Material and Methods

Strains: Deletion strains in the BY4741 background, wild-type genotype MATa;his3D1;leu2D0;met15D0;ura3, mutant genotype MATa;his3D1;leu2D0;met15D0;ura3, ORF::kanMX4 were provided by the EUROSCARF stock centre. Strains were stored in 15% (w/v) glycerol solution at -80°C.

Media and growth conditions: Strains were inoculated in 350ml of SD medium (0.14% yeast nitrogen base (YNB; Difco) without amino acids, 0.5% ammonium sulphate and 1% succinic acid; 2% (^w/_v) glucose; 20mg/l histidine, 20mg/l methionine, 20mg/l uracil, 20mg/l lysine and 100mg/l leucin, pH 5.8) and incubated for ~72h at 30C° on a micro-titre plate shaker (500rpm). This procedure was repeated once (second inoculation~48h). For experimental runs, strains were inoculated to an OD of 0.03 - 0.1 in 350ml of SD medium and cultivated for 48h in a Bioscreen analyzer C (Labsystems Oy , Finland ). Optical density was measured using a wide band (450-580nm) filter. Incubation as at 30.0°C (+/- 0.1°C) with ten minutes preheating time. Plates were subjected to shaking at highest shaking intensity with 60s of shaking every other minute. OD measurements were taken every 20 minutes during a 47h period. Strains were run in duplicates, duplicates on separate plates, 4 wildtype strains in randomised positions on each plate.

Calculation of growth variables: Data was processed and analysed using the software Prophecy. The growth variables length of lag phase, rate of growth and stationary phase OD increment were calculated in the following way: The average blanking value of 0.067 (representing the background absorption of SD medium) was subtracted from each OD measurement. All OD values were calibrated according to the formula calibrated OD (OD_c) =OD + 0.8324*OD³. Growth curves were smoothened by averaging over 1h (3 measurements) to prevent digital noise from affecting calculations. The effects of catastrophic events were reduced by removing negative slopes; if any calculated slope between consecutive OD values the latter value was adjusted as to equal the higher. Furthermore, if end OD values did not equal at least a doubling of the initial OD no growth was considered to occur. No rate of growth or stationary phase OD increment were calculated and the length of the lag phase for that curve were set to 48h. None such curves were found among the NaCl data.

Length of lag phase was calculated in the following way: Derived OD values were log transformed. 1 was added to avoid negative values. Initial OD was taken as the mean of the five OD values. Slopes were calculated between every 8:th value along the curve. Intercepts were calculated between every slope and the initial OD. The mean of the 2 highest slopes was taken as the length of the lag phase.

Rate of growth was calculated in the following way: Derived OD values were log transformed. Slopes were calculated between every third consecutive OD value. No slopes were calculated from the 8 first measurement points or from OD_c < 0.1 to avoid possible contribution from digitalisition effects. Of the seven highest slopes, the top two were discarded as possible artefacts and a mean was calculated from the following five. Generation time was calculated as log 2 divided by the mean.

Stationary phase OD increment was calculated in the following way: Initial OD was determined as for length of lag phase. Last measurement value was taken as the End OD. The difference between End OD and initial OD was taken as the stationary phase OD increment value. If the standard deviation for the last six measurements was more than 2% of End OD the culture was not considered to have reached stationary phase and no stationary phase OD increment was calculated from that curve.

Calculation of Phenotypic Index (PI): In each Bioscreen Analyzer C and in each run, 8 wild-types (wt) were grown in the same condition as the deletion strains. All 8 wt were used in the calculations of the average wt values below. For the growth variable rate of growth, a logarithmic strain coefficient, LSC, for each strain, in each environment was calculated as:

where wt_kj  is the doubling time of the k:th measurement of the wildtype in environment j, x_kj  is the doubling time of strain i in environment j and r indicates the run.

The logarithmic phenotypic index for each strain in NaCl, LPI_NaCl, for rate of growth was calculated as:

LSC and LPI_NaCl, for length of lag phase and LSC for the stationary phase OD increment was calculated correspondingly. LPI_NaCl, for stationary phase OD increment was calculated as

Strain coefficient , SC, and phenotypic index , PI, was calculated in a similar manner except that log transformed values were not used.

Significance tests: We performed tests of the null hypothesis that LPI_NaCl, equals 0 separately for lag phase, rate of growth and stationary phase and for each environment/strain combination. Statistical significance of the data was calculated using a two-tailed, two sample Students T-test (a=0.05, df =2). In order not to reject the null hypothesis only because of a too small variance estimate, a threshold of three mean standard deviations (a=0.0025) was also applied (standard deviation was considered to be 0.04 for rate of growth, 0.065 for stationary phase OD increment and to 0.08 for length of lag phase).