Article Figures & Data
Figures
- FIG. 1.
(A) Schematic representation of the SPE3-LYS9, spe3-lys9::NAT1, spe3-LYS9, and SPE3-lys9 alleles. (B) Southern blot analysis confirming the spe3-lys9::NAT1, spe3-LYS9, and SPE3-lys9 disruptions and subsequent reconstitution by SPE3-LYS9. Genomic DNA from strains H99 (SPE3-LYS9), H99-24 (spe3-lys9), H99-25 (spe3-lys9 SPE3-LYS9), H99-29 (spe3-LYS9), H99-44 (spe3-LYS9 SPE3-LYS9), H99-41 (SPE3-lys9), and H99-49 (SPE3-lys9 SPE3-LYS9) was digested with the restriction enzymes indicated and probed with genomic SPE3-LYS9 DNA.
- FIG. 2.
(A) Complementation of S. cerevisiae lys9 auxotrophy by C. neoformans SPE3-LYS9 cDNA. S. cerevisiae S157 transformed with pYES2.0 (LYS9) and S. cerevisiae S313 transformed with plasmid pYES2.0 (lys9) or pCnSPE3-LYS9 (lys9 + CnSPE3-LYS9) were plated on minimal medium with dextrose (SD) or galactose (SG) as the sole carbon source for 3 days. (B) Complementation of S. cerevisiae spe3 auxotrophy by C. neoformans SPE3-LYS9 cDNA was tested on minimal medium without pantothenate and with dextrose (SD-pantothenate) or galactose (SG-pantothenate) as the sole carbon source, with incubation for 4 days. Strains (clockwise from the top) included S. cerevisiae S157 transformed with pYES2.0 (SPE3) and S3545 transformed with plasmids pYES2.0 (spe3), pCnSPE3-LYS9 (spe3 + CnSPE3-LYS9), pCnATGG-SPE3-LYS9 (spe3 + CnATGG-SPE3-LYS9), pCnSPE3 (spe3 + CnSPE3), or pScSPE3 (spe3 + ScSPE3). Cn, C. neoformans; Sc, S. cerevisiae.
- FIG. 3.
Auxotrophic satisfaction of C. neoformans spe3-lys9, spe3-LYS9, and SPE3-lys9 mutants. Strains included C. neoformans H99 (SPE3-LYS9), H99-24 (spe3-lys9), H99-29 (spe3-LYS9), and H99-41 (SPE3-lys9). The medium was SD (ammonium as the nitrogen source) or YNB plus proline (proline as the nitrogen source), except when ethanol and glycerol were the carbon sources, in which case the medium was synthetic ethanol-glycerol medium (ammonium as the nitrogen source) or YNB-ethanol-glycerol plus proline medium (proline as the nitrogen source). Media were supplemented, as indicated, with various combinations of the following: spermidine, lysine, Ala-Lys dipeptide, and spermine. Plates were incubated for 4 days at 30°C.
- FIG. 4.
Temperature sensitivity of C. neoformans spe3-lys9, spe3-LYS9, and SPE3-lys9 mutants. Strains included C. neoformans H99 (SPE3-LYS9), H99-24 (spe3-lys9), H99-25 (spe3-lys9 + SPE3-LYS9), H99-29 (spe3-LYS9), H99-44 (spe3-LYS9 + SPE3-LYS9), H99-41 (SPE3-lys9), and H99-49 (SPE3-lys9 + SPE3-LYS9). (A) Five-microliter volumes of 10-fold dilutions of strains were spotted onto YPD or YNB plus proline medium and incubated for 2 days at 30 or 37°C, as indicated. (B) Growth rates of strains incubated in YPD broth at 30 or 37°C.
- FIG. 5.
Survival of mice infected with C. neoformans SPE3-LYS, spe3-LYS9, SPE3-lys9, and SPE3-LYS9-reconstituted strains. Strains included H99 (SPE3-LYS9), H99-24 (spe3-lys9), H99-25 (spe3-lys9 + SPE3-LYS9), H99-29 (spe3-LYS9), H99-44 (spe3-LYS9 + SPE3-LYS9), H99-41 (SPE3-lys9), and H99-49 (SPE3-lys9 + SPE3-LYS9).
- FIG. 6.
Northern analysis of SPE3-LYS9 transcription. RNA was isolated from strains H99 (SPE3-LYS9), H99-29 (spe3-LYS9), and H99-41 (SPE3-lys9) grown in the presence or absence of spermidine (SPE) and/or lysine (LYS). The GPD transcript was a control for RNA loading.
Tables
- TABLE 1.
Strain list
Strain Genotype Source S. cerevisiae S157 MATα ura3Δ 54 S313 MATα ura3Δ lys9Δ::natMX4 This study S3545 MATα ura3Δ spe3Δ::kanMX4 This study C. neoformans CDC1, CDC2 Serotype A J. Xu, personal communication B4495, B4496 Serotype B J. Xu, personal communication ATCC 34880, ATCC 34883 MATα serotype C 4, 40 JEC20 MAT a serotype D 18, 25 JEC21 MATα serotype D 18, 25 H99 MATα serotype A 35 H99-24, H99-26 MATα serotype A, spe3-lys9::NAT1 This study H99-25, H99-44, H99-49 MATα serotype A, SPE3-LYS9 This study H99-29, H99-30 MATα serotype A, spe3-LYS9 This study H99-41, H99-43 MATα serotype A, SPE3-lys9 This study
