DdcA antagonizes a bacterial DNA damage checkpoint

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/147820/1/mmi14151.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/147820/2/mmi14151_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/147820/3/mmi14151-sup-0001-Supinfo.pd

Discovery of a dual protease mechanism that promotes DNA damage checkpoint recovery

<div><p>The DNA damage response is a signaling pathway found throughout biology. In many bacteria the DNA damage checkpoint is enforced by inducing expression of a small, membrane bound inhibitor that delays cell division providing time to repair damaged chromosomes. How cells promote checkpoint recovery after sensing successful repair is unknown. By using a high-throughput, forward genetic screen, we identified two unrelated proteases, YlbL and CtpA, that promote DNA damage checkpoint recovery in <i>Bacillus subtilis</i>. Deletion of both proteases leads to accumulation of the checkpoint protein YneA. We show that DNA damage sensitivity and increased cell elongation in protease mutants depends on <i>yneA</i>. Further, expression of YneA in protease mutants was sufficient to inhibit cell proliferation. Finally, we show that both proteases interact with YneA and that one of the two proteases, CtpA, directly cleaves YneA <i>in vitro</i>. With these results, we report the mechanism for DNA damage checkpoint recovery in bacteria that use membrane bound cell division inhibitors.</p></div

Tn-seq yields many false positive results.

<p>The forty genes with the lowest relative fitness in the second growth period of MMC Tn-seq experiment are listed. Each gene was deleted and the deletion mutants were tested for sensitivity to MMC using a spot titer assay and a range of MMC concentrations. Genes labeled as not sensitive had no difference in growth relative to the WT strain on MMC containing media, with the exception of <i>ylbK</i>, which resulted in a polar effect on <i>ylbL</i> (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007512#pgen.1007512.s003" target="_blank">S3 Fig</a>).</p

Forward genetic screen experimental design and data analysis.

<p><b>(A)</b> A plot of the log₁₀ of insertion coverage on the y-axis and genomic position in nucleotides on the x-axis. <b>(B)</b> Experimental design for Tn-seq experiments. The transposon library was used to inoculate starter cultures to allow cultures to reach exponential phase. Cultures were split into control and treatment and grown for three growth periods. <b>(C)</b> Equations used to calculate relative fitness (W, fitness; G, generations, N, number of cells at the start (N₀) or end (N_f) of growth period; F, insertion frequency at the start (F₀) or end (F_f) of growth period; n, number of insertions used to calculate average). <b>(D)</b> The mean gene relative fitness is plotted as a bar graph for the genes indicated for all three Tn-seq experiments, error bars represent the 95% confidence interval. <b>(E)</b> A Venn diagram depicting overlaps of the 200 genes with the lowest fitness and an adjusted p-value less than 0.01 for all three Tn-seq experiments in growth period two.</p

DNA damage checkpoint recovery in <i>Bacillus subtilis</i>.

<p><b>(A)</b> Protease assay incubating purified CtpA or CtpA-S297A with purified YneA for the indicated time followed by SDS-PAGE and staining with coomassie blue. <b>(B)</b> Protease assay incubating purified CtpA or CtpA-S297A with commercially available lysozyme for 3 hours followed by SDS-PAGE and staining with coomassie blue. <b>(C)</b> Bacterial two hybrid assay using T18 fusions (rows) and T25 fusions (columns) co-transformed into <i>E</i>. <i>coli</i>. The T25 plasmids are: the empty vector (T25), T25-YlbL-S234A (T25-YlbL), and T25-CtpA-S297A (T25-CtpA). The T18 plasmids are: the empty vector (T18), T18-YneA, and T18-YneAΔN (which lacks the transmembrane domain). <b>(D)</b> A model for YlbL- and CtpA-dependent DNA damage checkpoint recovery. YlbL and CtpA are present as membrane proteases, and when high amounts of DNA damage are present, YneA production overwhelms both proteases resulting in delayed cell division. After DNA repair is complete and YneA expression decreases, YneA is cleared and cell division proceeds.</p

<i>yneA</i> is required for DNA damage sensitivity and cell elongation phenotypes.

<p><b>(A)</b> Spot titer assay using the indicated genotypes and media. <b>(B)</b> Cell length distributions plotted as histograms. The number of cells scored in each distribution is indicated as “n =” and the genotype of each strain is indicated above the distributions. The dotted vertical line in the “Vehicle” distributions is at the approximate mean of 2.25 μm. The dotted vertical line in the remaining distributions is at 6.75 μm. The y-axis in all graphs is normalized by the sample size yielding the density, and the x-axis is the cell length in μm. <b>(C)</b> The proportion of cells greater than 6.75 μm from the distributions in panel B is plotted as a bar graph. The error bars represent the standard deviation, , where p represents the proportion and n is the sample size. The asterisk indicates a p-value less than 0.05. <b>(D)</b> Spot titer assay testing the effect of over production of YneA using the indicated concentration of the inducer IPTG. <b>(E)</b> Western blot analysis of cell lysates using 100 μM IPTG for YneA expression with the indicated genotypes, using the indicated antiserum.</p

DNA damage delays cytokinesis in cells with protease deletions.

<p><b>(A)</b> Representative micrographs of cells with the indicated genotypes at the indicated time points. Membranes were stained with FM4-64. Scale bar is 5 μm. <b>(B)</b> Cell length distributions plotted as histograms. The y-axis in all graphs is normalized by the sample size yielding the density, and the x-axis is the cell length in μm. The number of cells scored in each distribution is indicated as “n =” and the genotype of each strain is indicated. The dotted vertical line in the “Vehicle” distributions is plotted at 2.25 μm, the approximate mean for all strains. The dotted vertical line in the remaining distributions is at 6.75 μm, which is three times the average length of untreated cells. <b>(C)</b> The proportion of cells represented by the histograms in panel B with length greater than 6.75 μm is plotted as a bar graph. The error bars represent the standard deviation, , where p represents the proportion and n is the sample size. The asterisk indicates a p-value less than 0.05.</p

YneA accumulates in protease mutants.

<p><b>(A)</b> A model for the function of YlbL and CtpA in regulating cell division. <b>(B)</b> Proteomics data plotted as fold change (Double Mutant/WT) vs. the p-value. Points plotted in black have a fold change less than one or a p-value greater than or equal to 0.05, and points plotted in red have a fold change greater than one and a p-value less than 0.05. <b>(C)</b> Western blot analysis of cell lysates from strains with the indicated genotypes at the indicated time points from the MMC recovery assay (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007512#sec014" target="_blank">methods</a>), using YneA or DnaN antiserum.</p

YlbL and CtpA have overlapping functions.

<p><b>(A)</b> Spot titer assay using the indicated genotypes and media. <b>(B)</b> Western blot analysis of cell lysates from the genotypes in panel A, using the indicated antiserum. <b>(C & D)</b> Spot titer assay using the indicated genotypes and media. <b>(E)</b> Western blot analysis of cell lysates from the genotypes indicated in panel D, using the indicated antiserum.</p