Expression of firefly luciferase in Candida albicans and its use in the selection of stable transformants.
The infectious yeast Candida albicans is a model organism for understanding the mechanisms of fungal pathogenicity. We describe the functional expression of the firefly luciferase gene, a reporter commonly used to tag genes in many other cellular systems. Due to a non-standard codon usage by this yeast, the CUG codons were first mutated to UUG to allow functional expression. When integrated into the chromosome of C. albicans with a strong constitutive promoter, cells bioluminesce when provided with luciferin substrate in their media. When fused to the inducible promoter from the HWP1 gene, expression and bioluminescence was only detected in cultures conditioning hyphal growth. We further used the luciferase gene as a selection to isolate transformed cell lines from clinical isolates of C. albicans, using a high-density screening strategy that purifies transformed colonies by virtue of light emission. This strategy requires no drug or auxotrophic selectable marker, and we were thus able to generate stable transformants of clinical isolates that are identical to the parental strain in all aspects tested, other than their bioluminescence. The firefly luciferase gene can, therefore, be used as a sensitive reporter to analyze gene function both in laboratory and clinical isolates of this medically important yeast.
Doyle
Timothy C
TC
Xenogen Corporation, 860 Atlantic Avenue, Alameda, CA 94501, USA. timbo@cmgm.stanford.edu
Nawotka
Kevin A
KA
Purchio
Anthony F
AF
Akin
Ali R
AR
Francis
Kevin P
KP
Contag
Pamela R
PR
D015139:Blotting, Southern / Q000379:methods; D002176:Candida albicans / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification* / Q000502:physiology; D003062:Codon / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D004271:DNA, Fungal / Q000032:analysis; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D023061:Gene Order; D005818:Genetic Engineering / Q000379:methods*; D005819:Genetic Markers / Q000502:physiology; D005822:Genetic Vectors / Q000235:genetics; D049409:Luciferases, Firefly / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics*; D008163:Luminescent Measurements; D016297:Mutagenesis, Site-Directed / Q000379:methods; D010957:Plasmids / Q000235:genetics; D013997:Time Factors; D014170:Transformation, Genetic / Q000235:genetics
D003062:Codon; D017931:DNA Primers; D004271:DNA, Fungal; D005819:Genetic Markers; D049409:Luciferases, Firefly