Cartoon of the uptick mechanism.

<p>The top portion of the figure shows a graphical representation of the Rok and OA∼P concentrations, as well as the availability of RNAPol and the rate of <i>comK</i> basal transcription during the transition to stationary phase. (RNAPol availability is used as a plausible stand-in for the cause of the global increase in transcription we have observed). The peak rate of transcription coincides with T₀, the time of departure from exponential growth. When the concentrations of available RNAPol and of OA∼P are low (1) Rok is dominant and the rate of <i>comK</i> transcription is also low. As the concentration of OA∼P increases further, Rok is antagonized at sites A1, A2 and A3 and at the same time RNAPol becomes more available. As a result the rate of <i>comK</i> transcription increases (2). Finally, the OA∼P concentration reaches a level that is able to repress at R1 and R2 and <i>comK</i> transcription slows (3). In reality, of course, three demarcated periods of time do not exist. Note that the concentration of Rok remains constant throughout and both RNAPol and OA∼P work to counteract its effects. Rok works at an unidentified site in addition to A1–3, shown here between A3 and R1. For simplicity, the availability of RNAPol is shown as constant after T₀, although our data would suggest that it varies somewhat.</p

Rok and Spo0A bind at <i>A2</i>.

<p>Panel A shows the effect of an <i>A2</i> mutation on <i>comK</i> expression, alone and in combination with a null-mutation in <i>rok</i>. The effect of a <i>spo0A</i> knockout mutation is shown for comparison and panel B compares the effect of a <i>rok</i> mutation alone.</p

Co-expression of P<i>comK-cfp</i>.

<p>With P<i>sdpA-yfp</i> (panel A) or with P<i>spo0A-yfp</i> (panel B). Cells were segmented microscopically and the average pixel intensities in the CFP and YFP channels were recorded for each cell. The green boxes surround competence-expressing cells, with the lower limit of the boxes set from the threshold value (36) derived from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002586#pgen-1002586-g008" target="_blank">Figure 8</a>. These cells comprise 12.6% of the 11,238 cells measured for panel A and 14.2% of the 14,369 cells measured for panel B. The numbers of cells with CFP values in excess of the threshold were deposited into bins and displayed as a histogram, normalized to the total number of cells within each bin of YFP values. These data have been plotted on a histogram as a percent of total competent cells, with similar results (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002586#pgen.1002586.s007" target="_blank">Figure S7</a>).</p

Organization of the <i>comK</i> promoter region.

<p>(A) Putative Spo0A binding sites for activation (<i>A1</i>, <i>2</i> and <i>3</i>) or inhibition (<i>R1</i> and <i>2</i>) are shown, as are the two ComK binding sites <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002586#pgen.1002586-Hamoen1" target="_blank">[27]</a>, the −35 and −10 promoter motifs, the start site (+1) of transcription and the initiation codon for translation. The Spo0A binding sites are predicted based on the published consensus <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002586#pgen.1002586-Molle1" target="_blank">[30]</a>. (B) Schematic representation of the <i>comK</i> promoter showing the putative Spo0A binding sites in relation to the ComK boxes and the region in which Rok binds <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002586#pgen.1002586-Smits2" target="_blank">[28]</a>. (C) Mutagenesis of the Spo0A binding sites. For each box the mutagenized sequences are shown below the wild type sequences. In panels A and C residues that differ from the Spo0A binding consensus sequence are shown in lower case.</p

Stochastic simulation data match the experimentally observed <i>comK</i> expression.

<p>(A) The average <i>comK</i> output of 5000 Gillespie simulations reproduces the relative amplitudes of the uptick in <i>ΔcomK</i>, <i>ΔcomKΔrok</i>, <i>ΔcomKΔrokΔspo0A</i> and <i>ΔcomKΔspo0A</i> mutants (compare to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002586#pgen-1002586-g001" target="_blank">Figure 1C</a>). (B) The curves are normalized to a peak amplitude of 1 and plotted on the same axes to emphasize the sharp downturn in expression in the simulated Δ<i>comK</i> strain (compare to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002586#pgen.1002586.s005" target="_blank">Figure S5</a>). The details of the simulation are presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002586#pgen.1002586.s013" target="_blank">Text S1</a>.</p

Transcription rates from the <i>comK</i> promoter.

<p>(A) The relative luminescence readings corrected for OD for the <i>comK</i> promoter and the OD readings for the growth curves are presented for the wild type (purple dotted curves) and <i>ΔcomK</i> (red curves) strains. Each pair of curves is connected to its Y-axis by a black arrow. (B) Expression from the comK promoter in a <i>ΔcomK</i> background compared with expression from the same promoter in a <i>ΔcomK Δspo0A</i> background. (C) Expression from P<i>comK</i> in <i>ΔcomK</i>, <i>ΔcomK Δspo0A</i>, <i>ΔcomK Δrok</i> and the <i>ΔcomK Δrok Δspo0A</i> backgrounds.</p

Effect of combined mutation of <i>A1</i>, <i>2</i>, and <i>3</i> on P<i>comK</i> expression.

<p>Panel A shows the effect of this triple mutation. Panel B shows its effect in <i>Δrok</i> and <i>Δspo0A</i> backgrounds.</p

Effect of mutations in the putative Spo0A binding sites on <i>comK</i> expression.

<p>The effects of mutations in <i>R1</i> (panel A), <i>R2</i> (panel B), <i>A3</i> (panel C) or <i>A1</i> (panel D) in the <i>ΔcomK</i> background are compared with the expression from the wild type <i>comK</i> promoter.</p

Demonstration of Turbo SNP FISH.

<p>A. Demonstration of SNP FISH efficacy under Turbo FISH and conventional RNA FISH conditions in WM983b cells. We targeted BRAF mRNA with guide probes, and then used detection probes that targeted either the V600E mutation for which BRAF is heterozygous in this cell line (top panels) or a common region for which BRAF is homozygous in this cell line (bottom panels). Left panels show the signals from the guide probe (that labels the mRNA), the middle panel shows the detection probe that detects the wild-type sequence, and the right panel shows the detection probe that detects the mutant sequence. B. Quantification of RNA as being either mutant or wild type in this cell line. Each bar corresponds to data from a single cell.</p

Comparison of signal from Turbo RNA FISH (5 minutes; red) to conventional RNA FISH (blue).

<p>A. Comparison of RNA FISH signal sensitivity at a range of hybridization times. Error bars reflect standard error of the mean. At 5 minutes, we found a statistically significant difference in signal sensitivity between Turbo FISH and conventional FISH for <i>TBCB</i> gene and <i>TOP2A</i> gene (p = 4.75×10⁻¹¹ and p = 1.19×10⁻⁷⁴, respectively; two-tailed t-test). B. Comparison of RNA FISH spot count at a variety of hybridization times. Error bars reflect standard deviation. At 5 minutes, we found a statistically significant difference in RNA FISH spot count between the Turbo FISH and conventional FISH for <i>TBCB</i> gene and <i>TOP2A</i> gene (p = 1.69×10⁻⁶⁸ and p = 2.07×10⁻²⁰, respectively; two-tailed t-test). For all conditions, we analyzed spot counts and calculated sensitivity on 100–150 cells. Data shown represents one of two replicate experiments.</p