RESUMO
BACKGROUND: Ion homeostasis is an essential property of living organisms. The yeast Saccharomyces cerevisiae is an ideal model organism to investigate ion homeostasis at all levels. In this yeast genes involved in high-affinity phosphate uptake (PHO genes) are strongly induced during both phosphate and potassium starvation, indicating a link between phosphate and potassium homeostasis. However, the signal transduction processes involved are not completely understood. As 14-3-3 proteins are key regulators of signal transduction processes, we investigated the effect of deletion of the 14-3-3 genes BMH1 or BMH2 on gene expression during potassium starvation and focused especially on the expression of genes involved in phosphate uptake. RESULTS: Genome-wide analysis of the effect of disruption of either BMH1 or BMH2 revealed that the mRNA levels of the PHO genes PHO84 and SPL2 are greatly reduced in the mutant strains compared to the levels in wild type strains. This was especially apparent at standard potassium and phosphate concentrations. Furthermore the promoter of these genes is less active after deletion of BMH1. Microscopic and flow cytometric analysis of cells with GFP-tagged SPL2 showed that disruption of BMH1 resulted in two populations of genetically identical cells, cells expressing the protein and the majority of cells with no detectible expression. Heterogeneity was also observed for the expression of GFP under control of the PHO84 promoter. Upon deletion of PHO80 encoding a regulator of the transcription factor Pho4, the effect of the BMH1 deletion on SPL2 and PHO84 promoter was lost, suggesting that the BMH1 deletion mainly influences processes upstream of the Pho4 transcription factor. CONCLUSION: Our data indicate that that yeast cells can be in either of two states, expressing or not expressing genes required for high-affinity phosphate uptake and that 14-3-3 proteins are involved in the process(es) that establish the activation state of the PHO regulon.
Assuntos
Proteínas 14-3-3/genética , Proteínas Inibidoras de Quinase Dependente de Ciclina/genética , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Simportadores de Próton-Fosfato/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas 14-3-3/deficiência , Transporte Biológico/genética , Fosfatos/metabolismo , Potássio/metabolismoRESUMO
BACKGROUND: The filamentous fungi Paecilomyces variotii and Penicillium roqueforti are prevalent food spoilers and are of interest as potential future cell factories. A functional CRISPR/Cas9 genome editing system would be beneficial for biotechnological advances as well as future (genetic) research in P. variotii and P. roqueforti. RESULTS: Here we describe the successful implementation of an efficient AMA1-based CRISPR/Cas9 genome editing system developed for Aspergillus niger in P. variotii and P. roqueforti in order to create melanin deficient strains. Additionally, kusA- mutant strains with a disrupted non-homologous end-joining repair mechanism were created to further optimize and facilitate efficient genome editing in these species. The effect of melanin on the resistance of conidia against the food preservation stressors heat and UV-C radiation was assessed by comparing wild-type and melanin deficient mutant conidia. CONCLUSIONS: Our findings show the successful use of CRISPR/Cas9 genome editing and its high efficiency in P. variotii and P. roqueforti in both wild-type strains as well as kusA- mutant background strains. Additionally, we observed that melanin deficient conidia of three food spoiling fungi were not altered in their heat resistance. However, melanin deficient conidia had increased sensitivity towards UV-C radiation.