YPD liquid medium consisted of 1% yeast extract, 2% bactopeptone, and 2% glucose. Chemically defined synthetic complete media lacking inositol and choline (I−C− media) used in this study was described by (Jesch et al. 2005 (link)), except that threonine was omitted. I−C− media contains (per liter): 20 g of glucose, 5 g of ammonium sulfate, 1 g of potassium phosphate, 0.5 of g magnesium sulfate, 0.1 g of sodium chloride, 0.1 g of calcium chloride, 0.5 mg of boric acid, 0.04 mg of cupric sulfate, 0.1 mg of potassium iodide, 0.2 mg of ferric chloride, 0.4 mg of manganese sulfate, 0.2 mg of sodium molybdate, 0.4 mg of zinc sulfate, 2 µg of biotin, 400 µg of calcium pantothenate, 2 µg of folic acid, 400 µg of niacin, 200 µg of p-aminobenzoic acid, 400 µg of pyridoxine hydrochloride, 200 µg of riboflavin, 400 µg of thiamine hydrochloride, 20 mg of adenine sulfate, 20 mg of arginine, 20 mg of histidine, 60 mg of leucine, 230 mg of lysine, 20 mg of methionine, 20 mg of tryptophan, and 40 mg of uracil. Where indicated, I− media was supplemented with 75 µM myo-inositol (I+) and/or 1 mM choline (C+). For example, I+C+ medium contains 75 µM inositol and 1 mM choline, whereas I−C− medium lacks both inositol and choline. Solid media contained 2% agar.
In preliminary experiments leading to the final screening, we observed that the presence of threonine in the I+C− medium as described by Jesch et al. (2005) (link) increased the lag phase of growth of a large number of mutant strains derived from S288C, including those in the BY4743 background, by about 10–12 h at 30°C. When threonine was omitted, this lengthening of the lag phase was not observed. Therefore, in order to compare growth of strains over comparable time intervals with and without inositol, threonine was excluded from all synthetic media used in this study. Similar inhibition of growth in the presence of threonine in synthetic complete media was previously reported by Shirra et al. (2001) (link).
The standard protocol used in the mutant screening was as follows: Each frozen master-plate was thawed completely, and cells were resuspended to homogeneity. A 96-pin microplate replicator (V & P Scientific, Inc) was used to transfer a 2-µl aliquot from each well in a master plate to a corresponding well containing 800 µl of YPD, plus G418 (200 µg/ml) in a deep-well microtiter plate. The deep-well plate was incubated for 2 days at 30°C. A 2-µl aliquots from each well from the YPD + G418 cell culture were then inoculated into a second deep-well plate containing 800 µl of YPD per well and incubated at 30°C for 15 h. A 1:50 dilution of the 15-h culture was carried out by transferring 2-µl aliquots from each well into 100 µl of I−C− media to dilute any carryover of inositol or choline from the YPD culture. Finally, 2 µl from each 100 µl dilution was transferred to Nunc Omni plates containing the following solid media: YPD, I+C+, I+C−, I−C−, I− C+, and 2% agar. Plating was carried out in duplicate to control for variability in inoculation volumes, and plates were incubated for 4 days at 30°C or 37°C. Each plate was photographed on days 2 and 4 using a digital camera to create a permanent record. In all cases, only wells that gave the same result on duplicate plates were scored in the final tally of Ino− phenotypes shown on Tables 1 and 2. Questionable cases and mutants of interest were reassessed in subsequent spotting assays. Growth of each strain on I−C− or I−C+ medium was scored visually relative to growth of the same strain on I+C− or I+C+ medium at the equivalent growth temperature, conducted independently by two different investigators in two separate blind screenings. On Tables 3 and S1, a score of “S” (strong) indicates no visible growth on media lacking inositol. A score “W” (weak) indicates some residual growth on I− medium, but substantially less than on I+ medium, while “VW” (very weak) indicates some growth on I− medium, but still visibly less than on I+ medium. Some strains had strong Ino− phenotypes at 30°C, but failed to grow at all on I+C+ media, I+C− media, and/or YPD at 37°C. Such strains are denoted as “NG” (no growth) at 37°C. This scoring system is described in detail in the legend of Table 3 and Table S1, where the assigned scores of all Ino− mutants identified in this study are listed. Several examples of Ino− phenotypes, as detected in the original screening, are shown in Fig. 1, as are several examples of variable growth on I−C+ medium that were not validated in the duplicates plates and/or in the subsequent spotting assays.