Figure 1. Identification of the small molecule that induces [GAR⁺] in a concentration dependent manner.

(A) S. gallinarum spotted next to yeast on GLY + GlcN (YP with 2% glycerol and 0.05% glucosamine) induced growth of naïve S. cerevisiae in a spatially dependent manner. An extract prepared from a S. gallinarum conditioned medium induced [GAR⁺] with similar strength. Each panel shows two biological replicates of yeast colonies spotted in five-fold serial dilutions from saturated cultures. (Bacterial spots are undiluted.) (B) Multiple-reaction monitoring-mass spectrometry (m/z 89.0200, 43.1000) traces of pure lactic acid (top), the purified active fraction from S. gallinarum conditioned medium (middle) and a co-injection of active fraction spiked with pure lactic acid (bottom). Results indicate that lactic acid is present in the active fraction (see Materials and methods). Note that y-axis scale differs between the top two traces due to the difference of lactic acid concentration in the pure standard, but they are normalized here for ease of viewing. (C) Plating assay (see Materials and methods) showing that both L- and D- isomers of lactic acid strongly induce [GAR⁺] in a concentration dependent manner. Plotted are the fraction of total colony forming units (CFUs) that grew on GLY + GlcN (mean with standard deviation from three biological replicates). Ten-fold dilutions were used for plates lacking lactic acid; 1000-fold and 10,000-fold dilutions were used for plates containing 0.025% LA and 0.625% LA, respectively.

DOI: http://dx.doi.org/10.7554/eLife.17978.003

Figure 1—figure supplement 1. Flow chart depicting the activity guided fractionation strategy used to identify the small molecule that induces [GAR⁺].

DOI: http://dx.doi.org/10.7554/eLife.17978.004

Figure 1—figure supplement 2. Growth curves—of cells that start as [gar⁻] at the outset of the experiment—in synthetic complete media (Sigma-Aldrich) containing glucosamine, with or without 0.1% L-lactic acid added.

DOI: http://dx.doi.org/10.7554/eLife.17978.005

Figure 1—figure supplement 3. Growth rates of [GAR⁺] and [gar⁻] cells do not differ in the presence of lactic acid.

Four biological replicates of [GAR⁺] or [gar⁻] cells were grown in GLY or GLY + 0.35% D-lactic acid liquid media, with OD₆₀₀ measurements taken every six minutes over four days using an automated Eon microplate reader (BioTek Instruments, Winooski, VT). [GAR⁺] cells neither grew faster than [gar⁻] cells in glycerol alone nor when lactic acid was added. (Note that [GAR⁺] cells did have a slightly longer lag time, i.e. slightly more time passed before they entered exponential phase, even though their growth rate during exponential phase was equivalent to that of [gar⁻] cells.) Solid lines mark mean values, dashed lines standard deviation.

DOI: http://dx.doi.org/10.7554/eLife.17978.006