The role of <i>GAT1</i> in cell growth.

<p>A) <i>GAT1</i> is not associated with the growth rate of both species. Cell suspensions of 2 OD₆₀₀ were spotted on YPD agar in three 10 fold dilutions and incubated for three days at the designated temperatures B) Similar spot assays were carried out on YPD with or without caffeine and plates were incubated at 30°C for 3 days. Experiments were carried out in duplicates.</p

Virulence of <i>H99gat1Δ</i> was minimally enhanced while of <i>R265gat1Δ</i> was significantly reduced in murine inhalation model.

<p>50,000 cells from each strain were inoculated intrapharyngeally to 10 Balb/c mice per group. Mice were fed with food and water ad libitum.</p

Comparison of expression levels of the genes encoding glycine cleavage enzymes in H99 and R265.

<p>RNA levels of genes encoded enzymes in the glycine cleavage decarboxylase complex were determined in cells growing in ammonium sulfate (NH₄), glycine or glycine plus ammonium sulfate (glycineNH4) as the sole nitrogen source. Expression is presented in folds of wild type levels growing in ammonium sulfate. A) Expression levels of the four genes in the wild type strains. B) Effect of <i>GAT1</i> deletion on the expression of glycine decarboxylase complex genes. The experiment was carried out in triplicates. Bars = standard error, wt = wild type.</p

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Utilization of nitrogen sources by H99 and R265.

<p>5 µl of cell suspensions at OD₆₀₀nm of 10, 0.1 and 0.001 were spotted on 2% glucose YNB agar with 10 mM of the indicated nitrogen source and incubated for 5–7 days at 30°C. Amino acids differentially utilized by the two species are italicized and underlined.</p

Examples of positive and negative growth on each nitrogen source.

<p>Cells were grown on 2% glucose YNB with 10 mM of each nitrogen source for 5–7 days at 30°C. 2 µl cell suspensions at an OD₆₀₀nm of 0.02 were spotted on the media. Limitted growth on L-phenylalanine and L-tryptophan media was designated as negatives.</p

Disruption of <i>GAT1</i> reduced ability to utilize all nitrogen sources.

<p>Wild type and <i>gat1Δ</i> strains were grown for 5–7 days at 30°C on 2% glucose YNB with 10 mM of different nitrogen sources. 5 µl cell suspensions at OD600nm of 10, 0.1 and 0.001 were spotted on the media. YPD and Blank (no nitrogen source) served as positive and negative control respectively.</p

Disruption of <i>GAT1</i> enhanced melanin synthesis and <i>LAC1</i> expression in both species but capsule size increased only in R265.

<p>A) Cells were grown in melanin induction media using 10 mM proline as the nitrogen source. Melanin production was assayed by measuring the absorbance at 475 nm and B) <i>LAC1</i> expressions were quantified by real time PCR. C) Micrograph of cell grown in capsule induction media. D) The relative capsule size was measured in >100 cells for each strain grown in capsule induction media. Experiments were carried out in triplicates. * <i>p</i>≤0.05 by the student t-test. Error bars = 2 standard deviation.</p

Utilization of different nitrogen sources according to molecular type.

<p><i>C. n.</i> = <i>Cryptococcus neoformans</i> and <i>C.g.</i> = <i>Cryptococcus gattii</i>. Bold italic are test giving 100% correlation with either species.</p

List of strains and their ability to utilize different nitrogen sources.

<p>List of strains and their ability to utilize different nitrogen sources.</p