<b>Genes manipulated to limit essential cellular processes.</b>

<p><b>Genes manipulated to limit essential cellular processes.</b></p

Nutrient-mediated growth rate regulation of DNA replication initiation in <i>B. subtilis</i>.

<p>(<b>A</b>) Culturing <i>B. subtilis</i> in a different media generates a range of steady-state growth rates and affects the frequency of DNA replication initiation. A wild-type strain (HM222) was grown overnight at 37°C in minimal media supplemented with succinate and amino acids (20 µg/ml). The culture was diluted 1∶100 into various media to generate a range of steady-state growth rates and grown at 37°C until an A₆₀₀ of 0.3–0.4. Genomic DNA was harvested from cells and marker frequency analysis was determined using qPCR. The <i>ori:ter</i> ratios are plotted versus growth rate (error bars indicate the standard deviation of three technical replicates). Representative data are shown from a single experiment; independently performed experiments are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004731#pgen-1004731-g004" target="_blank">Figures 4</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004731#pgen.1004731.s005" target="_blank">S5</a>. (<b>B</b>) Culturing <i>B. subtilis</i> at different temperatures generates a range of steady-state growth rates but does not affect the frequency of DNA replication initiation. A wild-type strain (HM715) was grown overnight at 23°C in LB. The culture was diluted 1∶100 into LB and incubated at different temperatures to generate a range of steady-state growth rates until an A₆₀₀ of 0.2–0.3. Genomic DNA was harvested from cells and marker frequency analysis was determined using qPCR. The <i>ori:ter</i> ratios are plotted versus growth rate (error bars indicate the standard deviation of three technical replicates). Representative data are shown from a single experiment; an independently performed replicate of the experiment is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004731#pgen.1004731.s001" target="_blank">Figure S1</a>. (<b>C</b>) Measurement of DnaA protein levels at various growth rates in wild-type <i>B. subtilis</i> (HM715). Cultures were grown at 37°C overnight as in (A) and diluted 1∶100 into various media (succinate, glycerol, glycerol + amino acids, LB). to generate a range of steady-state growth rates until an A₆₀₀ of 0.6–0.8. Cells were lysed and DnaA protein was detected using Western blot analysis (FtsZ protein was likewise detected and used as a loading control). For each culture media the average amount of DnaA (+/− standard deviation) from at least three biological replicates was determined using densitometry; values were normalized to LB.</p

Analysis of <i>oriC</i>-independent growth rate regulation through genetic targeting of essential cellular activities.

<p>Strains were grown and data presented as described for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004731#pgen-1004731-g005" target="_blank">Figure 5</a>, except that the depletion of PgsA required supplementation with 1 mM IPTG to overexpress the xylose repressor. The <i>ori:ter</i> ratios are plotted versus growth rate and the percentage change in the <i>ori:ter</i> ratios comparing each deletion/depletion is indicated (error bars indicate the standard deviation of three technical replicates). Representative data are shown from a single experiment; an independently performed replicate of the experiment is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004731#pgen.1004731.s008" target="_blank">Figure S8</a>. (<b>A</b>) P_spac-<i>pykA</i> (HM1176), P_spac-<i>pykA</i> Δ<i>oriC oriN⁺</i> (HM1186); (<b>B</b>) P_xyl-<i>pgsA</i> (HM1365), P_xyl-<i>pgsA</i> Δ<i>oriC oriN⁺</i> (HM1374); (<b>C</b>) Wild-type (HM715), Δ<i>rpsU</i> (HM1150), Δ<i>rplA</i> (HM1151), Δ<i>rplW</i> (HM1152), Δ<i>rpmJ</i> (HM1154), Δ<i>oriC oriN⁺</i> (HM950), Δ<i>rpsU</i> Δ<i>oriC oriN⁺</i> (HM1156), Δ<i>rplA</i> Δ<i>oriC oriN⁺</i> (HM1157), Δ<i>rplW</i> Δ<i>oriC oriN⁺</i> (HM1158), Δ<i>rpmJ</i> Δ<i>oriC oriN⁺</i> (HM1160). (<b>D</b>) P_spac-<i>pykA</i> (HM1176), P_spac-<i>pykA</i> Δ<i>dnaA oriN⁺</i> (HM1425); (<b>E</b>) P_xyl-<i>pgsA</i> (HM1365), P_xyl-<i>pgsA</i> Δ<i>dnaA oriN⁺</i> (HM1433); (<b>F</b>) Wild-type (HM715), Δ<i>rpsU</i> (HM1150), Δ<i>rplA</i> (HM1151), Δ<i>rpmJ</i> (HM1154), Δ<i>dnaA oriN⁺</i> (HM1423), Δ<i>rpsU</i> Δ<i>dnaA oriN⁺</i> (HM1429), Δ<i>rplA</i> Δ<i>dnaA oriN⁺</i> (HM1430), Δ<i>rpmJ</i> Δ<i>dnaA oriN⁺</i> (HM1432).</p

Analysis of <i>oriC</i>-dependent and <i>oriC</i>-independent growth rate regulation through small molecule targeting of fatty acid synthesis and protein synthesis.

<p>Strains were grown overnight at 37°C in LB medium. Overnight cultures were diluted 1∶1000 into fresh LB medium either without or with antibiotics (2 µg/ml cerulenin (<b>A</b>), 1 µg/ml chloramphenicol (<b>B</b>)) and grown at 37°C until they reached an A₆₀₀ of 0.3–0.5. For datapoints “+” indicates the presence of the small molecule inhibitor and “−” indicates the absence. Genomic DNA was harvested from cells and marker frequency analysis was determined using qPCR. The <i>ori:ter</i> ratios are plotted versus growth rate and the percentage change in the <i>ori:ter</i> ratios comparing each deletion/depletion is indicated (error bars indicate the standard deviation of three technical replicates). Representative data are shown from a single experiment; an independently performed replicate of the experiment is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004731#pgen.1004731.s009" target="_blank">Figure S9</a>. Wild-type (HM715), Δ<i>oriC oriN⁺</i> (HM950), Δ<i>dnaA oriN⁺</i> (HM1423). (<b>C</b>) Summary of growth rate control systems affecting DNA replication described in this report.</p

Multiple Regulatory Systems Coordinate DNA Replication with Cell Growth in <i>Bacillus subtilis</i>

<div><p>In many bacteria the rate of DNA replication is linked with cellular physiology to ensure that genome duplication is coordinated with growth. Nutrient-mediated growth rate control of DNA replication initiation has been appreciated for decades, however the mechanism(s) that connects these cell cycle activities has eluded understanding. In order to help address this fundamental question we have investigated regulation of DNA replication in the model organism <i>Bacillus subtilis</i>. Contrary to the prevailing view we find that changes in DnaA protein level are not sufficient to account for nutrient-mediated growth rate control of DNA replication initiation, although this regulation does require both DnaA and the endogenous replication origin. We go on to report connections between DNA replication and several essential cellular activities required for rapid bacterial growth, including respiration, central carbon metabolism, fatty acid synthesis, phospholipid synthesis, and protein synthesis. Unexpectedly, the results indicate that multiple regulatory systems are involved in coordinating DNA replication with cell physiology, with some of the regulatory systems targeting <i>oriC</i> while others act in a <i>oriC</i>-independent manner. We propose that distinct regulatory systems are utilized to control DNA replication in response to diverse physiological and chemical changes.</p></div

Nutrient-mediated growth rate regulation of DNA replication initiation is independent of Soj, YabA, and (p)ppGpp.

<p>(<b>A</b>) Growth rate regulation of DNA replication initiation is maintained in either Δ<i>soj</i> or Δ<i>yabA</i> mutants. Strains were grown overnight at 37°C in minimal media supplemented with succinate and amino acids (20 µg/ml). The culture was diluted 1∶100 into various media (succinate, glycerol, glycerol + amino acids, LB) to generate a range of steady-state growth rates and grown at 37°C until an A₆₀₀ of 0.3–0.4. Genomic DNA was harvested from cells and marker frequency analysis was determined using qPCR. The <i>ori:ter</i> ratios are plotted versus growth rate (error bars indicate the standard deviation of three technical replicates). Representative data are shown from a single experiment; an independently performed replicate of the experiment is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004731#pgen.1004731.s005" target="_blank">Figures S5A-B</a>. Wild-type (HM222), Δ<i>soj</i> (HM227), Δ<i>yabA</i> (HM739), Δ<i>soj</i> Δ<i>yabA</i> (HM741). (<b>B</b>) Growth rate regulation of DNA replication initiation does not require (p)ppGpp. Strains were grown overnight at 37°C in minimal media supplemented with succinate and amino acids (200 µg/ml). The culture was diluted 1∶100 into various media (succinate + amino acids, glycerol + amino acids, LB, PAB) to generate a range of steady-state growth rates and grown at 37°C until an A₆₀₀ of 0.2–0.6. Genomic DNA was harvested from cells and marker frequency analysis was determined using qPCR. The <i>ori:ter</i> ratios are plotted versus growth rate (error bars indicate the standard deviation of three technical replicates). Representative data are shown from a single experiment; an independently performed experiment is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004731#pgen.1004731.s005" target="_blank">Figure S5C</a>. Wild-type (HM222), Δ(p)ppGpp (HM1230).</p

Analysis of <i>oriC</i>-dependent growth rate regulation through genetic targeting of essential cellular activities.

<p>Strains were grown overnight at 37°C in LB medium; strains harbouring plasmids integrated into the genome by single-crossover were supplemented with appropriate antibiotics and inducer (0.1 mM IPTG or 0.1% xylose). Overnight cultures were diluted 1∶1000 into fresh LB medium and grown at 37°C until they reached an A₆₀₀ of 0.3–0.5; strains harbouring plasmids integrated by single-crossover were supplemented with appropriate antibiotics either without or with the appropriate inducer (1 mM IPTG or 1% xylose). For datapoints “+” indicates the presence of either the wild-type gene (when comparing with knockout mutants) or the inducer; “−” indicates the absence of either the gene (when comparing with wild-type) or the inducer. Genomic DNA was harvested from cells and marker frequency analysis was determined using qPCR. The <i>ori:ter</i> ratios are plotted versus growth rate and the percentage change in the <i>ori:ter</i> ratios comparing each deletion/depletion is indicated (error bars indicate the standard deviation of three technical replicates). Representative data are shown from a single experiment; an independently performed replicate of the experiment is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004731#pgen.1004731.s007" target="_blank">Figure S7</a>. (<b>A</b>) Wild-type (HM715), Δ<i>ndh</i> (HM1318), Δ<i>oriC oriN⁺</i> (HM957), Δ<i>ndh</i> Δ<i>oriC oriN⁺</i> (HM1319); (<b>B</b>) P_spac-<i>gapA</i> (HM1208), P_spac-<i>gapA</i> Δ<i>oriC oriN⁺</i> (HM1221); (<b>C</b>) Cultures were supplemented with 0.2% sodium acetate. Wild-type (HM715), Δ<i>pdhB</i> (HM1248), Δ<i>oriC oriN⁺</i> (HM950), Δ<i>pdhB</i> Δ<i>oriC oriN⁺</i> (HM1266); (<b>D</b>) P_spac-<i>fabHA</i> (HM964), P_spac-<i>fabHA</i> Δ<i>oriC oriN⁺</i> (HM966); (<b>E</b>) P_xyl-<i>plsC</i> (HM1080), P_xyl-<i>plsC</i> Δ<i>oriC oriN⁺</i> (HM1086); (<b>F</b>) Wild-type (HM715), Δ<i>ltaS</i> (HM1168), Δ<i>oriC oriN⁺</i> (HM957), Δ<i>ltaS</i> Δ<i>oriC oriN⁺</i> (HM1244).</p

Nutrient-mediated growth rate regulation of DNA replication initiation requires <i>oriC</i> and DnaA.

<p>(<b>A</b>) <i>oriC</i> is required for growth rate regulation of DNA replication initiation. Strains were grown overnight at 37°C in minimal media supplemented with succinate and amino acids (20 µg/ml). The culture was diluted 1∶100 into various media (succinate, glycerol, glycerol + amino acids, LB, PAB) to generate a range of steady-state growth rates and grown at 37°C until an A₆₀₀ of 0.3–0.4. Genomic DNA was harvested from cells and marker frequency analysis was determined using qPCR. The <i>ori:ter</i> ratios are plotted versus growth rate (error bars indicate the standard deviation of three technical replicates). Representative data are shown from a single experiment; an independently performed replicate of the experiment is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004731#pgen.1004731.s006" target="_blank">Figure S6A</a>. Wild-type (HM222), Δ<i>oriC oriN⁺</i> (HM228). (<b>B</b>) Integration of <i>oriN</i> into the <i>B. subtilis</i> chromosome does not eliminate growth rate regulation of DNA replication initiation. Strains were grown as in (A) and the overnight culture was diluted 1∶100 into various media (succinate, glycerol, glycerol + amino acids, LB). Genomic DNA was harvested from cells and marker frequency analysis was determined using qPCR. The <i>ori:ter</i> ratios are plotted versus growth rate (error bars indicate the standard deviation of three technical replicates). Representative data are shown from a single experiment; an independently performed replicate of the experiment is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004731#pgen.1004731.s006" target="_blank">Figure S6B</a>. Wild-type (HM715), <i>oriC</i>⁺<i>oriN⁺</i> (HM949). (<b>C</b>) DnaA activity is required for growth rate regulation of DNA replication initiation. Strains were grown as in (B). Genomic DNA was harvested from cells and marker frequency analysis was determined using qPCR. The <i>ori:ter</i> ratios are plotted versus growth rate (error bars indicate the standard deviation of three technical replicates). Representative data are shown from a single experiment; an independently performed replicate of the experiment is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004731#pgen.1004731.s006" target="_blank">Figure S6C</a>. Wild-type (HM715), DnaA^R264A<i>oriN⁺</i> (HM1122). (<b>D</b>) Measurement of DnaA protein levels at various growth rates in a Δ<i>oriC oriN⁺</i> strain (HM950). Cultures were grown as described in (B). Cells were lysed and DnaA protein was detected using Western blot analysis (FtsZ protein was likewise detected and used as a loading control). For each culture media the average amount of DnaA (+/− standard deviation) from at least three biological replicates was determined using densitometry; values were normalized to LB. (<b>E</b>) Subcellular localization of DNA over a range of growth rates in the wild-type (HM715) and Δ<i>oriC oriN⁺</i> (HM950) strains. Cells were grown as in (B) and the overnight culture was diluted 1∶100 into various media (succinate, glycerol, or glucose + amino acids). Samples were taken at an A₆₀₀ of 0.3–0.5 at which point membranes and DNA were stained. Arrows indicate cells without DNA and asterisks indicate space within the cell that does not contain DNA. Scale bar represents 3 µm.</p

Positions of two origins visualized simultaneously.

<p><b>A</b>. The origins of two replicons are plotted relative to the distance from the pole nearest any focus. Insets show numbers of cells with two foci of the replicon correspondingly coloured in the plot, comprising those with either one or two foci of the other replicon visualized; cells lacking foci of either replicon were excluded from the analysis. Red and green discs indicate respectively Chfp and Gfp fusions used to mark the origins shown. <b>B</b>. Examples of cells with origins of two replicons co-visualized. The separate components of the overlays are shown as examples of the images on which length measurements were made. Scale bar is 1 μm. <b>C</b>. Frequencies of focus combinations, shown as percentage of total cells scored.</p

Replication characteristics determined by high-throughput sequencing.

<p><b>A</b>. Base-pair frequency gradients. Data points are averages of binned read numbers representing successive blocks of 10kbp for c1 and c2 and 1kbp for c3. The top panels show the raw data for DNA from exponentially-growing cells, the third row shows the same data after division by the correspondingly binned data obtained from stationary-phase cells (second row). Nucleotide positions on the abscissa are reversed to conform to the intuitive sense of right and left chromosome arms. Because the data are plotted as raw read frequencies, relative copy numbers of the replicons can be read from the ordinates. <b>B</b>. Calculation of chromosome replication period, C, and speed. Origin/terminus ratios were used to calculate the time taken to replicate each chromosome arm from <i>ori</i>/<i>ter</i> = 2^C/τ. In the case of c1 and c2, the concavity of the stationary-phase base-pair frequency curves would falsify calculation of origin/terminus ratios from normalized data, necessitating use of the raw data plot. For this, raw data <i>ori</i>- and <i>ter</i>-proximal points that corresponded to points intersected by the linear regression plot of the normalized data were connected by lines whose upper extremities and intersection were taken as the <i>ori</i> and <i>ter</i> values respectively. The c3 stationary-phase bp frequency curve is essentially flat, validating the normalized data. The * values for c3 replication speed are calculated on the basis of terminus displacement creating arms estimated to be 340 and 530 kb long.</p