Genetic and Molecular Dissection of the Integration of Galactose and Glucose Signaling in Saccharomyces Cerevisiae Strains

Cells need to sense the environment in order to survive, in particular they need to detect nutrients which will provide different building blocks and energy for the cell. This task is complicated by the fact that there can be multiple sources for the same type of nutrient available for the cell. A classical example of how cells sense multiple signals is given by carbon catabolite repression in the budding yeast S. cerevisiae. In this model the preferred carbon source glucose represses the genes used to metabolize an alternative source such as galactose. This means that the preferred carbohydrate glucose is thought to inhibit the induction of galactose genes when above a threshold concentration. Instead, we show that galactose metabolic genes (GAL) induction depends on the ratio of galactose and glucose. Surprisingly, we find that a critical portion of information processing occurs upstream of the canonical components of the GAL pathway. We then explore how cells choose between different responses to the environment. Specifically, we set out to characterize the variability in the response to combinations of galactose and glucose between several natural yeast isolates. To elucidate the genetic basis of this phenotypic variation we use QTL mapping on these strains. Our study reveals that a signal transducer GAL3 plays a central role in establishing variation in GAL gene induction.Lastly, we focus on the control of transcription in the cell. Many promoters in the cell produce both a coding transcript and a divergent transcript. To identify mutants that affect transcriptional directionality we use a bidirectionalfluorescent protein reporter in the yeast nonessential gene deletion collection. We determine that chromatin assembly can regulate divergent transcription. Moreover, mutations in the chromatin assembly factor CAF-I can lead to genome wide derepression of nascent divergent transcription.Systems Biolog

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 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 7

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

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

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

Preparation for glucose exhaustion has upfront cost and delayed benefit.

<p>(A) Log2-ratio of BC187 cell number versus YJM978 cell number versus time during diauxic growth in two replicate co-cultures. A positive value on the vertical axis at any given moment indicates that BC187 has divided more times than YJM978 since time = 0, and therefore has a net fitness advantage. Raw data (black circles) and smoothing splines (gray curves) are shown for two replicates. (B) Median GAL1pr-YFP expression of BC187 (blue lines) and YJM978 (red lines), glucose concentration (purple circles and lines), and galactose concentration (orange circles and lines) from (A). Vertical dotted gray lines in (A) and (B) demarcate four phases of relative fitness and GAL1pr-YFP expression during the experiment (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002041#sec002" target="_blank">Results</a>). (C) Comparison of growth rate differences during diauxic growth versus steady-state sugar conditions. Data points on shaded backgrounds and labeled “diauxic growth” represent the slope of the data in (A) during Phase II (pink background) and Phase III (blue background). Positive values indicate that BC187 grows faster than YJM978. Data are the mean and SEM of <i>n</i> = 6 (Phase II) or <i>n</i> = 14 (Phase III) discrete derivatives in the shaded regions from (A). Points on a white background and labeled “steady-state” are computed from the same data as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002041#pbio.1002041.s012" target="_blank">S12C Fig.</a>, and represent the mean and SEM of 3–6 replicates. <i>p</i>-Values are computed by two-sample <i>t</i>-test.</p