CaNAT1 , a Heterologous Dominant Selectable Marker for Transformation of Candida albicans and Other Pathogenic Candida Species

Abstract

A dominant selectable marker for Candida albicans and other Candida species, which confers resistance to nourseothricin, was characterized. In a heterologous promoter system and a recyclable cassette, the marker efficiently permitted deletion and complementation of C. albicans genes. Neither growth nor filamentous development was affected in strains expressing this marker.Candida albicans is the most commonly isolated invasive fungal pathogen. Molecular genetic analysis of this fungus to date is principally based on the marker URA3, a biosynthetic gene that complements uridine auxotrophy (5), and on a dominant selectable marker, MPA r or IMH3 r , developed from a mutant C. albicans gene (2, 9).We developed a heterologous dominant marker to investigate morphogenesis of C. albicans. We chose a gene conferring resistance to nourseothricin, since wild-type C. albicans is susceptible to moderate nourseothricin concentrations (250 to 450 g/ml). Codon usage of the Streptomyces noursei nat1 gene, encoding nourseothricin acetyltransferase, was adapted to that of C. albicans to generate CaNAT1 (6, 8). De novo synthesis was performed by Bionexus (Oakland, Calif.). CaNAT1 was placed under the control of the ACT1 promoter in vector pAU34, which contains URA3 as a selectable marker (15), generating pJK850. We transformed strain CAI4 (ura3/ura3) (5) with pJK850 to be uridine prototrophic. The resulting strains JKC435 and JKC436 were resistant to 250 g of nourseothri-FIG. 1. CaNAT1 confers nourseothricin resistance on C. albicans. The ura3/ura3 strain CAI4 was transformed with the empty vector pAU34, which bears the selectable marker URA3, giving rise to JKC347 and JKC348, and with pAU34 containing CaNAT1, giving rise to JKC345 and JKC346. Transformants were streaked onto medium to select for uridine prototrophy (sc-ura). They were then replica plated onto YPD containing 250 g of nourseothricin/ml (YPDϩnat250). 1, JKC437; 2, JKC438; 3, JKC436; 4, JKC435. FIG. 2. Cells expressing CaNAT1 do not exhibit a growth disadvantage. (A)Competition between wild-type SC5314 and the DLH1/dlh1:: CaNAT1 strain JKC336. Fresh cultures of the two strains were inoculated in equal parts into rich medium at a density of ca. 1 cell/ml. The numbers of total colonies and nourseothricin-resistant colonies were determined at 48 h, and the proportion of CaNAT1-expressing cells to wild-type cells was calculated. The cultures were diluted in fresh medium after 48 h to the starting density of ca. 1 cell/ml. This process was repeated three times. Two separate experiments were performed. Per time point, three samples were taken to determine numbers of CFU. Bars represent standard deviations. (B) Competition between strains CAF2-1 (URA3/ura3) and CAI4 (ura3/ura3). To control for the sensitivity of the competition experiment protocol, the ura3/ura3 homozygote CAI4 was tested in competition with the URA3/ura3 CAF2-1 heterozygote. For colony counts, cultures were plated onto YPD. They were replica plated onto medium to select for uridine prototrophy to determin...

The GPI-modified proteins Pga59 and Pga62 of Candida albicans are required for cell wall integrity

“…CIpSAT1 and CIpSAT2 are derivatives of CIp10 (Murad et al, 2000) where the URA3 gene has been respectively substituted by the caSAT1 (Reuss et al, 2004) or CaNAT1 (Shen et al, 2005) markers that confer resistance to nourseothricine (Reuss et al, 2004).…”

Selective Advantages of a Parasexual Cycle for the YeastCandida albicans

“…To direct the integration of the resistance cassette to a noncoding region of the C. albicans genome, two targeting sequences, TS1 and TS2 (bp 367394-368202 and 366038-367194, respectively, on chr 7 of strain SC5314 as per genome assembly 21; http:// www.candidagenome.org/) were PCR amplified with primers TS1pf/TS1pr and TS2pf/TS2pr and cloned into plasmid pGemT-Easy (Promega). TS1 and TS2 were removed from pGemT using NotI and HindIII, respectively, and cloned into pNZ3 to obtain pNZ4.The NAT-resistance vector pNZ11 (designed so that integration would occur in chr 1; Figure 1B) was constructed as follows: The cassette containing a CaNAT-resistance gene flanked by an ACT1 promoter and the ACT1 terminator was PCR amplified from plasmid pJK850 (Shen et al 2005) using primers M13pr and Sacpf1. The PCR product was purified and then digested with both KpnI and SacI and inserted into KpnI/SacI-digested vector pBlueScript KS(+) to obtain pNZ11.…”

A versatile set of Lifeact-RFP expression plasmids for live-cell imaging of F-actin in filamentous fungi

“…Therefore, pALxLifeact transformant selection with nourseothricin was the preferred option, as it is much more stringent than ignite per se and has no reported problems in conferring cross-resistance to other selection markers, such as hygromycin B (Kück and Hoff, 2006). Nourseothricin has been successfully used for the transformation of several yeast species including Saccharomyces cerevisiae (Goldstein and McCusker, 1999), Schizosaccharomyces pombe (Hentges et al, 2005), Candida albicans (Shen et al, 2005) and Cryptococcus neoformans (McDade and Cox, 2001), and more recently in an increasing number of filamentous fungal species, including Acremonium chrysogenum and Sordaria macrospora (Kück and Hoff, 2006), as well as Neurospora crassa (Maerz et al, 2009;Maerz et al, 2008).…”