Tn-seq screen for new sporulation genes.

<p><b>(A)</b> Flow diagram of the screen. A Mariner-based transposon library was grown in sporulation medium. At the onset of starvation (T0), a sample was removed. The remaining culture was allowed to sporulate for 24 h (T24). Heat treatment at 80°C for 20 min killed vegetative cells and sporulation-defective mutants (pink circles). The surviving spores (blue circles) were germinated on LB agar plates and the resulting colonies pooled. The transposon insertion sites and their abundance were determined for the T0 sample and the T24 heat-resistant colony forming units (CFU) by deep sequencing and mapping onto a reference genome (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002341#sec017" target="_blank">Methods</a>). Transposon insertion profiles from two regions of the genome are depicted. The height of each vertical line reflects the number of sequencing reads at this position. Transposon insertions in previously identified sporulation genes such as <i>spoIIGA</i>, <i>sigE</i>, and <i>sigG</i> were statistically underrepresented after sporulation. A new sporulation gene, <i>spoIIT</i> (formerly <i>ywmB</i>) had 5.8-fold fewer transposon insertions after sporulation. Insertions in the essential <i>murAA</i> gene were not tolerated under either condition. <b>(B)</b> Scatterplot showing the extent of decrease in transposon insertions in all genes at T24. Previously identified sporulation genes are highlighted in pink. For each gene, the–log of the Mann Whitney <i>U p</i>-value comparing the T0 and T24 libraries is plotted against the fold reduction in transposon insertions at T24. The statistical significance thresholds (<i>p</i> <0.05, fold reduction >2) are highlighted by dashed red lines. All sporulation genes, including those outside the statistical significance thresholds, are listed in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002341#pbio.1002341.s014" target="_blank">S1 Table</a>. <b>(C)</b> Scatterplot showing the extent of decrease in transposon insertions in all genes at T5. The data are displayed as in (B). A more stringent significance threshold for fold reduction (>5-fold) was used for this dataset. The complete list of hits can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002341#pbio.1002341.s015" target="_blank">S2 Table</a>. The raw underlying numerical data for Fig 1B and 1C can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002341#pbio.1002341.s001" target="_blank">S1 Data</a>.