(A) A commercial Tn5 transposon was modified by inclusion of a Gateway conversion cassette (with the ccdB selection gene and chloramphenicol resistance gene (Cm^R)), a kanamycin resistance gene (Kan^R), and the UAU1 marker cassette at the multiple cloning site (MCS). A reaction with the modified Tn5, a pool of TagModules, and LR clonase induces recombination at the att sites, placing the TagModule within the transposon mosaic ends (Tn5L and Tn5R). (B) To generate the heterozygous disruption strains, multiple genomic DNA libraries were constructed and then mutagenized in vitro with the pool of tagged Tn5 transposons from (A). Plasmids containing individual insertions were recovered from E. coli and sequenced (arrow) to determine the disrupted gene and its corresponding tag. Results were sorted to maximize the unique gene insertions/tag pairs. Select insertions were amplified, followed by excision of the genomic DNA containing the tagged transposon insertion. Transformation of these inserts into C. albicans was mediated by homology of the genomic sequence flanking the transposon insertion, and integration was selected for based on the arginine prototrophy conferred by the Arg+ marker. Homologous recombination results in a gene disruption with a tagged Tn5 transposon. (C) Screening methodology. Approximately equivalent numbers of tagged transposon mutants are combined to create a tagged pool. Pools are then screened in diverse conditions, generally over 20 generations of competitive growth with pools being harvested and diluted every 5 generations (5 g, 10 g, 15 g, 20 g). The genomic DNA is then extracted from the pool and the uptags and downtags amplified separately using the common priming sites. After the pooled growth assay, the amplicons are then hybridized to an Affymetrix TAG4 microarray to calculate tag intensity as a proxy for strain abundance.

(A) Pooled growth of 3633 strains was measured in rich (YPD), semi-rich (SC), minimal (YNB), and low-nitrogen (SLAD) media. Samples were collected every 5 generations, after which tags were amplified and hybridized to an array. A linear regression was fitted to the log₂ of the normalized tag intensity values as a function of generation time. Strains were categorized as haploinsufficient if the regression slope of their time course was statistically significantly less than 0 (adjusted p<0.05). The total number of haploinsufficient strains was determined for each media condition. (B) The 269 haploinsufficient strains from the four media conditions were separated into bins depending on whether they showed haploinsufficiency in one, two, three, or all media conditions. (C) Comparison between C. albicans and S. cerevisiae haploinsufficient genes in rich media. Orthologs were retrieved for the 145 haploinsufficient genes in YPD (http://www.candidagenome.org/), then compared against results from haploinsufficiency profiling of S. cerevisiae in YPD [29].

Strains defined from pooled screens as haploinsufficient in reduced nutrient conditions (YNB or SLAD) media, but not haploinsufficient in rich media (YPD or SC) were grown individually on agar media that induces filamentation (Spider media). Duplicates of control wild-type C. albicans strains BWP17 and parent strain SC5314 are in the bottom right; strains with a filamentous defect are indicated with an arrow.

(A) Comprehensive view of the fitness profiles for 25 compounds. Z-scores for each of 3573 strains were calculated by comparing the intensity values for each strain under 20 generations of compound treatment to a no-drug control set. The dot plot distribution of the z-scores is useful in identifying outliers for each compound treatment. These 25 compounds represent the 44% of those tested that resulted in a small number of significant outliers. The most sensitive strain for each screen (highlighted in red) is listed on the right. (B) Comparison of haploinsufficiency in S. cerevisiae and C. albicans for nigericin and brefeldin A. Z-scores for S. cerevisiae were calculated as for C. albicans, except only the 1150 essential heterozygotes of S. cerevisiae genes were screened. Top, chemical structures of these two compounds. Bottom, dotplots of the z-scores calculated for the chemical treatments, which were screened in the pooled growth assay at a concentration that produces ∼10% inhibition of wild-type. Strains marked with a red triangle are those involved in Golgi vesicle transport. Unlabeled leftmost red triangles for nigericin (C. albicans) represent orf19.6558, orf19.2078, and TFP1, orf19.1265 (from left).

(A) Distribution of log₂ tag intensities of the 4239 successfully transformed strains when pooled and tags amplified and hybridized to a TAG4 array. Log₂ of background intensity  =  ∼6; cutoff for detection was 3X background (log₂  =  ∼7). 3633 strains were detected with microarray signal intensity 3X above background. (B) Biological replicates of C. albicans pool are highly correlated even when using one tag per strain. Independent pools representing the 4239 heterozygous disruption strains were grown for 20 generations in YPD +1% DMSO. Tags were amplified from each pool and hybridized to a TAG4 array. Pearson correlation of unnormalized tags is indicated in the upper left.

(A) Each C. albicans ORF was cloned into an S. cerevisiae CEN/ARS expression vector with expression controlled by the constitutive GPD promoter (pAG416GPD, [60]). Overexpression clones were then transformed into the corresponding S. cerevisiae strain containing a deletion of the essential ortholog and a Magic Marker cassette [32]. The Magic Marker cassette allowed selection of null haploid mutants via sporulation followed by selection for matA cells containing a knockout. 4.5×10⁻⁴ OD₆₀₀ of sporulated culture was plated. Left, negative control (vector-only); center, complementation with C. albicans ORF; right, positive control (complementation with corresponding S. cerevisiae ORF). TEM1 (upper panel) is an example of complementation of the C. albicans ORF; RXT3 (lower panel) does not complement. (B) Selected results from (A) were verified by tetrad dissection. For each box: left, negative control (vector-only); center, positive control (complementation with corresponding S. cerevisiae ORF), and right, complementation with C. albicans ORF. The left box is an example of full complementation, and on the right, a case of partial complementation. Tetrads were then replica-plated onto media containing 5-fluoroorotic acid (5-FOA) or geneticin (G418) to confirm that the overexpression clone was the source of complementation.

(A) Comparison of compound inhibition between S. cerevisiae and C. albicans. Wild-type S. cerevisiae (HO-1) and wild-type C. albicans (BWP17) were grown in YPD +1% DMSO (control) or with one of 1521 unique chemical compounds. A growth rate ratio was calculated by averaging 12 controls per 96-well plate and dividing that by the drug-treated growth rate. Highlighted in red are 40 compounds that inhibited C. albicans more than S. cerevisiae (measured by difference in growth rate ratio). (B) Representative dose response curves for compounds with different levels of inhibition between S. cerevisiae and C. albicans. X-axis represents drug concentration/dilution: initially 2 µL of compound of stock concentration was diluted into 100 µL of YPD, then compounds were serially diluted across the plate so that the final concentration was 1/64^th of that at the start. Growth rate ratio is as described in (A). Wild-type S. cerevisiae dose response curve are in black; wild-type C. albicans dose response curves are in red. Compounds labeled xxxx-xxxx are synthetic, previously uncharacterized chemicals (Chemical Diversity Labs, Inc.); 5-fluorouridine and brefeldin A are well-characterized compounds.

(A) Comparison of haploinsufficiency in S. cerevisiae and C. albicans for two Chemical Diversity Lab compounds, 1187–1561 (top) and 0136–0228 (bottom); description is as in (B). Leftmost red triangles in 0136–0228 (C. albicans) represent, from left, orf19.1376, orf19.6558, orf19.2078, and orf19.645.1. (B) Single strain growth curves for control (1% DMSO) and drug-treated TFP1 (0136–0228), right, and SYN8 (1187–1561), left. Wild-type C. albicans (BWP17) is included as a control for both plots (black & blue). (C) Overexpression of Tfp1p in a heterologous system alleviates growth defects resulting from treatment with 0136–0228. S. cerevisiae strain background is marked in the upper left corner. TFP1 was cloned into an S. cerevisiae 2 micron expression vector under a constitutive GPD promoter (pAG426GPD, [60]). Overexpression clones were then transformed into one of the three S. cerevisiae backgrounds (wild-type BY4743, a Δtfp1 null, or a drug-sensitive Δerg6 null) and grown in the presence (18.3 µM, red) or absence (1% DMSO, blue) of 0136–0228. As a control, an empty pAG426GPD plasmid was transformed and grown in parallel (black, green).