Generation of Viable Candida albicans Mutants Lacking the "Essential" Protein Kinase Snf1 by Inducible Gene Deletion.

Austin Mottola,Sonja Schwanfelder,Joachim Morschhäuser,Aaron P Mitchell

doi:10.1128/msphere.00805-20

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. albicansIMPORTANCE 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.

Highlights

The protein kinase Snf1, a member of the highly conserved AMPactivated protein kinase family, is a central regulator of metabolic adaptation
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
The catalytic subunit Snf1 seems to be essential in C. albicans, which is not the case in S. cerevisiae, as numerous attempts by different research groups to construct homozygous snf1Δ mutants have failed

Summary

Introduction

The protein kinase Snf, a member of the highly conserved AMPactivated protein kinase family, is a central regulator of metabolic adaptation. Either of the two SNF1 alleles, which were distinguished by a restriction site polymorphism, could be deleted, no homozygous snf1Δ mutants were obtained after transformation of the heterozygous mutants with the same deletion cassette and selection for nourseothricin-resistant mutants on rich medium, as expected for an essential gene While these studies provided strong evidence for the essentiality of Snf in C. albicans, the reason for this remained unresolved (but see below). After passage of the conditional mutants in SAP2-inducing medium, the deletable copy is excised by FLP-mediated recombination from the vast majority of the cells, generating an almost pure population of null mutants that can be phenotypically studied [9] This strategy provides firm proof for the essentiality (or not) of a gene, because true null mutants are obtained whose viability can be tested under any desired condition (as opposed to conditional mutants that have to be observed under specific, nonpermissive conditions)

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