Dataset 5

STEADY-STATE FITNESS OF BC187 AND YJM978 (Figure 5C, S12). Contains .fcs files of raw flow cytometry data

Dataset 11

TIMELAPSE MICROSCOPY (Figure S13). Contains .tiff files of raw microscopy data

Dataset 1

GROWTH CURVES (Figure 1, S1-3, S11A-B; Also used in figure 3, 4, 7C). Contains .csv files of raw OD600 readings from plate reader

Dataset 3

DIAUXIC GROWTH TIMECOURSE ON MULTIPLE STRAINS (Figure 3, S7A-B,D-F,I, S9A). Contains .fcs files of raw flow cytometry data

Dataset 7

STEADY-STATE FITNESS OF MULTIPLE STRAINS (Figure 7, S14). Contains .fcs files of raw flow cytometry data

Tradeoff between costs and benefits of preparation underlies natural variation in GAL pathway expression.

<p>(A) Illustration of how galactose cost (top) and the minimum mid-diauxic growth rate (bottom) are defined (see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002041#pbio.1002041.s002" target="_blank">S2</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002041#pbio.1002041.s014" target="_blank">S14</a> Figs., and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002041#sec004" target="_blank">Materials and Methods</a>). Glucose and glucose + galactose conditions indicate 0.03125% glucose and 0.3125% glucose + 0.25% galactose media, respectively. (B) Scatterplot of galactose cost versus mean GAL1pr-YFP expression at steady state in glucose + galactose. Data points are mean and SEM of <i>n</i> = 3 replicates. (C) Scatterplot of galactose cost versus minimum mid-diauxic growth rate. The latter is computed from the growth curves shown in Figs. <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002041#pbio.1002041.g001" target="_blank">1</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002041#pbio.1002041.s001" target="_blank">S1</a>. Data points are the mean and SEM of <i>n</i> = 3 replicates (galactose cost) or mean and range of <i>n</i> = 2 replicates (minimum rate).</p

A short-lag strain induces GAL genes hours before the diauxic shift.

<p>(A) Top: Schematic of GAL1pr-YFP transcriptional reporter and cartoon of fluorescence distribution as measured by flow cytometry. Bottom: Schematic of diauxic growth GAL gene induction experiment. (B) Definitions of induction metrics, <i>t</i>_low and <i>t</i>_high, when reporter expression is at low but above-basal or near-maximal levels, respectively. Diauxic growth for strains (C) YJM978 and (D) BC187, with GAL reporter expression distributions (gray shading), GAL reporter median (red line), glucose concentration (purple circles), and galactose concentration (orange circles). Time is defined relative to the moment when the culture achieves a density of 10⁶ cells/ml (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002041#pbio.1002041.s004" target="_blank">S4 Fig.</a>). Purple and orange lines are smoothing-spline fits to glucose and galactose measurements. Dotted purple line indicates time of glucose exhaustion, calculated using a local linear fit (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002041#sec004" target="_blank">Materials and Methods</a>). Data shown in (B) and (C) represent two replicate experiments. GAL reporter expression distribution is shown for only one of the two replicates. (E) Comparison of induction start time, <i>t</i>_low, and near-maximal induction time, <i>t</i>_high, for YJM978 (red bars) and BC187 (blue bars) cultures. Bars and error bars represent the mean and range, respectively, of two replicates.</p

Dataset 6

SYNTHETIC INDUCTION (Figure 6). Contains .fcs files of raw flow cytometry data

MATLAB analysis code

MATLAB scripts for reproducing the analysis and generating all the figures in the paper. Scripts are organized into subfolders according to which dataset they are meant to analyze. See README.txt for entire repository for details and usage instructions

Synthetic induction of GAL genes is costly in glucose but beneficial during transition to galactose.

<p>(A) Strains S288C (WT) and S288C-GEV, a congenic strain expressing the GEV protein, were used. Both WT and GEV strains induce GAL genes in response to galactose; strain GEV also induces GAL genes in response to β-estradiol. For technical reasons, two variants of the WT strain were used; each strain is haploid and its HO locus has been replaced with either a GAL1pr-YFP reporter (SLYA39) or a constitutive mCherry segmentation marker (SLYA32). (B) GAL1pr-YFP expression histograms of strains WT (SLYA39) (black) and GEV (green) at steady state in 2% glucose, 2% glucose + 30 nM β-estradiol, or 2% galactose. The same concentrations were used in the following experiments. (C) Top: log₂ ratio of GEV to WT (SLYA32) cell counts during steady-state co-culture in glucose (purple) or glucose + β-estradiol (black). Bottom: median GAL1pr-YFP expression of strain WT during this experiment. (D) Top: log₂ ratio of GEV to WT (SLYA32) cell counts upon sudden shift to galactose, after pre-growth in glucose (purple) or glucose + β-estradiol (black). Bottom: median GAL1pr-YFP expression of strain GEV during this experiment. (E) Top: log₂ ratio of cell counts of WT strain SLYA39 and SLYA32 after pre-growth in different conditions and shift to galactose medium. Before the medium shift, the strains were either both preconditioned in glucose (purple), or the query strain (SLYA39; numerator of log ratio) was preconditioned in galactose while the reference strain (SLYA32; denominator of log ratio) was preconditioned in glucose (black). The black line from (D) is reproduced in gray in (E) to compare synthetic and “natural” pre-induction of GAL genes. Bottom: median GAL1pr-YFP expression of the query strain (SLYA39) during this experiment. Data in (C–E) are mean and SEM of three replicates. *<i>p</i> = 0.008, **<i>p</i> = 0.01 for change in log₂ strain ratio by two-sample <i>t</i>-test. n.s., not significant (<i>p</i> > 0.05).</p