Tn-seq identifies mutants that initiate sporulation prematurely.

<p><b>(A)</b> Transposon insertion profiles for two regions of the genome at T0, T5, and T24. The number of sequencing reads in these profiles is scaled 10–20 times higher than those in Figs <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002341#pbio.1002341.g001" target="_blank">1</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002341#pbio.1002341.g002" target="_blank">2</a>. Insertions upstream of the <i>kinA</i> and <i>helD</i> genes are significantly enriched at T5. <b>(B)</b> Co-culture assay to assess premature spore formation of mutants identified by Tn-seq. Indicated strains were co-cultured at a 1:1 ratio with a wild-type (WT) strain constitutively expressing <i>lacZ</i>. At the onset of starvation (T0) and after heat-treatment at T5, cultures were plated on LB agar containing X-gal to determine the ratio of mutant to WT CFU. Representative examples of plates showing T0 CFU and T5 spores (heat-resistant CFU) from three mixed cultures (WT/WT, <i>helD</i> over-expression (OE)/WT, and Δ<i>sda</i>/WT) are shown. <b>(C)</b> Bar graph showing the competitive indices of indicated mutants and over-expression strains. The competitive index represents the ratio of mutant to WT spores at T5 divided by the ratio of mutant to WT CFU at T0. Error bars represent standard error of the mean from two biological replicates. The raw underlying numerical data for Fig 3C 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>. <b>(D)</b> A strain over-expressing <i>helD</i> (<i>helD</i> OE) initiates sporulation prematurely. The <i>helD</i> OE strain and WT constitutively expressing mCherry were mixed at a 1:1 ratio and cultured in sporulation medium. The cells were monitored by fluorescence microscopy for entry into sporulation as assayed by σ^F activity in the forespore. Both strains contain a σ^F-responsive promoter (P_{<i>spoIIQ</i>}) fused to <i>yfp</i> (<i>helD</i> OE) or <i>cfp</i> (WT). The percentage of <i>helD</i> OE and WT cells that have activated σ^F are indicated. More than 1,000 cells were counted for each time point. Scale bar indicates 3 µm. Additional examples can be seen in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002341#pbio.1002341.s004" target="_blank">S3B Fig</a>. <b>(E)</b> The experiment in (D) was performed using a 1:1 co-culture of two WT strains.