Generation of Viable <named-content content-type="genus-species">Candida albicans</named-content> Mutants Lacking the “Essential” Protein Kinase Snf1 by Inducible Gene Deletion

Abstract

ABSTRACT The protein kinase Snf1, a member of the highly conserved AMP-activated protein kinase family, is a central regulator of metabolic adaptation. In the pathogenic yeast Candida albicans, Snf1 is considered to be essential, as previous attempts by different research groups to generate homozygous snf1Δ mutants were unsuccessful. We aimed to elucidate why Snf1 is required for viability in C. albicans by generating snf1Δ null mutants through forced, inducible gene deletion and observing the terminal phenotype before cell death. Unexpectedly, we found that snf1Δ mutants were viable and could grow, albeit very slowly, on rich media containing the preferred carbon source glucose. Growth was improved when the cells were incubated at 37°C instead of 30°C, and this phenotype enabled us to isolate homozygous snf1Δ mutants also by conventional, sequential deletion of both SNF1 alleles in a wild-type C. albicans strain. All snf1Δ mutants could grow slowly on glucose but were unable to utilize alternative carbon sources. Our results show that, under optimal conditions, C. albicans can live and grow without Snf1. Furthermore, they demonstrate that inducible gene deletion is a powerful method for assessing gene essentiality in C. albicans. IMPORTANCE Essential genes are those that are indispensable for the viability and growth of an organism. Previous studies indicated that the protein kinase Snf1, a central regulator of metabolic adaptation, is essential in the pathogenic yeast Candida albicans, because no homozygous snf1 deletion mutants of C. albicans wild-type strains could be obtained by standard approaches. In order to investigate the lethal consequences of SNF1 deletion, we generated conditional mutants in which SNF1 could be deleted by forced, inducible excision from the genome. Unexpectedly, we found that snf1 null mutants were viable and could grow slowly under optimal conditions. The growth phenotypes of the snf1Δ mutants explain why such mutants were not recovered in previous attempts. Our study demonstrates that inducible gene deletion is a powerful method for assessing gene essentiality in C. albicans.

Keywords

• Candida albicans
• Snf1
• conditional mutants
• essential genes
• protein kinases