amdS as a dominant recyclable marker in Cryptococcus neoformans

•The amdS gene from A. nidulans enables the use of acetamide as a nitrogen source.•Heterologous expression of amdS in C. neoformans does not influence virulence.•The presence of amdS makes C. neoformans sensitive to fluoroacetamide.•Our amdS marker enables the deletion of multiple genes in one strain.•amdS can seamlessly tag proteins with fluorescent and validate genes as essential. While the fungal pathogen Cryptoccocus neoformans is a leading cause of death in immunocompromised individuals, the molecular toolkit currently available to study this important pathogen is extremely limited. To enable an unprecedented level of control over manipulation of the genome, we have developed a dominant recyclable marker by expanding on the classic studies of the amdS gene by Michael J. Hynes and John Pateman. The ascomycete Aspergillus nidulans employs the acetamidase AmdS to hydrolyse acetamide to ammonium and acetate, which serve as a nitrogen and carbon source, respectively. Acetamidase activity has never been reported in the Basidiomycota. Here we have successfully demonstrated that acetamide can be utilized as a good nitrogen source in C. neoformans heterologously expressing amdS and that this activity does not influence virulence, enabling it to be used as a basic dominant selectable marker. The expression of this gene in C. neoformans also causes sensitivity to fluoroacetamide, permitting counterselection. Taking advantage of this toxicity we have modified our basic marker to create a comprehensive series of powerful and reliable tools to successfully delete multiple genes in the one strain, generate markerless strains with modifications such as fluorescent protein fusions at native genomic loci, and establish whether a gene is essential in C. neoformans.