Complete induction medium contained 0.2% glucose, antibiotics, and 200 uM acetosyringone and was buffered to pH 5.3 with MES. GANT61 chemical structure bacteria
were collected by centrifugation and resuspended in induction medium to an optical density at 600 nm of 1.5 (corresponding to roughly 1.5 × 109 bacteria/ml). Histoplasma WU15 yeast were harvested Blebbistatin datasheet from solid HMM + uracil medium seeded 3 days earlier with 4 × 105 yeast/cm2. Yeast were collected by flooding plates with 5 mls HMM medium and scraping with a sterile spreader. Yeast were collected by centrifugation (1000 × g) and resuspended in induction medium at a density of 5 × 108 yeast/ml as determined by hemacytometer counts. For co-cultivation, 1.5 × 108 Agrobacterium cells were mixed with 5 × 107 Histoplasma yeast in a total volume of 400 ul and spread on Whatman #5 filter paper placed on top of solid induction medium supplemented with 0.7 mM cystine and 100 ug/ml uracil. Plates were incubated for 48 hrs at 25°C after which filters were transferred to selection medium (HMM + uracil + hygromycin + 200 uM cefotaxime) PARP inhibitor and incubated at 37°C with 5% CO2/95% air until Histoplasma transformants became visible (10-14 days). Six cm diameter plates were used so that roughly 50-100 transformants were obtained per plate. PCR-based screening of T-DNA insertion mutants Hygromycin-resistant transformants of Histoplasma were collected by flooding plates with HMM and
suspending cells with a sterile spreader. Suspensions
from individual plates were combined to obtain pools representing 100-200 independent transformant colonies. Yeast suspensions were diluted 1:10 into 10 mls HMM + uracil and grown for 24-48 hours. Two milliliters SDHB of culture were collected for nucleic acid isolation and the remaining culture frozen in 1 ml aliquots for later recovery of yeast. To purify Histoplasma nucleic acid for PCR, cells were collected by centrifugation (2000 × g) and nucleic acids released by mechanical disruption of yeast in the presence of detergents and organic solvent . 250 ul of lysis buffer (20 mM Tris pH 8.0, 200 mM NaCl, 2 mM EDTA, 2% SDS, 4% Triton X-100) and 250 ul of phenol:chloroform:isoamyl alcohol (25:24:1) were added to cells and nucleic acids released by bead beating cells with 0.5 mm-diameter acid-washed glass beads. Phases were separated by centrifugation (5 minutes at 14,000 × g) and the aqueous phase transferred to new tubes. Nucleic acids were recovered by precipitation of the aqueous phase with 2.5 volumes of ethanol. As no efforts were taken to remove RNA co-purifying with the DNA, total nucleic acids were quantified by spectrophotometric readings at 260 nm Screening of pools was done by two sequential PCR steps. Primers used are listed in Table 2. For the primary PCR, 50 ng of total nucleic acid was used as template in a 25 ul reaction with either a left border (e.g., LB6) or right border primer (e.g.