Landscape of essential growth and fluconazole-resistance genes in the human fungal pathogen Cryptococcus neoformans
Fig 5
Regulatory inserts enable assays of essential gene function.
(A) Plot showing transposon insert frequencies in the region surrounding the ERG11 gene at either time zero or after two passages in IC50 levels of fluconazole in YPD. Shaded area shows the upstream region where inserts are less fit in fluconazole. (B) Boxplot displaying distribution of log10-adjusted fold changes in insert density (i.e., frequency in fluconazole/frequency in DMSO). Each column shows inserts only within a 300 base pair region either immediately upstream of the start codon or downstream of the stop codon. Boxplots show first quartile, median, third quartile. The whiskers show the range to a maximum of 1.5 times the interquartile range above and below the first and third quartile, respectively. Outliers are displayed as individual datapoints. (C) Spot dilution assays with 5 μL spots plated. The initial leftmost spot is of OD600 = 20 culture and each successive spot is a 10-fold dilution, so that the final spot should be 105 less concentrated than the first. Both plates were spotted on the same day with the same dilution series. YPD plates were imaged after 48 h at 30°C and fluconazole plates were imaged after 72 h at 30°C. (D) Schematic of model for 5′ transposon insertions. Wildtype cells (top row) should grow well on YPD and be moderately impaired (approximately 50%) by an IC50 level of fluconazole. Cells with a transposon insertion in the 5′ regulatory region of ERG11 should grow well on YPD but be highly sensitive to IC50 levels of fluconazole. (E) Volcano plot of 1,251 predicted essential genes with 5 or more insert sites in the 300 base pairs upstream of the start codon (out of 6,975 C. neoformans genes) displaying the mean log10 (Fluconazole/DMSO) value on the x-axis and the −log10 (Bonferroni corrected p-value) on the y-axis. Individual genes are shaded orange if the distribution of inserts is statistically different from the distribution of inserts into noncoding regions genome-wide (p < 0.05 via Mann–Whitney U test after Bonferroni correction). Genes that are not statistically different are shaded blue. (F) Spot dilution assays with 5 μL spots plated. The initial leftmost spot is of OD600 = 20 culture and each successive spot is a 10-fold dilution, so that the final spot should be 105 less concentrated than the first. All plates were spotted on the same day with the same dilution series. YPD and YNB plates were imaged after 48 h at 30°C and drug plates were imaged after 72 h at 30°C. Data underlying A and B can be found in S1 Data at 10.5281/zenodo.15264486. Data underlying E can be found in S1 Table. Original images in panels C and F can be found in the Stowers Original Data Repository at http://www.stowers.org/research/publications/libpb-2480.