI; S, SphI. (C) Growth pattern of A. gossypii wild type and Δbas1 grown in liquid MA2 rich medium with (+ade) or without adenine supplementation (0.1 g/liter). (D) Microscopic phenotype of A. gossypii wild type and Δbas1 grown on liquid MA2 rich medium for 12 and 36 h. The germination delay of Δbas1 is restored by the addition of adenine (+ade; 0.1 g/liter). WT, wild type.

FIG. 3.

AgBas1p is localized to the nuclei of A. gossypii. An A. gossyppi spore (left) and a hypha (right) expressing the GFP-Bas1p were stained with the DNA-selective dye Hoechst (HO) 33342 and viewed under epifluorescence optics. Upper images are the differential interference contrast (DIC) pictures; middle images represent the GFP channel, and lower images correspond to the HO channel of the same field.

FIG. 4.

Identification of the DNA-binding site of AgBas1p by EMSA. (A) Structure of the AgADE4 promoter. Gray boxes indicate the Bas1-binding sites. The different probes used in the assays are depicted. (B) EMSA analyses using different probes: probe A, lanes 1 to 3; probe B, lanes 4 to 6; probe A1, lanes 7 to 9; probe A2, lanes 10 to 12, probe A3, lanes 13 to 15. An excess of the corresponding unlabeled probe was used in lanes 3, 6, 8, 11, and 14. No Bas1 DNA-binding domain peptide was included in lanes 1, 4, 7, 10, and 13.

FIG. 5.

Comparison of the steady-state transcription levels of purine and riboflavin genes in wild-type and Δbas1 strains. (A) Differences in the transcription levels of the genes included in the assays between wild-type and Δbas1 strains grown in MA2 rich medium. (B) Relative transcription levels of the indicated genes measured in the wild-type (gray bars) and Δbas1 (white bars) strains grown in MA2 rich medium with (+ade) or without (−ade) an excess of adenine (0.1 g/liter). Transcription levels are normalized to the level of AgACT1 mRNA. The ratio of relative transcription of the target gene in panel A was calculated as 2^−ΔΔCt, where ΔΔCt = ΔCt_Δbas1 − ΔCt_{wild type}. The ratio of the relative transcription of the target gene in panel B was calculated as 2^−ΔΔCt, where ΔΔCt = ΔCt_{MA2 + ade} − ΔCt_{MA2 − ade}. An average of three separate cDNA dilutions from each target gene were obtained, and the relative transcription levels were expressed as log₂ ± SD. vs, versus; WT, wild type.

FIG. 6.

Transcription profiles of purine and riboflavin genes in wild-type and Δbas1 strains during the trophic and productive phases. Transcription profiling of the wild-type (WT) and Δbas1 strains grown in MA2 rich medium was achieved at different time points. In the wild-type strain (upper panel), the transcription of ADE4 and SHM2 decreased along the growth curve, whereas the transcription of RIB1 and RIB3 increased during the productive phase. In the Δbas1 strain (lower panel), ADE4 and SHM2 show a constitutive deregulated transcription. Transcription levels are normalized with the transcription rate of AgACT1.Data are representative of four experiments with similar results and are presented as log₂ of the 2^−ΔΔCt (ΔΔCt = ΔCt_T − ΔCt_{12 h}). T, incubation time (h).

FIG. 7.

Characterization of the ΔC631BAS1 strain. (A) Growth pattern of ΔC631BAS1 strain grown in liquid MA2 rich medium in comparison with the wild-type and Δbas1 strains. (B) Relative transcription levels of purine and riboflavin genes in the wild-type (gray bars) and the ΔC631BAS1 (white bars) strains grown in MA2 rich medium with (+ade) or without (−ade) an excess of adenine (0.1 g/liter). Transcription levels are normalized to the level of AgACT1 mRNA. The ratio of the relative transcription of the target gene was calculated as 2^−ΔΔCt, where ΔΔCt = ΔCt_{MA2 + ade} − ΔCt_{MA2 − ade}. An average of three separate cDNA dilutions from each target gene was obtained, and the relative transcription levels are expressed as log₂ ± SD.