Fig. 5.

Imaging of TPP biosynthesis in living cells using the Spinach riboswitch. (A) Kinetics of TPP biosynthesis in E. coli. Cells expressing the Spinach riboswitch were cultured briefly in thiamine-free medium for 2 h and then imaged every 15 min for 3 h after the addition of 10 µM thiamine. Adenosine is an inhibitor of de novo TPP biosynthesis. Adenosine (300 μg/mL) was preadded to the thiamine-free medium to reduce basal TPP levels. Spinach riboswitch activation was monitored as an increase in fluorescence signal over time. Images are pseudocolored to show the fold increase in fluorescence at each time point relative to 0 min. The color scale represents 0- to 10.0-fold changes (black to yellow) in fluorescence signal. (Scale bar, 5 µm.) (B) The fluorescence increase in Spinach riboswitch-expressing cells is caused by TPP biosynthesis. In this experiment, we used pharmacologic treatments to determine if Spinach riboswitch fluorescence reflects binding to TPP and not to other cell metabolites. In each experiment, Spinach-riboswitch–expressing cells were cultured in thiamine-free medium for 2 h and then were treated with 10 µM thiamine and the indicated compounds. Adenosine is an inhibitor of de novo TPP biosynthesis. Cells pretreated for 2 h with adenosine (300 μg/mL) exhibited markedly reduced Spinach riboswitch fluorescence at 0 min, reflecting reduced basal TPP levels. Adenosine induces the expression of thiamine kinase (40); the increase in initial fluorescence was 2.3-fold faster in adenosine-treated cells than in cells not treated with adenosine. In another experiment, 1 mM oxythiamine was added 10 min before the addition of 10 µM thiamine and was present throughout imaging. Oxythiamine is a competitive inhibitor of thiamine kinase; the increase in initial fluorescence was 3.1-fold slower in cells treated with oxythiamine than in cells treated with thiamine only. As a control, the addition of 500 µM oxythiamine did not activate the fluorescence of the Spinach riboswitch (gray line, pretreated for 2 h with 300 μg/mL adenosine). Each data point represents the average fluorescence value of 250 cells from three experimental replicates. (C) Distribution of Spinach riboswitch fluorescence levels within a population of E. coli cells. The fluorescence in each cell was normalized to control for cell-to-cell variability in Spinach riboswitch expression levels. To do so, we used a plasmid that expresses both the Spinach riboswitch and the eqFP670 far-red fluorescent protein. Cells were imaged after 3 h of treatment with 10 µM thiamine, and the fluorescence of the Spinach riboswitch in individual cells was normalized for the fluorescence signal from eqFP670 (red) in the same cells. The average normalized fluorescence value (blue) was set to 100%, and individual cells were binned according to relative brightness. The percentage of cells in each bin is plotted. A total of 250 cells were quantified from three experimental replicates. (Scale bar, 10 µm.) (D) Representative examples of fast, moderate, and slow TPP-synthesizing cells. Imaging of Spinach riboswitch fluorescence revealed different cellular populations based on the rate of TPP biosynthesis. Cells that have a rate of increase within two SDs of the average (black) are considered to have moderate biosynthesis rates (gray). Cells that have a rate of increase greater or less than two SDs from the average are termed “fast” (red) or “slow” (blue), respectively. Time points after the addition of thiamine are indicated. The color scale represents 0- to 10.0-fold changes (black to yellow) in fluorescence signal. (Scale bar, 3 µm.)