Development of a Candida glabrata dominant nutritional transformation marker utilizing the Aspergillus nidulans acetamidase gene (amdS)

• Published in: FEMS yeast research 2016-05, Vol.16 (3), p.fow023
• Main Authors: Fu, Jianmin, Blaylock, Morganne, Wickes, Cameron F., Welte, William, Mehrtash, Adrian, Wiederhold, Nathan, Wickes, Brian L.
• Format: Article
• Language: English
• Subjects: Amidohydrolases - genetics; Amidohydrolases - metabolism; Aspergillus nidulans - enzymology; Aspergillus nidulans - genetics; Candida albicans - genetics; Candida glabrata - genetics; Candida glabrata - metabolism; Cloning, Molecular; Genetics, Microbial - methods; Molecular Biology - methods; Plasmids; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Regulatory Sequences, Nucleic Acid; Selection, Genetic; Transcription, Genetic; Transformation, Genetic
• ISSN: 1567-1364; 1567-1356; 1567-1364
• DOI: 10.1093/femsyr/fow023

The gene encoding Aspergillus nidulans acetamidase (amdS) was placed under control of Candida albicans ACT1 promoter and terminator sequences and then cloned into a plasmid containing C. glabrata ARS10, CEN8 or ARS10+CEN8 sequences. All plasmids transformed C. glabrata wild-type cells to acetamide+, with the ARS-only containing plasmid transforming cells at the highest frequencies (>1.0 × 104 transformants μg−1). Plasmids were rapidly lost under non-selective conditions with the frequency dependent on chromosomal element, thus recycling the acetamide– phenotype. The amdS plasmid was used to transform a set of clinical isolates resistant to a variety of antifungal drugs. All strains were successfully transformed to the acetamide+ phenotype at high frequency, confirming that this plasmid construct could be used as a simple dominant marker on virtually any strain. Gap repair experiments demonstrated that just as in Saccharomyces cerevisiae, gap repair functions efficiently in C. glabrata, suggesting that C. glabrata has numerous similarities to S. cerevisiae with regard to ease of molecular manipulation. The amdS system is inexpensive and efficient, and combined with existing C. glabrata plasmid elements, confers a high transformation frequency for C. glabrata with a phenotype that can be easily recycled. The Aspergillus nidulans amdS gene was successfully used as a heterologous dominant marker for the yeast pathogen, Candida glabrata. Graphical Abstract Figure. The Aspergillus nidulans amdS gene was successfully used as a heterologous dominant marker for the yeast pathogen, Candida glabrata.