Transformation of Candida albicans with a synthetic hygromycin B resistance gene

Abstract

Synthetic genes that confer resistance to the antibiotic nourseothricin in the pathogenic fungus Candida albicans are available, but genes conferring resistance to other antibiotics are not. We found that multiple C. albicans strains were inhibited by hygromycin B, so we designed a 1026 bp gene (CaHygB) that encodes Escherichia coli hygromycin B phosphotransferase with C. albicans codons. CaHygB conferred hygromycin B resistance in C. albicans transformed with ars2-containing plasmids or single-copy integrating vectors. Since CaHygB did not confer nourseothricin resistance and since the nourseothricin resistance marker SAT-1 did not confer hygromycin B resistance, we reasoned that these two markers could be used for homologous gene disruptions in wild-type C. albicans. We used PCR to fuse CaHygB or SAT-1 to approximately 1 kb of 5' and 3' noncoding DNA from C. albicans ARG4, HIS1 and LEU2, and introduced the resulting amplicons into six wild-type C. albicans strains. Homologous targeting frequencies were approximately 50-70%, and disruption of ARG4, HIS1 and LEU2 alleles was verified by the respective transformants' inabilities to grow without arginine, histidine and leucine. CaHygB should be a useful tool for genetic manipulation of different C. albicans strains, including clinical isolates.

Figure 1

Restriction map of the ACT1-regulated expression plasmid pYM70. Abbreviations: CaARS2 = autonomously-replicating sequence; ACT1pt = ACT1 promoter; TEF2tm = TEF2 terminator; ori = origin of replication; bla = beta lactamase; TEF2pt = TEF2 promoter; CaHygB = synthetic hygromycin B resistance gene; and ACT1tm = ACT1 terminator.

Figure 2

Growth of C. albicans on hygromycin B and nourseothricin. C. albicans CAI4, C. albicans YPT1 (which contains the SAT-1 marker), and C. albicans CAI4 transformed with pAU34, pAU34-CaHygB, pAU15, pAU15-CaHygB, pYM70, or both pYM70 and pSEC4 were incubated at 30°C for 48 h on solid YP-glucose, YP-maltose or buffered YNB-glucose containing either no antibiotic, hygromycin B, or nourseothricin. Strains in which CaHygB was expressed constitutively (pAU34-CaHygB, pYM70) or was induced by maltose (pAU15-CaHygB) grew in the presence of hygromycin B, and there was no cross-resistance between hygromycin B and nourseothricin.

Figure 3

Targeted disruption of ARG4, HIS1 and LEU2. When the two ARG4, HIS1 and LEU2 alleles were replaced in C. albicans SC5314, WO-1 and B311 with the CaHygB and SAT-1 markers, the resulting mutants acquired the ability to grow on rich medium (YP-glucose) + hygromycin B + nourseothricin. Homologous replacement of the genes of interest was verified by PCR (not shown) and by inability of the arg4 Δ/Δ, his1 Δ/Δ and leu2 Δ/Δ mutants, respectively, to grow on minimal medium (buffered YNB-glucose) containing hygromycin B and nourseothricin without arginine (CSM-Arg), histidine (CSM-His) or leucine (CSM-Leu). Abbreviations: Hyg. = hygromycin B; Nours. = nourseothricin; CSM = complete synthetic medium.